GA4 DNA, protein and methods of use

FIELD OF THE INVENTION
The invention relates to the field of molecular biology and plant growth hormones, and especially to gibberellins.
BACKGROUND OF THE INVENTION
Gibberellins are a large family of tetracyclic triterpenoid plant growth hormones which promote various growth and developmental processes in higher plants, such as seed germination, stem elongation, flowering and fruiting (Stowe, B. B. et al., Annu. Rev. Plant Physiol. 8:181-216 (1957)). A number of GA responsive dwarf mutants have been isolated from various plant species, such as maize, pea, and Arabidopsis (Phinney, B. O. et al., "Chemical Genetics and the Gibberellin Pathway" in Zea mays L. in Plant Growth Substance, ed., P. F. Waering, New York: Academic (1982) pp. 101-110; Ingram, T. J. et al., Planta 160:455-463 (1984); Koornneef, M., Arabidopsis Inf. Serv. 15:17-20. (1978)). The dwarf mutants of maize (dwarf-1, dwarf-2, dwarf-3, dwarf-5) have been used to characterize the maize GA biosynthesis pathway by determining specific steps leading to biologically important metabolites (Phinney, B. O. et al., "Chemical Genetics and the Gibberellin Pathway" in Zea mays L. in Plant Growth Substance, ed., P. F. Waering, New York: Academic (1982) pp. 101-110; Fujioka, S. et al., Plant Physiol. 88:1367-1372 (1988)). Similar studies have been done with the dwarf mutants from pea (Pisum sativunz L.) (Ingram, T. J. et al., Planta 160:455-463 (1984)). GA deficient mutants have also been isolated from Arabidopsis (ga1, ga2, ga3, ga4, ga5) (Koornneef, M., et al., Theor. Appl. Genet. 58:257-263 (1980)). The Arabidopsis ga4 mutant, induced by ethyl methanesulfonate (EMS) mutagenesis, is a germinating, GA responsive, semidwarf whose phenotype can be restored to wild type by repeated application of exogenous GA (Koomneef, M. et al., Theor. Appl. Genet. 58:257-263 (1980)).
In Arabidopsis, the ga4 mutant allele blocks the conversion of 3-.beta.-hydroxy GAs, reducing the endogenous levels of GA.sub.1, GA.sub.8 and GA.sub.4 and increasing the endogenous levels of GA.sub.19, GA20 and GA.sub.9 (Talon, M. et al., Proc. Natl. Acad. Sci. USA 87:7983-7987 (1990)). The reduced levels of the 3-.beta.-hydroxy GAs is the cause of the semidwarf phenotype of the ga4 mutant. It has been suggested that the pea le mutant also encodes an altered form of 3-,.beta.-hydroxylase (Ross, J. J. et al., Physiol. Plant. 76:173-176 (1989)).
SUMMARY OF THE INVENTION
The invention is first directed to GA4 DNA and the protein encoded by the GA4 DNA.
The invention is further directed to GA4 antisense DNA, and to the GA4 antisense RNA transcribed from it.
The invention is further directed to vectors containing GA4 encoding DNA and to the expression of GA4 protein encoded by the GA4 DNA in a host cell.
The invention is further directed to vectors containing GA4 antisense DNA and to the expression of GA4 antisense RNA by the GA4 antisense DNA in a host cell.
The invention is further directed to host cells transformed with the GA4 encoding DNA of the invention, and to the use of such host cells for the maintenance of the GA4 DNA or expression of the GA4 protein of the invention.
The invention is further directed to host cells transformed with the GA4 antisense DNA of the invention, and to the use of such host cells for the maintenance of the GA4 DNA or inhibition of expression of the GA4 protein of the invention.
The invention is further directed to transgenic plants containing the GA4 encoding or GA4 antisense DNA of the invention.
The invention is further directed to a method for altering plant growth, using the GA4 encoding or GA4 antisense DNA of the invention
The invention is further directed to a method for altering plant growth, using the recombinantly made GA4 protein of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1: T-DNA tagged mutant (T) is an allele of the ga4 locus. Both the T-DNA tagged allele, ga4-2 (T) and the EMS-induced allele, ga4-1 (ga4), respond to GA.sub.3 treatment with shoot elongation (T+GA.sub.3 and ga4+GA.sub.3, respectively). W, canonical wild type, Landsberg er; T, ga4-2; and ga4, ga4-1.
FIG. 2: DNA gel blot hybridization analysis showing cosegregation of the T-DNA insert with the ga4 mutation. DNA, isolated from leaf tissue of F3 progeny of individual F2 (ga4-2.times.tt2 ) plants exhibiting the semidwarf phenotype, is shown in lanes 1-8, (8 samples). Four fragments associated with the T-DNA insert were visible in DNA from all plants. Molecular weight DNA size markers are shown in Kb. L, canonical, wild type, Landsberg er.
FIGS. 3A-3B: Restriction map of the genomic clones (.lambda.T1-5 and .lambda.WT6) and subclones (pT12, pT34, and pWT32) used to isolate the GA4 gene. H, HindIII restriction site.
FIG. 4: Nucleotide [SEQ ID No. 1] and deduced amino acid sequence [SEQ ID No. 2] of the GA4 cDNA clone. The position of the intron as deduced from a comparison of cDNA and genomic sequences is indicated with a down arrowhead .tangle-soliddn. above the relevant line. The EMS-induced mutation at nucleotide 725 is indicated with a star (*) above that position. The underlined area indicates the sequence of the PCR labeled probe used for RNA gel blot analysis.
FIGS. 5A-5B: Nucleotide sequence [SEQ ID No. 3] of GA4 genomic DNA. The intron is underlined. The ATG initiation codon is indicated with a down arrowhead .tangle-soliddn. above and in front of the "A." The TGA stop codon is indicated with a star (*) above and after the "A.".
FIG. 6: Amino acid sequence comparison of GA4 and barley flavanone-3-hydroxylase (F3H) [SEQ ID No. 4]. Identical residues are shown in bold type.
FIG. 7: RNA gel blot analysis of GA4 gene expression in different tissues (silique, flower, root and leaf) of Arabidopsis.
FIG. 8: RNA gel blot analysis of ga4 and GA4 gene expression in Arabidopsis in 4-week-old rosette leaves of T-ga4 (ga4-2), ga4 (ga1) and Lan (Landsberg, er).
FIG. 9: RNA gel blot analysis of GA4 gene expression in Arabidopsis in ga4-1 with (+) or without (-) exogenous GAS. The ga4-1 plants were sprayed with 10.sup.-5 M GA.sub.3 and leaf samples were taken 8 and 24 hours after the treatment.
Definitions
Italicized, uppercase names, such as "GA4," refer to the wild type gene while italicized, lower case names, such as "ga4," refer to the mutant gene.
Uppercase names, such as "GA4," refer to the protein, DNA or RNA encoded by the GA4 gene, while lowercase names, such as "ga4," refer to the protein DNA or RNA encoded by the mutant ga4 gene.
"GA.sub.n " (with a number subscripted), refers to the "gibberellin A.sub.n " compound. The chemical structures of some of the gibberellin A.sub.n 's are presented in Moritz, T. et al., Planta 193:1-8 (1994).
Plant should be understood as referring to a multicellular differentiated organism capable of photosynthesis including angiosperms (monocots and dicots) and gymnosperms.
Plant cell should be understood as referring to the structural and physiological unit of plants. The term "plant cell" refers to any cell which is either part of or derived from a plant. Some examples of cells encompassed by the present invention include differentiated cells that are part of a living plant; differentiated cells in culture; undifferentiated cells in culture; the cells of undifferentiated tissue such as callus or tumors.
Plant cell progeny should be understood as referring to any cell or tissue derived from plant cells including callus; plant parts such as stems, roots, fruits, leaves or flowers; plants; plant seed; pollen; and plant embryos.
Propagules should be understood as referring to any plant material capable of being sexually or asexually propagated, or being propagated in vivo or in vitro. Such propagules preferably consist of the protoplasts, cells, calli, tissues, embryos or seeds of the regenerated plants.
Transgenic plant should be understood as referring to a plant having stably incorporated exogenous DNA in its genetic material. The term also includes exogenous DNA which may be introduced into a cell or protoplast in various forms, including, for example, naked DNA in circular, linear or supercoiled form, DNA contained in nucleosomes or chromosomes or nuclei or parts thereof, DNA complexed or associated with other molecules, DNA enclosed in liposomes, spheroplasts, cells or protoplasts.
A fragment of a molecule should be understood as referring to a shortened sequence of an amino acid or nucleotide genetic sequence that retains some desired chemical or biological property of the full-length sequence such that use of the full-length sequence is not necessary to achieve the desired purpose.
A mutation should be understood as referring to a detectable change in the genetic material which may be transmitted to daughter cells and possibly even to succeeding generations giving rise to mutant cells or mutant organisms. If the descendants of a mutant cell give rise only to somatic cells in multicellular organisms, a mutant spot or area of cells arises. Mutations in the germ line of sexually reproducing organisms may be transmitted by the gametes to the next generation resulting in an individual with the new mutant condition in both its somatic and germ cells. A mutation may be any (or a combination of) detectable, unnatural change affecting the chemical or physical constitution, mutability, replication, phenotypic function, or recombination of one or more deoxyribonucleotides; nucleotides may be added, deleted, substituted for, inverted, or transposed to new positions with and without inversion. Mutations may occur spontaneously and can be induced experimentally by application of mutagens. A mutant variation of a nucleic acid molecule results from a mutation. A mutant polypeptide may result from a mutant nucleic acid molecule.
A species should be understood as referring to a group of actually or potentially interbreeding natural populations. A species variation within a nucleic acid molecule or protein is a change in the nucleic acid or amino acid sequence that occurs among species and may be determined by DNA sequencing of the molecule in question.
A preparation that is substantially free of other A. thaliana DNA (or protein) should be understood as referring to a preparation wherein the only A. thaliana DNA (or protein) is that of the recited A. thaliana DNA (or protein). Though proteins may be present in the sample which are homologous to other A. thaliana proteins, the sample is still said to be substantially free of such other A. thaliana DNA (or protein) as long as the homologous proteins contained in the sample are not expressed from genes obtained from A. thaliana.
A DNA construct should be understood as referring to a recombinant, man-made DNA, linear or circular.
T-DNA (transferred DNA) should be understood as referring to a segment or fragment of Ti (tumor-inducing) plasmid DNA which integrates into the plant nuclear DNA.
Stringent hybridization conditions should be understood to be those conditions normally used by one of skill in the art to establish at least a 90% homology between complementary pieces of DNA or DNA and RNA. Lesser homologies, such as at least 70% homology or preferably at least 80% may also be desired and obtained by varying the hybridization conditions.
There are only three requirements for hybridization to a denatured strand of DNA to occur. (1) There must be complementary single strands in the sample. (2) The ionic strength of the solution of single-stranded DNA must be fairly high so that the bases can approach one another; operationally, this means greater than 0.2M. (3) The DNA concentration must be high enough for intermolecular collisions to occur at a reasonable frequency. The third condition only affects the rate, not whether renaturation/hybridization will occur.
Conditions routinely used by those of skill in the art are set out in readily available procedure texts, e.g., Current Protocol in Molecular Biology, Vol. 1, Chap. 2.10, John Wiley & Sons, Publishers (1994) or Sambrook et al., Molecular Cloning, Cold Spring Harbor (1989), incorporated herein by reference. As would be known by one of skill in the art, the ultimate hybridization stringency reflects both the actual hybridization conditions as well as the washing conditions following the hybridization, and one of skill in the art would know the appropriate manner in which to change these conditions to obtain a desired result.
For example, a prehybridization solution should contain sufficient salt and nonspecific DNA to allow for hybridization to non-specific sites on the solid matrix, at the desired temperature and in the desired prehybridization time. For example, for stringent hybridization, such prehybridization solution could contain 6.times. single strength citrate (SSC) (1.times.SSC is 0.15 M NaCl, 0.015M Na citrate; pH 7.0), 5.times. Denhardt's solution, 0.05% sodium pyrophosphate and 100 .mu.g per ml of herring sperm DNA. An appropriate stringent hybridization mixture might then contain 6.times. SSC, 1.times. Denhardt's solution, 100 .mu.g per ml of yeast tRNA and 0.05% sodium pyrophosplhate.
Alternative conditions for DNA--DNA analysis could entail the following:
1) prehybridization at room temperature and hybridization at 68.degree. C.;
2) washing with 0.2.times. SSC/0. 1% SDS at room temperature;
3) as desired, additional washes at 0.2.times. SSC/0.1% SDS at 42.degree. C. (moderate-stringency wash); or
4) as desired, additional washes at 0.1.times. SSC/0.1% SDS at 68.degree. C. (high stringency).
Known hybridization mixtures, e.g., that of Church and Gilbert, Proc. Natl. Acad. Sci. USA 81:1991-1995 (1984), comprising the following composition may also be used: 1% crystalline grade bovine serum albumin/1 mM EDTA/0.5M NaHPO.sub.4, pH 7.2/7% SDS. Additional, alternative but similar reaction conditions can also be found in Sambrook et al., Molecular Cloning, Cold Spring Harbor (1989). Formamide may also be included in prehybridization/hybridization solutions as desired.
It should be understood that these conditions are not meant to be definitive or limiting and may be adjusted as required by those of ordinary skill in the art to accomplish the desired objective.
A vector should be understood to be a DNA element used as a vehicle for cloning or expressing a desired sequence, such as a gene of the invention, in a host.
A host or host cell should be understood to be a cell in which a sequence encoding a GA4 DNA of the invention is incorporated and expressed. A GA4 gene of the invention or the antisense of the gene may be introduced into a host cell as part of a vector by transformation. Both the sense and the antisense DNA sequences are present in the same host cell since DNA is double stranded. The direction of transcription, however, as directed by an operably linked promoter as designed by the artisan, dictates which of the two strands is ultimately copied into RNA.
DETAILED DESCRIPTION OF THE INVENTION
The process for genetically engineering GA4 protein sequences, according to the invention, is facilitated through the cloning of genetic sequences that are capable of encoding the GA4 protein and through the expression of such genetic sequences. As used herein, the term "genetic sequences" is intended to refer to a nucleic acid molecule (preferably DNA). Genetic sequences that are capable of encoding GA4 protein can be derived from a variety of sources. These sources include genomic DNA, cDNA, synthetic DNA, and combinations thereof. The preferred source of the ga4 genomic DNA is a plant genomic library and most preferably an Arabidopsis thaliana genomic library. A more preferred source of the GA4 cDNA is a plant cDNA library and most preferably an Arabidopsis thaliana cDNA library made from silique mRNA, although the message is ubiquitously expressed in the root, leaf and flower of plants.
The recombinant GA4 cDNA of the invention will not include naturally occurring introns if the cDNA was made using mature GA4 mRNA as a template. Genomic DNA may or may not include naturally occurring introns. Moreover, such genomic DNA may be obtained in association with the homologous (isolated from the same source; native) 5' promoter region of the GA4 gene sequences and/or with the homologous 3' transcriptional termination region. Further, such genomic DNA may be obtained in association with the genetic sequences which provide the homologous 5' non-translated region of the GA4 mRNA and/or with the genetic sequences which provide the homologous 3' non-translated region.
In plants, the GA4 sequences of the invention can be identified using T-DNA insertion mutants. In a T-DNA insertion mutant, the mutant phenotype is a result of the T-DNA insertion. A genomic library from such a mutant can be screening for the T-DNA element, and the flanking sequence analyzed to determine the native sequence that was disrupted by the T-DNA and thus led to the phenotype of the mutant plant.
The T-DNA generally carries a resistance selection marker, such as that for kanamycin, that is used to identify outcrosses that retain the T-DNA. This confirms co-segregation of the mutant phenotype and the T-DNA insert. Having identified a T-DNA mutant with the T-DNA inserted at the site of the GA4 gene, the T-DNA then becomes a tag with which the ga4 mutant gene (flanking both sides of the T-DNA insertion) can be isolated and used to identify other GA4 genes in libraries from nonmutants of the same species or in libraries made from other species, Walden et al., Plant J., 1: 281-288 (1991). Additional tests, such as DNA gel blot analysis can then be used to confirm that the T-DNA insert is present in the gene of interest, here the ga4 gene.
As exemplified herein from Arabidopsis thaliana, the Arabidopsis ga4 mutant plant used to identify the GA4 (wild type) and ga4 (mutant) genetic sequences of the invention is deficient in an enzyme of the gibberellin biosynthetic pathway called 3-3-hydroxylase. Accordingly, it is believed that the site of T-DNA insertion in the ga4 mutants of the invention is in the GA4 gene that encodes the 3-.beta.-hydroxylase of the gibberellin biosynthetic pathway. The genomic sequence of GA4, including introns, is shown in FIGS. 5A-5B [SEQ ID No. 3]. The cDNA sequence of GA4 is shown in FIG. 4 [SEQ ID No. 1] as is the sequence of the GA4 protein encoded by the sequence [SEQ ID No. 2]. A single base mutation of G to A occurs at base 725 in a ga4 mutant that was produced by chemical (EMS) mutation, as described in the Examples. This results in an amino acid change from cystein to tyrosine.
Due to the degeneracy of nucleotide coding sequences, and to the fact that the DNA code is known, all other DNA sequences which encode the same amino acid sequence as depicted in FIG. 4 [SEQ ID No. 2] can be determined and used in the practice of the present invention. Additionally, those sequences that hybridize to sequence ID Nos. 1 or 3 under stringent conditions are also useful in the practice of the present invention.
A DNA sequence encoding GA4 protein or GA4 antisense RNA can be inserted into a DNA vector in accordance with conventional techniques, including blunt-ending or staggered-ending termini for ligation, restriction enzyme digestion to provide appropriate termini, filling in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and ligation with appropriate ligases. In one embodiment of the invention, expression vectors are provided that are capable of expressing GA4 mRNA or antisense RNA. Vectors for propagating a given sequence in a variety of host systems are well known and can readily be altered by one of skill in the art such that the vector will contain DNA or RNA encoding the desired genetic sequence and will be propagated in a desired host. Such vectors include plasmids and viruses and such hosts include eukaryotic organisms and cells, for example plant, yeast, insect, plant, mouse or human cells, and proka-yotic organisms, for example E. coli and B. subtilus. Shuttle vectors in which the desired genetic sequence is "maintained" in an available form before being extracted and transformed into a second host for expression are also useful DNA constructs envisioned as carrying the DNA of the invention.
A nucleic acid molecule, such as DNA, is said to be "capable of expressing" a polypeptide or antisense sequence if it contains a nucleotide sequence that encodes such polypeptide or antisense sequence and transcriptional and, if necessary, translational regulatory information operably linked to the nucleotide sequences that encode the polypeptide or antisense sequence.
Two DNA sequences (such as a promoter region sequence and the ga4 or GA4 gene encoding or antisense sequence) are said to be operably linked if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region sequence to direct the transcription of the desired sequence, or (3) interfere with the ability of the desired sequence to be transcribed by the promoter region sequence. Thus, a promoter region would be operably linked to a desired DNA sequence if the promoter were capable of effecting transcription of that DNA sequence.
Preferred prokaryotic hosts include bacteria such as E. coli, Bacillus, Streptomyces, Pseudomonas, Salmionella, Serratia, etc. The most preferred prokaryotic host is E. coli. The procaryotic host must be compatible with the replicon and control sequences in the expression plasmid.
Preferred eukaryotic hosts include plants, yeast, fungi, insect cells, mammalian cells. These hosts can be utilized for production of the desired genetic sequence, or GA4 or ga4 protein, in conventional methods, such as by growth in shake flasks, fermentors, tissue culture plates or bottles. Alternatively, multicellular organisms such as a plant might be used.
In one embodiment, a vector is employed that is capable of integrating the desired gene sequences into the host cell chromosome. Cells that have stably integrated the introduced DNA into their chromosomes can be selected by also introducing one or more markers which allow for selection of host cells which contain the expression vector. The marker may provide for prototrophy to an auxotrophic host, biocide resistance, e.g., antibiotics, or heavy metals, such as copper, or the like. The selectable marker gene sequence can either be directly linked to the DNA gene sequences to be expressed, or introduced into the same cell by co-transfection. In another embodiment, the introduced sequence will be incorporated into a plasmid or viral vector capable of autonomous replication in the recipient host. Any of a wide variety of vectors may be employed for this purpose. Factors of importance in selecting a particular plasmid or viral vector include: the ease with which recipient cells that contain the vector may be recognized and selected from those recipient cells which do not contain the vector; the number of copies of the vector which are desired in a particular host; and whether it is desirable to be able to "shuttle" the vector between host cells of different species.
DNA encoding the desired protein is preferably operably linked to a promoter region, a transcription initiation site, and a transcription termination sequence, functional in plants. Any of a number of promoters which direct transcription in a plant cell is suitable. The promoter can be either constitutive or inducible. Some examples of promoters functional in plants include the nopaline synthase promoter and other promoters derived from native Ti plasmids, viral promoters including the 35S and 19S RNA promoters of cauliflower mosaic virus (Odell et al., Nature 313:810-812 (1985)), and numerous plant promoters.
Alternative promoters that may be used include nos, ocs, and CaMV promoters. Overproducing plant promoters may also be used. Such promoters, operably linked to the GA4 gene, should increase the expression of the GA4 protein. Overproducing plant promoters that may be used in this invention include the promoter of the small subunit (ss) of ribulose-1,5-biphosphate carboxylase from soybean (Berry-Lowe et al., J. Molecular and App. Gen. 1:483-498 (1982), and the promoter of the chlorophyll a/b binding protein. These two promoters are known to be light-induced in eukaryotic plant cells (see, for example, Genetic Engineering of Plants, an Agricultural Perspective, A. Cashmore, Plenum, New York 1983, pages 29-38; Corruzi, G. et al., J. of Biol. Chem. 258:1399 (1983); and Dunsmuir, P. et al., J. of Mol. and Applied Genet. 2:285 (1983)).
Genetic sequences comprising the desired gene or antisense sequence operably linked to a plant promoter may be joined to secretion signal sequences and the construct ligated into a suitable cloning vector. In general, plasmid or viral (bacteriophage) vectors containing replication and control sequences derived from species compatible with the host cell are used. The cloning vector will typically carry a replication origin, as well as specific genes that are capable of providing phenotypic selection markers in transformed host cells, typically antibiotic resistance genes.
General methods for selecting transgenic plant cells containing a selectable marker are well known and taught, for example, by Herrera-Estrella, L. and Simpson, J. (1988) "Foreign Gene Expression in Plants" in Plant Molecular Biology, A Practical Approach, Ed. C. H. Shaw, IRL Press, Oxford, England, pp. 131-160.
In another embodiment, the present invention relates to a transformed plant cell comprising exogenous copies of DNA (that is, copies that originated outside of the plant) encoding a GA4 gene expressible in the plant cell wherein said plant cell is free of other foreign marker genes (preferably, other foreign selectable marker genes); a plant regenerated from the plant cell; progeny or a propagule of the plant; and seed produced by the progeny.
Plant transformation techniques are well known in the art and include direct transformation (which includes, but is not limited to: microinjection (Crossway, Mol. Gen. Genetics 202:179-185 (1985)), polyethylene glycol transformation (Krens et al., Nature 296:72-74 (1982)), high velocity ballistic penetration (Klein et al., Nature327:70-73 (1987)), fusion of protoplasts with other entities, either minicells, cells, lysosomes, or other fusible lipid-surfaced bodies (Fraley et al., Proc. Natl. Acad. Sci. USA 79:1859-1863 (1982)), electroporation (Fromm et al., Proc. Natl. Acad. Sci. USA 82:5824 (1985)) and techniques set forth in U.S. Pat. No. 5,231,019)) and Agrobacteziim tumefaciens mediated transformation as described herein and in (Hoekema et al., Nature 303:179 (1983), de Framond et al., Bioltechnology 1 .262 (1983), Fraley et al. WO84/02913, WO84/02919 and WO84/02920, Zambryski et al. EP 116,718, Jordan et al., Plant Cell Reports 7:281-284 (1988), Leple et al. Plant Cell Reports 11:137-141 (1992), Stomp et al., Plant Physiol. 92:1226-1232 (1990), and Knauf et al., Plasmid 8:45-54 (1982)). Another method of transformation is the leaf disc transformation technique as described by Horsch et al. Science 227:1229-1230 (1985).
The transformation techniques can utilize a DNA encoding the GA4 amino acid sequence of FIG. 4 [SEQ ID No. 2], including the GA4 DNA sequence of FIG. 4 [SEQ ID No. 1], the GA4 genomic sequence of FIG. [SEQ ID No. 3], fragments thereof or the antisense sequence, expressible in plants. Included within the scope of a gene encoding the GA4 amino acid sequence of FIG. 4 [SEQ ID No. 2] are functional derivatives of the GA4 sequence of the invention, as well as variant, analog, species, allelic and mutational derivatives.
As used herein, modulation of GA4 expression entails the enhancement or reduction of the naturally occurring levels of the protein. Specifically, the translation of RNA encoding GA4 can be reduced using the technique of antisense cloning.
In general, antisense cloning entails the generation of an expression module which encodes an RNA complementary (antisense) to the RNA encoding GA4 (sense). By expressing the antisense RNA in a cell which expresses the sense strand, hybridization between the two RNA species will occur resulting in the blocking of translation. Alternatively, overexpression of the GA4 protein might be accomplished by use of appropriate promoters, enhancers, and other modifications. Those of skill in the art would be aware of references describing the use of antisense genes in plants (van der Krol et al., Gene 72:45-50 (1988); van der Krol et al., Plant Mol. Biol. 14:467-486 (1990); Zhang et al., Plant Cell 4:1575-1588 (1992)).
Other foreign marker genes (i.e., exogenously introduced genes) typically used include selectable markers such as a neo gene (Potrykus et al., Mol. Gen. Genet 199:183-188 (1985)) which codes for kanamycin resistance; a bar gene which codes for bialaphos resistance; a mutant EPSP synthase gene (Hinchee et al., Bioltechnology 6:915-922 (1988)) which encodes glyphosate resistance; a nitrilase gene which confers resistance to bromoxynil (Stalker et al., J. Biol. Chem. 263:6310-6314 (1988)); a mutant acetolactate synthase gene (ALS) which confers imidazolinone or sulphonylurea resistance (EP application number 154,204); a methotrexate resistant DHFR gene (Thillet et al., J. Biol. Chem. 263:12500-12508) and screenable markers which include B3-glucuronidase (GUS) or an R-locus gene, alone or in combination with a C-locus gene (Ludwig et al., Proc. Natl. Acad. Sci. USA 86:7092 (1989); Paz-Ares et al., EMBO J. 6:3553 (1987)).
Alternatively, the genetic construct for expressing the desired protein can be microinjected directly into plant cells by use of micropipettes to mechanically transfer the recombinant DNA. The genetic material may also be transferred into plant cells using polyethylene glycol to form a precipitation complex with the genetic material that is taken up by cells. (Paszkowski et al., EMBO J. 3:2717-22 (1984)). The desired gene may also be introduced into plant cells by electroporation. (Fromm et al., "Expression of Genes Transferred into Monocot and Dicot Plant Cells by Electroporation," Proc. Nat'l. Acad. Sci. U.S.A. 82:5824 (1985)). In this technique, plant protoplasts are electroporated in the presence of plasmids containing the desired genetic construct. Electrical impulses of high field strength reversibly permeabilize biomembranes allowing the introduction of plasmids. Electroporated plant protoplasts reform cell walls, divide, and form plant calli. Selection of the transformed plant cells expressing the desired gene can be accomplished using phenotypic markers as described above.
Another method of introducing the desired gene into plant cells is to infect the plant cells with Agrobacterium tumefaciens transformed with the desired gene. Under appropriate conditions well-known in the art, transformed plant cells are grown to form shoots, roots, and develop further into plants. The desired genetic sequences can be joined to the Ti plasmid of Agrobacterium tumefaciens. The Ti plasmid is transmitted to plant cells on infection by Agrobacterium tumefaciens and is stably integrated into the plant genome. Horsch et al., "Inheritance of Functional Foreign Genes in Plants," Science 233: 496-498 (1984); Fraley et al., Proc. Nat'l Acad. Sci. U.S.A. 80: 4803 (1983)); Feldmann, K. A. et al., Mol. Gen. Genet., 208: 1-9 (1987); Walden, R. et al., Plant J., 1: 281-288 (1991).
Presently there are several different ways to transform plant cells with Agrobacterium:
(1) co-cultivation of Agrobacterium with cultured, isolated protoplasts, or
(2) transformation of cells or tissues with Agrobacterium.
Method (1) requires an established culture system that allows culturing protoplasts and plant regeneration from cultured protoplasts. Method (2) requires that the plant cells or tissues can be transformed by Agrobacterium and that the transformed cells or tissues can be induced to regenerate into whole plants. In the binary system, to have infection, two plasmids are needed: a T-DNA containing plasmid and a vir plasmid.
Routinely, however, one of the simplest methods of plant transformation is explant inoculation, which involves incubation of sectioned tissue with Agrobacterium containing the appropriate transformation vector (Plant Genetic Transformation and Gene Expression, A Laboratory Manual, Oxford: Blackwell Scientific Publications (1988); Walden, Genetic Transformation in Plants, Milton Koynes: Open University Press (1988)).
All plants from which protoplasts can be isolated and cultured to give whole regenerated plants can be used for the expression of the desired gene. Suitable plants include, for example, species from the genera Fragaiia, Lotus, Medicago, Onobrychis, Tfifolium, Tiigonella, Vigna, Citrus, Linum, Geranium, Manicot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersion, Nicotiana, Solanum, Petunia, Digitalis, Majorana, Cichorium, Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Hemerocallis, Nemesia, Pelargonium, Panicum, Pennisetum, Ranunculus, Senecio, Salpiglossis, Cucumis, Browallia, Glycine, Lolium, Zea, Triticum, Sorghum, and Datura. Additional plant genera that may be transformed byAgrobacterium includeIpomoea, Passiflora, Cyclamen, Malus, Prunus, Rosa, Rubus, Populus, Santalum, Alliuni, Liliunz, Narcissus, Ananas, Arachis, Phaseolus, and Pisum.
Plant regeneration techniques are well known in the art and include those set forth in the Handbook of Plant Cell Culture, Volumes 1-3, Eds. Evans et al. Macmillan Publishing Co., New York, N.Y. (1983, 1984, 1984, respectively); Predieri and Malavasi, Plant Cell, Tissue, and Organ Culture 17: 133-142 (1989); James, D. J., et al., J. Plant Physiol. I32:148-154 (1988); Fasolo, F., et al., Plant Cell, Tissue, and Organ Culture 16:75-87 (1989); Valobra and James, Plant Cell, Tissue, and Organ Culture 21:51-54 (1990); Srivastava, P. S., et al., Plant Science 42:209-214 (1985); Rowland and Ogden, Hort. Science 27:1127-1129 (1992); Park and Son, Plant Cell, Tissue, and Organ Culture 15:95-105 (1988); Noli and Minocha, Plant Cell Reports 5:464-467 (1986); Brand and Lineberger, Plant Science 57: 173-179 (1988); Bozhkov, P. V., et al., Plant Cell Reports 11:386-389 (1992); Kvaalen and von Arnold, Plant Cell, Tissue, and Organ Culture 27:49-57 (1991); Tremblay and Tremblay, Plant Cell, Tissue, and Organ Culture 27:95-103 (1991); Gupta and Pullman, U.S. Pat. No. 5,036,007; Michler and Bauer, Plant Science 77:111-118 (1991); Wetzstein, H. Y., et al., Plant Science 64:193-201 (1989); McGranahan, G. H., et al., Bio/Technology 6:800-804 (1988); Gingas, V. M., Hort. Science 26:1217-1218 (1991); Chalupa, V., Plant Cell Reports 9:398-401 (1990); Gingas and Lineberger, Plant Cell, Tissue, and Organ Culture 17:191-203 (1989); Bureno, M. A., et al., Phys. Plant. 85:30-34 (1992); and Roberts, D. R., et al., Can. J. Bot. 68:1086-1090 (1990).
Plant regeneration from cultured protoplasts is described in Evans et al., "Protoplast Isolation and Culture," in Handbook of Plant Cell Culture 1:124-176 (MacMillan Publishing Co., New York, 1983); M. R. Davey, "Recent Developments in the Culture and Regeneration of Plant Protoplasts," Protoplasts, 1983--Lecture Proceedings, pp. 19-29 (Birkhauser, Basel, 1983); P. J. Dale, "Protoplast Culture and Plant Regeneration of Cereals and Other Recalcitrant Crops," in Protoplasts 1983--Lecture Proceedings, pp. 31-41 (Birkhauser, Basel, 1983); and H. Binding, "Regeneration of Plants," in Plant Protoplasts, pp. 21-37 (CRC Press, Boca Raton, 1985).
Techniques for the regeneration of plants varies from species to species but generally, a suspension of transformed protoplasts containing multiple copies of the desired gene is first provided. Embryo formation can then be induced from the protoplast suspensions, to the stage of ripening and germination as natural embryos. The culture media will generally contain various amino acids and hormones, such as auxins and cytokinins. It is also advantageous to add glutamic acid and proline to the medium, especially for such species as corn and alfalfa.
Mature plants, grown from transformed plant cells, are selfed to produce an inbred plant. The inbred plant produces seed containing the recombinant DNA sequences promoting increased expression of GA4.
Parts obtained from regenerated plants, such as flowers, seeds, leaves, branches, fruit, and the like are covered by the invention provided that these parts comprise the herbicidal tolerant cells. Progeny and variants, and mutants of the regenerated plants are also included within the scope of this invention. As used herein, variant describes phenotypic changes that are stable and heritable, including heritable variation that is sexually transmitted to progeny of plants, provided that the variant still comprises a herbicidal tolerant plant through enhanced rate of acetylation. Also, as used herein, mutant describes variation as a result of environmental conditions, such as radiation, or as a result of genetic variation in which a trait is transmitted meiotically according to well-established laws of inheritance.
Plants which contain the GA4 encoding DNA of the invention and no other foreign marker gene are advantageous in that removal of the foreign marker gene, once inserted into the plant, may be impossible without also removing the GA4 gene. Absence of the foreign marker gene is sometimes desired so as to minimize the number of foreign genes expressed. This can be achieved by providing the GA4-encoding DNA between Ti-plasmid borders.
The T-DNA insertion mutant, ga4-2 and the EMS-induced mutant, ga4-1 both contain sequence alterations in the gene. The changes in the mutant alleles interfere with normal transcription. The deduced amino acid sequence of the GA4 protein shows similarity to the sequences of flavanone-3-hydroxylase and ACC oxidase from a variety of plant species (Meldgaard, M., Theor. Appl. Genet. 83: 695-706 (1992); Britsch, L. et al., J. Bio. Chem. 8: 5380-5387 (1992); Deikmann, J. et al., EMBO J. 7: 3315-3320 (1988)). The GA4 gene product is believed to be a 3-.beta.-hydroxylase. The 3-.beta.-hydroxylase is critical for controlling stem growth (Ingram et al., Planta 160: 455-463 (1984). Accordingly, the GA4 of the invention may be applied to crops to enhance and facilitate such stem elongation, flowering and fruiting. Alternatively, the DNA encoding GA4 may be genetically inserted into the plant host.
All plants which can be transformed are intended to be hosts included within the scope of the invention preferably, dicotyledonous plants). Such plants include, for example, species from the genera Fragana, Lotus, Medicago, Onobrychis, Tfifolium, Tfigonella, Vigna, Citrus, Linum, Geranium, Manicot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersion, Nicotiana, Solanum, Petunia, Digitalis, Majorana, Cichorium, Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Hemerocallis, Nemesia, Pelargonium, Panicum, Pennisetum, Ranunculus, Sencia, Salpiglossis, Cucumis, Browatia, Glycine, Lolium, Zea, Triticum, Sorghum, Malus, Apium, Datura, the le mutant in peas, the ga4 mutant in Arabadopsis, and the dwarf-1 mutant in Monocotyledonous plants such as corn.
Examples of commercially useful agricultural plants useful in the methods of the invention as transgenic hosts containing the GA4 DNA or antisense sequence of the invention include grains, legumes, vegetables and fruits, including but not limited to soybean, wheat, corn, barley, alfalfa, cotton, rapeseed, rice, tobacco, rye, tomatoes, beans, peas, celery, grapes, cabbage, oilseed, apples, strawberries, mulberries, potatoes, cranberries and lettuce.
Having now generally described the invention, the same will be more readily understood through reference to the following examples which are provided by way of illustration, and are not intended to be limiting of the present invention, unless specified.

EXAMPLES
Example 1--Methods
Plants and RNA and DNA Isolation
The ga4 mutant was obtained from M. Koornneef (Agricultural University, Wageningen, The Netherlands). T-DNA tagged ga4 mutant was generated by Agrobacterium root transformation with the pBIN19 vector (Bevan, M., Nuct. Acids. Res. 12:8711-8721 (1984)) (Clontech, Palo Alto, Calif. as "pBin19 in MC1022"). A description of T-DNA tagging and insertional mutagenesis is found in Walden et al., Plant J., 1: 281-288 (1991); Meinke, Dev. Gen., 12: 382-392 (1991). Plants were grown under greenhouse conditions using a 16-hr light/8-hr dark cycle. Tissue for DNA and RNA isolation was harvested at approximately 3-4 weeks after planting and before bolting, frozen into liquid nitrogen and stored at -70.degree. C. Genomic DNA was isolated using the methods of Watson, J. C. et al., DNA. Methods. Enzynzol. 118:57-75 (1986). Total RNA was isolated using the methods of Ausubel, F. M. et al., Current protocols in Molecular Biology, New York: Green Publishing Associates Wiley Interscience (1989).
Library Construction and Screening
The genomic library for the T-DNA insertion mutant, ga42, was constructed in .lambda. FIX II vectors (Stratagene, La Jolla, Calif. --see Stratagene Undigested Lambda FIXII Vector Cloning Kit Instruction Manual) and packaged using Gigapack II Gold packaging extracts (Stratagene). The ga4-2 and Landsberg genomic libraries and Landsberg cDNA library were plated on E. coli strain ER 1458 (New England Biolabs (Beverly, Mass.)--Cat. No. 401-C, pp. 202-203.) (Also see Raleigh, E. A., Meth. Enzymol., 152: 130-141 (1987) and Bullock, W. O. et al., BioTechniques, 5: 376-378 (1987).) Alternatively, Arabidopsis genomic and cDNA libraries may be obtained from the Arabidopsis Biological Resource Center, Ohio State University. The genomic library can be plated on E. coli strain NM554 and the cDNA library can be plated on E. coli strain Y1090 (both from Stratagene).
The DNA genomic library may be obtained as follows. One begins with a CsCl DNA preparation and partially digests it with Sau3AI. After digestion, a partial fill-in reaction is performed. The reaction mixture for the partial fill-in is as follows.
40 .mu.l DNA
6 .mu.l Sau3AI buffer 10.times.
2.5 .mu.l 0.1 M DTT
1 .mu.l 100 mM dATP
1 .mu.l 100 mM dGTP
5 .mu.l Klenow enzyme
4.5 Al H.sub.2 O
After 30 minutes at 37.degree. C. the reaction is terminated with phenol-chloroform and the DNA is obtained. The DNA is then loaded on a 0.7% low melting point agarose gel and after electrophoresing, bands between 10 and 23 kb are cut out from the gel. The gel with the cut-out bands is then melted at 67.degree. C. The isolated DNA is then placed in the following ligation mixture:
2 .mu.l Lambda Fix II, pre-digested arms (2 .mu.g)
1 .mu.g genomic DNA, partial fill-in
0.5 .mu.l 10.times. ligation buffer
0.5 .mu.l 10 mM ATP (pH 7.05)
0.5 .mu.l T4 DNA ligase
.about.1.5 .mu.l H.sub.2 O (to 5 .mu.l final volume)
Following ligation overnight at 4.degree. C., the DNA is packaged using GIGAPACK II GOLD.
Plaque lifts were made using Hybond filters (Amersham Corp.), which were then autoclaved for 2 min. Filters were hybridized with probes as described for DNA and RNA gel blot analysis below.
DNA Subcloning and Sequencing
Bacteriophage .lambda. DNA was prepared from ER1458 lysates according to the mini-prep method of Grossberger, D., Nucl. Acids. Res. 15:6737 (1987). DNA fragments were subcloned into pBluescript KS.sup.- vectors (Stratagene) and used to transform JM109.
Double stranded DNA was isolated from plasmid clones and purified by CsCl banding. Sequencing was performed using .alpha.-.sup.33 S-dATP and Sequenase (United States Biochemical Corp.) according to the manufacture's protocol for double stranded DNA sequencing. Sequence analysis was performed using the Sequence Analysis Software package (Genetics Computer Group, Inc., Madison, Wis.) and the Blast network service of the National Center for Biotechnology Information (Bethesda, Md.).
DNA and RNA Gel Blot Analysis
Electrophoresis of DNA was in Tris-Acetate-EDTA buffer with subsequent transfer in 25 mM NaHPO.sub.4 to Biotrans filters (International Chemical and Nuclear Corp.). Electrophoresis of RNA samples was in agarose gels containing RNAase inhibitor using MOPS/EDTA buffer and transferred to filters as for DNA. Filters were UV-crosslinked using a Stratalinker (Stratagene) and baked for I hr at 80.degree. C.
Radioactive probes were separated from unincorporated nucleotides using a 1-ml Sephadex G-50 spin column and denatured in a microwave oven (Stroop, W. G. et al., Anal. Biochem 2. 182:222-225 (1989)). Prehybridization for 1 hr and hybridization overnight were performed at 65.degree. C. in the hybridization buffer described by Church, G. M. et al., Proc. Natl. Acad. Sci. USA 81:1991-1995 (1984)). Filters were washed once for 15 min in 2.times.SSC at room temperature, then two times for 30 min in 0.1.times.SSC and 0.1%SDS at 60.degree. C. The damp filters were autoradiographed at -80.degree. C. using intensifying screens. Filters were stripped twice in 2mM Tris-HCl, pH8.0, 1 mM EDTA, 0.2% SDS at 70.degree. C. for 30 min prior to reprobing (Church, G. M. et al., Proc. Natl. Acad. Sci. USA 81:1991-1995 (1984)).
Example 2
Characterization of a Semidwarf T-DNA Insertion Mutant Allelic to ga4
A semidwarf mutant was generated from Arabidopsis thaliana (Landsberg erecta) as a result of Agrobacterium tumefaciens-mediated root transformation (Valvekens, D. et al., Proc. Natl. Acad. Sci. USA 85:5536-5540 (1988)). This mutant transgenic plant elongates its shoots in response to exogenously added GA.sub.3 (FIG. 1). The complementation analysis of the ga4-2 plant with ga4-1 plant (ga4.times.T) revealed that the transgenic plant has an insertion mutation that is an allele of the ga4 locus. There are several different gibberellin-responsive mutants in Arabidopsis, and therefore to test for allelism the transgenic plant was crossed to them in pairwise combination. Complementation analysis with the other genetically characterized semidwarf mutants in Arabidopsis revealed that the cross between the transgenic plant and the EMS-induced ga4 plant (Koornneef, M. et al., Theor. Appl. Genet. 58:257-263 (1980)) does not complement the mutant phenotype (FIG. 1). Therefore the mutation in the transgenic plant is an allele of the ga4 locus.
To test for co-segregation of the mutant phenotype and the T-DNA insert, the Ti progeny of the transgenic mutant that exhibited the semidwarf trait were outcrossed to either an Arabidopsis tt2 plant or to wild type C24 (Arabidopsis Biological Resource Center--Ohio State University). One of skill in the art, however, would know that any Arabidopsis thaliana could be used to perform the out-cross to ga4-2 to obtain the F1 progeny. The self-fertilized F2 progeny from those two crosses were tested for segregation of the kanamycin resistance marker encoded by the T-DNA. Progeny were grown on sterile medium containing 50 mg/L kanamycin, and the ratio of kanamycin resistant plants to sensitive plants was determined by their viability. As approximately three quarters of the F2 progeny from both crosses are resistant to kanamycin (Table I) the data indicates that there is one T-DNA insertion site in the transgenic plant.
TABLE I______________________________________Segregation Ratios of the F2 Progeny from ga4-2 (T-DNA tagged allele) Plants Crossed to tt2 Plants or Crossed to C24 Wild Type Plants Approximate T-DNA F2 Plant Kan.sup.r :Kan.sup.s Segregation Ratios Insertions______________________________________ga4-2 .times. tt2 163:56 3:1 1 (P > 0.8) ga4-2 .times. C24 104:29 3:1 1 (P > 0.3)______________________________________ Progeny were grown on sterile mineral nutrient medium containing 50 mg/L kanamycin; the ratio of kanamycin resistant plants to sensitive plants wa determined from their viability. The number of TDNA insertion sites predicted from the 3:1 segregation ratio and their probabilities from the Chisquare test are shown.
The self-fertilized F2 progeny from the two crosses were also tested for segregation of the mutant phenotype. The result from both crosses (Table II) shows a quarter of the resulting F2 progeny exhibit the semidwarf phenotype, indicating that the semidwarf phenotype is inherited as a single recessive mutation.
TABLE II______________________________________Segregation Ratios of the F2 Progeny from ga4-2 (T-DNA tagged allele) Plants Crossed to tt2 Plants or Crossed to C24 Wild Type Plants Approximate F2 Plant Wild Type:Dwarf Segregation Ratios Mutant Loci______________________________________ga4-2 .times. tt2 151:53 3:1 1 (P > 0.5) ga4-2 .times. C24 74:25 3:1 1 (P > 0.9)______________________________________ Progeny were soil grown and the ratio of plants that showed wild type compared to semidwarf phenotype were determined. The number of mutant loc predicted from the 3:1 segregation ratio and their probabilities from the Chisquare test are shown.
Although the data from these two independent tests are indicative, they are not sufficient to conclude that the ga4 allele is tagged by the T-DNA insert. The presence of the insert and its linkage with the mutant trait was therefore further tested by DNA gel blot analysis.
Example 3
DNA Gel Blot Analysis
Twenty F3 progeny from self-fertilized F2 plants (transgenic plant.times.tt2) were selected for their semidwarf phenotype and were then further tested for linkage of the T-DNA insert and the mutant phenotype by DNA gel blot analysis. DNA was isolated from leaf tissue of the individual F3 progeny, digested with HindIII and, after separation on an agarose gel and transfer, the DNA gel blot was probed with .sup.32 P-labeled pBIN19 plasmid containing the T-DNA border sequences (Bevan, M., Nucl. Acids. Res. 12:8711-8721 (1984)). The probe hybridizes to DNA from all the representative transgenic plants confirming the presence of the T-DNA insert (FIG. 2). For the results shown in FIG. 2, the DNA was digested with HindIII, separated by electrophoresis, bound to nylon filters, and then hybridized to .sup.32 P-labeled pBIN19 plasmid which contains the T-DNA border sequences. The hybridization pattern correlates with the T-DNA insert and the T-DNA/plant junctions. Four fragments associated with the T-DNA insert were visible in all plants (lanes 1-8) (FIG. 2) and cosegregate with the semidwarf phenotype. Therefore, the insertion site contains a complex T-DNA unit There is no hybridization with the wild type (Landsberg er) control. Thus, analysis from both the segregation test (Tables I and II) and the DNA gel blot analysis (FIG. 2), indicate that the T-DNA insert is the cause of the semidwarf mutation in the transgenic plant (the T-DNA tagged allele will be referred to as ga4-2) and that the T-DNA insert is tightly linked to the ga4 locus (the EMS-induced allele will be referred to as ga4-1).
Example 4
Isolation of the GA4 Gene
A genomic library was constructed with DNA isolated from F4 progeny of the ga4-2 plant. All constructs were subcloned into pBluescript KS.sup.-. The genomic clone, .lambda.T1-5, was derived by screening the ga4-2 genomic library using .sup.32 P-labeled pBIN19 vector as a probe. After plaque purification, clone .lambda.T1-5 was characterized by restriction enzyme analysis (FIGS. 3A-3B). The 1.2-kb HindIII fragment subclone, pT12, contains the T-DNA/plant DNA junction and was used to identify the insertion site by sequencing into the T-DNA insertion break point.
The genomic clone, WT6, was derived as follows. The subclone pWT32, which was deposited in a bacterial host with the American Type Culture Collection ("ATCC"), 12301 Park Lawn Drive, Rockville, Md. 20852, on Apr. 4, 1997 (ATCC Deposit No. 98394), and which corresponds to the T-DNA insertion site in XTI-5 was used as a probe to screen the leaf cDNA library and the ga4-1 genomic library.
To identify the region that corresponds to the T-DNA insertion site, the HindIII fragments of the genomic clone were subcloned into the plasmid vector pBluescript KS.sup.-. The 1.2 Kb HindIII fragment subclone, pT12, contains the T-DNA/plant DNA junction and was used to identify the insertion site by sequencing into the T-DNA insertion break point. The plant sequences from the flanking 3.4 Kb HindIII fragment subclone, pT34, which was deposited in a bacterial host with the American Type Culture Collection ("ATCC"), 12301 Park Lawn Drive, Rockville, Md. 20852, on Apr. 4, 1997 (ATCC Deposit No. 98393), were used to isolate the corresponding wild type genomic clone, .lambda.WT6 (FIGS. 3A-3B). The 3.2 Kb HindIII subclone from WT6 contains the sequences corresponding to the T-DNA insertion site in .lambda.T1-5 and was used as a probe to screen the leaf cDNA library and the ga4-1 genomic library. The isolated full length ga4 genomic and cDNA clones span sequences contained in both clone pT34, which was deposited in a bacterial host with the American Type Culture Collection ("ATCC"), 12301 Park Lawn Drive, Rockville, Md. 20852, on Apr. 4, 1997 (ATCC Deposit No. 98393), and pWT32, which was deposited in a bacterial host with the American Type Culture Collection ("ATCC"), 12301 Park Lawn Drive, Rockville, Md. 20852, on Apr. 4, 1997 (ATCC Deposit No. 98394).
Example 5
Nucleotide and Amino Acid Sequences
The GA4 cDNA is 1288 nucleotides with an open reading frame of 358 amino acids (FIG. 4; SEQ ID No. 1 and SEQ ID No. 2). There is a single 433-base-pair intron whose position was deduced from a comparison of the cDNA and genomic sequences (SEQ ID No. 3). Sequence analysis of the T-DNA/plant DNA junction indicates that the T-DNA insertion is within the intron. Analysis of the sequence revealed two possible AUG initiation codons (nucleotide position 67 and nucleotide position 76) within the open reading frame, both of which have weak homology to the "Kozak" consensus sequence for translation initiation (Kozak, M., Nuc. Acids Res. 15:8125-8148 (1987); Luitcke, H. A. et al., EMBO J. 6:43-48 (1987).
To confirm that the sequence determined is indeed the GA4 locus, genomic fragments from the other allele, ga4-1, were isolated and sequenced. The ga4-1 allele was generated by EMS mutagenesis in the same genetic background, Landsberg er. Sequence analysis of ga4-1 indicates that the EMS-induced mutation occurs at nucleotide 725 (FIG. 4) resulting in a single nucleotide change from G to A and a corresponding amino acid change from cysteine to tyrosine. This nucleotide change in the coding region, leading to the amino acid change, is presumably responsible for the ga4-1 mutation.
An alignment of the amino acid sequence of GA4 to barley flavanone-3-hydroxylase (F3H) [SEQ ID No. 4-1 exhibits a 24% amino acid identity (FIG. 6). FIG. 6 shows this alignment for the deduced amino acid sequences of the GA4 gene from Arabidopsis and flavanone-3-hydroxylase (F3H) from barley (Meldgaard, M., Theor. Appl. Genet. 83:695-706 (1992)). In addition, alignment to the amino acid sequence of 1-aminocyclopropane-1-carboxylate oxidase (ethylene-forming enzyme) from petunia shows 30% amino acid identity (data not shown).
On the basis of this sequence similarity, it is concluded that the GA4 gene encodes an hydroxylase involved in GA biosynthesis, and specifically a 3-.beta.-hydroxylase. This conclusion agrees with information based on biochemical studies (Talon, M. et al., Proc. Natl. Acad. Sci. USA 87:7983-7987 (1990)) that showed that the Arabidopsis ga4 mutant had reduced levels of the 3-hydroxy- and 3,13-hydroxy-GAs, and that it accumulates the 13-hydroxy-GAs and the non-3, 13-hydroxy-GAs, with some exceptions. Due to the ubiquitous nature of gibberellin growth factors, it is likely that a similar activity and gene sequence will be found for the cognate genes corresponding to GA4 in agronomically important crop plants, such as, for example, corn, peas, barley, potato, radish, rapeseed, alfalfa, celery, grapes, cabbage, lettuce, carrots, cucumber, squash, watermelon, rice and beans.
Example 6
The ga4 Mutant Overexpresses ga4 mRNA
To study the pattern of GA4 gene expression, total RNA was isolated from different tissue types and RNA gel blots were hybridized with a .sup.32 P-labeled PCR GA4 specific probe. A 1.4 Kb transcript is seen in root, flower, and siliques (FIG. 7). The same size transcript was detected in leaves when more RNA is loaded on the gel. This data is shown in FIG. 8--the "Lan" sample. The gene is expressed ubiquitously in the different tissues examined (root, leaf, flower and silique), but the message is most abundant in the silique.
There is differential expression in 4-week-old rosette leaves between the wild type and mutants. There is 3 to 4 fold more message expressed in the EMS induced ga4-1 plants as compared to wild type, but no message is detected in the T-DNA tagged ga4-2 plants (FIG. 8). The over-expression of ga4 message, detected in the ga4-1 plants, can be repressed by the application of 10.sup.-5 M exogenous GA.sub.3 on the rosette leaves of Arabidopsis. The transcriptional repression can be detected at 8 hours after the initial treatment and lasts for up to 24 hours (FIG. 9).
The over-expression of ga4 message in the EMS-induced ga4-1 mutant and transcriptional regulation by exogenous GA.sub.3 is a novel finding as regards the regulation of the gibberellin biosynthesis pathway. The terminal gibberellins in Arabidopsis are GA.sub.1 and GA.sub.4, which are effective in causing stem elongation (Talon, M. et al., Proc. Natl. Acad. Sci. USA 87:7983-7987 (1990)). GA.sub.3 has been shown to be present at low levels in vegetative tissue of maize. GA.sub.3 is biosynthesized from GA.sub.20 via GA.sub.5 ; and GA.sub.1 is the product of GA.sub.3 in Maize (Fujioka, S. et al., Plant Physiol. 94: 127-131 (1990)). There is no evidence of GA.sub.3 biosynthesis in Arabidopsis, but experiments show that exogenous GA.sub.3 is active in promoting stem elongation in Arabidopsis and in other species, for example, maize, cabbages, beans, rice, peas, watermelons, squash and cucumbers. The biological activity may be induced by either GA.sub.3 itself or the terminal GAs, such as GA.sub.1, as shown in the proposed pathway in maize (Fujioka, S. et al., Plant Physiol. 94:127-131 (1990)). In wild type plants, the concentrations and proportions of the cellular gibberellins are maintained by the balance between synthesis and utilization. In the ga4-1 plant, this balance is perturbed by the mutation and the concomitant reduction in the catalytic activity of the 3-.beta.-hydroxylase which leads to the accumulation of GA.sub.9 and GA.sub.20 and the reduction in GA.sub.4 and GA.sub.1, respectively. The mutated gene would either lead to translation of the mutant form of the protein (presumably inactive or less active) or to no translation at all. The over-expression of ga4 message as detected in the ga4-1 plants and the repression of transcription by exogenous GA.sub.3 indicates a transcriptional feedback regulatory mechanism. One hypothesis to explain these results in the ga4-1 plants is that the regulatory domain of the GA4 protein is intact but the reduced levels of endogenous GA.sub.4 and GA.sub.1 diminish the feedback control by the terminal GAs and the application of exogenous GA.sub.3, which leads to the accumulation of terminal GAs in Arabidopsis, restores the feed-back mechanism.
It has been previously established that 3-.beta.-hydroxylation is important in the regulation of stem growth (Ingram, T. J. et al., Planta 160:455-463 (1984)). Our results indicate that, in addition to the critical roles the properties and compartmentalization of the active GAs play in stem growth, molecular regulatory mechanisms also play an important part in the control of gibbereilin biosynthesis.
Example 7
Expressing the GA4 Protein
The GA4 protein is expressed by transforming a host with the DNA construct of SEQ ID No. I or SEQ ID No. 3 or a DNA construct comprising DNA encoding the amino acid sequence of SEQ ID No. 2 operably linked to a promoter. The GA4 protein is expressed from the construct in the transformed host cell.
Example 8
Gene Expression in a Plant
The expression of a gene in a plant is directed such that the gene has the same temporal and spatial expression pattern of GA4. The gene is operably linked to the regulatory sequences of GA4 DNA to create an expression module, and a plant is then transformed with the expression module.
Example 9
Modulating the Translation of RNA Encoding GA4 Protein
The translation of RNA encoding GA4 protein in a plant is modulated by generating an expression vector encoding antisense GA4 RNA. The plant is then transfected with the expression vector encoding the antisense GA4 RNA.
Example 10
Cloning DNA Encoding GA4 Protein
A DNA molecule encoding the GA4 protein is cloned by hybridizing a desired DNA molecule to the sequences or antisense sequences of DNA SEQ ID No. 1 or DNA SEQ ID No. 3 under stringent hybridization conditions. Those DNA molecules hybridizing to the probe sequences are selected and transformed into a host cell. The transformants that express GA4 are selected and cloned.
Example 11
Hybridization Conditions for Cloning DNA Encoding GA4 Protein
One possible set of hybridization conditions for the cloning of the DNA encoding GA4 protein is as follows:
1) prehybridizing for 1 hour;
2) hybridizing overnight at 65.degree. C. in the hybridization buffer; and
3) washing once for 15 minutes in 2.times.SSC at room temperature, then two times for 30 minutes in 0.1.times.SSC and 0.1% SDS at 60.degree. C.
Example 12
Stimulating Plant Stem Elongafion
Plant stem elongation is stimulated by inserting the DNA construct encoding the amino acid sequence shown in FIG. 4 [SEQ ID No. 2] into a transgenic plant. The transgenic plant is produced by any of several methods known in the art including those previously described in this specification.
The stem elongation may be stimulated in Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linuni, Geranium, Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersion, Nicotiana, Solanum, Petunia, Digitalis, Majorana, Cichorium, Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Hemerocallis, Nemesia, Pelargonium, Panicum, Pennisetum, Ranunculus, Sencia, Salpiglossis, Cucumis, Browallia, Glycine, Lolium, Zea, Triticum, Sorghum, Malus, Apium, and Datura.
Example 13
Producing Dwarf Plants
Dwarf plants are produced by blocking the GA4 gene by homologous recombination, or by transforming with a GA4 anti-sense DNA in order to produce transgenic plants. A cDNA sequence can be used to construct the antisense construct which is then transformed into a plant by using an Agrobacterium vector. (Zhang et al., Plant Cell 4: 1575-1588 (Dec. 1992)). Even partial antisense sequences can be used as antisense and can interfere with the cognate endogenous genes (van der Krol et al., Plant Mol. Biol. 14: 457-466 (1990)). The plant is transformed with the antisense construct according to the protocol of Valvekens et al., Proc. Natl. Acad, Sci, USA 85:5536-5540 (1988).
Dwarf plants are known to be commercially valuable. For example, dwarf trees for apples, cherries, peaches, pears and nectarines are commercially available (Burpee Gardens Catalogue 1994, pages 122-123).
Example 13
Molecular Weight Markers
The GA4 protein produced recombinantly is purified by routine methods in the art (Current Protocol in Molecular Biology, Vol. 2, Chap. 10, John Wiley & Sons, Publishers (1994)). Because, the deduced amino acid sequence is known, the molecular weight of this protein can be precisely determined and the protein can be used as a molecular weight marker for gel electrophoresis. The calculated molecular weight of the GA4 protein based on the deduced amino acid sequence is 39.5 kDa.
Conclusions
We have obtained full length genomic and cDNA clones and the sequences for the GA4 protein. It is believed that the GA4 locus encodes an hydroxylase involved in gibberellin biosynthesis.
All references mentioned herein are incorporated by reference in the disclosure.
Having now fully described the invention by way of illustration and example for purposes of clarity and understanding, it will be apparent to those of ordinary skill in the art that certain changes and modifications may be made in the disclosed embodiments and such modifications are intended to be within the scope of the present invention.
__________________________________________________________________________# SEQUENCE LISTING - - - - (1) GENERAL INFORMATION: - - (iii) NUMBER OF SEQUENCES: 4 - - - - (2) INFORMATION FOR SEQ ID NO:1: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1228 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: cDNA - - (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 67..1140 - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: - - ATAAGAAAAA AAACACAAAC ATCTATCAAA TTTACAAAGT TTTAAAACTA AT -#TAAAAAAG 60 - - AGCAAG ATG CCT GCT ATG TTA ACA GAT GTG TTT - #AGA GGC CAT CCC ATT 108 Met Pro Ala Met Leu Thr As - #p Val Phe Arg Gly His Pro Ile 1 - # 5 - # 10 - - CAC CTC CCA CAC TCT CAC ATA CCT GAC TTC AC - #A TCT CTC CGG GAG CTC 156 His Leu Pro His Ser His Ile Pro Asp Phe Th - #r Ser Leu Arg Glu Leu 15 - # 20 - # 25 - # 30 - - CCG GAT TCT TAC AAG TGG ACC CCT AAA GAC GA - #T CTC CTC TTC TCC GCT 204 Pro Asp Ser Tyr Lys Trp Thr Pro Lys Asp As - #p Leu Leu Phe Ser Ala 35 - # 40 - # 45 - - GCT CCT TCT CCT CCG GCC ACC GGT GAA AAC AT - #C CCT CTC ATC GAC CTC 252 Ala Pro Ser Pro Pro Ala Thr Gly Glu Asn Il - #e Pro Leu Ile Asp Leu 50 - # 55 - # 60 - - GAC CAC CCG GAC GCG ACT AAC CAA ATC GGT CA - #T GCA TGT AGA ACT TGG 300 Asp His Pro Asp Ala Thr Asn Gln Ile Gly Hi - #s Ala Cys Arg Thr Trp 65 - # 70 - # 75 - - GGT GCC TTC CAA ATC TCA AAC CAC GGC GTG CC - #T TTG GGA CTT CTC CAA 348 Gly Ala Phe Gln Ile Ser Asn His Gly Val Pr - #o Leu Gly Leu Leu Gln 80 - # 85 - # 90 - - GAC ATT GAG TTT CTC ACC GGT AGT CTC TTC GG - #G CTA CCT GTC CAA CGC 396 Asp Ile Glu Phe Leu Thr Gly Ser Leu Phe Gl - #y Leu Pro Val Gln Arg 95 - #100 - #105 - #110 - - AAG CTT AAG TCT GCT CGG TCG GAG ACA GGT GT - #G TCC GGC TAC GGC GTC 444 Lys Leu Lys Ser Ala Arg Ser Glu Thr Gly Va - #l Ser Gly Tyr Gly Val 115 - # 120 - # 125 - - GCT CGT ATC GCA TCT TTC TTC AAT AAG CAA AT - #G TGG TCC GAA GGT TTC 492 Ala Arg Ile Ala Ser Phe Phe Asn Lys Gln Me - #t Trp Ser Glu Gly Phe 130 - # 135 - # 140 - - ACC ATC ACT GGC TCG CCT CTC AAC GAT TTC CG - #T AAA CTT TGG CCC CAA 540 Thr Ile Thr Gly Ser Pro Leu Asn Asp Phe Ar - #g Lys Leu Trp Pro Gln 145 - # 150 - # 155 - - CAT CAC CTC AAC TAC TGC GAT ATC GTT GAA GA - #G TAC GAG GAA CAT ATG 588 His His Leu Asn Tyr Cys Asp Ile Val Glu Gl - #u Tyr Glu Glu His Met 160 - # 165 - # 170 - - AAA AAG TTG GCA TCG AAA TTG ATG TGG TTA GC - #A CTA AAT TCA CTT GGG 636 Lys Lys Leu Ala Ser Lys Leu Met Trp Leu Al - #a Leu Asn Ser Leu Gly 175 1 - #80 1 - #85 1 -#90 - - GTC AGC GAA GAA GAC ATT GAA TGG GCC AGT CT - #C AGT TCA GAT TTAAAC 684 Val Ser Glu Glu Asp Ile Glu Trp Ala Ser Le - #u Ser Ser Asp Leu Asn 195 - # 200 - # 205 - - TGG GCC CAA GCT GCT CTC CAG CTA AAT CAC TA - #C CCG GTT TGT CCT GAA 732 Trp Ala Gln Ala Ala Leu Gln Leu Asn His Ty - #r Pro Val Cys Pro Glu 210 - # 215 - # 220 - - CCG GAC CGA GCC ATG GGT CTA GCA GCT CAT AC - #C GAC TCC ACC CTC CTA 780 Pro Asp Arg Ala Met Gly Leu Ala Ala His Th - #r Asp Ser Thr Leu Leu 225 - # 230 - # 235 - - ACC ATT CTG TAC CAG AAC AAT ACC GCC GGT CT - #A CAA GTA TTT CGC GAT 828 Thr Ile Leu Tyr Gln Asn Asn Thr Ala Gly Le - #u Gln Val Phe Arg Asp 240 - # 245 - # 250 - - GAT CTT GGT TGG GTC ACC GTG CCA CCG TTT CC - #T GGC TCG CTC GTG GTT 876 Asp Leu Gly Trp Val Thr Val Pro Pro Phe Pr - #o Gly Ser Leu Val Val 255 2 - #60 2 - #65 2 -#70 - - AAC GTT GGT GAC CTC TTC CAC ATC CTA TCC AA - #T GGA TTG TTT AAAAGC 924 Asn Val Gly Asp Leu Phe His Ile Leu Ser As - #n Gly Leu Phe Lys Ser 275 - # 280 - # 285 - - GTG TTG CAC CGC GCT CGG GTT AAC CAA ACC AG - #A GCC CGG TTA TCT GTA 972 Val Leu His Arg Ala Arg Val Asn Gln Thr Ar - #g Ala Arg Leu Ser Val 290 - # 295 - # 300 - - GCA TTC CTT TGG GGT CCG CAA TCT GAT ATC AA - #G ATA TCA CCT GTA CCG 1020 Ala Phe Leu Trp Gly Pro Gln Ser Asp Ile Ly - #s Ile Ser Pro Val Pro 305 - # 310 - # 315 - - AAG CTG GTT AGT CCC GTT GAA TCG CCT CTA TA - #C CAA TCG GTG ACA TGG 1068 Lys Leu Val Ser Pro Val Glu Ser Pro Leu Ty - #r Gln Ser Val Thr Trp 320 - # 325 - # 330 - - AAA GAG TAT CTT CGA ACA AAA GCA ACT CAC TT - #C AAC AAA GCT CTT TCA 1116 Lys Glu Tyr Leu Arg Thr Lys Ala Thr His Ph - #e Asn Lys Ala Leu Ser 335 3 - #40 3 - #45 3 -#50 - - ATG ATT AGA AAT CAC AGA GAA GAA TGATTAGATA AT - #AATAGTTG TGATCTACTA 1170 Met Ile Arg Asn His Arg Glu Glu 355 - - GTTAGTTTGA TTAATAAATT GTTGTAAATG ATTTCAGCAA TATGATTTGT TT - #GTCCTC 1228 - - - - (2) INFORMATION FOR SEQ ID NO:2: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 358 amino - #acids (B) TYPE: amino acid (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: protein - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: - - Met Pro Ala Met Leu Thr Asp Val Phe Arg Gl - #y His Pro Ile His Leu 1 5 - # 10 - # 15 - - Pro His Ser His Ile Pro Asp Phe Thr Ser Le - #u Arg Glu Leu Pro Asp 20 - # 25 - # 30 - - Ser Tyr Lys Trp Thr Pro Lys Asp Asp Leu Le - #u Phe Ser Ala Ala Pro 35 - # 40 - # 45 - - Ser Pro Pro Ala Thr Gly Glu Asn Ile Pro Le - #u Ile Asp Leu Asp His 50 - # 55 - # 60 - - Pro Asp Ala Thr Asn Gln Ile Gly His Ala Cy - #s Arg Thr Trp Gly Ala 65 - # 70 - # 75 - # 80 - - Phe Gln Ile Ser Asn His Gly Val Pro Leu Gl - #y Leu Leu Gln Asp Ile 85 - # 90 - # 95 - - Glu Phe Leu Thr Gly Ser Leu Phe Gly Leu Pr - #o Val Gln Arg Lys Leu 100 - # 105 - # 110 - - Lys Ser Ala Arg Ser Glu Thr Gly Val Ser Gl - #y Tyr Gly Val Ala Arg 115 - # 120 - # 125 - - Ile Ala Ser Phe Phe Asn Lys Gln Met Trp Se - #r Glu Gly Phe Thr Ile 130 - # 135 - # 140 - - Thr Gly Ser Pro Leu Asn Asp Phe Arg Lys Le - #u Trp Pro Gln His His 145 1 - #50 1 - #55 1 -#60 - - Leu Asn Tyr Cys Asp Ile Val Glu Glu Tyr Gl - #u Glu His Met LysLys 165 - # 170 - # 175 - - Leu Ala Ser Lys Leu Met Trp Leu Ala Leu As - #n Ser Leu Gly Val Ser 180 - # 185 - # 190 - - Glu Glu Asp Ile Glu Trp Ala Ser Leu Ser Se - #r Asp Leu Asn Trp Ala 195 - # 200 - # 205 - - Gln Ala Ala Leu Gln Leu Asn His Tyr Pro Va - #l Cys Pro Glu Pro Asp 210 - # 215 - # 220 - - Arg Ala Met Gly Leu Ala Ala His Thr Asp Se - #r Thr Leu Leu Thr Ile 225 2 - #30 2 - #35 2 -#40 - - Leu Tyr Gln Asn Asn Thr Ala Gly Leu Gln Va - #l Phe Arg Asp AspLeu 245 - # 250 - # 255 - - Gly Trp Val Thr Val Pro Pro Phe Pro Gly Se - #r Leu Val Val Asn Val 260 - # 265 - # 270 - - Gly Asp Leu Phe His Ile Leu Ser Asn Gly Le - #u Phe Lys Ser Val Leu 275 - # 280 - # 285 - - His Arg Ala Arg Val Asn Gln Thr Arg Ala Ar - #g Leu Ser Val Ala Phe 290 - # 295 - # 300 - - Leu Trp Gly Pro Gln Ser Asp Ile Lys Ile Se - #r Pro Val Pro Lys Leu 305 3 - #10 3 - #15 3 -#20 - - Val Ser Pro Val Glu Ser Pro Leu Tyr Gln Se - #r Val Thr Trp LysGlu 325 - # 330 - # 335 - - Tyr Leu Arg Thr Lys Ala Thr His Phe Asn Ly - #s Ala Leu Ser Met Ile 340 - # 345 - # 350 - - Arg Asn His Arg Glu Glu 355 - - - - (2) INFORMATION FOR SEQ ID NO:3: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1663 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: cDNA - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: - - ATAAGAAAAA AAACACAAAC ATCTATCAAA TTTACAAAGT TTTAAAACTA AT -#TAAAAAAG 60 - - AGCAAGATGC CTGCTATGTT AACAGATGTG TTTAGAGGCC ATCCCATTCA CC -#TCCCACAC 120 - - TCTCACATAC CTGACTTCAC ATCTCTCCGG GAGCTCCCGG ATTCTTACAA GT -#GGACCCCT 180 - - AAAGACGATC TCCTCTTCTC CGCTGCTCCT TCTCCTCCGG CCACCGGTGA AA -#ACATCCCT 240 - - CTCATCGACC TCGACCACCC GGACGCGACT AACCAAATCG GTCATGCATG TA -#GAACTTGG 300 - - GGTGCCTTCC AAATCTCAAA CCACGGCGTG CCTTTGGGAC TTCTCCAAGA CA -#TTGAGTTT 360 - - CTCACCGGTA GTCTCTTCGG GCTACCTGTC CAACGCAAGC TTAAGTCTGC TC -#GGTCGGAG 420 - - ACAGGTGTGT CCGGCTACGG CGTCGCTCGT ATCGCATCTT TCTTCAATAA GC -#AAATGTGG 480 - - TCCGAAGGTT TCACCATCAC TGGCTCGCCT CTCAACGATT TCCGTAAACT TT -#GGCCCCAA 540 - - CATCACCTCA ACTACTGGTA TATCTTTTAT ACACTCGATC CTATATACTT GT -#ACTTGTGT 600 - - TTATTAGACC TTTTTCTACA TTAACAAAAA ACATATACAT AAGGACACAA TG -#TTTACATT 660 - - TAAGGTAGAA CATCCACAAA CGTTGGACGC CCTATAGGTA GTAACAAGGG GC -#ATAGATAA 720 - - CAGAAGCAAC CGAAATTTGC CTTGTCCTCG GAGTTTAGTG GATTTAAGAG TT -#AAGTGCAT 780 - - AATGAAATCT AGTGTAGTAG TGGACCCAAC TCAAAGATTT TGAAGATATG TA -#TTCTTTTA 840 - - ATCTTATCGG AGAAAACAAA ACAAAAAAAC AACAACTTGC TTTTCTATTT TA -#TTTAAAGG 900 - - TCGTACAAAT ATTTAATGTA TGTATATGCA AATTGTGTCT AAATCTCATC TG -#TACTAATT 960 - - AGATGAATAC AATTCGTTTT TAATTAACAG CGATATCGTT GAAGAGTACG AG -#GAACATAT 1020 - - GAAAAAGTTG GCATCGAAAT TGATGTGGTT AGCACTAAAT TCACTTGGGG TC -#AGCGAAGA 1080 - - AGACATTGAA TGGGCCAGTC TCAGTTCAGA TTTAAACTGG GCCCAAGCTG CT -#CTCCAGCT 1140 - - AAATCACTAC CCGGTTTGTC CTGAACCGGA CCGAGCCATG GGTCTAGCAG CT -#CATACCGA 1200 - - CTCCACCCTC CTAACCATTC TGTACCAGAA CAATACCGCC GGTCTACAAG TA -#TTTCGCGA 1260 - - TGATCTTGGT TGGGTCACCG TGCCACCGTT TCCTGGCTCG CTCGTGGTTA AC -#GTTGGTGA 1320 - - CCTCTTCCAC ATCCTATCCA ATGGATTGTT TAAAAGCGTG TTGCACCGCG CT -#CGGGTTAA 1380 - - CCAAACCAGA GCCCGGTTAT CTGTAGCATT CCTTTGGGGT CCGCAATCTG AT -#ATCAAGAT 1440 - - ATCACCTGTA CCGAAGCTGG TTAGTCCCGT TGAATCGCCT CTATACCAAT CG -#GTGACATG 1500 - - GAAAGAGTAT CTTCGAACAA AAGCAACTCA CTTCAACAAA GCTCTTTCAA TG -#ATTAGAAA 1560 - - TCACAGAGAA GAATGATTAG ATAATAATAG TTGTGATCTA CTAGTTAGTT TG -#ATTAATAA 1620 - - ATTGTTGTAA ATGATTTCAG CAATATGATT TGTTTGTCCT CAA - # 166 - #3 - - - - (2) INFORMATION FOR SEQ ID NO:4: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 377 amino - #acids (B) TYPE: amino acid (C) STRANDEDNESS: Not R - #elevant (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: peptide - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: - - Met Ala Pro Val Ser Asn Glu Thr Phe Leu Pr - #o Thr Glu Ala Trp Gly 1 5 - # 10 - # 15 - - Glu Ala Thr Leu Arg Pro Ser Phe Val Arg As - #p Glu Asp Glu Arg Pro 20 - # 25 - # 30 - - Lys Val Ala His Asp Arg Phe Ser Asp Ala Va - #l Pro Leu Ile Ser Leu 35 - # 40 - # 45 - - His Gly Ile Asp Gly Ala Arg Arg Ala Gln Il - #e Arg Asp Arg Val Ala 50 - # 55 - # 60 - - Ala Ala Cys Glu Asp Trp Gly Ile Phe Gln Va - #l Ile Asp His Gly Val 65 - #70 - #75 - #80 - - Asp Ala Asp Leu Ile Ala Asp Met Thr Arg Le - #u Ala Arg Glu Phe Phe 85 - # 90 - # 95 - - Ala Leu Pro Ala Glu Asp Lys Leu Arg Tyr As - #p Met Ser Gly Gly Lys 100 - # 105 - # 110 - - Lys Gly Gly Phe Ile Val Ser Ser His Leu Gl - #n Gly Glu Ala Val Gln 115 - # 120 - # 125 - - Asp Trp Arg Glu Ile Val Thr Tyr Phe Ser Ty - #r Pro Val Lys Ala Arg 130 - # 135 - # 140 - - Asp Tyr Gly Arg Trp Pro Glu Lys Pro Ala Gl - #y Trp Cys Ala Val Val 145 1 - #50 1 - #55 1 -#60 - - Glu Arg Tyr Ser Glu Arg Leu Met Gly Leu Se - #r Cys Asn Leu MetGly 165 - # 170 - # 175 - - Val Leu Ser Glu Ala Met Gly Leu Glu Thr Gl - #u Ala Leu Ala Lys Ala 180 - # 185 - # 190 - - Cys Val Asp Met Asp Gln Lys Val Val Val As - #n Phe Tyr Pro Arg Cys 195 - # 200 - # 205 - - Pro Gln Pro Asp Leu Thr Leu Gly Leu Lys Ar - #g His Thr Asp Pro Gly 210 - # 215 - # 220 - - Thr Ile Thr Leu Leu Leu Gln Asp Leu Val Gl - #y Gly Leu Gln Ala Thr 225 2 - #30 2 - #35 2 -#40 - - Arg Asp Gly Gly Lys Asn Trp Ile Thr Val Gl - #n Pro Ile Ser GlyAla 245 - # 250 - # 255 - - Phe Val Val Asn Leu Gly Asp His Gly His Ph - #e Met Ser Asn Gly Arg 260 - # 265 - # 270 - - Phe Lys Asn Ala Asp His Gln Ala Val Val As - #n Gly Glu Ser Ser Arg 275 - # 280 - # 285 - - Leu Ser Ile Ala Thr Phe Gln Asn Pro Ala Pr - #o Asp Ala Arg Val Trp 290 - # 295 - # 300 - - Pro Leu Ala Val Arg Glu Gly Glu Glu Pro Il - #e Leu Glu Glu Pro Ile 305 3 - #10 3 - #15 3 -#20 - - Thr Phe Thr Glu Met Tyr Arg Arg Lys Met Gl - #u Arg Asp Leu AspLeu 325 - # 330 - # 335 - - Ala Lys Arg Lys Lys Gln Ala Lys Asp Gln Le - #u Met Gln Gln Gln Leu 340 - # 345 - # 350 - - Gln Leu Gln Gln Gln Gln Ala Val Ala Ala Al - #a Pro Met Pro Thr Ala 355 - # 360 - # 365 - - Thr Lys Pro Leu Asn Glu Ile Leu Ala 370 - # 375__________________________________________________________________________

Number	Date	Country
WO 9303616	Mar 1993	WOX
WO 9316096	Aug 1993	WOX
WO 9318142	Sep 1993	WOX
WO 9403606	Feb 1994	WOX
WO 9428141	Dec 1994	WOX

GA4 DNA, protein and methods of use

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