AU6552599A - Tissue-specific and developmentally regulated transcriptional sequences and uses thereof - Google Patents

Description

S&F Ref: 266146D2

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT

ORIGINAL

*u

S

Name and Address of Applicant: Actual Inventor(s): Address for Service: Invention Title: Smart Plants International, Inc.

PO Box 26908 San Diego California 92196-0908 United States of America Leona Claire Fitzmaurice, T. Erik Mirkov, Kathryn Jane Elliott, Gregory Clyde Holtz Craig Duane Dickinson Spruson Ferguson St Martins Tower 31 Market Street Sydney NSW 2000 Tissue-specific and Developmentally Regulated Transcriptional Sequences and Uses Thereof The following statement is a full description of this invention, including the best method of performing it known to me/us:- 5845c TISSUE-SPECIFIC AND DEVELOPMENTALLY REGULATED TRANSCRIPTIONAL SEQUENCES AND USES THEREOF FIELD OF THE INVENTION This invention relates generally to plant biotechnology and specifically to DNA sequences capable of directing tissue-specific and developmentally regulated expression of gene fusion constructs in transgenic plants.

INTRODUCTION

Development of new crop plants through traditional plant breeding methods relies upon the observation of plant characteristics (phenotypes) and studies of their inheritance. Plant breeders have identified numerous desirable phenotypes and, through controlled breeding efforts, transferred these phenotypes into commercial plant varieties. However, in the process of transferring desirable phenotypes, undesirable phenotypes also can be transferred. Breeders must then perform numerous procedures which eventually remove all but the desired phenotype. As a result, the effort required to transfer a single trait may take from five to twenty-five years. This 20 major limitation of conventional breeding techniques can be overcome by applying the techniques of molecular biology and plant tissue culture.

Plants are highly evolved multicellular organisms. The hereditary material of plants, deoxyribonucleic acid or DNA, is contained within chromosomes which are comprised of genes encoding proteins. The specificity of expression of each gene is controlled by a regulatory region a transcriptional initiating sequence or promoter) associated with it. The gene-is •transcribed into ribonucleic acid (RNA) which is then translated into protein.

Proteins are key molecules in the plant cell, comprising enzymes which control 3biochemical events and structural molecules which provide a framework for cell components.

The production of transgenic plants begins with the introduction of new genetic material into a single plant cell. The next step, the production of a whole, transgenic plant, is greatly facilitated by the fact that plants, unlike most animals, can be regenerated asexually from such a single cell or a small piece of tissue.

Genetic engineering of plants is accomplished .by isolating and characterizing genes of interest, splicing them to desirable promoters, 'and transferring them to plant cells or tissues which are then regenerated to produce transgenic plants. As a result of this process, the transgenic plants contain the transferred genetic information in their chromosomes. This genetic information is inherited in subsequent generations and confers a new phenotype upon the progeny plants.

It is frequently desirable for the promoters used in the production of transgenic plants to be capable of conferring specificity of expression upon the transgenic construct. One aspect of this desirability is the ability to manipulate phenotypes in fruit in order to produce fruit which will have improved characteristics such as solids content, flavor, texture, processing qualities, and the like.

It is an object of this invention to provide transcriptional sequences that are useful in the production of transgenic plants, and are also capable of conferring specificity of expression upon the transgenic construct. It 20 is another object of the invention to provide transcriptional sequences that can be used to manipulate phenoiypes in fruit in order to produce fruit which will have improved characteristics such as solids content, flavor, texture, processing qualities, and the like.

SUMMARY OF THE INVENTION Novel DNA transcriptional sequences are provided which are capable of conferring upon gene fusion constructs the characteristics of tissue specific and developmentally regulated expression. In particular, DNA transcriptional sequences are provided which cause expression to occur in a tissue-specific manner, a fruit-specific manner, at specific times during 30 development, during fruit ripening. The transcriptional sequences are exemplified by sequences from clone )UC82-3.3. which is disclosed and claimed herein as SEQ ID NO. 2. The invention also provides sequences from clone pTOMUC82.1, which is disclosed and claimed herein as SEQ ID NO. 1.

Clone PTOMUC82.1 encodes a histidine decarboxylase-like protein (HDClike). Sequences from SEQ ID NO. 1, are useful, for example, as probes fdr identifying and isolating genes that may have tissue-specific, developmentally regulatable promoters. Once identified, these promoter regions can'be isolated. The invention provides gene fusion constructs containing the novel DNA sequences of SEQ ID NO. 2, thus enabling the production of high levels of RNA and, as appropriate, polypeptides reporter proteins, enzymes, etc.) in specific tissues, and at specific times during development, for example, during formation and ripening of fruit. The invention also provides transgenic plants and plant materials which contain gene fusion constructs containing the novel sequences of the invention operatively linked to at least one structural gene.

DESCRIPTION OF THE DRAWING Figure 1 shows restriction enzyme maps illustrating the derivation of the pUC82-3.3SB (S=Sstl, B=BglII) insert from the insert of AUC82-3.3 (SEQ ID NO. At the bottom of the figure is a schematic diagram of the nucleotide sequence of PUC82-3.3SB, with exons indicated by filled boxes. The percent sequence similarity between this genomic sequence 20 and the HDC-like coding sequence (PTOMUC82.1: SEQ ID NO. 1) is indicated below each exon.

DEFINITIONS

In the present specification and claims, reference will be made to phrases and terms of art which are expressly defined for use herein as follows: As used herein, promoter refers to a non-coding region of DNA involved in binding of RNA polymerase and other factors that initiate or Smodulate transcription whereby an RNA transcript is produced. Promoters can be naturally occurring or synthetically produced. Promoters, depending upon the nature of the regulation, may be constitutive or regulated. A constitutive promoter is always turned on. A regulatable promoter requires specific signals.in order for it to be turned on or off. A developmentally regulated promoter is one that is turned on or off as.a function of 4 development. A tissue-specific promoter is one that is turned on or off as a function of the tissue in which it is present.

In the present specification and claims, the terms promoter, transcriptional sequences, transcriptional initiating sequences and gene regulatory region are used interchangeably.

As used herein, the terms operatively linked, functionally linked or associated, or grammatical variations thereof, are equivalent terms that are used interchangeably. In particular these terms refer to the linkage of a promoter or a non-coding gene regulatory sequence to an RNA-encoding DNA sequence, and especially to the ability of the regulatory sequence or promoter to induce production of RNA transcripts corresponding to the DNA-encoding sequence when the promoter or regulatory sequence is recognized by a suitable polymerase. All three terms mean that linked DNA sequences promoter(s), structural gene reporter gene(s)), terminator sequence(s), etc.) are operational or functional, work for their intended purposes.

Stated another way, operatively or functionally linked, or associated, means that after the respective DNA segments are joined, upon appropriate activation of the promoter, the structural gene will be expressed.

As used herein, suitable plant material means and expressly 20 includes, plant protoplasts, plant cells, plant callus, plant tissues, developing plantlets, immature whole plants and mature whole plants.

As used herein, transgenic plants or plant compositions refer to plants or plant compositions in which heterologous or foreign DNA is expressed or in which the expression of a gene naturally present in the plant has been altered. Such DNA will be in operative linkage with plant regulatory signals and sequences. Expression may be constitutive or may be regulatable.

The DNA may be integrated into a chromosome or integrated into an episomal element, such as the chioroplast, or may remain as an episoma! element. In creating transgenic plants or plant compositions, any method for introduction of such DNA known to those of skill in the art may be employed.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS In accordance with the subject invention, DNA sequences and constructs are provided which allow for tissue-specific and/or developmentally regulated modification of gene expression, for example, during fruit maturation and ripening. Preferred sequences and constructs include transcriptional sequences which are activated at or shortly after the climacteric, so that in the early ripening of the fruit, they provide the desired level of transcription of the sequence of interest. Normally, the sequences of interest will be involved in affecting the expression of genes during ripening of the fruit or providing a property which is desirable following the growing (expansion) period of the fruit, or at or after harvesting. Desirably, the transcriptional sequences maintain their activity during the ripening or red fruit period, although the levels of their activity may also change during ripening.

As indicated above, the DNA sequences and constructs of the invention provide a regulated transcriptional sequence, which in one aspect is associated with fruit development and ripening. In one embodiment, the transcriptional sequence is one that is active upon or shortly after the onset of ripening in tomato fruit. In some DNA constructs of the invention, a sequence encoding a protein of interest is located downstream from and under the 20 transcriptional control of the fruit-related transcriptional sequence. The protein can be a marker protein such as GUS, CAT, LUX, etc., or an enzyme such as beta-fructofuranosidase, which provides for modification of the phenotype of the fruit.

The transcriptional regions may be native or homologous to the "0:0 25 host or foreign or heterologous to the host. By foreign it is intended that the transcriptional sequence is not found in the wild-tyrpe host into which the transcriptional sequence is introduced. Of particular interest are developmentally regulated and tissue-specific fruit) transcriptional initiation regions of clone .UC82-3.3 (SEQ ID NO. In tomato for example, 30 this transcriptional region is activated upon or shortly after the onset of fruit ripening and remains active during the red fruit stage, peaking approximately midway during the ripening process. Expression of a gene coding for a protein 6 of interest can be developmentally controlled and made fruit-specific as well as protoxylem-specific by operatively linking the sequence or gene of interest to this transcriptional sequence from XUC82-3.3 (SEQ ID NO. 2).

The transcriptional sequence of the invention may, for example, be employed for varying the phenotype of the fruit. For example, the pattern of expression of genes which affect the movement and storage of fixed carbon within the plant may be modified by operatively linking these genes to heterologous promoters. For example, a transcriptional cassette may be constructed which will include in the direction of transcription, a transcriptional sequence, a translational initiation region, a DNA sequence encoding a protein of interest, and a transcriptional and translational termination region functional in plants. One or more introns may be also be present. The DNA sequence encoding a protein of interest may have any open reading frame encoding the peptide of interest, an enzyme, or a sequence complementary to a genomic sequence, where the genomic sequence may be an open reading frame, an intron, a non-coding leader sequence, or any other sequence where the complementary sequence will inhibit transcription, messenger RNA processing, splicing, or translation. The DNA sequence of interest may be synthetic, naturally derived, or combinations 20 thereof. Depending upon the nature of the DNA sequence of interest, it may be desirable to synthesize the sequence with plant preferred codons. The plant preferred codons may be determined from the codons of highest frequency in the proteins expressed in the largest amount in the particular plant species of interest.

The termination region which is employed may be native with respect to the transcriptional sequence, may be native with respect to the coding DNA sequence, or may be derived from another source. Examples of termination regions from other sources include the octopine synthase and Snopaline synthase termination regions derived from the Ti-plasmid ofA.

tumefaciens.

The transcription construct will normally be joined to a marker for selection in plant cells. Conveniently, the marker may be resistance to a biocide, particularly an antibiotic, such as kanamycin, G418, bleomycin, hygromycin, chloramphenicol, or the like. The particular marker employed will be one which will allow for selection of transformed cells as compared to cells lacking the DNA which has been introduced.

Of particular interest for the subject invention is a tissue-specific developmentally regulated transcriptional sequence (promoter) from clone AUC82-3.3 (SEQ ID NO. The coding region of clone XUC82-3.3 (SEQ ID NO. 2) has homology to a bacterial histidine decarboxylase. Clone 3 .UC82- 3.3 was obtained by screening on L. esculentum cv. UC82 genomic DNA library for clones containing sequences which hybridized to ptomUC82.1, a cDNA of tomato fruit ripening. ptomUC82.1 was identified by screening a L.

esculentum cv. UC82 fruit cDNA library with labeled RNAs expressed either at an early green stage or at the "turning" to "pink" ripening stage of tomato fruit development; clones that hybridized strongly to the labeled RNAs were isolated and used to probe northern blots of fruit RNAs. ptomUC82.1 was identified as a cDNA corresponding to a gene expressed at low levels in early stages of fruit ripening, high levels at intermediate fruit ripening stages and decreased levels in fully ripened fruit. In addition, ptomUC82.1 was used to screen RNA from root, stem and leaf tissue. The mRNA complementary to 20 ptomUC82.1 was not present in these tissues, nor was it detectable in green fruit.

In order to isolate the regulatory sequences associated with the developmentally expressed gene corresponding to ptomUC82.1, a tomato genomic DNA library was screened by hybridization to ptomUC82.1. A DNA fragment was selected which hybridized to the subject cDNA. The fragment is referred to herein as XUC82-3.3 (SEQ ID NO. The 5' and 3' non-coding regions were isolated and manipulated for insertion of a foreign sequence reporter genes, enzymes, etc) to be transcribed under the regulation of the AXUC82.1 promoter, thus creating expression cassettes.

The DNA constructs provided herein are introduced into plants, plant tissues, or into plant protoplasts, particularly tomato plants, plant tissues, and protoplasts, to produce transgenic plants.

Numerous methods for producing or developing transgenic plants are available to those of skill in the art. The method used is primarily a function of the species of plant. These methods include, but are not limited to, the use of vectors, such as the modified Ti plasmid system of Agrobacterium tumefaciens, the Ri plasmid system of Agrobacterim rhizogenes and the RNA virus vector, satellite tobacco mosaic virus (STMV). Other methods include direct transfer of DNA by processes such as PEG-induced DNA uptake, microinjection, electroporation, microporjectile bombardment, and direct and chemical-induced introduction of DNA (see, Uchimiya et al. J. Biotech.

12:1-20, 1989, for a review of such procedures).

The resulting plants may then be grown, and flowers pollinated with pollen either from the same transformed strain or different strains. The resulting hybrid, having the desired phenotypic characteristic, may then be identified. Two or more generations of homozygous transgenic plants may be grown to ensure that the subject phenotypic characteristic is stably maintained and inherited. Seeds or plant tissue then may be harvested for use in producing plants with the new phenotypic property.

In addition to SEQ ID No. 2, the invention includes sequences :able to hybridize to SEQ ID No. 2, under standard high stringency conditions, 20 (such conditions being well known to those skilled in the art of molecular plant biology), as long as those hybridizing, sequences function as developmentally regulatable transcriptional sequences.

The following examples are offered by way of illustration and not by limitation.

EXPERIMENTAL

EXAMPLE 1 ISOLATION OF A DEVELOPMENTALLY REGULATED GENE 1. Construction of a cDNA library in plasmid pBR322 Isolation of RNA Tomato fruit at the 3-inch intermediate stage, fruit at the "turning" to "pink" stage of development, was collected from greenhouse-grown L. esculentum cv. UC82 (grown from seeds obtained from Hunt-Wesson Foods, Fullerton, CA), and frozen in liquid nitrogen. Polysomes were prepared from of pulverized frozen tissue (Schroder et al, Eur. J. Biochem. 67:527-541, 1976), and RNA was extracted from the polysomes using an SDS-phenolchloroform procedure similar to that described by Palmiter (Biochemistry 13:3606-3615, 1974). Poly(A)+ RNA was selected by affinity chromatography on oligo(dT)-cellulose columns using the procedure of Aviv and Leder (Proc.

Natl. Acad. Sci. USA 69:1408-1412, 1972), except that LiCI was used instead of NaCl.

b. Preparation of cDNA and construction of cDNA library A cDNA library was prepared by methods similar to those reported by Willa-Komaroff et al. (Proc. Nal. Acad. Sci. 75:3727-3731, 1978).

USA Ten ig of poly(A)+ RNA were collected by centrifugation and resuspended in 5 pl HO, brought to a final concentration of 2.7 mM

CH

3 HgOH, and incubated at room temperature for 5 minutes (Payver and Schimke J. Biol. Clem. 254:7636-7642, 1979). The first strand of cDNA was synthesized by reverse transcriptase (Molecular Genetic Resources, Tampa, Florida), and mRNA was removed by treatment with NaOH. The cDNA molecules were made double-stranded by DNA polymerase I, Klenow fragment (New England BioLabs, Beverly, MA). To ensure completion of the second 20 strand synthesis, the DNA molecules were incubated with reverse transcriptase (Molecular Genetic Resources, Tampa, 76). Following ethanol precipitation, the double-stranded molecules were digested with S1 nuclease (Boehringer Mannheim Biochemicals, Indianapolis, IN). The blunt-ended molecules were then tailed with d(C) in a reaction mixture containing terminal transferase buffer (Bethesda Research Laboratories, Inc., Rockville, MD), a- 2

P-JCTP,

dCTP, and terminal transferase (Ratliff Biochemicals, Los Alamos, NM).

The d(C)-tailed DNAs were annealed to pBR322 DNA which had been digested at the Pstl site and tailed with d(G) (New England Nuclear, Boston, MA). The recombinant plasmid DNA molecules were used to transform LE392 E. coli cells which were then plated on LB-tetracycline pg/ml) plates. The resultant cDNA library was stored by the procedure of Hanahan and Meselson (Gene 10:63-67, 1980).

2. Library screening with RNA probes a. Preparation of P-labeled RNA probes Twelve grams each of 1-inch green and 3-inch intermediate L.esculentum cv. UC82 fruit were pulverized in the presence of liquid nitrogen, and total RNA was prepared using a phenol extraction procedure conducted at pH 9.0. Total RNAs were subjected to oligo-dT cellulose chromatography for the selection of poly(A)+ RNA essentially as described by Aviv and Leder (Proc. Natl. Acad. Sci. USA 69:1408-1412, 1972), except that LiCI was used instead of NaCI.

Poly(A)+ RNAs prepared from the 1-inch green and 3-inch intermediate stages of L.esculentum cv. UC82 tomato fruit development were fractionated on linear sucrose gradients, 5-20% sucrose, to facilitate enrichment and identification of mRNAs encoding proteins ranging in size from 30 to 60 kilodaltons.

Samples of RNA from gradient fractions were translated in an mRNA-dependent rabbit reticulocyte translation system by the method of Pelham and Jackson (Eur. J. Biochem. 67:247-256, 1976). The lysate and reaction conditions were as provided by New England Nuclear (Boston, MA; October 1979 Manual) to produce peptides labeled with L-(S)-methionine.

20 Protein synthesis was assayed by determining the incorporation of TCAprecipitable label (Pelham and Jackson, Eur. J. Biochem. 67:247-256, 1976).

The translation products were then subjected to electrophoresis on a 12.5% SDS acrylamide gel (Laemmli, Nature 227:680-685, 1970) and fluorography.

b. Library screening Replica filters were prepared and the plasmids amplified (Hanahan and Meselson, Gene 10:63-67, 1980) using 200 Mg/ml chloramphenicol. DNA from cDNA clones was denatured, neutralized, and fixed to nitrocellulose filters (Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New 30 York, 1982).

RNAs from a gradient fraction of one-inch green fruit RNA encoding proteins with a molecular mass of approximately 30-60 kDa and from a similar gradient fraction of three-inch intermediate fruit RNA were labeled with "P in a polynucleotide kinase reaction. These labeled fruit RNAs were then hybridized to approximately 10,000 cDNA clones (a fraction of the complete cDNA library) bound to nitrocellulose filters as described above. Of 313 clones which yielded strong hybridization signals, 36% contained insert sequences which appeared to be expressed differentially at the two different stages of development. Included in this set of cDNA clones were ptomUC82- 2, ptomUC82-3, ptomUC82-6, ptomUC82-9, ptomUC82-10, and ptomUC82-22.

c. Identification of clone ptomUC82-3 as encoding a developmentally regulated sequence Clones which yielded strong hybridization signals in the abovedescribed hybridization experiments were used to prepare plasmid DNA which was then used to probe northern blots of total RNA prepared from the 1-inch green and 3-inch intermediate stages of L. esculentum cv. UC82 fruit development. Plasmid DNA was labeled with 32 P by nick translation.

Total RNA was prepared from 1-inch green and 3-inch intermediate developmental stages of L. esculentum cv. UC82 fruit as described above. The RNAs were subjected to electrophoresis on a agarose gel prepared in 1.1 M formaldehyde, 10 mM NaPO,, pH 7.4, and 20 electrophoresed in the same buffer. The RNA was transferred to a nitrocellulose filter essentially as described by Thomas (Proc. Natl. Acad. Sci.

USA 77:5201-5205, 1980). This filter was then cut into separate panels and hybridized with 32 P-labeled insert DNA from cDNA clones ptomUC82-2, 1 ptomUC82-3, ptomUC82-6, ptomUC82-9, ptomUC82-10, and ptomUC82-22.

After a 4-day exposure with an intensification screen, the autoradiographic patterns of hybridization indicated that clone ptomUC82-3 encodes a developmentally regulated, fruit-specific sequence which hybridized to a single RNA band with an apparent mobility of 1.7 kb on a 1.5% agarose gel.

Additional northern hybridization data, as well as mRNA 30 hybridization/selection analyses, indicated that cDNA clone ptomUC82-3 corresponds to a gene which is expressed at low levels at early stages of fruit ripening, high levels at intermediate fruit ripening stages, and decreased levels in fully ripened fruit. Sequences complementary to cDNA clone ptomUC82-3 were not detectable in RNA prepared from L. esculentum green fruit, leaves, stems, or roots. The insert in ptomUC82-3, which was restriction-enzyme mapped and sequenced by the dideoxynucleotide chain termination method, contained the ATG start codon and some of the coding sequence of Sequence I.D. No. 1 as well as 5' untranslated sequence.

4. Construction of a cDNA Library in Agtll and selection of clone ltomUC82-14 a. Total RNA extraction and poly(A) mRNA isolation Tomato fruit at the 3-inch intermediate stage was collected from greenhouse-grown L. esculentum cv. UC82 as described above. Procedures used for total RNA extraction and poly(A) mRNA isolation were essentially as described in section 2.a. (supra).

b. cDNA preparation and library construction in Xgtll 15 cDNA was prepared using reverse transcriptase, made doublei stranded with DNA polymerase 1, and made blunt-ended by treatment with S1 nuclease. Oligonucleotide adapter molecules as described by Wood et al.

(Nature 312:330-337, 1984) were used to join the blunt-ended, double-stranded DNA to the gtIl vector DNA. Ligation of the blunt-ended, double-stranded DNA product from the cDNA reactions to the adapter molecules was accomplished by incubating the DNA with a 50-fold excess of adapters.

The DNA products of this ligation reaction were phosphorylated by incubating them in a polynucleotide kinase reaction. Phosphorylated DNA "i with an apparent size greater than 1 kilobase pair was collected following fractionation on a Sepharose CL-4B column. The phosphorylated DNA Smr.olecules were then ligated to Agtl 1 arms and packaged using a Gigapack' lambda packaging extract obtained from Stratagene (La Jolla, CA). The resultant cDNA library contained 2 x 10 the library was amplified prior to screening.

c. Screening of the library and identification of AtomUC82-14 Approximately 2 x 10' p.f.u. of the amplified library were plated on E. coli Y1088 cells and screened with "P-labeled insert DNA derived from 13 ptomUC82-3. To prepare this probe, ptomUC82-3 plasmid DNA was digested with PstI, the -800 bp insert fragment was fractionated on an agarose gel and purified, and the fragment was labeled with 3 2 P in a nick translation reaction.

Plaques hybridizing to this probe were identified and plaque-purified.

Following plaque purification, the insert sizes of these recombinant bacteriophage were determined, and the clone containing the largest insert was selected for restriction enzyme analysis and sequencing by the dideoxynucleotide method. This clone was named XtomUC82-14, and the sequence of the insert DNA contained within this clone extends from nucleotides 7 through 1576 of Sequence I.D. No. 1.

EXAMPLE 2 ISOLATION OF TOMATO HDC PROMOTER 1. Construction and screening of genomic library.

A genomic library was constructed in X FIXMII (Stratagene, La 15 Jolla, CA) using DNA isolated from seedling tissue of L. esculentnu cv. UC82.

The genomic library was screened with a 32 P-labeled probe prepared from the 800 bp insert purified from cDNA ptomUC82-3 following digestion with PstI.

The hybridizations were conducted overnight at 42'C in 50% formamide, SSPE, 5X Denhardt's solution, 0.1% SDS, and 200 pg denatured salmon sperm 20 DNA. The screening resulted in the identification and plaque-purification of 13 clones which hybridized to the insert.

2. Isolation of HDC Promoters One of the clones isolated from the genomic DNA library, ).UC82-3.3, containing nucleic acids 1-4032 of Sequence I.D. No. was shown 25 by restriction enzyme mapping to contain putative regulatory regions upstream of the translation start site. A 3.7 kb Sstl-Bgll fragment from the 5' end of this clone was subcloned. Sequence analysis of the insert of this subclone revealed that it contains six exons that have 95-100% identity with comparable positions of ptomUC82-3 cDNA, and appears to include a promoter region. A fragment containing the 347 bp upstream from the Sstl restriction site near the end of the XUC82-3.3 insert was subcloned and sequenced.

The results of a sequence similarity search through the GenBank database release 67.0 and EMBL database release 26.0 (Devereaux et al., Nucl.

Acids Res. 12:387-395, 1984) indicate a 60% similarity between the amino acid sequences predicted from cDNA clone ptomUC82-3 and the Morganella morganii bacterial histidine decarboxylase gene. Thus, the L. esculehtum gene identified by hybridization to the cDNA clone ptomUC82-3 probe is considered to be a histidine decarboxylase-like (HDC) gene.

The promoter-containing region of AUC82-3.3, nucleotides 1-888 of SEQ ID No. 2, is herein referred to as the HDC promoter.

EXAMPLE 3 HDC-PROMOTER/TOMATO FRUIT INVERTASE CONSTRUCTS 1. HDC/3-L1.1 Construct HDC/3-LI.1 contains 538 bp of the HDC promoter region from .UC82-3.3 (nucleotides 349 to 886 of Sequence I.D. No. 2) fused 15 to the coding sequence of L. esculentum cv. UC82 invertase cDNA, which is fused at the 3' end to the NOS (nopaline synthase) terminator, as shown in Figure 2.

u" pTOM3-LI was digested with XhoI, made blunt-ended with T4 DNA polymerase, then digested with Notl to yield a 2202-bp fragment 20 containing 3 nucleotides from the vector polvlinker (AGC) plus the complete L. esculenrum cv. UC82 invertase cDNA coding sequence.

The above fragment prepared from pTOM3-LI and the 538 bp fragment of the HDC promoter (nucleotides 349 to 886 of Sequence I.D. No.

2) were purified and ligated with Notl- and SstI-digested pGEM-11Zf(-) (Promega Corporation, Madison, WI). The resultinQ plasmid was called 540/3-L1.

The NOS terminator is contained in plasmid pBI101 (Clontech, Palo Alto, CA). Plasmid pBl101 was digested vwih Ssrl and Hindlll, made blunt-ended with T4 DNA polymerase, yielding an -10-kb vector fragment.

The purified vector fragment was ligated to the DNA insert of -540/3-L1 which had been prepared by digestion with Notl and made blunt-ended with T4 DNA polymerase, to produce construct HDC/3L-1.1.

2. HDC/3-L1.2 Construct HDC/3-L1.2 contains 886 bp of the HDC promoter region from XUC82-3.3 (nucleotides 1 to 886 of Sequence I.D. No. 2) fused to the L. esculentum cv. UC82 invertase cDNA, which is fused at the 3' end to the NOS (nopaline synthase) terminator, as shown in Figure 2.

3. HDC/3-L1.3 Construct HDC/3-L1.3 contains 690 bp of the HDC promoter region from AUC82-3.3 (nucleotides 1 to 690 of Sequence I.D. No. 2) fused to the L. esculentum cv. UC82 invertase cDNA which is fused at the 3' end to the NOS (nopaline synthase) terminator, as shown in Figure 2.

EXAMPLE 4 HDC-PROMOTER/GUS

CONSTRUCTS

1. HDC/GUS.1 Construct HDC/GUS.1 contains the promoter fragment from 15 AUC82-3.3 which extends from 794 to 3 bp upstream of the ATG start codon (nucleotides 94 to 886 in Sequence I.D. No. 2) fused to the E. coli pglucuronidase (GUS) gene as shown in Figure 3.

Plasmid pUC82-3.3NH was digested with DdeI, the ends of the resultant fragment were filled in with DNA polymerase I, Klenow fragment, 20 and the 792 bp fragment was isolated and purified. Plasmid pUC82-3.3NH was constructed by inserting the 3.4-kb HindIII fragment, which extends from the NotI site in the vector polylinker to the first HindIII site from the 5' end of the XUC82-3.3 insert, into the Notl and HindIIl sites of pGEM-11Zf(-) (Promega Corporation, Madison, WI) to produce pUC82-3.3NH.

25 Plasmid pBI101.3/pUC was made by inserting the 2200 bp EcoRI-HindIII fragment of pB101.3 (Clontech, Palo Alto, CA) into EcoRI and HindIll-digested pUC119 (Vieira and Messing, in Methods in Enzymolog', R.

Wu and L. Grossman, eds. Vol. 153, pp. 3-11, Academic Press, New York, 1987). The 792 bp fragment was ligated to pBl101.3/pUC which had been digested with HindIII and BamHI, and the resulting plasmid was called 790/GUS.

The 3 kb EcoRI-HindIll fragment containing the HDC promoter- GUS fusion was isolated from -790/GUS and ligated to EcoRI- and HindIIIdigested pBIN19 (Clontech, Palo Alto, CA) to produce HDC/GUS.1.

2. HDC/GUS.2 Construct HDC/GUS.2 contains 690 bp of the HDC promoter region from .UC82-3.3 (nucleotides 1 to 690 of Sequence I.D. No. 2) fused to the E. coli GUS gene, as shown in Figure 3.

Plasmid pUC82-3.3NH was digested with Xbal and SspI, and the 710-bp fragment was isolated on a 1% agarose gel and purified. The fragment was ligated to gel-purified Xbal- and Snial-digested pBI101.3/pUC to create 690/GUS.

The 2.9-kb EcoRI-HindIll fragment containing the HDC promoter-GUS fusion was isolated from -690/GUS and ligated to EcoRI- and HindIII-digested pBIN19 (Clontech, Palo Alto, CA) to produce HDC/GUS.2.

15 EXAMPLE TRANSFORMATION OF TOMATO PLANTS WITH HDC PROMOTER CONSTRUCTS 1. Transformation of L. esculentum seedlings The transformation of seedlings of L. esculenrum cv. UC82 20 (grown from seeds obtained from Ferry Morse Seed Co., Modesto, CA) was done essentially according to the protocol of Fillatti et al. (Bio/Technology 5:726-730, 1987). Plasmids were inserted into Agrobacterium tumefaciens strain S" LBA4404 (Clontech, Palo Alto, CA; see also Ooms et al., Plasmid 7:15-19, 1982) through triparental mating for transfer into L. esculentum tissue.

25 The cultures were incubated at 27 C with 16 hours of light per day under 4,000 lux of light intensity. When kanamycin-resistant shoots reached a height of one inch, they were rooted on rooting medium. The transgenic shoots were then grown into fruit-bearing transgenic tomato plants.

2. Assays for Recombinant Gene Expression Since the HDC promoter sequences are developmentally regulated and fruit-specific, tomato fruit tissues are assayed for invertase or GUS expression at various stages of fruit development.

17 Invertase activity is assayed at 30 C on 50mM sucrose in 13.6M citric acid and 26.4 mM disodium phosphate (pH The reaction is stopped with the alkaline copper reagent of Somogyi Biol. Cliem. 160:61-68, 1945).

The liberated reducing sugars are measured according to Nelson Biol.

Chem. 153:375-380, 1944). Substrate specificity is determined by reacting samples (for example, -4 jg of protein obtained following Concanavlin A- Sepharose column chromatography) with 90 mg/ml of substrate (sucrose or raffinose), in 40 mM citric acid-NaHPO 4 buffer, pH 4.8, at 30'C for minutes. The products of these reactions are then analyzed by thin layer paper chromatography using isobutanol:pyridine:H0O:acetic acid (12:6:4:1) as the solvent for ascending chromatography (Gordon et cl., J. Chromatog. 8:44-59, 1962). The positions of the carbohydrates are detected with alkaline silver nitrate (Chaplin, "Monosaccharides", in Carbohydrate Analysis, A Practical Approach, Chaplin and Kennedy, eds; IRL Press, Washington, DC, pp. 1-36, 15 1986).

1. GUS activity was determined according to the protocols provided by Jefferson (Plant Mol. Biol. Rep. 5:387-405, 1987). Histochemical analysis of 3-inch intermediate ("turning" to "pink") L. esculentum cv. UC82 transgenic fruit indicated that GUS expression (under the control of the HDC promoter) 20 was localized to protoxylem tissue. This result was observed in plants transformed with either HDC/GUS.I or HDC/GUS.2.

The above results demonstrate the ability to identify inducible regulatory sequences in a plant genome, isolate the sequences and manipulate them. In this way, the production of transcription cassettes and expression 25 cassettes can be produced which allow for differentiated cell production of the desired product. Thus, the phenotype of a particular plant part may be modified, without requiring that the regulated product be produced in all tissues, which may result in various adverse effects on the growth, health, and production capabilities of the plant. Particularly, tissue specific fruit specific) transcription initiation capability is provided for modifying the phenotypic properties of a variety of fruits to enhance properties of interest such as processing, organoleptic properties, storage, yield, or the like.

18 All publications and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

19 SUMMARY OF SEQUENCES SEQUENCE ID NO. 1: a cDNA clone of pTOMUC82.1 from Lycopersicon esculentum SEQUENCE ID NO. 2: a genomic clone of UC82-3.3 from Lycopersicon esculentum SEQUENCE

LISTINGS

GENERAL INFORMATION: APPLICANT: Fitzmaurice Ph.D., Leona C.

Mirkov Ph.D., T. Erik Elliot Ph.D., Kathryn Holtz, Greg Dickinson, Craig (ii) TITLE OF INVENTION: Tissue-Specific Developmentally Regulated Transcriptional Sequences and Uses Thereof (iii) NUMBER OF SEQUENCES: 2 (iv) CORRESPONDENCE

ADDRESS:

ADDRESSEE: McCubbrey, Bartels, Meyer, Ward STREET: One Post St.

CITY: San Francisco STATE:

CA

COUNTRY:

USA

ZIP: 94104-5231 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: Patentln Release Version #1.25 (vi) CURRENT APPLICATION

DATA:

APPLICATION

NUMBER:

FILING DATE:

CLASSIFICATION:

(vii) PRIOR APPLICATION

DATA:

APPLICATION NUMBER: US 07/635,820 FILING DATE: 02-JAN-1991 (vii) PRIOR APPLICATION

DATA:

APPLICATION NUMBER: US 07/352,658 FILING DATE: 18-MAY-1989 (vii) PRIOR APPLICATION

DATA:

APPLICATION NUMBER: US 07/343,466 FILING DATE: 26-APR-1989 (vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: US 07/197,122 FILING DATE: 20-MAY-1988 (viii) ATTORNEY/AGENT

INFORMATION:

NAME: Meyer Esq., Virginia H.

REGISTRATION NUMBER: 30089 REFERENCE/DOCKET NUMBER: 51651M 00 (ix) TELECOMMUNICATION

INFORMATION:

TELEPHONE: (415) 391-6665 TELEFAX: (415) 391-6663 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 1576 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Lycopersicon esculentum (vii) IMMEDIATE SOURCE: CLONE: pTOMUC82.1 21 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:: ATGGAAATTC AAAAGGAGTT TGATTTAACG GTAGTTCCAA CAGAAGGTGA AATTGATGCA a a a.

a a 4* a a.

a a *a.a a 4* 4a a. a.

a .a.a a. a a a.

CCATTATCGC

ACGTCTTTTC

AAhGA-AGTATC

CCACTTTTGC

TTTCATTCA.A

GAGAAAGATG

CTTTTGGTTG

TACTCAATTT

GTTAATGGGG

CCAGCTATCA

CTTGAAAATT

GGGCTkATTC

AGTATTTC.A

ACAAGGAAA

ACAATTTCTG

AAAGGACATG

AA-AGATCGAC

CAAGGALAGAA

!AAAACTTGA

ATATAGGTTA

.ATTTCATTT

AAGATTTTGA

A.ATATTGGGG

GhAGAGAGCT

TCAAAGCAGC

AAATGATTA

TCA.ATATCPJ\

GTGGTTATTC

TCCCATTTAT

TTTCAGGGCA

CTTATGTTAG

GTAGTCGAALA

CTAGATTGCA

TTCTTGAAGC

TTTATGTCTC

CATGATTTTG

TCCAACAAAC

GAACAATTTT

AGTGGCTGTA

ATACATTACT

ACTTCCAAGT

AAGAATGTAT

TGAAGATTTA

TATTG GAG CT AAkATGACA.AT

CAAACATGCA

CAAkATTCTTG

TACCCACTCA

TGGATTTACA

ACAAGATTCC

AGGALATTAGT

AGTGTGbTGG

ACTCAATATT

ATGCATTATG

GGAGACCCCT

TTAGATTGGT

AGTGGTGGCA

GGGATATTAT

CGAATGG AG C CAATCkkAGT

ATTGATGACC

TATTA-TATCC

AAAAkAATTA

GGATGTCCAA

AAAATTGAGT

CCAATATTCT

ATAACATG CA

GTTATGCTGA

TAGAGACATT

AG CATCATG C

TTGCTCAGCA

TTGCACAACT

CTGAGGGCALA

ATG CATCAAA

TACAAACTAT

TACTTGTCA-A

TCGATTTCGT

ATTGCGATGC

CCTTCALAGA

TGCCTTGTGG

ATATTAATTC

TATGGTACTG

TTGAAA.ATGC

ATGAGTTTAG

GTCAAAACGG

CA CTTTAG CC

CCCTACAGAT

CTGGGAAATA

TCTCCATGGC

AGATTCACAT

CAACACTTTA

CAAGk.ACAAA C ATA CAAA CA

AGCATTATGT

GCCAATTGGT

CATTCAGATA

CA CAGATG CT TTTA.AG CAAG

TCGGTATTTG

TATTACTGTT

AATCCGATAT AAAAAATGAA 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 22 ATTTTTGAAC GATCTTGTGA CCATAA TTC ATTCATCGTT GGAACTTGTG TTACTTAAG GGCATGGCAC ATGTTGTGGT TATGCCAGGT ATTACAAGAG AAACTATAGA CAGTTTCTT AAAGATCTAA TGCAAGAGAG GAAGAGGTGG TTTCAGGATG GAAAAAACTC AGCCTCCTT TCTAGCAGAT GAGTTTGGAT CTCAAAATTG TATGTGCTCC CATA-ACAAGA TGCATAACTI AACtCCTTGG AACCATGACT TGAAATGGTC ATGATTATCA AGTATGTTTT TGATGCNAG) GTGACTCAAT AAAATTTATG ATCTAAATCG ATCTATAGTT TTCTAATAAA TTTATATGTI TACTTTCTTT GTTGTGCTTT TACACGALATG TTACTCA-ATA AAATTTGTAA TATAGAGTCA TTTAGAGTTT TCAAATCAAT TTTTATGTAT ACGTTGTTTA CAAATTTTGT AATTTAACCC TTGACCGTAA GACATG INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 4032 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA enmc (vi) ORIGINAL SOURCE: ORGANISM: Lycopersicori esculentum (vii) IMMEDIATE SOURCE: CLONE: lambda UC82-3.3 (ix) FEATURE: NAME/KEY: prim transcrijD! LOCATION: 889 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: GATCAAATTT AGTTTTGACA TCTTCTTCAC ATTTCAAkGCA TTAAAkACCArAj TTkACACTGT

A

C

1140 1200 1260 1320 1380 1440 1500 1560 1576

S

S. S S

S.

0

S

S S 555 5

S.

S S 0*5e 5005

S

S

*5Uc o 0 S. 0 *5

S

0 S 0 5050 @550 S. 05

OS

TTTATTATTA

ATTTTATATA

ATATAACATT

ATATGTACTT

AAATTA.AGTC

AGTTTGACTT

AAACAAATAG

ATACACCAAA

GTA.ACTA.ATG

AAAACATGTG

ATTAATTTTA

ATAAAACTAT

TT CAA C C A-A

TGGAGGAGCC

AAC-GTATCAT

TPATACTCATG

TATTTATTTA

ATTGATGCAC

AAAP-ATGAA

TTATTATATT

CTTTTCATTG

CTTTGCAGAC

CCTTCCTTTC

ACATATTA.AG

TAAGTCTCCA

TTATAAAAAG

ATTTTACATC

TTTTTTTTTT

TAkATATAAA.A

GCTTGAAA-AT

TTAACTTAAT

AAAAAAAAGA

AAkAGATAGTG

AGTTTCTA-AT

ATTA-ATTTAT

TTTTTATA-AC

CATCATCGCC

CGTCTTTTCA

AATTTAAkATT

AAAAATTGCA

TTCATTTATG

A.AATTGATTA

CATAACTTTA

AATTTGTTA-A

TAATTTTATT

CTTTATCATA

CAAACACTGT

CA-AATATAAA

AAAAATTAAT

TGGATGCACC

AG GTTAAAAC

AGATTCACAA

ATTTTTTTTA

TGATCATTTT

ATAGGAGTTT

AAGGAAGAAT

AGA-ACTCGAC

23

TTCTGAGTTT

TTGTTTATAT

AAATTACACT

AATTGTTA.AG

A-ATTTTTACA

TACAGACCTG

CAATTTTATA

C CAAAkATTTA AAAkACA CCAA

AGAGTCCACG

ATTTTTTTAT

AACTTTG'ACA

G CA-AAGCA-AC k-ACTTT!ATAT ATTATAT2ATG AAA C A-,,TC PA

GATTTAACGA

TTATGTCTCA

ATGATTTTGA

AATTTTATTA

TCTTACTTCA

ATAGAATAAT

GTGTTTCACA

AAAATAAGCAG

AAkAGAGTGTA GAATTU'-AkAG TAG k-AIATA AAAAATTG CT

TC-CATCGCAT

TTCAAACACT

GGTGTTLATT

TTAATTCATT

CTCTPJAnkAT TCTATCT TPA

ACATATOCTTA

TAC-TTCCAAC

GTGTG!-ATGGA

CTCAAkTA-TTT

TTCTAACATT

TAATGTACGT

A.ATTTGATTT

CATTTAAA-AT

CTCTCTATAA

GGAGCTAACA

CTATATGTC

AA-ATAkATTT AkATGTGTAAG

GAGTACCTAT

ACCA.ATTATT

CACTTCAATA

TGTTATAAkAT C GAAATTCA.A GTTTCkTTCA GATTTAAkTTT AC- AG GTGA-A

ATCTGATATT

AC-AGACATTG

120 180 240 300 360 420 480 600 660 720 780 840 900 960 1020 1080 1140 1200

TCCGAGCGAA

ACATAGTTAC

TCTCAATTTT

AGTATTTATA

TTTACATATC

ATTATATCTT

GCCACTTTAG

CACCCTACAG

CTCTGGGAAA

AATCTCCATG

GTGGTAGTTT

ATGGTCAAAA

AATCAATGAC

ATTTCTTTCT

ATACAGTACG

AGGTGAGTTT

ATATTCAATC

ATTC-ATAkATA

AAGTATCA

TTTTTACTCA

ATTTAAATAA

AGAAAAATTA

TTTTAAGTAT

TGGTTATTTA

CCCCACTTTT

ATTTCCATTC

TAGAGAAAGA

GCCTTTTGGT

TTGAATCATA

TCATTACGAG

AA-ATTTGTAT

TTTTCCCTTT

ATTAGCCATT

TGACATATAT

CATAATGATT

CTCATCACA-A

TATAGGTAKAG

ACGAAATTAT

ATACATTTTC

ATCAAAAGTT

TTTTGATTTG

ATTTACAGGT

C CAATTT CAT

AAAAGATTTT

TGAATATTGG

TGGGCAGGTA

TGAAAAATCA

CA-AGACGTCT

TG CCACACAAk

GCGGTAGTTC

TTTG CCAAAT

TATTTTTTCT

TTATCATATC

GTCACATTCT

24

GATATACATA

ACAAGCATTT

ATTTATTTTT

ATTCATTAAT

GCCAAATAAT

TATCCA-ATTA

TTGPLACAATT

GAXAGTGGCTG

GGNTACATTA

TCATTTTCAA

AAAAATTAA

TACTTTACTT

TAATGACCAC

ACAATAAACA

AAAATTTATT

CAAkACCTCCT

TTGACCCATT

CTTCAAG GA

TGTATAGTCT

TAGTGATCGA

ACGTGTGTAA

AAATCATCCC

ATTTTACGAT

ACATGTGTTA

GTGGAGATCC

TTTTAGATTG

CTAGTGGTGG

C AA.ACG G T

TOGGCGTA.ATC

TTGTTC TAC C

AATCCTTCTA

TACCATAGTC

TTCTCTCAAA

ATAGAGGTGA

CTCTTATACA

TTCATAkATT

TTCCATACAA

GGTAATTTAA

TAAACATAAA

TAACTTTATT

TTTATTCATA

CGAACATCAT

CTTTACTCAG

GTTTGCACPA

CACTNACCCC

GGAGAG

AGCCATTCTC

ATACOTACAC

TGCA.AGAGCT

ACO CATAAAC

CCTCCCCTAC

GTTOAC-ACAT

ATGCTCGAGC

TGTATTATA

1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 CTACATTGTC ATGGTTCTAA AATTCATTAT ATTTCCATGA CACACCTCAA CATCACTTTO

TTGTCTCATG

AAAGATCAAG TGTACCATCA S S

S*

S. S

TCAAATTGGG

ATTCTCTTCA

ATCACAATGA

TTGTCATCTT

TCAACAGATG

CAAATTTAAT

ATCATCTTAT

CACATTAGC

AACTCGAATT

CAAATATTTT

TTTGAGTTTT

GAACAATATT

AAAGATTCAC

ATCA-ACACTT

AAkCAAGA.ACA

CTTACATCTT

TCAAAGGAGC

CAAATGACAJ-.

TCGACAACAT

CATTTTGA.AG

TGACTATTAT

TCAGACGA.AT

GATTTCAAAG

ATGATGAATT

CATCAATTTG

CATAATTCTA

GATGTCTTAC

TCATAATGAA

TTTCTTATGG

ATATATTTGT

ATTACTCGAT

TAGTTAATGG

AACCAGCTAT

ATAACATCAC

TATTGATGAC

TTATTATATC

CTTTATCTTC

TCAGAAGTCC

GACTATTTGG

AATTTCGTCC

TATTTGTTGC

TTATCACATG

TCAATACTTT

GTGCATGGAG

TGGGACTTGA

CCTCTTGGTG

TGACATAAAT

TTAAATGATG

GCAGAAGAGA

TTTCA.AAGCA

GGAAATTGAT

CATCX-ATATC

TTTTGGTA-AA

CTCGATTTCG

CATTO CGATG

AATGACCTTT

AGAACCCATA

CTTCACGCCC

TACTACATTC

TTGCTTCCAT

TCATCAAAAA

ACTCAAA-CTC

ACCCAATACC

CGATAGAACT

GAGTCTTTTT

CAAGTGCTTC

GCTACTTCCT

GCAAGAATGT

TATGA-ACATT

AAkTATTC GTA

TTAGTATATA

TCATACAAAkC

CAGCATTATG

ATAGAGGATT TATAAAGTTG

CATACCATTT

GTGTTCTTCC

TTATTCATAT

ATATAATTGA

ATTCTTTTCA

TATATTATTT

AkATGTCTTAT

TTATCATTAT

TGAAPTCTACC

TAAkACAAATT

ATCACTTTCA

A-ATGGATATT

ATCGALATGGA

TACA.ATCAA

AAAATACATA

TGTGTTTATA

ACTTG; AAAT

TGGGCTA-ATT

TATAATAAAA

TTTTATALATT

CATTAATTAT

ATC GAG CAAG

AGGAATGGAG

TGCCTCAGTC

CCATACAAGG

GGTGCATC7A

GTCTTACCCT

TGATAACATA

TAAAGCATTT

ATATGCATCA

GCTACA.A.ACT

GTTACTTGTC

CATATATATT

GGAACAAkCCT

TGTGGTTATT

CTCCCATTTA

2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3 120 3180 3 2 3 300 3360 3420 3480 TCAAACATGT AAGCTTATTT ACATAGTTTC TTTCTATGGT TTGGAAGTAT TTCAATTTCA AGATAACAAG GAGAAGTTAC ATGCTACA.AT TTCTGGTAGT GCAAGAAJAGG ACATGCTAGA ATTTGAA.AGA TCGACTTCTT CTGTTGTTTT TGAACGACCT TAAGAGGCAT GGCACATGTT TCTTCAAAGA TC 26 TTATTCAATT TTCCTTCA.AC ATTTGACAAT AGGCAAAAA-A GGGCACAAAT TCTTGGGATG GTTAGCACcc TCTCAAAAAT CGAAATGGAT TTACACCA.AT TTGCALACAAG ATTCCATAAC GAAGCAGGAA TTAGTGTTAT TGTGACCATA AATTCATTCG GTAATTATGC CAGGTATTAC GCTCGATCGA AGTTACAATG A-ATTACCTTC AAGAAACCAA TCCAATGTCT TGTGGCGTTC TGAGTATATT AATTCCGCAG ATTCTTATGG TACTGTTTAA ATGCATTGAA AATGCTCGGT GCTGAATGAT TTTAGTATTA TCGTTGGAAC TTGTGTTGCT AAGAGAAACT ATAGATAGTT 3540 3600 3660 3720 3780 3840 3900 3960 4020 4032

Claims (12)

1. .DNA comprising SEQ ID NO. 2 (.UC82-3.3).

2. DNA able to hybridize under standard high stringency conditions with the DNA of Claim 1, wherein said hybridizing DNA functions as a developmentally regulatable transcriptional DNA sequence.

3. A DNA construct comprising in the direction of transcription, a transcriptional region from SEQ ID NO. 2, or a sequence able to hybridize thereto under standard high stringency conditions wherein said hybridizing DNA functions as a developmentally regulatable transcriptional DNA sequence; operatively linked to a DNA sequence of interest, wherein said DNA sequence of interest is other than the wild-type sequence normally associated with said transcriptional region, and wherein said S" DNA sequence is under the transcriptional regulation of said region or said hybridizing sequence; and a transcriptional termination region. 15 4. A DNA construct according to Claim 3, wherein said DNA sequence of interest encodes protein(s) which directly or indirectly gives rise to a phenotypic trait wherein said phenotypic trait is selected from the group consisting of tolerance or resistance to: herbicide, fungus, virus, bacterium, insect, nematode or arachnid; production of secondary metabolites, male or female sterility, and production of an enzyme or reporter compound. A DNA construct according to Claim 3, wherein said DNA sequence of interest encodes reporter protein(s) selected from the group consisting of chloramphenicol acetyltransferase (CAT), neomycin phosphotransferase (NPT), nopaline synthase (NOS), octopine synthase (OCS), /-1,3-glucuronidase (GUS), acetohydroxyacid synthase (AHAS), ,P- galactosidase (pGAL), and luciferase (LUX).

6. A DNA construct for integration into a plant genome comprising at least the right T-DNA border joined to a DNA construct according to Claim 3. A DNA vector comprising a broad spectrum prokaryotic replication system and a DNA construct according to Claim 3. 28

8. A plant transformed with a DNA construct according to Claim 3.

9. A tomato plant transformed with a DNA construct according to Claim 3.

10. A method for modifying the phenotype of fruit in a plant, said method comprising: transforming a suitable plant host cell with a DNA construct according to Claim 3, under genomic integration conditions, whereby said DNA construct becomes integrated into the genome of said plant host cell; regenerating a plant from said transformed plant host cell; and growing said plant to produce fruit of the modified phenotype.

11. A method for modifying the phenotype of a tomato fruit, said method comprising: transforming a suitable tomato plant host cell with a DNA construct, under genomic integration conditions, wherein said DNA construct comprises in the direction of transcription, a tomato HDC-like transcriptional region from SEQ ID NO. 2, or a sequence able to hybridize thereto under standard high stringency conditions wherein said hybridizing DNA functions as a developmentally regulatable transcriptional DNA sequence; operatively linked to a DNA sequence of interest, wherein said DNA sequence of interest is other than the wild-type sequence normally 20 associated with said transcriptional region, and wherein said DNA sequence is under the transcriptional regulation of said region or said hybridizing sequence, and further wherein said DNA sequence is capable of modifying the phenotype of fruit cells upon transcription; and a transcriptional termination region; whereby said DNA construct becomes integrated into the genome of said plant host cell; regenerating a plant from said transformed plant host cell; and growing said plant to produce fruit of the modified phenotype.

12. DNA comprising SEQ ID NO. 1 (pTOMUC82.1).

13. A DNA construct, substantially as hereinbefore described with reference to Example 3 or Example 4.

14. A DNA vector comprising a broad spectrum prokaryotic replication system and a DNA construct which is substantially as hereinbefore described with reference to Example 3 or Example 4. A plant transformed with a DNA construct which is substantially as hereinbefore described with reference to Example 3 or Example 4.

16. A tomato plant transformed with a DNA construct, substantially as hereinbefore described with reference to Example io Dated 21 December, 1999 Smart Plants International, Inc. Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON .dl 4 @o [R:\LIBAA]07792.doc:tab