AU765258B2 - Novel method of regulating seed development in plants and genetic sequences therefor - Google Patents

Description

WO 00/16609 PCT/AU99/00805 -1- NOVEL METHOD OF REGULATING SEED DEVELOPMENT IN PLANTS AND GENETIC SEQUENCES THEREFOR FIELD OF THE INVENTION The present invention relates generally to a method of inducing autonomous (i.e.

fertilisation independent) seed development in plants, including but not limited to the induction of autonomous endosperm development and/or partial autonomous embryo development. The invention further provides genes which are capable of regulating seed development in -plants and pertains to their use in preventing fertilizationdependant seed production or reducing the frequency thereof. More particularly, the present invention provides isolated nucleic acid molecules comprising nucleotide sequences which encode or are- complementary to.nucleotide sequences which encode regulatory polypeptides involved in the progressive development of an ovule into a seed in plants. The isolated nucleic acid molecules of the invention are useful for the production of plants having a wide range of novel phenotypes including, but not limited to, the ability to reproduce asexually, develop seed in the absence of fertilization, and the ability to produce parthenocarpic fruit or seedless fruit or fruits with soft seed traces such that the fruit are marketable as less seedy than wild-type fruit or seedless. The isolated nucleic acid molecules are further -useful in the detection of proteins and genetic sequences which interact with the polypeptides encoded by said nucleic acid molecules in the regulation of seed development in plants, thereby producing a novel range of products for the genetic modification of seed development.

GENERAL

Those skilled in the art will be aware that the invention described herein is subject to variations and modifications otherthan those specifically described. It is to be understood thatthe invention described herein includes all such- variations and modifications. The invention also includes all such steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

Throughout this specification, unless the context requires otherwise the word WO 00/16609 PCT/AU99/00805 -2- "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

Bibliographic details of the publications referred to by author in this specification are collected at the end of the description.

This specification contains nucleotide and amino acid sequence information prepared using the programme Patentln Version 2.0, presented herein after the bibliography.

Each nucleotide or amino acid sequence is identified in the sequence listing by the numeric indicator <210> followed by the sequence identifier <210>1, <210>2, etc). The length, type of sequence (DNA, protein (PRT), etc) and source organism for each nucleotide or amino acid seqeunce are indicated by information provided in the numeric indicator fields <211>, <212> and <213>, respectively. Nucleotide and amino acid sequences referred to in the specification are defined by the information provided in numeric indicator field <400>followed by the sequence identifier (eg. <400>1, <400>2, etc).

The designation of nucleotide residues referred to herein are those recommended by the IUPAC-IUB Biochemical Nomenclature Commission, wherein A represents Adenine, C represents Cytosine, G represents Guanine, T represents thymine, Y represents a pyrimidine residue, R represents a purine residue, M represents Adenine or Cytosine, K represents Guanine or Thymine, S represents Guanine or Cytosine, W represents Adenine or Thymine, H represents a nucleotide other than Guanine, B represents a nucleotide other than Adenine, V represents a nucleotide other than Thymine, D represents a nucleotide other than Cytosine and N represents -any nucleotide residue. The designation of amino acid residues referred to herein are also those recommended by the IUPAC-IUB Biochemical Nomenclature Commission, as indicated in Table 1. For those sequences comprising the variable residue Xaa X), it will be known to those skilled in the art that two or more consecutive Xaa residues WO 00/16609 PCT/AU99700805 -3in an amino acid sequence may be identical or non-identical residues, and the present invention is not limited by any particular configuration of such sequences unless specifically stated otherwise in the specification. The amino acid designation B (Asx) is also known by those skilled in the art to indicate an occurrence of Aspartate or Asparagine at a particular position in an amino acid sequence. The amino acid designation Z (GIx) is also known by those skilled in the art to indicate an occurrence of Glutamate or Glutamine at a particular position in an amino acid sequence.

As used herein, the term "derived from" shall be taken to indicate that a particular integer or group of integers has originated from the species specified, but has not necessarily been obtained directly from the specified source.

BACKGROUND TO THE INVENTION In plants which reproduce by sexual means, the endosperm and embryo of the developing seed are normally formed from the megagametophyte the embryo sac) which is contained within the central region of the ovules, whilst the integument(s) and other surrounding structures which enclose the megagametophyte differentiate into a seed coat. The development of the embryo sac in flowering plants can be divided into two stages, megasporogenesis and megagametogenesis. During megasporogenesis the female archesporial cells undergo meiosis and four megaspore cells are formed.

The polygonum-type of embryo sac formation is the most common type observed in flowering plants occurring, for example in Arabidopsis thaliana (Mansfield et al., 1991).

Polygonum-type embryo sacs form from the megaspore situated in the chalazal end of the ovule, after the three non-functional megaspores in the micropylar end degenerate. The remaining functional chalazal megaspore undergoes three successive mitotic divisions to produce the female gametophyte containing eightnuclei.

The embryo sac develops sexual competence within the gynoecium; following nuclear migration and cellularization events. The polygonum-type embryo sac has one egg cell, two synergids, three antipodal cells and a central cell containing two nuclei. The egg cell is located at the micropylar end of the embryo sac and, following fertilization, WO 00/16609 PCT/AU99/00805 -4the egg nucleus ultimately fuses with one of the male sperm nuclei to produce a zygote, the progenitor of the embryo. The egg is adjacent to two synergids which may play an important role in fertilisation by aiding in pollen tube attraction and guidance and facilitating the incorporation of the sperm nuclei into the egg and central cells.

The polar nuclei are fertilised by the other sperm nucleus, generating the triploid primary endosperm nucleus and completing the double fertilisation event characteristic of angiosperms. The mature endosperm nucleus undergoes several rounds of division without cytokinesis to generate a large number of free nuclei organised at the periphery of the central cell. Cytokinesis then ensues, progressing centripetally, until the endosperm becomes entirely cellular.

The fate of the endosperm can vary between plant species. In Arabidopsis thaliana, the endosperm is utilised during embryo development, whilst in cereals the endosperm persists.

The function of three antipodal cells located at the chalazal end of the embryo sac is not known, however they are thought to be involved in the import of-nutrients to the embryo sac. In some plants, for example Arabidopsis thaliana, the antipodal cells degenerate prior to fertilisation, whilst in other plants, such as cereal crop plants, they can proliferate.

A summary of embryogenesis in Arabidopsis thaliana is presented in Figure 1.

Little is-known of the mechanism or biochemistry of ovule development or the mechanism or biochemistry of the subsequent development of the ovule into a seed.

Specific regulatory mechanisms controlling such processes remain to be elucidated.

Many higher plants are capable of forming seed in the absence of fertilisation, a process known as apomixis (Asker and Jerling, 1992). Studies of fertilizationindependent seed production indicates that, in such plants embryos may-form inside embryo sacs derived from cells that have not undergone meiosis apospory or WO 00/16609 PCT/AU99/00805 diplospory) or the embryos may form directly from other maternal ovule cells. For example, in orchids, citrus and mango plants, adventitious embryos arise from the cells of the nucellus or inner integuments.

In plants such as Poa spp. and Pennisetum spp., aposporous embryo sacs may arise via mitosis from cells that differentiate from the nucellus following megaspore mother cell differentiation, wherein the aposporous embryo sac may develop more rapidly than the sexual embryo sac present in the same ovule, possibly because they are not delayed by meiosis (Koltunow, 1993). In many such cases, the development of the sexual embryo sac is often terminated (Asker and Jerling, 1992). lIn plants that undergo aposporous embryo sac formation, endosperm development usually, but not always, requires pseudogamy pollination and fusion of the sperm cell with only theunreduced polar cell or equivalent), however autonomous endosperm development following aposporous embryo sac formation does occur in Hieracium spp (Asker and Jerling, 1992).

Furthermore, in diplosporous plants, meiosis may be inhibited or aberrant or aborted at an early stage during megasporogenesis at the time the spores are formed).

In Antennaria spp., the megaspore mother cell is prevented from entering meiosis or undergoes an aberrant meiosis which resembles mitosis, such that the embryo sac produced has the same number of cells as a sexual embryo sac for that species. On the other hand, in Taraxacum spp., meiosis is aborted at an early stage and mitosislike divisions give rise to dyads, in the absence or presence of recombination.

Diplospory has also been observed in Ixerisspp and in the cruciferous plant Arabis holboellii (Asker and Jerling, 1992; Bocher, 1951; Roy and Reiseberg, 1989).

Genetic control of seed development and in particular, fertilisation-independent seed development, may involve only a few genes. Adventitious embryony in citrus appears to be controlled by a single dominant locus (Parlevliet and Cameron 1959; Iwamasa et al., 1967; Asker and Jerling, 1992). Recent reports on genetic control of apospory in Pennisetum species indicate that apospory may be controlled by a single dominant gene locus (Ozias-Akins et al., 1993; 1998). Work in Panicum and Ranunculus also WO 00/16609 PCT/AU99/00805 indicate similar control (Reviewed by Koltunow, 1993). The trait of apospory observed in Pennisetum squamulatum has.been introduced to a sexual species pearl millet and the resulting apomictic line has been shown to contain a single supernumerary chromosome containing the apomictic gene from P. squamulatum. The transferred chromosome can be detected by RFLPs and molecular markers linked to apospory have recently been identified on the transferred chromosome (Ozias-Akins et al., 1993; 1998).

There have not been many reports on studies of the genetic control of diplospory, however a recent study of diplospory in Taraxacum suggests that the control of female meiosis or apomixis may reside on a single chromosome and probably at a single locus (Reviewed by Koltunow, 1993) however, the gene(s) controlling diplosporous apomixis remain to be elucidated in this species.

Regulating seed development in plants has enormous economic utility in the horticulture and agriculture industries. For example, producing soft-seeded fruit (i.e.

fruit that lack an embryo and/or are shrivelled or shrunken or degenerate during development) or fruit having no seed, which fruit are more appealing to consumers, in particular with regard to edible fruits such as stone fruits, citrus fruits, grapes and melon varieties, amongst others. Additionally, plants that are capable-of autonomous seed formation in the absence of fertilisation are highly desirable products. Because plants which undergo autonomous seed formation do not require fertilisation to reproduce, such plants may express desirable characteristics stably between generations.

SUMMARY OF THE INVENTION In work leading up to the present invention, the inventors sought to elucidate the regulatory mechanisms involved in seed and fruit development in higher plants. The inventors developed a visual screen to facilitate the identification of genes which are capable of being used to regulate the development of the ovule into seed and may be used to produce fruit having soft seed, especially in the absence of fertilization.

WO 00/16609 PCT/AU99/00805 -7- In particular, the inventors have chemically-mutagenised a male-sterile, butfully female-fertile plant line which is incapable of forming seed in the absence of a pollen donor, to produce plants which are both capable of forming seed in the absence of a pollen donor- and capable of producing soft-seeded fruit or seedless fruit in the absence of a pollen donor. By characterising a transposon-tagged mutant which belongs to the same complementation group as the chemically-induced mutant, the inventors were able to isolate genomic DNA from the tagged mutant in the region surrounding the transposon and to demonstrate that the homologous genomic DNA derived from a wild-type plant is able to complement the mutation in geneticallytransformed mutant plants. The mutated gene which has been complemented using this approach has been designated.as the FIS2 gene.

The inventors have identified two additional genes, designated FIS1 and FIS3, which are also. capable of regulating autonomous endosperm-development -and/or autonomous embryogenesis and/or autonomous seed development in plants and in particular, in Arabidopsis thaliana._ In summary, the FIS family of genes described herein have been shown by the present inventors to be at least partial negative regulators of autonomous endosperm development and/or autonomous embryogenesis.

Accordingly, one aspect of the present invention provides a method of inducing autonomous endosperm development in a plant, said method at least comprising the step of inhibiting, interrupting or otherwise reducing the expression of a negative regulator of seed formation in one or more female reproductive cells, tissues or organs of said plant or a progenitor cell, tissue or organ thereof. According to this embodiment of the invention, the reduced expression of the negative regulator is achieved by the introduction of a transgene which comprises a FIS genetic sequence in the sense or antisense orientation as described herein.

Preferably, the inventive method provides in part or whole for autonomous embryogenesis and more preferably, for autonomous seed development in plants.

WO 00/16609 PCT/AU99/00805 -8- In a particularly preferred embodiment, the negative regulator of seed formation is a FIS polypeptide which comprises an amino acid sequence which is at least about identical to any one of <400>1 or <400>2 or <400>3, or alternatively or in addition which is capable of being encoded by a nucleotide sequence which is at least about identical to the nucleotide sequence set forth in any one of <400>4,-<400>5, <400>6, <400>7, <400>8 or <400>9, or a sequence complementary thereto.

A second aspect of the invention provides isolated nucleic acid molecules which are used to inhibit, prevent or interrupt the expression of a FIS polypeptide in a plant according to the inventive method, including those genomic equivalents of the Arabidopsis thaliana FIS polypeptides exemplified herein.

A third aspect of the invention provides a transgenic plant or a plant cell, tissue, organ produced according to the method described herein, including the seed produced by said plant and progeny plants derived therefrom which are capable of forming softseed in the absence of fertilisation or alternatively, which are capable of forming fullyfertile seed in the absence of fertilisation.

A further aspect of the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence which encodes or is complementary to a nucleotide sequence which encodes a FIS polypeptide, protein or enzyme which is capable of regulating seed development in plants. Preferably, the subject nucleic acid molecule is involved in regulating the development of the ovule into seed in the absence of fertilization, such as by acting as a repressor of autonomous embryogenesis and/or a partial repressor of autonomous endosperm development.

In one embodiment, the isolated nucleic acid molecule of the invention encodes FIS1, a member of the E(z) class of proteins which also comprises novel amino acid sequence motifs not normally associated with this class of protein, in particular a TNFR/NGFR protein domain, an R-G-D tripeptide domain and a novel domain designated the WCA motif. The FIS1 polypeptide preferably comprises an amino acid WO 00/16609 PCT/AU99/00805 -9sequence which is at least about 50% identical to the amino acid sequence set forth in <400>1.

In another embodiment, the isolated nucleic acid molecule of the invention encodes FIS2, a zinc-finger or zinc-finger-like protein. The invention clearly extends to isolated nucleic acid molecules which encode zinc-finger or zinc-finger-like proteins which comprises an amino acid sequence which is at least about 50% identical to the amino acid sequence set forth in <400>2.

In yet another embodiment, the isolated nucleic acid molecule of the invention encodes FIS3 and is capable of hybridizing under at least low stringency hybridization conditions to that region of chromosome 3 of Arabidopsis thaliana which maps between the markers m317 and DWF1 as set forth in Figure 9B, or which is at least about 50% identical to the amino acid sequence set forth in <400>3.

In an alternative embodiment, the isolated nucleic acid molecule of the invention comprises a nucleotide sequence which is at least about 50% identical to the nucleotide sequences set forth in any one of <400>4, <400>5, <400>6, <400>7, <400>8, or <400>9, or a complementary nucleotide sequence thereto.

In a further alternative embodiment, the isolated nucleic acid molecule of the invention comprises a nucleotide sequence which is capable of hybridizing under at least low stringency-hybridization conditions to the nucleotide sequences set forth in any one of <400>4, <400>5, <400>6, <400>7, <400>8, or <400>9, or a complementary nucleotide sequence thereto.

In a particularly preferred embodiment,-the isolated nucleic acid molecule of the invention comprises the nucleotide sequence set forth in any one of <400>4, <400>5, <400>6, <400>7, <400>8, or <400>9, or a complementary nucleotide sequence thereto or a homologue, analogue or derivative of said nucleotide sequences.

A further aspect of the invention provides a cell which has been transformed or WO 00/16609 PCT/AU99/00805 transfected with the subject nucleic acid molecule or a dominant-negative sense molecule or an antisense molecule or a ribozyme molecule or a gene-targeting molecule or a co-suppression molecule which is derived from a nucleic acid molecule comprising a FIS gene, preferably in an expressible form. The present invention clearly extends to transformed tissues, organs and whole organisms comprising the subject nucleic acid molecule or a dominant-negative sense molecule or an antisense molecule or a ribozyme molecule or a gene-targeting molecule or a co-suppression molecule which is derived from said nucleic acid molecule.

in a particularly preferred embodiment, the invention provides a plant cell, tissue, organ or whole plant which comprises the nucleic acid molecule described herein or a dominant-negative sense molecule or an antisense molecule or a ribozyme molecule or a gene-targeting molecule or a co-suppression molecule which is derived from said nucleic acid molecule. The invention extends to the progeny of such a plant, the only requirement being that said progeny also contain said nucleic acid molecule, dominant-negative sense molecule, antisense molecule, ribozyme molecule, genetargeting molecule or a co-suppression molecule.

A still further aspect of the invention provides an isolated promoter sequence which is capable of conferring expression at least in one or more female reproductive cells, tissues or organs of said plant or a progenitor cell, tissue or organ thereof.

A still further aspect of the present invention provides an isolated or recombinant FIS.

polypeptide or a homologue, analogue, derivative or epitope thereof.

The recombinant FIS polypeptides or derivatives thereof comprising FIS protein domains which are involved in forming protein:protein interactions are particularly useful in the isolation of further peptides and polypeptides which are normally regulated by said FIS polypeptides. By appropriate strategies described herein, the nucleic acid molecules encoding said peptides and polypeptides may also be isolated and expressed in the cells under the control of suitable promoter sequences, such as a FIS gene promoter, to induce autonomous endosperm development and/or WO 00/16609 PCT/AU99/00805 S- 11 autonomous embryogenesis and/or autonomous or pseudogamous seed development in plants.

A further aspect of the invention extends to an a monoclonal or polyclonal antibody molecule which is capable of binding to a FIS polypeptide or an epitope thereof.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic representation showing female gametophyte, fertilisation and embryogenesis of Arabidopsis thaliana embryogenesis. The ovule contains the female gametophyte composed of an egg, a 2n central cell, two synergids next to the egg, and three antipodal cells in the chalazal end. Pollen tube enters the ovule through the micropyle and delivers two sperm cells that fuse with the egg and the central cell. Following fertilisation, a-zygote and a primary endosperm cell are produced. During embryogenesis, embryo and endosperm development occurs.

At the end of embryogenesis a mature embryo is formed.

Figure 2 is a schematic representation of a genetic screen used to detect autonomous endosperm mutants in Arabidopsis thaliana, showing threedifferent types of readily distinguishable flower morphologies. Morphology type 1 is the pistillata homozygous type in which the siliques are short and there are no stamens or pollen. Morphology type 2 indicates self-fertile plants with stamens and siliques that are longer than Type 1. Morphology type 3 is the putative fis mutant. In this type, although the siliques are long, there are no petals or stamens, indicating that pistillata has not reverted (from Peacock et al., 1995).

Figure 3 is a copy of a photographic representation showing wild-type arid fisseed Sdevelopment. Seed development ofwild-type Arabidopsis thaliana and fis mutants are compared at developmental phases (Bowman and Koornneef, 1994). Phase 1 shows ovules connected to the ovary wall by the funiculus; in the subsequent phases, only the developing seed is shown. The relative size of the ovule compared with the developing seed is shown by the Inset. The lengths of siliques at the different phases are: phase 1:0.29 0.04 mm (0 HAF); phase 2:0.60 0.08 mm (36 HAF); phase WO 00/16609 PCT/AU99/00805 -12- 3:1.00 ±0.07 mm (72 HAF); and phase 4 1.26 ±-0.07 mm (120 HAF). a, b, and c represent different developmental types seen in the fis mutants. X, Y, and Z-represent postulated genes other than FIS1, FIS2, and FIS3.

Figure 4 is a photocopy of a photographic representation of cryoscanning electron micrographs of ovules and seeds of fis mutants and fertilized wild-type plants.

Developing ovules [nucellar column protruding from the inner integument (ii) and the outer integument (oi) as-shown in B] of wild-type, fisl/fisl homozygotes, (C) fis2/fis2 homozygote, and FIS3/fis3 heterozygote. Sexually fertilized seeds (s) of pi/pi FIS/FIS plants 7 days after fertilization. Unfertilized ovules shrivel (arrow).

Seeds developing without fertilization(s) of fis1/fisl homozygote, fis2/fis2 homozygotes, and FIS3/fis3 heterozygote. Collumella on the surface of (I) sexually fertilized seeds of wild type and autonomously-developing fis2/fis2 homozygous seeds. (Bar: 20/,m for A-C, G, I, and J; 100 pm for D-F; and 200 Hm for H) (from Chaudhury etal.,1997).

Figure 5 is a copy of a photographic representation showing various stages of embryo development in wild-type plants and fis mutant plants, as follows. Panel 1, 7-day old wild type embryo; panel 2, 7-day old fisl mutant embryo (Ler background) arrested at the heart stage; panel 3, 7-day old fis2 mutant embryo (Ler background) arrested at the heart stage; panel 4, 7-day old fis3 mutant embryo (Ler background) arrested at the heart stage; panel 5, 7-day old fis2/fis2 homozygous mutant embryo (Col background) arrested at the heart stage; panel 6, fis2/fis2 homozygous mutant embryo (Col background) arrested at the torpedo stage; panel 7, 7-day old fisl/fis2-2 double homozygous mutant embryo arrested at the heart stage; and panel 8, welldeveloped embryo of fis1/fis2-2 double homozygous mutant.

Figure 6 is a graphical representation showing the localization of the fisl allele and the mea allele on chromosome 1 of Arabidopsis thaliana. The BAC clones 14010 and 14J10 were isolated using the mea probe. The position of the BACs and marker genes is based on the information from the AbtD.

WO 00/16609 PCT/AU99/00805 13.- Figure 7 is a graphical representation of the position of fis2 locus on chromosome 2.

The relative position of the fis2 locus and RFLP markers YUP11D2R end, 11A7L end, and BAC26D2 fragment 5BC was established by examining the segregation of RFLPs' in plants with recombination breakpoints in either the er-fis2 or the fis2-as interval.

YUP9D3, and 11D2 were originally identified based on their location, shown in the WEB site describing the Arabidopsis thaliana-mapped YACs. 11A7L end showing tight linkage with fis2 was used to isolate cosmid pOCA18H1 (in vector pOCA18). The length of YAC, BAC, and cosmid clones are shown in parenthesis.

Figure 8 is a graphical representation showing the localisation of the fis3 locus on chromosome 3, between the morphological markers hy and gl. The position of the SSLP marker nga162 and the RFLP marker ve039 are also indicated. The position of the transposable Ds elerfient in a transposon-tagged fis3 mutant line is also indicated- (DT51). Numbers in brackets refer to recombination distance (cM).

Figure 9A is a graphical representation showing the localisation of morphological markers, cosmid clones, BAC clones, YAC clones and RFLP markers on chromosome 3 of Arabidopsis thaliana.

Figure 9B is a graphical representation showing the localisation of-morphological markers, cosmid clones, BAC clones, YAC clones and RFLP markers around the RFLP marker ve039 fis3 locus on chromosome 3 of Arabidopsis thaliana.

Figure 10A is a graphical representation of the F1 plant P19 resulting from the cross DSG X Ac. Two sectors (branches) of this plant show fis-like phenotype, as indicated by the black circles whilst the normal phenotype is indicated by the white circles Figure I0B is a photographic representation of a Southern blot of BamHI digested genomic DNA from the transposon-tagged plant P19 and a wild type plant. The probe used correspond to a fragment of approximately 10kb in length (3BB) from cosmid WO 00/16609 PCT/AU99/00805 -14cosl8H1 which contains fragment E2 (Figure 11).

Figure 11 is a schematic representation of the physical map of the cosmid pOCA18H1. The genetic loci indicated are; LB, left border repeat; NOS-NPT-OCS, a chimeric gene which is expressed in plant cells and confers resistance to kanamycin; plAN7, contains a ColE1 plasmid origin of replication and a bacterial supF tRNA gene; COS, the cos region from phage lambda; RB, right border repeat; TET, a bacterial tetracycline resistance gene. The direction of transcription for the NOS-NPT-OCS gene is indicated by the arrow. The restriction sites indicated are: B, BamHI; C, Clal; E, EcoRI; H, EcoRV, V; Hindlll; K, Kpnl; P, Pstl; and S, Sail. The A. thaliana genomic DNA partially digested with Taql was ligated in the C/al digested pOCA18. The corresponding site of insertion ofthe DSG transposon in DNA obtained from the fis2-2 tagged mutant is indicated by the open triangle.

Figure 12 is a schematic representation of a silique from fis2/FIS2 heterozygote and a silique from the cross of fis2/fis2 homozygote with transgenic A. thaliana ecotype C24 containing the T-DNA from cosmid pOCA18H1. Black circles correspond to good fertile seeds and open circles correspond to sterile seeds.

Figure 13A is a schematic representation of the single base pair changes occurring in the fis2 gene of mutant fis2-1 plants. The amino acid sequence (SEQ ID NO: <400>211) is shown below the nucleotide sequence (SEQ ID NO: <400>210).

Numbers on the left hand side correspond to the nucleotide sequence and numbers on the right hand side correspond to the amino acid sequence. The localization of the fis2-1 mutation (deletion of T) is shown with the resulting- frame-shift. The stop codon is indicated with an asterisk Lower case letters show the intron sequence.

Figure 13B is a schematic representation of the single base pair changes occurring in the fis2 gene of mutant fis2-3 plants. The amino acid sequence (SEQ ID NO: <400>212) is shown below the nucleotide sequence of the-wild-type gene (SEQ ID NO: <400>213). Numbers on the left hand side correspond to the nucleotide sequence WO 00/16609 PCT/AU99/00805 and numbers on the right hand side correspond to the amino acid sequence. The nucleotide sequence around the fis2-3 mutation (G to A) at the junction of intron 5 and exon 6 is also shown.

Figure 14 is a graphical representation of the FIS2 amino acid sequence(SEQ ID NO: <400>2), showing the locations of the acidic regions (single underlined); the putative nuclear localization signal (NLS; double-underlined) identified by functional expression studies; and the C2H2 zinc finger motif (triple underlined) including conserved cysteine and histidine residues.

Figure 15 is a graphical representation of a bi-dimensional plot of-a C-terminal region of the FIS2 predicted protein sequence showing the tandem repeats between residue 120 and 520 thereof. The dot matrix was obtained using the software Antherprot V3.2 with a window size of 19 amino acids and a identity threshold of 10. The principle of the method is described in (Staden, 1982).

Figure 16 is a photographic representation of a Southern.blot showing A. thaliana FIS2 genome organisation. Genomic DNA was digested with either BamHI, Bglll, or Clal prior to electrophoresis. The DNA was transferred onto nylon membranes and hybridized with the Fis2 cDNA insert.

Figure 17 is a photographic representation of the expression pattern of the Fis2 transcript in root, shoot, leaf, bolt, flower and silique of wild type Arabidopsis as detected by RT-PCR analysis.

Figure 18 is a representation showing the FIS1 nucleotide sequence (SEQ ID NO: <400>4) and deduced amino acid sequence of thewild-type MEDEA/FIS1 polypeptide (SEQ ID NO: <400>1). The acidic region is underlined. The C5 domain is in boldface.

The cysteines of the CXC domain are are in boldface and underlined. Basic residues of a putative bi-partite nuclear localization signal are indicated by asterisks under the amino acid residues. The 115-amino acid SET domain is boxed. The position of nucleotide changes in the fisl mutant allele and the point of insertion of the transposon WO 00/16609 PCT/AU99/00805 -16in the medea mutant are indicated by the arrows.

Figure 19 is a schematic representation showing three polycomb group polypeptides from Arabidopsis thaliana (FIS1, EZA1 and CURLY LEAF), the Drosophila melanogaster Enhancer of zeste polypeptide and the Caenorhabditis elegans Maternal-Effect Sterile-2 (MES-2) polypeptide. The SET domain is shown as a shaded box. The CXC domain is shown as a hatched box. Positions-of the acidic-domain putative nuclear localization signal and C5 domain are indicated. The arrows on the FIS1 protein indicate the positions of mutations in the corresponding gene which produce the fisl mutant phenotype (black arrow) and the mea mutant phenotype (open arrow). Numbers on the right refer to the protein length in amino acid residue.

Figure 20 is a schematic representation showing the amino acid sequence alignment of various Enhancer of zeste E(z)-like proteins around the C5 cysteine-rich domain.

The asterisks indicate the positions of the five conserved cysteine residues. The numbers on the right refer to amino acid positions.

Figure 21 is a schematic representation showing the amino acid sequence alignment of various Enhancer of zeste E(z)-like proteins. Darker shading represents highly conserved regions.The numbers on the right refer to amino acid positions in each amino acid sequence.

Figue..211s a schematic representation showing the amino acid sequence alignment of the TNFR/NGFR domains of various Enhancer of zeste E(z)-like proteins. The first 2 sequences (tnfr-rl and tnfr-r2) are both found in the human TNFR typel protein (Genbank P19348). The remaining 5 sequences are derived from E(z)-like proteins of Arabidopsis thaliana (FIS1, EZA1 and CURLY LEAF), Drosophila melanogaster and Caenorhabditis elegans (MES-2). The six conserved cysteine residues are indicated by asterisks. The numbers on the right refer to amino acid positions in each amino acid sequence.

Figure 23 is a schematic representation showing the amino acid sequence alignment WO 00/16609 PCT/AU99/00805 -17of the WCA domains of various Enhancer of zeste E(z)-like proteins. The sequences are derived from Arabidopsis thaliana (FIS1, EZA1 and CURLY LEAF), Drosophila melanogaster human (EZH2) and murine (Ezhl) E(z)-like proteins. The alignment was obtained using the computer program Clustalw and was viewed with the computer program Genedoc. The numbers on the right refer to amino acid positions in each amino acid sequence.

Figure 24 is a schematic representation of the FIS1/GUS and FIS2/GUS fusion constructs, showing the positions of the FIS1 and FIS2 promoter regions (open boxes), predicted translation start site (ATG), exons (black boxed regions), and introns (thin lines). There is a further translation start site in the FIS2 gene which the inventors have foundmay be used to produce a FIS2 polypeptide, located at nucleotide positions 364 to 366 of SEQ ID NO:<400>6. The location of the C2H2 zinc finger motif in the FIS2 polypeptide is indicated. Numbers to the left of the schematic indicate the length of the region derived from the FIS1 and FIS2 genes, respectively that has bneen fused to the GUS open reading frame in these fusion constructs.

Figure 25 is a copy of a photographic representation showing the expression of the FISI/GUS fusion constructs depicted in Figure 24, in the central nucleus (Panel 1); two endosperm nuclei (Panel three endosperm nuclei (Panel six endosperm nuclei (Panel 32 endosperm nuclei (Panel and endosperm cyst (Panel 6).

Figure 26 is a copy of a photographic representation showing the expression of the FIS2/GUS fusion constructs depicted in Figure 24, in the unfused nuclei of the central cell (Panel fused nucleus of the central cell (PAnel two free endosperm nuclei (Panel four free endosperm nuclei (Panel eight free endosperm nuclei (Panel 15 free endosprem nuclei (Panel 30 free endosperm nuclei (Panel and endosperm cyst (Panel 8).

Figure 27 is a copy of a photographic representation showing the interaction between FIS1 and FIS3 polypeptides in a yeast two-hybrid assay system. Left panel, formation of FIS1/FIS1 homodimers. Right panel, formation of FIS1/FIS3 heterodimers. Below, WO 00/16609 PCT/AU99/00805 -18- a schematic representation of the constructs used, as described in the Examples.

Figure 28 is a copy of a photographic representation showing the interaction between FIS1, FIS2 and FIS3 polypeptides in a yeast two-hybrid assay system. Left panel, formation of FIS1/FIS2 and FIS1/FIS2 heterodimers. Right panel, formation of EzA1/FIS3 and FIS1/FIS3 heterodimers.

Figure 29 is a copy of a photographic representation showing the relative degree of interaction between FIS1, FIS2, FIS3 and EzA1 polypeptides in a yeast two-hybrid assay system, wherein yeast growth under adenine selection requires binding between the proteins expressed from both the pGBT vector and the pGAD vector, and wherein the number of symbols is proportional to the degree of yeast growth observed under adenine selection and indicates no yeast growth. The proteins expressed from each vector are also indicated.

Figure 30 is a copy of a schematic representation of a screening method for the isolation of MOF repressor genes that regulate FIS gene expression.

DETAILED DESCRIPTION OF THE INVENTION One aspect of the present invention provides a method of inducing autonomous endosperm development in a plant, said method at least comprising the step of inhibiting, interrupting or otherwise reducing the expression of a negative regulatorof seed formation in one or more female reproductive cells, tissues or organs of said plant or a progenitor cell, tissue or organ thereof.

Preferably, the inventive method provides in part or whole for autonomous embryogenesis and more preferably, for autonomous seed development in plants. It this regard, it will be apparent to those skilled in the art from the description provided herein that, in order for autonomous embryogenesis or autonomous seed development to occur, the methods and reagents described herein may, in certain circumstances, represent a minimum requirement and that additional unspecified integers or steps may be required. The present invention clearly extends to the use of the specific WO 00/16609 PCT/AU99/00805 19reagents and steps described herein to produce autonomous embryogenesis and/or autonomous seed development.

The word "autonomous" as used herein means in the absence of fertilization or by the process of pseudogamy. Accordingly, the terms "autonomous endosperm -development" and "autonomous embryogenesis" or similar term, shall be taken to mean endosperm development and embryogenesis respectively, in the absence of fertilization or by the process of pseudogamy.

Similarly, the term "autonomous seed development" shall be taken -to refer to the development of seed independent of fertilization or by the process of pseudogamy, wherein said seed comprise one or more organs of a seed, including anyone or more of female gametophyte, endosperm, embryo and a seed coat, irrespective of whether or not said seed structure is fertile or infertile. Accordingly, autonomous seed development clearly includes the process of "apomixis" wherein viable seed are produced either in the absence of fertilisation or by the process of pseudogamy.

Where the production of fertile seed is required, it is essential that autonomous seed development leads to the formation of at least an endosperm and an embryo, notwithstanding that the endosperm may subsequently degenerate. In certain commercial applications involving the production of soft-seeded or parthenocarpic fruit varieties, autonomous endosperm formation may comprise the formation of non-viable seed wherein the embryo crushes down, leaving only soft seed comprising an endosperm. Alternatively, the endosperm may commence development autonomously and later degenerate, leaving seedless fruit.

In the present context, the word "seed" shall be taken to refer to any plant structure which is formed by continued differentiation of the ovule of the plant, following its normal maturation point at flower opening, irrespective of whether it is formed in the presence or absence of fertilization and irrespective of whether or not said seed structure is fertile or infertile. Fertile seed will generally require all tissues and organs required for development of a plant, including a storage tissue such as a haploid female gametophyte or a triploid maternally-derived endosperm, an embryo and a WO 00/16609 PCT/AU99/00805 seed coat. Infertile seed may lack one or more of the tissues or organs present in a fertile seed and may not give rise-to a plant in the next generation. It will be kinown to those skilled in the art that not all seed comprise' an endosperm and that someangiosperm seeds comprise only an embryo and seed coat, whilst many gymnosperm seed comprise a female gametophyte as storage tissue (rather than an endosperm), in addition to a seed coat and an embryo.

The word "expression" as used herein shall be taken in its widest context to refer to the transcription of a particular genetic sequence to produce sense or antisense mRNA or the translation of a sense mRNA molecule to produce a peptide, polypeptide, oligopeptide, protein or enzyme molecule. In the case of expression comprising the production of a sense mRNA transcript, the word-"expression" may also be construed to indicate the combinati6n-of transcription and translation processes, with or without subsequent post-translational events which modify the biological activity, cellular or sub-cellular localization, turnover or steady-state level of the peptide, polypeptide, oligopeptide, protein or enzyme molecule.

By "inhibiting, interrupting or otherwise reducing the expression" of a stated integer is meant that transcription and/or translation post-translational modification of the integer is inhibited or prevented or interrupted such that the specified integer has a reduced biological effect on a cell, tissue, organ or organism in which it would otherwise be expressed. Alternatively or in addition, the term "inhibiting, interrupting or otherwise reducing the expression" of:a stated integer-shall be takento mean that the rate or steady-state level of transcription ofthe integer is reduced and/or the rate or steadystate level of translation of the integer is reduced and/or that the biological activity or steady-state level of the peptide, polypeptide, oligopeptide, protein or enzyme molecule is reduced, such that the stated .integer has a reduced biological effect on a cell, tissue, organ or organism in which it would otherwise be expressed. Alternatively or in addition, the term "inhibiting, interrupting or otherwise reducing the expression" of a stated integer shall be taken to mean that a post-translational event which modifies the biological activity of the stated integer is modified such that the stated integer has a reduced biological effect on a cell, tissue, organ or organism in which it WO 00/16609 PCT/AU99/00805 -21would otherwise be expressed, including a modification to the cellular or sub-cellular localization of the stated integer and/or increased turnover of the stated integer.

Those skilled in the art will be aware of how whether expression is inhibited, interrupted or reduced, without undue experimentation.

For example, the level of expression of a particular gene may be determined by polymerase chain reaction (PCR) following reverse transcription of an mRNA template molecule, essentially as described by McPherson et al. (1991). Alternatively, the expression level of a genetic sequence may be determined by northern hybridisation analysis or dot-blot hybridisation analysis or in situ hybridisation analysis or similar technique, wherein mRNA is transferred to a membrane support and hybridised to-a "probe" molecule which comprises a nucleotide sequence complementary to the nucleotide sequence of the mRNA transcript encoded by the gene-of-interest, labelled with a suitable reporter molecule such as a radioactively-labelled dNTP (eg [a- 32 P]dCTP or [ca-SS]dCTP) or biotinylated dNTP, amongst others. Expression of the gene-of-interest may then be determined by detecting the appearance of a signal produced by the reporter molecule bound to the hybridised probe molecule.

Alternatively, the rate of transcription of a particular gene may be determined by nuclear run-on and/or nuclear run-off experiments, wherein nuclei are isolated from a particular cell or tissue and the rate of incorporation of rNTPs into specific mRNA molecules is determined. Alternatively, the expression of the gene-of-interest may be determined by RNase protection assay, wherein a labelled RNA probe or "riboprobe" which is complementary to the nucleotide sequence of mRNA encoded by said geneof-interest is annealed to said mRNA for a time and under conditions sufficient for a double-stranded mRNA molecule to form, after which time the sample is subjected to digestion by RNase to remove single-stranded RNA molecules and in particular, to remove excess unhybridised riboprobe. Such approaches are described in detail by Sambronk et al. (1989) and Ausubel (1987)..

Those skilled in the art will also be aware of various immunological and enzymatic methods for detecting the level of expression of a particular gene at the protein level, WO 00/16609 PCT/AU99/00805 22 for example using rocket immunoelectrophoresis, ELISA, radioimmunoassay and western blot immunoelectrophoresis techniques, amongst others.

The term "negative regulator" shall be taken to mean any peptide, oligopeptide, polypeptide, protein, enzyme, RNA, mRNA, tRNA or DNA molecule, secondary metabolite, macromolecule or small molecule which is capable of delaying, interrupting or preventing a biological process in a-cell, tissue, organ or organism.

Those skilled in the art will be aware that the term 'female reproductive cells, tissues or organs" refers to cells and tissues and organs comprising the gynoecium, ovule, female gametophyte, nucellus or integument, wherein each integer is considered collectively or in isolation.

A "progenitor cell, tissue or organ" refers to a cell, tissue or organ which is capable of developing into a cell, tissue or organ which comprises a stated integer. In the present context, a progenitor cell, tissue or organ refers to a cell, tissue or organ which is capable of developing into a female reproductive cell, tissue or organ as defined herein.

Accordingly, the term "negative regulator of seed formation" refers to a peptide, oligopeptide, polypeptide, protein, enzyme, RNA, mRNA, tRNA or DNA molecule, secondary metabolite, macromolecule or small molecule which is capable of delaying, interrupting or preventing the formation :of-seedo ,aseed organ in a plant. With particular reference to the presently described invention, a "negative regulator Of seed formation" refers to any peptide, oligopeptide, polypeptide, protein, enzyme, RNA, mRNA, tRNA or DNA molecule, secondary metabolite, macromolecule or small molecule which is capable of delaying, interrupting or preventing autonomous endosperm development in a plant.

Preferred negative regulators of seed formation in the present context are peptides, oligopeptides, polypeptides, proteins or enzymes which are capable of delaying, interrupting or preventing autonomous seed development in a plant. Such negative WO 00/16609 PCT/AU99/00805 23regulators may be repressors of one or more steps in autonomous fertilizationindependent) seed development in the plant.

For the purposes of nomenclature, the terms "fertilisation-independent seed gene product", "FIS gene product", "FIS protein", "FIS polypeptide" or "FIS peptide" or similar term shall be used to refer to a negative regulator of seed formation. The term "FIS gene" shall be taken to refer to the gene which encodes such a negative regulator of seed formation. In this context, specific FIS peptides, FIS polypeptides, FIS proteins and FIS genes are referred to by numerical descriptors, as are the alleles of such peptides, polypeptides, proteins and genes. For example, the FIS genes are described herein as FIS1, FIS2 and FIS3, etc., whilst the allelic variants at each gene locus are referred to as FIS1-1, FIS1-2, FIS1-3, FIS2-1, FIS2-2, FIS3-3, etc.

As will be known to those skilled in the art, mutated forms of a specific wild-type FIS gene product or gene encoding same, are indicated herein in lower case, for example as fisl polypeptide, fisl gene, etc.

Without being bound by any theory or mode of action, such negative regulators may, when expressed in the plant, prevent autonomous endosperm development from being initiated or alternatively, prevent autonomous endosperm development from progressing once it has been initiated, thereby optionally promoting a "default" pathway wherein seed comprising an endosperm are produced by sexual means via fertilization. Negative regulators of autonomous endosperm formation are also most likely to be expressed normally in maternally-derived cells, tissues and organs of the plant, because an implicit feature of autonomous endosperm development is the absence of a genetic contribution from the male gametophyte. Additionally, -as exemplified herein, plants in which the expression of one or more negative regulators of autonomous endosperm development has been prevented or reduced in the maternal tissues are capable of reproducing sexually in the presence of a pollen donor, indicating that the negative regulator is not derived from the male gametophyte.

Accordingly, in a preferred embodiment, the negative regulator of seed formation is a WO 00/16609 PCT/AU99/00805 -24peptide, polypeptide or protein which, when expressed in maternal tissues of a plant, completely or partially inhibits or prevents the autonomous development of the ovule into a seed it prevents or at least reduces the frequency fertilization-independent seed development) and more preferably, a peptide, polypeptide or protein which, when expressed in maternal tissues of a plant, completely or partially inhibits or prevents autonomous embryogenesis and/or partial autonomous endosperm development in the plant.

A particularly preferred embodiment of the present invention provides a method of inducing autonomous endosperm development in a plant, said method at least comprising the step of inhibiting, interrupting or otherwise reducing the expression of a negative regulator of seed formation in one or more female reproductive cells, tissues or organs of said plant or a progenitor cell, tissue or organ thereof, wherein the negative regulator of seed formation is a FIS polypeptide selected from the list comprising: a FIS1 polypeptide which comprises an amino acid sequence having at least about 50% overall amino acid sequence identity to the amino acid sequence set forth in <400>1; a FIS2 polypeptide which comprises an amino acid sequence having at least about 60-70% amino acid sequence-identity-to theamino acid sequence set forth in <400>2; (iii) a FIS3 polypeptide which comprises an amino acid sequence having at least about 60-70% amino acid sequence identity to the amino acid sequence set forth in <400>3; and (iv) a FIS3 polypeptide encoded by a nucleotide sequence which is capable of hybridizing under at least low stringency conditions to that region of chromosome 3 of Arabidopsis thaliana which maps between the markers m317 and DWF1 as set forth in Figure 9B.

Preferably, a FIS1 polypeptide which is at least 50% identical to the amino acid sequence set forth in <400>1 further comprises: a cysteine-rich domain designated C5, comprising the consensus amino WO 00/16609 PCT/AU99/00805 acid sequence motif: c-X 2 x x 25-35 -C-X 3 (as represented herein by the individual sequences set forth in <400>-10 to <400>20), wherein numerical values indicate the number of consecutive multiple occurrences of a particular amino acid residue; (ii) a cysteine-rich domain designated the CXC domain which comprises at least about 14 cysteine residues within a sequence of 61-67 consecutive amino acids and located C-terminal to the C5 domain; and (iii) a consensus amino acid sequence motif designated SET and located C-terminal to the CXC domain and comprising the-amino acid sequence: S-

-X-G-X-G-X-F-X

6 G-E-X-I-

-X

2 -X 2

-X

2

-X

6 -D-X

X

2

-F-X-N-H-X

3 4-P-X-C-Y-A- X2 E-L-X-F-D-Y-X-Y ,(as represented herein by the individual sequences set forth in <400>21 to <400>22), wherein numerical values indicate the number of consecutive multiple occurrences of a particular amino acid residue.

More preferably, the C5 domain comprises the amino acid sequence: C-X 2

X

4

X

2 X 22-32 -C-X 3 (as represented herein by the individual sequences set forth in <400>23 to <400>33), and more preferably, the amino acid sequence C-R-R-C- X 2

-H-X

22 3 2

-C-X

3 (as represented herein by the individual sequences set forth in <400>34 to <400>44) and still more preferably the amino acid sequence C-R-R-C-X 2 -F-D-C-X-M-H-X 22 -3 2

-C-X

3 (as represented herein by the individual sequences set forth in <400>45 to <400>55) or a homologue, analogue or derivative of said amino acid sequence or a fragment comprising at least contiguous amino acids thereof wherein numerical values indicate the number of consecutive multiple occurrences of a particular amino acid residue.

WO 00/16609 PCT/AU99/00805 -26- In a most particularly preferred embodiment, a FIS1 polypeptide will comprise a domain having an amino acid sequence which corresponds to amino acid residues 269-309 of <400>1 or a homologue, analogue or derivative of said amino acid sequence.

More preferably, the cysteine-rich domain designated CXC comprises the consensus amino acid sequence, C-X6-1o-C-X-C-X9_l

C-X-C-X

3 -XC- X -C-CX 3

-C-X

4

-C-X-C-X

6

-C-X

4 c-x 2 -c (as represented herein by the individual sequences set forth in <400>56 to <400>75) and more preferably the amino acid sequence, C-X6- 10 -C-X-C-X9-o 1

-C-X-C-X

3

-C-X

2

-R-F-X-G-C-X-C-X

2 3

-Q-C-X

4

-C-X-

C- -X-A-X 2

-P-X

2 -C-D-X-C (as represented herein by the individual sequences set forth in <400>76 to <400>95) and still more preferably, the amino acid sequence, C-X6- 1 o-C-X-C-X 9 _io-C-X-C-X 3

-C-X

2

-R-F-X-G-C-X-C-X

2 3-Q-C-X4_-C-X

C-F-X-A-X

2

-E-C-D-P-X

2 -C-D-X-C (as represented herein by the individual sequences set forth in <400>96 to <400>115) or a homologue, analogue or derivative of said amino acid sequence or a fragment comprising at least 5 contiguous amino acids thereof, wherein numerical values indicate the number of consecutive multiple occurrences of a particular amino acid residue.

In a most particularly preferred embodiment, a FIS1 polypeptide will comprise a CXC domain which comprises an amino acid sequence which corresponds to amino acid residues 450- 515 of <400>1 or a homologue, analogue orderivative of said amino acid sequence.

Preferably, the SET domain will comprise a sequence of amino acids which is at least about 50-60% identical to amino acid residues 551-665 of <400>1, more preferably at least about 60-70% identical to amino acid residues 551-665 of <400>1 and still more preferably at least about 70-80% identical to amino acid residues 551-665 of <400>1.

WO 00/16609 PCT/AU99/00805 -27 In a particularly preferred embodiment, the SET domain of a FIS1 polypeptide will comprise an amino acid sequence which is substantially identical or identical to amino acid residues 551-665 of <400>1 ora homologue, analogue or derivative of said amino acid sequence.

Alternatively or in addition, the FIS1 polypeptide will further comprise a cysteine-rich domain designated TGNF/NGFR which comprises the consensus amino acid sequence motif c,-xii_, -cb -x1_ 2 -c -X2-3Cd-XX_-_-ce-x 7-9 -Cf (as represented herein by the individual sequences set forth in <400>116 to <400>180), wherein Ca ,Cb ,Cc,Cd ,e and C, represent successive cysteine residues in said sequence motif and numerical values indicate the number of consecutive multiple occurrences of a particular amino acid residue.

The TGNF/NGFR domain set forth in any one of <400>116 to <400>180 may include an additional one or two or three amino acids immediately before the C-terminal Cysteine residue.

Preferably, the TGNF/NGFR domain set forth in any one of <400>116 to <400>180, with or without additional C-terminal residues referred to supra, comprises Phenylalanine or Tyrosine or Histidine at position six from the N-terminus.-Altematively or in addition, the TGNF/NGFR domain set forth in any one of <400>116 to <400>180, with or without additional C-terminal residues referred to supra, comprises Glutamine or Asparagine or Aspartate or Serine in the third-to-last amino acid position of said consensus. Even more preferably, the TGNF/NGFR domain set forth in any one of <400>116 to <400>180, with or without additional C-terminal residues referred to supra, will comprise a Histidine residue at position six from the N-terminus and an Asparagine residue in the third-to-last amino acid position of said consensus (i.e.

three amino acids from the C-terminus).

In a particularly preferred embodiment, the TGNF/NGFR domain comprises an amino acid sequence which corresponds to amino acid residues 460-498 of <400>1 or a homologue, analogue or derivative thereof.

WO 00/16609 PCT/AU99/00805 -28- In a further embodiment, the cysteine-rich domain designated TGNF/NGFR may further be capable of forming the intrachain disulfide bonds Ca -Cb and/or Cc-Ce and/or Cd-Cf.

In a still further embodiment, the TGNF/NGFR domain may be contained within the CXC domain of a FIS1 polypeptide, such as in the case of the Arabidopsisthaliana FIS1 polypeptide exemplified herein as-<400>1.

Alternatively or in addition, the FIS1 polypeptide, and more particularly the SET domain of the FIS1 polypeptide, may further comprise the amino acid sequence motif R-G-D. Those skilled in the art will be-aware of the structure of the R-G-D motif and its occurrence in proteins which are involved in cell adhesion (Ruoslahti and Piersbacher, 1986; d'Souza et 1991). Without being bound by any theory or mode of action, the tripeptide motif R-G-D (<400>181) may play a role in binding of the FIS1 polypeptide to a cognate receptor molecule, thereby modulating or initiating a signal transduction pathway which is relevant to autonomous seed development. For example, it is possible that the FIS1 polypeptide binds to its cognate receptor to inhibit binding of an activator molecule thereto, wherein said activator molecule would, if bound to the receptor, activate autonomous seed development in the maternal tissues.

Alternatively or in addition, a FIS1 polypeptide which is at least 50% identical to the amino acid sequence set forth in <400>1 further comprises an amino acid sequence comprising 12-13 amino acid residues wherein at least about 5-12 of said residues, more preferably at least about 8-12 of said residues, are the acidic amino acids glutamate and/or aspartate. In an even more preferred embodiment, at least 12 of the amino acids in the 12-13 amino acid long sequence will be acidic residues. In.a particularly preferred embodiment, the FIS1 polypeptide will comprise the amino acid sequence set forth in <400>182 as follows:

E-E-D-E-E-D-E-E-E-D-E-E-E,

or a homologue, analogue or derivative of said amino acid sequence. According to this embodiment, it is particularly preferred that the acidic domain is located in the Nterminal region of the FIS1 polypeptide, more preferably N-terminal to the C5 domain.

WO 00/16609 PCT/AU99/00805 -29- Whilst not being bound by any theory or mode of action, this acidic region may be required for forming an interaction with other proteins.

Alternatively or in addition, a FIS1 polypeptide which is at least 50% identical to the amino acid sequence set forth in <400>1 further comprises an amino sequence which is at least about 50% identical to the consensus amino acid sequence motif set forth in <400>183, and designated "WCA motif" as follows: W-X-

-X

2

-X-

or alternatively (<400>184 to <400>186) W-X-

-X

2 -X 3

-XI_

3

-C

and more preferably the amino acid sequence set forth in <400>187, as follows: W-X-P-X-E-K-X-L-Y-L-K-G-X-E-I-F-G-X-N-S-C-X-

L-X-G-X-K-T-C,

and even more preferably the amino acid sequence set forth in <400>188, as follows:

W-X-P-X-E-K-X-L-Y-L-K-G-X-E-I-F-G-X-N-S-C-X-V-A-X-N-I-L-X-

G-X-K-T-C,

or a homologue, analogue or derivative of said amino acid sequence or a fragment comprising at least 5 contiguous amino acids thereof located C-terminal to the domain and N-terminal to the CXC domain, subject to the proviso that the first cysteine residue and the alanine residue are always present, the amino acid residue at position 1 in said consensus is a hydrophobic amino acid residue and the amino acid residue at positions 27 and 28 in said consensus is either L or M.

In a particularly preferred embodiment, the FIS1 polypeptide will further comprise a WCA motif which comprises the amino-acid sequence set forth in <400>189, as follows:

W-T-P-V-E-K-D-L-Y-L-K-G-I-E-I-F-G-R-N-S-C-D-V-A-L-N-I-L-R-

G-L-K-T-C,

or a homologue, analogue or derivative of said amino acid sequence or a fragment WO 00/16609 PCT/AU99/00805 comprising at least 5 contiguous amino acids thereof located C-terminal to the domain and N-terminal to the CXC domain.

Optionally, the FIS1 polypeptide further comprises a nuclear localisation domain located C-terminal to the C5 domain and N-terminal to the CXC domain. As used herein, the term "nuclear localisation domain" shall be taken to refer to an amino acid sequence which is at least postulated to be capable of targeting a- polypeptide comprising said domain to the nucleus of a cell. Those skilled in the art will be aware of the specific requirements of a domain which is postulated to be involved in nuclear localisation. Preferably, a nuclear localisation domain comprises an amino acid sequence which is rich in lysine and/or arginine residues. More preferably, the nuclear localisation signal of a FIS1 polypeptide will include the amino acid sequence motif set forth in <400>190 to <400>191, as follows: K-K-XI-2

-K

and more preferably, the amino acid sequence set forth in <400>192 to <400>193, as follows: K-K-Xi2

-K-X

2 -R-X 2

-R-K-K-X-R-X-R-K

and still more preferably,the amino acid sequence set forth in <400>193, as follows:

K-K-X

2

-K-X

2 -R-X 2

-R-K-K-X-R-X-R-K

or a homologue, analogue or derivative of said amino acid sequence or a fragment comprising at least 5 contiguous amino acids thereof, wherein numerical values indicate the number of consecutive multiple occurrences of a particular amino acid residue.

In a particularly preferred embodiment, the nuclear localisation signal of a FIS1 polypeptide will include the amino acid sequence motif set forth in <400>194, as follows: K-K-V-S -R-K-S-S-R-S-V-R-K-K-S-R-L-R-K or a homologue, analogue or derivative of said amino acid sequence or a fragment comprising at least 5 contiguous amino acids thereof which retains the potential to target a polypeptide to the nucleus.

WO 00/16609 PCT/AU99/00805 -31- In a particularly preferred embodiment of the invention, a FIS1 polypeptide having at least about 50% amino acid sequence identity to the amino acid sequence set forth in <400>1 will further comprise all of the amino acid sequence motifs and protein domains described supra.

For the purposes of further describing the FIS1 polypeptide, it is preferred that the percentage identity to the amino acid sequences set forth in <400>1 is at least about 60-70% overall, more preferably at least about 70-80% overall, still more preferably at least about 80-90% overall and still even more preferably at least about 90-99% identity overall. In a particularly preferred embodiment, the negative regulator of seed formation will comprise an amino acid sequence sharing absolute identity to the amino acid sequence set forth in <400>1 or a homologue, analogue or derivative of said amino acid sequence.

For the purposes of nomenclature, the amino acid sequence set forth in <400>1 is a polycomb protein (Goodrich et al., 1997) having homology to the Enhancer of zeste family of proteins (Laible et a.(1997), which was derived from Arabidopsis thaliana and described initially by Grossniklaus et al. (1998). Those skilled in the art will be aware of the structure and function of the polycomb group of-proteins and in particular, the E(z) class of proteins. By way of background, the E(z) proteins generally comprise a SET-like domain, in addition to a CXC-like domain and a C5-like domain.

Whilst not being bound by any theory or mode of action, proteins which contain a SET domain are generally involved in regulating gene expression by controlling chromatin structure and thereby modulating the accessibility of the chromatin to transcription factors. The C5 domain and CXC domain appear to be necessary for the function of the Drosophila E(z) polypeptide, which also comprises a SET domain. Accordingly, the possibility exists that the FIS1 polypeptide may interact with nuclear chromatin to prevent positive regulatory factors which would otherwise be capable of inducing autonomous seed development and/or partial autonomous endosperm development and/or autonomous embryogenesis from interacting with the chromatin and inducing such autonomous developmental patterns.

WO 00/16609 PCT/AU99/00805 -32- For the present purpose of inducing autonomous seed development, the step of inhibiting, interrupting or otherwise reducing the expression of the FIS1 polypeptide in one or more female reproductive cells, tissues or- organs of said plant or a progenitor cell, tissue or organ thereof, requires more than the mere disruption of the SET domain present in said protein. In this regard, Grossniklaus et al. (1998) demonstrated that a mutation in nucleotide sequence encoding the FIS1 polypeptide, known as medea (mea), produces 50% embryo lethality in the seed produced following self-fertilization of MEA/mea plants plants which are heterozygous for the mutant allele), however these authors did not demonstrate autonomous seed development and/or partial autonomous endosperm development and/or autonomous embryogenesis. The mea mutant allele at this locus comprises a Ds transposable element inserted within or Nterminal to the SET domain of FIS1 which is present in the E(z) protein family, thereby resulting in the translation of a fisl mutantpolypeptide designated medea (mea) which lacks the SET domain, however comprises all protein domains N-terrinal to the site of insertion of Ds.

Accordingly, this aspect of the invention, in so far as it relates to the inhibition, interruption or reduction in expression of a negative regulator of seed formation which comprises the amino acid sequence set forth in <400>1, does not exclusively utilise the mutation or disruption of the SET domain of <400>1 amino acid residues 551 to 665 of <400>1) or the mimicking the mea mutant allele. Such exclusive mutation or disruption of the SET domain does not, in any event, produce a plant which is capable of autonomous seed formation, autonomous embryogenesis or autonomous endosperm development.

As exemplified herein, the present inventors have discovered that mutations in the FIS1 gene which eliminate one or more of the amino acid sequences upstream of the SET domain and optionally including the SET domain are capable of conferring autonomous seed formation on plants.

Accordingly, in performing the present invention, the expression of the FIS1 polypeptide may be inhibited, disrupted, prevented or otherwise reduced by preventing WO 00/16609 PCT/AU99/00805 33 the synthesis of a polypeptide which comprises any one or more of the FIS1 protein domains or amino acid sequence motifs described herein, subject to the proviso that said FIS1 protein domain or amino acid sequence motif does not comprise exclusively the SET domain.

Accordingly, the present invention clearly encompasses the mutation or disruption of the SET domain of <400>1 in conjunction with other means for inhibiting, interrupting or otherwise reducing the expression of the amino acid sequence set forth in <400>1, for example the mutation or disruption of one or more other regions of said amino acid sequence, the only requirement being that said other means produces-a plant which is capable of autonomous seed formation, autonomous embryogenesis or autonomous endosperm development.

In a particularly preferred embodiment, all of the FIS1 protein domains are prevented from being expressed in the performance of the invention, including the production of a null allele.

For the purposes of nomenclature, the amino acid sequence set forth, in <400>2 relates to the Arabidopsis thaliana FIS2 polypeptide, a putative C2H2 zinc-finger protein or zinc-finger-like protein which is involved in regulating autonomous embryogenesis and partially-regulating autonomous endosperm development, at least in that plant.

Accordingly, it is particularly preferred that a FIS2 polypeptide which is at least about 50% identical to the amino acid sequence set forth in <400>2 will further comprise a zinc-finger protein motif or zinc-finger-like protein motif which comprises about 20 to about 25 amino acid residues in length, containing the amino acid sequence motifs set forth in <400>195 and <400>196, as follows: <400>195: C-X 2 and <400>196: X-H-X 4

-H.

More preferably, a FIS2 polypeptide will comprise a zinc-finger protein motif or zinc- WO 00/16609 PCT/AU99/00805 34 finger-like protein motif which comprises the amino acid sequence set forth in <400>197,_as follows:

C-X

2

-C-X

6 -H-Xs-H-X 4

-H,

and even more particularly, the amino acid sequence set forth in <400>198, as follows:

C-X

2

-C-X

3

-C-X

2 -H-Xs-H-X 4

-H.

In a more particularly preferred embodiment,-a FIS2 polypeptide will comprise a zincfinger protein motif or zinc-finger-like protein motif which comprises the amino acid sequence set forth in <400>199, as follows: C-P-F-C-L-I-P-C-G-G-H-E-G-L-Q-L-H-L-K-S-S-H; or (ii) a homologue, analogue or derivative of said amino acid sequence.

As used herein, the term "zinc-finger protein motif" shall be taken to refer to a primary amino acid sequence which is capable of forming a secondary protein structure which is characteristic of the class of transcription factors known in the art as "zinc-finger" proteins, wherein said secondary protein structure is formed by the formation of disulfide bridges between cysteine residues in the primary amino acid sequence.

The term "zinc-finger-like protein motif" shall be taken to refer to a primary amino acid sequence which shows amino acid sequence similarity to a zinc-finger protein motif, notwithstanding that it is not capable of forming a secondary protein structure characteristic of zinc-finger proteins by the formation of disulfide bridges between cysteine residues in the primary amino acid sequence.

For the purposes of further describing the FIS2 polypeptide, it is preferred that the percentage identity to the amino acid sequences set forth in <400>2 is at least about 60-70% overall, more preferably at least about 70-80% overall, still more preferably at least about 80-90% overall and still even more preferably at least about 90-99% identity overall. In a particularly preferred embodiment, the negative regulator of seed formation will comprise an amino acid sequence sharing absolute identity to the amino acid sequences set forth in <400>2 or a homologue, analogue or derivative thereof.

WO 00/16609 PCT/AU99/00805 For the purposes of nomenclature, the amino acid sequence set forth in <400>3 relates to the Arabidopsis thaliana FIS3 polypeptide, a protein which is involved in regulating autonomous endosperm development, at least in that plant.

For the purposes of further describing the FIS3 polypeptide, it is preferred that the percentage identity to the amino acid sequence set forth in <400>3-is-at least about 60-70% overall, more preferably at least about 70-80% overall, still more preferably at least about 80-90% overall and still even more preferably at least about 90-99% identity overall. In a particularly preferred embodiment, the negative regulator of seed formation will comprise an amino acid sequence sharing absolute identity to the amino acid sequences set forth in <400>3 ora homologue, analogue or derivative thereof.

In an-alternative embodiment, the FIS3 polypeptide will be encoded by a nucleic acid moelcule that is capable of hybridising under at least low stringency hybridisation conditions to the fis3 mutant allele.

As exemplified herein, the present inventors have identified a mutant phenotype designated fis3 which is at least capable of autonomous endosperm development and/or autonomous seed formation. The present inventors have mapped the fis3 mutant allele to chromosome 3 of Arabidopsis thaliana, at a region which lies between the morphological markers hy3 and gll. Further mapping localized the fis3 mutant allele to a region between the RFLP markers m317 and DWF1. The fis3 allele has been shown further to map to a region on chromosome 3 of A. thaliana which is approximately 6 cM from the-SSLP marker nga162 and approximately 1 cM from the RFLP marker ve039.

Those skilled in the art will be aware that the close genetic linkage between the FIS3 locus on chromosome 3 of A. thaliana and the RFLP marker ve039 indicates that said RFLP marker is useful in identifying plants which comprise the FIS3 gene and in isolating the FIS3 gene.

Accordingly, it is preferred that a FIS3 polypeptide will be encoded by a nucleotide WO 00/16609 PCT/AU99/00805 36sequence which is capable of hybridizing under at least low stringency conditions to the RFLP marker designated ve039 which maps approximately 1 cM from the FIS3 locus on chromosome 3-of Arabidopsis thaliana.

For the purposes of defining the stringency, a low stringency is defined herein as being a hybridisation and/or a wash carried out in 6xSSC buffer, 0.1% SDS at 28 0

C.

Generally, the stringency is increased by reducing the concentration of SSC buffer, and/or increasing the concentration of SDS and/or increasing the temperature of the hybridisation and/or wash. Conditions for hybridisations and washes are well understood by one normally skilled in the art. For the purposes of clarification of parameters affecting hybridisation between nucleic acid molecules, reference can conveniently be made to pages 2.10.8 to 2.10.16. of Ausubel et al. (1987), which is herein incorporated by reference.

-Those skilled in the art will be aware that confirmation of the identity of the FIS3 gene may be carried out by complementation of the fis3 mutant phenotype using YAC, BAC or cosmid clones or fragments thereof which hybridize to the RFLP marker ve039. The nucleotide sequence of the FIS3 gene may then be determined by sequencing the genes present in those clones which successfully complement the fis3 mutant phenotype.

Accordingly, the present inventors have further created a map of contiguous YAC and pl cosmid clones in the region surrounding the RFLP marker ve039, which indicates that the fis3 mutant allele (and thus the wild-type FIS3 gene) is localized on the YACS and/or p1 -lones MCB22 and/or MNH5 and/or CIC7E1.

Accordingly, in a further preferred embodiment of the invention the FIS3 polypeptide is encoded by a nucleic acid molecule which is capable of hybridising under at least low stringency hybridisation conditions to one or more of the YACS and/or pl clones designated MCB22 and/or MNH5 and/or CIC7E1.

For the purposes of nomenclature, the RFLP marker ve039 and the YAC clone WO 00/16609 PCT/AU99/00805 37- CIC7E1 and the p1 clones MCB22 and MNH5 are all publicly available from the following internet sites: http://www.Kazusa.or.JP/arabi/chr3/ http://genome-www:stanford.edu/Arabidopsis/chr3-INRA/ More preferably, FIS3-encoding genetic sequences are preferably isolated by hybridisation under medium or more preferably, under high stringency conditions, to a probe which comprises at least about 30 contiguous nucleotides derived from the region of chromosome 3 of Arabidopsis thaliana which maps between the markers m317 and DWF1 as set forth in Figure 9B.- It will be apparent from the preceding description that the present invention clearly extends to themodulation of expression of negative regulators of seed development which comprise homologues, analogues and derivatives of a FIS polypeptide, including the FIS1 and FIS2 amino acid sequences set forth in <400>1 and <400>2 respectively, and the FIS3 polypeptide encoded by a nucleotide sequence which is capable of hybridizing under at least low stringency conditions to that region of chromosome 3 of Arabidopsis thaliana which maps between the markers m317 and DWF1.

In the present context, "homologues" of a FIS polypeptide refer to those amino acid sequences or peptide sequences which are derived from polypeptides, enzymes or proteins of the present invention or alternatively, correspond substantially to the polypeptides and amino acid sequences listed supra, notwithstanding any naturallyoccurring-amino acid substitutions, additions or deletions thereto.

For example, amino acids may be replaced by other amino acids having similar properties, for example hydrophobicity, hydrophilicity, hydrophobic moment, antigenicity, propensity to form or break a-helical structures or P-sheet structures, and so on. Alternatively, or in addition, the.amino acids of a homologous amino acid sequence may be replaced by other amino acids having similar properties, for example hydrophobicity, hydrophilicity, hydrophobic moment, charge or antigenicity, and so on.

Naturally-occurring amino acid residues contemplated herein are described in Table 1.

WO 00/16609 PCT/AU9900805 -38- A homologue may be a synthetic peptide produced by any method known to those skilled in the art, such as by using Fmoc chemistry.

Alternatively, a homologue of a FIS polypeptide may be derived from a natural source, such as the same or another species as the polypeptides, enzymes or proteins of the present invention. Preferred sources of homologues of the amino acid sequences listed supra include any of the sources contemplated herein.

"Analogues" of a FIS polypeptide encompass those amino acid sequences which are substantially identical to the amino acid sequences listed supra notwithstanding the occurrence of any non-naturally occurring amino acid analogues therein.

Preferred non-naturally occurring amino acids contemplated herein are listed below in Table 2.

The term "derivative" in relation to a FIS polypeptide shall be taken to refer hereinafter to mutants, parts, fragments or polypeptide fusions of said polypeptides. Derivatives include modified amino acid sequences or peptides in which ligands are attached to one or more of the amino acid residues contained therein, such as-carbohydrates, enzymes, proteins, polypeptides or reporter molecules such as radionuclides or fluorescent compounds. Glycosylated, fluorescent, acylated or alkylated forms of the subject peptides are also contemplated by the present invention. Additionally, derivatives may comprise fragments or parts of an amino acid sequence disclosed herein and -are within the scope of the invention, as are homopolymers or heteropolymers comprising two or more copies of the subject sequences.

Procedures for derivatizing peptides are well-known in the art.

Substitutions encompass amino acid alterations in which an amino acid is replaced with a different naturally-occurring or a non-conventional amino acid residue. Such substitutions may be classified as "conservative", in which case an amino acid residue is replaced with another naturally-occurring amino acid of similar character, for WO 00/16609 PCT/AU99/00805 -39example Gly-Ala, Val-lle-Leu, Asp+-Glu, Lys*-Arg, AsnGIn or Phe*-+Trp-+Tyr.

Substitutions encompassed by the present invention may also be "non-conservative", in which an amino acid residue which is present in a repressor polypeptide is substituted with an amino acid having different properties, such as a naturallyoccurring amino acid from a different group (eg. substituted a charged or hydrophobic amino acid with alanine), or alternatively, in which a naturally-occurring amino acid is substituted with a non-conventional amino acid.

Amino acid substitutions are typically of single residues, but may be of multiple residues, either clustered or dispersed.

Amino acid deletions will usually be of the order of about 4-10 amino acid residues, while insertions may be of any length. Deletions and insertions may be made to the N-terminus, the C-terminus or be internal deletions or insertions. Generally, insertions within the amino acid sequence will be smaller than amino-or carboxyl-terminal fusions and of the order of 1-4 amino acid residues.

Preferred homologues, analogues and derivatives of the FIS polypeptides described herein, including the amino acid sequences set forth in <400>1 and/or <400>2 and/or <400>3, will comprise at least about 5-10 contiguous amino acids of said polypeptide or preferably at least about 10-20 contiguous amino acid residues or more preferably at least about 20-50 contiguous amino acid residues. Accordingly, such homologues, analogues and derivatives may be full-length or less than full-length sequences compared to the full-length A. thaliana FIS polypeptides.

It will be apparent to those skilled in the art that the expression of a homologue, analogue or derivative of a FIS polypeptide whicb_ targeted prevented, interrupted or otherwise reduced) using the inventive method described herein must be capable of functioning in vivo as a negative regulator of seed development in a plant and preferably in the maternal cells, tissues or organs thereof.

WO 00/16609 PCT/AU99/00805 40 In other embodiments of the invention described herein, homologues, analogues and derivatives of a FIS polypeptide may be useful as a tool in performing the inventive method. For example, homologues, analogues and derivatives of the FIS polypeptide, including those which are shorter than the full-length sequence and do not possess the same activity as the full-length sequence, will at least be useful in the preparation of antibody molecules capable of binding to the full-length sequence for use in diagnostic assays or as inhibitor molecules. Alternatively such homologues, analogues and derivatives may be useful as nhibitors of the full-length FIS1 and/or FIS2 and/or FIS3 polypeptides, by preventing binding of the full-length polypeptides to a protein or nucleic acid molecule with which they interact in vivo. For example,-homologues, analogues or derivatives of the FIS2 polypeptide may comprise the zinc-finger motif and act as a non-functional competitive inhibitor of the full-length polypeptide.

Alternatively or in addition, a homologue, analogue or derivative of the FIS polypeptides described herein will be catalytically equivalent to the naturally-occurring FIS polypeptide exemplified herein and comprise an amino acid sequence which is at least about 60-70% identical thereto. Preferably, the percentage identity to <400>2 will be at least about 70-80%, more preferably at least about 80-90% and even more preferably at least about 90-95% or at least about 98 or 99%.

In determining whether or not two amino acid sequences fall within defined percentage identity or similarity limits, those skilled in the art will be aware that it is necessary to conduct a side-by-side comparison of amino acid sequences. In such comparisons or alignments, differences will arise in the positioning of non-identical amino acid residues depending upon the algorithm used to perform the alignment. In the present context, references to percentage identities and similarities between two or more amino acid sequences shall be taken to refer to the number of identical and similar residues respectively, between said sequences as determined using any standard -algorithm known to those skilled in the art. In particular, amino acid identities and similarities are calculated using the GAP programme of the Computer Genetics Group, Inc., University Research Park, Madison,.Wisconsin, United States of America (Devereaux et al, 1984), which utilizes the algorithm of Needleman and Wunsch (1970) WO 00/16609 PCT/AU99/00805 -41or alternatively, the CLUSTAL W algorithm of Thompson et al (1994) for multiple alignments, to maximise the number of identical/similar amino acids and to minimise the number and/or length of sequence gaps in the alignment.

Means for inhibiting, interrupting or otherwise reducing the expression of a negative regulator of seed formation in one or more female reproductive cells, tissues-or organs of a plant or a progenitor cell, tissue or organ thereof include any means known to those skilled in the art in so far as said means are applicable to the FIS polypeptides described herein or a homologue, analogue or derivative thereof.

Such means include mutagenesis of the gene(s) which encode(s) the FIS polypeptide(s) described herein, such that it is no longer capable of being expressed at a biologically-effective level in the maternal cells, tissues or organs of the plant.

Means for performing such mutagenesis of a FIS gene include the use of chemical mutagens, radiation and insertional inactivation by molecular means, amongst others and the present invention clearly encompasses the use of all such methods.

As used herein, the term biologically-effective level" shall be taken to mean a level of expression of a FIS polypeptide which is sufficient to delay, inhibit, interrupt or prevent autonomous seed development and/or partial autonomous -endosperm development and/or autonomous embryogenesis in a plant.

Reference herein to a "gene" is to be taken in its broadest context and includes: a classical genomic gene consisting of transcriptional and/or translational regulatory sequences and/or a coding region and/or non-translated sequences (i.e.

introns, and untranslated sequences);or (ii) mRNA or cDNA corresponding to the coding regions exons) and and 3'untranslated sequences of the gene.

The term "gene" is also used to describe synthetic or fusion molecules encoding all or part of a functional product. Preferred seed formation genes of the present invention may be derived from a naturally-occurring seed formation gene by standard WO 00/16609 PCT/AU99/00805 42 recombinant techniques. Generally, an seed formation gene may be subjected to mutagenesis to produce single or multiple nucleotide substitutions, deletions and/or additions.

Nucleotide insertional derivatives include 5' and 3' terminal fusions as well as intrasequence insertions of single or multiple nucleotides. Insertional nucleotide sequence variants are those in which one or more nucleotides are introduced into a predetermined site in the-nucleotide sequence although random insertion is also possible with suitable screening of the resulting product.

Deletional variants are characterised by-the removal of one or more nucleotides from the sequence.

Substitutional nucleotide variants are those in which at least one nucleotide in the sequence has been removed and a different nuicleotide inserted in its place. Such a substitution may be "silent" in that the substitution does not change the amino acid defined by the codon. Alternatively, substituents are designed to alter one amino acid for another similar acting amino acid, or amino acid of like charge, polarity, or hydrophobicity As used herein, the term "FIS gene" and variants such as "FIS1 gene", "FIS2 gene" and "FIS3 gene" shall be taken to refer to a wild-type or functional gene as hereinbefore defined which encodes a functional FIS polypeptide at a biologicallyeffective level. Consistent with nomenclature known to those skilled in the art, a FIS1 polypeptide is encoded by a FIS1 gene, a FIS2.polypeptide is encoded by a FIS2 gene and a-FIS3 polypeptide is encoded by a FIS3 gene.

Preferred F/S genes, the expression of which is intended to be modified by the performance of the invention, include the FIS1, FIS2 and FIS3 genes exemplified herein and homologues, analogues and derivatives thereof- For the purposes of nomenclature, the FISI gene comprises a sequence of WO 00/16609 PCT/AU99/00805 -43nucleotides which is at least about 50% identical to the nucleotide sequence set forth in <400>4 or <400>5. The nucleotide sequence set forth in <400>4 relates to the FIS1 cDNA and the nucleotide sequence set forth in <400>5 relates to the FISI genomic gene sequence.

For the purposes of nomenclature, the FIS2 gene comprises a sequence of nucleotides which is at least about 50%identical to the nucleotide sequence set forth in <400>6 or <400>7. The nucleotide sequence set forth in <400>6 relates to the FIS2 cDNA and the nucleotide sequence set forth in <400>7 relates to the FIS2 genomic gene sequence.

For the purposes of nomenclature, the FIS3 gene comprises a sequence of nucleotides which is at least about 50% identical to the nucleotide sequence set forth in <400>8 or <400>9. The nucleotide sequence set forth in <400>8 relates to the FIS3 cDNA and the nucleotide sequence set forth in <400>9 relates to the FIS3 genomic gene sequence.

The FIS3 gene comprises either the nucleotide sequence set forth in <400>8 or <400>9, or a complemetnary sequence thereto, or a sequence of nucleotides which is at least capable of hybridizing under at least low stringency conditions to that region of chromosome 3 of Arabidopsis thaliana which maps between the markers m317 and DWF1 as set forth in Figure 8B and which encode a FIS3 polypeptide which is capable of modulating autonomous seed development and/or partial autonomous endosperm development and/or autonomous embryogenesis in a plant.

WO 00/16609 WO 0016609PCT/AU99/00805 44 TABLE 1 Amino Acid Three-letter One-letter Abbreviation Symbol Alanine Ala

A

Arginine Arg

R

Asparagine Asn

N

Aspartic acid Asp

D

Cysteine Cys

C

Glutamine Gin

Q

Glutamic acid Glu

E

Glycine Gly

G

Histidine His

H

Isoleucine le I Leucine Leu

L

Lysine Lys

K

Methionine Met

M

Phenylalanine Phe

F

Proline Pro

P

Serine Ser

S

Threonine Thr

T

Tryptophan Trp,

W

Tyrosine Tyr -Y Valine Val

V

Any amino acid as above Xaa

X

WO 00/16609 WO 0016609PCT/AU99/00805 45 TABLE 2 Non-conventional Code Non-conventional Code amino acid amino acid a-aminobutyric acid ax-amnino-a-methylbutyrate aminocyclopropanecarboxylate, amnino isobutyric acid aminonorbornyl-.

carboxylate cyclohexylalanine cyclopentylalanine D-alanine D-arginine D-aspartic acid D-cysteine D-glutamine D-glutamic acid -D-hi-stidine D-isoleucine D-leucine -D--ysine D-methionine D-ornithine D-phenylalanine D-proline D-serine D-threonine D-tryptophan Abu Mgabu Cpro Aib Norb Chexa Cpen Dal Darg Dasp Dcys Dgln Dglu Dhis Dule Dleu Dlys Dmet Dorn Dphe Dpro Dser Dthr Dtrp L-N-methylalanine L-N-methylarginine L-N-methylasparagine L-N-methylaspartic acid L-N-methylcysteine L-N-methylglutamine L-N-methylglutamic acid L-N-methylhistidine L-N-methylisolleucine L-N-methylleucine L-N-methyllysine L-N-methylmethionine L-N-methylnorleucine L-N-ni~thylnorvaline L-N-methylornithine L-N-methylphenylalanine L:N--methylproline- L-N-methylserine L-N-methylIthreonine L-N-methyltryptophan L-N-methyltyrosin e L-N-methylvaline L-N-methylethylglycine L-N-methyl-t-butylglycine L-norleucine L-norvaline Nmala Nmarg Nmasn Nmasp Nmcys Nmgln-- Nmglu Nmhis Nmile NmIleu.

Nm-lys Nnimet Nmnle N mnva Nmorn Nmphe Nmpro Nmser Nmthr Nmtrp Nmtyr Nmval Nmetg Nmtbug NMe Nva WO 00/16609 WO 0016609PCT/AU99/00805 46 D-tyrosine D-valine D-a-methylalanine D-a-methylarginine D-a-methylasparagine D-a-methylaspartate D-a-methylcysteine D-a-methylglutamine D-a-methylhistidine D-a-methylisoleucine D-a-.methylleucine D-a--methyllysine D-a-methylmethionine D-a-methylornithine D-a-methylphenylalanine D-ix-methylproline D-a-methylserine D-oa-methylthreonine D-cx-methyltryptophan D-a-methyltyrosine D-a-methylvaline D-N-methylalanine D-N-methylarginine D-N-methylasparagine D-N-methylaspartate D-N-methylcysteine Dtyr Dval Dmala Dmarg Dmasn Dmasp Dmcys Dmgln Dmhis Dmile Dmleu Dmldys Dmmet Dmorn Dmphe Dmpro Dmser Dmthr Dmtrp Dmty D-mval Dnmala Dnmarg Dnmasn- Dnmasp Dncys ox-methyl-aminoisobutyrate ca-methy1-y -aminobutyrate a-methylcyclohexylaianine a-methylcylcopentylalaninea-methyl-ct-napthylalanine a-methylpenicillamine N-(4-aniinobutyl)glycine N-(2-aminoethyl)glycine N-(3-aminopropyl)glycine N-amino-a-methylbutyrate a-napthylaianine N-benzylglycine -N-(27carbamylethyl)glycine N-(carbamylmethyl)giycine N-(-2-carboxyethyl)glycine N-(carboxymethyl)glycine N-cyclobutylglycine- N-cycloheptylglycine- N-cyclohexylglycine N-cyclodecylglycine N-cylcododecylglycine N-cyclooctylglycine N-cyclopropylglyciie N-cycloundecylglycine N-(2 ,2-diphenylethyl) glycine N-(3 ,3-diphenyipropyl) glycine Maib Mgabu Mchexa Mcperi Manap Mpen Nglu Naeg Norn Nmaabu Anap Nphe Ngln Nasn Nglu- Nasp Ncbut Nchep Nchex Ncdec Nccod Ncoct Ncpro Ncund-- Nbhm Nbhe WO 00/16609 WO 0/1669 PCT/AU99/00805 47 D-N-methylglutamine D-N-methylglutamate D-N-methylhistidine D-N-methylisoleucine D-N-methylleucine ~D-N-methyllysine N-methylcycl-ohexylalanine D-N-methylornithine,- N-methylglycine N-methylamninoisobutyrate 1-methylpropyl)glycine N-(2-methylpropyl)glycine D-N-methyltryptophan D-N-mrethyltyrosine D-N-methylvaline y-aminobutyric acid L-t-butylglycine L-ethylglycine L-homnophenylalanine L-a-methylarginine L-ax-methylaspartate L-a-methylcysteine L-a-methylglutamine L-ax-methiylhistidine L-cx-methylisoleucine Dnmngln Dnmglu Dinihis Dnile Dnleu Dnlys Nmchexa Drimorn Nala Nmaib Nile Nleu Dnmtrp Drntyr Dnmval Gabu Tbug Etg Hphe Marg Masp Mcys Mgln Mhis Mile 3-guanidinopropyl) glycine 1-hydroxyethyl)glycine N-(hydroxyethyl))glycine N-(imidazolylethyl)) glycine N-(3-indolylyethylI glycine N-methyl-y -aminobutyrate D-N-methylmethionine N-methylcyclopentylalanine D-N-methylphenylalanine D-N-methylproline D-N-methyl'serine D-N-methylthreonine 1-methylethyl)glycine N-methyla-napthylalanine N-methylpenicillamnine N-(p-hydroxyphenyl)glycine N-(thiomethyl)glycine penicillamine, L-a-methylalanine L-a-methylasparagine L-a-methyl-t-butylglycine L-methylethylglycine L-o-methylglutamate L-a-methylhomo phenylalanine N-(2-methylthioethyl) gl-ycine L-a-methyllysine Narg Nthr Nser Nhis Nhtrp Nmgabu Drnmet Nmcpen Dnimphe Dnmpro Dnser Dnmthr Nval Nmanap Nrnpen Nhtyr Ncys Pen Mala Masn Mtbug Metg Mglu Mhphe Nmet Mlys L-a-methylleucine Mleu WO 00/16609 PCT/AU99/00805 -48-- L-a-methylmethionine L-a-methylnorvaline L-a-methylphenylalanine L-a-methylserine L-a-methyltryptophan L-a-methylvaline N-(N-(2,2-diphenylethyl) carbamylmethyl)glycine 1-carboxy-l-(2,2-diphenylethylamino)cyclopropane Mmet Mnva- Mphe Mser Mtrp Mval Nnbhm Nmbc L-a-methylnorleucine L-a-methylornithine L-a-methylproline L-a-methylthreonine L-a-methyltyrosine L-N-methylhomo phenylalanine N-(N-(3,3-diphenylpropyl) carbamylmethyl)glycine Mnle Morn Mpro Mthr Mtyr Nmhphe Nnbhe As used herein, the term "fis gene" shall be taken to refer to a mutantor biologicallyineffective allele of a FIS gene as hereinbefore defined.

By "biologically-ineffective" is meant that a stated integer is not capable of performing its normal biological role in the cell with respect to autonomous seed development and/or partial autonomous endosperm development and/or autonomous embryogenesis.

Particularly preferred chemical mutagens include EMS and methanesulfonic acid ethyl ester. As will be known to those skilled in the art, EMS generally introduces point mutations into the genome of a cell in a random non-targeted manner, such that the number of point mutations introduced into any one genome is proportional to the concentration of the mutagen used. Accordingly, in order to identify a particular mutation, large populations of seed are generally treated with EMS and the effect of the mutation is screened in the M2 seed. Notwithstanding that this is the case, the fis2 and fis3 mutant alleles described herein were identified in EMS-mutagenised tines of Arabidopsis thaliana. Methods for the application and use of chemical mutagens such as EMS are well-known to those skilled in the art WO 00/16609 PCT/AU99/00805 -49- Preferred irradiation means include ultraviolet and gamma irradiation of whole plants, plant parts and/or seed to introduce point mutations into one or more of the FIS genes present in the genome thereof or alternatively, to create chromosomal deletions in the region of said FIS genes. Methods for the application and use of such mutagens are well-known to those skilled in the art.

Insertional inactivation by molecular means may be achieved by introducing a DNA molecule into one or more of the FIS genes present in the genome of a plant such that Sthe regulatory region and/or reading frame of the F/S gene is disrupted, thereby resulting in either no FIS polypeptide being expressed or a mutant fis polypeptide (i.e.

a truncated or biologically ineffective polypeptide) being expressed in the maternallyderived cells, tissues or organsof the plant. Alternatively, a nucleic acid molecule which is capable of insertionally-inactivating a FIS gene may not be inserted directly into the regulatory region or structural regions of said gene, but in-the chromatin-which is adjacent thereto, such that the insertion promotes a change in chromatin structure which prevents or inhibits expression of the F/S gene or at least reduces expression of the FIS gene to a biologically-ineffective level in the maternally-derived cells, tissues or organs of the plant.

Preferred DNA molecules for insertional inactivation of a FIS gene include gene targeting molecules, transposon molecules, T-DNA molecules and other nucleic acid molecules which comprise one or more translation stop codons or are capable of altering the reading frame of a FIS gene when inserted therein or alternatively, are capable of disrupting one or more regulatory regions essential for expression of a FIS gene in the maternal cells, tissues or organs of the plant. The use of gene targeting molecules, transposon molecules, T-DNA molecules and nucleic acid molecules which comprise one or more translation stop codons is particularly preferred as such molecules may be introduced at any appropriate site within the open reading frame of a FIS gene to prevent the expression of a biologically effective FIS polypeptide.

As used herein, a "gene-targeting molecule" is an isolated nucleic acid molecule which is capable of being introduced into a target genetic sequence within the genome of a WO 00/16609 PCT/AU99/00805 plant by homologous recombination, wherein said nucleic acid molecule comprises one or more nucleotide sequences to facilitate said homologous recombination linked to additional nucleotide sequences which are non-homologous to the target genetic sequence, such that the nucleotide sequence of the target genetic sequence is altered following insertion of the gene-targeting molecule. In the present context, a genetargeting molecule will preferably comprise nucleotide sequences capable of disrupting the open reading frame of a FIS gene-when inserted into the homologous region thereof, flanked by one or more nucleotide sequences which are homologous to said FIS gene to facilitate insertion of the gene-targeting molecule into said FIS gene by means of homologous recombination.

Additional means for inhibiting, interrupting or otherwise reducing the expression of a FIS polypeptide include means which target transcription and/or mRNA stability and/or mRNA turnover and/or accessibility of mRNA to ribosomes or polysomes. Such means include the use of antisense molecules, ribozyme molecules, gene silencing molecules and the like introduced into the cell in an expressible format and expressed therein.

In the context of the present invention, an antisense molecule is an RNA molecule which is transcribed from the complementary strand of a nuclear FIS gene to that which is normally transcribed to produce a "sense" mRNA molecule capable of being translated into a FIS polypeptide. The antisense molecule is therefore complementary to the sense mRNA, or a part thereof. Although not limiting the mode of action of the antisense molecules of the present invention to any specific mechanism, the antisense RNA molecule possesses the capacity to form a double-stranded mRNA by base pairing with the FIS-encoding sense mRNA, which may prevent translation of the sense mRNA and subsequent synthesis of a FIS polypeptide product.

Ribozymes are synthetic RNA molecules which comprise a hybridising region complementary to two regions, each of at least 5 contiguous nucleotide bases in the target sense mRNA. In addition, ribozymes possess highly specific endoribonuclease activity, which autocatalytically cleaves the target sense mRNA. A complete description of the function of ribozymes is presented by Haseloff and Gerlach (1988) WO 00/16609 PCT/AU99/00805 -51and contained in International Patent Application No. W089/05852. The present invention extends to ribozymes which target a sense mRNA encoding a polypeptide involved in seed formation; such as the fis2 polypeptide described herein, thereby hybridising to said sense mRNA and cleaving it, such that it is no longer capable of being translated to synthesise a functional polypeptide product.

In the context of the present invention, gene silencing molecules are molecules which comprise nucleotide sequences complementary to the nucleotide sequence of an antisense mRNA which is complementary to a FIS sense mRNA encoding a FIS polypeptide, linked in head-to-head or tail-to-tail configuration to a part or region of said sense mRNA such that the gene silencing molecule is capable of being transcribed into mRNA which has self-complementarity. Whilst not being bound by any theory or mode of action, a gene silencing molecule has the potential to form a secondary structure such as a hairpin loop in the nucleus and/or cytosol of a cell and to sequester sense mRNA which is transcribed therein, such that single-stranded regions of the sequestered mRNA are rapidly degraded and/or a translationally-inactive complex is formed.

According to this embodiment, the present invention provides a ribozyme, antisense or gene silencing molecule comprising a sequence of contiguous nucleotide bases which are able to form a hydrogen-bonded complex with a sense mRNA encoding a fis polypeptide described herein, to reduce translation of said mRNA. Although the preferred antisense and/or ribozyme and/or gene silencing molecules hybridise to at least about 10 to 20 nucleotides of the target molecule, the present invention extends to molecules capable of hybridising to at least about 50-100 nucleotide bases in length, or a molecule capable of hybridising to a full-length or substantially full-length mRNA.

In yet a further embodiment of the invention, expression of a FIS polypeptide may be inhibited, interrupted or otherwise reduced by introducing to the cell a sense molecule, for example a co-suppression molecule or dominant-negative sense molecule in an expressible format and expressing said molecule therein.

WO 00/16609 PCT/AU99/00805 -52 The term "sense molecule" as used herein shall be taken to refer to an isolated nucleic acid-molecule which encodes or is complementary to an isolated nucleic acid molecule which encodes a FIS polypeptide involved in autonomous seed development, in particular a FIS1, FIS2 or FIS3 polypeptide or a homologue, analogue or derivative thereof, wherein said nucleic acid molecule is provided in a format suitable for its expression to produce a recombinant polypeptide when said sense molecule is introduced into a host cell by transfection or transformation.

A "co-suppression molecule" is a sense molecule which is capable of producing cosuppression when introduced and optionally, expressed in a cell.

Co-suppression is the reduction in expression of an endogenous gene that occurs when one or more copies of said gene, or one or more copies of a substantially similar gene are introduced into the cell. The present invention clearly extends to the use of co-suppression to inhibit the expression of a FIS gene as described herein.

In the present context, the term "dominant-negative sense molecule" shall be taken to mean a sense molecule as defined herein which comprises a nucleotide sequence which encodes a polypeptide which is capable of inhibiting, preventing or reducing the biological action of a FIS polypeptide, thereby enhancing or facilitating autonomous seed development and/or autonomous endosperm development and/or autonomous embryogenesis.

As will be known to those skilled in the art, a dominant negative sense molecule derived from a FIS polypeptide of the invention will lack the biological activity of the fulllength FIS polypeptide.

Preferred dominant-negative sense molecules of the invention will comprise at least one or more functional protein domains of the wild-type FIS protein. For example, a dominant-negative sense molecule which is capable of reducing expression of the FIS1 polypeptide may comprise only an acidic region and/or putative receptor binding domain TNFR/NGFR domain or RGD tripeptide, etc.) such that it is capable of WO 00/16609 PCT/AU99/00805 -53 competing with a biologically-active FIS1 polypeptide for binding to another protein or receptor, thereby inhibiting the effect of said biologically-active FIS1 polypeptide.

Similarly, a dominant-negative sense molecule which is capable of reducing expression of the FIS1 polypeptide may comprise a zinc-finger domain of the FIS2 polypeptide as described herein, such that it is capable of competing with the biologically-active FIS2 polypeptide for binding. The present invention clearly extends to the use of isolated nucleotide sequences encoding any and all combinations of the protein domains which are present in the FIS poypeptides described herein for the purpose of producing such dominant-negative sense molecules.

It is understood in the art that certain modifications, including nucleotide substitutions amongst others, may be made to the dominant-negative sense molecule, cosuppression molecule, gene-targeting- molecule, transposon molecule, T-DNA molecule, antisense, ribozyme or gene-silencing molecule of the present invention, without destroying the efficacy of said molecules in inhibiting the expression of the FIS gene. It is therefore within the scope of the present invention to include any nucleotide sequence variants, homologues, analogues, or fragments of.the said gene encoding same. However, in the case of gene-silencing molecules, ribozymes and-antisense molecules, those skilled in the art will be aware that it is necessary for such nucleotide sequence variants to be capable of hybridising to the biologically active FIS gene sequence or to sense mRNA encoded therefor.

A dominant-negative sense molecule or an antisense molecule or a ribozyme molecule or a gene-targeting molecule or transposon molecule or T-DNA molecule or a cosuppression molecule or gene-silencing molecule capable of targeting expression of a FIS gene in a plant will preferably comprise a nucleotide sequence having at least about 60-70% identity, more preferably at least about 70-80% identity, still more preferably at least about 80-90% identity or a tleast about 95-99% identity to the nucleotide sequence of a FIS1 or FIS2 gene set forth in any one of <400>4, <400>5, <400>6, <400>7, <400>8 or <400>9 or a complementary nucleotide sequence thereto.

In an alternative embodiment, a dominant-negative sense molecule or an antisense WO 00/16609 PCT/AU99/00805 -54 molecule or a ribozyme molecule or a gene-targeting molecule or transposon molecule or T-DNA molecule, or a co-suppression molecule or gene-silencing molecule capable of targeting expression of a FIS gene in a plant will preferably comprise a nucleotide sequence which is capable of hybridizing under at least low stringency conditions, more preferably under at least moderate stringency conditions and even more Spreferably under at least high stringency conditions, to any one of <400>4, <400>5, <400>6, <400>7, <400>8 or <400>9 or to that region of chromosome 3 of Arabidopsis thaliana which maps between the markers m317 and DWF1 as set forth in Figure 9B and which encode a FIS3 polypeptide which is capable of modulating autonomous seed development and/or partial autonomous endosperm development and/or autonomous embryogenesis in a plant.

In a further alternative embodiment, the dominant-negative sense molecule or an antisense molecule or a ribozyme molecule or a gene-targeting molecule or a cosuppression molecule is derived from the genomic equivalent of the Arabidopsis thaliana FIS1, FIS2 or FIS3 gene exemplified herein.

The present invention further extends to the mutation or insertional inactivation of such genomic equivalents in order to produce crop and horticultural plants capable of autonomous endosperm development and/or autonomous embryogenesis and/or autonomous seed development and/or apomictic development.

By "genomic equivalent" is meant a homologue of a FIS gene which is derived from another plant species. Such genomic equivalents may be isolated without undue experimentation, using any of the methods known to those skilled in the art, for example by hybridization, PCR, expression screening using antibodies or by functional assays.

Preferred genomic equivalents of the Arabidopsis thaliana FIS genes described herein are derived from crop plants which produce fruit having seed, especially crop plants which produce fruits having large numbers of seed or stone fruit.

WO 00/16609 PCT/AU99/00805 More preferably, the genomic equivalents of the Arabidopsis thaliana FIS genes are derived from mango, pawpaw, olives, apple, cherry, plum, peach, apricot, grape, passionfruit, date, fig, tomato, pear, tamarillo, quince, strawberry, blackberry, gooseberry, loganberry, Capsicum spp. and citrus plants, amongst others.

As will be known to those skilled in the art, the efficacy of a dominant-negative sense molecule or an antisense molecule or a ribozyme molecule or a gene-targeting molecule or transposon molecule or T-DNA molecule or a co-suppression molecule or gene-silencing molecule is dependent upon it being introduced and preferably, expressed in the maternal cell, tissue or organ or a progenitor cell, tissue or organ thereof. Such introduction and expression may be facilitated by presenting said dominant-negative sense molecule or an antisense molecule or a ribozyme molecule or a gene-targeting molecule or transposon molecule or T-DNA molecule or a cosuppression molecule or gene-silencing molecule in a genetic construct.

The present invention clearly extends to the use of genetic constructs designed to facilitate the introduction and/or expression of a dominant negative sense molecule, antisense molecule, ribozyme molecule, co-suppression molecule or gene-targeting molecule or transposon molecule or T-DNA molecule or gene-silencing molecule in a plant cell-and preferably in a maternal cell, tissue or organ or a progenitor cell, tissue or organ thereof.

Those skilled in the art will also be aware that expression of a dominant-negative sense, antisense, ribozyme, gene-targeting, co-suppression or gene-silencing molecule may require said molecule to be placed in operable connection with a promoter sequence. The choice of promoter for the present purpose may vary depending upon the level of expression required and/or the tissue, organ and species in which expression is to occur.

Reference herein to a "promoter" is to be taken in its broadest context and includes the transcriptional regulatory sequences of a classical eukaryotic genomic gene, including the TATA box which is required for accurate transcription initiation, with or without a WO 00/16609 PCT/AU99/00805 -56- CCAAT box sequence and additional regulatory elements upstream activating sequences, enhancers and silencers) which alter gene expression in response to developmental and/or external stimuli, or in a tissue-specific manner. In the context of the present invention, the term "promoter" also includes the transcriptional regulatory sequences of a classical prokaryotic gene, in which case it may include a box sequence and/or a -10 box transcriptional regulatory sequences. In the present context, the term "promoter" is also used to-describe a synthetic or fusion molecule, or derivative which confers, activates or enhances expression of said sense molecule in a cell. Preferred promoters may contain additional copies of one or more specific regulatory elements, to-further enhance expression and/or to alter the spatial expression and/or tempral expression of a nucleic acid molecule to which-it is operably connected. For example, copper-responsive regulatory elements may be placed adjacent to a heterologous promoter sequence driving expression of a nucleic acid molecule to confer copper inducible expression thereon.

Placing a nucleic acid molecule under the regulatory control of a promoter sequence means positioning said molecule such that expression is controlled by the promoter sequence. A promoter is usually, but not necessarily, positioned upstream or 5' of a nucleic acid molecule which it regulates. Furthermore, the regulatory elements comprising a promoter are usually positioned within 2 kb of the start -site of transcription of a sense, antisense, ribozyme, gene-targeting molecule or cosuppression molecule or chimeric gene comprising same. In the construction of heterologous promoter/structural gene combinations it is generally preferred to position the promoter at a distance from the gene transcription start site that is approximately the same as the distance between that promoter and the gene it controls in its-natural setting, the gene from which the promoter is derived. As is known in the art, some variation in this distance can be accommodated without loss of promoter function.

Similarly, the preferred positioning of a regulatory sequence element with respect to a heterologous gene to be placed under its control is defined-by the positioning of the element in its natural setting, the genes from which it is derived. Again, as is known in the art, some variation in this distance can also occur.

WO 00/16609 PCT/AU99/00805 -57- Examples of promoters suitable for use in genetic constructs of the present invention include promoters derived from the genes of viruses, yeasts, moulds, bacteria, insects, birds, mammals and plants which are capable of functioning in isolated plant cells, preferably in the maternally-derived cells of a plant or the cells, tissues and organs derived therefrom. The promoter may regulate the expression of the sense, antisense, ribozyme, gene-targeting molecule, co-suppression or gene-silencing -molecule constitutively, or differentially with respect to the tissue in which expression occurs or, with respect to the developmental stage at which expression occurs, or in response to external stimuli such as physiological stresses, pathogens, or metal ions, amongst others.

Promoters suitable for use according to this embodiment are further capable of functioning in cells derived from both monocotyledonous and dicotyledonous plants, including broad acre crop plants or horticultural crop plants.

Examples of promoters useful in performing this embodiment include the CaMV promoter, NOS promoter, octopine synthase (OCS) promoter, Arabidopsis thaliana SSU gene promoter, the meristem-specific promoter (meril),napin seed-specific promoter, and the like. In addition to the specific promoters identified herein, cellular promoters for so-called housekeeping genes are useful.

In a particularly preferred embodiment, the promoter may be derived from a genomic clone comprising a seed formation gene, in particular derived from the genomic gene equivalents of the A. thaliana FIS1, FIS2 OR FIS3 gene referred to herein.

The genetic construct may further comprise a terminator sequence and be introduced into a suitable host cell where it is capable of being expressed to produce a recombinant dominant-negative polypeptide gene product or alternatively, a cosuppression molecule, a ribozyme, gene silencing or antisense molecule.

The term "terminator" refers to a DNA sequence at the end of a transcriptional unit which signals termination of transcription. Terminators are 3 '-non-translated

DNA

WO 00/16609 PCT/AU99/00805 -58sequences containing a polyadenylation signal, which facilitates the addition of polyadenylate sequences to the 3'-end of a primary transcript. Terminators active in cells derived from viruses-, yeasts, moulds, bacteria, insects, birds, mammals and plants are known and described in the literature. They may be isolated from bacteria, fungi, viruses, animals and/or plants.

Examples of terminators particularly suitable for use in the genetic constructs of the present invention include the nopaline synthase (NOS) gene terminator of Agrobacterium tumefaciens, the terminator of the Cauliflower mosaic virus (CaMV) gene, the zein gene terminator from Zea mays, the Rubisco small subunit (SSU) gene terminator sequences and subclover stunt virus (SCSV) gene sequence terminators,.

amongst others.

Those skilled in the art will be aware of additional promoter sequences and terminator sequences which may be suitable for use in performing the invention. Such sequences may readily be used without any undue experimentation.

The genetic constructs of the invention may further include an origin of replication sequence which is required for replication in a specific cell type, for example a bacterial cell, when said genetic construct is required to be maintained as an episomal genetic element (eg. plasmid or cosmid molecule) in said cell.

Preferred origins of replication include, but are not limited to, the fl-ori and co/El origins of replication.

The genetic construct may further comprise a selectable marker gene or genes that are functional in a cell into which said genetic construct is introduced.

As used herein, the term "selectable marker gene" -includes any ger.e which confers.

a phenotype on a cell in which it is expressed to facilitate the identification and/or selection of cells which are transfected or transformed with a genetic construct of the invention or a derivative thereof.

WO 00/16609 PCT/AU9/00805 .59- Suitable selectable marker genes contemplated herein include the ampicillin resistance tetracycline resistance gene bacterial kanamycin resistance gene (Kan), phosphinothricin resistance gene, neomycin phosphotransferase gene (nptil), hygromycin resistance gene, 1-glucuronidase (GUS) gene, chloramphenicol acetyltransferase (CAT) gene and luciferase gene, amongst others.

In a preferred embodiment, the subject method comprises the additional first step of transforming the cell, tissue, organ or organism with a nucleic acid molecule which comprises the sense, antisense, ribozyme, co-suppression or gene-targeting molecule or transposon or T-DNA molecule. As discussed supra this nucleic acid molecule may be contained within a genetic construct. The nucleic acid molecule or a genetic construct comprising same may be introduced into a cell using any known method for the transfection or transformation of said cell. Wherein a cell is transformed by the genetic construct of the invention, a whole organism may be regenerated from a single transformed cell, using any method known to those skilled in the art.

By "transfect" is meant that the introduced nucleic acid molecule is introduced into said cell without integration into the cell's genome.

By "transform" is meant that the introduced nucleic acid molecule or genetic construct comprising same or a fragment thereof comprising a FIS gene sequence is stably integrated into the genome of the cell.

Means for introducing recombinant DNA into plant tissue or cells include, but are not limited to, transformation using CaCI, and variations thereof, in particular the method described by Hanahan (1983), direct DNA uptake into protoplasts (Krens et al, 1982; Paszkowski etal, 1984), PEG-mediated uptake to protoplasts (Armstrong et al, 1990).

microparticle bombardment, electroporation (Fromm et al., 1985), microinjection of DNA (Crossway et al., 1986), microparticle bombardment of tissue explants or cells (Christou et al, 1988; Sanford, 1988), vacuum-infiltration of tissue with nucleic acid, or in the case of plants, T-DNA-mediated transfer from Agrobacterium to the plant tissue as described essentially by An et a/.(1985), Herrera-Estrella et al. (1983a, 1983b, WO 00/16609 PCT/AU99/00805 60 1985).

For microparticle bombardment of cells, a microparticle is propelled into a cell to produce a transformed cell. Any suitable biolistic cell transformation methodology and apparatus can be used in performing the present invention. Exemplary apparatus.and procedures are disclosed by Stomp et al. Patent No. 5,122,466) and Sanford and Wolf Patent No. 4,945,050). When using biolistic transformation procedures, the genetic construct may incorporate a plasmid capable of replicating in the cell to be transformed.

Examples of microparticles suitable for use in such systems include 1 to 5 /m gold spheres. The DNA construct may be deposited on the microparticle by any suitable technique, such as by precipitation.

Alternatively, wherein the cell is derived from a multicellular organism and where relevant technology is available, a whole organism may be regenerated from the transformed cell, in accordance with procedures well known in the art.

Plant tissue capable of subsequent clonal propagation, whether by organogenesis or embryogenesis, may be transformed with a genetic construct of the present invention and a whole plant regenerated therefrom. The particular tissue chosen will vary depending on the clonal propagation systems available for, and best suited to, the particular species being transformed. Exemplary tissue targets include leaf disks, pollen, embryos, cotyledons, hypocotyls, megagametophytes, callus tissue, existing meristematic tissue apical meristem, axillary buds, and root meristems), and induced meristem tissue cotyledon meristem and hypocotyl meristem).

The term "organogenesis", as used herein, means a process by which shoots and roots are developed sequentially from meristematic centres.

The term "embryogenesis", as used herein, means a process by which shoots and roots develop together in a concerted fashion (not sequentially), whether from somatic WO 00/16609 PCT/AU99/00805 -61cells or gametes.

The regenerated transformed plants may be propagated by a variety of means, such as by clonal propagation or classical breeding techniques. For example, a first generation (or T1) transformed plant may be selfed or crossed to another T1 plant and homozygous second generation (or T2) transformants selected.

In the case of woody -fruit crops such as citrus and grapes which are highly heterozygous and propagated vegetatively from cuttings, the genes to be introduced must be dominant in action and the cultivar identity must be maintained by using the primary transformants directly, for example by generating clonal derivatives of primary transformants. It is preferred in the commercial application of the invention to the production of softseeded fruits that transgenic plants having reduced expression of FIS knock-out plants) are further made male-sterile by any means known to those skilled in the art, preferably by the expression of a gene construct which induces male-sterility in plants as a dominant phenotype, such as by the expression of a barnase gene or a gene encoding a cytotoxin under control of an anther-specific or tapetum-specific gene promoter. Where the barnase gene or a gene encoding a cytotoxin is used to induce male-sterility, this should only need to be present in the heterozygous state to observe the male-sterile phenotype. In this way, there is no initiation of seed formation from those cells of the primary transformant which do not contain or express the introduced gene. This strategy is particularly relevant to the application of the invention in cases where fruits comprise multiple seeds, such as citrus fruits, grapes, berries, pears, apples and tomato, amongst others. In the case of-stone fruit, although some fruit having normal seed may initiate in the absence of male-sterility, it may be possible to screen and select for those fruit having soft seed.

In applications of the invention to the production of apomictic plants by an autonomous seed development mechanism (as opposed to a pseudogamous mechanism which requires pollination to initiate seed development), it is also preferred that plants are WO 00/16609 PCT/AU99/00805 -62made male-sterile to reduce or prevent any "leakiness" in the downregulation of endogenous FIS gene expression, thereby ensuring that all seed which are produced by transgenic plants are the products of apomixis and not hybrid seed.

In the case of woody plants such as citrus and grapes which are generated by cuttings, it is particularly preferred to employ a strategy wherein dominant-acting male-sterilityinducing gene constructs and the gene construct capable of down-regulating expression of the negative regulator of seed formation are introduced into plant material and primary transformants selected which contain both genes integrated into their genome. As with all transformation strategies, a large number of primary transformants should be generated to facilitate elimination of those transformants wherein the introduced gene constructs are inserted into housekeeping genes -or otherwise have an adverise effect on the plant, including an adverse effect on the quality or yield of the plant products derived therefrom. Primary transformants are propagated by cuttings to generate lines of transgenic plant material which either contain single or multiple copies of the introduced gene construct(s) and the mature plants derived therefrom assayed for product quality.

Plants may be made male-sterile before or ifter the gene construct targeting fis gene expression is introduced into plants or alternatively, at the same timeLas-the gene construct targeting fis gene expression is introduced into plants. Wherein the plants are made male-sterile before or after introducing the gene construct targeting FIS gene expression, this is best achieved by making such plants homozygous for one or both of the introduced genes the male-sterility gene and/or the gene construct targeting FIS gene expression). Persons skilled in the art will be aware of the most preferred means for making plants homozygous for one or both of the introduced genes for any particular plant species-of-interest. Clearly, in the case of vegetativelypropagated species, such an approach is not viable.

Preferably, plants are made male-sterile at the same time as the gene construct targeting fis gene expression is introduced into plants. Such an approach is particularly preferred in the case of woody plants which are propagated vegetatively. In such cases WO 00/16609 PCT/AU99/00805 -63 it is even more preferable to include the male-sterility-inducing gene on the same vector as the gene construct which downregulates FIS gene expression in the plant.

Those skilled in the art will also be aware of the advantage of having the male-sterile phenotype cosegregate with the introduced gene construct which targets fis gene expression. This advantage may be derived advantageously by having both gene "cassettes" located on the same gene construct such that they are-closely linked,-to prevent recombination therebetween occurring at a high frequency, in the primary transformants and in the progeny plants derived therefrom Methods for the production of male-sterile plants will be known to those skilled in the art and the present invention is not limited by such means.

The regenerated transformed organisms contemplated herein may take a variety of forms. For example, they may be chimeras of transformed cells and non-transformed cells; clonal transformants all cells transformed to contain the expression cassette); grafts of transformed and untransformed tissues in plants, a transformed root stock grafted to an untransformed scion The above-mentioned dominant-negative sense molecules, antisense molecules, ribozyme molecules, gene-targeting molecules, transposons, T-DNA molecules, gene silencing molecules and co-suppression molecules are particularly useful for reducing or eliminating the expression of particular FIS genes in plants, to produce plants which at least exhibit autonomous endosperm development.

A transformed plant comprising the introduced-nucleic acid molecule contemplated herein to reduce the expression of FIS polypeptide will preferably exhibit a phenotype which is substantially identical to the autonomous seed formation phenotype of the fis1, fis2 or fis3 mutant described herein.

Arrested embryo development which results from inhibition of expression of the FIS gene may be concomitant with autonomous endosperm development in the plant into which the subject dominant-negative sense molecule or an antisense molecule or a WO 00/16609 PCT/AU99/00805 -64ribozyme molecule or a gene-targeting molecule or a co-suppression molecule is introduced and expressed. As exemplified herein, in the absence of FIS2 expression or expression of any of the protein domains of the FIS1 polypeptide referredto herein, Arabidopsis thaliana ecotype Landsberg plants produce autonomous seed or seed-like structures which lack a functional embryo and are softer than wild-type seed.

In fact, the invention is particularly useful to produce parthenocarpic fruit or "seedless fruit" which lacks a fully-developed embryo not normally produced by wild or naturallyoccurring organisms belonging to the same genera or species as the genera or species from which the transfected or transformed cell is derived. Such seedless fruit may, in fact, include fruits having soft seed which are present at a level which allows the fruit to be marketed as "less seedy" than wild-type fruit.

Preferred target plants in which the invention may be performed include stone fruits such as apricots and peaches, citrus fruits such as oranges, lemons, grapefruits, mandarins and tangelos, amongst others, in addition to grapes, apples, melons, pears, and berries, amongst others.

Preferably, the inventive method is used to develop plants which autonomously form seed comprising an embryo and an endosperm.

Alternatively or in addition, such plants may be apomictic, in which case they will autonomously develop fully-fertile seed. As the presently described genes have been shown to at least be capable of repressing autonomous embryogenesis and partial autonomous endosperm development in vivo, the application of such genes to the development of fully-fertile apomictic seeds, those skilled in the art will also be aware of the particular utility of the presently-described FIS genes in producing plants which are capable of autonomously forming fully-fertile seed apomictic plants).

Preferred target plants in which this embodiment of the invention may be performed include monocotyledonous or dicotyledonous broadacre or horticultural crop plants, are those plants which produce seed of agronomic value, such as grain crop plants, WO 00/16609 PCT/AU99/00805 in particular rice, wheat, maize, rape, rye, safflower, sunflower, millet and barley, amongst others.

The present inventors are aware of the possible existence of one or more modifier genes which, in combination with the dominant-negative sense molecule, antisense molecule, ribozyme molecule, gene-targeting molecule, transposon, T-DNA molecule, gene-silencing molecule or co-suppression molecule which comprise the FIS gene sequences described herein, interact to produce plants capable of complete autonomous embryogenesis in addition to -complete autonomous endosperm development, wherein the mature seed are fully-fertile. It is clearly within the scope of the present invention to include the optional use of nucleotide sequences derived from the presently-described FIS genes in combination with any other gene(s) or alternatively, any sense molecule, dominant-negative sense molecule, antisense molecule, ribozyme molecule, gene-targeting molecule, transposon, T-DNA molecule, gene-silencing molecule or co-suppression molecule comprising said other gene(s), to perform the inventive method.

As an alternative to the introduction of specific modifier genes in combination with the dominant-negative sense molecule, antisense molecule, ribozyme molecule, genetargeting molecule, transposon, T-DNA molecule, gene-silencing molecule or cosuppression molecule of the invention, it is also within the capabilities of the skilled artisan to introduce a dominant-negative -sense molecule, antisense molecule, ribozyme molecule, gene-targeting molecule, transposon, T-DNA molecule, genesilencing molecule or co-suppression molecule into a genetic background which expresses the modifier gene at a level which is such that introduction of said inventive molecules thereto will be sufficient to produce a plant which is capable of autonomous seed development and/or autonomous-endosperm development and/or autonomous embryogenesis and preferably, an apomictic plant.

A second aspect of the invention clearly extends to the isolated nucleic acid molecules which are used to inhibit, prevent or interrupt the expression of a FIS polypeptide in a plant according to the inventive method, including those genomic equivalents of the WO 00/16609 PCT/AU99/00805 66- Arabidopsis thaliana FIS polypeptides exemplified herein.

-Preferably, the nucleic acid molecule according to this aspect of the invention will comprise a dominant-negative sense molecule or an antisense molecule or a ribozyme molecule or a gene-targeting molecule or a co-suppression molecule or a gene silencing molecule which comprises a nucleotide sequence which is derived from a FIS gene as described herein or a genomic equivalent thereof.

A third aspect of the invention clearly extends to a transgenic plant or a plant cell, tissue, organ produced according to the method described herein, including the seed produced by said plant and progeny plants derived therefrom which are capable of reproducing by apomictic means.

According to this aspect, the invention provides a cell which has been transformed or transfected with the subject nucleic acid molecule or a dominant-negative sense molecule or an antisense molecule or a ribozyme molecule or a gene-targeting molecule or a co-suppression molecule which is derived from a FIS gene, preferably in an expressible form.

A further aspect of the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence which encodes or is complementary to a nucleotide sequence which encodes a polypeptide, protein or enzyme which is- capable of regulating autonomous endosperm development in a plant.

Preferably, the polypeptide, protein or enzyme is further capable of regulating autonomous embryogenesis and more preferably, autonomous seed development in a plant.

By "capable of regulating endosperm development" means that the polypeptide, protein or enzyme is involved in asexual seed. development in plants at least to the extent that a disruption of expression or reduction in the level of expression of said polypeptide, protein or enzyme in the plant induces-at least partial autonomous WO 00/16609 PCT/AU99/00805 -67- endosperm development therein.

By "capable of regulating embryogenesis" means that the polypeptide, protein or enzyme is involved in asexual seed development in plants at least to the extent that a disruption of expression or reduction in the level of expression of said polypeptide, protein or enzyme in the plant induces at least partial autonomous embryogenesis therein.

By "capable of regulating seed development" means that the polypeptide, protein or enzyme is involved in asexual seed development in plants at least-to the extent that a disruption of expression or reduction in the level of expression of said polypeptide, protein or enzyme in the plant induces at least partial autonomous endosperm development and partial autonomous embryogenesis therein and preferably induces the autonomous development of fully-fertile seeds.

In one alternative embodiment, the nucleic acid molecule of the invention encodes or is complementary to a nucleic acid molecule which encodesa FIS polypeptide, protein or enzyme or a protein domain thereof according to any one or more embodiments described herein or a genomic equivalent thereof.

Alternatively or in addition, the isolated nucleic acid molecule of the invention comprises a FIS gene which is involved in fertilization-independent seed production in a plant.

In the context of the present invention, "fertilization-independent seed production" means the autonomous formation of fertile seed or seed-like structures comprising an embryo and/or endosperm with or without a seed coat, from any of the organs forming the gynoecium or contained within the gynoecium. More particularly, fertilizationindependent seed production results in the autonomous formation of fertile seed or seed-like structures from the megaspore and/or non-archesporial cells such as those forming the nucellus or integument.

WO 00/16609 PCT/AU99/00805 -68- Accordingly, the present invention clearly encompasses those isolated genes which are expressed to regulate autonomous seed formation in any plant species, regardless of whether or not that gene is capable of resulting in the formation of fully-fertile seed or seed-like structures. Those skilled in the art will recognize that the isolated gene described herein does however perform a critical role in autonomous seed production in plants. The inventors have characterised the FIS (Fertilization Independent Seed) family of genes, at least three genes of which are exemplified herein, designated FIS1, FIS2 and FIS3 and which encode different polypeptide repressors capable of inhibiting autonomous embryogenesis and partial autonomous endosperm development in plants.

Those skilled in the art may readily assay for-FIS gene activity of an isolated nucleic acid molecule by determining the ability of an inhibitor of the expression of said nucleic acid molecule, such as a mutagen, an antisense molecule, dominant-negative sense molecule, ribozyme molecule, co-suppression molecule, transposon, T-DNA, gene silencing molecule or gene-targeting molecule as described herein, to induce autonomous endosperm development and/or autonomous embryogenesis and/or autonomous seed formation in a plant.

Alternatively, the activity of the polypeptide encoded by a FIS gene maybe inhibited using a ligand which specifically binds thereto, such as an antibody molecule or a peptide, oligopeptide, polypeptide, enzyme or chemical compound which binds to its active site, and the autonomous induction of formation of seed or seed-like structures is assayed. For convenience, the plant being assayed may first be made male-sterile to reduce background self-fertilization events.

Preferably, the isolated nucleic acid molecule of the invention comprises a FIS gene which comprises the sequence of nucleotides set forth in any one of <400>4, <400>5, <400>6, <400>7, <400>8 or <400>9 or a homologue, analogue or derivative thereof or a complementary nucleotide sequence thereto.

For the present purpose, "homologues" of a nucleotide sequence shall be taken to WO 00/16609 PCT/AU99/00805 -69refer to an isolated nucleic acid molecule which is substantially the same as the nucleic acid molecule of the present invention or its complementary nucleotide sequence, notwithstanding the occurrence within said sequence, of one or more nucleotide substitutions, insertions, deletions, or rearrangements.

"Analogues" of a nucleotide sequence set forth herein shall be taken-to refer to an isolated nucleic acid molecule which is substantially the same as a nucleic acid molecule of the present-invention or its complementary. nucleotide sequence, notwithstanding the occurrence of any non-nucleotide constituents not normally present in said .isolated nucleic acid molecule, for example carbohydrates, radiochemicals including radionucleotides, reporter molecules such as, but not limited to DIG, alkaline phosphatase or horseradish peroxidase, amongst others.

"Derivatives" of a nucleotide sequence set forth herein shall be taken to refer-to any isolated nucleic acid molecule which contains -significant sequence identity to said sequence or a part thereof. Generally, the nucleotide sequence of the present invention may be subjected to mutagenesis to produce single or multiple nucleotide substitutions, deletions and/or insertions. Nucleotide insertional derivatives of the nucleotide sequence of the present invention include 5' and 3' terminal fusions as well as intra-sequence insertions of single or multiple nucleotides or nucleotide analogues.

Insertional nucleotide sequence variants are those in which one or more nucleotides or nucleotide analogues are introduced into a predetermined site in the nucleotide sequence of said sequence, although random insertion is also possible with suitable screening of the resulting-product being performed. Deletional variants are characterised. by the removal of one or more nucleotides from the nucleotide sequence. Substitutional nucleotide variants are those in which at least one nucleotide in the sequence has been removed and a different nucleotide or nucleotide analogue inserted in its place.

Particularly preferred homologues, analogues or derivatives of the nucleotide sequences set forth in any one of <400>4, <400>5, <400>6, <400>7, <400>8 or <400>9 include any one or more of the isolated nucleic acid molecules selected from WO 00/16609 PCT/AU99/00805 the following: an isolated nucleic acid molecule which comprises a nucleotide sequence which is at least about 60% identical to any one of <400>4,. <400>5, <400>6, <400>7, <400>8 or <400>9 or a complementary sequence thereto; (ii) an isolated nucleic acid molecule which comprises a nucleotide sequence which is at least about 60% identical to at least about 30 contiguous nucleotides of any one of <400>4, <400>5, <400>6, <400>7, <400>8 or <400>9 or a complementary sequence thereto; (iii) an isolated nucleic acid molecule which is capable of hybridising under at least low stringency conditions to at least about 25-30 contiguous nucleotides of any one of <400>4, <400>5, <400>6, <400>7, <400>8 or <400>9 or a complementary sequence thereto; and (iv) an isolated nucleic acid molecule which is capable of hybridising under at least low stringency conditions to at least about 25-30 contiguous nucleotides of the RFLP marker designated ve039 or the YAC clone CC7E1 or the pl clones MCB22 or MNH5 or a complementary sequence thereto; Such homologues, analogues and derivatives may be obtained by any standard procedure known to those skilled in the art, such as by nucleic acid hybridization (Ausubel et al, 1987), polymerase chain reaction (McPherson et al, 1991) screening of expression libraries using antibody probes (Huynh et al, 1985) or by functional assay as exemplified herein.

In nucleic acid hybridizations, genomic DNA, mRNA or cDNA or a part of fragment thereof, in'isolated form or contained within a suitable cloning vector such as a plasmid or bacteriophage or cosmid molecule, is contacted with a hybridization-effective amount of a nucleic acid probe derived from any one of <400>4, <400>5, <400>6, <400>7, <400>8 or <400>9 or alternatively, from the RFLP marker designated ve039 or the YAC clone CC7E1 or the pl clones MCB22 or MNH5, for a time and under conditions sufficient for hybridization to occur and the hybridized nucleic acid is then detected using a detecting means.

WO 00/16609 PCT/AU99/00805 -71- Detection is performed preferably by labelling the probe with a reporter molecule capable of producing an identifiable signal, prior to hybridization. Preferred reporter molecules include radioactively-labelled nucleotide triphosphates and biotinylated molecules.

Preferably, variants of the FIS genes exemplified herein, including genomic equivalents, are isolated by hybridisation under medium or more preferably, under high stringency conditions, to the probe.

In the polymerase chain reaction (PCR), a nucleic acid primer molecule comprising at least about 14 nucleotides in length derived from a FIS gene is hybridized to a nucleic acid template-molecule and specific nucleic acid molecule copies of the template are amplified enzymatically as described in McPherson et al, (1991), which is incorporated herein by reference.

In expression screening of cDNA libraries or genomic libraries, protein- or peptideencoding regions are placed operably under the control of a suitable promoter sequence in the sense orientation, expressed in a prokaryotic cell or eukaryotic cell in which said promoter is operable to produce a peptide or polypeptide, screened with a monoclonal or polyclonal antibody molecule or a derivative thereof against one or more epitoles of a FIS polypeptide and the bound antibody is then detected using a detecting means, essentially as described by Huynh et al (1985) which is incorporated herein by reference. Suitable detecting means according to this embodiment include 25 1-labelled antibodies or enzyme-labelled antibodies capable of binding to the firstmentioned antibody, amongst others.

The nucleic acid molecule of the invention or a homologue, analogue or derivative thereof may be obtained from any plant species.

A still further aspect of the invention provides an isolated promoter sequence which is -capable of conferring expression at least in one or more female reproductive cells, tissues or organs of said plant or a progenitor cell, tissue or organ thereof.

WO00/16609 PCT/AU99/00805 72- Preferably, the promoter is capable of conferring expression in the ovule or a progenitor cell thereof or a derivative cell, tissue or organ thereof.

More preferably, the promoter sequence is isolatable as a DNA fragment which is capable of hybridising under at least low stringency conditions to any one or more of the nucleotide sequences set forth in <400>4, <400>5, <400>6, <400>7, <400>8 or <400>9 or a complementary nucleotide sequence thereto and even more preferably to the 5'-region of any one or more of said nucleotide sequences and still even more preferably to the 5'-untranslated regions-of any one of <400>4, <400>5, <400>6, <400>7, <400>8 or <400>9 or a complementary nucleotide sequence thereto.

In a particularly preferred embodiment, the promoter at least comprises a nucleotide sequence which corresponds to nucleotide residues 1 to 3142 of <400>5 or a part thereof; or nucleotide residues 1785 to 3142 of <400>5 or a part thereof; or nucleotide residues 1 to 2851 of <400>7 or a part thereof; or nucleotide residues 1531 to 2851 of <400>7 or a part thereof; or nucleotide residues 1 to 1200 of <400>9 or a part thereof.

Alternatively or in addition, the promoter sequence may further comprise the exonl and/or intronl sequence of a FIS gene described herein, in particular a FIS gene as described in <400>5 or <400>7 or <400>9.

The present invention clearly extends to the promoter sequence and/or exonl and/or intronl sequences in operably connection with a structural gene region derived from the same or a different genetic sequence, optionally in a genetic construct.

A still further aspect of the present invention provides an isolated or recombinant

FIS

polypeptide or a homologue, analogue, derivative or epitope thereof.

Particularly preferred derivatives of a FIS polypeptide include those peptides, oligopeptides and polypeptides which comprise at least about 5-10 contiguous amino acids derived from any one of <400>1 or <400>2 or <400>3 or which comprise any WO 00/16609 PCT/AU99/00805 -73 one of the protein domains of the FIS1 or FIS2 or FIS3 polypeptides described herein or a fragment thereof comprising at least about 5 amino acids in length.

As used herein, the term "epitope" refers to a peptide or derivative of a FIS polypeptide which is at least useful for the preparation of antibody molecules, including recombinant antibodies, polyclonal or monoclonal antibody molecules.

It will be apparent from the description provided herein that a recombinant FIS polypeptide or an epitope thereof may be produced by standard means by expressing a sense molecule which comprises a nucleotide sequence which encodes said polypeptide operably under the control of a suitable promoter sequence in a host cell for a time and under conditions sufficient for translation to occur.

As will be known to those skilled in the art, expression of a sense fiolecule may be carried out in a prokaryotic cell such as a bacterial cell, for example an Escherichia coli cell. Alternatively, such expression may be performed in a eukaryotic cell such as an insect cell, mammalian cell, plant cell or yeast cell, amongst others. In any case, unless the sense molecule is expressed under the control of a strong universal promoter, it is important to select a promoter sequence which is capable of regulating expression in the cell comprising the sense molecule in an expressible format. Persons skilled in the art will be in a position to select appropriate promoter sequences for expression of the sense molecule without undue experimentation.

Examples of promoters useful in performing this embodiment include the CaMV promoter, NOS promoter, octopine synthase (OCS) promoter, Arabidopsis thaliana SSU gene promoter, napin seed-specific promoter, P 32 promoter, BK5-T imm promoter, lac promoter, tac promoter, phage lambda A or A, promoters, CMV promoter (U.S.

Patent No. 5,168,062), T7 promoter, lacUV5 promoter, SV40 early promoter (U.S.

Patent No. 5,118,627), SV40 late promoter Patent No.-5,118,627), adenovirus promoter, baculovirus P10 or polyhedrin promoter Patent Nos. 5,243,041, 5,242,687, 5,266,317, 4,745,051 and 5,169,784), and the like. In addition to the specific promoters identified herein, cellular promoters for so-called housekeeping WO 00/16609 PCT/AU99/00805 -74genes are useful.

In a preferred embodiment, the recombinant FIS polypeptide or a homologue, analogue, derivative or epitope thereof is provided in a sequencably-pure format or a substantially pure format.

By "sequencably pure" is meant that the subject polypeptide or a homologue, analogue, derivative or epitope thereof is purified sufficiently to facilitate amino acid sequence determination.

Preferably, said polypeptide or a homologue, analogue, derivative or epitope is at least about 20% pure; more preferably at least about 40% pure, even more preferably at least about 60% pure and even more preferably at least about 80% pure or pure on a weight basis.

It is apparent from the description provided herein that the FIS polypeptides are likely to be involved -in a range of biological interactions in the regulation of seed development in plants (see for example, the description in Example 16); in particular protein:protein interactions, such as via the acidic region of the FIS1 polypeptide or the repeat structure of the FIS2 polypeptide, amongst others and/or protein:nucleic acid molecule interactions, such as via one or more of the cysteine-rich regions of the FIS1 polypeptide or the zinc-finger motif of the FIS2 polypeptide, amongst others. Such interactions-are well known for their effects in regulating gene expression in both prokaryotic and eukaryotic cells, in addition to being critical for DNA replication and in the case of certain viruses, RNA replication.

As used herein, the term "interaction" shall be taken to refer to a physical association between two or more molecules or "partners", one of which comprises a FIS polypeptide or a protein domain thereof as described herein or a p3ptide derivative thereof. The association is involved in one or more cellular processes involved in seed development in plants and preferably occurs at least in the maternal cells, tissues or organs, such as in the process of imprinting.

WO 00/16609 PCT/AU99/00805 The "association" may involve the formation of an induced magnetic field or paramagnetic field, covalent bond formation such as a disulfide bridge formation between polypeptide molecules, an ionic interaction such as occur in an ionic lattice, a hydrogen bond or altematively, a van der Waals interaction such as a dipole-dipole interaction, dipole-induced-dipole interaction, induced-dipole-induced-dipole interaction or a repulsive interaction or any combination of the above forces of attraction.

As used herein, the term "FIS partner" shall be taken to mean any amino acid sequence which is derived from a FIS polypeptide and which is capable of directly interacting with one or more peptides, oligopeptides, polypeptides, proteins, RNA molecules and DNA molecules to confer or regulate autonomous endosperm development and/or autonomous embryogenesis and/or autonomous or pseudogamous seed development in plants.

The present invention clearly extends to those peptides, oligopeptides, polypeptides, proteins, RNA molecules and DNA molecules which interact with a FIS partner.

Preferably, the peptides, oligopeptides, polypeptides, proteins, RNA molecules and DNA molecules which interact with a FIS partner are normally regulated by one or more FIS polypeptides.

By appropriate strategies described herein, the peptides, oligopeptides, polypeptides, proteins, RNA molecules and DNA molecules which interact with a FIS partner and the nucleic acid molecules encoding said interacting peptides, oligopeptides, polypeptides and proteins are isolated.

Conventional one-hybrid, two-hybrid and three-hybrid assays may be used to identify and isolate the peptides, oligopeptides, polypeptides, proteins, RNA molecules and DNA molecules which interact with a FIS partner. Such assays are described in detail by Poutney et al. (1997), Bendixen et a/.(1994), Vidal et al. (1996a,b), Yang et al.

(1995) and Zhang et al. (1996), which are incorporated herein by way of reference.

WO 00/16609 PCT/AU99/00805 -76- In such assays, recombinant cells are produced which are capable of expressing both binding partners. Inscreening applications, a representative random library is generally produced in a cellular host, such that each cell expresses a different peptide, oligopeptide, polypeptide or protein or RNA molecule or DNA molecule, in addition to expressing the FIS partner. The transformed cells of the library may further contain a nucleotide sequence which comprises or encodes a reporter molecule,the expressionof which is capable of being modified by the interaction between the binding partners.

The cells are cultured for-a time and under conditions sufficient for expression of said second nucleotide sequences encoding the partners to occur and cells wherein expression of said reporter molecule is modified are selected.

Alternatively or in addition, the binding partners are further expressed as a fusion protein with a nuclear targeting motif capable of facilitating targeting of said peptide to the nucleus of said host cell where transcription occurs, in particular the yeast-operable SV40 nuclear localisation signal.

The FIS partner and/or its cognate binding partner may also be expressed constitutively on the surface of a bacteriophage, such as by phage display, a process well-known in the art.

In the case of nucleic acid molecule binding partners which interact with the FIS partner, it is preferred that the nucleotide sequences of the random library are placed in operable connection with a nucleic acid molecule which encodes the reporter molecule. Wherein the FIS partner inhibits activity of the other binding partner in vitro, expression of the reporter molecule will preferably be inhibited. In such cases, it is advantageous for the selection of cells in which the interaction has occurred for the expression of the reporter molecule to be toxic to the cell. For example, the CYH2 gene encodes a product which is lethal to yeast cellsin the presence of the drug cycloheximide or the LYS2 gene which confers lethality in the presence of the drug a-aminoadipate In this case, only those cells in which the interaction between the binding partners has occurred will survive selection. Alternatively, if the FIS partner activates activity of the other binding partner in vitro, it is preferable for WO 00/16609 PCT/AU99/00805 77 expression of the the reporter molecule to be activated by the interaction between the binding partners. In such cases, it is advantageous for the selection of cells in which the interaction has occurred for the expression of the reporter molecule to encode resistance to a toxic compound, for example an antibiotic compound or herbicide. As with other embodiments described herein, only those cells in which the interaction between the binding partners has occurred will survive selection on the selective medium.

In the case of protein-based binding partners which interact with the FIS partner, the expression of the reporter molecule may be linked to the interaction between the binding partners by expressing both binding partners as fusion polypeptides with different regions derived from a known transcription factor, such that their interaction.

reconstitutes a functional transcription factor which is capable of regulating expression of the reporter molecule in the cell. As with the other embodiments described herein, the selection of reporter molecule and the selection means will depend upon whether or not the interaction between the binding partners has a positive or negative effect on expression of a structural gene in the cell to which the interaction is operably connected.

Examples of suitable reporter genes include but are not limited to HIS3 (Larson et al.,1996; Condorelli et al.,1996; Hsu et al.,1991; and-Osada et al.,1995) and LEU2 (Mahajan et al., 1996) the protein products of which allow cells expressing these reporter genes to survive on appropriate cell culture medium. Conversely, the reporter gene is the URA3 gene, wherein URA3 expression is toxic to a cell expressing this gene, in the presence of the drug 5-fluoro-orotic acid (5FOA). Other counterselectable reporter genes include CYH1 and LYS2, which.confer lethality in the presence of the drugs cycloheximide and a-aminoadipate respectively.

The cells used to perform this embodiment may be any cell capable of supporting the expression of exogenous DNA, such as a bacterial cell, insect cell, yeast cell, mammalian cell or plant cell. In a particularly preferred embodiment of the invention, the cell is a bacterial cell, mammalian cell or a yeast cell. In a particularly preferred WO 00/16609 PCT/AU99/00805 -78embodiment of the invention, the cell is a yeast cell.

The promoter which is used to regulate expressiorf of the binding partners and/or the reporter molecule must be operably in the cell line used. In the case of yeast andlor bacterial cells, it is particularly preferred that the promoter is selected from the list comprising GALl, CUP1, PGK1, ADH2, PH05, PRB1, GUTI, SP013, ADHI, CMV, or T7 promoter sequences. Wherein the promoter is intended to regulate expression of the reporter molecule, it is further preferred that said promoter include one or more recognition sequences for the binding of a DNA binding domain derived from a transcription factor, for example a GAL4 binding site or LexA operator sequence.

Any standard means may be used to introduce the nucleic acid moleculeswhich encode the binding partners and reporter molecule into the cell, including cell mating, transformation or transfection procedures. The nucleotide sequences encoding the binding partners may be each contained within a separate genetic construct and introduced into the cell together or by sequential transformation. Alternatively, these nucleotide sequences may be introduced into separate populations of host cells which are subsequently mated and those cell populations containing both nucleotide sequences selected on media permitting growth of host cells successfully transformed with both nucleic acid molecules. Alternatively, these nucleotide sequences may be contained on a single genetic construct and introduced into the host cell population in a single-step.

Cells in which the interaction-between the binding partners has occurred are selected and the nucleic acid molecule which encodes the other partner the non-FIS partner) may be recovered from the cell and the nucleotide sequence and derived amino acid sequence encoded therefor are determined using standard procedures.

Techniques for such methods are described, for example by Ausubel et al (1987 et seq), amongst others.

Accordingly, a still further aspect of the present- invention contemplates peptides, WO 00/16609 PCT/AU99700805 -79oligopeptides and polypeptides and isolated nucleic acid molecules identified by-the method of the present invention.

The isolated nucleotide sequences which encode nucleic acid binding partners capable of interacting with a FIS partner may be expressed directly in a transgenic plant cell, tissue or organ under the control of a suitable promoter sequence, to confer autonomous or pseudogamous phenotypes thereon. Because the FIS polypeptide is a negative regulator of autonomous seed development, -these non-FIS partners are likely to represent DNA-binding sites in the promoter region of a gene the expression of which is required for seed development to occur. Accordingly, removal of the FISbinding domains from such genetic sequences, such as by expressing the genetic sequence under the control of a heterologous promoter which is not-recognised by FIS will confer the autonomous seed phenotype on the cell. Similarly, in the case of polypeptide non-FIS partners, mutagenesis to remove the FIS recognition domains therefrom will also remove or reduce the ability of the FIS polypeptide to inhibit, or otherwise reduce autonomous seed development in the plant.

A further aspect of the invention extends to an a monoclonal or polyclonal antibody molecuile which is capable of binding to a FIS polypeptide or an epitope thereof.

Standard methods may be used to prepare the antibodies. By using a FIS peptide, oligopeptide or polypeptide -described herein, polyclonal antisera or monoclonal antibodies can be made using standard methods. For example, a mammal, a mouse, hamster, or rabbit) can be immunized with an immunogenic form of the FIS peptide, oligopeptide or polypeptide which elicits an antibody response in themammal.

Techniques for conferring immunogenicity on a peptide include conjugation to carriers or other techniques well known in the art. For example, the peptide can be administered in the presence of adjuvant. The progress of immunization can be monitored by detection of antibody titres in plasma.or serum. Standard ELISA or other immunoassay can be used with the immunogen as antigen to assess the levels of antibodies. Following immunization, antisera can be obtained and, if desired IgG molecules correspond to the polyclonal antibodies isolated from the sera.

WO 00/16609 PCT/AU99/00805 To produce monoclonal antibodies, antibody producing cells (lymphocytes) can be harvested from an immunized animal and fused with myeloma cells by standard somatic cell fusion procedures thus immortalizing these cells and yielding hybridoma cells. Such techniques are well known in the art. For example, the hybridoma technique originally developed by Kohler and Milstein (1975) as well as other techniques such as the human B-cell hybridoma technique (Kozbor et al., 1983), the EBV-hybridQma technique to produce human monoclonal antibodies (Cole et al., 1985; Roder, 1986), and screening of combinatorial antibody libraries (Huse et al., 1989).

Hybridoma cells can be screened immunochemically for production of antibodies which are specifically reactive with the peptide and monoclonal antibodies isolated.

As with all immunogenic compositions for eliciting antibodies, the immunogenically effective amounts of the peptides of-the-invention must be determined empirically.

Factors to be considered include the immunogenicity of the native peptide, whether or not the peptide will be complexed with or covalently attached to an adjuvant or carrier protein or other carrier and route of administration for the composition, i.e. intravenous, intramuscular, subcutaneous, etc., and the number of immunizing doses to be administered. Such factors are known in the vaccine art and it is well within the skill of immunologists to make such determinations without undue experimentation.

Antibodies can be fragmented using conventional techniques and the fragments screened for utility in the same manner as described above for whole antibodies. For example, F(ab')2 fragments can be generated by treating antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges to produce Fab' fragments.

It is within the scope of this invention to include any second antibodies (monoclonal, polyclonal or fragments of antibodies) directed to the first mentioned antibodies discussed above. Both the first and second antibodies may be used in detection assays or a first antibody may be used with a commercially available antiimmunoglobulin antibody.

WO 00/16609 PCT/AU99/00805 -81 The polyclonal, monoclonal or chimeric monoclonal antibodies can be used to detect the peptides of the invention, parts thereof, analogues, or homologues in various biological materials, for example they can be used in an ELISA, radioimmunoassay or histochemical tests.

EXAMPLE 1 -Plant Material and growth conditions The wild type Colombia, C24, Landsberg erecta, pistillata2 (pi2) mutant, and CHII were provided by Arabidopsis Biological Resource Center (Ohio State University, Ohio, USA). DSG line and AC1 line were provided by Dr. Sundaresan, Singapore.

Arabidopsis thaliana was grown either in pots containing a mixture of 50% sand and 50% compost, or aseptically in petri dishes containing a modified Murashige and Skoog (MS) media (Langridge, 1957). All plants were grown in artificially lit cabinets at 23 0 C, under long day (16 h light, 8 h dark), or continuous light (24 h light) conditions at a light intensity of 200 mmol m" 2 sec 1 EXAMPLE 2 A Visual Screen for determining autonomous endosperm development in plants I. Background A visual screen was developed to determine whether a particular plant has the capacity for autonomous or pseudogamous development of seeds and seed-like structures. Our visual genetic screen is based on the difference in silique length between sterile (short silique) and fertile (long silique) Arabidopsis thaliana plants.

Arabidopsis thaliana is a self-fertilising hermaphrodite plant. The fused carpel or silique is surrounded by the male sexual organs consisting of six stamens topped by anthers that.release pollen during anthesis. In self-fertile plants, anthesis and pollination is complete even before the flowers are completely opened. As fertilisation takes place and seeds are formed, the siliques elongate about five-fold giving rise to full-length seed pods. In the absence of seed formation, the siliques remain short.

WO 00/16609 ,PCT/AU99/0005 -82- Mutants of Arabidopsis thaliana are known which have either impaired male structural organs (for example, the stamenless or antherless mutants) or microspore development (such as the pollenless mutant). In particular, the recessive mutation pistillata (pi) produces a mutant plant when expressed in the homozygous state (i.e.

pi/pi) which is devoid of petals and stamens, has short siliques, but undiminished female-fertility. When exogenous pollen is used to pollinate the stigma-of the pi/pi mutant, siliques are elongated to the level seen in wild-type plants.

Material derived from such an approach may comprise plants capable of dominant or recessive autonomous endosperm formation, or partially-dominant or recessive pseudogamous endosperm formation. These may be distinguished from each other according to the following experimental design.--- II. Experimental Design Visual screen for partially dominant and recessive autonomous endosperm development in plants This screen comprised the mutagenesis of plants containing the pistillata mutation and the subsequent selection of those plants in which silique elongation was observed in the absence of fertilization by a pollen donor. Plants which were putatively characterised as being capable of autonomous endosperm development were identified by their ability to produce elongated siliques in the absence of fertilisation, without concomitant reversion of the male reproductive apparatus.

Heterozygous Pl/pi seeds were made by pollinating a female pi/pi homozygote with pollen from a wild-type homozygous PI/PI plant. The Pi/pi heterozygous seeds produced from this cross were then mutagenised using ethyl methane sulfonate (EMS). The M1 plants were grown and self-fertilised and M2 seeds were harvested and planted.

Four types of plants, heterozygous Pl/pi (fully-fertile), homozygous wild-type PI/PI (fully-fertile), homozygous recessive pi/pi (male-sterile amphimictic plants having only WO 00/16609 PCT/AU99/00805 -83short siliques) and homozygous recessive pi/pi apo/apo(male-sterile soft-seeded plants having elongated siliques) were present in the-M2 generation. The pi/pi plants do not produce normal stamens or petals and were readily distinguished from the fully-fertile plants.

Those plants which were self-fertile with normal stamens and petals-(i.e. PI/PI and PI/pi plants) were uprooted and discarded as soon as they were identified. Among the pi/pi homozygotes, those plants which are putative soft-seeded mutants were identified as stamenless plants having long siliques.

B. Visual screen for partially-dominant and recessive pseudogamous endosperm development Plants (pi/pi) were subjected to a pseudogamy test as follows: The pi/pi M2 plants were pollinated with pollen derived from wild type PI/PI plants. Silique elongation was monitored in the pollen recipients to ascertain that the crosses were successful.

Seeds were harvested, planted and the resulting plants were screened for the maternally-derived (pi/pi) phenotype which, following such cross-pollination, is indicative of partially-dominant or recessive pseudogamous endosperm development having occurred. Absent complete penetrance of the soft-seeded phenotype, dominant pseudogamous mutants are alsodetecte'hn'this screen.

C. Visual Screen for dominant pseudogamous endosperm development To distinguish dominant pseudogamous mutants from partially-dominant and recessive pseudogamous mutant plants, pi/pi M1 plants were screened directly after mutagenesis for sectors having elongated siliques. To test for pseudogamy, pi/pi plants after-mutagenesis were crossed with wild-type PI/PI plants as described for recessive autonomous endosperm development. Silique elongation was monitored in the pollen recipients to ascertain that the crosses were successful. Seeds were harvested, planted and the resulting plants were screened for the maternally-derived (pi/pi) phenotype which, following such cross-pollination, is indicative of dominant pseudogamous endosperm development having occurred.

WO 00/16609 PCT/AU99/00805 84- EXAMPLE 3 Mutagenesis, mutant identification and analysis Heterozygous PI/pi seeds were generated by pollinating a homozygous pi/pi mutant plant with pollen from a wild-type PI/PI plant. For each mutagenesis, 2 gram of F1 seed (P1/pi) was mutagenized as described previously (Chaudhury et al., 1994) and germinated in pots to produce the M1 generation. The M1 plants were allowed to self-fertilize and set seed. Seed from each pot of the M1 plants were harvested separately by collecting at least 10 mature siliques from each plant to ensure that sufficient seeds were obtained from each M1 plant. In the M2 population, 1/4 of the progeny plants were homozygous for the pistillata mutation (pi/pi). Fully-fertile PI/pi and PI/PI plants were identified by the presence of petals and stamens and were removed. Mutants were detected in the pi/pi population, on the basis of elongation of siliques without formation of stamens (Figure-2).

I. Identification and analysis of mutants showing partially dominant and recessive autonomous endosperm development All EMS-generated mutants were crossed with wild-type plants and the F1 plants were selfed to produce F2 seeds, in order to observe dominant, recessive and partiallydominant mutations in the next generation.

In the screen described herein for autonomous mutants, a total of six mutants were identified in which silique elongation and seed development was observed in the absence of pollination. These mutants were designated as fis fertilisation independent seed) mutants. More particularly, these six mutants fell into three complementation groups, designated fisl, fis2 and fis3. Three of the six mutants are allelic to fis2 and were designated fis2-1, fis2-3 and fis2-4.

The six fis mutants obtained so far are from different M1 seed families and thus represent independent mutations. The developmental analyses done so far has been carried out using plants obtained from a primary mutant screen.

A comparison of seed morphology and development in the fis mutants, compared to WO 00/16609 PCT/AU99/00805 85 wild-type Arabidopsis thaliana plants is presented in Figures 3, 4 and X.

A. Seed morphology and development in the absence of fertilisation Based on the analyses of seed size and shape by scanning electron microscopy (SEM) studies, the seed morphology and development are not significantly altered in the mutants compared to wild-type seeds. Detailed sectioning and Nomarski optics studies have been done in one of these mutants.

In unpollinated heterozygotes of the fis mutants, one-third to one-half of the ovules in the elongated siliques were transformed into seed-like structures resembling normal, sexually produced seed in external morphology and size. Endosperm cells develop normally and aborted embryo-like structures develop. The seeds of such plants were initially white, however became shrivelled and brown as they matured. Accordingly, such mutants exhibit an autonomous partial seed (APS) phenotype and are at least capable of autonomous endosperm development. In control pi/pi plants, no endosperm or embryo-like structures were formed.

B. Seed morphology and development following fertilisation Fertilized ovules of pi/pi plants developed into seeds. All sexually-fertilized seeds from wild-type plants turn green and mature after pollination, whereas seeds from pollinated FIS/fis heterozygotes contained green (mature) and white (embryo-arrested seed) at a 1:1 ratio. The fis ovules were similar to FIS ovules in early stages of ovule development. Both inner and outer integuments and the nucellar tissues of the fis mutants were indistinguishable from those of FIS plants.

When siliques containing the white seed were pollinated, some seeds developed which became green and eventually brown. Other seeds remain white but develop embryos which are clearly past the globular stage. This result suggests that the mutation conferring the APS trait is co-dominant. We are currently investigating the possibility that the partially-developed embryos are pseudogamous.

In one mutant at least, analysis of the progeny suggest that the white seed phenotype WO 00/16609 PCT/AU99/00805 86is controlled by the female gamete; rather than the sporophyte. The gametophytic control may be indicative of diplospory in this mutant. This question may be resolved by following the transmission of the mutant phenotype via the pollen. In the instant case, such an analysis is possible because the M2 seed were obtained in families and the gametophytic mutants may be identified in fertile plants.

Embryo sac, embryo, and endosperm development in ovules from the fis mutants were compared with those of ovules of the cogenic Ler-FIS plants. In pi/pi ovules, no embryo or endosperm cells were seen.. Three days after pollination of the pi/pi plant with pollen from a PI/PI plant, the ovules contained an embryo and free nuclear endosperm cells, and each ovule had expanded to the size of the mature seed. In the mutant ovules from a FIS2/fis2 heterozygous plant, the ovule development was equivalent to the development of pi/pi ovules 3 days after pollination, and endosperm cells occasionally were accompanied by an embryo-like structure at the micropylar end (Figure 4).

When the fis2/fis2 homozygous mutant plants were pollinated with pollen from a FIS/FIS plant, embryos developed further than they did in the unpollinated fis2/fis2 plants.

Homozygous fis2 plants were pollinated with pollen from a FIS/FIS plant homozygous for a 35S-GUS reporter gene. The resulting torpedo-stage embryos were stained to detect the product of the GUS gene. All of the embryos resulting from self-pollination of the FIS/FIS 35S-GUS/35S-GUS plant stained blue, as did the embryos resulting from a pollination of a pi/pi FIS/FIS plant with pollen from a 35S-GUS/35S-GUS plant.

In contrast, when 35S-GUS pollen was used to pollinate fis2/fis2 homozygotes, the resulting torpedo stage embryos were either GUS-positive or GUS-negative, suggesting that both zygotic and maternal embryos were present. The presence of GUS sequences in the blue embryos and their absence in the white embryos has. been confirmed by PCR using primers from the GUS genes.

After fertilization, the outer integuments of the Arabidopsis wild-type ovule develop WO 00/16609 PCT/AU99/00805 -87polygonal structures with a central elevation called the columella (Mansfield, 1994).

These structures were not seen in unfertilized ovules that did not develop any mature seed characters before they atrophied: Although the fis seeds were not fertilized, they did form the columella in the outer integument cells, and they were indistinguishable from normal zygotic seeds before they shrivelled.

C. Ploidy of the endosperm The ploidy of the endosperm cells from fis2 mutant was determrined by measuring the fluorescence intensity of nuclei in 4',6-diamidino-2-phenylindole-stained sections. The average brightness of autonomous fis2 endosperm nuclei was found to be 79.4 14.4 and that of wild-type control nuclei was 108 23.1 The background value was 35.5 6.2. The results are consistent with the autonomous endosperm being diploid in contrast to the triploid condition of the sexual endosperm nuclei.

II. Identification and analysis of pseudogamous mutants Approximately 15,000 homozygous recessive pi/pi M2 plants were bulk-pollinated with pollen from L. erecta parent and 90,000 plants were screened for maternal pi/pi phenotype as an indication of pseudogamy.

Approximately 0.1% of plants produced progeny having the recessive maternal phenotype. The possibility existed that these plants may be the result of an extremely rare self-pollination in plants having a very low level of reversion of the pistillata allele to wild-type. As a consequence, those progeny having the recessive maternal phenotype were progeny-tested in the next generation. These progeny are analysed as described supra and pseudogamous mutants are retained and analysed further.

III. Further analysis of mutants Embryo sac development The autonomous and pseudogamous mutants obtained to date were analysed further with respect to determining the nature of embryo sac development therein. We have developed a clearing technique which enables female meiosis and embryo sac development to be observed in wild-type plants and this technology is also used to WO 00/16609 PCT/AU99/00805 -88analyse female meiosis and embryo sac development in each of the mutants.

The present inventors observed an embryo sac with a two cell embryo in sections of fis3-2 mutant seed-like structures.

Effects of genetic background in modifying mutant phenotypes The embryos derived from the mutant embryo sacs are arrested mainly at heart stage irrespective of paternal contributions for all fis mutants in the Ler genetic background (Figure 5, panels In fisl, fis2-1, and fis2-2 homozygous mutants, the proportion of embryos arrested at various stages were investigated in the Ler background. In the case of fis1/fisl homozygotes, 140/155 seeds arrested at heart stage, 4/155 seeds were not arrested, and the remaining seeds were arrested beyond the torpedo stage of development. Similar numbers were obtained for fis2-1 and fis2-2 homozygous mutants in the Ler background. However, no fis3 homozygous plants were generated (see below).

In contrast, when the fisl and fis2 mutants were crossed to the ecoptype Col, the proportion of mutant embryos in the progeny which were arrested at later stages increased, compared to that observed in the Ler background.

In particular, the proportion of mutant seeds with torpedo embryo or beyond was determined for the mature seeds of Colx fis1, Col x fis2 and Col x fis3 crosses. In the progeny of the Col x fisl cross, the proportion of homozygous fisl mutant seeds with embryos arrested at the torpedo stage or beyond was 10.5% in the F2 generation [i.e.

(Col x fisl) F2] compared to only 3.2% in the Ler background. In the progeny of the Col x fis2 cross, the proportion of homozygous fis2 mutant seeds with embryos arrested at the torpedo stage or beyond was 15% in the F2 generation (Col x fis2) F2] compared to only 4.5% in the Ler background. In the progeny of the Col x fis3 cross, the proportion of heterozygous fis3 mutant seeds with embryos arrested at the torpedo stage or beyond was 4.5% in the F2 generation (Col x fis3) F2] compared to only 2.8% in the Ler background.

WO 00/16609 PCT/AU99/00805 89 Given the difference of embryo development for the fisl and fis2 mutants between Ler and Col.backgrounds, it is likely that there exists a modification system in Col that allows the mutant embryos to develop further than in Ler. To determine the genetic basis of this modification, fis2-1/fis2-1 and fis2-2/fis2-2 homozygous mutants were screened from the (Col x fis2) F2 population (Figure 5, panels 5 and Some homozygous mutants showed much better embryo development than others. For example, one (Col x fis2) F2 plant produced 42/117 wild-type looking seeds, compared to only 9/159 fis2-1/fis2-1 seeds in the Ler background. In some extreme cases we could observe up to 100% seeds looking normal in some part of the plants.

An unmodified fisl/fisl, an/an (Ler) mutant was crossed to one modified fis2-2/fis2-2 (Col) plant. From the progeny of this cross,-double homozygous mutants were constructed as described above and some lines showed further embryo development later arrest). One double mutant line produced up to 40/195 wild type looking seed. These data suggest that fisl and fis2 may share the same modification system.

To investigate the role of the modification system in embryo development, the modified seeds were sectioned and compared to the same stage of the unmodified fis2-1 in the Ler ecotype background. Data indicated that endosperm cellularisation in modified seeds was similar to that of wild-type seeds, while most fis2-1 seeds in the-Ler ecotype lacked endosperm cellularisation or were only partially cellularised. Without being bound by any theory or mode of action, these data suggest that the modification system may involve an endosperm cellularisation process.

In order to understand the influence of the modification system on the seedlings derived from the mutant seeds, we germinated the arrested seeds from the F2 seeds from the crosses between Col and all three fis mutants. The seedlings from the arrested seeds displayed a wide range of morphological phenotypes. The seedlings can be divided in three groups based on the ability to regenerate into viable plants; as follows: normal looking seedlings that show no obvious difference from wild type; (ii) seedlings that display abnormalities at early stages of development and WO 00/16609 PCT/AU99/00805 later become viable and form wild type looking plants; and (iii) morphologically-deformed seedlings that can not develop into viable seedlings.

In this grouping, type (ii) seedlings have fewer abnormalities than type (iii) seedlings, particularly in respect of the cotyledons and the bottom rosette leaves which usually become thicker, longer and deformed in type (iii) plants. The upper rosette leaves were gradually restored-to wild type appearance in type (ii)-plants. The upper part of type (ii) plants is completely normal and can produce flowers and seeds. Type (iii) seedlings are dramatically deformed with accumulation of anthocyanins in the thickened cotyledon, an no green- rosette leaves form in these plants, possibly explaining why these seedlingsdo not develop into viable plants.

To correlate seed phenotype to the stage of embryo arrest; we arranged the modified fis2-1 homozygous mutant seeds into three groups, as follows: normal looking mutant seeds; (ii) seeds with torpedo or further developed embryo; and (iii) completely flat seeds or seeds with heart stage embryo.

Type seeds produced only wild type plants and 80% of these seed germinated.

Type (ii) seeds produced all three types of seedlings listed supra, in the ratios of wild type seedlings; 15% type (ii) seedlings; and 3% type (iii) seedlings. Type (iii) seeds germinated at a rate of 9/120 seeds and only produced Type (iii) non-viable seedlings.

Studies of homozygous mutant plants In spite of several attempts to identify homozygous mutants for both the fis3-1 and fis3- 2 mutant alleles, no homozygote was obtained in Leror Col ecotype backgrounds. In contrast, it is easy to obtain fisl and fis2 homozygotes for all fis2 alleles. In an attempt to generate fis3-1 and fis3-2 homozygous mutants, about 2,000 arrested seeds for each of (Col x fis3-1)F1 and (Col x fis3-2) F1 plants were germinated on MS plates.

Those seeds were derived from mutant embryo sacs which had been fertilized by either wild type or mutant pollen with equal chance as the mutation does not affect the WO 00/16609 PCT/AU99/00805 91 fertility of pollen. Theoretically, FIS3/fis3 and fis3/fis3 should be obtained with equal numbers among the germinated plants if the fis3 mutation does not affect embryo development. However, for fis3-1 we could obtain only 28 heterozygous plants and for fis3-2, we could only obtain 23 heterozygous, thereby showing the conditional lethality of the mutation in fis3-1/fis3-1 and fis3-2/fis3-2 homozygotes. In contrast, fisl and fis2 homozygotes accounted for 50% of the total surviving plants-in similar screening in the Col x fisl and Col x fis2 crosses. These data suggest that the FIS3 gene may have a function in the embryo.

Gene interactions Double mutant studies are important genetic strategies to define independent pathways of gene action. If two genes act in the same pathway, the double mutant phenotype is often the same as the phenotype of the single mutant, in which case the gene of the single mutant is epistatic over-the other gene which is mutated in the double mutant. However, the effect of each allele in a double mutant may be enhanced or even synergistic, giving rise to a qualitatively novel phenotype in the double mutant compared to what would be expected from the parental phenotypes.

Double mutants are produced by standard genetic procedures which are well-known in the art.

Because the APS phenotype obtained in at least one of our fis mutants appears to be co-dominant from the point of view of autonomous endosperm development, double mutants are produced which comprise combinations between this mutant and the other five single mutants described herein, to clarify the pathways that control autonomous seed production and to produce.mutant plants having a higher degree of penetrance of the autonomous seed phenotype. Double mutants between each of the other fis mutants are also produced.

In particular, a double an/an, fis1/fisl mutant was crossed to the Ds-induced fis2- 2/fis2-2 mutant in a Col background a fis2-2/fis2-2 modified mutant). The F1 plants with 75% mutant seeds were harvested and germinated on MS plates with kanamycin WO 00/16609 PCT/AU99/00805 -92selection to select for the fis2-2 allele. Because these plants were kanamycin resistant, they must at least contain one copy of fis2-2 gene. The surviving plants were also screened to isolate those showing the an/an marker phenotype, and the DNA from these plants was sequenced to select those homozygous for the fisl mutation. To detect homozygous fis2-2 mutants, we designed three primers for use in PCR screening as follows: a first pair of primers derived from the Ds-interrupted FIS2 sequence in the fis2-2 mutant, which in use provides a positive PCR product only when there is no Ds insertion; and (ii) a second pair of primers, comprising a Ds-specific primer derived from the nucleotide sequence of Ds and a second primer derived from the FIS2 sequence in the fis2-2 mutant, which in use provides a positive PCR product when the fis2-2 mutant allele is present.

This screening strategy was used to generate three fisl/fis2-2 double homozygous plants. There are no morphological abnormaties in these double mutants except in the an/an selection marker. After emasculation, these plants still produced seeds similar to those observed for the single fisl or fis2 mutant plants. In the double homozygotes, the seeds were arrested in the same way as for the fis2-2/fis2-2 modified mutant (Figure 5, panels 7 and In the F2 generation, some plants exhibited a lesser degree of modification than the fis2-2/fis2-2 modified mutant, producingmainly seeds having a heart stage embryo.

Conditionality of the mutant phenotype The possibility that the autonomous development of seeds in the fis mutant is influenced by environmental conditions is tested by growing the six fis mutants at a constant temperature of 16°C and under photoperiods comprising either 8 hr light or 16 hr light, compared to the conditions under which the mutations were first-detected 22°C under continuous light). Plants having a higher degree of penetrance of the autonomous seed phenotype are retained for further analysis.

WO 00/16609 PCT/AU99/00805 -93 Gene dosage effects In many of the autonomous fis mutants described herein, sexual transmission of the mutant fis allele following cross-pollination with a pollen donor may occur at a low frequency, indicating a degree of female sterility is associated with the mutation.

Heterozygous plants are isolated by screening for the mutation in fertile plants. The heterozygous plants are then used to construct genetic lines of plants in which the mutation is in homozygous condition, such that all seeds produced therefrom are autonomous. Genetic lines in which the level of penetrance of autonomous seed production is increased are retained forfurther analysis.

EXAMPLE 4 Mapping of FIS alleles To map the FIS loci, pollen from each of the FIS/fis PI/PI plants was used to pollinate W100F, a male-fertile derivative of W 00 that contains 10 morphological mutations distributed on the arms-of the five Arabidopsis chromosomes (Koornneef et al, 1987).

Among the F2 progeny of FIS/fis W100F/+, plants which were homozygous for the different recessive morphological mutations were scored for FIS/FIS (all seeds in the siliques were normal) and for FIS/fis (the siliques contained a mixture of fully developed and embryo-arrested seeds).

I. The FIS1 allele Genetic data showed that the morphological marker an was closely linked to the fisl allele. The genetic distance between an and FIS1 is 1 cM (Figure As FIS1 was localized to the end of chromosome 1, two flanking markers were used to further map the FIS1 gene.

One such marker comprised the kanamycin-resistance gene NPTII, which is present in this region of chromosome 1 of a genetic line of Arabidopsis thaliana ecotype No-0 designated E12, as part of a genetic construct containing the Ds transposable element.

The E12 line was crossed to the fisl mutant and F1 progeny were back-crossed to WO 00/16609 PCT/AU99/00805 -94 wild-type Arabidopsis thaliana ecotype Landsberg erecta (Ler). Recombinants between fisl and NPTII were selected from the backcrossed F1 lines. Following this approach, the genetic distance between fisl and NPTII was determined to be 17 cM (Figure 6).

To identify the closest molecular marker to the FIS1 gene, SSLP markers from contiguous BAC clones in the region of the morphological marker an were designed, based on the released sequence data from Arabidopsis data base.

The SSLP marker designated F26B7 (Figure 6) was used first to test recombinants between the FISl and NPTII genes. From 87 plants produced from such recombination events, 23 plants were identified in which a crossover had occurred between F26B7 and the FIS1 gene, a recomniination frequency of-26.4%.

The SSLP markers athacs and the left-end and right-end rescue fragments derived from the BAC clone T7123 were also used to test these 87 plants. No plants were identified in which a crossover had occurred between FIS1 and the SSLP markers, indicating that FIS1 is tightly linked to these markers on chromosome 1 (Figure 6).

The BAC clone T5P2 which contains athacs, the BAC clone T7123 and the BAC clone F26B7 map to the-same contiguous region on chromosome 1. Accordingly, data indicated that the FIS1 gene was located either within the BAC clone T7123 or within the BAC clone which maps immediately to the left of T7123 Figure 6).

The MEDEA (syn. MEA) gene described by Grossniklaus etal (1998) was shown to map in this region of chromosome- 1. Plants expressing the mea phenotype exhibit embryo lethality Grossniklaus et al (1998), however do not exhibit autonomous seed development. The mea mutant is a Ds-tagged gametophytic maternal mutant. To determine how closely the MEA gene mapped to the FIS1 gene on chromosome 1, a PCR-generated probe derived from the nucleotide sequence of the MEA gene was WO 00/16609 PCT/AU99/00805 hybridized to clones on an IGF filter. Five positive clones were identified, which mapped to the left of the BAC clone T7123 (Figure indicating a tight linkage.

DNA derived from the fisl homozygous mutant was also sequenced using MEA gene primers and a single base change was found in fisl mutant compared to the wild-type MEA gene sequence. This base change introduced a translation stop codon in the region of the open reading frame of the MEA gene, thereby resulting in early termination of translation-and the synthesis of a truncated polypeptide. These data indicate that the fisl mutant gene is an allele-of the MEA gene. However, the different phenotype of the fisl mutant compared-to the mea mutant, indicates that the point mutation in fisl is-critical to reduce expression of the wild-type MEA/FIS1 gene to a biologically inactive level which is sufficient to facilitate autonomous seed development.

I. The FIS2 alleles Mapping studies on the FIS2 gene utilised the fis2-1 mutant line where appropriate.

The fis2-py recombination frequency of 9,28 1.56 (map distance of 10.26; n 345) and the fis2-er recombination frequency of 13.07 2.73 (map distance of 15.14; n 153) positioned fis2 between erand py on chromosome 2.

The heterozygous FIS2/fis2 was crossed to wild-type Arabidopsis thaliana ecotype Colombia (Cross No.1) or CHII (Cross No.2) and the F2 progeny were obtained..Foreach selected individual F2 plant derived from these crosses, a pool of F3 plants was grown to facilitate determination of the genotype of the corresponding F2 plant. In the F2 population derived from Cross No.1, er/er FIS2/fis2 recombinants were isolated and allowed to self-fertilize. In the F2 population derived from Cross No. 2, FIS2/fis2 as/as plants were isolated and allowed to self-fertilize.

DNA from the F3 pools were prepared for RFLP analysis. Three types of RFLP probes were used in this analysis. Clones such as mi277, m323, and ve017 which appear on WO 00/16609 PCT/AU99/00805 96- the RI map, the left and right ends of YAC clones and fragments derived from cosmid clones or BAC clones were used. Total DNA extraction and DNA gel blot analysis were performed as described by Church and Gilbert (1984).

The RFLP markers ve017, mi277 and m323 were mapped relative to the ER, FIS2 and as loci using the recombinant F2 plants er/erFIS2/fis2 and FIS2/fis2 as/as. Marker ve017 mapped between AS and FIS2 genes. Of 8 plants tested, five showed a recombination break point in the FIS2-ve017 interval. On the other hand, out of er/er FIS2/fis2 plants tested, 10 plants had a recombination break points in the mi277- FIS2 interval and 5 plants had a recombination break point in the m323-FIS2 intervals.

These data indicate that the markers mi227 and m323 map on the ER-proximal side of FIS2, in the order ER-mi277-m323-FIS2.

Based on a map of contiguous YAC clones for chromosome 2,-the YAC clone designated Y9D3 (Figure 7) was selected and its left and right ends were rescued and used as RFLP markers to test for linkage to the FIS2 locus in the F2 population. The Y9D3 left end-FIS2 interval showed no recombination break point out of 65 er/er FIS2/fis2 plants tested. However, a recombination break point was observed in 3 plants out of 9 FIS2/fis2 as/as F2 plants. These data indicate that the left-end of the YAC clone Y9D3 maps on the as proximal side of Fl2 (Figure 7).

Using the Y9D3 left-end as a probe, two other YAC clones, designated Y11D2 and Y11A7 in Figure 7, were isolated from the same YAC library. The Y11D2 right-end and the Y11A7 left-end were used as RFLP markers to test their position on chromosome 2 relative to the FIS2 gene. The Y1.1 D2 right-end mapped on the er proximal side of FIS2 whilst the Y11A7 left-end showed no recombination break point in its interval with. These data indicate that the Y11A7 left-end is tightly linked to the FIS2 gene (Figure 7).

WO 00/16609 PCT/AU99/00805 -97- I. The FIS3 allele The FIS3 gene was located on chromosome 3, between the morphological markers hy3 and gl1 (Figure The fis3 mutant was crossed to wild-type Arabidopsis thaliana ecotype Columbia, to facilitate detailed mapping. In the F2 population, 107 plants were harvested and DNA prepared. One SSLP marker, designated nga162 (Figures 8 and 9) was used to determine that the nga162 marker was about 6-cM north of the FIS3 gene. An even closer RFLP marker, designated ve039 (syn. veo39) was identified which mapped cM north of the FIS3 gene (Figures-8 and Analysis of the F2 population from a cross between the triple mutant hy/hy FIS3Ms3 gll/gl1 and wildtype Columbia and in particular, analysis of the recombinants, for example the singlecrossover mutants hy/hy FIS3/fis3 GLI/gl1 and Hy/hy FIS3/fis3 gll/gll, provide for accurate localization of the FIS3 gene.

A contiguous map of YAC clones and pl clones was constructed around the ve039 marker (Figure Data suggest that the FIS3 gene is present in the p1 clones MCB22 and/or MNH5 and/or the YAC clone CIC7E1, to the left of ve039.

EXAMPLE Transposon tagging of the FIS2 gene A clone containing a transposon carrying a promoterless reporter gene was-also used to tag the FIS2 gene. In the DSG tagged line, the transposon was found to be closely linked to the molecular marker m323 (see Example A line containing an Ac element was crossed into the DSG line fis2-2 and F1 plants were screened for sectors that show fertilization independent silique elongation and which segregate in a 1:1 ratio of normal:-fis2-2 in the seeds. In the F1 of the DSG X Ac cross, one chimeric plant designated P19, was observed which showed both of these properties, indicating that the DSG transposon had possibly integrated into the FIS2 gene in that line (Figure The line containing the transposon inserted into the fis2 gene was designated fis2-2.

WO 00/16609 PCT/AU99/00805 -98- EXAMPLE 6 Cloning the EIS2 gene To clone the F/S2 gene, the left-end of Y11A7 was used to screen a cosmid library provided by Dr. Neil Olszewiski (University of Minnesota, USA) and a BAC library. One 110 kb BAC clone (B26D2.in Figure 7) and a 16 kb cosmid clone (cosl8H1 in Figure 7) were isolated, both of which contain the Fis2 gene.

A physical map of the cosmid clone cosl8H1 was obtained, using the restriction enzymes BamHI EcoRI and EcoRV (Figure 11).

Additionally, a bacteriophage A genomic library (see Example 9) was prepared using DNA derived from the DSG-tagged fis2-2 mutant described in the preceding Example.

Since the FIS2 gene mapped to the BAC clone B26D2, DSG must have transposed into a location covered by one of the sub-fragments of B26D2. The sub-fragments of B26D2 (Figure 11) were used as probes to test the tagged mutants. DNA covered by one of the EcoRI fragments, designated E2 in Figure 11, was interrupted by DSG. The DSG transposon also hybridized to the E2 fragment. Accordingly, the genomic library was screened using a BamHI fragment containing the DSG 5'-end and the E2 probe (see Example 9).

By sequencing the DSG-containing DNA and the corresponding wild type sequence from cosmid pOCA18H1 (Figure 11), the.position of the DSG insertion was determined to lie within the FIS2 gene.

EXAMPLE 7 Cosmid pOCA18H1 complements the fis2 mutant phenotype To confirm the presence of the FIS2 gene in the cosmid clone pOCA18H1 (Figure 11), complementation tests were performed wherein this clone was introduced into the Arabidopsis thaliana fis2 mutant line.

WO 00/16609 PCT/AU99/00805 -99- Agrobacterium-mediated transformation of Arabidopsis thaliana root explants was performed as described by Valvekens (1988) with some modifications. Timentin was used instead of vancomycin. Bacto agar TM was replaced by 0.3% (w/v) Phytoagar TM. Bacto agar TM is the trade mark of Difco Company and Phytoagar TM is the trademark of Sigma Chemical Company. Constructs were introduced into Agrobacterium tumefaciens strain AGL1 by the triparental mating procedures with pRK2013 as a helper plasmid (Ditta, 1980). Stability of the plasmid insert in AGL1 was tested by restriction digestion and gel electrophoresis of plasmid DNA.

Fresh overnight cultures of Agrobacterium tumefaciens strain AGL1 carrying individual plasmids were used to infect root explants derived from 4-week old Arabidopsis thaliana plants. Kanamycin-resistant transgenic plants were regenerated as described previously (Valvekens, 1988). Transformed shoots were transferred to Murashige and Skoog (MS)-containing agar, supplemented with 50 pg/ml kanamycin and 100 pg/ml timentin. Seeds of transgenic plants were germinated either in soil or on MScontaining agar plates supplemented with 50 pg/ml kanamycin.

Cosmid pOCA18H1 (Figure 11) was introduced into the Agrobacterium tumefaciens AGL1 strain by triparental mating using E. coli RK2013 as a helper strain. A.

tumefaciens transconjugants were selected on LB containing rifampicin (50 pg/ml) and tetracyclin (3.5 pg/ml). Spurious rearrangements in the cointegrates were determined by re-transformation of the cosmid clone into E. coli strain D5Ha and restriction mapping of the plasmid DNA derived therefrom.

Arabidopsis thaliana ecotype C24 root-explants were transformed with A. tumefaciens containing cosmid pOCA18H1 and regenerated as described by Valvekens et al, (1988). For each T1 plant, T2 seeds were sown on media containing kanamycin pg/ml) to determine the segregation ratio for kanamycin resistance. Kanamycinresistant T2 plants were crossed to the fis2 mutant and the ratio of arrested seeds in F1 plants were scored.

WO 00/16609 PCT/AU99/00805 100- The ratios of arrested seeds were scored. The ratio of fis:FIS seeds was predicted to shift from the 1:1 ratio expected in the absence of complementation, to a ratio of 1:3 -expected following complementation. In the seed of six independent kanamycinresistant F1 lines, a segregation ratio of 3:1 (FIS:fis) was in fact observed (Figure 12).

In contrast, the same ratio shift was not observed in kanamycin-sensitive plants of the same cross.

These data indicate that the cosmid clone pOCA18H1 complements the fis2 mutant Sphenotype and contains the FIS2 gene.

EXAMPLE 8 Isolation of the FIS2 cDNA clone DNA probes derived from the EcoRI fragments El and E2 were used to screen 200,000 plaques from an Arabidopsis late silique cDNA library obtained from Anna Koltunow (CSIRO, Div. of Plant Industry, Adelaide, Australia). Prehybridisation and hybridisation were performed in 10% PEG 600, 7% SDS, 0.25 M NaCI, 0.05 M NaPO 4 at pH 7.2, 1% bovine serum albumin, 1 mM EDTA at 65°C for 2 hrs and 16 hr, respectively. The filters were washed at room temperature (once-in 2XSSC, 1% SDS for 30 min each) and exposed O/N on X-ray film with 2 intensifying screens at 700C.

A total of 4 positive cDNA clones were obtained, two of which hybridised to DNA probe derived from the left hand side of the DSG insertion and the two others hybridised to DNA probe derived from the left hand side of the DSG insertion. These 4 plaques were purified, excised, analysed by restriction mapping and sequenced.

The DNA isolated from positive plaques of the Arabidopsis late silique cDNA library from were sub-cloned in vivo from the LambdaZap® vector using the ExAssist® interference resistant helper phage.

WO 00/16609 PCT/AU99/00805 -101- Sequencing was performed by double-stranded sequence analysis on an Applied Biosystems Model 370A DNA Sequencer using a fluorescent dye-labelled dideoxy terminator kit. The sequence data were analysed using computer software DNA Strider for Macintosh (Marck, 1988),and the GCG Sequence Analysis Package software (Devereux, 1984).

The nucleotide sequence of the full-length FIS2 cDNA clone is presented in <400>6.

The derived amino acid sequence of this cDNA clone is presented in <400>2.

The cDNA inserts which hybridised to the right hand side of the DSG insertion in the transposon-tagged line-had the same 3'-end sequence, indicating that they both came from the same gene and that the longest cDNA clone was potentially full length. The longest cDNA was designated CTF1. The 5'-end of CTF1 was about 750 bp to the right of the DSG insertion. Almost 400 bp at the 3'-end of CTF1 were not on the E2 fragment (Figure 11) but on the adjacent EcoRI fragment, designated E4 inFigure 11.

Those cDNA inserts which hybridised to the left hand side of the DSG insertion were both about 1.7 kb long. One clone, designated CTF2a, shared 100% nucleotide sequence identity with the genomic sequence of the El fragment (Figure 11). The second clone, designated CTF2b, shared 85% nucleotide sequence identity with CTF2a, indicating that CTF2a and CTF2b contained related cDNAs which are variants of the same gene family. CTF2a is in the same orientation as CTF1, indicating that the 3'-end of CTF2a was located 500 bp from the junction between the EcoRI fragments El and E2 and, as a consequence, more than 2 kb from the DSG insertion.

EXAMPLE 9 Construction and screening of a genomic library to isolate the fis2-2 gene Genomic DNA from the DSG-tagged mutant fis2-2 was digested using the enzyme Sau3Al and size-fractionated on a glycerol gradient. The 10-12 kb fraction was then ligated into bacteriophage AEMBL4 BamHI-digested and dephosphorylated arms. The-.: WO 00/16609 PCT/AU99/00805 -102ligated DNA was packaged into sonicated extract BHB2690 and freeze-thaw lysate from induced packaging proteins BHB2688. The number of plaque-forming units (PFU) of the recombinant bacteriophage was determined by plating the bacteriophage onto solid media plates using Escherichia coli strain K803 cells. Approximately 9 x 104 PFU were transferred from plates onto nylon filter membranes and screened using a BamHI fragment containing the 5'-end of DSG and E2 as probes. Prehybridization and hybridization were performed at 42 0 C for 45 min and overnight, respectively, in a solution comprising formamide, 3XSSC, 21.5X Denhardt's Solution, 0.1% SDS and 0.5 mg/ml salmon sperm DNA. The filters were washed at room temperature twice in 2XSSC, 0.1% SDS for 15 min each wash and twice in 0.1XSSC, 0.1% SDS for 15 min each wash, before exposing the filters to X-ray film with an intensifying screen at -80 C.

Positive-hybridizing plaques were plaque-purified in subsequent screening rounds and sequenced as described in Example 8.

The nucleotide sequence of the wild-type FIS2 gene is presented herein as <400>7.

Nucleotide sequence analysis of the 5'-region of the FIS2 gene sequence was performed, using www.NETGENE2, to predict intron-exon splice junctions. Data obtained from the WWW.NETGENE2 server in relation to the confidence of the predicted splice sites in the FIS2 gene are presented in Table 3.

WO 00/16609 PCT/AU99/00805 103- TABLE 3 Confidence for the predicted intron splice sites of the FIS2 gene Position Acceptor/ Confidence Seq id Nucleotide Sequence Donor Level' no: 590 Donor 1.00 200 AAAAAACAAC gtatgcattc 875 Acceptor 0.56 201 gtttattcag CCATATTTCC 932 Donor 0.88 202 CTACAGGGAT gtgagtaaca 1228 Acceptor 0.86 203 ttttgcttag GTCAAATTCA 1300 Donor 1.00 204 AAAGCTGAAG gtgagccttt 1401 Acceptor nd* 205 ccaaatgcag TAGTGGAAAA 1454 Donor 0.94 206 AGGTCACGAG gtaggcacta 1582 Acceptor nd 207 ttgtgccacag GGCTTGCAAC Intron sequences are shown in lower case and exons in upper case.

nd, not determined.

1, The cutoff value for each confidence level is as follows: Highly confident donor sites: 95% Highly confident acceptor Nearly all true donor sites: 50% Nearly all true acceptor sites: The present inventors have further analysed the genomic structure of the FIS2 gene present in Arabidopsis thaliana ecotype Columbia. Compared to the nucleotide sequence of the FIS2 gene present in the Landsberg ecotype, a 180 bp deletion occurs in exon 8 of the Columbia ecotype, producing a 60 amino acid deletion in the derived amino acid sequence of the FIS2 polypeptide encoded therefor. PCR analysis of the same region in the Arabidopsis thaliana ecotypes C24 and WS indicated that the deletion was ecotype-specific and only present in the Columbia ecotype.

Additionally, the FIS2 gene of Arabidopsis thaliana ecotype Columbia comprises a 26 bp deletion in intron 7 compared to Arabidopsis thaliana ecotype Landsberg.

WO 00/16609 PCT/AU99/00805 104- EXAMPLE The fis2 mutant phenotype results from single basepair changes In order to determine the nucleotide sequence the fis2 mutant gene, seven amplification primer pairs were designed, based upon the nucleotide sequence of the CTF1 cDNA clone. These primers were synthesized using an Applied Biosystems automatic DNA synthesizer Model 394.- The primer pairs were used to amplify and sequence the mutant fis2 gene from genomic DNA derived from fis2-1, fis2-2, and fis2-3 homozygous mutant plants. Each primer pair amplified a 500-600 base pair fragment from genomic DNA.

PCR was carried out in 20 ml of 50 mM KC1, 10 mM Tris-HC1 pH 9.0, 0.1% (v/v) Triton X-100, 2 mM of each primer, 0.4 mM dNTP, 1.5 mM MgC1, and 2 units/reaction Taql DNA polymerase. The PCR conditions comprised a first denaturation step of min duration at 94°C, followed by thirty cycles, each cycle comprising: denaturation at 94°C for 20 sec; (ii) annealing at 55°C for 30 sec: (iii) polymerisation at 72°C for 30 sec; and a final incubation at 25°C for 1 min. Reactions -V--re performed using a Corbett Research Capillary Thermal Sequencer Model FTS-1S.

PCR products were purified using Wizard Prep and sequenced directly. If necessary, PCR products were purified from 1% agarose gels following electrophoresis thereon, prior to being sequenced.

Sequencing reactions were carried out as described in Example 8.

The nucleotide sequence of the fis2-1 mutant allele revealed a 1 bp deletion in exon 8, in the region corresponding to position 1835 in the wild-type FIS2 cDNA (<400>6).

WO 00/16609 PCT/AU99/00805 105- This mutation produced a frame-shift in the mutant fis2-1 allele compared to the wildtype allele, thereby terminating translation of the FIS2-1 polypeptide four amino acids downstream of the deletion point (Figure 13A).

The nucleotide sequence of the fis2-3 mutant allele revealed a single base change at the splice junction of intron 5, producing the mutation of AG to AA (Figure 13B).

Similar single base changes in intron splice junctions have been reported for other EMS-induced mutants (Sun and Kamiya,-1994).

EXAMPLE 11 The FIS2 polypeptide is a putative transcription factor The derived amino acid sequence of the FIS2 polypeptide is presented herein as <400>2. In this regard, there are three in-frame putative translation start sites in the FIS2 cDNA, commencing at nucleotide positions 1 and 37 and 364 of SEQ ID NO:<400>6.

A search for known protein motifs in derived amino acid sequence of the FIS2 polypeptide revealed a putative C2H2 zinc-finger motif within-the first 151 residues of the polypeptide, and several putative nuclear localization signals (NLS) distributed between residues 1 to 661 of the FIS2 protein (Figure 14). However, as stated in Example 15 below, in vivo expression data suggest that the true NLS is localised within the first 121 amino acids of the FIS2 polypeptide (shaded region in Figure 14).

Amino acid sequences which contain zinc finger motifs are generally nucleic acid binding proteins in which the finger structures are maintained by the cysteine and/or histidine residues of the C2H2 zinc-finger motif being organized around a zinc metal ion (Stanojevic et al., 1989; Berg, 1993). Several members of the C2H2 zinc-finger proteins, also known as the TFIIIA/Kruppel-like zinc-finger protein gene family, play important and diverse roles in growth and development in Drosophila melanogaster (Stanojevic et al, 1989; Treisman and Desplan, 1989). Recently, C2H2 zinc-finger WO 00/16609 PCT/AU99/00805 -106proteins have been identified in plants (Meissner and Michael, 1997; Takatsuji, et al., 1994); Takatsuji et al, 1991; Sakai et al, 1995; Tague and Goodman, 1995).

The presence of both the zinc finger motif and the NLS suggests that the FIS2 polypeptide may well be a transcription factor belonging to the TFIIIA or Kruppel-like zinc-finger protein gene family.

Another characteristic of the FIS2 polypeptide is a high content of serine residues a characteristic feature of other C2H2 zinc-finger proteins (Tague and Goodman, 1995).

Additionally, the FIS2 polypeptide comprises highly repetitive amino acid sequences, located between residues 243 and 642 of <400>2 (Figure 14). The repeat comprises a core of 22 amino acid residues in length, which is repeated 12 times Although the core sequence is not 100% identical armong the 12 repeats, the homology is easily detectable using sequence analysis and dot matrix computer program (Figure The repeated region is likely to be involved in protein-protein interactions, suggesting that the FIS2 polypeptide may be one component of a protein complex.

EXAMPLE 12 The FIS2 gene is a single copy gene Genomic DNA from Arabidopsis seedlings was prepared by the CTAB protocol (Taylor, 1982; Dellaporta, 1983). Genomic DNA (5 was digested with restriction enzymes prior to electrophoresis on 1% agarose gels. The DNA was then transferred to a HybondN membrane, prehybridized for 1 hr, hybridized and the filters were washed according to Church and Gilbert (1984). Probes were labelled with [a-32P]-dCTP using the random primer method (Feinberg and Vogelstein, 1983). This analysis revealed that the FIS2 gene is a single copy gene (Figure 16).

WO 00/16609 PCT/AU99/00805 107- EXAMPLE 13 Expression of the FIS2 gene in plants Total RNA was prepared individually from Arabidopsis thaliana roots, shoots, leaves, stems, and flowers according to Dolferus (1994). Total RNA was also prepared from siliques using the phenol extraction method.

Total RNAs were DNase-treated and RT-PCR (McPherson, 1991) was performed on 2 mg of RNA using .the primers 1F (SEQ ID NO: <400>208: TCATCTCTTCCTTATGAAGTT- and 2R (SEQ ID NO: <400>209: TGTTGATAATGTCCCATCG-3') which anneal in the region of exon 12 and exon 8, respectively. First strand cDNA was synthesized for 1 hr at 370C in 50 mM Tris-HC1 at pH8.3, 10 mM MgCI 2 75 mM KC1, 10 mM DTT, 0.5 mM dNTP, 4 units RNasin (Promega) and 5 units MMLV reverse transcriptase (Epicentre). PCR amplification was then carried out on 5 pl of RT reaction in a final volume of 20 pl, containing 50 mM KC1, 10 mM Tris-HC1 pH 9, 0.1% Triton X-100, 1 mM of each primer, 0.4 mM dNTP, 1.5 mM MgC1 2 and 2 units of Taql DNA polymerase (Perkin-Elmer). The amplification reaction comprised a first denaturation step of 5 min duration at 940C, followed by thirty cycles, each cycle comprising: a 20 sec denaturation step performed at 94 0

C;

(ii) a 20 sec annealing step performed at 55°C; and (iii) a 1 min elongation step performed at 720C, followed by a final cycle comprising incubation for 2 min at 72°C, followed by 1 min at 28°C. Amplification reactions were-performed using a Corbett Research Capillary Thermal Sequencer Model FTS-1S. RT-PCR products were separated by agarose gel (1%)electrophoresis.

Amplification products corresponding to the FIS2 transcript were present at least in shoots, leaves, bolts and siliques, with a much weaker signal present in flowers (Figure 17).

WO 00/16609 PCT/AU99/00805 108- EXAMPLE 14 Nucleotide sequence of the FISl gene and structure of the FIS1 polypeptide The nucleotide sequence of the cDNA encoding the FIS1 polypeptide is presented in <400>4.

Genomic clones encoding the FIS1. polypeptide were obtained and nucleotide sequences were obtained as described herein. The nucleotide sequence of the FIS1 gene is presented in <400>5.

The fisl mutation maps to the same locus as the mea mutation. Accordingly, the amino acid sequence of the FIS1 polypeptide set forth in <400>1 corresponds to the sequence disclosed by Grossniklaus et al. (1998).

DNA derived from the fisl homozygous mutant was sequenced using MEA gene primers and a single base change was found in fisl mutant compared to the wild-type MEA gene sequence disclosed by Grossniklaus et al (1998).--This single base change introduced a translation stop codon in the 5'-region of the open reading frame of the MEA gene, thereby resulting in early termination of translation and the synthesis of a truncated polypeptide (Figure 18). Accordingly, the fisl allele is a presumptive null allele. In particular, the single base change comprised the substitution of a thymidine residue for a cytidine residue at position 320 of <400>4, producing a stop codon TAA in this region which results in translation being terminated at amino acid 102 in <400>1 of the FIS1 polypeptide.

In contrast, the mea mutation comprises a Ds transposon inserted into the C-terminal region of the gene, in particular at the junction between nucleotide positions 1756 and 1757 in <400>4. Accordingly, in the medea mutation the insertion is such that a polypeptide with a short truncation in the carboxyl terminal results.

The fisl mutant gene is an allele of the MEA gene. The different phenotype of the fis1 WO 00/16609 PCT/AU99/00805 -109mutant compared to the mea mutant, indicates that the point mutation in fisl is critical Sto reduce expression of the wild-type MEA/FIS1 gene to a biologically inactive level which is sufficient to facilitate autonomous seed development.

The MEDEA/FIS1 polypeptide (<400>1) comprises at least the following peptide motifs or protein domains: an acidic domain, presumably required for interaction with other polypeptides; (ii)a C5 motif comprising five conserved cysteine residues and having an unknown function; (iii) a putative nuclear localization signal; (iv)a CXC domain comprising a stretch of cysteine residues, of unknown function; and a SET domain, which is shared by some of the polycomb group of proteins, including E(z) enhancer of zeste).

The Arabidopsis thaliana Polycomb group proteins designated EZA1 and CURLY LEAF and the Drosophila melanogaster E(z)polypeptide and the Caenorhabditis elegans MES-2 polypeptide also comprise the SET domain, the CXC domain, domain and a nuclear localisation signal (Figure 19).

Comparison of the fisl and mea alleles indicates that in the fisl mutant, none of these five structural motifs are present, whilst in the mea mutant all domains except the SET domain are present. The phenotypic difference between fis1 mutant and mea suggests that the structural motifs present in the MEDEA/FIS1 polypeptide may be biologically significant in regulating fertilization independent seed development in plants, whilst the SET domain alone may be important in embryogenesis.

Sequence alignment of various E(z)-like proteins around the C5 cysteine-rich domain using program ClustalW (Thompson et al., 1994; Figure 20) revealed the WO 00/16609 PCT/AU99/00805 S11-0 following consensus sequence, as represented by the amino acid sequences contained in any one or more of SEQ ID NO:<400> 10 to SEQ ID NO:<400> 55

C-R-R-C-X

2 -H-X( 22 32 )-C-X3-C-Y, wherein numerical values indicate the number of consecutive amino acids in the consensus sequence.

Additional motifs have been identified within the E(z) class of polypeptides, including the FIS1 polypeptide, by aligning the amino acid sequence of MEDEA/FIS1 to the amino acid sequences of-several E(z) polypeptides, using the multiple sequence alignment program ClustalW (Thompson et al., 1994). The aligned amino acid sequences of MEDEA/FIS1, EZA1, CURLY LEAF, E(z) and MES-2 are presented in Figure 21.

This analysis revealed strong homology in the SET domain, CXC domain, C5 domain, in addition to a putative TNFR/NGFR motif (Figure 22) and an RGD motif which had not been previously identified for this class of proteins.

The TNFR/NGFR domain overlaps the previously-described CXC domain in MEDEA and other E(z)-like proteins. This consensus domain consists of about 40 amino acids, containing 6 conserved cysteine residues. The TNFR/NGFR domain is defined by a general consensus sequence as represented by any one or more of the amino acid sequences set forth in SEQ ID NO:<400>116 to SEQ ID NO:<400>180, as follows:

-X(

5 10-C-X( 0 2 2 1-C-X( 4

-X

2

-C,

wherein numerical values indicate the number of consecutive amino acids in the consensus sequence. The motif may be found from 1 to 4 times in a given protein sequence. TNFR family members regulate processes that range from cell proliferation to programmed cell death. This domain is also found in cytokine receptor CD27, CD30), in FAS antigen, the receptor for FASL, a protein involved in apoptosis, and other cytokine receptor proteins. The TNFR/NGFR motif is also present in the WO 00/16609 PCT/AU99/00805 111 proteins designated TNFR-R1 and TNFR-R2 (Figure 22).

Of all the E(z) proteins analysed, only the MEDEA/FIS1 polypeptide comprised a close match to the TNFR/NGFR motif found in the MOTIF database at 100%. The other E(z)-like proteins shown in Figure 22 do not match this amino acid sequence motif at 100% using the MOTIF program. Although the CXC domain found in all the E(z)-like sequences contains the 6 conserved cysteine of the TNFR/NGFR domain with the correct spacing between each of them, at least one of the other conserved residues is different in these other protein sequences.

The sequence Arg-Gly-Asp (SEQ ID NO:<400> 181) which is present in the MEDEA/FIS1 polypeptide, is also found in fibronectin where it is crucial for its interaction with its cell surface receptor, an integrin Ruoslahti and Piersbacher (1986).

The motif is also found in other proteins collagen, vitronectin, fibrinogen and snake disintegrin), where it has been shown to play a role in cell adhesion. The role of this motif in the FIS1 polypeptide in unclear.

A further novel motif was identified C-terminal to the C5 domain and N-terminal to the CXC domain in the MEDEA/FIS1-polypeptide, designated as the WCA motif (Figure 23), which comprises the amino acid sequence set forth in SEQ ID NO:<400>189:

W-T-P-V-E-K-D-L-Y-L-K-G-I-E-I-F-G-R-N-S-C-D-V-A-L-N-I-L-R-G-L-K-T-C.

Alignment of the E(z) polypeptide to the E(z)-like polypeptides MEDEA/FIS1, CURLY, EZA1 and MES-2 reveals the consensus sequence-as respresented by the amino acid sequence set forth in SEQ ID NO:<400>185, as follows W-X- (E/A/Di) -X 2 -X-

-X-G-X-K-

or alternatively, the consensus sequence as respresented bythe amino acid sequence set forth in SEQ ID NO:<400>186, as follows WO 00/16609 PCT/AU99/00805 -112- W-X-

-X

2 -X 3 X-N-X-C-X-

-X

1 3

-C.

EXAMPLE FIS1 and FIS2 promoter GUS fusions show similar pattern of expression We studied the expression pattern of the FIS1 and FIS2 genes, by fusing their promoter sequences to the GUS reporter gene, introducing the FIS promoter/ GUS fusion constructs into plant cells, regenerating whole plants therefrom and determining the GUS staining pattern in the transgenic plants.

In particular, two different the FIS1 promoter/ GUS fusion constructs were produced as follows, and introduced into A. thaliana using standard procedures for the transformation of this plant species: A 1357 bp FIS1 promoter GUS construct, including nucleotides from 440 bp upstream of the translation initiation site of the FIS1 gene, to about 917 bp downstream of the translation initiation site of the FIS1 gene about nucleotides 1785 to 3143 of <400>5); and (ii) a 2987 bp FIS1 promoter GUS construct, including nucleotides from 2070 bp upstream of the translation initiation site of the FIS1 gene,to about 917 bp downstream of the translation initiation site of the FIS1 gene about nucleotides 156 to 3143 of <400>5).

Each FIS1/GUS fusion construct contained the complete sequence of exons 1 and 2, and 80 bp of exon 3, including the first 2 introns of the FIS1 gene nucleotide sequence (<400>5).

Two different the FIS2 promoter/ GUS fusion constructs were also produced as follows, and introduced into A. thaliana using standard procedures for the transformation of this plant species: A 1620 bp FIS2 promoter GUS construct, including nucleotides from WO 00/16609 PCT/AU99/00805 -113- 1281 bp upstream of the translation initiation site of the FIS2 gene, to about 339 -bp downstream of the translation initiation site of the FIS2 gene about nucleotides 1908 to about nucleotides 3528 of <400>7); and (ii) a 3528 bp FIS2 promoter GUS construct, including nucleotides from 3189 bp upstream of the translation initiation site of the FIS1 gene, to about 339 bp downstream of the translation initiation site of the FIS1 gene about nucleotides 1 to 3528 of <400>7).

Each FIS2/GUS fusion construct contained the complete sequence of exons 1, 2 and 3, and 39 bp of exon 4, including the first 3 introns of the FIS2 gene nucleotide sequence The putative zinc-finger protein motif found in the FIS2 polypeptide was also included the FIS2/GUS fusion protein products of these two FIS2/GUS fusion constructs.

The FIS1/GUS and FIS2/GUS fusion constructs described herein are represented schematically in Figure 24.

For the transformation of A. thaliana with each of the above FIS1/GUS and FIS2/GUS fusion constructs, 10 independent transformants were investigated for expression of the FIS1/GUS and FIS2/GUS fusion proteins, respectively, using standard histochemical methods. Both the FIS1/GUS and FIS2/GUS fusion proteins were found to express exclusively in the female gametophyte before and after pollination (Figures and 26, respectively). Fusion protein expression was not detected elsewhere in the plants. Fusion protein expression was also observed in the nucleus-of central cell, in the absence of fertilisation and when no nuclear division had yet occured.

FIS2/GUS fusion protein expression (Figure 26) was first observed particularly in the two polar nuclei in mature embryo sac initially before fusion into a central cell nucleus.

Expression was then detected in the homodiploid central cell nuclei. After pollination, fusion protein expression was observed through each of the nuclear divisions that WO 00/16609 PCT/AU99/00805 S- -114produce the endosperm, up to the stage of a 32 free endosperm nucleus. Later-in development, fusion protein expression decreased, except in the endosperm nuclei at the chalazal end. Several nuclei at the chalazal end, or endosperm cysts, expressed the FIS2/GUS fusion protiens until the heart stage was reached, when the endosperm start cellularising. All expression was restricted to within the nucleus and likely to result from the putative nuclear localization domain in the FIS2 gene sequence being included in this construct. Presumably, this signal guided the FIS2/GUS fusion protein into the nucleus, as iin the case of the wild-type FIS2 protein.

The FIS1/GUS fusion showed more diffused expression than FIS2/GUS (Figure probably because this construct did not contain any nuclear localization_ signal.

However, the pattern of FIS1/GUS fusion protein expression pattern was similar to that observed for the FIS2/GUS fusion protein. FIS1/GUS fusion protein expression was observed at the position of the central cell, however it is unclear whether FIS1/GUS expression initiated in the fused nuclei before or after nuclear fusion had occurred.

After fertilization, two or four free endosperm nuclei expressing the FIS1/GUS fusion protein were detected, however expression was more diffused than for FIS2/GUS at this stage. In some cases, six free endosperm nuclei could be observed to express FIS1/GUS fusion protein, suggesting that the wild-type FIS1 protein has a similar pattern of expression to the FIS2 protein. As with the expression of the FIS2/GUS fusion protein, FIS1/GUS expression finally became localised to the chalazal end endosperm nuclei until the heart stage was reached, and declined in the other parts of endosperm.

When wild-type A. thaliana plants were pollinated using pollen derived from transgenic plants containing the expressible FIS1/GUS and FIS2/GUS fusion constructs, no FIS1/GUS or FIS2/GUS fusion protein expression detectable in the fertilized endosperm, suggesting that expression of FIS1 and FIS2 genes might occur in the maternal genome and/or that said expression may be triggered before pollination occurs.

WO 00/16609 PCT/AU99/00805 -115 Several putative nuclear localisation signals (NLS) were identified in the amino acid sequence of the FIS2 polypeptide (Example 11). In this regard, since both FIS2 promoter constructs directed FIS2/GUS fusion protein expression to the nucleus in the preceding Example, the FIS2 coding sequence included in these constructs must contain a functional nuclear localisation signal (NLS). However, further analysis of the FIS2 genes sequences included in these FIS2/GUS fusion constructs revealed that only the N-terminal putative NLS was present in both constructs, suggesting that this sequence is the functional NLS.

EXAMPLE 16 -Transposon tagging of the FIS3 gene The method of tagging the FIS3 gene was the same as that described in Example for tagging the FIS2 gene. In the DSG tagged line designated DT51,-the transposon was found to be closely linked to fis3, between the SSLP marker designated nga162 and the RFLP marker designated ve039 (Figure The line DT51, containing Ds closely linked to fis3, was crossed with pollen from a plant containing Ac and approximately 2,000 F1 plants were screened for sectors that produced a 50:50 ratio of normal to fertilization-independent silique elongation (Figure 10). Since the DSG element was known to be closely-linked to FIS3 in the orginal DT51 line and this element transposes to closely-linked sites on the chromosome, it is highly likely that the appearance of the fis3 mutant phenotype in these progeny lines was the result of the FIS3 gene being tagged.

The FIS3 gene is then isolated using standard procedures. First, DNA flanking the insertion site of the DSG element (Figure 8) in the fis3-tagged mutant is cloned. A genomic DNA library is produced from the DNA of the tagged line and screened using the Ds element as a probe. Alternatively, or in addition, the gene sequences flanking the Ds element may be isolated using inverse PCR and/or tailed PCR to amplify sequences from genomic DNA or cloned genomic DNA, The nucleotide sequences of the flanking DNA may then be used to isolate the corresponding FIS3 gene sequences from a genomic library constructed using DNA derived from wild-type plants. The WO 00/16609 PCT/AU99/00805 116clones isolated from the wild-type library are subsequently used to complement the mutation in the EMS-mutagenised fis3 lines, to confirm the identity of the isolated FIS3 DNA sequences.

EXAMPLE 17 Isolation and nucleotide sequence of the Fis3 gene The present inventors isolated a 1372 bp full-length FIS3 cDNA from an Arabidopsis thaliana late silique cDNA library. The nucleotide sequence of this cDNA (<400>8) corresponded to the nucleotide sequence of the recently-described FIE gene (Ohad et al., 1999). and determined if our two alleles of fis3 (fis3-1 and 3-2) contained mutations in their FIE gene. The derived amino acid sequence of the FIS3 polypeptide is set forth herein as <400>3.

The cDNA clone was used to isolate a FIS3 genomic clone, by identifying the corresponding nucleotide sequence in the database of the Arabidopsis Genome Initiative (PI clone MOE17; Accession Number AB025629). The nucleotide sequence of the FIS3 genomic clone is set forth herein as <400>9.

-Nucleotide sequence analysis of the corresponding fis3-1 and fis3-2 mutant alleles indicated that these genes were allelic to the FIE gene. In the fis3-1 mutant allele, a G to A substitution was observed at the border of the third intron, modifying the acceptor donor site from AG to AA. In the fis3-2 mutant allele, a G to A substitution resulted in the amino acid substitution of glycine at position 104 to glutamate.

EXAMPLE 18 Identification of protein-protein interaction between FIS proteins using a yeast two hybrid system The FIS1, FIS2, and FIS3 cDNAs were inserted them into the yeast two-hybrid vectors pGBT9 and pGAD424, to determine whether the polypeptides encoded therefor form homodimers and/or heterodimers.

WO 00/16609 PCT/AU99/00805 117- In particular, the full-length FIS1 cDNA sequence, encoding a 689 amino acid polypeptide comprising the A, C5, N, CXC and SET domains, and the deletion mutants designated: ABgl, encoding a 513 amino acid polypeptide and lacking the C-terminal SET domain-encoding region; ABcl, encoding a 320 amino acid polypeptide and lacking the C-terminal N, CXC and SET domain-encoding regions; APst, encoding a 62 amino acid polypeptide and lacking the C-terminal portion of FIS1 comprising the five domain-encoding regions; and A160, lacking 160 bp at the end of the FIS1 cDNA, were constructed (Figue 27). The full-length FIS2 and FIS3 cDNAs were also used. Control constructs, employing the empty vectors pGBT9 and pGAD424, or alternatively the EzA1 cDNA, were also used. Each cDNA was cloned into each vector and yeast were transformed with vectors expressing different FIS polypeptides, in the presence of adenine selection and 3-Galactosidase activation, to select for cells expressing from both constructs.

Data presented in Figure 27 to 29 indicate that the FIS1, FIS2 and FIS3 polypeptides are capable of forming certain homodimers or heterodimers.

In particular, data presented in the left panel of Figure 27 indicates that the full-length FIS1 polypeptide is capable of forming homodimers with the full-length FIS1 polypeptide, or with truncated versions thereof comprising the A and C5 regions only having the C-terminal 369 amino acids containing the N, CXC and SET domains deleted).

Similarly, data presented in the right panel of Figure 27 indicates that the full-length FIS3 polypeptide is capable of forming heterodimers with the full-length FIS1 polypeptide, or alternatively, heterodimers with truncated versions of FIS1 comprising the A and C5 regions only having the C-terminal 369 amino acids containing the N, CXC and SET domains deleted). Accordingly, the A and/or C5 regions appear to be the minimum requirement for FIS1 homodimer or FIS1/FIS3 heterodimer formation.

WO 00/16609 PCT/AU99/00805 -118- Data presented in the left panel of Figure 28 also support the conclusion that FIS1 and FIS3 interact to an extent that is similar to FIS1/FIS, however there is only a weak interaction between FIS1 and FIS2 polypeptides in the yeast two-hybrid assay.

Data presented in the right panel of Figure 28 indicate that EzA1 and FIS1 polypeptides both interact with the FIS3 polypeptide, however the is-no significant interaction apparent in the yeast two-hybrid assay between the FIS2 and FIS3 polypeptides.

These data are also supported by the data obtained for a separate experiment, presented in Figure 29.

The data presented herein support the hypothesis (see below) that the FIS1, FIS2 and FIS3 proteins form a complex to repress seed -development in vivo.

EXAMPLE 19 A screen to isolate genes which regulate FIS gene expression Based upon the results obtained for FISIGUS fusion constructs described herein, genes which regulate FIS gene expression Mother of FIS (herinafter "MOF genes")] may encode either repressor proteins MOF repressor genes) which inhibit expression of FIS proteins in the male gametophyte or alternatively, activator proteins MOF activator genes) which activate or enhance expression of FIS proteins in the female gametophyte In the repressor model (Figure 30), wild-type MOF represses FIS gene promoter function and thus, FIS gene expression is inhibited in the male gametophyte, so that FIS protein is not expressed in the pollen. Without being bound by any theory or mode of action, when a MOF gene is mutated and rendered non-functional or alternatively, encodes a non-functional MOF repressor protein, FIS protein is expressed in the male gametophyte. Asa consequence, variations in the pattern of FIS protein expression WO 00/16609 PCT/AU99/00805 -119in the male gametophyte will assist in identifying putative MOF gene mutants, which are useful as molecular tags to isolate the correpsonding wild-type genes using standard hybridisation and polymerase chain reaction approaches.

In the activator model, MOF proteins normally activate the expression of FIS proteins in the female gametophyte. In plants containing the FIS2/GUS reporter construct described herein, we showed that FIS-GUS was expressed in the female gamete, presumably as a consequence of the activity of MOF activator proteins.

MOF genes which regulate enhance, activate, up-regulate, repress or downregulate) FIS gene expression are isolated using the following procedure: seeds derived from transgenic plants containing a functional FIS2 promoter/GUS fusion construct are mutagenised; (ii) GUS gene expression is assayed in the mutagenised lines; and (iii) those plants having altered GUS gene expression compared to the nonmutagenized transgenic parent are selected, wherein, if the selected plant has a mutated-MOF gene or expresses an aberrant MOFgene product GUS reported gene expression is altered.- In the performance of the subject method, those plants having a mutant MOF gene, FIS protein express the GUS reporter gene in the male gametophyte. By looking at GUS staining pattern, putative MOF repressor mutants are identified and the corresponding MOF repressor genes are isolated.

The subject method can also be used to identify MOF activator genes which, when mutated, decrease GUS gene expression in the female gamete. As with the identification of MOF repressor genes described supra, putative MOF activator mutants are identified and the corresponding MOF activator genes are isolated WO 00/16609 PCT/AU99/00805 -120- EXAMPLE Discussion Without being bound by any theory or mode of action, the FIS1, FIS2 and FIS3 polypeptides may form a complex which negatively-regulates the expression of genes that are required for the transformation of ovules into seeds or alternatively, these polypeptides may act in concert to prevent such a developmental transformation from occurring in the maternal tissues. Since seed development is linked to a diverse array of phenotypes having profound implications in agronomy, (parthenocarpy), this complex and the mode of action and regulation thereof will be pivotal to seed development.

The FIS1 and FIS2 polypeptides at least are putative transcription factors which have the potential for forming zinc-finger or zinc-binding secondary structures and, as a consequence, are likely to regulate the expression of other genes. Genes which may be regulated by FIS1-FIS2-FIS3 are likely to comprise a set of genes whose increased expression in a diverse set of organisms initiate seed development. Inappropriate activation of these genes presumably via a down regulation of FIS1-FIS2-FIS3 would initiate- seed development without fertilization, producing autonomous and/or pseudogamous endosperm development.

The homology of FIS1 to polycomb group of proteins suggest that this polypeptide at least or alternatively, a FIS1-FIS2-FIS3 complex, might be involved in interacting with chromatin to maintain a status of chromatin that leads to gene inactivation. Thus, FIS1-FIS2-FIS3 may mediate epigenetic gene silencing by altering chromatin structure or methylation status.

Epigenetic gene silencing, when occurring differentially in the paternal and the maternal genome of an organism is known as "imprinting" and it is possible that the action of FIS1-FIS2-FIS3 is mediated via such a process. FIS1-FIS2-FIS3 may control silencing of a number of genes in the female gamete in the absence of pollination.

Mutation in either of these genes would lead to an activation of the silenced genes WO 00/16609 PCT/AU99700805 121giving rise to the fertilization independent seed phenotype. The genes controlled by the FIS1-FIS2-FIS3 complex, or a subset of such a complex, may be a subset of the imprinted genes in the female gamete that are kept silent by the combined action of these FIS polypeptides.

During normal seed development following pollination, the expression of genes derived from the paternal parent which are not silenced facilitate endosperm development in a manner similar to that which occurs in the fis mutants.

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81. Marck, et al. (1988) 82. Dellaporta et al. (1983) 83. Roder et (1986) 84. Ohad et a! (1999) Ozias-Akins (1998) EDITORIAL NOTE APPLICATION NUMBER 61824/99 The following Sequence Listing pages 1 to 94 are part of the description. The claims pages follow on pages 126 to 142.

PCT/AU99/00805a P:\OPER\MRO\AUTSEED2.PCr 21/9/99 -1I- <110> Commonwealth Scientifi <120> Novel method of reguia genetic sequences ther <130> p: \oper\mro\autseed.pc <140> <141> <150> US 60/101184 <151> 1998-09-21 <150> AU PP6063 <151> 1998-09-22 <150> AU PP6062 <151> 1998-09-22 <150> AU PP6061 <151> 1998-09-22 <150> AU PQ1345 <151> 1999-07-01 <150> AU PQ1346 <151> 1999-07-01 <160> 213 <170> Patentln Ver. <210> 1 <211> 689 <212> PRT <213> Arabidopsis thaliana <400> 1 Met Glu Lys Glu Asn His Glu 1 5 Leu Asn Gin Ile Lys Giu Gin Lys Arg Lys Phe Giu Leu Arg Ser His His Gin Ser The Asp Asn Gly Gly Asp Asn Lys Ser Arq His Phe Ser Ala Ser Ser Tyr Vai Leu Asp Giu Asp Gin 100 Leu Phe Leu Asp Giu Asp Val 115 Ile Val Glu Lys Leu Pro Arg 130 135 Ser Gin Leu Met Aia Giu Ser 145 150 SEQUENCE LISTING .c and Industrial Research Organisation Lting seed development in plants and efor Gly Lys Pro Gin Ser As n Ala Leu Thr Val1 Pro Leu Thr Giu Asn Asp Asp 110 Lys Thr Arg PCT/AU99/00805L P:\OPERXMRO\AUTSEED2.PCr 21/9/99 -2- Tyr Giu Phe Giy Val 225 Asn Thr Met Se r Cys 305 His Asp Lys Ile Giu 385 Arg Val1 Leu Pro His 465 Asn Gin Arg Tyr Asp Ser Leu 210 Asp Asp Thr Ile Se r 290 Ser Val1 Pro Giy Leu 370 Gin His Arg Lys Cys 450 Giu Arg Cys Se r Leu Giu Giu 195 Asp Vali Giy Ala Phe 275 Giu Giu Met Asn Ile 355 Arg Asp As n Lys Lys 435 Thr Asn Giu 180 Asp Asp Ser Thr Phe 260 Asp Asp His Asp Asn 340 Glu Giy Gin Gin Lys 420 Thr Cys Giu Aia Asp Giu Asp Arg Vai Vai 215 Ile Leu 230 Gly Giu Asp Phe His Met Ser Ser 295 Tyr Leu 310 Asp Asn Met Trp Phe Giy Lys Thr 375 Thr Met 390 Thr Lys Arq Leu Ser Giy Ser Lys 455 Giu Lys Leu Giu Phe 200 Arg Giu Ala Ala His 280 Leu Lys Se r Thr Arg 360 Cys Ser Lys Arg Glu 440 Cys Giu Giu 185 Ile Arg Arg Ser Asp 265 Glu Phe Vali Ile Pro 345 Asn Leu Leu Val Lys 425 Aia Giy Ser Lys Val Al a 220 Giu Thr His Giu Glu 300 Val Lys Lys Asp Tyr 380 As n Lys Arg Tyr Cys 460 Se r Gin Asp Leu Asp 175 Cys Asp Leu Leu Thr 255 Arg Se r Gin Aia Vali 335 Tyr Leu Met Thr Arg 415 Pro Tyr Leu Giu Giu Tyr Glu Lys 240 Ile Cys Arg Pro Asp 320 Ser Leu Asn Arg Gin 400 Ser Ala Thr Thr Asn 480 Arg Cys Pro Asn Cys Cys Tyr Cys Gly Cys Aia Ile 490 Asn Arg Giu 505 Giy Asp Gly 520 Cys 475 Giy Cys Thr Lys Asp Cys 515 525 Vai Gin Ile Gin Cys Lys Asn Met Gin Phe Leu Leu Gin Thr Asn Lys PCT/AU99/00805a P:\OPER\MRO\AUTSEED2.PCT 21/9/99 -3- 530 535 540 Lys Ile Leu Ile Gly Lys Ser Asp Val His Gly Trp Gly Ala Phe Thr 545 550 555 560 Trp Asp Ser Leu Lys Lys Asn Glu Tyr Leu Gly Glu Tyr Thr Gly Glu 565 570 575 Leu Ile Thr His Asp Glu Ala Asn Glu Arg Gly Arg Ile Glu Asp Arg 580 585 590 Ile Gly Ser Ser Tyr Leu Phe Thr Leu Asn Asp Gin Leu Glu Ile Asp 595 600 605 Ala Arg Arg Lys Gly Asn Glu Phe Lys Phe Leu Asn His Ser Ala Arg 610 615 620 Pro Asn Cys Tyr Ala Lys Leu Met Ile Val Arg Gly Asp Gin Arg Ile 625 630 635 640 Gly Leu Phe Ala Glu Arg Ala Ile Glu Glu Gly Glu Glu Leu Phe Phe 645 650 655 Asp Tyr Cys Tyr Gly Pro Glu His Ala Asp Trp Ser Arg Gly Arg Glu 660 665 670 Pro Arg Lys Thr Gly Ala Ser Lys Arg Ser Lys Glu Ala Arg Pro Ala 675 680 685 Arg <210> 2 <211> 813 <212> PRT <213> Arabidopsis thaliana <400> 2 Met Ala Arg Lys Ser Ile Arg Gly Lys Glu Val Val Met Val Ser Asp 1 5 10 Asp Asp Asp Asp Asp Asp Asp Val Asp Asp Asp Lys Asn Ile Ile Lys 25 Cys Val Lys Pro Leu Thr Val Tyr Lys Asn Leu Glu Thr Pro Thr Asp 40 Ser Asp Asp Asn Asp Asp Asp Asp Asp Asp Val Asp Val Asp Glu Asn 55 Ile Ile Lys Tyr Ile Lys Pro Val Ala Val Tyr Lys Lys Leu Glu Thr 70 75 Arg Ser Lys Asn Asn Pro Tyr Phe Leu Arg Arg Ser Leu Lys Tyr Ile 90 Ile Gin Ala Lys Lys Lys Lys Lys Ser Asn Ser Gly Gly Lys Ile Arg 100 105 110 Phe Asn Tyr Arg Asp Val Ser Asn Lys Met Thr Leu Lys Ala Glu Val 115 120 125 Val Glu Asn Phe Ser Cys Pro Phe Cys Leu Ile Pro Cys Gly Gly His 130 135 140 Glu Gly Leu Gin Leu His Leu Lys Ser Ser His Asp Ala Phe Lys Phe 145 150 155 160 PCT/AU99/00805a P:\OPER\MRO\AUTSEED2.PCr 21t9/99 -4- Giu Val1 Asn Arg Leu 225 Ser Ser Ala Ser His 305 Leu Pro Asp Ser Pro 385 Tyr Lys His Ile Phe Tyr Lys Ser Pro Gin 195 Arg Gin 210 Met Giu Thr His Ser Giu Giu Ser 275 Ser Pro 290 Vai Asn Giu Lys Pro Lys Glu Asp 355 Asp Ile 370 Lys Val Lys Ala Vai Leu Ser Leu 435 Aia Giu 450 Arg Asp 180 Leu Arg Leu Val1 Lys 260 Ser Pro Asp Asn Aia 340 Val1 Leu Arg Arg His 420 Giu Ser Aia 165 Thr Ser Asp Asp Asn 245 Ile Giu Arg Asp Giu 325 His Ser Thr Arg Giu 405 Val Lys Ser Giu Ile Pro Asp Leu 230 Asp Ser Pro Ala Asp 310 Se r Ser Phe Thr Giy 390 Thr Asn Aia Giu Lys Lys Leu Ser 215 Asp Asp Asp Lys His 295 Asp Thr Ser Pro Thr 375 Ser Gin Asp Ser Pro 455 Asp Phe Thr 200 Asn Asp Asn Ile Vali 280 Ser Val His Lys Pro 360 Gin Arg Pro Giu Asp 440 Lys His Gly 185 Phe Asn Leu Val1 Leu 265 Pro Ser Se r Vali Lys 345 Arg Pro Arg Ala Asn 425 Ile Vali Gi y 170 Val1 Cys Val Pro Ser 250 Thr His Aia Ser Asn 330 Asn Thr Ala Lys Ile 410 Val1 Leu Pro Pro Leu Ser Lys Arg 235 Ser Thr Vai Giu Pro 315 Giu Giu Arg Ile Gin 395 Aia Ser Thr His Giu Lys Lys Lys 220 Gi y Pro Thr Asn Lys 300 Pro Asp Se r Ser Val1 380 Leu Giu Ser Thr Val1 460 Vali Asp Asn 205 Leu Thr Pro Gin Asp 285 Asn Arg Asn Thr Ser 365 Giu Tyr Se r Pro Thr 445 Asn Asp Asp 190 Arg Asn Giu Arg Leu 270 Gly Giu Ala Ile His 350 Lys Pro Ala Se r P ro 430 Gir Asp Val 175 Val Asn Val1 Asn Ala 255 Ala Asn Ser His Se r 335 Met Giu Ser Lys Giu 415 Giu Pro Glu Ser Gly Gin Leu Asp 240 His Ile Val1 Thr Se r 320 Ser Asn Thr Giu Arg 400 Pro Ala Ala Asn Val Ser Ser Thr Pro Arg Ala His Ser Ser Lys Lys Asn Lys Ser Thr 465 470 475 480 PCT/AU99/00805& P:\OPER\MRO\AUTSEED2.PCT 21/9/99 Arg Lys Asn Val Asp Asn Val Pro Ser 485 Lys Ser Thr Asp 545 Ser Lys His Ser Ala 625 Arg Arg Gin Glu Arg 705 Asn T rp Ser Asn Lys Thr Ser Glu 515 Pro Arg 530 Asp Asn Asn Ile Val Pro Ser Ser 595 Leu Pro 610 Thr Thr Val Ser Leu Glu Pro Met 675 Thr Asp 690 Leu Val Ile Phe Ala Cys Ser Ser 755 Asn Gly 770 Ser 500 Pro Aia Ile Leu His 580 Lys Pro Gin Arg Arg 660 Thr Asp Gly Val1 Giu 740 Phe Leu Asp Lys His Pro Thr 565 Val1 Lys Lys Pro Arg 645 Leu Phe Tyr Val1 Arg 725 Giu Asp Ile Ile Val1 Ser Ser 550 Arg Asn Asn Thr Ala 630 Lys Lys Giu Ala Se r 710 Lys Phe T rp Cys Leu Arg Ser 535 Pro Thr Asp Lys Arg 615 Lys Giu Gly Gin Leu 695 Lys Gin Ala T rp Ala 775 Thr His 520 Lys Pro Gin Asp Ser 600 Ser Ala Leu Arg Val 680 Asp Glu Arg Lys T rp 760 Lys Thr 505 Val Lys Lys Pro Lys 585 rhr Ser Giu His Gin 665 Met Ile Glu Val Leu 745 Arg Thr Pro 490 Thr Asn Asn Thr Ala 570 Val1 His Lys Pro Al a 650 Phe Ser Se r Lys Ile 730 His Met Phe Pro Gin Asp Lys Arg 555 Ile Ser Lys Lys Ser 635 Giu Tyr Asn Giu Arg 715 Ala Lys Phe His Lys Pro Asp Ser 540 Ser Ala Ser Lys Thr 620 Glu Arg His Glu Arg 700 Tyr Asp Glu Arg Lys 780 Thr Thr PAsn 525 Thr Ser Glu rhr A\sp 605 Se r Pro Cys Ser Asp 685 Leu Met Gly Glu Ile 765 Cys Arg Ile 510 Val Arg Lys Ser Pro 590 Asp Asp Lys Glu Gin 670 Se r Arg Tyr His Met 750 Lys Thr Ser 495 Ala Ser Lys Lys Giu 575 Arg Asn Ile Val1 Ala 655 Thr Giu Leu Leu Val1 735 Lys Leu Thr Ser Glu Ser Asn Thr 560 Pro Al a Ala Leu Thr 640 Lys Met Asn Giu T rp 720 Pro Asn T rp Ile Leu Leu Ser Asn Ser Asp 785 790 Glu Ala Gly Gin Phe Thr Ser Gly Ser 795 PCT/AU99/00805, 4 P:\OPER\MRO\AtJTSEED2.PCT 2119/99 -6- Ala Asn Ala Asn Asn Gin Gin Ser Met Giu Vai Asp Giu 805 810 <210> 3 <211> 369 <212> PRT <213> Arabidopsis thaliana <400> 3 Met Ser Lys Ile Thr Leu Giy 1 Pro Lys Phe Cys Asp Gi y Ile Asp Ile 145 Gi y Val1 Cys Thr Pro 225 Asn Ser Lys Asp Leu Lys Asn Asp Se r 130 Thr Ile Leu Gly Tyr 210 Thr Tyr Asn Pro Val1 Gly Giu Pro Val 115 Val1 Ala Cys Se r Met 195 Val1 Lys Val Lys Leu Phe Asp Glu Tyr 100 Asn Asn Ser Ile Val1 180 Asp Giu Phe Asp 5 Lys Tyr Val1 Gly Ser Val Ser Glu Lys Leu 165 Asp Thr Lys Val Cys 245 Ser Ala Thr Ala 70 Phe Ala Giu Ile Asp 150 Ile Phe Thr Se r Gin 230 As n Tyr VTal kla 55 Ilie Tyr Ala Thr Arg 135 Giu Phe His Ile Phe 215 Phe Arg Asn Lys Val 40 Gly Ser Thr Gly Ile 120 Thr Ser Ala Pro Lys 200 Thr Pro T rp Giu Val 25 Phe Gly Alia Val1 Gly 105 His Gin Val Gly Ser 185 Ile T rp Val1 Phe Ser Ile 10 Thr Asn Asn Phe Asn Arg Leu Gin 75 Ser Trp 90 Val Lys Lys Ser Pro Leu Arg Leu 155 Ala Gly 170 Asp Ile Trp Ser Thr Asp Phe Thr 235 Gly Asp 250 Val1 A.rg Leu Ile Ser Ala Gly Leu Lys 140 T rp Gly Tyr Met Asp 220 Ala Phe Gly Ile Asp Thr Tyr Cys Ile Val 125 Pro Asn His Arg Lys 205 *Pro *Ser Ile Ser Gln Ala Leu Ala Gly Ile 110 Gly Gin Val Arg Phe 190 G iu Ser Ile Leu Leu Giu Arg Tyr Asp Val Arg His Leu Giu Tyr 175 Ala Phe Lys His Ser 255 Thr Gly Phe Asn Asn Val Gly Val1 Thr 160 Giu Se r T rp Phe Thr 240 Lys Ser Val Asp Asn 260 Giu Ile Leu Leu Trp Giu Pro Gin Leu Lys Giu Asn 265 270 PCT/AU99/00805a P:\OPER\MRO\AUTSEED2.PCT 21/9/99 -7- Ser Pro Gly Glu Gly Ala Ser Asp Val Leu Leu Arg Tyr Pro Val Pro 275 280 285 Met Cys Asp Ile Trp Phe Ile Lys Phe Ser Cys Asp Leu His Leu Ser 290 295 300 Ser Val Ala Ile Gly Asn Gin Glu Gly Lys Val Tyr Val Trp Asp Leu 305 310 315 320 Lys Ser Cys Pro Pro Val Leu Ile Thr Lys Leu Ser His Asn Gin Ser 325 330 335 Lys Ser Val Ile Arg Gin Thr Ala Met Ser Val Asp Gly Ser Thr Ile 340 345 350 Leu Ala Cys Cys Glu Asp Gly Thr Ile Trp Arg Trp Asp Val Ile Thr 355 360 365 Lys <210> 4 <211> 2309 <212> DNA <213> Arabidopsis thaliana <220> <221> CDS <222> (14)..(2080) <400> 4 aggcgagtgg tta atg gag aag gaa aac cat gag gac gat ggt gag ggt 49 Met Glu Lys Glu Asn His Glu Asp Asp Gly Glu Gly 1 5 ttg cca ccc gaa cta aat cag ata aaa gag caa atc gaa aag gag aga 97 Leu Pro Pro Glu Leu Asn Gin Ile Lys Glu Gin Ile Glu Lys Glu Arg 20 ttt ctg cat atc aag aga aaa ttc gag ctg aga tac att cca agt gtg 145 Phe Leu His Ile Lys Arg Lys Phe Glu Leu Arg Tyr Ile Pro Ser Val 35 gct act cat get tca cac cat caa tcg ttt gac tta aac cag ccc get 193 Ala Thr His Ala Ser His His Gin Ser Phe Asp Leu Asn Gin Pro Ala 50 55 gca gag gat gat aat gga gga gac aac aaa tca ctt ttg tcg aga atg 241 Ala Glu Asp Asp Asn Gly Gly Asp Asn Lys Ser Leu Leu Ser Arg Met 70 caa aac cca ctt cgt cat ttc agt gcc tca tct gat tat aat tct tac 289 Gin Asn Pro Leu Arg His Phe Ser Ala Ser Ser Asp Tyr Asn Ser Tyr 85 gaa gat caa ggt tat gtt ctt gat gag gat caa gat tat get ctt gaa 337 Glu Asp Gin Gly Tyr Val Leu Asp Glu Asp Gin Asp Tyr Ala Leu Glu 100 105 gaa gat gta cca tta ttt ctt gat gaa gat gta cca tta tta cca agt 385 Glu Asp Val Pro Leu Phe Leu Asp Glu Asp Val Pro Leu Leu Pro Ser 110 115 120 PCT/AU99/008054 P:\OPER\MRO\AUTSEED2.PCT 2119199 -8gtc Val1 125 ttc Phe aag Lys gaa Giu gaa Giu ggg Gi y 205 aag Lys ctc Leu tcc Ser tgc Cys ccc Pro 285 gat Asp aca Thr att Ile aag ctt Lys Leu acc aaa Thr Lys aga caa Arg Gin gaa gat Glu Asp 175 aaa tgc Lys Cys 190 cag gac Gin Asp tac ctc Tyr Leu aag ctt Lys Leu aag aca Lys Thr 255 cgt cgt Arg Arg 270 gag tct Giu Ser aga caa Arg Gin gaa gct Giu Ala gtg gtc Val Val 335 cca Pro agt Ser atc Ile 160 gag Giu gaa Giu tat Tyr gaa Giu aag Lys 240 ata.

Ile tgc Cys aga Arg cca Pro gat Asp 320 t ca Ser att Ile agc Ser 145 tat Tyr gaa Giu ttt Phe ggt Gly gtg Val 225 aat Asn act Thr atg Met tcc Ser tgc Cys 305 cat His gat Asp gtt Val1 130 cag Gin tat Tyr gat Asp tct Ser ttg Leu 210 gat Asp gat Asp act Thr ata Ile agc Ser 290 agt Ser gtg Val1 cca Pro gag Giu ctg Leu ttg Leu gaa Giu gaa Glu 195 gat Asp gtt Val gga Gly gct Ala ttc Phe 275 gaa Glu gag Glu atg Met aac Asn aag Lys atg Met aat Asn gaa Giu 180 gat Asp gat Asp t cg Ser act Thr ttc Phe 260 gat Asp gac Asp cat His gat Asp aac Asn 340 cta Leu gct Ala ggt Gly 165 gaa Glu gta Val ctg Leu gac Asp gct Ala 245 cag Gin tgt Cys aaa Lys tgt Cys aat Asn 325 act Thr cca cga Pro Arg 135 gaa Giu 150 gag Giu gat Asp gac Asp gtc Val1 ata Ile 230 ggt Gly gat Asp cat His tct Ser tac Tyr 310 gat Asp atg Met agt Se r gca Ala gag Giu cga Arg gtg Val 215 ttg Leu gag Glu ttt Phe atg Met agt Ser 295 ctc Leu aac Asn tgg T rp tcc Ser gat Asp cta Leu gaa Giu ttt Phe 200 cgg Arg gaa Giu gct Ala gct Ala cat His 280 ttg Leu aag Lys tct Se r acg Thr att Ile tct Ser gaa.

Giu gaa Glu 185 ata Ile cgt Arg aga Arg tct Ser gat Asp 265 gag Giu ttt Phe gtq Val1 ata Ile cct Pro 345 aca Thr gtg Val ttg Leu 170 atc Ile tgg Trp gct Ala tac Tyr gat Asp 250 aga Arg aag Lys gag Giu agg Arg t ca Se r 330 gta Val1 tgg Trp att Ile 155 agc Ser aag Lys acg Thr ctc Leu aat Asn 235 ttg Leu cgt Arg tat Tryr gat Asp agt Ser 315 aac Asn gag Glu gtc Vl 140 ggt Gly agt Ser ~aaa Lys gtt Val gcc kla 220 gaa G1u aca rhr cat His gag Giu gaa Glu 300 gtg Val1 aag Lys aag Lys 433 481 529 577 625 673 721 769 817 865 913 961 1009 1057 1105 gat ctt tac ttg aaa gga att gag ata ttt ggg aga aac agt tgt gat Asp Leu Tyr Leu Lys Gly 350 Giu Ilie Phe Gly Asn Ser Cys Asp PCT/AU99/00805#L P:\OPER\MRO\AUTSEED2.PCT 21/9/99 -9gca tta aac ata Ala Leu Asn Ile egg ggg ctt aag Arg Gly Leu Lys tge eta gag att Cys Leu Giu Ile 1153 1201 aat tac atg ege Asn Tyr Met Arg caa gat caa tgt Gin Asp Gin Cys atg tea tta gae Met Ser Leu Asp ett aae Leu Asn 395 aaa act aca Lys Thr Thr agt agt agg Ser Ser Arg 415 aga cac aat cag Arg His Asn Gin ace aaa aaa gta Thr Lys Lys Val tet ega aaa Ser Arg Lys 410 tat get egt Tyr Ala Arg 1249 1297 teg gte ege aaa Ser Val Arg Lys teg aga etc cga Ser Arg Leu Arg tat ccg Tyr Pro 430 cet get tta aag Pro Ala Leu Lys aca act agt gga gaa get aag ttt tat Thr Thr Ser Gly Giu Ala Lys Phe Tyr 440 1345 eac tac aca eca His Tyr Thr Pro act tge aag tea Thr Cys Lys Ser aaa tgt gga cag eaa tge Lys Cys Gly Gin Gin Cys 455 460 ect tgt tta act Pro Cys Leu Thr gaa aat tge tge Giu Asn Cys Cys aaa tat tge ggg Lys Tyr Cys Giy tge tea Cys Ser 475 1393 1441 1489 1537 aag gat tge Lys Asp Cys tge aca aat Cys Thr Asn 495 aat ege ttt gga Asn Arg Phe Gly gga tgt aat tgt gea att ggc eaa Gly Cys Asn Cys Ala Ile Gly Gin 485 490 ega eaa tgt ect Arg Gin Cys Pro ttt get get aat Phe Ala Ala Asn gaa tge gat Giu Cys Asp eca gat Pro Asp 510 ett tgt egg agt Leu Cys Arg Ser ect ett age tgt Pro Leu Ser Cys gat gge act ett Asp Gly Thr Leu ggt Gly 525 eaa Gin gag aca eca gtg Giu Thr Pro Vai ace aat aaa aag Thr Asn Lys Lys 545 ate caa tgc aag Ile Gin Cys Lys atg eaa tte etc Met Gin Phe Leu 1585 1633 1681 att etc att gga Ile Leu Ile Gly tet gat gtt eat Ser Asp Val His gga tgg Gly Trp 555 ggt gca ttt Gly Ala Phe tat act gga Tyr Thr Giy 575 ata gaa gat Ile Giu Asp .590 tgg gac tet ctt Trp Asp Ser Leu aag aat gag Lys Asn Giu gaa etg ate act Giu Leu Ile Thr gat gaa get aat Asp Giu Ala Asn tat ete gga gaa Tyr Leu Gly Giu 570 gag egt ggg aga Giu Arg Gly Arg 585 ttg aat gat eag Leu Asn Asp Gin 1729 1777 egg att ggt Arg Ile Gly tee tac ete ttt Ser Tyr Leu Phe 1825 PCT/AU99/00805a P:\OPER\MRO\AUTSEED2.PCr 21/9/99 gaa atc gat gct Glu Ile Asp Ala cgt aaa gga aac gag ttc aaa ttt ctc Arg Lys Gly Asn Glu Phe Lys Phe Leu 615 tca gca aga Ser Ala Arg tgc tac gcc Cys Tyr Ala ttg atg att gtg Leu Met Ilie Val aga gga Arg Gly 635 gat cag agg Asp Gin Arg gag ctt ttc Giu Leu Phe 655 cgt ggt cga Arg Gly Arg 670 cta ttt gcg Leu Phe Ala aga gca atc gaa Arg Ala Ile Glu ttc gac tac tgc Phe Asp Tyr Cys gga cca gaa cat Gly Pro Giu His gaa ggt gag Giu Gly Giu 650 gat tgg tcg Asp Trp Ser tct aag gaa Ser Lys Giu 1873 1921 1969 2017 2065 gaa cct aga aag act ggt gct tct Giu Pro Arg Lys Thr Gly Ala Ser 675 aaa agg Lys Arg 680 gcc cgt cca gct cgt Ala Arg Pro Ala Arg tagtttttga tctgaggaga agcagcaatt caagcagtcc tttttttatg ttatggtata tcaattaata atgtaatgct aacttaagtt tctgttttat ttgttttagg gtgttttgtt tttcaaagtt ttctttttgt atttcaattt aaaaacaatg aaaaaaaaa <210> <211> 6534 <212> DNA <213> Arabidopsis thaliana <400> attttgtgtt actaaaccaa tgtatcatat gtgtcttaac tttatgttgt taaaaaaaaa 2120 2180 2240 2300 2309 ctcgagagct atattatgta tgtaatggtc ttttttcata ccggctgtgt ttggacatgt ggaaccttgg gaaaagatca gctttaatat cattttcttt cacaaaagaa tgaatttatc caacacacat aaacactaga tctattattt tcatcggtaa ttttataatt tggtgattqt aacctttgag agatctttct cttcttctca cacaagaaac ctcttttcca aaaaattatt ttatttttaa tctatttata ttaatcttaa tatgttggga gtttcaaaga acctatattt tgatgtatag accaaaacag ctagatgacc tncatcctct gtgtttcaat agctggtccc aaaaataaag acgttggatc aaaaaaaaac taaaactggt tgtagaccaa aaatcattat atagacgtgg ggtccttcca ctttgcgaat tgattcgacc atatcaatga cattattttt acacgacgat gggctttcat aaaatagaga actttatcca aaaaatcaaa tatccatctg taccataagt aataagaaag 120 180 240 300 360 420 480 540 600 660 attgaatcat tcgaaaagac ctgccaattc cggtcttaat ttcagnaacc agtatcatcc taagatngaa catgaagaaa atttcaaatg atctccatta PCT/AU99/008050L P:\OPERl\MRO\AUTSEED2.PCT 2119/99 -11 Igctttcttag cctcctcaac gagtcttata gaaaaccaga tcgcccaagc cagacaaaaa ttttggggct acaacactat tgacctttgg tggttacggc ggtgtatgta attttgtgaa tttttgatga cttgaatgtt gtatatatgt ccattgaaca aaaaacttgg gttttaaatg gaagatttaa tagctaggaa aaatgtatat aaatatgagt aaacgtcaag ttcttaaatt agattataat accctttgca ggttaatgga catatatttt atctattatg ggaaaaccat cctcctcctc tccggtcacc cacgatcttg atgctctgtt ttgtgccttt cttgtttctt acacgaagtg atatggggtc tataatgtgg ggatagactt ttacatctgt tacgttgact tatgtgtaat tttgagattt cacaaaaata ttaatttaag agatttaatt ccgtataatt actcatttgg tgaatctaat attgatctat taacgccttt caaacccaac tcccatatta tttccttaat ttatcctttt gaaggttagt aattgtgtgt ttattcgtcc gaggacgatg cctctatctt gtctccggcg tttctcatca ctccattccc gggatcaaaa gcgacacatg atgctgatgn tacgtggagg tggtggaggc aacctcccca gttggaattg ttgagcatta gatgatcatg ttttattttt gtagaatatc tctttttata aacgttataa aaatttacga ttcttccata atatataatg caacattttt ttttttataa atctacatac ttttcctttt aaggttttct caccaacatc ttcactccaa ttatgataga catgcatgat gtgagggttt tcctctttct tttcctctgt tcgtcttcac atctctacat gaaccatgag aaagatggat ggtttagagt agaggtggtc taagagatga ttcgtttgga gtttaatgaa agaggtcgag tgtaagatat attttctatt agaaagcaaa attatatttt atagtaaaaa attgaataat tatgcataat ccattaatat tcattggttt tattgtatat atatagtact aaagcaagca acaaaaaaaa agagaagcga acatatatga tcaataacat ccataaaact ttcacgtctc tattcaccag tttaatccac ctgctggatc cacgaccatg gttggttagg cgtggttaag cgagagagcc ggtagaaagt gggtctcaat gatcttcaaa aaaaggctgt ttgacatatt tcttgctagg aatattttat tataacacac atatccggat atagccatat aagtcttacc actaaaccaa gaatttaaac atattttgaa agtccaaata atcaacttct gaaaaaaaga attgactagg ttagggtgaa tttatttttg tctctctata gcagatgtcg agtctctcag gttctcttca tctataaatc gttttgntct cctgtggttg gttgtggcag cttgtggtgg tttgtgaact gggtttttgg gaatcatgtg atactcatct aatttaaccc ctaaaaataa cctttttaag ttacgtagaa atatattttt aaatagaaac ggacactcca gttgtcacat ttgagctgtc aattaaaatt cgtttctt~c tatttaaaaa agaggcgagt ttatgaaatc ttttcttgtg aatttgtcta taaaagagca 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 gccacccgaa ctaaatcaga PCT/AU99/00805t P:\OPER\MRO\AUTSEED2.PCT 21/9/99 12 aatcgaaaag tattctcttc acgattccaa aaacctaaaa cgaatcgata aatatattta gatttttttg actaaatagg atatatcaag agagtccagt aaattcgagc gacttaaacc agaatgcaaa caaggttatg cttgatgaag cgatccatta aaaatattaa attgcagtag atttgaatgg atgaggaaga ggttagtttt tgctcaagaa gcgtgctctc ataaaaactt tcgtgagaaa taagaatgat tttccaggat ttttttaatt gagagatttc tgaagttttt agagatgtta agagaatttt acgacatcca taatataaag tttttgtcaa tccaaattgt aaatcgaaat ttctgaaatt tgagatacat agcccgctgc acccacttcg ttcttgatga atgtaccatt catgggtctt tcatatacta ccagctgatg tgaggcacta aatcaagaaa tgcattacat attttcagga gccaagtacc catacgtcga tgttttaaat gqaactgctg tttgctgata tagccacaaa tgcatatcaa ctgaaaatta agatcatcta ccaaactttt gatctgtcgg ctggaactta acaaaatagt ttttttttta caaaactttt taaaaaatca tccaagtgtg agaggatgat tcatttcagt ggatcaagat attaccaagt caccaaaagg tatagtaatc gctgaaagtg gaattgagca gaaaaatgcg atgttcttga cggttgggca tcgaagtgga tcaataactt tttcaaatct ggtaagagac aggagaggag ataaacagtt aaattaaaac gtatccaaaa ggttataaaa aatacaattt acattcagcc gtattcaagt tttacctata gctactcatg aatggaggag gcctcatctg tatgctcttg gtcaagcttc catgtgtgtt actcatagtg attctgtgat gtgaagaaga aattttctga ttattaattt ggactatggt tgtttcggac tcctgcttat aatgtaggaa atttgggtgc aggacttatc atncattagt cagaatttag cttagaataa taaaattgaa gtt t tttt ta aaaaaagcca attctagttt tattacttga cttcacacca acaacaaatc attataattc aagaagatgt caattgttga ttttgtttcg catatataca tggtaagaga tgaggaagat agatgtagac qtagtccata ttggatgatc atattggtaa ttaatttttg agatacaatg tccaagacaa atggtaactt gtttttattt tttaatattt 2520 tcataactat 2580 cataatcctt 2640 aaaatgccag 2700 aaaaataatt 2760 aataatagta 2820 gtacaaagaa 2880 agattgatgc 2940 cactatatat 3000 ttaacagaga 3060 tcaatcgttt 3120 acttttgtcg 3180 ttacgaagat 3240 accattattt 3300 gaagctacca 3360 tactagtttc 3420 tttctttaac 3480 caaatctatt 3540 gaagaagaag 3600 cgatttatat 3660 tttaataaac 3720 tggtcgtgcg 3780 caatattcga 3840 ttgtttttcg 3900 aactcaagct 3960 taactactgc 4020 tgaatctttc 4080 tattttatca 4140 gtgaggcttc tgatttgaca gacgtcattg aaagggagat ccgtcgttgc gatcatacat tttgttttac agatattcga ttgtcatatg catgagaagt atgagcccga gtctagatcc gtaagcatta aattcattta aattattttg ttagtttcac aacccttata tataaggtta 4200 4260 PCT/AU99/00805a P:\OPER\MRO\AUTSEED2.PCT 21/9/99 13 agtgattaac ttaattagat gaggatgaag atagacaacc cctctctctc tcaatttttt attaaatagg tgaggagtgt tcaaacaaga ttgtggtctc ctttacttga aaggaattga aatgcattaa tatatatact gttgcattaa acatacttcg gaacaagatc aatgtactat gtacactaac ctatgtcgta cctatatgtg ttgatggttt gtcggtccgc aaaaaatcga aacaactagt ggagaagcta atgtggacag caatgccctt tgtcattcaa tttttcctaa attttttgtt caggtgctca ttggccaatg cacaaatcga atctttgtcg gagttgtcct tctataatca aagtaattca gagatggcac tcttggtgag ttcaaaccaa taaaaaggta acgaaagaca tttaactatc gttcatggat ggggtgcatt atagttattg gggcattnaa cattaaatat attgtggata ctcggagaat atactggaga gaagatcgga ttggttcttc ttttgaagta acggttttaa ttattttaca gctcgaaatc actcagcaag acctaactgc tgctttggct atgcagtgag tgtctattcc gacagaagct agatccaaac gatatttggg tacactgtat ggggcttaag gtcattagac attattctca tatcacaggt gactccgaaa agttttataa gtttaactca gccggaagat aaggattgca caatgtcctt cttaggtaac aaccaaaagt acaccagtgc atcaacgtca atttcccgta tacatgggta aacccttttt tggtttgacc actgatcact ctacctcttt tgtcagagcg cattgttac ttaattacgt gatcatgtga aacactatgt agaaacaggt tccttgatta acgtgcctag cttaacaaaa tgacatgtat taccaaaaaa atatgctcgt gcactacaca cgaaaattgc ccatgagatt acaatcgctt gttttgctgc actttcactt cttataaaaa aaatccaatg aatccgtacc ttttactaga agcaatcatg tttttttaaa cgtcaggact catgatgaag aagacaaatc tcaaggtctc ttattagtta tggataatga ggacgcctgt aaaaaaataa tgctggttcg agatttacaa ctacacaaag gttaaaaaca gtatctcgaa tatccgcctg ccatgcactt tgcgagaaat taatttgaac tggaggatgt taatcgtgaa caatatctct aaactttata caagaacatg gaaaatttaa ttctcattgg taaatataag aaaggtttaa ctcttaaaaa ctaatgagcg tagtttgttt tatctctctc ctggtttaat taactctata agagaaggat aaatagattt cagttgtgat ttacatgcgc acacaatcag catgaagttt aaagtagtag ctttaaagaa gcaagtcaaa attgcgggta atgagtttgt aattgtgcaa tgcgatccag ttatacaaat tatagctgtg caattcctcc aactaattat aaagtctgat aataagttta aactttaglc gaatgagtat tgggagaata 4320 4380 4440 4500 4560 4620 4680 4740 4800 4860 4920 4980 5040 5100 5160 5220 5280 5340 5400 5460 5520 5580 5640 5700 5760 5820 5880 accttgaatg atcaggtaac ttcagaataa tcattcqcgg gttacacatc tattcgaatc aaagtaacat 5940 gatgctcgcc gtaaaggaaa cgagttcaaa tttctcaatc 6000 tacgccaagg tactaagccg ttatacttta tcttgaacaa 6060 PCT/AU99/008051j P:\OPER\MROAUTSEED2.PCT 21t9/99 -14atactaacat actttactcg ttaaatgatt gttgatgatt aggtgaggag tcgagaacot gtttttgatc aattaataat tatacaaaca tcgttgattg ttttcttgtc gtgagaggag cttttcttcg agaaagactg tgaggagaag gtaatgctat aaaatactta gttttcatat gagaaaattt atoagaggat actactgcta gtgcttctaa cagcaattca tttgtgttac tgttagtttc tgaagatatt aggttacgaa tggtctattt tggaccagaa aaggtctaag agcagtcctt taaaccaaaa tttagttaaa ccaagaaact aatttatggt goggagagag catgcggatt gaagcccgtc tttttatgtt cttaagtttc tcgtgtatca caaactoatt ttcgtgtgca caatcgaaga ggtogcgtgg cagctcgtta atggtatatc tgtt 6120 6180 6240 6300 6360 6420 6480 6534 <210> 6 <211> 2640 <212> DNA <213> Arabidopsis thaliana <220> <221> CDS <222> (1)..(2439) <400> 6 atg got agg aag t Met Ala Arg Lys S 1 gat gat gat gat g Asp Asp Asp Asp A ata ogg ggg aag gaa gtg gta atg gtt tot gat Ile Arg Gly Lys Giu Val Val Met Val Ser Asp gat gat gtt Asp Asp Val gat aaa aat Asp Lys Asn atc ato aaa Ile Ile Lys oca acg gat Pro Thr Asp tgt gto aaa Cys Val Lys tct gat gat Ser Asp Asp cot ctt aca gta tao aag aat ott gaa Pro Leu Thr Val Tyr Lys Asn Leu Giu aat gat gat Asn Asp Asp gat gat gat gtt Asp Asp Asp Val gtt gat gaa aao Val Asp Giu Asn ato aaa tat ato Ile Lys Tyr Ile oot gtt goa gta Pro Val Ala Val aag aaa ott gaa Lys Lys Leu Glu ogo tca aaa aao Arg Ser Lys Asn atc caa gca Ile Gin Ala tto aao tao Phe Asn Tyr 115 aag aaa Lys Lys 100 agg gat Arg Asp oca tat tto ota Pro Tyr Phe Leu aaa aag aag toa Lys Lys Lys Ser 105 gtg agt aao aaa Val Ser Asn Lys 120 agg tot ttg aag Arg Ser Leu Lys tac ata Tyr Ile aat toa ggt ggg Asn Ser Gly Gly atg aoa ota aaa Met Thr Leu Lys 125 ttg att coa tgt aaa ata aga Lys Ile Arg 110 got gaa gta Ala Giu Val gga ggt cac gtg gaa aat ttt tot tgo oca ttt tgo PCT/AU99/00805y P:\OPER\MRO\AUTSEED2.PCT- 21/9/99 Val Glu Asn Phe Ser Cys Pro Phe Cys Leu Ile Pro Cys Gly Gly His 130 135 140 gag ggc ttg caa ctt cat ttg aag tca tca cat gac gcc ttt aaa ttt 480 Glu Gly Leu Gin Leu His Leu Lys Ser Ser His Asp Ala Phe Lys Phe 145 150 155 160 gag ttt tat cgg gca gag aaa gat cac gga ccg gaa gtt gat gtc tcc 528 Glu Phe Tyr Arg Ala Glu Lys Asp His Gly Pro Glu Val Asp Val Ser 165 170 175 gtg aaa agt gat aca ata aaa ttt ggg gtt cta aag gat gat gta gga 576 Val Lys Ser Asp Thr Ile Lys Phe Gly Val Leu Lys Asp Asp Val Gly 180 185 190 aat ccc caa ttg agc cct ttg acg ttt tgc tcg aaa aat cgt aac caa 624 Asn Pro Gin Leu Ser Pro Leu Thr Phe Cys Ser Lys Asn Arg Asn Gin 195 200 205 aga aga caa aga gat gat agc aat aac gtt aag aaa ctt aat gta ctc 672 Arg Arg Gin Arg Asp Asp Ser Asn Asn Val Lys Lys Leu Asn Val Leu 210 215 220 ctt atg gag ttg gat tta gat gac tta cct cgt ggc aca gaa aat gat 720 Leu Met Glu Leu Asp Leu Asp Asp Leu Pro Arg Gly Thr Glu Asn Asp 225 230 235 240 tct act cat gtg aat gat gat aat gtc tca tcg cca cca aga gct cac 768 Ser Thr His Val Asn Asp Asp Asn Val Ser Ser Pro Pro Arg Ala His 245 250 255 tct tcc gag aag att agc gac att tta acc acg act caa cta gca ata 816 Ser Ser Glu Lys Ile Ser Asp Ile Leu Thr Thr Thr Gin Leu Ala le 260 265 270 get gaa tec tct gaa cct aag gtg cct cat gtg aat gat ggt aat gtc 864 Ala Glu Ser Ser Glu Pro Lys Val Pro His Val Asn Asp Gly Asn Val 275 280 285 tca tcg cca cca aga gct cac tct tcg gcc gag aag aat gaa tct act 912 Ser Ser Pro Pro Arg Ala His Ser Ser Ala Glu Lys Asn Glu Ser Thr 290 295 300 cat gtg aat gat gat gat gat gtc tca tca cca cct aga get cac tct 960 His Val Asn Asp Asp Asp Asp Val Ser Ser Pro Pro Arg Ala His Ser 305 310 315 320 ttg gag aag aat gaa tct act cat gtg aat gag gat aat att tea tcg 1008 Leu Glu Lys Asn Glu Ser Thr His Val Asn Glu Asp Asn Ile Ser Ser 325 330 335 cca cca aaa get cac tct tcg aag aag aat gaa tcg act cat atg aat 1056 Pro Pro Lys Ala His Ser Ser Lys Lys Asn Glu Ser Thr His Met Asn 340 345 350 gat gaa gat gtc tca ttt cca cca aga act cgc tct tcg aag gag acg 1104 Asp Glu Asp Val Ser Phe Pro Pro Arg Thr Arg Ser Ser Lys Glu Thr 355 360 365 age gac att tta acc aca act caa cca gca ata gtt gaa cct tct gaa 1152 PCT/AU99/00805ek P:\OPER\MRO\AUTSEED2.PCT 21/9199 16- Ser Asp 370 cct aag Pro Lys 385 tac aag Tyr Lys aag gtg Lys Val cac tct His Ser ata gct Ile Ala 450 gta tca Val Ser 465 cgt aag Arg Lys aag aag Lys Lys tct tct Ser Ser aca cca Thr Pro 530 gat gat Asp Asp 545 agc aac Ser Asn aag gtg Lys Val cac tct His Ser Ile Leu gtg cgt Val Arg gct aga Ala Arg ctg cat Leu His 420 ttg gag Leu Glu 435 gag tc Glu Ser tcg aca Ser Thr aat gtt Asn Val act agc Thr Ser 500 gaa cct Glu Pro 515 aga gct Arg Ala aat att Asn Ile att tta Ile Leu cct cat Pro His 580 tca aag Ser Lys Thr cgt Arg gag Giu 405 gtg Val aag Lys tct Ser cca Pro gat Asp 485 gac Asp aag Lys cac His cca Pro act Th r 565 gtg Val1 aag Lys Thr ggt Gly 390 act Thr aat Asn gct Ala gaa Giu aga Arg 470 aat Asn ata Ile gtg Val1 tct Se r tcg Se r 550 agg Arg aat Asn aat Asn Thr 375 agt Ser caa Gin gat Asp agc Ser cct Pro 455 gct Ala gtc Val tta Leu cgt Arg tca Ser 535 cca Pro act Thr gat Asp aaa Lys Gin aga Arg cca Pro gaa Giu gac Asp 440 aag Lys cac His ca Pro act Thr cat His 520 aag Lys c ca Pro caa Gin gat Asp tct Se r Pro Ala aga aaa Arg Lys gca ata Ala Ile 410 aat gtc Asn Val 425 att tta Ile Leu gtg cct Val Pro tct tca Ser Ser tcg rca Ser Pro 490 acg act Thr Thr 505 gtg aat Val Asn aag aat Lys Asn aaa act Lys Thr rca gca Pro Ala 570 aaa gtc Lys Val 585 act cat Thr His Ile Val 41lu Pro Se~qr Giu cag Gin 395 gct Ala tca Ser acc Thr cat His aag Lys 475 cca Pro caa Gin gat Asp aaa Lys cgc Arg 555 ata Ile tca Ser aag Lys tta Leu gag Giu tcg Ser acg Thr gtg Val1 460 aag Lys aaa Lys rca Pro gat Asp tct Ser 540 tct Se r gct Ala t cg Ser aaa Lys tat Tyr tct Ser ca Pro act Thr 445 aat Asn aat Asn act Thr aca Thr aat Asn 525 act Thr tcg Ser gag Giu aca Thr gat Asp 605 gca Ala tct Ser cca Pro 430 caa Gin gat Asp aaa Lys cgc Arg ata Ile 510 gtc Val1 cgt Arg aag Lys tct Ser rca Pro 590 gat Asp aag Lys gaa Glu 415 gaa Glu cca Pro gaa Glu tct Ser tct Ser 495 gct Ala tca Ser aag Lys aag Lys gaa Giu 575 aga Arg aat Asn Cgg Arg 400 rca Pro gct Ala gca Ala aat Asn act Th r 480 tcg Ser gag Giu tcg Se r aat Asn act Thr 560 cct Pro gct Ala gcc Ala 1200 1248 1296 1344 1392 1440 1488 1536 1584 1632 1680 1728 1776 1824 595 600 tca ttg cca rca aaa act cgc tct tcg aag aag act agc gac att tta 17 1872 PCT/AU99/008054.

P:\OPER\MRO\AUTSEED2.PCT 21/9/99 17- Lys I cctI Pro gca Ala 650 ttc I Phe tct Ser I agc Ser aag Lys I atc Ile 1 730 cat HisI atg I MetI ttc PheI caa Gin gaa Thr 620 gaa Glu cgg Arg cac His gag Glu cgc Arg 700 tac Tyr gat Asp gaa Giu agg Arg aaa Lys 780 acc Thr gat Asp Asp Ile Leu aag gtg act Lys Val Thr 640 gag gct aaa Glu Ala Lys 655 caa aca atg Gin Thr Met 670 agc gag aat Ser Giu Asn aga ctt gaa Arg Leu Glu tat ctt tgg Tyr Leu Trp 720 cat gtt cct His Val Pro 735 atg aag aat Met Lys Asn 750 aaa ctg tgg Lys Leu Trp act acc atc Thr Thr Ile ggc agt gct Giy Ser Ala 800 taacagtggt 1920 1968 2016 2064 2112 2160 2208 2256 2304 2352 2400 2449 aat gcc aac Ala Asn Ala Asn Asn Gin Gin Ser Met Glu Val 805 810 tagtcgccat ggagatctcg agatcttttt cttagtagta gggatcaaca aggctgagat 2509 ctctatctcg tttattacat ttctttctat ttactgtgtc gtaaccttta agtttaccct 2569 cttactagtt tgtgaatctg tgacattcag atcaataagg ttaagcttga aatttaaaat acttgagaag g <210> 7 2629 2640 PCT/AU99/00805a- P:\OPER\MRO\AUTSEED2.PCT 2119/99 18 <211> 6458 <212> DNA <213> Arabidopsis thaliana <400> 7 aagcttgacc taatcaaagt ctgtcttcca ccatgtcaat ctactgtgaa gagattctcg tcgaaaacat cgttctcggc acctaggcaa atggtgatgg atcgacgcat atatagaatg gcgcccttcc agaaattgga agaaaaacaa gatcttggaa ccttctttag tgtttgagag tgtcttggat ctttagtatt cttaaactta caataaaacg caacaataat aatatgttat gtttctagct gcaacataaa tgcgaaatca aqatatttta tttttctttt ttttaacaaa caaatcagac aacatgaaca ccaaagtatg ggatctgaat aagaaggtgg cgaaggagaa ctttttttct aatatggttt cggcaaatta aaaaaaaaat ataaaaatgt gtaattgata aacaacacct attgttgact tagtgattct caaatttgag aaataaaata attttattgg taaagtccaa ataggtttct aataagatat ttgttcttcg taaagtaaat ttaaaatgta tctaattttc atttagtctg aatagaaata gaggatttgt actgcgcaga cgcgatttgt ctatcatccg ttcgctgaga tctttctact ttgaattgac gtgtcgagtg tggatttttt aaaattaaac acattttcaa ccaagtgacg ctcctcattg tttgcattct atatccttct aatttcgagt acttaaaatg tcgcatattt cggaaactca aatctctctg tttgatcatt cgaaatctct ggaatttcaa tattttcttt tttatcaaaa caagtcacaa ctccgaggaa gaatgagtac tcttcttccc aatctgcgga gttggtcgga tttacgcgct tgttttaccc tcctaagttc tttatcaatt ctttgtgtag tgactggatt tttatcaaac tgctatttgg tatcttatgt tcttttttaa tgattaccaa ggccatatta atttagggtt gaaatacgcc atactagttt cctttgcgaa atagattaaa caatatatat tcttataatc ttttaatata ttgtcactgt ccaaaacctt ttgaatttgg gatcggaatg gggatgagag ggttttaatt aactatggag tcacaaggag ttttgaaagt tcataccaag ttaaattttt aatgagtgag agtttcacta ctacagaaat cattttgcct agacttattc tctgatttta acccgaagaa acttttaaaa tcgggtattt gcaccaacac tgtgttaaag atcaaaaacg tatgcatgct cttgttgttt tctactgcca gatgattcaa ttgatctttg aaaaattaca ttacctggat aaatggatag ttgggatcgt cgcggcggag gcgctcgccg aaacttctat caaacttcac aacactcgga aattggataa actgttttga tgttacatta ctcaagtatg cttttagaat tttggtttta ttaaatcatt gaaaaattta ttacgaaatt catctctata caaaaatgaa tggtgaaggc atcattccgc tagattcagt ccatgatttc tcaaaaattt tattttcatt 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 tttaagagca ttttgtctgc aaattttaaa tgatatacat aatagatttt tattaatctc 1680 PCT/AU99/00805& P:\OPER\MRO\AUTSEED2.PCTr 21/9/99 -19aaaatttggt tcaatctcaa atgtaaatat ttcttatttg gaagttatgt cactattctt ctagtttata gatggttaga gatatttctg agcttataga ggtttctgat tgtcaaacct tgatgatgat agtatacaag tttggtaata cagaaaccat tggttccgtt tagactaatt tagttttttc aagcaaagaa tatgctttca ttatgttttg aaacttaaaa atattacttt cttatatttt gtaacaaaat aaattctctc aaatgcaqta ggtaggcact gtattcagaa tgtgaagttg aatataagac attcagcttt tagtcttgta gtagagattc tactctttat tatcgcaatt cgatcattcg ctaattaaaa ttaatcaagc gatgatgatg cttacagtat gatgatgttg aaacttgaaa tgtgcattgt tttagtatat tttatttttt ggaggaaatg tttgtttatt aaaaaagaag gaagtgatat ttttctaaaa ttcactagta tctcttttgc gcttaggtca gacactaaaa gttgtgattt gtggaaaatt aattaattag gtttcatgct tatctacttc tctttcttac tatatattct gataacgttc tattatgttt aatgggtcaa gggcagctaa gagtttcgcc ttgcgtggat agatggctag atgatgatga acaagaatct atgttgatga ctcgctcaaa gtttaatttc tgacatagca ttttggtatg tgttgttgta cagccatatt tatgaatctc catttcaaag tcaccaccaa ttttttggaa taatacatga aattcaggtg gctgaaggtg cttctgacag tttcttgccc aatcaagctt catttagtct tgtgatgaag ttgtgtcttt tcaacaaaaa tcttctattt catattggga aggtctgccg cattaaatgg aatgatctat cacagtctaa gaagtccata tgttgatgat tgaaactcca aaacatcatc aaacaacgta ttcatcaaac attgttcttc aaaactggtt tttgtatcta tcctacgaag ttctatatca aaaaattctc aattaagcca ctaaatttgt aatgacgact ggaaaataag agcctttaat tttatcatct attttgcttg tctaataata atctttgcta cacacctaat tcgtagtcat aaaaataaca tttcttttaa aacatgcaca ttacagtttt ctatttcatg acatgtttca ttcaatgttt cgggggaagg gataaaaata acggattctg aaatatatca tgcattctct actttttatt attttcatgt cggttttact tagttttttt gtctttgaag cttttgttta aaaattaaat tatgtgaaat tttgtggtat tcttcgcttc attcaactac tggttgtttc acatatacat attccatgtg tctttcattt aaatacaatg caaagaagag 1740 atttttaaag 1800 ataagaagat 1860 aaaaaattat 1920 ttttccagtc 1980 accttcctta 2040 cacagtgaaa 2100 ttgatttctt 2160 atggcgtgac 2220 aagtggtaat 2280 tcatcaaatg 2340 atgataatga 2400 aacctgttgc 2460 tttttgtttg 2520 atttctagct 2580 attaattagt 2640 aattagagaa 2700 cattattaat 2760 tacataatcc 2820 tcattttttt 2880 accatatctt 2940 gacccttcct 3000 tgtaattagg 3060 tcatgccaca 3120 agggatgtga 3180 ctttcaaaaa 3240 ttctgtatcc 3300 gaggtcacga 3360 ttaacaacgt 3420 tcttatgttt 3480 PCT/AU99/00805* P:\OPER\MRO\AUTSEED2.PCTr 21t9/99 20 gtgccacagg tatgtaagta taacaagagt tgtctccgtg gcaatgttag gttctaaagg gaatgccttt aatctatgca aagaaactta aatgattcta gagaagatta aaggtgcctc gagaagaatg cactctttgg aaagctcact ccaccaagaa atagttgaac aagcggtaca ctgcatgtga agcgacattt catgtgaatg tctactcgta actagcgaca cgtcatgtga aaatctactc aagactagca cctcatgtga aaatctactc aagactagcg gtgactcgtg gcttgcaact aaatttttta actatttatc aaaagtgata tcataatgtt atgatgtagg ctctagttgc gctcgaaaaa atgtactcct ctcatgtgaa gcgacatttt atgtgaatga aatctactca agaagaatga cttcgaagaa ctcgctcttc cttctgaacc aggctagaga atgatgaaaa taaccacgac atgaaaatgt agaatgttga tattaactac atgatgataa gtaagaatga acattttaac atgatgataa ataagaaaga acattttagc ttagtagaag tcatttgaag gtgatctaat tattttagcg caataaaatt gaactcacca aaatccccaa taagatatgt tcgtaaccaa tatggagttg tgatgataat aaccacgact tggtaatgtc tgtgaatgat atctactcat gaatgaatcg gaaggagacg taaggtgcgt gactcaaccn tgtctcatcg tcaaccagca atcatcgaca taatgtccca gactcaacca tgtctcatcg tgataatatt taggactcaa agtctcatcg tgataatgcc tacgactcaa aaaagagtta tcatcacatg tttgtttatg ggcagagaaa tggggttagt tgatgttatt ttgagccctt ttcagcatca agaagacaaa gatttagatg gtctcatcgc caactagcaa acgcctttaa tttttgcatg gatcacggac agtaaactcg ttttttaatt tgacgttttg tcttctaaaa gagatgatag acttacctcg caccaagagc tagctgaatc tcatcgccac caagagctca gatgatgatg tctcatcacc gtgaatgagg ataatatttc actcatatga atgatgaaga agcgacattt taaccacaac cgtggtagta -gaagaaaaca gcaatagctg agtcttctga ccaccagaag ctcactcttt atagctgagt cctctgaacc ccaagagctc actcttcaaa tcgccaccaa aaactcgctc acaatagctg agtcttctga acaccaaa ctcactcttc atttgagttt aaatagtatg cggaagttga atacataaat tatttttcag gtaaaatttc gccaaaccat caataacgtt tggcacagaa tcactcttcc ctctgaacct ctcttcggcc acctagagct atcgccacca tgtctcattt tcaaccagca gttatatgca accaaaggtg ggagaaggct taaggtgcct gaagaataaa ttcgaagaag acctaaggtg aaagaagaat ctcttcgaag acctaaggtg aaagaagaat ctcttcgaag tgaacctaag taaaaggtta 3540 3600 3660 3720 3780 3840 3900 3960 4020 4080 4140 4200 4260 4320 4380 4440 4500 4560 4620 4680 4740 4800 4860 4920 4980 5040 5100 5160 5220 5280 ccatcgccac ccagcaatag acaccaagag tcattgccac ccagcaaaag caaaaactcg ctgagtctga ctcactcttc caaaaactcg ctgagccttc catgcagagc ggtgcgaggc PCT/AU99/00805# P:%OPER\MRO\AUTSE2.PCT 21/9/99 -21ttttcttttg tacagattgg taattttctt ccaaaagctg agaatgagac cttgcttttt tattgttttt gaaaagcgat ttctatttta t tggacgggt gatggacatg aattcttcat tactcatcat tgatgcttgc cggtctcatc tgaagcagga agttgatgaa gggatcaaca gtaaccttta ttaagcttga <210> 8 <211> 1372 <212> DNA atttatttgc agcgtcttaa catgtctttg cacggtgaag tgatgattat tcttgatttn tttgaaaata acatgtatct cttgcataca tttagctagc ttccttgggC ctttcgattg gtgatacgaa gtgctcgtga tgcgccaaga caattcacct taacagtggt aggctgagat agtttaccct aatttaaaat tcaaagttat gggtcgacag atttatgtaa ccaatgactt gctttagata tattgaattg ttcacagaga ttggaacata tgaattagaa tttgttaaaa atgtgaagag gtaatagtct acttgttgga gcaggtggtg ccttccacaa ctggcagtgc tagtcgccat ctctatctcg cttactagtt acttgagaag acataatcac tactaagatt attacttgtc ttctatcact caatgttttg ttgaacaagt ttagcgaacg ttacgaaaaa cttgaacgtc tttgtacgaa caatatgatc atgtggttct tttgcaaaac ttcatagaca gggataatca gagaatgttt atgcactacc tgctaatgcc ggagatctcg tttattacat tgtgaatctg gagagact cccaaacaat tatctatttt aatgtctaac cctggtaatt tcatactcac ttgtgggtgt aacaaaggta aaagtcaagt ttgggggcag ttcataagga tcaaactaat ctttatattt aggattaaac atcctcctca aacaatcaac agatcttttt ttctttctat gcaggtggtt atttcactaa gaggatagcg ttcttttctt tgaggatttt gagcaaagag gctttttact tgccaaattg ggtgatcgcg agagatgaag atctaactca gactttgcct tgtggaacaa gtaactcgga aatctatgga cttagtagta ttactgtgtc 5340 5400 5460 5520 5580 5640 5700 5760 5820 5880 5940 6000 6060 6120 6180 6240 6300 6360 6420 6458 tgacattcag atcaataagg <213> Arabidopsis thaliana <220> <221> CDS <222> (1138) <400> 8 gtcagacaga gagagagatt tcgaatatcg a atg tcg aag ata acc tta ggg Met Ser Lys Ile Thr Leu Gly 1 aac gag tca ata gtt ggg tct ttg act oca tcg aat aag aaa tcg tac Asn Glu Ser Ile Val Gly Ser Leu Thr Pro Ser Asn Lys Lys Ser Tyr 15 aaa gtg acg aat agg att cag gaa qgg aag aaa cct ttg tat gct gtt PCT/AU99/00805& P:\OPER\MRO\AUTSEED2.PC 21/9/99 -22- Lys Val Thr Asn Arg Ile Gin Giu Gly Lys Lys Pro Leu Tyr Ala Val 30 gtt ttc aac ttc ctt gat gct cgt ttc ttc gat gtc ttc gtt acc gct 196 Val Phe Asn Phe Leu Asp Ala Arg Phe Phe Asp Val Phe Val Thr Ala 45 50 ggt gga aat cgg att act ctg tac aat tgt ctc gga gat ggt gcc ata 244 Gly Giy Asn Arg Ile Thr Leu Tyr Asn Cys Leu Gly Asp Gly Ala Ile 65 tca gca ttg caa tcc tat gct gat gaa gat aag gaa gag tcg ttt tac 292 Ser Ala Leu Gin Ser Tyr Ala Asp Glu Asp Lys Glu Giu Ser Phe Tyr 80 acg gta agt tgg gcg tgt ggc gtt aat ggg aac cca tat gtt gcg gct 340 Thr Val Ser Trp Ala Cys Gly Val Asn Gly Asn Pro Tyr Val Ala Ala 95 100 gga gga gta aaa ggt ata atc cga gtc att gac gtc aac agt gaa acg 388 Gly Gly Val Lys Gly Ile Ile Arg Val Ile Asp Val Asn Ser Giu Thr 105 110 115 att cat aag agt ctt gtg ggt cat gga gat tca gtg aac gaa atc agg 436 Ile His Lys Ser Leu Val Gly His Gly Asp Ser Val Asn Giu Ile Arg 120 125 130 135 aca caa cct tta aaa cct caa ctt gtg att act gct agc aag gat gaa 484 Thr Gin Pro Leu Lys Pro Gin Leu Val Ile Thr Ala Ser Lys Asp Glu 140 145 150 tct gtt cgt ttg tgg aat gtt gaa act ggg ata tgt att ttg ata ttt 532 Ser Val Arg Leu Trp Asn Val Giu Thr Gly Ile Cys Ile Leu Ile Phe 155 160 165 gct gga gct gga ggt cat cgc tat gaa gtt cta agt gtg gat ttt cat 580 Ala Gly Ala Gly Gly His Arg Tyr Glu Val Leu Ser Val Asp Phe His 170 175 180 ccg tct gat att tac cgc ttt gct agt tgt ggt atg gac acc act att 628 Pro Ser Asp Ile Tyr Arg Phe Ala Ser Cys Gly Met Asp Thr Thr Ile 185 190 195 aaa ata tgg tca atg aaa gag ttt tgg acg tac gtc gag aag tca ttc 676 Lys Ile Trp Ser Met Lys Giu Phe Trp Thr Tyr Val Giu Lys Ser Phe 200 205 210 215 aca tgg act gat gat cca tca aaa ttc ccc aca aaa ttt gtc caa ttc 724 Thr Trp Thr Asp Asp Pro Ser Lys Phe Pro Thr Lys Phe Val Gin Phe 220 225 230 cct gta ttt aca gct tcc att cat aca aat tat gta gat tgt aac cgt 772 Pro Val Phe Thr Ala Ser Ile His Thr Asn Tyr Val Asp Cys Asn Arg 235 240 245 tgg ttt ggt gat ttt atc ctc tca aag agt gtg gac aac gag atc ctg 820 Trp Phe Gly Asp Phe Ile Leu Ser Lys Ser Val Asp Asn Glu Ile Leu 250 255 260 ttg tgg gaa cca caa ctg aaa gag aat tct cct ggc gag gga gct tca 868 PCT/AU99/00805et P:\OPER\MRO\AtJTSEED2.PCT 21/9/99 23 Leu Trp 265 gat gtt Asp Val Giu Pro Gin Leu Glu Asn Ser Pro Giu Gly Ala Ser cta tta aga Leu Leu Arg gtt cca atg Val Pro Met att tgg ttt Ile Trp Phe ttt tct tgt Phe Ser Cys cat tta agt His Leu Ser gcg ata ggt Ala Ile Gly aat cag Asn Gin 310 gaa gga aag Giu Gly Lys att aca aag Ile Thr Lys 330 goc atg tct Ala Met Ser gto tgg gat Val Trp Asp agt tgc oct Ser Cys Pro cct gtt ttg Pro Val Leu 325 agg caa aca Arg Gin Thr 916 964 1012 1060 1108 1158 tca cac aat Ser His Asn aag tct gta Lys Ser Vai gto gat gga Val Asp Gly 345 act ata Thr Ile 360 acg att ctt got tgo tgo gag gao ggg Thr Ile Leu Ala Cys Cys Giu Asp Gly 355 att aoo aag tagoggtotg agtcttgtag Ile Thr Lys tgg ogo tgg Trp Arg Trp gaattgatga attaggagtg ogaagaaatg agatatocat oatgttgota ctcootgaga oottgagatg otctttgtag gtaooaoagt gtatacott tctggagatt ttgtottatt gotgtatoot ggagotttat tgcaaaaaaa aaaa tcttttattg taattotgat ccttgttaao gtcoacott ctcttagttc aataoaoaag 1218 1278 1338 1372 <210> 9 <211> 4643 <212> DNA <213> Arabidopsis thaliana <400> 9 ttotaatttt ottttttgat aatgtgaott aaaooottta aaaatooatt aaataoattt tatoatggta aataaoaaca gtgagaggat aaataggogg aoatoaaacc taottagoao totattagtt gttgcatcta tgttttttaa ggtgtatota aaatotattt tagttaaagt gagatgaaag taagotttaa ataaaaoaga togttootoo attttggctt aatgoaatat gtatogtatt aoooatggat aotatgatat atttggaaaa taaataagta aaaatgttaa aotttotatt ttcttatatc gcaagaaaat gcaottotat taoaagtaaa ataaaototo gtattooaaa aaatgctctc atoaatttat ttoaaattgg ggtgatottg aaaataaaaa ggtogattat tottataaaa ggaggtgtag gtattoaaat aaogaagaga ttacaacttc ttatggtttg attttgtttt cttaaggtaa agagooaagt otttcoataa tccagaagaa PCT/AU99/008054 P:\OPER\MRO\AUTSEED2.PCT 21/9/99 24 atgatccata tagctattga aaaagaatta aaaaacgtga atagtaatga tttcaatcaa tcgtatcaat aaaaacgtta tcaggtccaa acctgtgaat ttgtqagctt tcgaagataa tcgtacaaag aacttccttg gatctcgact cagattgatt gcctttagtg catttagttc atttaaagtg aactttggtt agtgagttat tttttttcta tattttgttt attcgtcgaa gttctcgggt ggtcgagcat gcaatgagta tgttgtgtgc atcttaacta gatttactag tttgcataca gtatcaaatt cataaaattg taccatttaa atggtcaata atctataata caaagaatat aatttgattt aataaaagtt ctgtgtctaa agattagtgt ccttagggaa tgacgaatag atgctcgttt ttcaattcga gtaacattaa agattaaaag aaactttctg aactcattac gatgttgtta gaagcgttta attttgaaac tcaatgtggg acatcgatcg tccatgaaaa ttgggattag gagttaactt ctaacagttt gttgtagttt ttagaaatat gtaaacttga tgagaaaaag agaaatgtat atccaaacct atataagatt ttcctttgaa atttattttt ttcttaccat tggaaaaaat gtaaaaaaat gtaattgaca tatatcattt acatattaaa aaaaaagaaa catcgtaacg ttttttctaa tgtcgtgagg tcaaaatcta tcgaaaatta caaaacacca gcgagagtca gacagagaga cgagtcaata gttgggtctt gattcaggaa gggaagaaac cttcgatgtc ttcgttaccg aatcactgtt ttcaattctg ggcctttcct tttgtgtttg attgaaactt tgcttgatgc gttttggaga ttttgtggaa cttatacact tgatttgcat gtctgcttat ctaaggaggt gtttaagctt tcctgtattg tagatagagt gaagtcatta ttccgaatgt acctgatagt gatacccgtt tgggcttagt accaatcggt aatgagtgga tgggctttga taccatgtga gtaatcttac acacttggtt ccgcactaag gttgtttggt ttttcatctt tcctagtttc ttgagtttct catagaagct atgtggtact atgtaaaatt aaataatgtt aaccattttt taattactac gacaaatagg tttaattcta aaggtgagtt cggttacagt gttgttgttg gagatttcga tgactccatc ctttgtatgc ctggtggaaa ggtctgttta attttggatt tatagtctaa gatatggttt tctgttctaa tccttttgaa gagattttgt ccttatacat ggctctttag aggctctgat gttaatttgt aagtccttgg aggtctcatt tgctcagtct cgttggattt ttaaccaagg gttggaattg 600 tgggtggaag 660 ttcaaaacat 720 agtaaaacta 780 tttcagaaaa 840 taaacttcga 900 agtcctatta 960 gtgtgttgtg 1020 aattttaata 1080 catgagagac 1140 atatcgaatg 1200 gaataagaaa 1260 tgttgttttc 1320 tcggqtaaaa 1380 ggttttgatt 1440 ctgatttcta 1500 gattatgtaa 1560 ttgttttcta 1620 aaaaaattga 1680 acggtcatca 1740 ggaagttatt 1800 tagactgctc 1860 gctcatttgt 1920 accgcgtaaa 1980 aatcgtcttc 2040 ggtccaatcg 2100 ctctttataa 2160 caatatactt 2220 taaattgaat 2280 ggttctttca 2340 PCT/AU99/00805a P:\OPERkMRO\AUTSEED2.PCr 21/9/99 tttaaccttt tgtagattac ctgatgaaga tttcactgag cgttttacac gagtaaaagg tgcattttta ctgtgctcgc cacaaccttt tttcttccta ttaattgaat gatatttgct ttttatctaa tcttattttg gtatggacac aaacttccat cttattcttt gatgatccat ccttgtcttg acttctatat ggtttggtga ttggtgtact aacgagatcc tcattgttgc tttttgtggt tatttggttt gagagctcgt attttgtgac ctgggatttg acttagctag tctgtacaat tgtaaggaag gccatttggt ggtaagttgg tataatccga tggatgttct aaatgtgcag aaaacctcaa aagtatcctg aaggatgaat ggagctggag ttcagttagt aaaattgtag cactattaaa tttaaaaaac ggctgtctat caaaattccc taacaacaag gtaggtattt ttttatcctc gttaaaacac tgttgtggga tccaaacaca ttgcagggag atcaagtttt tagatacaaa aaattactcg aaaagttgcc ttcatgaatc tgtctcggag catacatatt tatattttgt gcgtgtggcg gtcattgacg atgtatccta agtcttgtgg cttgtgatta acttcttttt ctgttcgttt gtcatcgcta tttctacaat gattttcatc atatggtcaa cttttgagaa agagttttgg cacaaaattt tgacatacaa acagcttcca tcaaaggtta tttactcttg accacaactg acataatcat cttcagatgt cttgtgacct tttgcattct ctggtttgtt ctcctgtttt tcattactgc atggtgccat agcttttcca ctatgtcctc ttaatgggaa tcaacagtga gcaaatgatt gtcatggaga ctgctagcaa tatttgttgg gtggaatgtt tgaagttcta aatatataga cgtctgatat tgaaaggtac aaatggcttg acgtacgtcg gtccaattcc atattggtga ttcatacaaa gtaagtcaat tgttgttcta aaagagaatt tcatttcatc tctattaaga ccatttaagt atagatagat atcaggtaat gattacaaag cattggtgta atcagcattg tcaaattaaa tggagagcag cccatatgtt aacgattcat ctatatcttt ttcagtgaac ggtatatctc tgattaagag gaaactggga agtgtggtga gacaatgtta ttaccgcttt gatcgagcac tggttcgttt agaagtcatt ctgtaagtat tggcctttgt ttatgtagat gatggttaag tcggatttta ctcctggcga acatatattt tacccggttc tctgttgcga tacttcaact caggaaggaa taagttaqtt tctcttatta 2400 caatcctatg 2460 gtaagtgatg 2520 aaggaagagt 2580 gcggctggag 2640 aaggtattat 2700 cttgtataat 2760 gaaatcagga 2820 ttggctttct 2880 ctgttacgtt 2940 tatgtatttt 3000 gccaatattg 3060 aggggaacca 3120 gctagttgtg 3180 atattgtaat 3240 gtatgatctt 3300 cacatggact 3360 tttgttttag 3420 aaataacatt 3480 tgtaaccgtt 3540 attaattcat 3600 gagtgtggac 3660 ggttaggatc 3720 acagttgaac 3780 caatgtgtga 3840 taggtaatca 3900 tttcttattc 3960 aggtttatgt 4020 tcggattcag 4080 atacaatgtt tgatctttaa gaaatgtttt agtcttgaca tgattttctg ttgccatata 4140 PCT/AU99/008051 P:\OPER\MRO\AUTSEED2.PCT 2119/99 26 ggttatcaca ggtataaatc tcaatagtta attgtagcac ccaagtagcg tccattcttt tgtagccttg ttattctctt ctttcataag caatcaatca catcttctct ctgtaaatca gattcttgct gtctgagtct tattgtaatt ttaacgtcca agttcaatac ctttctagta aagtctgtaa ctcaccaatg aaccaaactt tgctgcgagg tgtaggaatt ctgatcatgt cccttgtacc acaaggctgt ttC tcaggcaaac cagtgaaaat tggattctga acgggactat gatgaattag tgctactccc acagtgtata atcctggagc agccatgtct ttcttaatgt cacactgttt atggcgctgg gagtgcgaag tgagaccttg ccctttctgg tttattgcag gtcgatggaa tatttatgac cttccatggg gacgtgatta aaatgagata agatgctctt agattttgtc gaaccactct 4200 4260 4320 4380 4440 4500 4560 4620 4643 <210> <211> 39 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 25 Xaa Xaa Cys Xaa Xaa Xaa Cys <210> 11 <211> <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <c400> 11 Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 25 Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys PCT/AU99/OO8O5it P:\OPER\MRO\AUTSEED2.PCT 2119/99 27 <210> 12 <211> 41 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 12 Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 25 Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys <210> 13 <211> 42 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 13 Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 25 Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys <210> 14 <211> 43 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 14 Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 25 Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys <210> <211> 44 <212> PRT PCT/AU99/OO8O5i; P:\0PER\MRO\AUTSEED2.PT 21/9/99 -28- <213> Artificial sequence <220> <223> Description of Artificial Sequence:motif <400> Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 25 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys <210> 16 <211> <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 16 Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 25 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys 40 <210> 17 <211> 46 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 17 Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 25 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys 40 <210> 18 <211> 47 <212> PRT <213> Artificial Sequence <220> PCT/AU99/OO8O5a P:\OPER\MRO\AUTSEED2.PCT 21/9/99 29 <223> Description of Artificial Sequence:motif <400> 18 Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 25 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys 40 <210> 19 <211> 48 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 19 Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 25 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys 40 <210> <211> 49 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 25 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa 40 Cys <210> 21 <211> 110 <212> PRT PCT/AU99/00805& P:\OPER\MRO\AUTSEED2.PCr 21t9/99 <213> Artificial Sequence <220> <223> Description of Artificial <400> 21 Ser Xaa Xaa Xaa Gly Xaa Gly Xaa 1 5 Xaa Glu Xaa Xaa Xaa Glu Tyr Xaa Xaa Xaa Xaa Arg Gly Xaa Xaa Xaa Phe Xaa Xaa Xaa Xaa Xaa Xaa Xaa 55 Xaa Xaa Xaa Phe Xaa Asn His Xaa 70 Xaa Xaa Xaa Val Xaa Gly Xaa Xaa Xaa Xaa Xaa Xaa Xaa Glu Glu Leu 100 <210> 22 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial <400> 22 Ser Xaa Xaa Xaa Gly Xaa Gly Xaa 1 5 Xaa Glu Xaa Xaa Xaa Glu Tyr Xaa Xaa Xaa Xaa Arg Gly Xaa Xaa Xaa 40 Phe Xaa Xaa Xaa Xaa Xaa Xaa Xaa 55 Xaa Xaa Xaa Phe Xaa Asn His Xaa 70 Xaa Xaa Xaa Xaa Val Xaa Gly Xaa Xaa Xaa Xaa Xaa Xaa Xaa Glu Glu 1100 Sequence :motif Phe Xaa Xaa Xaa Gly Glu Xaa Ile Asp Xaa Xaa Xaa Xaa Asp Xaa Xaa Xaa Xaa Pro Xaa Arg Xaa Gly Xaa Xaa Phe Asp Tyr 105 Sequence :motif Phe Xaa Xaa Xaa Gly Glu Xaa Ile 25 Asp Xaa Xaa Xaa Xaa Asp Xaa Xaa Xaa Xaa Xaa Pro Xaa Arg Xaa Gly Leu Xaa Phe Asp 105 Xaa Xaa Xaa Xaa Xaa Ser Xaa Xaa Xaa Cys Xaa Xaa Tyr Xaa 110 <210> 23 <211> PCT/AU99/008056 P:\OPER\MRO\AUTSEED2.PCr 21/9/99 -31- <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 23 Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa His Xaa Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 25 Xaa Xaa Cys Xaa Xaa Xaa Cys Xaa <210> 24 <211> 41 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 24 Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa His Xaa Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 25 Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys Xaa <210> <211> 42 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequencemotif <400> Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa His Xaa Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 25 Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys Xaa <210> 26 <211> 43 <212> PRT <213> Artificial Sequence PCT/AU99/OO8O54 P\kOPER\MRO\AUTSEED2. PCT- 21/9/99 32 <2 <223> Description of Artificial Sequence:motif <400> 26 Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa His Xaa Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 25 Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys Xaa <210> 27 <211> 44 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 27 Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa His Xaa Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 25 Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys Xaa <210> 28 <211> <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequencemotif <400> 28 Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa His Xaa Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 25 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys Xaa 40 <210> 29 <211> 46 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif PCT/AU99/OO8O5)9 P:\OPER\MRO\AUTSEED2. PCT 21/9/99 33 <400> 29 Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa His Xaa Xaa xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 25 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys Xaa 40 <210> <211> 47 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa His Xaa Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 25 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys Xaa 40 <210> 31 <211> 48 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 31 Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa His Xaa Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 25 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys Xaa 40 <210> 32 <211> 49 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif PCT/AU99/OO8O5% P:\OPER\MROAUTSEED2.PCT 2119/99 34 <400> 32 Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa His Xaa Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 25 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys 40 Xaa <210> 33 <211> <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 33 Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa His Xaa Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 25 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa 40 Cys Xaa <210> 34 <211> <212> PRT <213> Artificial Sequence <220> <223> Description of Artificiai Sequence:motif <400> 34 Cys Arg Arg Cys Xaa Xaa Xaa Asp Cys Xaa Xaa His Xaa Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 25 Xaa Xaa Cys Xaa Xaa Xaa Cys Tyr <210> <211> 41 <212> PRT <213> Artificial Sequence PCT/AU99/OO8O5it P:\OPER\MROkAUTSEED2.PCT 21/9/99 35 <220> <223> Description of Artificial Sequence:motif <400> Cys Arg Arg Cys Xaa Xaa Xaa Asp Cys Xaa Xaa His Xaa Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 25 Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys Tyr <210> 36 <211> 42 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 36 Cys Arg Arg Cys Xaa Xaa Xaa Asp Cys Xaa Xaa His Xaa Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 25 Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys Tyr <210> 37 <211> 43 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 37 Cys Arg Arg Cys Xaa Xaa Xaa Asp Cys Xaa Xaa His Xaa Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 25 Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys Tyr <210> 38 <211> 44 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif PCT/AU99/00805ia P:kOPER\MRO\AUTSEED2.PCr 21/9/99 -36- <400> 38 Cys Arg Arg Cys Xaa Xaa Xaa Asp Cys Xaa Xaa His Xaa Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 25 Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys Tyr <210> 39 <211> <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 39 Cys Arg Arg Cys Xaa Xaa Xaa Asp Cys Xaa Xaa His Xaa Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 25 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys Tyr 40 <210> <211> 46 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> Cys Arg Arg Cys Xaa Xaa Xaa Asp Cys Xaa Xaa His Xaa Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 25 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys Tyr 40 <210> 41 <211> 47 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 41 Cys Arg Arg Cys Xaa Xaa Xaa Asp Cys Xaa Xaa His Xaa Xaa Xaa Xaa 1 -5 10 PCT/AU99/00805a P:\OPER\MRO\AUTSEED2.PCr 21/9/99 37 Xaa Xaa Xaa Xaa Xaa Xaa xaa Xaa Xaa xaa Xaa xaa xaa Xaa Xaa Xaa 25 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys Tyr 40 <210> 42 <211> 48 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequencemotif <400> 42 Cys Arg Arg Cys Xaa Xaa Xaa Asp Cys Xaa Xaa His Xaa Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 25 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys Tyr 40 <210> 43 <211> 49 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 43 Cys Arg Arg Cys Xaa Xaa Xaa Asp Cys Xaa Xaa His Xaa Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 25 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys 40 Tyr <210> 44 <211> <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif PCT/AU99/00805i P:\OPERMRO\AUTSEED2.PCT 21/9/99 1> -38- <400> 44 Cys Arg Arg Cys Xaa Xaa Xaa Asp Cys Xaa Xaa His Xaa Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 25 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa 40 Cys Tyr <210> <211> <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> Cys Arg Arg Cys Xaa Xaa Phe Asp Cys Xaa Met His Xaa Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 25 Xaa Xaa Cys Xaa Xaa Xaa Cys Tyr <210> 46 <211> 41 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequencemotif <400> 46 Cys Arg Arg Cys Xaa Xaa Phe Asp Cys Xaa Met His Xaa Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 25 Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys Tyr <210> 47 <211> 42 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif PCT/AU99/00805& P:\OPER\MR0\AUTSEED2P(r 21/9/99 -39- <400> 47 Cys Arg Arg Cys Xaa Xaa Phe Asp Cys Xaa Met His Xaa Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 25 Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys Tyr <210> 48 <211> 43 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 48 Cys Arg Arg Cys Xaa Xaa Phe Asp Cys Xaa Met His Xaa Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 25 Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys Tyr <210> 49 <211> 44 <212> FRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 49 Cys Arg Arg Cys Xaa Xaa Phe Asp Cys Xaa Met His Xaa Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 25 Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys Tyr <210> <211> <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> Cys Arg Arg Cys Xaa Xaa Phe Asp Cys Xaa Met His Xaa Xaa Xaa Xaa 1 5 10 PCT/AU99/00805q- P:\OPER\MRO\AUTSEED2.PCr 2119/99 40 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 25 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys Tyr 40 <210> 51 <211> 46 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequencemotif <400> 51 Cys Arg Arg Cys Xaa Xaa Phe Asp Cys Xaa Met His Xaa Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 25 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys Tyr 40 <210> 52 <211> 47 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 52 Cys Arg Arg Cys Xaa Xaa Phe Asp Cys Xaa Met His Xaa Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 25 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys Tyr 40 <210> 53 <211> 48 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 53 Cys Arg Arg Cys Xaa Xaa Phe Asp Cys Xaa Met His Xaa Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 25 PCT/AU99/OO8O54 P:\OPERMRO\AUTSEED2.PCT 21/9/99 -41- Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys Tyr 40 <210> 54 <211> 49 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequencemotif <400> 54 Cys Arg Arg Cys Xaa Xaa Phe Asp Cys Xaa Met His Xaa Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 25 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys 40 Tyr <210> <211> <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> Cys Arg Arg Cys Xaa Xaa Phe Asp Cys Xaa Met His Xaa Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 25 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa 40 Cys Tyr PCT/AU99/OO8O5ik P:\OPER\MRO\AUTSEED2.PCr 21/9t99 42 210> 56 <211> 61 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequencemotif <400> 56 Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa 25 Cys Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa 40 Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Cys 55 <210> 57 <211> 62 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequencemotif <400> 57 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa 25 Xaa Cys Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa 40 Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Cys 55 <210> 58 <211> 63 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 58 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa 25 Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa Cys PCT/AU99/00805#i P:kOPER\MRO\AUTSEED2.PCT 21/9/99 43 40 Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Cys 55 <210> 59 <211> 64 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 59 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa 25 Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Cys 55 <210> <211> <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequencemotif <400> Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa 25 Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys 40 Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa 55 Cys <210> 61 <211> 62 <212> PRT <213> Artificial Sequence WO 00/16609 WO 0016609PCT/AU99/00805 -44 <220> <223> Description of Artificial Sequence:motif <400> 61 Cys Xaa Xaa xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa 25 Xaa Cys Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa 40 Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Cys 55 <210> 62 <211> 63 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequencemotif <400> 62 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa 25 Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa Cys 40 Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Cys 55 <210> 63 <211> 64 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequencemotif <400> 63 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa'Xaa Xad 1 .5 10 Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa 25 Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa.

40 Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Cys 55 PCT/AU99/00805ia P:\OPER\MRO\AUTSEED2.P2T 21/9/99 <210> 64 <211> <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequencemotif <400> 64 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa 25 Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys 40 Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa 55 Cys <210> <211> 66 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequencemotif <400> Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Cys Xaa 25 Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa 40 Cys Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa 55 Xaa Cys <210> 66 <211> 62 <212> PRT <213> Artificial Sequence PCT/AU99/00805it P:\OPER\MRO\AUTSEED2.PCI' -21/9/99 -46 <220> <223> Description of Artificial Sequencemotif <400> 66 Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa 25 Cys Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa 40 Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Cys 55 <210> 67 <211> 63 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 67 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa 25 Xaa Cys Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa Cys 40 Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Cys 55 <210> 68 <211> 64 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequencemotif <400> 68 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa 25 Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa 40 Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Cys 55 PCT/AU99/00805* P:\0PER\MR0\AUTSEED2.PCT 21/9/99 47 <210> 69 <211> <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 69 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa 25 Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys 40 Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa 55 Cys <210> <211> 66 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequencemotif <400> Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa 25 Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa 40 Cys Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa 55 Xaa Cys <210> 71 <211> 63 <212> PRT <213> Artificial Sequence <220> PCT/AU99/00805i P:\OPER\MR\AUTSEED2.PCr 21/199 -48 <223> Description of Artificial Sequence:motif <400> 71 Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa 25 Xaa Cys Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa Cys 40 Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Cys 55 <210> 72 <211> 64 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:mot-i-F' <400> 72 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa 25 Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa 40 Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Cys 55 <210> 73 <211> <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 73 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa 25 Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys 40 Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa PCT/AU99/OO8O54 P:%OPER\MROAUTSEED2.PCT 21/9/99 -49 55 Cys <210> 74 <211> 66 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequencemotif <400> 74 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa 25 Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa 40 Cys Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa 55 Xaa Cys <210> <211> 67 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequencemotif <400> Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Cys Xaa 25 Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa 40 Xaa Cys Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys 55 Xaa Xaa Cys <210> 76 <211> 61 <212> PRT <213> Artificial Sequence PCT/AU99/00805 P:\OPER\MRO\AUTSEED2.PCr 21/9/99 <220> <223> Description of Artificial Sequence:motif <400> 76 Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Arg Phe Xaa Gly 25 Cys Xaa Cys Xaa Xaa Gin Cys Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa 40 Ala Xaa Xaa Glu Cys Asx Pro Xaa Xaa Cys Asp Xaa Cys 55 <210> 77 <211> 62 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 77 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Arg Phe Xaa 25 Gly Cys Xaa Cys Xaa Xaa Gin Cys Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa 40 Xaa Ala Xaa Xaa Glu Cys Asx Pro Xaa Xaa Cys Asp Xaa Cys 55 <210> 78 <211> 63 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 78 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Arg Phe 25 Xaa Gly Cys Xaa Cys Xaa Xaa Gin Cys Xaa Xaa Xaa Xaa Cys Xaa Cys 40 Xaa Xaa Ala Xaa Xaa Glu Cys Asx Pro Xaa Xaa Cys Asp Xaa Cys 55 PCT/AU99/ 00805 P:\OPER\MRO\AUTSEED2.PCT 21/9/99 -51 <210> 79 <211> 64 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 79 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Arg 25 Phe Xaa Gly Cys Xaa Cys Xaa Xaa Gin Cys Xaa Xaa Xaa Xaa Cys Xaa 40 Cys Xaa Xaa Ala Xaa Xaa Glu Cys Asx Pro Xaa Xaa Cys Asp Xaa Cys 55 <210> <211> <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa 25 Arg Phe Xaa Gly Cys Xaa Cys Xaa Xaa Gin Cys Xaa Xaa Xaa Xaa Cys 40 Xaa Cys Xaa Xaa Ala Xaa Xaa Glu Cys Asx Pro Xaa Xaa Cys Asp Xaa 55 Cys <210> 81 <211> 62 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif N PCT/AU99/00805 P:\OPER\MRO\AUTSEED2.PCI' 21/9/99 52 <400> 81 Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Arg Phe Xaa 25 Gly Cys Xaa Cys Xaa Xaa Gin Cys Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa 40 Xaa Ala Xaa Xaa Glu Cys Asx Pro Xaa Xaa Cys Asp Xaa Cys 55 <210> 82 <211> 63 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 82 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Arg Phe 25 Xaa Gly Cys Xaa Cys Xaa Xaa Gln Cys Xaa Xaa Xaa Xaa Cys Xaa Cys 40 Xaa Xaa Ala Xaa Xaa Glu Cys Asx Pro Xaa Xaa Cys Asp Xaa Cys 55 <210> 83 <211> 64 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 83 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Arg 25 Phe Xaa Gly Cys Xaa Cys Xaa Xaa Gin Cys Xaa Xaa Xaa Xaa Cys Xaa 40 Cys Xaa Xaa Ala Xaa Xaa Glu Cys Asx Pro Xaa Xaa Cys Asp Xaa Cys 55 PCT/AU99/00805 P:\OPER\MRO\AUTSEED2.PCT 21/9/99 -53- <210> 84 <211> <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 84 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa 25 Arg Phe Xaa Gly Cys Xaa Cys Xaa Xaa Gin Cys Xaa Xaa Xaa Xaa Cys 40 Xaa Cys Xaa Xaa Ala Xaa Xaa Glu Cys Asx Pro Xaa Xaa Cys Asp Xaa 55 <210> <211> 66 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Cys Xaa 25 Xaa Arg Phe Xaa Gly Cys Xaa Cys Xaa Xaa Gln.Cys Xaa Xaa Xaa Xaa 40 Cys Xaa Cys Xaa Xaa Ala Xaa Xaa Glu Cys Asx Pro Xaa Xaa Cys Asp 55 Xaa Cys <210> 86 <211> 62 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 86 PCT/AU99/00805 P:\OPER\MRO\AUTSEED2.PCT 21/9/99 54 Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xa-a Xaa Xaa Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Arg Phe Xaa Gly 25 Cys Xaa Cys Xaa Xaa Xaa Gin Cys Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa 40 Xaa Ala Xaa Xaa Glu Cys Asx Pro Xaa Xaa Cys Asp Xaa Cys 55 <210> 87 <211> 63 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 87 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Arg Phe Xaa 25 Gly Cys Xaa Cys Xaa Xaa Xaa Gin Cys Xaa Xaa Xaa Xaa Cys Xaa Cys 40 Xaa Xaa Ala Xaa Xaa Glu Cys Asx Pro Xaa Xaa Cys Asp Xaa Cys 55 <210> 88 <211> 64 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 88 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Arg Phe 25 Xaa Gly Cys Xaa Cys Xaa Xaa Xaa Gln Cys Xaa Xaa Xaa Xaa Cys Xaa 40 Cys Xaa Xaa Ala Xaa Xaa Glu Cys Asx Pro Xaa Xaa Cys Asp Xaa Cys 55 PCT/AU99/00805 P:\OPER\MRO\AUTSEED2.PCT 21/9199 55 <210> 89 <211> <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 89 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Arg 25 Phe Xaa Gly Cys Xaa Cys Xaa Xaa Xaa Gin Cys Xaa Xaa Xaa Xaa Cys 40 Xaa Cys Xaa Xaa Ala Xaa Xaa Glu Cys Asx Pro Xaa Xaa Cys Asp Xaa 55 Cys <210> <211> 66 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa 25 Arg Phe Xaa Gly Cys Xaa Cys Xaa Xaa Xaa Gin Cys Xaa Xaa Xaa Xaa 40 Cys Xaa Cys Xaa Xaa Ala Xaa Xaa Glu Cys Asx Pro Xaa Xaa Cys Asp 55 Xaa Cys <210> 91 <211> 63 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 91 Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa PCT/AU99/00805 P:\OPER\MRO\AUTSEED2.PCI' 21/9/99 -56- 1 5 10 Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Arg Phe Xaa 25 Gly Cys Xaa Cys Xaa Xaa Xaa Gin Cys Xaa Xaa Xaa Xaa Cys Xaa Cys 40 Xaa Xaa Ala Xaa Xaa Glu Cys Asx Pro Xaa Xaa Cys Asp Xaa Cys 55 <210> 92 <211> 64 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 92 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Xaa Xaa 10 Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Arg Phe 25 Xaa Gly Cys Xaa Cys Xaa Xaa Xaa Gin Cys Xaa Xaa Xaa Xaa Cys Xaa 40 Cys Xaa Xaa Ala Xaa Xaa Glu Cys Asx Pro Xaa Xaa Cys Asp Xaa Cys 55 <210> 93 <211> <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 93 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Arg 25 Phe Xaa Gly Cys Xaa Cys Xaa Xaa Xaa Gln Cys Xaa Xaa Xaa Xaa Cys 40 Xaa Cys Xaa Xaa Ala Xaa Xaa Glu Cys Asx Pro Xaa Xaa Cys Asp Xaa 55 Cys PCT/AU99/00805 P:OPERWMAUTSEED2.PCT 2119/99 -57- <210> 94 <211> 66 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 94 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa 25 Arg Phe Xaa Gly Cys Xaa Cys Xaa Xaa Xaa Gin Cys Xaa Xaa Xaa Xaa 40 Cys Xaa Cys Xaa Xaa Ala Xaa Xaa Glu Cys Asx Pro Xaa Xaa Cys Asp 55 Xaa Cys <210> <211> 67 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa 1 5 10 i Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Cys Xaa 25 Xaa Arg Phe Xaa Gly Cys Xaa Cys Xaa Xaa Xaa Gin Cys Xaa Xaa Xaa 40 Xaa Cys Xaa Cys Xaa Xaa Ala Xaa Xaa Glu Cys Asx Pro Xaa Xaa Cys 55 Asp Xaa Cys <210> 96 <211> 61 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif PCT/AU99/00805 P:k0PER\MRO\AUTSEED2.PCr 21/9/99 58 <400> 96 Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Arg Phe Xaa Gly 25 Cys Xaa Cys Xaa Xaa Gin Cys Xaa Xaa Xaa Xaa Cys Xaa Cys Phe Xaa 40 Ala Xaa Xaa Glu Cys Asp Pro Xaa Xaa Cys Asp Xaa Cys 55 <210> 97 <211> 62 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequencemotif <400> 97 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Arg Phe Xaa 25 Gly Cys Xaa Cys Xaa Xaa Gin Cys Xaa Xaa Xaa Xaa Cys Xaa Cys Phe 40 Xaa Ala Xaa Xaa Glu Cys Asp Pro Xaa Xaa Cys Asp Xaa Cys 55 <210> 98 <211> 63 <212> PRT <213> Artificial Sequence <220> <2?23> Description of Artificial Sequence:motif <400> 98 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Arg Phe 25 Xaa Gly Cys Xaa Cys Xaa Xaa Gln Cys Xaa Xaa Xaa Xaa Cys Xaa Cys 40 Phe Xaa Ala Xaa Xaa Glu Cys Asp Pro Xaa Xaa Cys Asp Xaa Cys 55 <210> 99 <211> 64 FI/AUv/VUO'fl' P:\OPER\MRO\AUTSEED2.PCT 21/9199 59 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 99 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Arg 25 Phe Xaa Gly Cys Xaa Cys Xaa Xaa Gin Cys Xaa Xaa Xaa Xaa Cys Xaa 40 Cys Phe Xaa Ala Xaa Xaa Glu Cys Asp Pro Xaa Xaa Cys Asp Xaa Cys 55 <210> 100 <211> <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequencemotif <400> 100 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa 25 Arg Phe Xaa Gly Cys Xaa Cys Xaa Xaa Gin Cys Xaa Xaa Xaa Xaa Cys 40 Xaa Cys Phe Xaa Ala Xaa Xaa Glu Cys Asp Pro Xaa Xaa Cys Asp Xaa 55 Cys <210> 101 <211> 62 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequencemotif <400> 101 Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 PCT/AU99/00805 P:\OPER\MRO\AUTSEED2.PCT 21/9/99 60 Xaa Xaa Xaa Xaa cys Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Arg Phe xaa 25 Gly Cys Xaa Cys Xaa Xaa Gin Cys Xaa Xaa Xaa Xaa Cys Xaa Cys Phe 40 Xaa Ala Xaa.Xaa Glu Cys Asp Pro Xaa Xaa Cys Asp Xaa Cys 55 <210> 102 <211> 63 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 102 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Arg Phe 25 Xaa Gly Cys Xaa Cys Xaa Xaa Gin Cys Xaa Xaa Xaa Xaa Cys Xaa Cys 40 Phe Xaa Ala Xaa Xaa Glu Cys Asp Pro Xaa Xaa Cys Asp Xaa Cys 55 <210> 103 <211> 64 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 103 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Xaa 1 510 Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Arg 25 Phe Xaa Gly Cys Xaa Cys Xaa Xaa Gin Cys Xaa Xaa Xaa Xaa Cys Xaa 40 Cys Phe Xaa Ala Xaa Xaa Glu Cys Asp Pro Xaa Xaa Cys Asp Xaa Cys 55 <210> 104 <211> <212> PRT PCT/AU99/00805 P:\OPERMRO\AUTSEED2.PC! 21/9/99 -61- <213> Artificial sequence <220> <223> Description of Artificial Sequence:motif <400> 104 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa 25 Arg Phe Xaa Gly Cys Xaa Cys Xaa Xaa Gin Cys Xaa Xaa Xaa Xaa Cys 40 Xaa Cys Phe Xaa Ala Xaa Xaa Glu Cys Asp Pro Xaa Xaa Cys Asp Xaa 55 Cys <210> 105 <211> 66 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 105 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Cys Xaa 25 Xaa Arg Phe Xaa Gly Cys Xaa Cys Xaa Xaa Gin Cys Xaa Xaa Xaa Xaa 40 Cys Xaa Cys Phe Xaa Ala Xaa Xaa Glu Cys Asp Pro Xaa Xaa Cys Asp 55 Xaa Cys <210> 106 <211> 62 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 106 Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Arg Phe Xaa Gly PCT/AU99/00805 P:\OPER\MRO\AUTSEED2.PCI 2119/99 62 25 Cys Xaa Cys Xaa Xaa Xaa Gin Cys Xaa Xaa Xaa Xaa Cys Xaa Cys Phe 40 Xaa Ala Xaa Xaa Glu Cys Asp Pro Xaa Xaa Cys Asp Xaa Cys 55 <210> 107 <211> 63 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 107 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Arg Phe Xaa 25 Gly Cys Xaa Cys Xaa Xaa Xaa Gin Cys Xaa Xaa Xaa Xaa Cys Xaa Cys 40 Phe Xaa Ala Xaa Xaa Glu Cys Asp Pro Xaa Xaa Cys Asp Xaa Cys 55 <210> 108 <211> 64 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 108 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Arg Phe 25 Xaa Gly Cys Xaa Cys Xaa Xaa Xaa Gln Cys Xaa Xaa Xaa Xaa Cys Xaa 40 Cys Phe Xaa Ala Xaa Xaa Glu Cys Asp Pro Xaa Xaa Cys Asp Xaa Cys 55 <210> 109 <211> <212> PRT <213> Artificial Sequence PCT/AU99/00805 P:\OPER\MRO\AUTSEED2.PCT 2119/"9 63 <220> <223> Description of Artificial Sequence:motif <400> 109 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Arg 25 Phe Xaa Gly Cys Xaa Cys Xaa Xaa Xaa Gin Cys Xaa Xaa Xaa Xaa Cys 40 Xaa Cys Phe Xaa Ala Xaa Xaa Glu Cys Asp Pro Xaa Xaa Cys Asp Xaa 55 Cys <210> 110 <211> 66 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequencemotif <400> 110 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa 25 Arg Phe Xaa Gly Cys Xaa Cys Xaa Xaa Xaa Gln Cys Xaa Xaa Xaa Xaa 40 Cys Xaa Cys Phe Xaa Ala Xaa Xaa Glu Cys Asp Pro Xaa Xaa Cys Asp 55 Xaa Cys <210> 111 <211> 63 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 111 Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Arg Phe Xaa 25 PCT/AU99/00805 P:\OPER\MRO\AUTSEED2.PCT 21/9/99 -64 Gly Cys Xaa Cys Xaa Xaa Xaa Gin Cys Xaa Xaa xaa Xaa Cys xaa Cys 40 Phe Xaa Ala Xaa Xaa Glu Cys Asp Pro Xaa Xaa Cys Asp Xaa Cys 55 <210> 112 <211> 64 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 112 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Arg Phe 25 Xaa Gly Cys Xaa Cys Xaa Xaa Xaa Gin Cys Xaa Xaa Xaa Xaa Cys Xaa 40 Cys Phe Xaa Ala Xaa Xaa Glu Cys Asp Pro Xaa Xaa Cys Asp Xaa Cys 55 <210> 113 <211> <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequencemotif <400> 113 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cyvs Xaa Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Arg 25 Phe Xaa Gly Cys Xaa Cys Xaa Xaa Xaa Gin Cys Xaa Xaa Xaa Xaa Cys 40 Xaa Cys Phe Xaa Ala Xaa Xaa Glu Cys Asp Pro Xaa Xaa Cys Asp Xaa 55 Cys <210> 114 <211> 66 PCT/AU99/00805 P:\OPER\MRO\AUTSEED2.PCT 21/999 65 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 114 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa 25 Arg Phe Xaa Gly Cys Xaa Cys Xaa Xaa Xaa Gin Cys Xaa Xaa Xaa Xaa 40 Cys Xaa Cys Phe Xaa Ala Xaa Xaa Glu Cys Asp Pro Xaa Xaa Cys Asp 55 Xaa Cys <210> 115 <211> 67 <212> PRT <213> Artificial Sequence <22 0> <223> Description of Artificial Sequencemotif <400> 115 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Cys Xaa 25 Xaa Arg Phe Xaa Gly Cys Xaa Cys Xaa Xaa Xaa Gin Cys Xaa Xaa Xaa 40 Xaa Cys Xaa Cys Phe Xaa Ala Xaa Xaa Glu Cys Asp Pro Xaa Xaa Cys 55 Asp Xaa Cys <210> 116 <211> <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 116 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa 1 5 10 PCT/AU99/00805 P:\OPER\MRO\AUTSEED2. PCI' 21/9/99 66 Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa 25 Xaa Xaa Cys <210> 117 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 117 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys 1 5 10 Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa 25 Xaa Xaa Xaa Cys <210> 118 <211> 37 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 118 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa 1 5 10 Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa 25 Xaa Xaa Xaa Xaa Cys <210> 119 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 119 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 1 5 10 Xaa Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa 25 PCT/AU99/00805 P:\OPER\MRO\AUTSEED2.PCT 21/9199 67 Xaa Xaa Xaa Xaa Xaa Cys <210> 120 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 120 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Cys 1 5 10 Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa 25 Xaa Xaa Xaa Cys <210> 121 <211> 37 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequencemotif <400> 121 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa 1 5 10 Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa 25 Xaa Xaa Xaa Xaa Cys <210> 122 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 122 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa 1 5 10 Xaa Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa 25 Xaa Xaa Xaa Xaa Xaa Cys PCT/AU99/00805 P:\OPER\MRO\AUTSEED2.PCT 2119/99 -68- <210> 123 <211> 39 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 123 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 1 5 10 Xaa Xaa Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa 25 Xaa Xaa Xaa Xaa Xaa Xaa Cys <210> 124 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequencemotif <400> 124 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa 1 5 10 Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa 25 Xaa Xaa Xaa Cys <210> 125 <211> 37 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequencemotif <400> 125 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys 1 5 10 Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa 25 Xaa Xaa Xaa Xaa Cys <210> 126 <211> 38 PCT/AU99/00805 P:\OPER\MRO\AUTSEED2.PCT 21/9/99 69 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequencemotif <400> 126 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa 1 5 10 Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa 25 Xaa Xaa Xaa Xaa Xaa Cys <210> 127 <211> 39 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequencemotif <400> 127 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 1 5 10 Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa 25 Xaa Xaa Xaa Xaa Xaa Xaa Cys <210> 128 <211> <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequencemotif <400> 128 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 1 5 10 Xaa Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 25 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys <210> 129 <211> 39 <212> PRT <213> Artificial Sequence PCT/AU99/00805 P:XOPER\MRO\AUTSEED2.PCT 21/9/99 70 <220> <223> Description of Artificial Sequence:motif <400> 129 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa 1 5 10 Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa 25 Xaa Xaa Xaa Xaa Xaa Xaa Cys <210> 130 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 130 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa 1 5 10 Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa 25 Xaa Xaa Xaa Xaa Xaa Cys <210> 131 <211> 37 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequencemotif <400> 131 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Cys 1 5 10 Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa 25 Xaa Xaa Xaa Xaa Cys <210> 132 <211> 37 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif PCT/AU99/00805 P:\OPER\MRO\AUTSEED2.PCT 2119/99 71 <400> 132 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys 1 5 10 Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa 25 Xaa Xaa Xaa Xaa Cys <210> 133 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequencemotif <400> 133 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa 1 5 10 Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa 25 Xaa Xaa Xaa Xaa Xaa Cys <210> 134 <211> 39 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequencemotif <400> 134 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 1 5 10 Xaa Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa 25 Xaa Xaa Xaa Xaa Xaa Xaa Cys <210> 135 <211> <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 135 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 1 5 10 PCT/AU99/00805 P:\OPERNMR0\AUTSEED2.PCTr 21/9/99 -72- Xaa Xaa Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 25 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys <210> 136 <211> 39 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 136 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa 1 5 10 Xaa Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa 25 Xaa Xaa Xaa Xaa Xaa Xaa Cys <210> 137 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 137 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa 1 5 10 Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa 25 Xaa Xaa Xaa Xaa Xaa Cys <210> 138 <211> 37 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 138 Cys Xaa xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Cys 1 5 10 Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa 25 PCT/AU99/00805 P:XOPER\MRO\AUTSEED2.PCT 21/9/99 73 Xaa Xaa Xaa Xaa Cys <210> 139 <211> 37 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 139 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa 1 5 10 Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa 25 Xaa Xaa Xaa Xaa Cys <210> 140 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 140 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa 1 5 10 Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa 25 Xaa Xaa Xaa Cys <210> 141 <211> 37 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 141 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa 1 5 10 Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa 25 Xaa Xaa Xaa Xaa Cys PCT/AU99/00805 P:\OPER\MRO\AUTSEED2.PCT 21/9/99 74 <210> 142 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 142 Cys Xaa Xaa Xaa Xaa Xaa Xaa: Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys 1 5 10 Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa 25 Xaa Xaa Xaa Xaa Xaa Cys <210> 143 <211> 39 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 143 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa 1 5 10 Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa 25 Xaa Xaa Xaa Xaa Xaa Xaa Cys <210> 144 <211> <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 144 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 1 5 10 Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 25 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys <210> 145 PCT/AU99/00805 P:k0PER\MR0\AUTSEED2.PC2T 21/9/99 75 <211> 41 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 145 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 1 5 10 Xaa Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 25 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys <210> 146 <211> <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 146 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa 1 5 10 Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 25 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys <210> 147 <211> 39 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 147 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa 1 5 10 Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa 25 Xaa Xaa Xaa Xaa Xaa Xaa Cys <210> 148 <211> 38 <212> PRT <213> Artificial Sequence PCT/AU99/00805 P:\OPER\MRO\AUTSEED2.PCT 21/9/99 76 <220> <223> Description of Artificial Sequence:motif (400> 148 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Cys 1 5 10 Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa 25 Xaa Xaa Xaa Xaa Xaa Cys <210> 149 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 149 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys 1 5 10 Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa 25 Xaa Xaa Xaa Xaa Xaa Cys <210> 150 <211> 39 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 150 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa 1 5 10 Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa 25 Xaa Xaa Xaa Xaa Xaa Xaa Cys <210> 151 <211> <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequencemotif PCT/AU99/00805 P:\OPER\MRO\AUTSEED2.PCr 21/9/99 77 (400> 151 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 1 5 10 Xaa Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 25 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys <210> 152 <211> 41 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 152 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 1 5 10 Xaa Xaa Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 25 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys <210> 153 <211> <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequencemotif <400> 153 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa 1 5 10 Xaa Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 25 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys <210> 154 <211> 39 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 154 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa PCT/AU99/00805 P:\OPER\MRO\AUTSEED2.PCT 21/9/99 -78- 1 5 10 Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa 25 Xaa Xaa Xaa Xaa Xaa Xaa Cys <210> 155 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequencemotif <400> 155 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Cys 1 5 10 Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa 25 Xaa Xaa Xaa Xaa Xaa Cys <210>'156 <211> 38 <212> FRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 156 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa 1 5 10 Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa 25 Xaa Xaa Xaa Xaa Xaa Cys 210> 157 21>37 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequencemotif <400> 157 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa 1 5 10 Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa PCT/AU99/00805 P:\OPER\MRO\AUTrSEED2.PCT 21/91%9 79 25 Xaa Xaa Xaa Xaa Cys <210> 158 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 158 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa 1 5 10 Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa 25 Xaa Xaa Xaa Xaa Xaa Cys <210> 159 <211> 39 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 159 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys 1 5 10 Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa 25 Xaa Xaa Xaa Xaa Xaa Xaa Cys <210> 160 <211> <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 160 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa 1 5 10 Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 25 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys PCT/AU99/00805 P:\OPER\MRO\AUTSEED2.PCr 21/9/99 80 <210> 161 <211> 41 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 161 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 1 5 10 Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 25 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys -210>~ 162 <211> 42 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 162 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 1 5 10 Xaa Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 25 Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys <210> 163 <211> 41 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 163 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa 1 5 10 Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 25 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys PCT/AU99/00805 P:\OPER\MRO\AtJTSEED2.PCT 21/9/99 <210> 164 <211> <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequencemotif <400> 164 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa 1 5 10 Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 25 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys <210> 165 <211> 39 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequencemotif <400> 165 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Cys 1 5 10 Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa 25 Xaa Xaa Xaa Xaa Xaa Xaa Cys <210> 166 <211> 39 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 166 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys 1 5 10 Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa 25 Xaa Xaa Xaa Xaa Xaa Xaa Cys <210> 167 <211> <212> PRT PCT/AU99/00805 P:kOPER\MRO\AUTSEED2.PCT 21/9/99 82 <213> Artificial sequence <220> <223> Description of Artificial Sequence:motif <400> 167 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa 1 5 10 Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 25 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys <210> 168 <211> 41 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 168 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 1 5 10 Xaa Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 25 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys <210> 169 <211> 42 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequencemotif <400> 169 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 1 5 10 Xaa Xaa Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 25 Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys <210> 170 <211> 41 <212> PRT <213> Artificial Sequence <220> PCT/AU99/00805 P\kOPER\MRO\AUTSEED2. PCTr 21/9/99 83 <223> Description of Artificial Sequencemotif (400> 170 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa 1 5 10 Xaa Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 25 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys <210> 171 <211> <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequencemotif <400> 171 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa= Xaa Xaa Xaa Xaa Cys Xaa Xaa 1 5 10 Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Cys 25 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys (210> 172 <211> 39 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 172 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa 1 5 10 Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa 25 Xaa Xaa Xaa Xaa Xaa Xaa Cys <210> 173 <211> 38 <212> PRT <213> Artificial Sequence <~220> <223> Description of Artifi'cial Sequencemotif <400> 173 PCT/AU99/00805- P:\OPER\MRO\AUTSEED2.PCr 21/9199 84 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa 10 Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa 25 Xaa Xaa Xaa Xaa Xaa Cys <210> 174 <211> 39 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequencemotif <400> 174 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa 1 5 10 Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa 25 Xaa Xaa Xaa Xaa Xaa Xaa Cys <210> 175 <211> <212> PRT <2i3> Artificial Sequence <220> <223> Description of Artificial Sequencemotif <400> 175 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys 1 5 10 Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 25 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys <210> 176 <211> 41 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 176 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa 1 5 10 PCT/AU99/00805 P:\OPER\MRO\AUTSEED2.PCr 21/9/99 85 Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 25 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys <210> 177 <211> 42 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 177 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 1 5 10 Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 25 Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys <210> 178 <211> 43, <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 178 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 1 5 10 Xaa Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 25 Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys <210> 179 <211> 42 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 179 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa 1 5 10 Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 25 PCT/AU99/00805 P:\OPER\MRO\AUTSEED2.PCT 21/9/99 -86- Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys <210> 180 <211> 41 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 180 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa 1 5 10 Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 25 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys <210> 181 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 181 Arg Gly Asp 1 <210> 182 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 182 Glu Glu Asp Glu Glu Asp Glu Glu Glu Asp Glu Glu Glu 1 5 <210> 183 <211> 34 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 183 Trp Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ile Phe 1 5 10 PCT/AU99/00805 P:\OPER\MRO\AUTSEED2.PCT 21/9/99 -87- Gly Xaa Asn Ser Cys Xaa Xaa Ala Xaa Xaa Xaa Xaa Xaa Gly Xaa Lys 25 Xaa Cys <210> 184 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 184 Trp Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 Xaa Xaa Asn Xaa Cys Xaa Xaa Ala Xaa Xaa Xaa Xaa Xaa Lys Xaa Cys 25 <210> 185 <211> 33 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 185 Trp Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 Xaa Xaa Asn Xaa Cys Xaa Xaa Ala Xaa Xaa Xaa Xaa Xaa Xaa Lys Xaa 25 Cys <210> 186 <211> 34 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 186 Trp Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 Xaa Xaa Asn Xaa Cys Xaa Xaa Ala Xaa Xaa Xaa Xaa Xaa Xaa Xaa Lys 25 PCT/AU99/00805 P:\OPER\MRO\AUTSEED2.PCT 21/9/99 -88- Xaa Cys <210> 187 <211> 34 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 187 Trp Xaa Pro Xaa Glu Lys Xaa Leu Tyr Leu Lys Gly Xaa Glu lie Phe 1 5 10 Gly Xaa Asn Ser Cys Xaa Xaa Ala Xaa Asn lie Leu Xaa Gly Xaa Lys 25 Thr Cys <210> 188 <211> 34 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 188 Trp Xaa Pro Xaa Glu Lys Xaa Leu Tyr Leu Lys Gly Xaa Glu Ile Phe 1 5 10 Gly Xaa Asn Ser Cys Xaa Val Ala Xaa Asn lie Leu Xaa Gly Xaa Lys 25 Thr Cys <210> 189 <211> 34 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 189 Trp Thr Pro Val Glu Lys Asp Leu Tyr Leu Lys Gly Ile Glu Ile Phe 1 5 10 Gly Arg Asn Ser Cys Asp Val Ala Leu Asn Ile Leu Arg Gly Leu Lys 25 Thr Cys PCT/AU99/00805 P:\OPER\MRO\AUTSEED2.PCT 21/9/99 -89- <210> 190 <211> <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 190 Lys Lys Xaa Xaa Lys 1 <210> 191 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 191 Lys Lys Xaa Xaa Xaa Lys 1 <210> 192 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 192 Lys Lys Xaa Xaa Lys Xaa Xaa Arg Xaa Xaa Arg Lys Lys Xaa Arg Xaa 1 5 10 Arg Lys <210> 193 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 193 Lys Lys Xaa Xaa Xaa Lys Xaa Xaa Arg Xaa Xaa Arg Lys Lys Xaa Arg 1 5 10 Xaa Arg Lys PCT/AU99/00805 P:\OPER\MRO\AUTSEED2.PCT 21/9/99 <210> 194 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 194 Lys Lys Val Ser Arg Lys Ser Ser Arg Ser Val Arg Lys Lys Ser Arg 1 5 10 Leu Arg Lys <210> 195 <211> <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 195 Cys Xaa Xaa Cys Xaa 1 <210> 196 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 196 Xaa His Xaa Xaa Xaa Xaa His 1 <210> 197 <211> <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 197 Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa His Xaa Xaa Xaa His Xaa 1 5 10 Xaa Xaa Xaa His <210> 198 <211> PCT/AU99/00805 P:\OPER\MRO\AUTSEED2.PCT 21/9/99 -91- <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 198 Cys Xaa Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa His Xaa Xaa Xaa His Xaa 1 5 10 Xaa Xaa Xaa His <210> 199 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 199 Cys Pro Phe Cys Leu lie Pro Cys Gly Gly His Glu Gly Leu Gin Leu 1 5 10 His Leu Lys Ser Ser His <210> 200 <211> <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:splice junction <400> 200 aaaaaacaac gtatgcattc <210> 201 <211> <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:splice junction <400> 201 gtttattcag ccatatttcc <210> 202 <211> <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif PCT/AU99/00805 P:\OPER\MRO\AUTSEED2.PCT 21/9/99 -92- <400> 202 ctacagggat gtgagtaaca <210> 203 <211> <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 203 ttttgcttag gtcaaattca <210> 204 <211> <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 204 aaagctgaag gtgagccttt <210> 205 <211> <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 205 ccaaatgcag tagtggaaaa <210> 206 <211> <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 206 aggtcacgag gtaggcacta <210> 207 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:motif <400> 207 ttgtgccaca gggcttgcaa c 21 <210> 208 <211> 21 PCT/AU99/00805 P:\OPER\MRO\AUTSEED2.PCT 21/9/99 93y--\il <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:primer <400> 208 tcatctcttc cttatgaagt t 21 <210> 209 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:primer <400> 209 tgttgataat gtcccatcg 19 <210> 210 <211> 180 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:fis2 gene fragment <400> 210 actagcaaca ttttaactag gactcaacca gcaatagctg agtctgaacc taaggtgcct catgtgaatg atgataaagt ctcatcgaca ccaagagctc actcttcaaa gaagaataaa 120 tctactcata agaaagatga taatgcctca ttgccaccaa aaactcgctc ttcgaagaag 180 <210> 211 <211> 170 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:peptide <400> 211 Thr His Arg Ser Glu Arg Ala Ser Asn Ile Leu Glu Leu Glu Thr His 1 5 10 Arg Ala Arg Gly Thr His Arg Gly Leu Asn Pro Arg Ala Leu Ala Ile 25 Leu Glu Ala Leu Ala Gly Leu Ser Glu Arg Gly Leu Pro Arg Leu Tyr 40 Ser Val Ala Leu Pro Arg His Ile Ser Val Ala Leu Ala Ser Asn Ala 55 Ser Pro Ala Ser Pro Leu Tyr Ser Val Ala Leu Ser Glu Arg Ser Glu 70 75 Arg Thr His Arg Pro Arg Ala Arg Gly Ala Leu Ala His Ile Ser Ser 90 PCT/AU99/00805 P:\OPER\MRO\AUTSEED2.PC 21/9/99 -94- Glu Arg Ser Glu Arg Leu Tyr Ser Leu Tyr Ser Ala Ser Asn Leu Tyr 100 105 110 Ser Ser Glu Arg Thr His Arg His Ile Ser Leu Tyr Ser Leu Tyr Ser 115 120 125 Ala Ser Pro Ala Ser Pro Ala Ser Asn Ala Leu Ala Ser Glu Arg Leu 130 135 140 Glu Pro Arg Pro Arg Leu Tyr Ser Thr His Arg Ala Arg Gly Ser Glu 145 150 155 160 Arg Ser Glu Arg Leu Tyr Ser Leu Tyr Ser 165 170 <210> 212 <211> 66 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:peptide <400> 212 Ala Arg Gly Ala Leu Ala Gly Leu Leu Tyr Ser Ala Ser Pro His lie 1 5 10 Ser Gly Leu Tyr Pro Arg Gly Leu Val Ala Leu Ala Ser Pro Val Ala 25 Leu Ser Glu Arg Val Ala Leu Leu Tyr Ser Ser Glu Arg Ala Ser Pro 40 Thr His Arg Ile Leu Glu Leu Tyr Ser Pro His Glu Gly Leu Tyr Val 55 Ala Leu <210> 213 <211> 241 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:fisl gene fragment <400> 213 gtaagtaaaa ttttttagtg atctaatttt gtttatgttt ttgcatgaaa tagtatgtaa caagagtact atttatctat tttaagcggg cagagaaaga tcacggaccg gaagttgatg 120 tctccgtgaa aagtgataca ataaaatttg gggttagtag taaactcgat acataaatgc 180 aatgttagtc ataatgttga actcaccatg atgttatttt ttttaattta tttttcaggt 240 t 241

Claims (14)

126- THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS: 1. A method of inducing the development of seed in the absence of fertilization comprising inhibiting, interrupting or otherwise reducing expression of a polypeptide that delays interrupts or prevents autonomous fertilization-independent) seed formation or autonomous embryogenesis or autonomous endosperm development in one or more female reproductive cells, tissues or organs of a plant or a progenitor cell, tissue or organ thereof, wherein the polypeptide is a member selected from the group consisting of: a FIS1 polypeptide which comprises an amino acid sequence having at least 50% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 1; (ii) a FIS2 polypeptide which comprises an amino acid sequence having at least 60% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 2; (iii) a FIS1 polypeptide that is encoded by a nucleotide sequence having at least 50% identity to the coding region of the nucleotide sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 5; and (iv) a FIS2 polypeptide that is encoded by a nucle6tide sequence having at least 60% identity to the coding region of the nucleotide sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 7; and wherein the FIS1 polypeptide comprises three amino acid sequence motifs: C-X 2 -C-X 4 -C-X( 25 35 )-C-X 3 -C, wherein numerical values indicate the number of consecutive multiple occurrences of a particular amino acid residue; (ii) a cysteine-rich domain (CXC) comprising at least about 14 cysteine residues within a sequence of 61-67 consecutive amino acids and located C- terminal to and (iii) an amino acid sequence located C-terminal to (ii) and comprising the amino acid sequence: 6 X 2 2 2 6 -D- P:\OPER\jMS\SPECIFICATIONS\6!824-99. DOC 7/7/03

127- X 2 2 4 2 Y-X-Y, wherein numerical values indicate the number of consecutive multiple occurrences of a particular amino acid residue. 2. The method of claim 1 wherein the FIS1 polypeptide further comprises a cysteine-rich domain which comprises the consensus amino acid sequence motif, Ca-X(1 1-14)-Cb-X(1-2)-Cc-X(2-3)-Cd-X(8 1 1 )-Ce-X(7-9)-Cf wherein each of the integers designated Ca ,Cb ,Cc,Ca ,Ce and Cf are successive cysteine residues in said sequence motif and numerical values indicate the number of consecutive multiple occurrences of a particular amino acid residue. 3. The method of claim 1 wherein the FIS1 polypeptide further comprises the amino acid sequence motif R-G-D. 4. The method of claim 1 wherein the FIS1 polypeptide further comprises an amino acid sequence of 12-13 amino acid residues in length wherein at least 5 of said residues are glutamate and/or aspartate. The method of claim 1 wherein the FIS1 polypeptide further comprises an amino sequence selected from the group consisting of: 2 S: C; and (ii) 2 3 3 6. The method of claim 1 wherein the FIS1 polypeptide further comprises a nuclear localisation signal. P:\OPER'jMS\SPECIFICATIONs\6 824-99.DOC 22/7/03

128- 7. The method of claim 6 wherein the nuclear localisation signal includes the amino acid sequence motif: wherein numerical values indicate the number of consecutive multiple occurrences of a particular amino acid residue. 8. The method of claim 1 wherein the FIS2 polypeptide comprises the amino acid sequence motif: C-X 2 -C-Xn-H-X 4 -H, wherein n 10 to 15 amino acid residues in length and wherein numerical values indicate the number of consecutive multiple occurrences of a particular amino acid residue. 9. The method according to any one of claims 1 to 8 wherein the polypeptide is a member selected from the group consisting of: a FIS2 polypeptide which comprises the amino acid sequence set forth in SEQ ID NO: 2 or a fragment thereof that delays interrupts or Sprevents autonomous seed development, autonomous embryogenesis or autonomous endosperm development in a plant; and (ii) a FIS2 polypeptide that is encoded by the coding region of the nucleotide sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 7. 10. The method according to any one of claims 1 to 9 wherein the expression of the polypeptide is reduced by a method comprising mutagenesis of a gene o* P:\OPER\jMS\SPECIFICATIONs\6 824-99DOC 7/7/03

129- encoding said polypeptide, subject to the proviso that said mutagenesis does not result in the expression of truncated FIS1 polypeptide that is encoded by a gene having a single mutation in a region encoding the following amino acid sequence and no other mutation in said gene: 6 X 2 2 2 6 -D- X 2 2 34 2 D-Y-X-Y, wherein numerical values indicate the number of consecutive multiple occurrences of a particular amino acid residue. 11. The method of claim 10 wherein the mutagenesis produces a null allele. 12. The method of claim 10 wherein the mutagenesis results in the expression of a FIS1 polypeptide having one or more functional protein domains disrupted. 13. The method of claim 12 wherein the functional protein domain comprises an amino acid sequence motif of the polypeptide selected from the group consisting of: C-X 2 -C-X 4 -C-X( 2 5 35 )-C-X 3 -C; (ii) a cysteine-rich domain (CXC) comprising at least about 14 cysteine residues within a sequence of 61-67 consecutive amino acids and located C- terminal to in the polypeptide; (iii) a domain located C-terminal to (ii) in the polypeptide and comprising the amino acid sequence: 6 (T/S)-X 2 2 2 F-X-(L/I)-X 6 -D-X 2 2 3 4 2 (iv) a cysteine-rich domain which comprises the consensus amino acid sequence motif, P.\OPER'jMS\SPECIFICATIONs\61824-99.DOC -7/7/03 -130- Ca-X(11-14)-Cb-X(1-2)-Cc-X(2-3)-Cd-X(8- 11)-Ce-X(7-9)-Cf wherein each of the integers designated Ca ,Cb ,CC ,Ce and Cf are successive cysteine residues in said sequence motif; the amino acid sequence motif R-G-D; (vi) a domain of 12-13 amino acid residues in length wherein at least 5 of said residues are glutamate and/or aspartate; (vii) the amino sequence: 2 C; (viii) the amino sequence: 2 3 3 (ix) the amino acid sequence motif C-X 2 -C-Xn-H-X 4 wherein n 10 to amino acid residues in length; and the nuclear localisation signal of said polypeptide, and wherein numerical values indicate the number of consecutive multiple occurrences of a particular amino acid residue of any one of said motifs. 14. The method of claim 10 wherein the mutagenesis is performed using a chemical mutagen. The method of claim 14 wherein the chemical mutagen is EMS. 16. The method of claim 10 wherein the mutagenesis is performed by inserting a -nucleic acid molecule into the gene. 17. The method of claim 16 wherein the nucleic acid molecule comprises a member selected from the group consisting of: T-DNA; a gene targeting molecule; and a transposon. P:\OPERjMS\SPECIFICATIONs\61 824-99. DO- 7/7/03 131 18. The method according to any one of claims 1 to 9 wherein the expression of the polypeptide is reduced by a method comprising expressing an antisense or ribozyme molecule in the plant for a time and under conditions sufficient to reduce or inhibit the expression of said polypeptide, wherein said antisense or ribozyme molecule comprises a nucleotide sequence that is complementary to the nucleotide sequence of mRNA encoding said polypeptide. 19. The method according to any one of claims 1 to 9 wherein the expression of the polypeptide is reduced by a method comprising expressing a nucleic acid molecule which encodes said polypeptide or a fragment thereof in the plant for a time and under conditions sufficient to reduce or inhibit the expression of said polypeptide, and wherein said nucleic acid molecule comprises at least nucleotides in length. The method according to any one of claims 1 to 19 wherein the seed comprises an endosperm. 21. The method according to any one of claims 1 to 19 wherein the seed lacks a functional embryo structure. 22. The method of claim 21 wherein the seed is a soft seed. 23. The method according to any one of claims 1 to 20 wherein the seed is able to germinate. 24. A method of producing seedless or soft-seeded fruit comprising inhibiting, interrupting or otherwise reducing expression of a polypeptide that delays interrupts or prevents autonomous fertilization-independent) seed formation or autonomous embryogenesis or autonomous endosperm development in one or more female reproductive cells, tissues or organs of a plant or a progenitor cell, P:\OPER'jMS\SPECIFICATIONS\61824-99DOC 7/7/03

132- tissue or organ thereof, wherein a polypeptide is a member selected from the group consisting of: a FIS1 polypeptide which comprises an amino acid sequence having at least 50% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 1; (ii) a FIS2 polypeptide which comprises an amino acid sequence having at least 60% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 2; (iii) a FIS1 polypeptide that is encoded by a nucleotide sequence having at least 50% identity to the coding region of the nucleotide sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 5; and (iv) a FIS2 polypeptide that is encoded by a nucleotide sequence having at least 60% identity to the coding region of the nucleotide sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 7; and wherein the FIS1 polypeptide comprises three amino acid sequence motifs: C-X 2 -C-X 4 -C-X( 25 35 )-C-X 3 wherein numerical values indicate the number of consecutive multiple occurrences of a particular amino acid residue; S(ii) a cysteine-rich domain (CXC) comprising at least about 14 cysteine residues within a sequence of 61-67 consecutive amino acids and located C- terminal to and (iii) an amino acid sequence located C-terminal to (ii) and comprising the amino acid sequence: 6 X 2 2 2 6 -D- X 2 2 3 4 2 D-Y-X-Y, wherein numerical values indicate the number of consecutive multiple occurrences of a particular amino acid residue. The method of claim 24 wherein the FIS1 polypeptide further comprises a P:\OPERiMS\SPECIFICATION\61 824-99. DOC- 7/7/03

133- cysteine-rich domain which comprises the consensus amino acid sequence motif, Ca-X(11-14)-Cb-X(1- 2 )-Cc-X(2- 3 )-Cd-X(8-11)-Ce-X(7-9)-Cf wherein each of the integers designated Ca ,Cb ,Cc ,Cd ,Ce and Cf are successive cysteine residues in said sequence motif and numerical values indicate the number of consecutive multiple occurrences of a particular amino acid residue. 26. The method of claim 24 wherein the FIS1 polypeptide further comprises the amino acid sequence motif R-G-D. 27. The method of claim 24 wherein the FIS1 polypeptide further comprises an amino acid sequence of 12-13 amino acid residues in length wherein at least 5 of said residues are glutamate and/or aspartate. 28. The method of claim 24 wherein the FIS1 polypeptide further comprises an amino sequence selected from the group consisting of: 2 C; and (ii) 2 3 3 29. The method of claim 24 wherein the FIS1 polypeptide further comprises a nuclear localisation signal. The method of claim 29 wherein the nuclear localisation signal includes the amino acid sequence motif K-K-X( 1 2 wherein numerical values indicate the number of consecutive multiple occurrences of a particular amino acid residue. 31. The method of claim 24 wherein the FIS2 polypeptide comprises the amino acid sequence motif P:\OPERVJMS\SPECIFICATIONS6 I 824-99.DOC 22/7/03

134- C-X 2 -C-Xn-H-X 4 -H, wherein n 10 to 15 amino acid residues in length and wherein numerical values indicate the number of consecutive multiple occurrences of a particular amino acid residue. 32. The method according to any one of claims 24 to 31 wherein the polypeptide is a member selected from the group consisting of: a FIS2 polypeptide which comprises the amino acid sequence set forth in SEQ ID NO: 2 or a fragment thereof that delays interrupts or prevents autonomous seed development, autonomous embryogenesis or autonomous endosperm development in a plant; and (ii) a FIS2 polypeptide that is encoded by the coding region of the nucleotide sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 7. 33. The method according to any one of claims 24 to 32 wherein the expression of the polypeptide is reduced by a method comprising mutagenesis of a gene encoding said polypeptide, subject to the proviso that said mutagenesis does not "result in the expression of truncated FIS1 polypeptide that is encoded by a gene having a single mutation in a region encoding the following amino acid sequence and no other mutation in said gene: 6 X 2 2 2 6 -D- X 2 2 3 4 2 D-Y-X-Y, wherein numerical values indicate the number of consecutive 4 4 P:\OPERjmS\SPECIFICATIONs\61824-99.DOC -7/7/03

135- multiple occurrences of a particular amino acid residue. 34. The method of claim 33 wherein the mutagenesis produces a null allele. The method of claim 33 wherein the mutagenesis results in the expression of a FIS1 polypeptide having one or more functional protein domains disrupted. 36. The method of claim 35 wherein the functional protein domain comprises an amino acid sequence motif of the polypeptide selected from the group consisting of: C-X 2 -C-X 4 -C-X( 2 5- 35 )-C-X 3 -C; (ii) a cysteine-rich domain (CXC) comprising at least about 14 cysteine residues within a sequence of 61-67 consecutive amino acids and located C- terminal to in the non-mutant polypeptide; (iii) a domain located C-terminal to (ii) in the non-mutant polypeptide and comprising the amino acid sequence: 6 (T/S)-X 2 2 2 2 (iv) a cysteine-rich domain which comprises the consensus amino acid sequence motif, Ca-X(11-14)-Cb-X(1-2)-Cc-X(2-3)-Cd-X( 8 1 )-Ce-X( 7 -9)-Cf wherein the integers designated Ca ,Cb ,C ,Cd ,Ce and Cf are successive cysteine residues in said sequence motif; the amino acid sequence motif R-G-D; (vi) a domain of 12-13 amino acid residues in length wherein at least 5 of said residues are glutamate and/or aspartate; (vii) the amino sequence: 2 P:\OPER\JMS\SPECIFICATIONs\61824-99DOC 7/7/03

136- C; (viii) the amino sequence: 2 3 13 (ix) the amino acid sequence motif C-X 2 -C-Xn-H-X 4 wherein n 10 to amino acid residues in length; and the nuclear localisation signal of said polypeptide, and wherein numerical values indicate the number of consecutive multiple occurrences of a particular amino acid residue of any one of said motifs. 37. The method according to claim 33 wherein the mutagenesis is performed using a chemical mutagen. 38. The method of claim 37 wherein the chemical mutagen is EMS. 39. The method according to claim 33 wherein the mutagenesis is performed by inserting a nucleic acid molecule into the gene. The method of claim 39 wherein the nucleic acid molecule comprises a member selected from the group consisting of: T-DNA; a gene targeting molecule; and a transposon. 41. The method according to any one of claims 24 to 32 wherein the expression of the polypeptide is reduced by a method comprising expressing an antisense or ribozyme molecule in the plant for a time and under conditions sufficient to reduce or inhibit the expression of said polypeptide, wherein said antisense or ribozyme 0" molecule comprises a nucleotide sequence that is complementary to the nucleotide Ssequence of mRNA encoding said polypeptide. sequence of mRNA encoding said polypeptide. P:\OPER\lhs\SPECIFICATIONs\6 824-9. DOC 22/7/03

137- 42. The method according to any one of claims 24 to 32 wherein the expression of the polypeptide is reduced by a method comprising expressing a nucleic acid molecule which encodes said polypeptide or a fragment thereof in the plant for a time and under conditions sufficient to reduce or inhibit the expression of said polypeptide, and wherein said nucleic acid molecule comprises at least nucleotides in length. 43. The method according to any one of claims 24 to 42 wherein the seed comprise an endosperm. 44. The method according to any one of claims 24 to 44 wherein the seed lack a functional embryo structure. An isolated nucleic acid molecule which is capable of inhibiting or reducing the expression of a FIS polypeptide that delays interrupts or prevents autonomous fertilization-independent) seed formation or autonomous embryogenesis or autonomous endosperm development in a plant, wherein said nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of: a nucleotide sequence encoding an amino acid sequence set forth in SEQ ID NO: 2; S.(ii) a nucleotide sequence selected from the group consisting of: SEQ ID NO: 6 and SEQ ID NO: 7; (iii) a nucleotide sequence that is complementary to or and a nucleotide sequence that hybridises under at least low stringency conditions to or (ii). *e 46. The isolated nucleic acid molecule of claim 45 selected from the group consisting of: an antisense molecule; a ribozyme; a co-suppression molecule; a gene-targeting molecule; a gene-silencing molecule; and a dominant-negative sense molecule. P:\OPER\jMS\SPECIFICATIONS\61 824-99DOc 22/7/03

138- 47. An isolated nucleic acid molecule selected from the group consisting of: a nucleic acid molecule comprising a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 2 or a fragment thereof that delays interrupts or prevents autonomous seed development, autonomous embryogenesis or autonomous endosperm development in a plant; (ii) a nucleotide sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 7; (iii) a nucleotide sequence that is complementary to or and (iv) a nucleotide sequence that hybridises under at least low stringency conditions to or (ii). 48. A gene construct comprising the isolated nucleic acid molecule according to any one of claims 45 to 47 operably linked to a promoter sequence that is operable in a plant cell, plant tissue or plant organ. 49. The gene construct of claim 48 wherein the promoter is operable in one or :i:o more female reproductive cells, tissues or organs of a plant. 50. The gene construct of claim 49 wherein the promoter is operable in the ovule. 51. The gene construct of claim 48 wherein the promoter is operable in the seed of a plant or a seed cell, seed tissue, seed organ or a progenitor cell of said seed. 52. The gene construct according to any one of claims 48 to 51 wherein the promoter comprises a nucleotide sequence selected from the group consisting of: nucleotides 1 to 3142 of SEQ ID NO: (ii) nucleotides 1785 to 3142 of SEQ ID NO: (iii) nucleotides 1 to 2851 of SEQ ID NO: 7; P:\OPERjtJSSPECIFICATIONS\6 824-99OC 22/7/03

139- (iv) nucleotides 1531 to 2851 of SEQ ID NO: 7; nucleotides 1 to 1200 of SEQ ID NO: 9; and (vi) a fragment of any one of to capable of conferring expression at least in one or more female reproductive cells, tissues or organs of a plant. 53. An isolated nucleic acid molecule that encodes a polypeptide that delays interrupts or prevents autonomous seed development, autonomous embryogenesis or autonomous endosperm development in a plant, wherein said nucleic acid molecule is isolated by the process comprising: amplifying or hybridising nucleic acid using a nucleic acid probe or primer of at least ten nucleotides in length derived from a nucleotide sequence selected from the group consisting of: SEQ ID NO: 6 or SEQ ID NO: 7; and (ii) isolating the amplified or hybridised nucleic acid. 54. The isolated nucleic acid molecule of claim 53 wherein the probe or primer comprises a nucleotide sequence selected from the group consisting of: the sequence set forth in SEQ ID NO: 208; (ii) the sequence set forth in SEQ ID NO: 209; and (iii) a complementary nucleotide sequence to or (ii). A cell that has been transformed or transfected with the isolated nucleic acid molecule according to any one of claims 45 to 47, 53 or 54, or a gene construct comprising said isolated nucleic acid molecule. 56. A plant that comprises the isolated nucleic acid molecule according to any one of claims 45 to 47, 53 or 54 introduced into its genome. 57. The plant according to claim 56 wherein said plant produces parthenocarpic fruit or soft-seeded fruit in the absence of fertilization by virtue of the presence of P:\OPER'jMS\SPECIFICATIONs\6 I824-99.DOC- 22/7/03

140- the isolated nucleic acid molecule in its genome. 58. A propagule of the plant according to claim 56 or 57, wherein said propagule comprises the introduced nucleic acid molecule in its genome. 59. The propagule of claim 58 comprising seed. Use of the isolated nucleic acid molecule according to any one of claims to 47, 53 or 54 in the manufacture of a member selected from the group consisting of: an antisense molecule; a ribozyme; a co-suppression molecule; a gene- targeting molecule; a gene-silencing molecule; and a dominant-negative sense molecule; wherein said member is for the production of a transformed plant that exhibits one or more autonomous fertilization-independent) phenotypes selected from the group consisting of: it is apomictic or produces seed; (ii) it produces seed endosperm; (iii) it produces seed embryo; (iv) it produces soft-seeded fruit; and it produces parthenocarpic fruit. 61. An isolated promoter which is capable of conferring expression at least in one or more female reproductive cells, tissues or organs of said plant or a progenitor cell, tissue or organ thereof, said promoter comprising a nucleotide sequence selected from the group consisting of: nucleotides 1 to 3142 of SEQ ID NO: (ii) nucleotides 1785 to 3142 of SEQ ID NO: (iii) nucleotides 1 to 2851 of SEQ ID NO: 7; (iv) nucleotides 1531 to 2851 of SEQ ID NO: 7; C* nucleotides 1 to 1200 of SEQ ID NO: 9; and (vi) a fragment of any one of to capable of conferring expression at P:\OPERjNIS\SPECIFICATIONs\6 824-99.OC 22/7/03 141 least on one or more female reproductive cells, tissues or organs of a plant. 62. An isolated promoter of plants which is capable of conferring expression at least in one or more female reproductive cells, tissues or organs of a plant or a progenitor cell, tissue or organ thereof, wherein said promoter is isolated by the process of: amplifying or hybridising nucleic acid using a nucleic acid probe or primer of at least ten nucleotides in length from a nucleotide sequence selected from the group consisting of: SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8 and SEQ ID NO: 9; (ii) hybridising the amplified or hybridised nucleic acid to plant DNA under at least low stringency hybridisation conditions for a time sufficient for hybridisation to occur; and (iii) isolating the hybridising nucleic acid and operably connecting same to a structural reporter gene to determine the ability of said molecule to induce gene expression in one or more female reproductive cells, tissues or organs of a plant or a progenitor cell, tissue or organ thereof. 63. An isolated or recombinant polypeptide that delays interrupts or prevents autonomous fertilization-independent) seed formation or autonomous embryogenesis or autonomous endosperm development in a plant, wherein said polypeptide comprises a member selected from the group consisting of: a polypeptide that comprises an amino acid sequence having at least identity to SEQ ID NO: 2; (ii) a polypeptide comprising the amino acid sequence set forth in SEQ ID "NO: 2; and (iii) a polypeptide encoded by a nucleotide sequence selected from the group consisting of: SEQ ID NO: 6 and SEQ ID NO: 7. 64. The isolated or recombinant polypeptide of claim 63 wherein the polypeptide having at least 50% identity to SEQ ID NO: 2 comprises the amino acid sequence P:\OPER\jMS\SPECIFCATIONs\61 824-99.DOC 22/7/03

142- motif: C-X 2 -C-Xn-H-X 4 -H, wherein n 10 to 15 amino acid residues in length and wherein numerical values indicate the number of consecutive multiple occurrences of a particular amino acid residue. The isolated or recombinant polypeptide of claim 63 or 64 wherein said polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 2 or a fragment of said sequence that delays interrupts or prevents autonomous seed development, autonomous embryogenesis or autonomous endosperm development in a plant. 66. The isolated or recombinant polypeptide according to any one of claims 63 to 65 wherein said polypeptide is encoded by the coding region of a nucleotide sequence selected from the group consisting of: SEQ ID NO: 6 and SEQ ID NO: 7. 67. A polypeptide ligand of the polypeptide according to any one of claims 63 to 66, wherein said ligand is capable of regulating embryogenesis or seed formation in .a plant in the absence of fertilization by virtue of its interaction with said polypeptide. 68. The polypeptide ligand of claim 67 wherein said ligand is identified by a screening method employing the polypeptide selected from the group consisting of one-hybrid assay; two-hybrid assay; and three-hybrid assay. Dated this 22 n d day of July 2003. Commonwealth Scientific and Industrial Research Organisation By its Patent Attorneys Davies Collison Cave