AU714791B2

AU714791B2 - Improvements in or relating to gene expression

Info

Publication number: AU714791B2
Application number: AU18014/97A
Authority: AU
Inventors: James Phillip Haseloff; Sarah Hodge
Original assignee: British Technology Group Ltd
Current assignee: BTG International Ltd
Priority date: 1996-02-14
Filing date: 1997-02-14
Publication date: 2000-01-13
Anticipated expiration: 2017-02-14
Also published as: ZA971272B; GB9603069D0; WO1997030164A1; JP2001501805A; EP0880593A1; NZ331220A; CA2246414A1; CN1216067A; AU1801497A

Description

WO 97/30164 PCT/GB97/00406 Title: Improvements in or Relating to Gene Expression Field of the Invention This invention relates to a nucleic acid sequence optimised for expression in a plant cell, and to vectors and plant cells comprising the nucleic acid sequence.

Background of the Invention In yeast there is a gene called GAL4, the product of which acts as a transcriptional activator (Johnston 1987 Microbiol. Rev. 51, 458-476). The GAL4 gene is quite large (3Kb) and the encoded polypeptide comprises an N-terminal DNA-binding domain, a C terminal domain having a transcriptional activator function, and an intervening glucoseresponsive element "GRE" (GAL4 being repressed in the presence of glucose).

The GAL4 protein has been quite well characterised. Amino acid residues 1-147 of the protein bind to DNA in a sequence-specific manner (Keegan et al 1986 Science 231, 699- 704). The transcriptional activation function is associated with two short portions of the C terminal domain (Ma Ptashne 1987 Cell 48, 847-853). The DNA sequence to which GAL4 binds has been identified as a 17mer, which must be present as a repeat for optimal GAL4 binding (Giniger et al, 1985 Cell 40, 767-774), and may be referred to as a GAL4responsive "upstream activation sequence" (UAS): GAL4 binds to the UAS 5' of a gene by means of the DNA-binding domain, and the C-terminal domain causes up-regulation of transcription of the gene.

Recently, a two-element system has been developed for directing gene expression in Drosophila melanogaster. Brand and Perrimon (1993 Development 118: 401-415,) randomly inserted the gene encoding the GAL4 into the Drosophila genome using a P- WO 97/30164 PCT/GB97/00406 2 element based vector.

A large number of stable Drosophila lines were generated which each express GAL4 in a particular pattern, dependent on adjacent genomic DNA sequences. A chosen target gene could then be cloned under the control of GAL4 upstream activation sequences (UAS), separately transformed, and maintained silently in the absence of GAL4. Genetic crossing of this single line with any of the library of GAL4-containing lines allowed activation of the target gene in many different tissue and cell types, and the phenotypic consequences of mis-expression, including those lethal to the organism, could be conveniently studied. In addition, the library of GAL4-containing flies has become an increasingly characterised and shared resource, and which provides a common entry point for various "reverse" genetic techniques.

A similar system operable in plants would be highly desirable. However, expression of heterologous eukaryotic genes in plants has proved highly problematical in the past (see, for example, Green Fluorescent Protein) and efficient expression of GAL4 appears equally difficult, and it has been suggested that this is due to inefficient translation of GAL4 mRNA in plants (Reichel et al, 1995 Plant Cell Reports 14, 773-776). Ma et al, (1988 Nature 334, 631-633) were able to obtain transient expression of modified, functional GAL4 derivatives in tobacco-leaf protoplasts but could not demonstrate the presence of functional, full-length wild-type GAL4. Similarly, transient expression of GALA derivatives has been demonstrated in maize protoplasts (McCarty et al, 1991 Cell 66, 895- 905) and when introduced by biolistic methods, in maize aleurone tissues or embryogenic calli (Goff et al, 1991 Genes Dev. 5, 298-309; Goff et al, 1992 Genes Dev. 6, 864- 875). Hitherto however, there have been no reports of stable, efficient expression of functional GAL4 or derivaties thereof in a plant cell.

It is an aim of the present invention to provide a novel expression system operable in plants, utilising a modified portion of the GAL4 gene.

Summary of the Invention In a first aspect the invention provides a nucleic acid sequence. expressible in a plant cell.

encoding at least an effective portion of a GAL4 DNA-binding domain, the sequence having an A/T base content substantially reduced relative to the wild-type yeast sequence.

The A/T content of the wild-type yeast sequence encoding the DNA-binding domain of GAL4 is about 59%. The A/T base content of the sequence of the invention encoding the effective portion of the GAL4 DNA-binding domain will be understood to be substantially reduced when it is less than 50%. Preferably the A/T content is less than and more preferably less than 40%. The sequence of the invention may be made, for example, by site-directed mutagenesis, or be made de novo by chemical synthesis.

An "effective portion" of the DNA-binding domain is a portion sufficient to retain most over 50%) of the DNA-binding activity of the full length DNA-binding domain.

Typically the "effective portion" will include at least two thirds of the full length sequence of the DNA-binding domain. Conveniently the nucleic acid sequence will encode substantially all of the GAL4 DNA-binding domain amino acid residues 1-147 of the yeast polypeptide), although a substantially smaller portion (about 75 amino acid residue) is quite adequate to retain most of the DNA-binding activity (Kraulis er al, 1992 Nature 356, 448-450: and Marmorstein er al, 1992 Nature 356, 408-414). In one particular embodiment, the sequence will include the nucleotide sequence shown in the portion (nucleotides 1 to about 460) of Figure 1, which sequence has a substantiallv reduced A/T content relative to the wild-type yeast sequence, yet encodes an identical amino acid sequence.

The GAL4 DNA-binding domain has no transcriptional activation function in its own right. Thus. in preferred embodiments, the sequence encoding the effective portion of the GAL4 DNA-binding domain will be operably linked to one or more other nucleic acid S* sequences, which sequences may be structural encode functional polypeptides) and/or regulatory. Typically the sequence encoding the DNA-binding domain will be operably linked fused in-frame) to a sequence encoding a peptide or poiypeptide with a regulatory function, preferably a transcriptional activator. The transcriptional activator may be the activation domain of GAL4 protein, the sequence encoding which should preferably be optimised for expression in plants by reducing the A/T content thereof). Alternatively, the transcriptional activator could be any one of a number of such proteins known to be active in plants, which will be well-known to those skilled in the art, such that the sequence of the invention encodes a chimeric polypeptide, including at least an effective portion of the GAL4 DNA-binding domain and a transcriptional activation domain. A particularly suitable transcriptional activator domain is that obtainable from herpes simplex virus (HSV) VP-16, (see Greaves and O'Hare 1989 J. Virol 63, 1641- 1650; VP, being also known as VMW65). Other transcriptional activation domains include certain peptides encoded by E. coli genomic DNA fragments (Ma Ptashne 1987 Cell 51, 113-119) or synthetic peptides designed to form amphiphilic a-helix (Giniger Ptashne 1987 Nature 330, 670-672). A common feature appears to be the requirement for excess charge, either positive (Gill Ptashne 1987 Cell 51, 121-126) or, more especially for plant activation domains, excess negative charge (Estruch et 1994 Nucl. Acids Res.

22, 3983-3989). With this in mind. the person skilled in the art could readily synthesise, or find naturally-occurring sequences, which encode peptides or polypeptides with transcriptional activation activity in plants. Such activator sequences may, in any event, be modified for optimal activity in a plant cell.

In a further aspect the invention provides a nucleic acid construct, comprising the nucleic acid sequence defined above. In one particular embodiment, the nucleic acid construct could be used as an "enhancer-trap" to "fish" for plant enhancer sequences (Cf.

Sundaresan et al, 1995 Genes Dey. 9, 1797-1810). In such an embodiment, the construct will preferably include right and left Ti-DNA, to allow for random, stable insertion into the genome of a plant cell host. The construct will preferably include a naive plant promoter sequence, by which term is meant a promoter (operable in a plant S cell) which requires the presence of a suitable enhancer sequence to cause substantial levels of transcription. Such a "naive" promoter may be thought of as an "enhancerdependent" promoter, and essentially corresponds to the TATA box region of known plant promoters. "Plant" promoters includes reference to viral and bacterial promoter sequences which are active in a plant cell nucleotides -48 to +1 of the CaMV 35S promoter).

The naive promoter is conveniently substantially adjacent to one Ti border, the promoter being in competent relationship with the sequence encoding the GAL4 DNA-binding domain fused with a transcription activation domain operable in plants such that. should the promoter become inserted in the plant host cell genome in functional relationship with an enhancer sequence, the promoter will direct the expression of the GAL4 DNA-binding domain and the transcriptional activation domain. The construct may additionally comprise a plant selectable marker Kanamycin resistance) for convenience.

Additionally, the construct may include a reporter gene'active in plants (such as Green Fluorescent Protein, or 0-glucuronidase "GUS") in operable linkage to a GAL4-responsive upstream-activation sequence (UAS), such that the reporter gene will be expressed in response to synthesis of the GAL4 DNA-binding domain/transcriptional activator fusion protein. Preferably the reporter gene is modified GFP as disclosed in WO 96/27675, or GUS (conveniently with a nuclear localisation signal). Preferably the UAS includes one or more repeats of the 17mer to which GAL4 binds (Giniger er al, 1985 Cell 40, 767- 774).

As an alternative method of introducing the GAL4 binding domain/transcriptional activator sequence into a plant host cell genome, the construct may include Ds ("dissociation") elements, which may then be moved about the genome by the action of a transientlvexpressed Ac (Activator) enzyme Cocherel er al, 1996 Plant Mol. Biol. 30, 539- 551) In a further aspect. the invention provides a plant or part thereof a plant host cell or cell line) comprising the construct defined above. Typically the construct will have become integrated into a plant cell genome and be stably maintained therein. In particular the invention provides a plurality of plants or parts thereof (such as isolated plant cells, or cell lines, or tissue cultures) comprising a library, each plant or part thereof including a stably maintained nucleic acid sequence encoding an effective portion of the GAL4 DNA-binding domain as defined above. Conveniently the nucleic acid sequence will be incorporated into the genome of plant cells present in the library.

The library of piants or parts thereof thus provides a very useful resource. The library may be e.g. Arabidoosis chaliana. or of any other plant which is routinely used in 6 research. Each plant or part thereof in the library may have a particular pattern of expression of the integrated reporter gene (see, for example, Sundaresan et al., 1995 Genes Dev. 9. 1797-1810; Klimyuk er al.. 1995 Molec. Gen. Genet. 249, 357-365).

Thus. introduction of a further gene. having a GAL4 responsive UAS into the cell line, will result in expression of the introduced gene in the same temporal/spatial pattern as the reporter gene, such that researchers can express a particular gene of interest in selected tissues and/or at selected times in a predictable manner.

The invehtion thus provides a method of expressing a gene of interest in a known (predictable) pattein in a plant or part thereof, the method including: introducing the gene of interest into the plant or part thereof, said gene of interest having a GAL4responsive upstream activation sequence (UAS), characterised in that said plant or part thereof comprises a reporter gene expressed in a known pattern under the influence of a transcriptional activator comprising an effective portion of a GALA DNA-binding domain encoded by a sequence in accordance with the invention defined above, such that binding of the transcriptional activator to the UAS causes transcriptional activation of the gene of S interest.

0 The temporal/spatial pattern of expression of the introduced gene or genes of interest will then mirror that already known for the reporter gene. Typically the plant or par thereof will be Arabidoosis rhaliana. but may be any other plant which is routinely studied (e.g.

maize, rice, potato etc).

o Typically the gene or genes of interest will be introduced by crossing GAL4-expressing plant cell lines with a plant cell line comprising the gene(s) of interest linked to a GAL4responsive UAS.

0 S. The nucleic acid sequence of the invention has other useful applications, in addition to use as an "enhancer trap". or to direct the expression of a gene or genes of interest in a predictable manner. The modified GAL4 DNA-binding domain/transcriptional activator may also be used to co-ordinate the expression of a plurality of genes of interest. In many circumstances it is desirable to express simultaneously a number of genes in a plant either for research purposes, or for agricultural/industrial motives to study metabolic pathways, or to manipulate the synthesis of desirable products such as plant dyes or lipids).

Thus, the invention provides a method for co-ordinating the expression of a plurality of genes of interest in a plant or part thereof, each of the genes of interest being functionally associated with a GAL4-responsive UAS. characterised in that the plant or part thereof includes a sequence in accordance with the first aspect of the invention defined above and is capable of expressing a transcriptional activator including an effective portion of a GAL4 DNA-binding domain, such that binding of the transcriptional activator to the UAS causes simultaneous transcriptional activation of all of the genes of interest.

Conveniently the plurality of genes of interest may all be associated, in a polycistronic arrangement, with a single UAS, which facilitates their introduction into the plant or part thereof (the genes of interest typically being genes which are not naturally found in the plant or part thereof e.g. mammalian or bacterial genes, or genes from a different plant or possibly natural plant genes which have been the subject of various modifications).

Alternatively one or more genes may be operably linked to a respective UAS. The transcriptional activator may be encoded by a sequence already present in the plant or part thereof, or may be introduced simultaneously with (or subsequent to) introduction of the plurality of genes of interest.

The invention will now be further described by way of illustrative examples and with reference to the accompanying drawings, of which: Figure 1 shows a nucleotide sequence in accordance with the invention encoding a modified G.AL4 DNA-binding domain. fused in frame to a sequence encoding a modified transcriptional activation domain from HSV VP16. the vertical line denotes the end of the GAL4 DNA-binding domain: Figure 2 is a schematic representation of a nucleic acid construct comprising a sequence s in accordance with the invention: WO 97/30164 PCT/GB97/00406 8 Figure 3 shows the nucleotide sequence of part of the nucleic acid construct represented schematically in Figure 2; and Figures 4a-4d show micrographs of roots of plants comprising a sequence in accordance with the invention.

Examples The inventors have used an approach similar to that described by Brand Perrimon (1993, cited above), but adapted it for use in Arabidopsis, using Agrobacterium tumefasciens mediated transformation to produce "enhancer trap" plant cell lines with a transcription activator comprising the GALA DNA-binding domain, fused to the activation domain of HSV VP16 (mGAL4-VP16).

Example 1 Construction of mGAL4-VP16 gene Eight oligonucleotides, which correspond to the modified sequence of the GAL4 DNA binding region, were synthesised.

(28-mer) GGC AAC AAT GAA GCT ACT GTC TTC TAT C (Seq. ID No. 3) VP16-3' (31-mer) GGC AGA TCT ACC CAC CGT ACT CGT CAA TTC C (Seq. ID No.4) MGAL1 (109-mer) GGC AAG CTT GGA TCC AAC AAT GAA GCT CCT GTC CTC CAT CGA GCA GGC CTG CGA CAT CTG CCG CCT CAA GAA GCT CAA GTG CTC CAA GGA GAA GCC GAA GTG CGC CAA G (Seq. ID No. MGAL2 (108-mer) TCC TCT CGA GGG AAG ATC AGG AGG AAG AGC TGC TCC AGG CGC TCC WO 97/30164 PCT/GB97/00406 9 AGG CGG GAC TCC ACT TCG GTG AGG TGG GCG CGG GTC AGC GGG GAG CGC TTG GTT TTG GGA GAG (Seq. ID No. 6) MGAL3 (81-mer) TTC CCT CGA GAG GAC CTC GAC ATG ATC CTG AAA ATG GAC TCC CTC CAG GAC ATC AAA GCC CTG CTC ACC GGC CTC TTC GTC (Seq. ID No. 7) MGAL4 (89-mer) GGG TGA GGG GCA TGT CGG TCT CCA CGG AGG CCA GGC GGT CGG TGA CGG CGT CTT TGT TCA CGT TGT CCT GGA CGA AGA GGC CGG TGA GC (Seq.

ID No. 8) GGA GAC CGA CAT GCC CCT CAC CCT GCG CCA GCA CCG CAT CAG CGC GAC CTC CTC CTC GGA GGA GAG CAG CAA CAA GGG CC (Seq. ID No. 9) MGAL6 (83-mer) AGT GGA GCT CGT CCC CCA GGC TGA CGT CGG TCG GGG GGG CCG TCG AGA CGG TCA ACT GGC GCT GGC CCT TGT TGC TGC TCT CC (Seq. ID No. The full-length oligonucleotides were purified by electrophoresis in a 5% polyacrylamide gel containing 7M urea and 90mM Tris-borate 1mM EDTA pH8.3 (TBE) buffer. The fractionated oligonucleotides were detected by brief staining with filtered 0.05% toluidine blue dye, excised and eluted overnight at 37'C in 0.5M ammonium acetate, 0.1% SDS and 0.1mM EDTA. The eluted DNAs were precipitated and washed with ethanol, and phophorylated using T4 plynucleotide kinase. DNAs (0.5ug each) were separately resuspended in 50mM Tris-HC1 pH 9.0, 10mM MgCl 2 10mM DTT with 1mM ATP and units T4 polynucleotide kinase, and incubated for 30 mins at 37 0

C.

The plasmid pCMVGal65 (Cousens et al, 1989 EMBO J. 8, 2337-2342) was used as a source of the GAL4-VP16 sequence with unmodified codon usage. Two restriction endonuclease fragments were isolated from the plasmid. A Sacl-SacI fragment, which WO 97/30164 PCT/GB97/00406 contains the sequence encoding the DNA binding element of the Saccharomyces cerevisiae GAL4 protein, and a SacI-KpnI fragment, which encodes the activation domain of the herpes simplex virus VP16 protein, were purified by fractionation and elution from a low gelling temperature (LGT) 1% agarose gel.

The 5' portion of the mGAL4-VP16 sequence was amplified by the polymerase chain reaction (PCR), using the GAL-5' and MGAL2 oligos as primers and the Sacl-SacI GAL4 DNA fragment as a template. Primers and template were incubated under standard conditions with VENT DNA polymerase (New England Biolabs) and subjected to cycles of 94 0 C for 30 secs, 50°C for 1 min, 72 0 C for 1 min. The product was cut with XhoI restriction endonuclease and purified after electrophoresis through a 1.5% LGT agarose gel.

The MGAL4 and MGAL5 oligonucleotides were annealed by heating to 65 0 C and slow cooling to room temperature. The MGAL3 and MGAL6 oligonucleotides were then added and also annealed to the mixture. The oligonucleotides were then incubated with Klenow fragment of DNA polymerase 1 and T4 DNA ligase in 50mM Tris-HCl pH 7.5, MgCl1, 10mM DTT, ImM dNTPs and 1mM ATP for 60 mins at 37°C. An excess of MGAL3 and MGAL6 oligonuceotides were added, and the mixture was subject to PCR amplification, as above. The product was cut with the restriction endonucleases XhoI and SacI, and purified after electrophoresis through a 1.5% LGT agarose gel.

The three gel-purified restriction fragments: the GAL-5'/MGAL2 PCR-amplified product, corresponding to the 5' portion of the GAL4 DNA binding domain, the MGAL3-6 annealed and amplified product, corresponding to the 3' portion of the GAL4 DNA binding domain, and the SacI-KpnI fragment from pCMVGaI65, corresponding to the VP16 activation domain; were mixed and ligated with T4 DNA ligase in Tris-HCI pH 7.5. 10mM MgCl 2 10mM DTT and 1mM ATP at 20°C overnight. The ligated product was PCR amplified (as above) using MGAL1 and VP16-3' as primers.

The product, containing the entire modified GAL4-VP16 sequence was cut with BamHI and ligated to PBI121 (Jefferson et al, EMBO J. 6:3907, 1987) which had been cut with SacI, blunt ended by treatment with T4 DNA polymerase, and re-cut with BamHI. The WO 97/30164 PCT/GB97/00406 11 recombinant plasmid (pBIN 35S-mGAL4-VP16) contained the mGAL4-VP16 gene downsteam of the constitutive 35S plant promoter.

In Figure 1, the lower DNA sequence is that of the fusion gene (Seq. ID No. encoding in the 5' portion the GAL4 DNA-binding domain, and encoding in the 3' portion the transcriptional activation domain from HSV VP16. The wild-type sequence is shown above for comparison. It will be seen that the wild type GAL4 DNA-binding domain coding sequence is A/T rich content is shown on the right for each line of sequence), and it is believed that this causes inefficient expression in plants. In particular, it is believed that the A/T rich DNA comprises one or more regions which, when transcribed, are recognized as mRNA splice sites in plant cells. The altered nucleotides are shown outlined in Figure 1. The encoded polypeptide sequence is shown below (Seq.

ID No. The modifications to the nucleotide sequence were carefully selected to ensure that there was no resulting change in the encoded amino acid sequence. The numbers above the sequences represent the number of the nucleotide or amino acid at that position.

Certain restriction endonuclease sites are also shown.

The 35S promoter and mGAL4-VP16 gene were excised by digestion with the restriction endonucleases HinDIII and EcoRI, purified by 1% LGT agarose gel electrophesis, and subcloned into a vector containing a GAL4-responsive mgfp5-ER marker gene (see Example 2 below). The resulting construct is shown in Figure 2, in which R and L represent right and left Ti plasmid border sequence. The GAL4-VP16 fusion gene is downstream of the TATA-box region (nucleotides -48 to of the CaMV 35S gene, were inserted adjacent to the right Ti border. The plasmid also contains the kanamycin resistance (Kan R) selectable marker, and a reporter gene (GUS or GFP) operably linked to a synthetic GAL4-UAS promoter sequence. Certain restriction endonuclease sites are also shown.

Figure 3 shows the nucleotide sequence (Seq. ID No. 15) of the plasmid across the junctions of the Ti right border, the CaMV35S TATA box, and the start of the GAL4 DNA-binding domain coding region.

WO 97/30164 PCT/GB97/00406 12 The mGAL4-VP 16 gene was directly assayed for activity in transformed Arabidopsis plants by Agrobacterium-mediated transformation (Valvekens et al Proc. Natl. Acad. Sci.

USA 85:5536-5540, 1988).

Example 2 Construction of GAL-GFP enhancer trap vector.

Modification of the T-DNA right border.

The plasmid pBIN19 (Bevan, M. Nuc. Acids Res. 12:8711-8721, 1984) was digested with the restriction endonuclease SacII, and a 2.6 Kb fragment containing the Agrobacterium T-DNA right border sequence was subcloned into SacII cut pGEM 5Zf(Genbank accession No. X65308; commercially available from Promega). The bacteria containing the recombinant phagemid were superinfected with helper phage to produce single strand phagemid DNA.

A synthetic mutagenic oligonucleotide ATA TCC TGT CAA ACA CTG GAT CCG AGC TCC AAT TCA TAG TTT AAA CTG AAG GCG GG; Seq. ID No. 11) was kinased and annealed to the purified phagemid DNA, extended on the template using T7 DNA polymerase, and ligated using T4 DNA ligase. The DNA was transformed into bacteria, and recombinant plasmids (pTr+) were screened for the insertion of new sites for the restriction endonucleases BamHI, SmaI and EcoRI immediately adjacent to the T- DNA right border.

Insertion of TATA box region.

The TATA box region of the 35S promoter was PCR amplified from pBI121 using oligonucleotides complementary to the -48 region of the promoter (oligonucleotide GGC AGA TCT TCG CAA GAC CCT TCC TCT ATA TAA GG; Seq. ID No. 12) and the downstream O-glucuronidase gene (oligonucleotide GUSSnaBI; CAC ACA AAC GGT GAT ACG TA; Seq. ID No. 13). The PCR product was cut with restriction endonucleases BglII and BamHI and ligated to BamHI cut pTr+. The resulting recombinant plasmid (pTr+A35S) contained a minimal (naive) promoter positioned WO 97/30164 PCT/GB97/00406 13 adjacent to the T-DNA right border.

Insertion of 35S promoter driven mGAL4-VP 16 gene.

Derivatives of the GAL4 with unmodified codon usage were inserted into the plasmid in unsuccessful trial experiments, and one of these constructions (the insertion of a truncated GAL4 gene derived from pMA236; Ma et al, 1988 Nature 334, 631-633) had resulted in the insertion of a unique HindII site immediately adjacent to the BamHI site in pTR+A35S. This plasmid, pTr+AGAL4, was digested with restriction endonucleases HindIII and EcoRI, and ligated with the HinDIII-EcoRI fragment from pBIN 35S-mGAL4- VP 16, to introduce the 35S-driven mGAL4-VP 16 gene. The Agrobacterium sequences from this plasmid (pTr+35S-mGAL4-VP 16) were then recloned into a binary plant transformation vector which contained a GAL4-responsive marker gene (see below), for testing in planta.

Construction of a synthetic GAL4-responsive promoter.

A HinDIII-XbaI restriction endonuclease fragment containing five optimised binding sites for GALA was excised from the plasmid pUAST (Brand Perrimon, 1993 Development, 118, 401-415), and ligated into HinDIII-XbaI cut pBI101 (Jefeerson et al., 1987 EMBO J. 6, 3901-3907) upstream of the GUS gene (to make pBINAUAS-GUS). A sequence corresponding to the -90 region of the 35S promoter was PCR amplified using, oligonucleotides DELAS1 (GAA CTC TAG AAG CTA CTC CAC GTC CAT AAG GGA CAC ATC ACA ATC CCA CTA TCC TTC GC; Seq. ID No. 14) and GUSSnaBI (see above). The product was XbaI-BamHI cut and cloned into similarly cut pBIN AUAS- GUS. The resulting plasmid (pBIN UAS(-90-AS1)GUS) contains a synthetic GALA promoter upsteam of the 0-glucuronidase (GUS) coding sequence. The GUS gene was then replaced with that of the green fluorescent protein (GFP).

GAL4-responsive mgfp5-ER gene.

We have extensively mutated the green fluorescent protein gene for optimised expression in plants. The optimised gene is called mgfp5-ER (Siemering et al, Current Biology 6:1653-1663, 1996; W096/27675).

WO 97/30164 PCT/GB97/00406 14 The mgfp5-ER gene has been cloned into the plant transformation vector pBI121, and we have excised a BamHI-SacI fragment, which contains the coding sequence, and have inserted this into BamHI-SacI cut pBIN UAS(-90-AS1)GUS to replace the GUS gene.

This plasmid (pBIN UAS(-90-AS1)mgfp5-ER) was cut with SacII, and ligated with the SacII fragment containing the modified T-DNA right border and mGAL4-VP16 gene from pTr+35S-mGAL4-VP16, to give pBIN 35S-GAL4-VP16+ UAS-mgfp5-ER (abbreviated to Testing of the pBIN35S-GAL-GFP vector.

The pBIN 35S-GAL-GFP plasmid was introduced into Agrobacterium tumefaciens strain LBA4044 (Jefferson et al., EMBO J. 6:3901-3907, 1987) by electroporation. followed by selection on kanamycin-containing plates. The Agrobacterium strain was used to transform Arabidopsis using the technique described below. Plants were scored for the GAL4dependent induction of GFP fluorescence after transformation.

Construction of GAL-GFP enhancer trap vector The pTR+35S-mGAL4-VP16 plasmid was treated with BamHI (to excise the 35S promoter sequences) and religated using T4 DNA ligase. This plasmid (pTR+mGAL4-VP16) contains the modified GAL4-VP16 positioned with a minimal promoter adjacent to the T- DNA right border. The T-DNA sequences were then excised by digestion with SacII, and ligated with similarly cut pBIN UAS(-90-AS1)mgfp5-ER. This produced pBIN GAL-GFP, which contains the highly active mGAL4-VP6 transcription activator, responsive to adjacent enhancer elements, and a GAL4-dependent GFP gene. GFP fluorescence is produced in response to GAL4-VP16 activation.

Enhancer trap screen.

The enhancer trap vector pBIN GAL-GFP was introduced into Agrobacterium tumefaciens (see details below) LBA4044, and has been used to generate over 7,500 transformed Arabidopsis seedlings. Transgenic seedlings have been screened directly for GAL4mediated GFP expression, and seeds were collected from expressing plants. We have a library of 'over 250 Arabidopsis lines which show stable inheritable patterns of GAL4- WO 97/30164 PCT/GB97/00406 mediated GFP expression in the root.

Transactivation.

To demonstrate GAL4-mediated transactivation of a foreign gene we have generated transgenic Arabidopsis that contain the T-DNA sequences from pBIN UAS(-90-AS 1)GUS).

In the absence of GAL4-VP16, these plants do not express the GUS gene. However, when genetically crossed with lines expressing the mGAL4-VP16 gene, the GUS gene is activated in a pattern of expression which accurately reflects that of the mGAL4-VP16 gene from the donor parent (see Example 3 below).

Example 3 A stable transformed line (J2302) of Arabidopsis thaliana, which formed part of the library described above, expressed modified GFP (under the influence of the mGAL4- VP16 activator) in the cells of the extreme root tip (see Figures 4a and 4b).

Figure 4a is a low power micrograph showing GFP-mediated epifluorescence (400nm excitation) at the root tip. Figure 4b is a higher magnification confocal micrograph (488nm excitation) showing the area of GFP-mediated fluorescence in greater detail.

The line J2302 was crossed (using standard techniques) with another Arabidopsis line which comprised a stable, silently-maintained GUS reporter gene operably linked to a GAL4-responsive UAS. The product of the cross, as expected, proceeded to express GUS, under the influence of the GAL4-VP16 transcriptional activator, in the same pattern as the GFP reporter gene in J2302 at the extreme root tip). Suitably stained samples, imaged by brightfield microscopy, are shown in Figures 4c and 4d, and demonstrate GUS reporter gene activity (darkest staining) at the root tip.

This illustrates that the modified GAL4 DNA-binding domain sequence can successfully be used in the construction of enhancer trap vectors; (ii) express genes of interest (as exemplified by GUS) in a predictable pattern; and (iii) co-ordinate the simultaneous expression of a plurality of genes of interest (as WO 97/30164 PCT/GB97/00406 16 exemplified by GFP and GUS).

Example 4 Transformation of Arabidopsis thaliana.

The methods employed are based on those originally disclosed by Valvekens et al. 1988.

Proc. Natl. Acad Sci. 85, 5536-5540).

Media Unless otherwise stated all procedures were carried out aseptically using sterile solutions and equipment. All media are used in standard plastic disposable petri dishes or, for later stages, Magenta GA 7 pots (Magenta Corp., USA). Hormones are dissolved in dimethyl sulfoxide (DMSO) as xl000 stocks. Hormones and antibiotics are added after autoclaving and cooling of the media to 65 0

C.

1. Germination medium (GM) comprised: lx Murashige and Skoog salt mixture; 1% sucrose; 100 mg/1 inositol; 1.0 mg/I thiamine; 0.5 mg/1 pyridoxine; 0.5 mg/1 nicotinic acid; 0.5 g/1 2-(N-morpholino)ethanesulfonic acid (MES); (adjust to pH 5.7 with 1M KOH); and 0.8% Difco Bacto agar (for solid GM medium).

la. GM As GM, but supplemented with: 50 mg/l kanamycin (Sigma) 2. Callus-inducing medium (CIM) comprised: lx Gamborg's B5 medium; 2% glucose; g/1 MES (pH 5.7, adjusted with 1N KOH); 0.5 mg/1 2,4-D (Sigma); 0.05 mg/1 kinetin (Sigma); and 0.8 agar (for solid CIM medium).

3. Shoot-inducing medium with Vancomycin (SIM V750 K50) comprised: Gamborg's medium; 2% glucose; 0.5 g/1 MES (pH 0.8% agar; 5 mg/l N6-(2isopentenyl)adenine (2ip); 0.15 mg/1 indole-3-acetic acid (IAA); 750 mg/l vancomycin; and 50 mg/1 kanamycin.

3a. SIM V500 WO 97/30164 PCT/GB97/00406 17 As SIM, but supplemented with: 500 mg/1 vancomycin; and 50 mg/1 kanamycin.

4. Root-inducing medium (RIM) comprised: Gamborg's B5 medium; 2% glucose: 0.5 g/1 MES (pH 0.8% agar; 12.5 mg/1 indole butyric acid (IBA); and 50 mg/1 cefotaxime.

Growth of plants Place seeds into a 15 ml polypropylene centrifuge tube.

(ii) Add ethanol for 2 min. Remove ethanol with pipette.

(iii) Replace with 5 commercial bleach 0.25 available chlorine) containing one drop of NP40 per 50 ml. Leave for 15 min, shaking regularly.

(iv) Wash seeds in sterile, distilled water for at least three times.

After last wash, add the sterile seeds to 100ml of GM in a conical flask. Grow with shaking for 2-4 weeks in a culture room at 20-25 0

C.

Agrobacterium-mediated transformation.

1. Day One Using a scalpel, cut-off all the green (upper) parts of the plantlets to leave only the root systems.

(ii) Lay out the roots on plates containing solid CIM. Gently press down on each bunch of roots to ensure that they are in contact with the surface of the agar.

(iii) Incubate for 3 d in growth room (22 C, continual light).

2. Day Four After ensuring that there is no visible sign of contamination, collect the callus-induced roots in an empty petri dish.

(ii) Cut the roots into 0.5 cm explants.

(iii) Add 3-5 ml of Agrobacterium culture which has been grown overnight at 28oC in Luria broth, and washed by centrifugation. Swirl with blunt-nosed forceps. Leave to cocultivate for 2min.

(iv) Blot the roots dry on double-thick sterile filter-paper Whatman No. 1) in a petri dish.

Place the explants onto solidified CIM medium in a petri dish. and gently press them onto the surface to ensure contact with the medium.

WO 97/30164 PCT/GB97/00406 18 (vi) Incubate plates in the growth room for 2 d to allow co-cultivation of Agrobacterium.

3. Day Six Transfer the explants to a petri dish in 20-25 ml of water.

Agitate with blunt-nosed forceps to wash off the agrobacteria. The bathing medium should become somewhat turbid. Transfer the root pieces to a sieve and repeat the washing. Then lift the sieve out of buffer and drain. Press down the explants down onto the mesh to remove as much buffer and agrobacteria as possible. Push the semi-dried explants together into a mass.

(Custom-made, autoclavable and re-usable, sieves are used to hold the explants during Agrobacterium infections and washings of root explants. These sieves are made from 100 ml plastic three-cornered beakers and 100 um nylon mesh. The top 3-4 cm is cut off the beaker and the mesh held in place across the bottom of this piece by pushing a ring 2 cm high cut from just above the base of the beaker into the lower portion of the top part.

The two pieces are sealed together by pushing a hot metal rod through them in several places.) (ii) Blot small bundles of root material on double-thick sterile filter-paper in a petri dish until dry and transfer to solidified SIM V750 K50 medium, taking care that root explants are in close contact with the medium.

(iii) Incubate in growth room.

Regeneration In growth room, tiny green kanamycin resistant calli appear on the yellowish rootexplants after 3 weeks.

(ii) Every 2 weeks, transfer explants to fresh SIM V750 K50. Shoots (often initially vitreous) intermittently appear from the green calli over the next several weeks.

(iii) Transfer these shoots to RIM, where roots will develop over a period of 2 to 3 weeks (iv) After the formation of roots, transfer the plantlets to GM in vented Magenta GA7 pots or directly to soil. Ensure that the humidity is kept low to ensure good seed set.

Harvest seed pods when they turn yellowish-brown and are therefore mature.

Germination and screening of Fl progeny.

To test for transformation, Fl seedlings are germinated on GM Put seeds on GM K50 (surface sterilise the seeds if necessary).

(ii) Put petri dishes in the dark at 4oC (refrigerator) for 3-5 d. to break seed dormancy.

This is not necessary if seeds were stored for more than a month.

(iii) Incubate petri dishes in growth room for 2 weeks. Sensitive seedlings form neither roots nor leaves, and have white cotyledons. Transformed seedlings are phenotypically normal.

(iv) Transgenic callus and shoots were screened for GFP expression using a inverted fluorescence microscope (Leitz DM-IL) fitted with a filter set (Leitz-D excitation BP355- 425, dichroic 455,- emission LP460 suitable for the main 395nm excitation and 509nm emission peaks of GFP. The use of a 7mm threaded extension tube with a 4x objective (EF 4/0.12) gave a greater working distance, and has allowed the convenient direct observation of tissue within inverted sealed petri dishes. A 100 Watt long wavelength hand-held UV lamp (UV Products, B100AP) was also used for routine monitoring of transgenic shoots and plants. Transgenic Arabidopsis Fl seedlings were grown in sterile agar culture for 5 days, and were mounted in water under glass coverslips for microscopy.

The specimens were examined using a BioRad MRC-600 laser-scanning confocal microscope equipped with a krypton-argon laser and filter sets suitable for the detection of fluorescein and texas red dyes (BioRad KI/K2), and a Nikon 60x PlanApo N.A. 1.2 water immersion objective.

The terms "comprise", "comprises", "comprised" and "comprising" when used in this specification are taken to specify the presence of stated features, integers, steps or components but does not preclude the presence or addition of one or more other features, i. integers, steps, components or groups thereof.

0 0 WO 97/30164 PCTIGB97/00406 SEQUENCE LISTING GENERAL INFORMATION:

APPLICANT:

NAME: Medical Research Council STREET: 20 Park Crescent CITY: London COUNTRY: United Kingdom POSTAL CODE (ZIP): WIN 4AL TELEPHONE: (0171) 636 5422 TELEFAX: (0171) 323 1331 (ii) TITLE OF INVENTION: Improvements in or Relating to Gene Expression (iii) NUMBER OF SEQUENCES: (iv) COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.30 (EPO) INFORMATION FOR SEQ ID NO: 1: SEQUENCE CHARACTERISTICS: LENGTH: 701 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ix) FEATURE: NAME/KEY: CDS LOCATION:17..694 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: AAGCTTGGAT CCAACA ATG Met 1 AAG CTC CTG TCC Lys Leu Leu Ser 5 TCC ATC GAG CAG Ser Ile Glu Gin GCC TGC Ala Cys GAC ATC TGC Asp Ile Cys TGC GCC AAG Cys Ala Lys CTC AAG AAG CTC Leu Lys Lys Leu

AAG

Lys TGC TCC AAG GAG Cys Ser Lys Glu

AAG

Lys CCG AAG Pro Lys TGT CTG AAG AAC Cys Leu Lys Asn

AAC

Asn TGG GAG TGT CGC Trp Glu Cys Arg TCT CCC AAA Ser Pro Lys 145 ACC AAG Thr Lys CGC TCC CCG CTG Arg Ser Pro Leu

ACC

Thr CGC GCC CAC CTC Arg Ala His Leu GAA GTG GAG TCC Glu Val Glu Ser 193 WO 97/30164 PCT/GB97/00406

CGC

Arg CTG GAG CGC CTG Leu Glu Arg Leu CAG CTC TTC CTC Gin Leu Phe Leu

CTG

Leu ATC TTC CCT CGA Ile Phe Pro Arg 241 289 GAC CTC GAC ATG Asp Leu Asp Met CTG AAA ATG GAC Leu Lys Met Asp

TCC

Ser CTC CAG GAC ATC Leu Gin Asp Ile AAA GCC Lys Ala CTG CTC ACC Leu Leu Thr

GGC

Gly CTC TTC GTC CAG Leu Phe Val Gin

GAC

Asp 100 AAC GTG AAC AAA Asn Val Asn Lys GAC GCC GTC Asp Ala Val 105 ACC CTG CGC Thr Leu Arg 337 ACC GAC Thr Asp CAG CAC Gin His 125

CGC

Arg 110 CTG GCC TCC GTG Leu Ala Ser Val

GAG

Glu 115 ACC GAC ATG CCC Thr Asp Met Pro

CTC

Leu 120 CGC ATC AGC GCG Arg Ile Ser Ala

ACC

Thr 130 TCC TCC TCG GAG Ser Ser Ser Glu AGC AGC AAC AAG Ser Ser Asn Lys 385 433 481 529

GGC

Gly 140 CAG CGC CAG TTG Gin Arg Gin Leu

ACC

Thr 145 GTC TCG ACG GCC Val Ser Thr Ala

CCC

Pro 150 CCG ACC GAC GTC Pro Thr Asp Val

AGC

Ser 155 CTG GGG GAC GAG Leu Gly Asp Glu CAC TTA GAC GGC His Leu Asp Gly

GAG

Glu 165 GAC GTG GCG ATG Asp Val Ala Met GCG CAT Ala His 170 GCC GAC GCG Ala Asp Ala TCC CCG GGG Ser Pro Gly 190

CTA

Leu 175 GAC GAT TTC GAT Asp Asp Phe Asp

CTG

Leu 180 GAC ATG TTG GGG Asp Met Leu Gly GAC GGG GAT Asp Gly Asp 185 TAC GGC GCT Tyr Gly Ala 577 CCG GGA TT ACC Pro Gly Phe Thr CAC GAC TCC GCC His Asp Ser Ala

CCC

Pro 200 625 CTG GAT Leu Asp 205 ATG GCC GAC TTC Met Ala Asp Phe

GAG

Glu 210 TTT GAG CAG ATG Phe Glu Gin Met

TTT

Phe 215 ACC GAT GCC CTT Thr Asp Ala Leu 673

GGA

Gly 220 ATT GAC GAG TAC Ile Asp Glu Tyr GGT GGG TAGATCT Gly Gly 225 INFORMATION FOR SEQ ID NO: 2: SEQUENCE CHARACTERISTICS: LENGTH: 226 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: WO 97/30164 PCT/GB97/00406 Met 1 Lys Lys Leu Glu Leu Phe Ser Ala Thr 145 His Asp Phe Lys Leu Leu Ser Ser Ile Glu Gin Ala Cys Asp Ile Cys Arg Leu Lys Asn Thr Gin Lys Val Val Thr 130 Val Leu Phe Thr Leu Asn Arg Leu Met Gin Glu 115 Ser Ser Asp Asp Pro Lys I Trp Ala Phe Asp Asp 100 Thr Ser Thr Gly Leu 180 His Cys Glu His Leu Ser Asn Asp Ser Ala Glu 165 Asp Asp Ser Cys Leu Leu 70 Leu Val Met Glu Pro 150 Asp Met Ser Lys Arg Thr 55 Ile Gin Asn Pro Glu 135 Pro Val Leu Ala Glu Tyr 40 Glu Phe Asp Lys Leu 120 Ser Thr Ala Gly Pro 200 Lys 25 Ser Val Pro Ile Asp 105 Thr Ser Asp Met Asp 185 Tyr Pro Pro Glu Arg Lys 90 Ala Leu Asn Val Ala 170 Gly Gly Lys Lys Ser Glu 75 Ala Val Arg Lys Ser 155 His Asp Ala Cys Thr Arg Asp Leu Thr Gin Gly 140 Leu Ala Ser Leu Ala Lys Leu Leu Leu Asp His 125 Gln Gly Asp Pro Asp 205 Lys Arg Glu Asp Thr Arg 110 Arg Arg Asp Ala Gly 190 Met Cys Ser Arg Met Gly Leu Ile Gin Glu Leu 175 Pro Ala SLeu SPro Leu Ile Leu Ala Ser Leu Leu 160 Asp Gly Asp 195 Phe Gly 225 (2) Glu 210 Gly Phe Glu Gin Met Thr Asp Ala Leu Ile Asp Glu Tyr INFORMATION FOR SEQ ID NO: 3: SEQUENCE CHARACTERISTICS: LENGTH: 28 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear WO 97/30164 PCT/GB97/00406 23 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: GGCAACAATG AAGCTACTGT CTTCTATC 28 INFORMATION FOR SEQ ID NO: 4: SEQUENCE CHARACTERISTICS: LENGTH: 31 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: GGCAGATCTA CCCACCGTAC TCGTCAATTC C 31 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 109 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: GGCAAGCTTG GATCCAACAA TGAAGCTCCT GTCCTCCATC GAGCAGGCCT GCGACATCTG CCGCCTCAAG AAGCTCAAGT GCTCCAAGGA GAAGCCGAAG TGCGCCAAG 109 INFORMATION FOR SEQ ID NO: 6: SEQUENCE CHARACTERISTICS: LENGTH: 108 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: TCCTCTCGAG GGAAGATCAG GAGGAAGAGC TGCTCCAGGC GCTCCAGGCG GGACTCCACT TCGGTGAGGT GGGCGCGGGT CAGCGGGGAG CGCTTGGTTT TGGGAGAG 108 INFORMATION FOR SEQ ID NO: 7: SEQUENCE CHARACTERISTICS: LENGTH: 81 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear WO 97/30164 PCT/GB97/00406 24 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: TTCCCTCGAG AGGACCTCGA CATGATCCTG AAAATGGACT CCCTCCAGGA CATCAAAGCC CTGCTCACCG GCCTCTTCGT C 81 INFORMATION FOR SEQ ID NO: 8: SEQUENCE CHARACTERISTICS: LENGTH: 89 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: GGGTGAGGGG CATGTCGGTC TCCACGGAGG CCAGGCGGTC GGTGACGGCG TCTTTGTTCA CGTTGTCCTG GACGAAGAGG CCGGTGAGC 89 INFORMATION FOR SEQ ID NO: 9: SEQUENCE CHARACTERISTICS: LENGTH: 80 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: GGAGACCGAC ATGCCCCTCA CCCTGCGCCA GCACCGCATC AGCGCGACCT CCTCCTCGGA GGAGAGCAGC AACAAGGGCC INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 83 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: AGTGGAGCTC GTCCCCCAGG CTGACGTCGG TCGGGGGGGC CGTCGAGACG GTCAACTGGC GCTGGCCCTT GTTGCTGCTC TCC 83 INFORMATION FOR SEQ ID NO: 11: SEQUENCE CHARACTERISTICS: LENGTH: 56 base pairs WO 97/30164 PCT/GB97/00406 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: ATATCCTGTC AAACACTGGA TCCGAGCTCC AATTCATAGT TTAAACTGAA GGCGGG 56 INFORMATION FOR SEQ ID NO: 12: SEQUENCE CHARACTERISTICS: LENGTH: 35 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: GGCAGATCTT CGCAAGACCC TTCCTCTATA TAAGG INFORMATION FOR SEQ ID NO: 13: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: CACACAAACG GTGATACGTA INFORMATION FOR SEQ ID NO: 14: SEQUENCE CHARACTERISTICS: LENGTH: 59 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: GAACTCTAGA AGCTACTCCA CGTCCATAAG GGACACATCA CAATCCCACT ATCCTTCGC 59 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 91 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear WO 97/30164 PCUIGB97/00406 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: TAGGTTTACC CGCCAATATA TCCTGTCAAA CACTGGATCT TCGCAAGACC CTTCCTCTAT ATAAGGAAGT TCATTTCATT TGGAGAGGAC A 91

Claims

1. A nucleic acid sequence, expressible in a plant cell, encoding at least an effective portion of a GAL4 DNA-binding domain, the sequence having a A/T base content substantially reduced relative to the wild-type yeast sequence, characterised in that the A/T base content is less than

2. A nucleic acid sequence is claimed in Claim 1 characterised in that the A/T base content is less than

3. A sequence according to Claim 1 or Claim 2, further including a structural and/or regulatory sequence in operable linkage to the sequence encoding the effective portion of the GAL4 DNA-binding domain.

4. A sequence according to any one of Claims 1 to 3 wherein the nucleic acid encodes a peptide or polypeptide, having transcriptional activation activity, in operable linkage with the sequence encoding the effective portion of the GAL4 DNA-binding domain. S 20

5. A sequence according to any one of Claims 1 to 4, wherein the sequence encodes a peptide or polypeptide, having transcriptional activation activity in a plant cell, which is a modified portion of GAL4, VP16, GCN4 or a designed sequence which does not naturally occur. 25

6. A nucleic acid construct including a sequence in accordance with any one of claims 1 to

7. A construct according to Claim 6, capable of inserting a sequence according to any one of Claims 1 to 5 into the genome of a plant host cell.

8. A construct according to Claim 6 or 7, further including a reporter gene in operable linkage with a GAL4-responsive upstream activation sequence (UAS). 28

9. A construct according to any one of Claims 6, 7 or 8, including a sequence encoding GUS or green fluorescent protein (GFP) suitable for expression in a plant cell.

10. A construct according to any one of Claims 6 to 9, including T-DNA borders or Ds elements.

11. A construct according to any one of Claims 6 to 10, wherein expression of the sequence according to any one of Claims 1 to 4 is under the control of an enhancer- dependent plant promoter.

12. A construct according to any one of Claims 6 to 11, further including a selectable marker gene.

13. A plant or part thereof including a nucleic acid sequence according to any one of Claims 1 to 5, or transformed with a construct according to any one of Claims 6 to 12.

14. A plant or part thereof according to Claim 13, wherein the sequence is stably 20 maintained in the plant genome.

15. A library including a plurality of plants or parts thereof, each plant or part thereof including a stably integrated nucleic acid sequence according to Claim 4 or Claim 5, which sequence causes the expression of a reporter gene in a known T 25 temporal and/or spatial pattern.

16. A library according to Claim 15, made by the introduction of a nucleic acid construct according to any one of Claims 7 to 12 into a plurality of plants or parts thereof. S

17. A library according to Claim 15 or 16 including a plurality of Arabidopsis thaliana plants. 29

18. A method of expressing a gene of interest in a known pattern in a plant or part thereof, the method including: introducing the gene or interest into the plant or part thereof, said gene of interest having a GAL4- responsive upstream activation sequence (UAS), characterised in that said plant or part thereof includes a reporter gene expressed in a known pattern under the influence of a transcriptional activator including an effective portion of a GAL4 DNA-binding domain encoded by a sequence according to any one of Claims 1 to 5, such that binding of the transcriptional activator to the UAS causes transcriptional activation of the gene of interest.

19. A method according to Claim 18, wherein the gene of interest is introduced into one or more plants or parts thereof from a library according to any one of Claims 15, 16 or 17.

20. A method for co-ordinating the expression of a plurality of genes of interest in a plant or part thereof, each of the genes of interest being functionally associated with a GAL4-responsive UAS, characterised in that the plant or part thereof includes a sequence according to any one of Claims 1 to 5 and is capable of 0.0 expressing a transcriptional activator including an effective portion of a GAL4 66*: 20 DNA-binding domain, such that binding of the transcriptional activator to the UAS causes simultaneous transcriptional activation of all of the genes of *e* :interest.

21. A method according to Claim 20, wherein at least some of the genes of interest 25 are arranged polycistronically and associated with a single GAL4-responsive UAS.

22. A method according to Claim 20, wherein genes of interest are functionally associated with respective GAL4-responsive UASs. DATED this 27 th day of October 1999 BTG INTERNATIONAL LTD WATERMARK PATENT TRADEMARK ATTORNEYS 290 BURWOOD ROAD SRA HAWTHORN VICTORIA 3122 SAUSTRALIA o/-7 LCG:KMH:MXM