AU2005202657B2 - Method and means for modifying gene expression using unpolyadenylated RNA - Google Patents

Description

141780590

AUSTRALIA

Patents Act 1990 (Cth) Complete Specification (Divisional) Commonwealth Scientific and Industrial Research Organisation Invention Title Method and means for modifying gene expression using unpolyadenylated RNA The invention is described in the following statement: Blake Dawson Waldron Patent Services Level 39, 101 Collins Street Melbourne VIC 3000 Telephone: 61 3 9679 3065 Fax: +61 396793111 17 June 2005 Ref: 03 1386 0488 MEANS AND METHODS FOR MODIFYING GENE EXPRESSION USING UNPOLYADENYLATD

RNA

Field of the invention.

The invention relates to methods for reducing the phenotypic expression of a nucleic acid of interest in plant cells by providing aberrant RNA molecules, preferably unpolyadenylated RNA molecules comprising at least one target specific nucleotide sequence homologous to the nucleic acid of interest, preferably a sense strand, into the nucleus of plant cells. The target-specific unpolyadenylated RNA molecules may be provided by introduction of chimeric DNAs which when transcribed under control of conventional promoter and 3' end formation and polyadenylation regions yield RNA molecules wherein at least the polyadenylation signal may be removed by the autocatalytic activity of a selfsplicing ribozyme comprised within the transcribed RNA molecules. Also provided are plant cells comprising such RNA molecules or chimeric

DNA

encoding such RNA molecules, as well as plants. Similar methods and means for reducing the phenotypic expression of a nucleic acid by co-suppression in eukaryotic cells are provided.

Background of the invention.

Post-transcriptional gene silencing (PTGS) or co-suppression, is a common phenomenon associated with transgenes in transgenic plants. PTGS results in sequence-specific removal of the silenced transgene RNA as well as homologous endogenous gene RNA or viral RNA. It is characterized by low steady-state mRNA levels with normal (usually high) rates of nuclear transcription of transgenes being maintained. There are a number of common features or characteristics for PTGS. PTGS is i) sequence-specific; ii) systemically transmissible; iii) often associated with the presence of multiple copies of transgenes or with the use of strong promoters; iv) frequently correlated with the presence of repetitive

DNA

structures, including inverted repeat T-DNA insertion patterns; v) often accompanied by de novo DNA methylation in the transcribed region, and vi) may be meiotically reset.

A number of hypothetical models have been proposed to explain PTGS (see e.g.

Wassenegger and Pdlissier, 1998). Typically, these models suggest the involvement of a host encoded enzyme (RNA-directed RNA polymerase (RdRP)) which is proposed to use aberrant RNA as templates to synthesize small copy RNA molecules (cRNA). These cRNAs would then hybridize with the target mRNA to form duplex structures, thereby rendering the mRNA susceptible to degradation by endoribonucleases. So far, there has been no direct evidence that RdRP is involved in PTGS in plants.

An important question arising from the existing models is what type of RNA is the aberrant RNA that would be used as a template by RdRP, and in which cellular compartment RdRP would function.

Several reports have described the accumulation of unproductive or unpolyadenylated transgene RNA in plants which are post-transcriptionally silenced (Lee et 1997; van Eldik et al. 1998; Covey et al., 1997; van Houdt et al., 1997; Metzlaff et 1997).

The following documents relate to methods and means for regulating or inhibiting gene expression in a cell.

US 5,190,131 and EP 0 467 349 Al describe methods and means to regulate or inhibit gene expression in a cell by incorporating into or associating with the genetic material of the cell a non-native nucleic acid sequence which is transcribed to produce an mRNA which is complementary to and capable of binding to the mRNA produced by the genetic material of that cell.

EP 0 223 399 Al describes methods to effect useful somatic changes in plants by causing the transcription in the plant cells of negative RNA strands which are io substantially complementary to a target RNA strand. The target RNA strand can be a mRNA transcript created in gene expression, a viral RNA, or other RNA present in the plant cells. The negative RNA strand is complementary to at least a portion of the target RNA strand to inhibit its activity in vivo.

is EP 0 240 208 describes a method to regulate expression of genes encoded for in plant cell genomes, achieved by integration of a gene under the transcriptional control of a promoter which is functional in the host and in which the transcribed strand of DNA is complementary to the strand of DNA that is transcribed from the endogenous gene(s) one wishes to regulate.

EP 0 647 715 Al and US patents 5, 034,323, 5,231,020 and 5,283,184 describe methods and means for producing plants exhibiting desired phenotypic traits, by selecting transgenotes that comprise a DNA segment operably linked to a promoter, wherein transcription products of the segment are substantially homologous to corresponding transcripts of endogenous genes, particularly endogenous flavonoid biosynthetic pathway genes.

Waterhouse et al. 1998 describe that virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and anti-sense RNA. The sense and antisense RNA may be located in one transcript that has self-complementarity.

Hamilton et al. 1998 describes that a transgene with repeated DNA, i.e. inverted copies of its 5' untranslated region, causes high frequency, post-transcriptional suppression of ACC-oxidase expression in tomato.

WO 98/53083 describes constructs and methods for enhancing the inhibition of a target gene within an organism which involve inserting into the gene silencing vector an inverted repeat sequence of all or part of a polynucleotide region within the vector.

WO 95/34688 describes methods for cytoplasmic inhibition of gene expression and provides genetic constructs for the expression of inhibitory RNA in the cytoplasm of eukaryotic cells. The inhibitory RNA may be an anti-sense or a cosuppressor RNA. The genetic constructs are capable of replicating in the cytoplasm of a eukaryotic cell and comprise a promoter region, which may be a plant virus subgenomic promoter in functional combination with the RNA encoding region.

W095/15394 and US 5908779 describe a method and construct for regulating gene expression through inhibition by nuclear antisense RNA in (mouse) cells.

The construct comprises a promoter, antisense sequences, and a cis-or transribozyme which generates 3'-ends independently of the polyadenylation machinery and thereby inhibits the transport of the RNA molecule to the cytoplasm.

Summary of the invention.

SThe present invention provides a method for reducing the phenotypic expression of a nucleic acid of interest, which is normally capable of being expressed in a plant cell, the method comprising the step of providing to the nucleus of that plant cell aberrant RNA comprising a target-specific nucleotide sequence, V 5 preferably unpolyadenylated RNA comprising a target specific nucleotide S sequence, particularly by producing aberrant RNA such as unpolyadenylated S RNA by transcription of a chimeric DNA comprised within the plant cell, the chimeric DNA comprising a plant-expressible promoter operably linked to a CM target specific DNA region encoding that RNA and optionally further comprising a DNA region involved in 3' end formation and polyadenylation, preceded by a self-splicing ribozyme encoding. DNA region.

The invention also provides a method for reducing the phenotypic expression of a nucleic acid of interest, which is normally capable of being expressed in a plant cell, the method comprising the step of introducing into the nuclear genome of the plant cell a chimeric DNA to generate a transgenic plant cell, the chimeric DNA comprising the following operably linked parts: a) a plant-expressible promoter region, preferably a constitutive promoter or an inducible promoter or a tissue-specific promoter; Q 2o b) a target-specific DNA region encoding a target-specific nucleotide sequence, preferably a target-specific DNA region comprising a nucleotide sequence of at least 10 consecutive nucleotides having at least about 70 sequence identity to about 100 sequence identity to the nucleic acid of interest or comprising a nucleotide sequence of at least 10 consecutive nucleotides having at least about 70 sequence identity to about 100 sequence identity to the complement of said nucleic acid of interest; c) a DNA region encoding a self-splicing ribozyme, preferably a self-splicing ribozyme comprising a cDNA copy of a self-splicing ribozyme from avocado sunblotch viroid, peach latent mosaic viroid, Chrysanthemum chlorotic mottle viroid, carnation stunt associated viroid, Newt satellite 2 transcript, Neurospora VS RNA, barley yellow dwarf virus satellite RNA,arabis mosaic virus satellite RNA, chicory yellow mottle virus satellite RNA S1, lucerne transient streak virus satellite RNA, tobacco ringspot virus satellite RNA, subterranean clover mottle virus satellite RNA, solanum nodiflorum mottle virus satellite RNA, velvet tobacco mottle virus satellite RNA, Cherry small circular viroid-like RNA or hepatitis delta virus RNA, particularly a DNA region comprising the nucleotide sequence of SEQ ID No 1 or SEQ ID No 2 or a ribozyme-effective part thereof; and d) a DNA region involved in 3' end formation and polyadenylation; wherein said chimeric DNA when transcribed produces a first RNA molecule comprising a target specific nucleotide sequence and a self-splicing ribozyme, which when cleaved by autocatalysis produces a second RNA molecule comprising a target specific nucleotide sequence wherein the 3' end of the first RNA molecule comprising the polyadenylation site has been removed.

Optionally, a transgenic plant may be regenerated from the transgenic plant cell.

Preferably, the DNA region encoding a self-splicing ribozyme is located immediately upstream of the DNA region involved in 3' end formation and polyadenylation.

It is another objective of the invention to provide a chimeric DNA molecule for reducing the phenotypic expression of a nucleic acid of interest, which is normally capable of being expressed in a plant cell, comprising a) a plant-expressible promoter region, preferably a constitutive promoter or an inducible promoter or a tissue-specific promoter, b) a target-specific DNA region encoding a target-specific nucleotide sequence, preferably a target-specific DNA region comprising a nucleotide sequence of at least 10 consecutive nucleotides having at least about 70 sequence identity to about 100 sequence identity to the nucleic acid of interest or comprising a nucleotide sequence of at least 10 consecutive nucleotides having at least about 70 sequence identity to about 100 sequence identity to the complement of said nucleic acid of interest; c) a DNA region encoding a self-splicing ribozyme, preferably a self-splicing ribozyme comprising a cDNA copy of a self-splicing ribozyme from avocado sunblotch viroid, peach latent mosaic viroid, Chrysanthemum chlorotic mottle viroid, carnation stunt associated viroid, Newt satellite 2 transcript, Neurospora VS RNA, barley yellow dwarf virus satellite RNA,arabis mosaic virus satellite RNA, chicory yellow mottle virus satellite RNA S1, lucerne transient streak virus satellite RNA, tobacco ringspot virus satellite RNA, subterranean clover mottle virus satellite RNA, solanum nodiflorum mottle virus satellite RNA, velvet tobacco mottle virus satellite RNA, Cherry small circular viroid-like RNA or hepatitis delta virus RNA, particularly a DNA region comprising the nucleotide sequence of SEQ ID No 1 or SEQ ID No 2 or a ribozyme-effective part thereof; and d) a DNA region involved in 3' end formation and polyadenylation; wherein said chimeric DNA when transcribed produces a first RNA molecule comprising a target specific nucleotide sequence and a self-splicing ribozyme, which when cleaved by autocatalysis produces a second RNA molecule comprising a target specific nucleotide sequence wherein the 3' end of the first RNA molecule comprising the polyadenylation site has been removed.

Preferably, the DNA region encoding a self-splicing ribozyme is located immediately upstream of the DNA region involved in 3' end formation and polyadenylation.

It is yet another objective. of the invention to provide plant cells and plants comprising a nucleic acid of interest which is normally capable of being phenotypically expressed, further comprising a chimeric DNA, preferably stably integrated into the nuclear genome, comprising a) a plant-expressible promoter region, preferably a constitutive promoter or an inducible promoter or a tissue-specific promoter, Sb) a target-specific DNA region encoding a target-specific nucleotide sequence, preferably a target-specific DNA region comprising a nucleotide sequence of at least 10 consecutive nucleotides having at least about 70 sequence identity to about 100 sequence identity to the nucleic acid of interest or n 5 comprising a nucleotide sequence of at least 10 consecutive nucleotides having at least about 70 sequence identity to about 100 sequence C identity to the complement of said nucleic acid of interest; Sc) a DNA region encoding a self-splicing ribozyme, preferably a self-splicing ribozyme comprising a cDNA copy of a self-splicing ribozyme from avocado sunblotch viroid, peach latent mosaic viroid, Chrysanthemum chlorotic mottle viroid, carnation stunt associated viroid, Newt satellite 2 transcript, Neurospora VS RNA, barley yellow dwarf virus satellite RNA,arabis mosaic virus satellite RNA, chicory yellow mottle virus satellite RNA S1, lucerne transient streak virus satellite RNA, tobacco ringspot virus satellite

RNA,

subterranean clover mottle virus satellite RNA, solanum nodiflorum mottle virus satellite RNA, velvet tobacco mottle virus satellite RNA, Cherry small circular viroid-like RNA or hepatitis delta virus RNA, particularly a DNA region comprising the nucleotide sequence of SEQ ID No 1 or SEQ ID No 2 or a ribozyme-effective part thereof; and d) a DNA region involved in 3' end formation and polyadenylation; wherein said chimeric DNA when transcribed produces a first RNA molecule comprising a target specific nucleotide sequence and a self-splicing ribozyme, which when cleaved by autocatalysis produces a second RNA molecule comprising a target specific nucleotide sequence wherein the 3' end of the first RNA molecule comprising the polyadenylation site has been removed.

The invention also provides a method for identifying a phenotype associated with the expression of a nucleic acid of interest in a plant cell, the method comprising: 1) selecting within the nucleic acid of interest a target sequence of at least consecutive nucleotides; 2) introducing a chimeric DNA into the nucleus of a suitable plant host cell comprising the nucleic acid of interest, the chimeric DNA comprising the following operably linked DNA fragments: a) a plant-expressible promoter region; b) a target-specific DNA region comprising a nucleotide sequence of at least about 70% to about 100% sequence identity to said target sequence or to the complement of said target sequence followed by c) a DNA region encoding a self-splicing ribozyme located immediately upstream of d) a DNA region involved in 3' end formation and polyadenylation; 3) observing the phenotype by a suitable method.

Yet another objective of the invention is to provide a method for reducing the phenotypic expression of a nucleic acid of interest, which is normally capable of being expressed in a eukaryotic cell, the method comprising the step of providing to the nucleus of said eukaryotic cell aberrant RNA, preferably unpolyadenylated RNA, comprising a target specific nucleotide sequence of at least 10 consecutive nucleotides with at least about 70% sequence identity to about 100% sequence identity to the nucleotide sequence of the nucleic acid of interest, paritucularly by producing aberrant RNA such as unpolyadenylated RNA by transcription of a chimeric DNA comprised within the eukaryotic cell, the chimeric DNA comprising a plant-expressible promoter operably linked to a target specific DNA region encoding that RNA and optionally further comprising a DNA region involved in 3' end formation and polyadenylation, preceded by a self-splicing ribozyme encoding DNA region.

Still another objective of the invention is to provide a method for reducing the phenotypic expression of a nucleic acid of interest, which is normally capable of O being expressed in a eukaryotic cell, comprising the step of introducing into the nuclear genome of the eukaryotic cell a chimeric DNA to generate a transgenic plant cell, comprising the following operably linked parts: a) a promoter region functional in the eukaryotic cell; g 5 b) a target-specific DNA region comprising nucleotide sequence of at least consecutive nucleotides with at least about 70% sequence identity to about 100% sequence identity to the nucleotide sequence of the nucleic acid of interest; c) a DNA region encoding a self-splicing ribozyme; and d) a DNA region involved in 3' end formation and polyadenylation wherein the chimeric DNA when transcribed produces a first RNA molecule comprising a target specific nucleotide sequence and a self-splicing ribozyme, which when cleaved by autocatalysis produces a second RNA molecule comprising a target specific nucleotide sequence wherein the 3' end of the first RNA molecule comprising the polyadenylation site has been removed.

The invention also provides a eukaryotic cell comprising a nucleic acid of interest, normally capable of being phenotypically expressed, further comprising a chimeric DNA comprising the following operably linked parts: a) a promoter region functional in the eukaryotic cell; b) a target-specific DNA region comprising nucleotide sequence of at least consecutive nucleotides with at least about 70% sequence identity to about 100% sequence identity to the nucleotide sequence of the nucleic acid of interest; c) a DNA region encoding a self-splicing ribozyme; and d) a DNA region involved in 3' end formation and polyadenylation wherein said chimeric DNA when transcribed in the eukaryotic cell produces a first RNA molecule comprising a target specific nucleotide sequence and a selfsplicing ribozyme, which when cleaved by autocatalysis produces a second

RNA

molecule comprising a target specific nucleotide sequence wherein the 3' end of the first RNA molecule comprising the polyadenylation site has been removed, as well as non-human eukaryotic organisms comprising or consisting essentially of such eukaryotic cells.

Brief Description of the Figures Figure 1. Schematic representation of the ribozyme-containing GUS chimeric gene (pMBW267 and pMBW259) the control construct (pMBW 265) and the GUS chimeric gene used for supertransformation (pBPPGH). 35S-P: CaMV promoter; GUS: region encoding fi-glucuronidase; SAT: cDNA copy of the satellite RNA of Barley Yellow Dwarf Virus (BYDV) in positive strand orientation or in minus strand orientation Ocs-T: region from the octopine synthase gene from Agrobacterium involved in 3' end formation and polyadenylation; 3' Sat: cDNA copy of the 3' end of the satellite RNA of BYDV; 5' SAT: cLNA copy of the 5' end of the satellite RNA of BYDV; PP2-P: 1.3 kb promoter region of a gene encoding the cucurbit phloem protein PP2; Nos-T: region from the nopaline synthase gene from Agrobacterium involved in 3' end formation and polyadenylation; C: autocatalytic cleavage site in the RNA molecule transcribed from the chimeric gene.

Detailed description of the preferred embodiments of the invention.

Although gene-silencing, either by anti-sense RNA or through co-suppression using sense RNA, is a commonly observed phenomenon in transgenic research, the intentional generation of gene-silenced transgenic eukaryotic cells and transgenic organisms, particularly plant cells and plants, still faces a number of problems. In particular the efficiency of gene-silencing is still amenable to improvement, both in number of transgenic lines exhibiting the phenomenon as -12well as in the level of reduction of transcription and ultimately the phenotypic expression of particular nucleic acid of interest in a particular transgenic line.

A number of improved methods for gene-silencing have already been described, e.g. the simultaneous use in one cell of anti-sense and sense RNA targeted to the nucleic acid of interest, preferably co-located on one transcript exhibiting self-complementarity. Novel methods for increasing the efficiency of genesilencing, preferably gene-silencing through co-suppression in a eukaryotic cell or organism, preferably plant cell or plant, and means therefore, are described in the different embodiments provided by the specification and claims.

The current invention is based on the unexpected observation by the inventors, that the provision or the introduction of aberrant target-specific RNA, preferably unpolyadenylated target-specific RNA, particularly an aberrant target-specific RNA comprising a nucleotide sequence essentially identical to the nucleic acid of interest in sense orientation, into the nucleus of a cell of a eukaryotic organism, particularly a cell of plant, resulted in an efficient reduction of the expression of the nucleic acid of interest, both in the level of reduction as well as in the number of transgenic lines exhibiting gene-silencing. The understanding of hypothetical mechanisms through which gene-silencing, particularly PTGS, is supposed to proceed did not allow to predict that among all variables potentially involved in initiation and maintenance of gene-silencing, the selection of this one parameter-i.e. providing aberrant, preferably unpolyadenylated RNA- would have been sufficient to significantly increase the efficiency of gene-silencing, particularly gene-silencing through co-suppression.

In one embodiment of the invention, a method is provided for reducing the phenotypic expression of a nucleic acid of interest, which is normally capable of being expressed in a plant cell, comprising the step of providing aberrant RNA such as unpolyadenylated RNA which includes a target-specific nucleotide sequence to the nucleus of that plant cell. Conveniently, the aberrant RNA such as unpolyadenylated RNA including the target-specific nucleotide sequence may be produced by transcription of a chimeric DNA or chimeric gene comprised within the plant cell, preferably incorporated, particularly stably integrated into the nuclear genome of the plant cell. In a particularly preferred embodiment, the s aberrant RNA is unpolyadenylated RNA which still exhibits other modifications characteristic of mRNA, such as, but not limited to, the presence of a capstructure at the 5' end.

As used herein, the term "expression of a gene" refers to the process wherein a DNA region which is operably linked to appropriate regulatory regions, particularly to a promoter region, is transcribed into an RNA which is biologically active which is either capable of interaction with another nucleic acid or which is capable of being translated into a polypeptide or protein. A gene is said to encode an RNA when the end product of the expression of the gene is biologically active RNA, such as e.g. an antisense RNA, a ribozyme or a replicative intermediate. A gene is said to encode a protein when the end product of the expression of the gene is a protein or polypeptide.

A nucleic acid of -interest is "capable of being expressed", when said nucleic acid, when introduced in a suitable host cell, particularly in a plant cell, can be transcribed (or replicated) to yield an RNA, and/or translated to yield a polypeptide or protein in that host cell.

The term "gene" means any DNA fragment comprising a DNA region (the "transcribed DNA region") that is transcribed into a RNA molecule a mRNA) in a cell operably linked to suitable regulatory regions, a plantexpressible promoter. A gene may thus comprise several operably linked DNA fragments such as a promoter, a 5' leader sequence, a coding region, and a 3' region comprising a polyadenylation site. A plant gene endogenous to a particular plant species (endogenous plant gene) is a gene which is naturally found in that plant species or which can be introduced in that plant species by conventional breeding. A chimeric gene is any gene which is not normally found in a plant species or, alternatively, any gene in which the promoter is not associated in nature with part or all of the transcribed DNA region or with at least one other regulatory region of the gene.

As used herein, "phenotypic expression of a nucleic acid of interest" refers to any quantitative trait associated with the molecular expression of a nucleic acid in a host cell and may thus include the quantity of RNA molecules transcribed or replicated, the quantity of post-transcriptionally modified RNA molecules, the quantity of translated peptides or proteins, the activity of such peptides or proteins.

A "phenotypic trait" associated with the phenotypic expression of a nucleic acid of interest refers to any quantitative or qualitative trait, including the trait mentioned, as well as the direct or indirect effect mediated upon the cell, or the organism containing that cell, by the presence of the RNA molecules, peptide or protein, or posttranslationally modified peptide or protein. The mere presence of a nucleic acid in a host cell, is not considered a phenotypic expression or a phenotypic trait of that nucleic acid, even though it can be quantitatively or qualitatively traced. Examples of direct or indirect effects mediated on cells or organisms are, agronomically or industrial useful traits, such as resistance to a pest or disease; higher or modified oil content etc.

As used herein, "reduction of phenotypic expression" refers to the comparison of the phenotypic expression of the nucleic acid of interest to the eukaryotic cell in the presence of the RNA or chimeric genes of the invention, to the phenotypic expression of the nucleic acid of interest in the absence of the RNA or chimeric genes of the invention. The phenotypic expression in the presence of the chimeric RNA of the invention should thus be lower than the phenotypic expression in absence thereof, preferably be only about 25%, particularly only about 10%, more particularly only about 5% of the phenotypic expression in absence of the chimeric RNA, especially the phenotypic expression should be completely inhibited for all practical purposes by the presence of the chimeric RNA or the chimeric gene encoding such an RNA.

A reduction of phenotypic expression of a nucleic acid where the phenotype is a qualitative trait means that in the presence of the chimeric RNA or gene of the invention, the phenotypic trait switches to a different discrete state when compared to a situation in which such RNA or gene is absent. A reduction of phenotypic expression of a nucleic acid may thus, be measured as a reduction in transcription of (part of) that nucleic acid, a reduction in translation of (part of) that nucleic acid or a reduction in the effect the presence of the transcribed RNA(s) or translated polypeptide(s) have on the eukaryotic cell or the organism, and will ultimately lead to altered phenotypic traits. It is clear that the reduction in phenotypic expression of a nucleic acid of interest, may be accompanied by or correlated to an increase in a phenotypic trait.

As used herein "a nucleic acid of interest" or a "target nucleic acid" refers to any particular RNA molecule or DNA sequence which may be present in a eukaryotic cell, particularly a plant cell.

As used herein "aberrant RNA" refers to polyribonucleotide molecules which have characteristic differing from mRNA molecules normally found in that cell.

The different characteristics include but are not limited to the absence or removal of a 5' cap structure, presence of persistent introns e.g. introns which have been modified in their splice sites so as to prevent splicing, or the absence of the polyA tail normally found associated with the mRNA ("unpolyadenylated

RNA").

$The term "target-specific nucleotide sequence" as used herein, refers to a nucleotide sequence (either DNA or RNA nucleotide sequence depending on the context) which can reduce the expression of the target nucleic acid of interest by gene-silencing. Preferably, only the expression of the target nucleic acid or gene, or nucleic acids or genes comprising essentially similar nucleotide c- sequence is reduced.

O

Preferably the target-specific nucleotide sequence comprises a nucleotide N sequence corresponding to the "sense" nucleotide sequence of the nucleic acid or gene of interest. In other words, a target-specific sense nucleotide sequence may be essentially similar to part of an RNA molecule transcribed or produced from the nucleic acid or gene of interest or to parts of the nucleic acid or gene of interest controlling the production of that transcribed or produced RNA molecule, when read in the same 5' to 3' direction as the transcribed or produced

RNA

molecule.

Preferably, the target specific nucleotide sequence corresponds to part of a nucleic acid region from which RNA is produced, particularly a region which is transcribed and translated. It is particularly preferred that the target sequence corresponds to one or more consecutive exons, more particularly is located within a single exon of a coding region. However, the target specific nucleotide sequence may also be corresponding to untranslated regions of the RNA molecule produced from the nucleic acid or gene of interest. Moreover, in the light of a recent publication by Mette et al. (1999), it is expected that the target specific nucleotide sequence may also correspond to the regions controlling the production or transcription of RNA from the nucleotide or gene of interest, such as the promoter region.

The length of the sense target-specific nucleotide sequence may vary from about 10 nucleotides (nt) up to a length equaling the length (in nucleotides) of the target nucleic acid. Preferably the total length of the sense nucleotide sequence is at least 10 nt, preferably 15 nt, particularly at least about 50 nt, more particularly at least about 100 nt, especially at least about 150 nt, more especially at least about 200 nt, quite especially at least about 550 nt. It is V 5 expected that there is no upper limit to the total length of the sense nucleotide Csequence, other than the total length of the target nucleic acid. However for practical reason (such as e.g. stability of the chimeric genes) it is expected that the length of the sense nucleotide sequence should not exceed 5000 nt, Cparticularly should not exceed 2500 nt and could be limited to about 1000 nt.

It will be appreciated that the longer the total length of the sense nucleotide sequence is, the less stringent the requirements for sequence identity between the total sense nucleotide sequence and the corresponding sequence in the target nucleic acid or gene become. Preferably, the total sense nucleotide sequence should have a sequence identity of at least about 75% with the corresponding target sequence, particularly at least about 80 more particularly at least about 85%, quite particularly about 90%, especially about more especially about 100%, quite especially be identical to the corresponding part of the target nucleic acid. However, it is preferred that the sense nucleotide sequence always includes a sequence of about consecutive nucleotides, particularly about 20 nt, more particularly about 50 nt, especially about 100 nt, quite especially about 150 nt with 100% sequence identity to the corresponding part of the target nucleic acid. Preferably, for calculating the sequence identity and designing the corresponding sense sequence, the number of gaps should be minimized, particularly for the shorter sense sequences.

As used herein, "sequence identity" with regard to nucleotide sequences

(DNA

or RNA), refers to the number of positions with identical nucleotides divided by the number of nucleotides in the shorter of the two sequences. The alignment of -18the two nucleotide sequences is performed by the Wilbur and Lipmann algorithm (Wilbur and Lipmann, 1983) using a window-size of 20 nucleotides, a word length of 4 nucleotides, and a gap penalty of 4. Computer-assisted analysis and interpretation of sequence data, including sequence alignment as described above, can, be conveniently performed using the programs of the Intelligenetics

T

M Suite (Intelligenetics Inc., CA). Sequences are indicated as "essentially similar" when such sequence have a sequence identity of at least about 75%, particularly at least about 80 more particularly at least about quite particularly about 90%, especially about 95%, more especially about 100%, quite especially are identical. It is clear than when RNA sequences are said to be essentially similar or have a certain degree of sequence identity with DNA sequences, thymine in the DNA sequence is considered equal to uracil in the RNA sequence.

It is expected however, that the target-specific nucleotide sequence may also comprise a nucleotide sequence corresponding to the uantisense" nucleotide sequence of the nucleic acid or gene of interest. In other words, a target-specific antisense nucleotide sequence may be essentially similar to the complement of part of an RNA molecule transcribed or produced from the nucleic acid or gene of interest or to the complement of parts of the nucleic acid or gene of interest controlling the production of that transcribed or produced RNA molecule, when read in the same 5' to 3' direction as the transcribed or produced RNA molecule.

The requirements for antisense target-specific nucleotide sequences with regard to length, similarity etc. are expected to be essentially similar as for sense target-specific nucleotide sequences as specified herein.

It will be clear to the person skilled in the art that the unpolyadenylated

RNA

molecule may comprise more than one target-specific nucleotide sequence and particularly that the unpolyadenylated RNA molecule may comprise sense and antisense target-specific nucleotide sequences wherein the sense and antisense nucleotide sequences are essentially complementary to each other and capable of forming an artificial hairpin structure as described in Waterhouse et al., 1998 or in PCT-application PCT/IB99/00606 (incorporated by reference).

It will also be clear that the unpolyadenylated RNA molecule may comprise one or more RNA stabilizing elements. As used herein, "an RNA stabilizing element" is a nucleotide sequence which when included into an RNA molecule prolongs the half-life time of that RNA molecule, i.e. protects it from being degraded.

Preferred RNA stabilizing elements include stable stem-loop sequences, such as the stem-loop sequences found in the mRNA encoded by the histone genes in mammalian cells, which are involved in conferring stability to the histone mRNA. An example of such a histone stem loop encoding sequence is included in SEQ ID No 7. Homologous sequences or functional equivalent sequences to the sequence of SEQ ID No 7, derived from other organisms particularly plants may also be used to the same effect.

Inclusion of such an RNA stabilizing element in an unpolyadenylated

RNA

molecule, or of a nucleotide sequence encoding such an RNA stabilizing element in a chimeric gene encoding the unpolyadenylated RNA molecule may further enhance the efficiency of gene-silencing of the target gene.

As indicated above, introduction of target-specific unpolyadenylated RNA into the nucleus of a plant cell can conveniently be achieved by transcription of a chimeric DNA encoding RNA introduced into the nucleus, preferably stably integrated into the nuclear genome of a plant cell.

In a preferred embodiment of the invention, the target-specific unpolyadenylated RNA may be produced in the nucleus of a plant cell by transcription of a chimeric DNA encoding a first target-specific RNA, which may be further processed by the action of a ribozyme also present, and preferably also encoded by a chimeric gene, in the plant cell to yield a second unpolyadenylated target-specific RNA. It will be clear for the person skilled in the art that the RNA processing need not be subsequently but can occur simultaneously. In a particularly preferred embodiment the ribozyme is a self-splicing ribozyme which is comprised within the generated target specific RNA transcript.

Thus, in a particularly preferred embodiment of the invention, a method is provided for reducing the phenotypic expression of a nucleic acid of interest, which is normally capable of being expressed in a plant cell, the method comprising the step of introducing into the nuclear genome of the plant cell a chimeric DNA to generate a transgenic plant cell, the chimeric DNA comprising the following operably linked parts: a plant-expressible promoter region; a target-specific DNA region; a DNA region encoding a self-splicing ribozyme; and a DNA region involved in 3' end formation and polyadenylation wherein the chimeric DNA when transcribed produces a first RNA molecule comprising a target specific nucleotide sequence and a selfsplicing ribozyme, which when cleaved by autocatalysis produces a second RNA molecule comprising a target specific nucleotide sequence wherein the 3' end of the first RNA molecule comprising the polyadenylation site has been removed.

The method may optionally further comprise the step of regenerating a the transgenic plant cell into a transgenic plant.

As used herein, "a ribozyme" is a catalytic RNA molecule that has the intrinsic ability to break and form covalent bonds in ribonucleic acids at specific sites in the absence of a cofactor other than a divalent cation.

As used herein a "self-splicing ribozyme" or "self-cleaving ribozyme" is a ribozyme capable of autocatalysis at a specific site within that ribozyme.

Preferred self-splicing ribozymes are self-splicing ribozymes with a so-called hammerhead structure. However, it is expected that self-cleaving ribozymes with another conformation such as the hairpin self-cleaving structures encountered in the minus strand of replication intermediates of e.g. the nepoviruses can also be used to the same effect.

Particularly preferred self-splicing ribozymes are those involved in the replication of small circular plant pathogenic RNAs, such as but not limited to the selfsplicing ribozyme from avocado sunblotch viroid, peach latent mosaic viroid, Chrysanthemum chlorotic mottle viroid, carnation stunt associated viroid, Newt satellite 2 transcript, Neurospora VS RNA, barley yellow dwarf virus satellite RNA,arabis mosaic virus satellite RNA, chicory yellow mottle virus satellite RNA S1, lucerne transient streak virus satellite

RNA,

tobacco ringspot virus satellite RNA, subterranean clover mottle virus satellite RNA, solanum nodiflorum mottle virus satellite RNA, velvet tobacco mottle virus satellite RNAvSCMoV or Cherry small circular viroid-like RNAcscRNAI. Table 1 lists different variant ribozymes suitable for the invention, as well as a reference to their nucleotide sequence.

The DNA regions encoding self-splicing ribozymes may be cDNA copies of part of the mentioned plant pathogenic RNAs comprising the ribozyme, or may be synthetic DNA. Also comprised are variants such as mutants including substitutions, deletions or insertions of nucleotides within the ribozyme nucleotide sequence in such a way that the autocatalytic capacity of the ribozymes is not substantially altered.

Preferably, the DNA region encoding the self-splicing ribozyme is located immediately upstream of the DNA region encoding the 3' end formation and polyadenylation signal. However, having read the specification, the person skilled in the art will immediately realize that the DNA region encoding the selfsplicing ribozyme may be comprised within the chimeric gene encoding the unpolyadenylated RNA at other locations, provided that a sufficiently large second RNA comprising a target-specific nucleotide wherein the polyadenylation site is removed may be generated.

It will be clear that when an RNA stabilizing element (or the DNA sequence encoding such RNA stabilizing element) is included, the RNA stabilizing element should also preferably immediately precede the DNA region encoding the selfsplicing ribozyme. However the RNA stabilizing element may be included at other locations, provided that it will be located in the unpolyadenylated

RNA

upon processing by the ribozyme.

2005202657 Table 1. Different self-cleaving ribozymes Avocado sunbioloch R1 Avo .de l1 GWh-F Oun oc visold varin 0- a b- and Sm

M

Avocado sunblotch virold variant 9-1 Rakowald and Symons 1989 V og17 Avocado sunblotch virold variant A-2 Rakowski and Spimons 1 V o 17 Avocado aunblolch viroid variant Rakowski and Symons 1989 173 Avocado aunblotch virold variant C-3 Rakowad and Symons 1989 V 173 Avocado surablotch virold variant C-4 Rakowaki and Symons 1989 V og173 Avocado subf th irl aata4E n pos u 335 3 352-356 3 352-356 3 352-356 3 352-356 352-356 352-356 352-356 Aodunthc vrald varian C4 Ilakowaki and Swmoras 1989 Rakowsk and vyrnons 1989 vocado~~~a suboc *r itB Rakowaki arid S s Rakoski nd Smons Avocado sunblotch virold variant C-5 Rakowuki and Symons 1989 Avocado sunblotch virold variant B-6 Rakowk and Symoa 1 Avcdsn blol hl,4 ~i da .i 1 Avocad sun varant C-6 fakolwmki and Sy s n Raowk and Avoca o sunblotch Yrd detC7 Raikowald and Svmons A vocado sunblotch irold variant C8 Virology 178 352-356 Virology 173 352-56 Virology 173 352-356 Virology 173 352-356 17 352-M VIrology 173352-356 VIrology 173 352-356 J. Gen VireL 75 1543-1549 J. Gen Vfrol. 75 1543-1549 Proc. Nall. Acad. Scl. 89 311-15 Proc. Nail. Acad. Sd. 89 3711-3715 M31100 M31086 M31085 M31092 M31088 M31 093 M3089 M1094 M31090 M31091 M31096 1097 574687 h Sh Ih

A

akowki and OVym s hanImerhead hmhad hammeuhead hanwnerhead hamerhead hammerhead hammerhd hammerhOd harmmnihead hammerhead hammerhead haniterhead ammerhead ammerhad ammerhead mmmrethead Iarmeriead ammerhead ammeihead unimerhead Immerhead vakowsk andb Symons 17 Jun 2005 hammerhead hmmerhed hamrhead hamrhead Avcd s oc vrl varant C-9 RakowsRki and Sy s 1989 A6 B Avocdo snbltch irod AS~d- SernanUU and ~7~~k r- -R vocad bl Av o unolh irld

V

Semanlck and Szychowski 1994 a1t P Ia-~ Is1.~I.L aa L I Pec Inten moi M dP Vd. I Henmandez and Fl m e

F

Hernandez,,, an I each atentmosai--- Peach latent Mosaic *W PL Hernandez and Flos ab. A.d

I

Fag latenit Mosaic Mid P"ch4t* anouea Peach S lawtmsacv I Ac Har 38W 52 Peach ery ltn QI MC Cana a Hadini at al.

Peach Intent mosift viroi variant gds2 Amro t al. 11998 J. rol. 72 7397-7406 Peach latent mocvirOld variant gds21 Amro t al. 1898 JI Virl. 72 7397-7406 Peach latent mosaic virold variant gda5 Ambros ot al. 1998 Viral. 72 7397-7400 Peach latent mosaic virold variant gds23 Ambros et al 1998 J. Viral. 72 7397-7406 Peach latent mosaic rald variant gdsi8 Ambros at al 1998 J. Viral. 72 7397-7406 Paad~i anu~l4..i. -7 AJ005294 AJ0052956 AJ005295 I A=5299 Kra-M Went momic Wold vd t d is ,vnuros61U at l.I UVlft 72739774AR PJUU~5UU IhmTJT~t~1 Peach latent Mamie virold variant d&3 mros at al.

J- VIrnL 72 7397.74Kl peach latent mosaic virold variant gda19 Ambros at al. 1998 Viral. 72 7397-7406 A Peach latent moaic virold variant gdal3 Ambros at al. 1998 J. ViraL 72 7397-7406 AJW5302 I hmmIorha Peach latent mosaic virold variant gds6 Ambros at al. 1998 J. Viral. 727397-7406 AJBO0 fhamnwhead Peach latent mosaic wirnad variant gdsal6 A.mbros. -t a 199 PUCh latent MOUIC WOld-wifiRdsI8 Vi Jf a. 72 7307-M46 ra 406 P r7270746 IA.1 005==5 2005202657 17 Jun 2005 Peach latent moaak vhrald variant ecl Anbrs at aL ion 1J1 O7397-740 Peach latent mosaic virod variant esc5 I. Anoros el at.

1998 lonI M ni M 7f1 wn Peach lnaentI mounln. d II-. .1wn Peach~~ latent moai l=2 Ambros f al.

Peachi latent ma11ic 1, r-Rq Peach Intent mosaic w1rold variant If) Ambro a t al.

P

eacht lataLai vi. i doa.ch.- laan mosaro ant -4 ArolDIs e al.

P

eachi Iatent maA si b Nachlatet moelc kW a s~ Ambrou at l.

h l t 1 J. VIral. 72 7397-7406 Viral. 72 7397-7406 1 Vlrol. 72 7397-7408 J- VMral. 72 7397-7408 JVra.72 7397-7406 Chic aSenmosatvtanic Wrb Anmros t al.

AJ005307 AJ005310 11 005312 AJ005313 AJ005314 00515 1 ach latent 9 v i Id Pec laten m r ih varant lab Ambros at al.

F

~mnt~h IaaM mnaal.. uI.dI .a.ma..t 5.4 4 4 Peach latent mosaic virold vi t I l AMuOr at ar.

i 72 73 -7400 Peach latent mosaic virod variant Isl8b jAmbros at al. 1998 J. Viral. 72 7397-7406 Peach latent mosaic virold variant l I broA at al. 1998 J. Viral. 72 7397-7406 Peach latent mosae virold vadant ho A mbros at al.

JViral 72 73977 6 .1vd72 739774Wa Peach latent mosaic virold variant isl~b Ambros et at. 1998 J. Viral. 72 7397-7406 Peach latent mosaic viroid variant 11b Ambros atl J. VItal. 72 7397.7406 8 Peach latent mosaic virod variant tailb Ambros stat. J. VIral. 72 73a77406 0039 Peach latent mosaic virold variant Islb Ambros at al. 1998 J. Viral. 72 7397-7406 Peach latent mosaic virold variant isl4b Ambros at al. 1996 J. Viral. 72 7397-7406 Crysanthemum chiorotic motle virold Navaro and Flres 1997 Proc. Nail. Acad. Sd. 94 1122-11 00 Barley yellow dwar virus selllte RNA Miller at al. 1991 Virology 163 711-720 Araspot mSacvrsstell N 3668 Alis Mosaic virus satellite RNA Kaper at al. 1988 Blochein. Blophys. Res. Com. 154 318-325 21212 Chcory ye matenir aellitRNA 81 -h h hammerhead harmmerhead hammerhead hammufeghead hammerhead hamrnrhead hammerhead hammnerted hammerhead hammnrnead hamrnerhead hammerhead hamnmerhad harnmerhead hamrnerhead halnerhead hammer head hammerhead 'ammerhead amrhead haramerhead hammerhead hammerhead harnmerhead hammerhead hammerhead d hamierhead hammerhead hammerhead hammerhead hammerhead hammerhead hammer head hammerhead hammerhead hammnerhead hamtmerhead hairpin harrnerhead alwy yellow mottle virus Satellite RNA 51 Rubino at al.

JGen Viral 71 18 3 J .Gen 002 Vld7 N10 Lucem 1ra '^W2 Lucerne transient 088k who satellite RNA LTSVN K ees& t a.

FEBS Lett 159 18 0 FEES Lett 159 IwiR*.

-Ii 59 I -WS*UU'IU .ucefm traniant streak virus satelite RNA LTSVC lhuhnaaw ah.4 1a~ 40 I rabaccornwtviuusgtelieRNA.1 fBuzoyan al al rTobacco ringpo virus satellite RNA.2 Iuam at 14879~ inmema 3utr~~n1Mrnthvirus satelite RNA.1I avEs stal. jigg0 Violg 173 21amm-224 1 Sleaiacoe xtvrs asteite RNA.2 Ibve t at. I la irolo 7 ~4A I__ olanumn nod MOMS vir RN hammerhead ariU no Symurm 1N82 NuclfIcidan4 sf40 1982 !Nudeld Res 10 36813091 PQzw ihwnmwhead Velvet tbaco motile virus cirlar vRroka AI I and Gymons 140es 3I8T vIw w IdU43 _ffJ1l4~5 uceo s e s.T 0814691 Cherr smsall ciroular virod-lke RNA D0i Sofeari .Vil71 WSW0O N12833 mnod. smre -mo& mo~ hhammerhead Camaion sMall virold-llke.RNA-1 Herandez at al. 1992 Nucleic Adds Res. 20 2 -62 ammehead Natoptek~svladescna(Nwl~szeitg~trnscrp: jpstaneta 188 J0Cel.hDil.merhe1a7-144arni78hamheaea Camation small virold-Ilke RNA-2 Hemandez ot al. 1992 Nucleic Acds Res. 20 6323-6329 hammerhead hammerhead Nuroporair vldescens (NwM) satellite 2 transcipt Epstein at al. 1996 Cll X04478 Neurospora VS RNA Seville and CoiWs -T990 Cael 1 8569 M32974V Aslievg 0 2005202657 17 Jun 2005 Scstosme atomsf te DNIebyeta.1g Ldesfn Riu j~irWU,4I p g I The use of ribozymes in transgenic organisms to generate RNA molecules with and or 3' termini of interest has been documented in the art. Rubio et al.

1999, describe broad-spectrum protection against Tombusviruses elicited bs defective interfering (DI) RNAs in transgenic plants. To produce RNAs with authentic 5' and 3' termini identical to those of native DI RNA, the DI RNA sequence transcribed from a DNA cassette was flanked by ribozymes.

Transgenic Nicotiana benthamiana plants were better protected than nontransgenic plants against infection by tomato bushy stunt virus and related tombusviruses. DI RNAs interfere drastically with virus accumulation through effective competition with the parental virus for transacting factors required for replication. Egli and Braus, 1994 describe uncoupling of mRNA 3' cleavage and polyadenylation by expression of a hammerhead ribozyme in yeast. Eckner et al. 1991 described that test gene transcripts which could obtain a mature histone 3' end by the RNA cleaving activity of a cis-acting ribozyme, thus circumventing the cellular 3' end processing machinery were found to be transport deficient and accumulated in the nuclear compartment. However, these documents in the art are not related to methods for inhibiting phenotypic expression by homology dependent gene-silencing, particularly by PTGS.

A particularly preferred self-splicing ribozyme is the ribozyme comprised with the Barley yellow dwarf virus (BYDV) satellite RNA, quite particularly the satellite RNA found in BYDV isolates of the RPV serotype.

It has been found that reduction of the phenotypic expression of the nucleic acid of interest using a chimeric gene according to the invention was most efficient using a cDNA copy of the ribozyme comprised within the minus strand of BYDV satellite RNA. Therefore, ribozymes which show an autocatalytic activity similar to the autocatalytic activity of the ribozyme comprised within the minus strand of -27- BYDV satellite RNA are especially suited for the methods of the invention.

Autocatalytic activity of ribozymes can be compared with the autocatalytic activity of the strand of BYDV satellite RNA as described by Miller et al. 1991.

The ribozyme motif within the strand of BYDV satellite RNA has been identified as the nucleotide sequence of SEQ ID No 1 from the nucleotide at position 194 to the nucleotide at position 236. The ribozyme motif within the strand of BYDV satellite RNA has been identified as the nucleotide sequence of SEQ ID No 2 from the nucleotide at position 310 to the nucleotide at position 322 followed by the nucleotide sequence of SEQ ID No. 2 from the nucleotide at position 1 to the nucleotide at position 89.

It goes without saying that more than one DNA region encoding a ribozyme may be comprised within the chimeric gene. These ribozymes may be clustered, e.g.

they may all be located the region immediately proceeding DNA region encoding the '3 end formation and polyadenylation signal.

However, it is expected that more than one DNA region encoding a ribozyme may be comprised within the chimeric gene in such a way that upon selfcleavage more than one unpolyadenylated RNA molecules each comprising a target-specific nucleotide sequence is generated. Such a chimeric DNA could thus comprise: a) a plant expressible promoter b) a first target-specific DNA region c) a DNA region encoding a first self-splicing ribozyme d) a second target-specific DNA region e) a DNA region encoding a second self-splicing ribozyme f) a DNA region encoding a 3' end formation and polyadenylation signal.

The first and second self-splicing ribozyme may be identical, essentially similar or different. Likewise, the first and second target-specific DNA region encoding the RNA with a target-specific nucleotide sequence may be identical, essentially similar or different.

For practical reasons, it is thought that the number of DNA regions encoding a ribozyme within a single chimeric gene should not exceed five.

In a preferred embodiment, the nucleic acid of interest, whose phenotypic expre3sion is targeted to be reduced, is a gene incorporated in the genome of a io eukaryotic cell, particularly a plant cell. It will be appreciated that the means and methods of the invention can be used for the reduction of phenotypic expression of a gene which belongs to the genome of the cell as naturally occurring, (an endogenous gene), as well as for the reduction of phenotypic expression of a gene which does not belong to the genome of the cell as naturally occurring, but has been introduced in that cell (a transgene). The transgene can be introduced stably or transiently, and can be integrated into the nuclear genome of the cell, or be present on a replicating vector, such as a viral vector.

In another preferred embodiment, the nucleic acid of interest, whose phenotypic expression is targeted to be reduced is a viral nucleic acid, particularly a viral RNA molecule, capable of infecting a eukaryotic cell, particularly a plant cell. In this case, the phenotype to be reduced is the replication of the virus, and ultimately, the disease symptoms caused by the infecting virus.

For the purpose of the invention, the term "plant-expressible promoter" means a promoter which is capable of driving transcription in a plant cell. This includes any promoter of plant origin, but also any promoter of non-plant origin which is capable of directing transcription in a plant cell. A whole range of plant -29expressible promoters, is available to direct the transcription of the chimeric genes of the invention. These include, but are not limited to strong promoters such as CaMV35S promoters Harpster et al., 1988). In the light of the existence of variant forms of the CaMV35S promoter, as known by the skilled artisan, the object of the invention can equally be achieved by employing these alternative CaMV35S promoters and variants. It is also clear that other plantexpressible promoters, particularly constitutive promoters, such as the opine synthase promoters of the Agrobacterium Ti- or Ri-plasmids, particularly a nopaline synthase promoter, or subterranean clover virus promoters can be used to obtain similar effects. Also contemplated by the invention are chimeric genes to reduce the phenotypic expression of a nucleic acid in a cell, which are under the control of single subunit bacteriophage RNA polymerase specific promoters, such as a T7 or a T3 specific promoter, provided that the host cells also comprise the corresponding RNA polymerase in an active form.

It is a further object of the invention, to provide methods for reducing the phenotypic expression of a nucleic acid in specific cells, particularly specific plant cells by placing the chimeric genes of the invention under control of tissue-specific or organ-specific promoters. Such tissue-specific or organspecific promoters are well known in the art and include but are not limited to seed-specific promoters W089/03887), organ-primordia specific promoters (An et al., 1996), stem-specific promoters (Keller et al., 1988), leaf specific promoters (Hudspeth et al. ,1989), mesophyl-specific promoters (such as the light-inducible Rubisco promoters), root-specific promoters (Keller et al.,1989), tuber-specific promoters (Keil et al., 1989), vascular tissue specific promoters Peleman et al., 1989 stamen-selective promoters WO 89/10396, WO 92/13956), dehiscence zone specific promoters WO 97/13865) and the like.

In another embodiment of the invention, the expression of a chimeric gene to reduce the phenotypic expression of a target nucleic acid can be controlled at will by the application of an appropriate chemical inducer, by operably linking the transcribed DNA region of the chimeric genes of the invention to a promoter whose expression is induced by a chemical compound, such as the promoter of the gene disclosed in European Patent publication 0332104, or the promoter of the gene disclosed in WO 90/08826.

It will be clear to the person skilled in the art that the same effect in reducing the phenotypic expression of a nucleic acid in a plant cell may be achieved using a trans-splicing ribozyme to remove at least the polyadenylation site from the RNA transcript of a chimeric gene comprising a plant expressible promoter, a targetspecific DNA region and a DNA region encoding a 3' end termination and polyadenylation signal to generate unpolyadenylated RNA comprising a targetspecific nucleotide sequence.

As used herein "a trans-splicing ribozyme" is an RNA molecule capable of catalyzing the breakage or formation of a covalent bond within another

RNA

molecule at a specific site.

The trans-splicing ribozyme should be chosen or designed in such a way that it recognizes a specific site preceding, preferably immediately preceding the polyadenylation signal of the RNA transcript comprising a target-specific nucleotide sequence. Methods to design such trans-splicing ribozyme with endoribonuclease activity are known in the art (see e.g. Haselhoff and Gerlach, 1988, WO 89/05852) The DNA region encoding a trans-splicing ribozyme may be comprised within the chimeric gene encoding the target-specific RNA. Upon transcription of the chimeric gene an RNA molecule comprising the trans-splicing ribozyme and the -31target-specific nucleotide sequence may then generated, wherein the transsplicing ribozyme is capable of cleaving a specific site preceding the polyadenylation site of another similar RNA molecule, to generate unpolyadenylated target-specific RNA molecules.

The trans-splicing ribozyme may also be provided by expression of another chimeric gene encoding an RNA molecule comprising the trans-splicing 0ribozyme in the same plant cell, according to methods and means available in Cl the art (see e.g. Vaish et al. 1998; Bramlage et al. 1998).

Alternative methods may exist to provide unpolyadenylated target-specific

RNA

to the nucleus of a plant cell. Such methods include e.g. transcription of a chimeric gene, integrated in the nuclear genome of a plant cell comprising a target-specific DNA region, by an DNA-dependent RNA polymerase different from RNA polymerase II, such that RNA transcripts are generated independent from the normal processing mRNA machinery (including intron-splicing, capping and polyadenylation). This can be achieved e.g. by operably linking the targetspecific DNA region to a promoter region, recognized by a single subunit

RNA

polymerase from a bacteriophage, such as but not limited to the T7 polymerase, and a DNA region comprising a terminator for such a polymerase. In this case, the plant cell needs to be provided with a chimeric gene encoding the corresponding RNA polymerase. Providing unpolyadenylated target-specific RNA to the nucleus of a plant cell can also be achieved e.g. by operably linking the target-specific DNA region to a promoter region, recognized by a eukaryotic RNA polymerase I or III, and a DNA region comprising a terminator for such a polymerase. The means and methods for constructing such chimeric genes and plant cells are described in detail in WO 97/49814 (incorporated by reference).

Another alternative to provide unpolyadenylated target-specific RNA to the nucleus of a plant cell may include transcription of a chimeric gene comprising a target -specific DNA region operably linked to a plant-expressible promoter and linked to a DNA region comprising a 3' end formation signal but not a polyadenylation signal.

Although not intending to limit the invention to a specific mode of action, it is expected that the trigger of the homology-dependent gene-silencing mechanisms of the cell, particularly the co-suppression mechanism, is the accumulation of target-specific RNA into the nucleus of that cell. Providing unpolyadenylated RNA to the nucleus of the cell may be one mechanism of causing accumulation of target-specific RNA in a nucleus of a cell, but other aberrations such as the absence of a cap-structure or the presence of persistent introns etc. may constitute alternative ways to cause the accumulation of targetspecific RNA in the nucleus of a cell.

Moreover, it is expected that other aberrations in the target-specific

RNA

molecules in addition to the absence of the polyA tail, including the absence of a cap-structure, or the presence of persistent introns or the presence of abnormal secondary structures, particularly the presence of giant hairpin structures, may have a cumulative effect on the inhibition of the normal transit of the RNA from the nucleus to the cytoplasm and hence have a cumulative or synergystic effect on the reduction of the phenotypic expression of a nucleic acid of interest.

The recombinant DNA comprising the chimeric gene to reduce the phenotypic expression of a nucleic acid of interest in a host cell, may be accompanied by a chimeric marker gene, particularly when the stable integration of the transgene in the genome of the host cell is envisioned. The chimeric marker gene can comprise a marker DNA that is operably linked at its 5' end to a promoter, functioning in the host cell of interest, particularly a plant-expressible promoter, preferably a constitutive promoter, such as the CaMV 35S promoter, or a light -33inducible promoter such as the promoter of the gene encoding the small subunit of Rubisco; and operably linked at its 3' end to suitable plant transcription 3' end formation and polyadenylation signals. It is expected that the choice of the marker DNA is not critical, and any suitable marker DNA can be used. For example, a marker DNA can encode a protein that provides a distinguishable colour to the transformed plant cell, such as the Al gene (Meyer et al., 1987), can provide herbicide resistance to the transformed plant cell, such as the bar gene, encoding resistance to phosphinothricin (EP 0,242,246), or can provide antibiotic resistance to the transformed cells, such as the aac(6') gene, encoding resistance to gentamycin (W0O94/01560).

A recombinant DNA comprising a chimeric gene to reduce the phenotypic expression of a gene of interest, can be stably incorporated in the nuclear genome of a cell of a plant. Gene transfer can be carried out with a vector that is a disarmed Ti-plasmid, comprising a chimeric gene of the invention, and carried by Agrobacterium. This transformation can be carried out using the procedures described, for example, in EP 0 116 718.

Alternatively, any type of vector can be used to transform the plant cell, applying methods such as direct gene transfer (as described, for example, in EP 0 233 247), pollen-mediated transformation (as described, for example, in EP 0 270 356, W085/01856 and US 4,684,611), plant RNA virus-mediated transformation (as described, for example, in EP 0 067 553 and US 4,407,956), liposomemediated transformation (as described, for example, in US 4,536,475), and the like.

Other methods, such as microprojectile bombardment as described for corn by Fromm et aL. (1990) and Gordon-Kamm et al. (1990), are suitable as well. Cells of monocotyledonous plants, such as the major cereals, can also be transformed using wounded and/or enzyme-degraded compact embryogenic tissue capable of forming compact embryogenic callus, or wounded and/or degraded immature embryos as described in WO92/09696. The resulting transformed plant cell can then be used to regenerate a transformed plant in a conventional manner.

The obtained transformed plant can be used in a conventional breeding scheme to produce more transformed plants with the same characteristics or to introduce the chimeric gene for reduction of the phenotypic expression of a nucleic acid of interest of the invention in other varieties of the same or related plant species, or in hybrid plants. Seeds obtained from the transformed plants contain the chimeric genes of the invention as a stable genomic insert.

The means and methods of the invention can also be used for the reduction of gene expression by co-suppression in eukaryotic cells and organisms.

In one embodiment the invention provides a method for reducing the phenotypic expression of a nucleic acid of interest, which is normally capable of being expressed in a eukaryotic cell, comprising the step of providing unpolyadenylated RNA comprising a target specific sense nucleotide sequence of at least 10 consecutive nucleotides with at least about 70% sequence identity to about 100% sequence identity to the nucleotide sequence of the nucleic acid of interest, to the nucleus of the eukaryotic cell.

In another embodiment, a method is provided for reducing the phenotypic expression of a nucleic acid of interest, which is normally capable of being expressed in a eukaryotic cell, comprising the step of introducing into the nuclear genome of the eukaryotic cell a chimeric DNA to generate a transgenic plant cell, DNA comprising the following operably linked parts: a promoter region functional in the eukaryotic cell; a target-specific DNA region comprising nucleotide sequence of at least 10 consecutive nucleotides with at least about sequence identity to about 100% sequence identity to the nucleotide sequence of the nucleic acid of interest; s a DNA region encoding a self-splicing ribozyme; and a DNA region involved in 3' end formation and polyadenylation wherein the chimeric DNA when transcribed produces a first RNA molecule comprising a target specific nucleotide sequence and a self-splicing ribozyme, which when cleaved by autocatalysis produces a second RNA molecule comprising a target specific nucleotide sequence wherein the 3' end of the first RNA molecule comprising the polyadenylation site has been removed.

Different preferred embodiments and definitions described in connection with the reduction of gene expression by homology dependent gene silencing in plant cells and plants also apply mutatis mutandis to the means and methods described for reduction of gene expression by co-suppression in eukaryotic cells and organisms. As used herein "eukaryotic cells" comprise plant cells, animal cells and human cells and cells from yeasts and fungi as well as cultures of such cells.

It is a further object of the invention to provide eukaryotic cells, preferably plant cells and organisms (preferably plants) comprising the chimeric genes for the reduction of the phenotypic expression of a target nucleic acid as described in the invention.

The methods and means of the invention can thus be used to reduce phenotypic expression of a nucleic acid in a eukaryotic cell or organism, particularly a plant cell or plant, for obtaining shatter resistance (WO 97/13865), for obtaining modified flower colour patterns (EP 522 880, US 5,231,020), for obtaining nematode resistant plants (WO 92/21757, WO 93/10251, WO 94/17194), for delaying fruit ripening (WO 91/16440, WO 91/05865, WO 91/16426,

WO

92/17596, WO 93/07275, WO 92/04456, US 5,545,366), for obtaining male sterility (WO 94/29465, W089/10396, WO 92/18625), for reducing the presence of unwanted (secondary) metabolites in organisms, such as glucosinolates (W097/16559) or chlorophyll content (EP 779 364) in plants for modifying the profile of metabolites synthesized in a eukaryotic cell or organisms by metabolic engineering e.g. by reducing the expression of particular genes involved in carbohydrate metabolism (WO 92/11375, WO 92/11376, US 5, 365, 016, WO 95/07355) or lipid biosynthesis (WO 94/18337, US 5, 530, 192), for delaying senescence (WO 95/07993), for altering lignification in plants (WO 93/05159, WO 93/05160), for altering the fibre quality in cotton (US 5, 597, 718), for is increasing bruising resistance in potatoes by reducing polyphenoloxidase

(WO

94/03607), etc.

The methods of the invention will lead to better results and/or higher efficiencies when compared to the methods using conventional sense or antisense nucleotide sequences and it is believed that other sequence-specific mechanisms regulating the phenotypic expression of target nucleic acids might be involved and/or triggered by the presence of the double-stranded

RNA

molecules described in this specification.

A particular application for reduction of the phenotypic expression of a transgene in a plant cell, inter alia, by antisense or sense methods, has been described for the restoration of male fertility, the latter being obtained by introduction of a transgene comprising a male sterility DNA (WO 94/09143, WO 91/02069). The nucleic acid of interest is specifically the male sterility DNA.

Again, the processes and products described in this invention can be applied to these methods in order to arrive at a more efficient restoration of male fertility.

It will be appreciated that the methods and means described in the specification can also be applied in High Throughput Screening (HTS) methods, for the identification or confirmation of phenotypes associated with the expression of a nucleic acid sequence with hitherto unidentified function in a eukaryotic cell, particularly in a plant cell.

Such a method comprises the steps of: 1. selecting a target sequence within the nucleic acid sequence of interest with unidentified or non-confirmed function/phenotype when expressed. Preferably, if the nucleic acid has putative open reading frames, the target sequence should comprise at least part of one of these open reading frames. The length of the target nucleotide sequence may vary from about 10 nucleotides up to a length equalling the length (in nucleotides) of the nucleic acid of interest with unidentified function.

2. Introducing a chimeric DNA into the nucleus of a suitable host cell, comprising the nucleic acid of interest, wherein the chimeric DNA comprises a promoter region suitable for expression in the host cell, a DNA region encoding the target-specific nucleotide sequence, and a DNA region encoding a self-splicing ribozyme located immediately upstream of a DNA region involved in 3' end formation and polyadenylation.

3. observing the phenotype by a suitable method. Depending on the phenotype expected, it may be sufficient to observe or measure the phenotype in a single cell, but it may also be required to culture the cells to obtain an (organized) multicellular level, or even to regenerate a transgenic organism, particularly a transgenic plant.

It is also clear that the methods and means of the invention are suited for the reduction of the phenotypic expression of a nucleic acid in all plant cells of all plants, whether they are monocotyledonous or dicotyledonous plants, particularly crop plants such as but not limited to corn, rice, wheat, barley, sugarcane, cotton, oilseed rape, soybean, vegetables (including chicory, brassica vegetables, lettuce, tomato), tobacco, potato, sugarbeet but also plants used in horticulture, floriculture or forestry. The means and methods of the invention will be particularly suited for plants which have complex genomes, such as polyploid plants.

It is expected that the chimeric RNA molecules produced by transcription of the chimeric genes described herein, can spread systemically throughout a plant, and thus it is possible to reduce the phenotypic expression of a nucleic acid in cells of a non-transgenic scion of a plant grafted onto a transgenic stock comprising the chimeric genes of the invention (or vice versa) a method which may be important in horticulture, viticulture or in fruit production.

The following non-limiting Examples describe the construction of chimeric genes for the reduction of the phenotypic expression of a nucleic acid of interest in a eukaryotic cell and the use of such genes. Unless stated otherwise in the Examples, all recombinant DNA techniques are carried out according to standard protocols as described in Sambrook et al. (1989) Molecular Cloning:

A

Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, NY and in Volumes 1 and 2 of Ausubel et al. (1994) Current Protocols in Molecular Biology, Current Protocols, USA. Standard materials and methods for plant molecular work are described in Plant Molecular Biology Labfax (1993) by R.D.D. Croy, jointly published by BIOS Scientific Publications Ltd (UK) and Blackwell Scientific Publications,

UK.

Throughout the description and Examples, reference is made to the following sequences: SEQ ID No 1: cDNA copy of the strand of BYDV RPV satellite RNA SEQ ID No 2: cDNA copy of the strand of BYDV RPV satellite RNA SEQ ID No 3: oligonucleotide for PCR amplification (SatPR1) SEQ ID No 4: oligonucleotide for PCR amplification (SatPR2) SEQ ID No 5: oligonucleotide for PCR amplification (SatPR3) SEQ ID No 6: oligonucleotide for PCR amplification (SatPR4) SEQ ID No 7: nucleotide sequence encoding a histone stem from mammalian histone genes.

The following examples are presented in order to more fully illustrate the preferred embodiments of the invention and should not be construed, however, as limiting the broad scope of the invention.

Examples Example 1 Experimental procedures 1.1 Chimeric DNA Constructs Ribozyme-containing GUS gene constructs and a control construct The ribozyme sequences used are the plus strand or negative strand selfcleavage sequences of the satellite RNA of the barley yellow dwarf virus (BYDV) RPV serotype, which was isolated in CSIRO Plant Industry (SEQ ID 1 and 2; Miller et al., 1991).

The two ribozyme-containing GUS constructs (pMBW259 and pMBW267) and one control GUS construct (pMBW265) are schematically drawn in Figure 1.

pMBW259 contains two plus strand cleavage sites, while pMBW267 contains the negative strand cleavage site.

To make these constructs, a A-glucuronidase (GUS) gene sequence was modified to contain a Ncol site around the translational start ATG and cloned into pART7 (Gleave, 1992) at the Xhol/EcoRI sites, forming pMBW258. The fulllength BYDV-RPV satellite sequence was amplified by PCR using primers SatPRI (SEQ ID No. 3) and SatPR4 (SEQ ID No. digested with BamHI and cloned into pMBW258 at the BamHI site, and the resulting cassette was excised and cloned into pART27 (Gleave, 1992), forming pMBW265. The same full-length satellite sequence was inserted into the BamHI site of pMBW258 but in the antisense orientation, and the resulting asSat-ocs was cloned into pART27 to give rise to pMBW267.

To make pMBW259, the 3' and 5' halfs of the satellite RNA sequences were amplified by PCR using primer pairs SatPR3 (SEQ ID No. 5) and SatPR4 (SEQ ID No. 6) and using SatPR1 (SEQ ID No. 3) and SatPR2 (SEQ ID No 4), -41respectively. Fusion of the full-length sequence with the 3' half and the 5' half sequences were made through ligation between the EcoRV and Hpal ends of the three PCR fragments. This fusion mimics the natural multimeric forms of the satellite RNA, and therefore maintains the plus strand cleavae property of the native forms. The fusion sequence was cloned into pGEM-3Z (Promega) at the Sacl/Pstl sites, excised with HindllVEcoRI, blunted, and inserted into pART7 at the Smal site, into which the GUS sequence described above was then cloned at the Xhol/EcoRI sites. The resulting 3SS-GUS-Sat-ocs was inserted into pART27 at the Notl site, forming pMBW259.

The super-transforming GUS construct The BamHI fragment was excised from plG121 Hm (Hiei et al.,1994) and cloned into pART7. The GUS-nos sequence was then excised by AccI, blunted, and inserted into pBluescript at the Hincl site. The 1.3 kb promoter region of a cucurbit phloem protein PP2 gene was excised with Notl/Hindll irom a lambda clone CPPI.3 and cloned into the above Bluescript plasmid. The resulting PP2- GUS-nos was excised with Notl/Kpnl and inserted into pWBVec2 (Wang et al., 1998), giving rise to pBPPGH (Fig. 1).

1.2 Tobacco transformation Nicotiana tobaccum cv. W38 was transformed and regenerated into whole plants essentially as described by Ellis et al. 1987. For constructs pMBW259, pMBW265 and pMBW267, 50 mg/L kanamycin was included in the media for selection of transformed tissue. For construct pBPPGH, 25 mg/L hygromycin B was used.

1.3 GUS assay GUS gene expression was assayed histochemically or fluorometrically according to Jefferson et al. 1987.

-42- Example 2: GUS expression in transgenic tobacco transformed with a single type of the GUS constructs.

Transgenic plants containing pMBW259 aid pMBW267 showed very low levels V 5 of GUS expression, as judged by lack of, or faint blue, GUS staining. Plants Ntransformed with pMBW265 showed more GUS expression than with pMBW259 and pMBW267, but the level was much lower than plants transformed with pBPPGH. The best pMBW265 lines expressed 13.3% of the GUS activity by an average PBPPGH line.

Example 3 GUS expression in super-transformed lines containing pBPPGH and one of the three other constructs of Example 1.

In order to promote silencing of a normal GUS gene by the presence of the ribozyme sequence near the 3' end of the GUS gene transcript, plants containing pMBW259, pMBW265 or pMBW267 and pBPPGH were constructed by re-transformation. Histochemical GUS assays of the super-transformants showed that the pMBW267 background gave substantially higher proportions of transformants than the pMBW259 or the pMBW265 background that showed Q 20 low levels of GUS expression as indicated by the lack of strong and uniform blue staining. Super-transformants containing pBPPGH and pMBW265 showed the best GUS expression.

Table 2 shows the result of fluorometric GUS (MUG) assay of the supertransformants. The lines (E and F) containing pBPPGH and pMBW267 showed uniformly low GUS expression compared with the other lines. The best

GUS

expression came from the C lines which contain pBPPGH and pMBW265.

Among the three constructs tested, pMBW265 does not contain the full-length functional ribozyme sequences of the BYDV satellite RNA in a continuous stretch, and is therefore expected to produce mainly poly(A)+ RNA. pMBW259 contains two copies of the plus strand riboz~ime sequence, and should give rise to RNA that have poly(A) tails removed by ribozyme cleavage. pMBW267 contain the negative strand ribozyme. The negative strand ribozyme was previously shown to be much (at least 10-fold) more efficient than the plus strand ribozyme (Miller et al., 1991), and therefore it is expected that pMBW267 produces poly(A)- RNA more efficiently. Our experiment showed that the supertransformed lines having the pMBW267 background expressed uniformly low levels of GUS activity in comparison with the lines having the pMBW259 or the pMBW265 background. The highest GUS expressing lines were from the pMBW265 background, which does not produce polyA- RNA.

Table 2. MUG assay of superrnfmeItbcoles Super- MUG Super- MUG Super-

MUG

transformed Readings transformed Readings transformed Readings lines lines Al 10.1 Cl 8.84 El 4.32 A2 15.8 C2 16.9 E2 3.15" A3 30.6 C3 17.9 E3 3.56 A4 47.3 C4, 22.8 E4 3.31 AS 0.29 CS 11.7 E5 3.68 A6 10.3 C6 14.5 E6 5.02 A7 5.8 C7 44.0 E7 263 A8 13.15S C8 19.0 E8 10.27 A9 7.34 C929.8 E9 10.81 AIO 9.76 CIO 32.1 EIO' .13.1 All 17.74 C11 37.1 Ell 5.10 A12 34.8 -C12 2.51 E12 2.86 A13 4.33 C13 14.5 E13 4.00 A14 3.41 C14 25.8 E14 16.8 11.2 cis 1.20 E15 4.02 A16 2.04 C16 30.2 E16 1.29 Al7 13.29 C17 9.70 E17 1.18 A18 14.6 C18 13.4 E18 3.51 A-19 0.14 C19 19.3 E19 0.43 11.2 C20 11.0 020 11.8 A21 9.22 DI 6.01 F1 5.73 A22 11.3 D2 12.9 F2 5.10 61 9.51 D3 0.19 F3 4.16 B2 44.7 D4 1.88 F4 4.69 83 11.7 D5 1.24 F5 0 B4 1.25 D6 0.44 F6 1.93 B5'35D7 14.1 Fl 3.21 B114D8 0.91 F8 Z.7 B6.8D9 5.49 F9 1.86 B248D10 1.30 NIO 3.21 B163D11 15.1 F11. 285 BI .2D12 6.63 F12 3.25 D313711l3 12.2 F13 2.11 B1 .8D14 15.8 F14 2.84 B1 06DIS 1.32 F15 3.11 614 11.9 016 2.2P F16 2.06 3.11 D17 3.5; F17 2.90 816 8.25 D18 22., F18 3.15 B74.12 1D19 1 3.0 F19 4.16 6.048 D20 14.37 F20 12.49 A and B, from super-transformation of two independent pMBW259 lines with pBPPGH; C and D, from super-transformation of two independent pMBW265 lines with pBPPGH; E and F, from super-transformation of two independent pMBW267 lines with pBPPGH.

Example 4. Additional chimeric DNA constructs Additional chimeric DNA constructs are made using conventional DNA cloning techniques and introduced in plants comprising the appropriate target genes GUS silencing constructs type 1 SRibozyme containing GUS constructs similar to pMBW259 and pMBW267 (see Example 1) are adapted to include a nucleotide sequence encoding an RNA stabilizing element (histone stem form mammalian histone genes; SEQ ID No 7) between the nucleotide sequence derived from the GUS gene and upstream of the ribozyme encoding DNA region.

GUS silencing constructs type 2 Ribozyme containing GUS constructs similar to pMBW259 and pMBW267 (see Example but wherein the nucleotide sequence derived from the GUS gene are in antisense orientation opposite to these homologous sequences in pMBW259 and pMBW267) are adapted to include a nucleotide sequence encoding an RNA stabilizing element (histone stem form mammalian histone genes; SEQ ID No 7) between the nudeotide sequence derived from the GUS gene and upstream of the ribozyme encoding DNA region.

GUS silencing constructs type 3 Chimeric Gus silencing genes are constructed similar to the chimeric GUS silencing genes described in WO 99/53050 (particularly page 36) comprising an -46additional DNA region encoding a ribozyme between the DNA region encoding the hairpin RNA and the DNA region encoding the transcription termination and polyadenytation. These constructs comprise the following elements a CaMV35S promoter (as described in Ekample 1) a nucleotide sequence of at least 500 bp derived from the GUS gene in sense orientation a spacer nucleotide sequence comprising about 700 bp of the PVY Nia gene, see W099/53050) the complement of the nucleotide sequence derived from the GUS gene (i.e.

part of the GUS gene in antisense orientation) a ribozyme encoding DNA region as in pMBW259 and pMBW267 (Example 1) an ocs-T terminator (as described in Example 1) PVY resistance constructs Chimeric PVY resistance genes are constructed comprising the following elements a CaMV35S promoter (as described in Example 1) a nucleotide sequence comprising about 700 bp of the PVY Nia gene, see W099/53050) in sense orientation a spacer nucleotide sequence part of the GUS gene) the complement of the nucleotide sequence derived from PVY part of the PVY sequence in antisense orientation) a ribozyme encoding DNA region as in pMBW259 and pMBW267 (Example 1) an ocs-T terminator (as described in Example 1) When Gus silencing constructs are analysed, the transgenic plants comprise a functional GUS transgene and the silencing constructs are introduced either by -47direct transformation of transgenic GUS gene containing plants or by crossing appropriate transgenic plants.

When PVY silencing constructs are used, tiinsgenic plants comprising the PVY silencing constructs are inoculated with PVY, according to standard-methods (see WO 99/53050).

In transgenic plants containing a GUS transgene, GUS expression is efficiently silenced upon introduction of the GUS silencing constructs in the majority of the obtained transgenic lines.

Transgenic plants containing the PVY resistance genes, are extremely resistant to infection by PVY in the majority of the obtained transgenic lines.

-48-

REFERENCES

An et al., 1996 The Plant Cell 8: 15-30 Bramlage et al. 1998 TIBTECH 16, 434-438 Covey et al., 1997 Nature 385:781-782 Eckner et al. 1991 EMBO J. 10: 3513-3522 Egli and Braus, 1994 J. Biol. Chem. 1994 269:, 27378-27383 Ellis et al. 1987 EMBO Journal, 6: 11-16 Fromm et al. j1 990 Bia/Technology 8: 833 Gleave, 1992 Plant Mol. Biol. 20: 1203-1207 Gordon-Kamm et al. .1990 The Plant Cell 2: 603 Hamilton et al. 1998 The Plant Journal 15(6): 737-746 Harpster et al., 1988 Mol. Gen. Genet 212, 182-190 Haselhoff and Gerlach, 1988 Nature 334 585-591 Hiei et al., 1994 Plant Journal 6: 271 -282 Hudspeth et al., 1989 Plant Mol Biol 12: 579-589 Jefferson et al., 1987 EMBO J. 6, 3901-3907 Keil et al., 1989 EMBO J. 8: 1323-1330 Keller et 1988 EMBO J. 7: 3625-3633 Keller et al., 1989 Genes Devel. 3: 1639-1646 Lee et al. 1997 Plant Journal 12: 1127-1137 Mette et al., 1999 EMBO J 18:241-248 Metzlaff et al. 1997 Cell 88, 845-854 Miller et al., 1991 Virology 183: 711-720, 1991 Peleman et al., 1989 Gene 84: 359-369 Rubio et al. 1999 J. Virology 73: 5070-5078 Vaish et al. 1998 Nucleic Acids Res. 26: 5237-5242 van Eldik et al. 1998 Nucleic Acids Res. 26: 5176-5181 van Houdt et al., 1997 Plant Journal 12: 379-392 -49- Wang et al., 1998 Acta Horticulturae 461:1-407 Wassenegger and P6lissier, 1998 Plant Mol. Biol. 37 349-362 Waterhouse et al. 1998 Proc. Nat. Acad. Sci USA 95:13959-13964 Wilbur and Lipmann, 1983 Proc. Nat."Acad-Sci. U.S.A. 80: 726

Claims (42)

1. A method for reducing the phenotypic expression of a nucleic acid of interest in an 0 animal cell wherein the nucleic acid is capable of being expressed in the animal cell, said method comprising the step of providing to the nucleus of said animal cell unpolyadenylated RNA comprising a target specific sense nucleotide sequence and a target specific antisense nucleotide sequence, wherein said target specific sense nucleotide sequence comprises at least about 20 consecutive nucleotides having a sequence at least about 85% identical to the IN sequence of a corresponding part of said nucleic acid of interest, wherein said target specific antisense nucleotide sequence comprises at least about 20 consecutive nucleotides having a sequence at least about 85% identical to the sequence of the complement of part of an RNA molecule transcribed or produced from said nucleic acid of interest, wherein said target Ni specific sense nucleotide sequence and said target specific antisense nucleotide sequence are essentially complementary to each other.

2. The method of claim I wherein said target specific sense nucleotide sequence is at least about 95% identical to said sequence of a corresponding part of said nucleic acid of interest and said target specific antisense nucleotide sequence is at least about 95% identical to said sequence of the complement of part of said RNA molecule transcribed or produced from said nucleic acid of interest.

3. The method of either claim 1 or claim 2 wherein the animal cell is a human cell.

4. The method according to any of the above claims wherein said animal cell is a cultured cell. The method according to any of the above claims wherein said target specific sense nucleotide sequence corresponds to part of said nucleic acid of interest from which RNA is produced.

6. The method of claim 5 wherein said target specific sense nucleotide sequence corresponds to one or more consecutive exons of said nucleic acid of interest.

7. The method according to either claim 5 or claim 6 wherein said target specific sense nucleotide sequence corresponds to a translated region of said nucleic acid of interest.

8. The method of claim 5 wherein said target specific sense nucleotide sequence corresponds to an untranslated region of the RNA produced from said nucleic acid of interest.

9. The method according to any one of the above claims wherein said unpolyadenylated RNA is produced by transcription of a chimeric DNA comprising a promoter operably linked to a target specific DNA region encoding said RNA. 201557536 The method of claim 9 wherein the promoter is a constitutive promoter. O

11. The method of claim 9 wherein the promoter is an inducible promoter. 0 12. The method according to any one of claims 9 to 11 wherein the promoter is a -Z tissue-specific promoter.

13. The method according to any one of claims 9 to 12 wherein the promoter is recognized by a single subunit RNA polymerase from a bacteriophage.

14. The method according to any one of claims 9 to 12 wherein the promoter is Srecognized by a eukaryotic RNA polymerase I or III and said chimeric DNA further comprises a terminator for said polymerase I or III. 10 15. The method according to any one of the above claims wherein the nucleic acid of interest is a gene incorporated in the genome of said animal cell.

16. The method according to any one of the above claims wherein the nucleic acid of interest is an endogenous gene of said animal cell.

17. The method according to any one of claims I to 15 wherein the nucleic acid of interest is a transgene that has been introduced into said animal cell.

18. The method according to any one of claims 1 to 15 or claim 17 wherein the nucleic acid of interest is a viral nucleic acid.

19. The method according to any one of the above claims wherein said unpolyadenylated RNA lacks a 5' cap structure.

20. The method according to any one of the above claims wherein said unpolyadenylated RNA comprises a persistent intron.

21. The method according to any one of the above claims wherein said target specific sense nucleotide sequence and said target specific antisense nucleotide sequence are capable of forming an artificial hairpin structure.

22. The method according to any one of the above claims comprising the steps of a) selecting within the nucleic acid of interest a target sequence of at least consecutive nucleotides b) introducing a chimeric DNA into the nucleus of said animal cell comprising the nucleic acid of interest, the chimeric DNA encoding said unpolyadenylated RNA, and 201557536 -52- c) observing a phenotype of said animal cell by a suitable method; O thereby identifying a phenotype associated with the expression of said nucleic acid of interest in said animal cell. O Z 23. The method of claim 22 wherein the phenotype associated with the expression of said nucleic acid of interest in said animal cell was hitherto unidentified.

24. The method according to either claim 22 or claim 23 wherein the phenotype is Vobserved after culturing of the animal cells. (N The method according to any one of claims 22 to 24 wherein the phenotype is a Vmodified profile of metabolites synthesized in said cell. S 10 26. An isolated unpolyadenylated RNA comprising a target specific sense nucleotide sequence and a target specific antisense nucleotide sequence, wherein said target specific sense nucleotide sequence comprises at least about 20 consecutive nucleotides having a sequence at least about 85% identical to the sequence of a corresponding part of a nucleic acid of interest, wherein said target specific antisense nucleotide sequence comprises at least about 20 consecutive nucleotides having a sequence at least about 85% identical to the sequence of the complement of part of an RNA molecule transcribed or produced from said nucleic acid of interest, wherein said target specific sense nucleotide sequence and said target specific antisense nucleotide sequence are essentially complementary to each other, wherein said nucleic acid of interest is normally present in an animal cell and wherein said unpolyadenylated RNA reduces expression of said nucleic acid of interest when provided to the nucleus of said cell.

27. The isolated unpolyadenylated RNA of claim 26 wherein the animal cell is a human cell.

28. The isolated unpolyadenylated RNA of either claim 26 or 27 wherein said target specific sense nucleotide sequence is at least about 95% identical to said sequence of a corresponding part of said nucleic acid of interest and said target specific antisense nucleotide sequence is at least about 95% identical to said sequence of the complement of part of said RNA molecule transcribed or produced from said nucleic acid of interest.

29. The isolated unpolyadenylated RNA of any one of claims 26 to 28 wherein the nucleic acid of interest is a gene incorporated in the genome of said animal cell. The isolated unpolyadenylated RNA of any one of claims 26 to 29 wherein the nucleic acid of interest is an endogenous gene of said animal cell. 201557536

31. The isolated unpolyadenylated RNA of any one of claims 26 to 29 wherein the 0 nucleic acid of interest is a transgene that has been introduced into said animal cell.

32. The isolated unpolyadenylated RNA of any one of claims 26 to 29 or claim 31 0 wherein the nucleic acid of interest is a viral nucleic acid. 5 33. The isolated unpolyadenylated RNA of any one of claims 26 to 32 which lacks a cap structure. 1^ V) 34. The isolated unpolyadenylated RNA of any one of claims 26 to 33 which Scomprises a persistent intron. The isolated unpolyadenylated RNA of any one of claims 26 to 34 wherein said target specific sense nucleotide sequence and said target specific antisense nucleotide sequence are capable of forming an artificial hairpin structure.

36. An animal cell comprising the unpolyadenylated RNA of claim 26.

37. The animal cell of claim 36 which is a human cell.

38. The animal cell of either claim 36 or claim 37 which is a cultured cell.

39. The cell according to any one of claims 36 to 38 which has a modified phenotype compared to a corresponding animal cell lacking said unpolyadenylated RNA or chimeric DNA. A non-human animal comprising the cell of either claim 36 or claim 37.

41. A chimeric DNA encoding the isolated unpolyadenylated RNA of any one of claims 26 to

42. A chimeric DNA of claim 41 wherein the encoded unpolyadenylated RNA comprises a target specific sense nucleotide sequence that is at least about 95% identical to said sequence of a corresponding part of said nucleic acid of interest and a target specific antisense nucleotide sequence that is at least about 95% identical to said sequence of the complement of part of said RNA molecule transcribed or produced from said nucleic acid of interest.

43. A chimeric DNA of either one of claim 41 or claim 42 wherein the encoded unpolyadenylated RNA comprises a target specific sense nucleotide sequence that corresponds to part of said nucleic acid of interest from which RNA is produced. 201557536

44. A chimeric DNA of any one of claims 41 to 43 wherein the encoded C unpolyadenylated RNA comprises a target specific sense nucleotide sequence that corresponds Sto one or more consecutive exons of said nucleic acid of interest. z 45. A chimeric DNA of any one of claims 41 to 44 wherein the encoded M€ 5 unpolyadenylated RNA comprises a target specific sense nucleotide sequence that corresponds to a translated region of said nucleic acid of interest.

46. A chimeric DNA of claim 43 wherein the encoded unpolyadenylated RNA V)0 comprises a target specific sense nucleotide sequence that corresponds to an untranslated region of the RNA produced from said nucleic acid of interest.

47. A chimeric DNA according to any one of claims 41 to 46 comprising a promoter operably linked to a target specific DNA region encoding said RNA.

48. A chimeric DNA of claim 47 wherein the promoter is a constitutive promoter.

49. A chimeric DNA of claim 47 wherein the promoter is an inducible promoter. A chimeric DNA of any one of claims 47 to 49 wherein the promoter is a tissue- specific promoter.

51. A chimeric DNA of any one of claims 47 to 50 wherein the promoter is recognized by a single subunit RNA polymerase from a bacteriophage.

52. A chimeric DNA of any one of claims 47 to 50 wherein the promoter is recognized by a eukaryotic RNA polymerase I or III and said chimeric DNA further comprises a terminator for said polymerase I or III.

53. An animal cell comprising the chimeric DNA of any one of claims 41 to 52.

54. The animal cell of claim 53 which is a human cell. The animal cell of either claim 53 or claim 54 which is a cultured cell.

56. The cell of any one of claims 53 to 55 which has a modified phenotype compared to a corresponding animal cell lacking said unpolyadenylated RNA or chimeric DNA.

57. A non-human animal comprising the cell of any one of claims 53, 54 and 56. 201557536