POLYNUCLEOTIDES CONTROLLING THE EXPRESSION OF AND CODING FOR GENE B IN TOMATO

Abstract: An isolated complementary or genomic DNA segment encoding lycopene cyclase of the B locus of tomato and its control elements which are responsible for its transient expression in chromogenic tissues such as fruit and flower are provided.

Description: POLYNUCLEOTIDES CONTROLLING THE EXPRESSION OF AND CODING FOR GENE B IN TOMATO FIELD AND BACKGROUND OF THE INVENTION The present invention relates to a novel polynucleotide sequences isolated from tomato and, more particularly, to a novel lycopene cyclase gene and novel control elements controlling its specific expression in chromogenic tissues of plants, e.g., fruit and flower. Carotenoids - functions and biosynthesis: Carotenoids comprise one of the largest classes of pigments in nature. In photosynthetic organisms carotenoids serve two major functions - as accessory pigments for light harvesting, and as protective agents against photooxidation processes in the photosynthetic apparatus. Another important role of carotenoids in plants, as well as in some animals, is that of providing distinctive pigmentation. Most of the orange, yellow, or red colors found in the flowers, fruits and other organs of many higher plant species are due to accumulation of carotenoids in the cells. The biosynthesis of carotenoids has been reviewed extensively (Britton, 1988; Sandmann, 1994a). Carotenoids are produced from the general isoprenoid biosynthetic pathway, which in plants takes place in the chloroplasts of photosynthetic tissues and chromoplasts of fruits and flowers. The first unique step in carotenoid biosynthesis is the head-to-head condensation of two molecules of geranylgeranyl pyrophosphate (GGPP) to produce phytoene (Figure 1). All the subsequent steps in the pathway occur in association with membranes. Four desaturation (dehydrogenation) reactions convert phytoene to lycopene via phytofluene, ζ-carotene, and neurosporene, as intermediates. Two cyclization reactions convert lycopene to β-carotene (Figure 1). Further reactions involve the addition of various oxygen-containing side groups which form the various xanthophyll species (not shown). It has been established in recent years that four enzymes in plants catalyze the biosynthesis of β-carotene from GGPP: phytoene synthase, phytoene desaturase, ζ-carotene desaturase and lycopene cyclase (reviewed in Sandmann, 1994b). All enzymes in the pathway are nuclear encoded. Genes for phytoene synthase and phytoene desaturase have been previously cloned from tomato (Ray et al., 1992; Pecker et al., 1992). The red color of ripe tomatoes is provided by lycopene, a linear carotene which accumulates during fruit ripening as membrane-bound crystals in chromoplasts (Laval-Martin et al., 1975). It is presumed to serve as an attractant of predators that eat the fruit and disperse the seeds. Accumulation of lycopene begins at the "breaker" stage of fruit ripening after the fruit has reached the "mature green" stage. In the "breaker" stage, which is indicated by the commencement of color change from green to orange, chlorophyll is degraded and chloroplasts turn into chromoplasts (Gillaspy et al., 1993; Grierson and Schuch, 1993). Total carotenoid concentration increases between 10 to 15-fold during the transition from "mature green" to "red". This change is due mainly to a 300-fold increase in lycopene (Fraser et al., 1994). The cDNA which encodes lycopene β-cyclase, CrtL-b, was cloned from tomato (Lycopersicon esculentum cv. VF36) and tobacco (Nicotiana tabacum cv. Samsun NN, Pecker et al., 1996, U.S. Pat. application No. 08/399,561 and PCT/US96/03044 (WO 96/28014) both are incorporated by reference as if fully set forth herein) and was functionally expressed in Escherichia coli. This enzyme converts lycopene to β-carotene by catalyzing the formation of two β-rings, one at each end of the linear carotene. The enzyme interacts with half of the carotenoid molecule and requires a double bond at the C-7,8 (or C-7,8') position. Inhibition experiments in E. coli indicated that lycopene cyclase is the target site for the inhibitor 2-(4-methylphenoxy)tri-ethylamine hydrochloride (MPTA, Pecker et al., 1996). The primary structure of lycopene cyclase in higher plants is significantly conserved with the enzyme from cyanobacteria but differs from that of the non-photosynthetic bacteria Erwinia (Pecker et al., 1996). Levels of mRNAs of CrtL-b and Pds, which encodes phytoene desaturase, were measured in leaves, flowers and ripening fruits of tomato. In contrast to genes that encode enzymes of early steps in the carotenoid biosynthesis pathway, whose transcription increases during the "breaker" stage of fruit ripening, the level of CrtL-b mRNA decreases at this stage (Pecker et al., 1996). Hence, the accumulation of lycopene in tomato fruits is apparently due to a down-regulation of the lycopene cyclase gene that occurs at the breaker stage of fruit development. This conclusion supports the hypothesis that transcriptional regulation of gene expression is a predominant mechanism of regulating carotenogenesis. The search for tissue specific control elements in plants is on going, however, only limited number of tissue specific control elements capable of specifically directing gene expression in chromogenic tissues (fruit, flower) have so far been isolated. These include the promoters of the genes E4 and E8 (Montgomery et al., 1993), which are up-regulated by increase in ethylene concentration during tomato fruit ripening, the tomato gene 2A11 gene (Van Haaren and Houck, 1991) and the poly gal acturonase (PG) gene (Nicholass et al., 1995; Montgomery et al., 1993), which are upregulated in tomato fruits during ripening. There is thus a widely recognized need for, and it would be highly advantageous to have, a novel tissue specific control elements capable of specifically directing gene expression in chromogenic tissues. The search for structural genes encoding enzymes associated with carotenogenesis is ongoing, and every new gene isolated not only provides insight into carotenogenesis, but also provides a tool to control and modify carotenogenesis for commercial purposes (Hirschberg et al. 1997, Cunningham FX Jr. and Gantt B, 1998). There is thus a widely recognized need for, and it would be highly advantageous to have, a novel lycopene cyclase capable of altering the composition of carotenoids in carotenoids producing organisms. SUMMARY OF THE INVENTION According to one aspect of the present invention there is provided an isolated complementary or genomic DNA segment comprising a nucleotide sequence coding for a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 18 and 19 and functional naturally occurring and man-induced variants thereof, with the provision that the polypeptide has a major lycopene cyclase catalytic activity. According to further features in preferred embodiments of the invention described below, the nucleotide sequence is selected from the group consisting of SEQ ID NOs: 8, 9, 10 and 11 and functional naturally occurring and man-induced variants thereof. According to still further features in the described preferred embodiments the nucleotide sequence is a cDNA or a genomic DNA isolated form tomato. According to another aspect of the present invention there is provided a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 18 and 19 and functional naturally occurring and man-induced variants thereof, the polypeptide having a major lycopene cyclase catalytic activity. According to another aspect of the present invention there is provided a transduced cell overexpressing a polypeptide including an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 18 and 19 and functional naturally occurring and man-induced variants thereof, the polypeptide having a major lycopene cyclase catalytic activity, the cell therefore over producing β-carotene on an expense of lycopene. According to still further features in the described preferred embodiments the transduced cell is selected from the group consisting of a prokaryotic cell and a eukaryotic cell. According to still further features in the described preferred embodiments the eukaryotic cell is of a higher plant. According to still further features in the described preferred embodiments the cell forms a part of a transgenic plant. According to yet another aspect of the present invention there is provided a method of down-regulating production of β-carotene in a cell comprising the step of introducing into the cell at least one anti-sense polynucleotide sequence capable of base pairing with messenger RNA coding for a polypeptide including an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 18 and 19 and functional naturally occurring and man-induced variants thereof, the polypeptide having a major lycopene cyclase catalytic activity, the cell therefore under producing β- carotene from lycopene. According to still further features in the described preferred embodiments the at least one anti-sense polynucleotide sequence includes a synthetic oligonucleotide. According to still further features in the described preferred embodiments the synthetic oligonucleotide includes a man-made modification rendering the synthetic oligonucleotide more stable in cell environment. According to still further features in the described preferred embodiments the synthetic oligonucleotide is selected from the group consisting of methylphosphonate oligonucleotide, monothiophosphate oligonucleotide, dithiophosphate oligonucleotide, phosphoramidate oligonucleotide, phosphate ester oligonucleotide, bridged phosphorothioate oligonucleotide, bridged phosphoramidate oligonucleotide, bridged methylenephosphonate oligonucleotide, dephospho internucleotide analogs with siloxane bridges, carbonate bridge oligonucleotide, carboxymethyl ester bridge oligonucleotide, carbonate bridge oligonucleotide, carboxymethyl ester bridge oligonucleotide, acetamide bridge oligonucleotide, carbamate bridge oligonucleotide, thioether bridge oligonucleotide, sulfoxy bridge oligonucleotide, sulfono bridge oligonucleotide and α-anomeric bridge oligonucleotide. According to still further features in the described preferred embodiments the at least one anti-sense polynucleotide sequence is encoded by an expression vector. According to still further features in the described preferred embodiments the cell is selected from the group consisting of a prokaryotic cell and a eukaryotic cell. According to still further features in the described preferred embodiments the eukaryotic cell is of a higher plant. According to still further features in the described preferred embodiments the cell forms a part of a transgenic plant. According to still another aspect of the present invention there is provided an expression construct for directing an expression of a gene in fruit or flower comprising a regulatory sequence selected from the group consisting of an upstream region of a B allele of tomato and an upstream region of a b allele of tomato. According to still further features in the described preferred embodiments the expression construct comprising a functional part of nucleotides 1-1210 of SEQ ID NO: 14 or nucleotides 1-1600 of SEQ ID NO: 15, or functional naturally occurring and man-induced variants thereof. According to still further features in the described preferred embodiments the expression construct comprising at least one control element having a sequence selected from the group consisting of SEQ ID NOs:21-24, all derived from SEQ ID NO: 1 1, and functional naturally occurring and man-induced variants thereof. According to still further features in the described preferred embodiments the expression construct is selected from the group consisting of plasmid, cosmid, phage, virus, bacmid and artificial chromosome. According to still further features in the described preferred embodiments the expression construct is designed to integrate into a genome of a host. According to yet another aspect of the present invention there is provided a transduced cell or transgenic plant transduced with the above described expression construct. According to still another aspect of the present invention there is provided a method of isolating a gene encoding a polypeptide having an amino acid sequence homologous to SEQ ID NOs: 17, 18 and 19 and having a major lycopene cyclase catalytic activity from a species, the method comprising the step of screening a complementary or genomic DNA library prepared from isolated RNA or genomic DNA extracted from the species with a probe having a sequence derived from SEQ ID NOs: 8, 9, 10 or 1 1 and isolating clones reacting with the probe. The present invention successfully addresses the shortcomings of the presently known configurations by providing novel polynucleotides controlling the expression of genes in fruit and flower in plant and a novel polynucleotide encoding lycopene cyclase. BRIEF DESCRIPTION OF THE DRAWINGS The invention herein described, by way of example only, with reference to the accompanying drawings, wherein: FIG. 1 presents the pathway of carotenoid biosynthesis in plants and algae. Enzymes are indicated by the their gene assignment symbols: aba2, zeaxanthin epoxidase; CrtL-b, Lycopene β-cyclase- CrtL-e, lycopene ε- cyclase,* CrtR-b, β-ring hydroxylase,* CrtR-e, ε-ring hydroxylase,* Pds, phytoene desaturase (crtP in cyanobacteria); Psy, phytoene synthase (crtB in cyanobacteria); Zds, ζ-carotene desaturase (crtQ) in cyanobacteria. GGDP, geranylgeranyl diphosphate. FIG. 2 shows fine genetic mapping and molecular organization of B on chromosome 6 of the tomato linkage map. The linkage map was adopted from Eshed and Zamir (1995). The relevant chromosomal segments from L. pennellii that were introgressed to L. esculentum lines IL 6-2 and IL 6-3 are represented by black bars. High-resolution genetic map around B is displayed with genetic distances in map units (cM). Positions of the YAC inserts are designated under the map. FIG. 3 demonstrates levels of mRNA (relative units) during fruit ripening of wild-type tomato L. esculentum . Data are derived from quantifying the DNA products in the RT-PCR analysis of total RNA extracted at different stages of fruit development. Ripening stages: IG, immature green; MG, mature green, B, breaker, O, Orange; P, pink; R, red. FIG. 4 demonstrates levels of mRNA (relative units) during fruit ripening of the tomato mutant High-beta. Data are derived from quantifying the DNA products in the RT-PCR analysis of total RNA extracted at different stages of fruit development. Ripening stages: G, green; MG, mature green, B, breaker, O, Orange; P, pink; R, red. DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention is of novel polynucleotide sequences isolated from tomato which can be used to control gene expression in plant chromogenic tissues, especially fruit and flower. The present invention is further of polynucleotide sequences isolated from tomato which encode a lycopene cyclase which can be used to alter carotenogenesis is carotenoids producing organisms. The principles and operation of the present invention may be better understood with reference to the drawings and accompanying descriptions. Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not limited in its application to the details of construction and the arrangement of the components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting. Fruit of the cultivated tomato (Lycopersicon esculentum) accumulate lycopene, a red carotenoid pigment. A dominant allele of gene B determines accumulation of β-carotene in the fruits of the tomato mutant 'high-beta ', at the expense of lycopene, resulting in a unique orange color. Conversion of lycopene to β-carotene in the biosynthesis pathway of carotenoids is catalyzed by the enzyme lycopene β-cyclase. Previously it was shown that CrtL-b, the gene for lycopene β-cyclase, does not map to the locus B in the tomato genetic map. This ruled out the possibility that a mutation in lycopene β-cyclase encoded by CrtL-b causes the phenotype in high-beta. The locus B was mapped to chromosome No. 6. The dominant allele B was found in the tomato introgression line IL 6-2. The DNA of B was identified and cloned by a map-based (positional) cloning method. The nucleotide sequence of this gene was determined and demonstrated a novel type of a lycopene cyclase enzyme. Its primary structure has some similarity to other lycopene cyclases and to the enzyme capsanthin- capsorubin synthase from pepper. In addition, nucleotide sequence was identified, which functions as a strong promoter during fruit development in the B allele of the mutant High-beta. Thus, according to one aspect of the present invention there is provided an isolated complementary or genomic DNA segment comprising a nucleotide sequence coding for a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 18 and 19 and functional naturally occurring and man-induced variants thereof. The polypeptide has a major lycopene cyclase catalytic activity. Polypeptides which share at least 70, 75, 80, 85, 90, 95 or more identical amino acid residues with SEQ ID NOs: 17, 18 or 19 are also within the scope of the present invention. As used herein in the specification and in the claims section below, the phrase "major lycopene cyclase catalytic activity" refers to catalytic activity mainly directed at the conversion of lycopene to β-carotene by catalyzing the formation of two β-rings, one at each end of the linear carotene, such that if introduced into lycopene-accumulating E. coli cells, such cells accumulate also β-carotene up to a range of at least few percent e.g., 5 %, to preferably about 15 %, or more, of total carotenoids therein by symmetric formation of two β-ionone rings on the linear lycopene molecules therein. According to a preferred embodiment of the invention the nucleotide sequence is as set forth in SEQ ID NOs: 8, 9, 10 or 1 1, or functional naturally occurring or man-induced variants thereof. As further shown below these sequences are genomic and complementary DNA sequences which were derived while reducing the present invention to practice from certain tomato cultivars or lines. However, nucleotide sequences which share 70, 75, 80, 85, 90, 95 or more identical nucleotides with SEQ ID NOs: 8, 9, 10 or 11 are also within the scope of the present invention. According to another aspect of the present invention there is provided a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 18 and 19 and functional naturally occurring and man-induced variants thereof, the polypeptide having a major lycopene cyclase catalytic activity. Homologous polypeptides as describe above and further detailed hereinunder are also envisaged. According to another aspect of the present invention there is provided a transduced cell overexpressing a polypeptide including an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 18 and 19, and functional naturally occurring and man-induced variants thereof, the polypeptide having a major lycopene cyclase catalytic activity, the cell therefore over producing β-carotene on an expense of lycopene. The cell according to the present invention can be of any type. For example, the cell can be a prokaryotic cell or a eukaryotic cell. Preferably the cell is of a higher plant. The cell preferably forms a part of a transgenic plant. Methods of transducing cells (and cells in organisms to form transgenic organisms) are well known in the art and do not require further description herein. Protocols are available, for example, in (Sambrook et al., 1989). As used herein in the specification and in the claims section below, the term "transduced" refers to the result of a process of inserting nucleic acids into cells. The insertion may, for example, be effected by transformation, viral infection, injection, transfection, gene bombardment, electroporation or any other means effective in introducing nucleic acids into cells. Following transduction the nucleic acid is either integrated in all or part, to the cell's genome (DNA), or remains external to the cell's genome, thereby providing stably transduced or transiently transduced cells. According to yet another aspect of the present invention there is provided a method of down-regulating production of β-carotene in a cell comprising the step of introducing into the cell at least one anti-sense polynucleotide sequence capable of base pairing with messenger RNA coding for a polypeptide including an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 18 and 19 and functional naturally occurring and man-induced variants thereof, the polypeptide having a major lycopene cyclase catalytic activity, the cell therefore under producing β- carotene from lycopene. Again, the cell can be of any type. For example, the cell can be a prokaryotic cell or a eukaryotic cell. Preferably the cell is of a higher plant. The cell preferably forms a part of a transgenic plant. As used herein in the specification and in the claims section below, the term "down regulating" means also reducing, lowering, inhibiting, etc., e.g., permanently or transiently reducing. As used herein in the specification and in the claims section below, the term "production" means also formation or generation. As used herein in the specification and in the claims section below, the term "introducing" means also providing with or inserting. The at least one anti-sense polynucleotide sequence according to the present invention can includes one or several synthetic oligonucleotides capable of base pairing with messenger RNA derived from the above- identified nucleotide sequences. The synthetic oligonucleotide preferably includes a man-made modification rendering the synthetic oligonucleotide more stable in cell environment. The modified oligonucleotide can be, for example, a methylphosphonate oligonucleotide, monothiophosphate oligonucleotide, dithiophosphate oligonucleotide, phosphoramidate oligonucleotide, phosphate ester oligonucleotide, bridged phosphorothioate oligonucleotide, bridged phosphoramidate oligonucleotide, bridged methylenephosphonate oligonucleotide, dephospho internucleotide analogs with siloxane bridges, carbonate bridge oligonucleotide, carboxymethyl ester bridge oligonucleotide, carbonate bridge oligonucleotide, carboxymethyl ester bridge oligonucleotide, acetamide bridge oligonucleotide, carbamate bridge oligonucleotide, thioether bridge oligonucleotide, sulfoxy bridge oligonucleotide, sulfono bridge oligonucleotide or an α-anomeric bridge oligonucleotide. For further details the reader is referred to Cook (1991). Alternatively, the anti-sense polynucleotide sequence is encoded by an anti-sense expression vector. Such vectors are well known in the art and are commercially available from, for example, pBIlOl, pBI121, pBI221 (commercially available from Colntech.) Further according to the present invention, there is provided an expression construct for directing an expression of a gene in fruit or flower of a plant. The expression vector according to the present invention includes a regulatory sequence selected from the group consisting of an upstream region of a B allele of tomato and an upstream region of a b allele of tomato. Thus, according to a preferred embodiment of the invention, the expression construct includes a functional part of nucleotides 1-1210 of SEQ ID NO: 14 or nucleotides 1-1600 of SEQ ID NO: 15, or functional naturally occurring and man-induced variants thereof. According to a preferred embodiment, the expression construct includes at least one control element having a sequence selected from the group consisting of SEQ ID NOs: 21-24, all derived from SEQ ID NO: l l, and functional naturally occurring and man-induced variants thereof. As further detailed in the Examples section hereinbelow, these sequence elements, which are 26, 13, 9, and 8 bp long and start at (5' end) nucleotides 859, 753, 479 and 306, respectively, of SEQ ID NOs: 11, 15, are located upstream to the initiator methionine codon in the B allele are the main difference between the B and b allele, and are therefore responsible for the differential expression of the B locus in tomato. The expression construct according to the present invention can be a plasmid, cosmid, phage, virus, bacmid or an artificial chromosome. Each of these constructs has unique sequences rendering the construct most applicable for some as opposed to other applications, as well known in the art. Regardless of its type, according to a preferred embodiment of the present invention the expression construct is designed to integrate into a genome of a host, such that stable transfectants are obtainable. However, the scope of the present invention is not limited to such constructs. In other words, constructs designed for transient transfection are also within the scope of the present invention. In any case, the construct preferably includes at least one positive and/or negative selection gene, and is suitable for transformation, transfection, transgenization and gene knock-in procedures. According to yet another aspect of the present invention there is provided a transduced cell or a transgenic plant transduced with the above described expression construct. Such a cell or plant is expressing the gene located downstream to the regulatory sequence in a controlled developmental manner, mimicking the expression of the lycopene cyclase gene of the B locus in b or B tomato plants. According to still another aspect of the present invention there is provided a method of isolating a gene encoding a polypeptide having an amino acid sequence homologous to SEQ ID NOs: 17, 18 and 19 and having a major lycopene cyclase catalytic activity from a species. The method is effected by executing the following method steps, in which a complementary or genomic DNA library prepared from isolated RNA or genomic DNA extracted from the species is screened with a probe having a sequence derived from SEQ ID NOs: 8, 9, 10 or 1 1 and clones reacting with the probe are isolated. Such clones are good candidates to include segments of genes homologous to SEQ ID NOs: 8, 9, 10 or 11, which genes are good candidates to encode a polypeptide having an amino acid sequence homologous to SEQ ID NOs: 17, 18 and 19. 5' cloning strategies, such as, but not limited to RACE protocols can be employed to isolate full length clones, as well known in the art. Thus, according to the present invention, the following uses of gene B of tomato are anticipated: (i) Increasing the content of β-carotene in tissues of transgenic plants over-expressing it. This is an advantageous attribute in fruits and vegetables because it will provide better nutritional value and enhanced color. (ii) Increasing the accumulation of lycopene in fruits and flowers of transgenic plants by reducing the activity of B using anti-sense inhibition, preferably via anti-sense expression. (iii) Achieving strong expression of transgenes specifically in fruits and flowers using the promoter sequence of the gene B from High- beta tomato cultivars. Each of the various and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the Examples section that follows. EXAMPLES Bacteria and plants: E. coli strain XL 1 -Blue was used in all experiments described herein. Tomato (Lycopersicon esculentum) CV M82 served as the 'wild-type' strain in the fruit ripening measurements. The introgression lines IL 6-2 and IL 6-3 (Eshed and Zamir, 1994) were used as a source for the B mutation and employed for fine mapping of the B locus. Fine mapping and cloning of the B locus: As a source to B mutation, the lines IL-6-2 or IL-6-3 (BB) were used (Eshed and Zamir, 1995). Each line was crossed with the cultivated tomato cv M-82 (bb), and the hybrids were selfed to create an F-2 population that segregated for both the B phenotype and the introgressed DNA segment. 1335 F-2 plants were scored for the RFLP using markers CT193 and TG578 (Pnueli et al., 1998; Tanksley et al., 1992) and for the B phenotype, and recombinant plants were collected. The 32 resulting recombinants were further screened with all the available RFLP probes surrounding B to accurately map the mutated locus (Figure 2). One RFLP marker, TM16 (Pnueli et al., 1998), was co- segregated with B in less than 0.0375 cM resolution. The tomato genomic library in YACs was screened with DNA of markers TM16 and TG275. Two overlapping YAC clones, designated 271 and 310, were identified by hybridization. DNA sequences from the ends of the inserts in these YACs were amplified by PCR as previously described (Pnueli et al., 1998) and were used as molecular probes to screen the 32 recombinant plants for Restriction Fragment Length Polymorphism (RFLP). The YAC ends were mapped as shown in Figure 2. It was established that YAC 310 overlaps the B locus, thus ensured that the 200 kb insert of YAC 310 contains the B gene. In contrast, recombination between the left end of YAC 271 (27 lie) and the B phenotype indicated that this YAC clone did not carry the B locus and defined its location in a relatively small region of YAC 310 that did not overlap with YAC 271 (Figure 2). The DNA insert of YAC 310 was cut with EcoRI and the resulting fragments were subcloned in the vector λ-gtl l . Two phage clones designated Bl and B3, co-segregated with the B locus and mapped to the end of YAC 310. The nucleotide sequence of the insert of Bl was determined. The B 1 fragment was further used to screen a genomic library of wild-type tomato (cv VF36) in the lambda vector EMBL3, and a cosmid library of L. pennellii. A single positive phage clone and a single positive cosmid clone were isolated, respectively. The Bl fragment was also used to screen 1.5 million plaques of a cDNA library from a tomato fruit and 3 identical clones were isolated. The ca. 1300 bp inserts in these clones contained an open reading frame that was lacking the 5' end, as determined by nucleotide sequence analysis. The full- length cDNAs were then obtained using reverse-transcription polymerase chain reaction (RT-PCR) method with RNA isolated from wild-type (VF- 36) and high-beta (IL 6-3)flowers and fruits. For the PCR reaction we used 5' primers based on the genomic sequence taken from the sequence of Bl insert and the 3' primers based on the cloned cDNA. The full coding region of the cDNA of the allele b of wild type tomato (cv. VF-36) and the allele B from L. pennellii were excised in pBluescript KS- vector which were designated pBESC and pBPENN, respectively. DNA sequence comparison between cDNA and genomic sequences revealed no introns interference in the genomic sequence of the b (and B). DNA blot hybridization was done according to conventional techniques (Sambrook et al., 1989, Eshed and Zamir, 1994) at low stringency in a buffer containing 10 x Denharts, 5 x SSC, 50 mM phosphate buffer (pH-7), 1 % SDS, 50 mg salmon sperm (sheared, autoclaved and boiled before adding to the mixture). Filters were washed with 5 x SSC at 65 °C. Genomic DNA of tomato was prepared from 5 grams of leaf as previously described (Eshed and Zamir, 1995). Amplification by the polymerase chain reaction (PCR) method of the full length cDNA of the b allele was carried out with the following oligonucleotide primers, whose sequence was derived from the genomic sequence of the Bl clone (see below): Forward: 5'- AATGGAAGCTCTTCTCAAGCCT-3 ' (SEQ ID NO: l), Reverse: 5'- CACATTCAAAGGCTCTCTATCGC-3' (SEQ ID NO:2). Total RNA was extracted from 1.5 grams of fruit or 0.1 gram of flower or leaf tissues as previously described (Pecker et al., 1996). Measurement of mRNA levels by the reverse transcription followed by polymerase chain reaction (RT-PCR) technique was carried out as previously described (Pecker et al., 1996) using the following oligonucleotides as primers for the PCR reaction. For amplification of the gene Psy the following primer were employed: Forwardl : 5'- TCGAGAACGGACGATG-3' (SEQ ID NO:3), Forward2 (internal): 5'- TGCAGAGAGACAGATG-3' (SEQ ID NO:4) and Reverse: 5'- ATTTCATGCTTTATCTTTGAAG-3' (SEQ ID NO:5). For amplification of allele B: Forward 5'- GCTGAAGTTGAAATTGTTGA-3' (SEQ ID NO: 6) and Reverse 5'- TCTCTTCCTCAATAACACTT-3' (SEQ ID NO:7). Sequence analysis: DNA sequence analysis was performed by the ABI Prism 377 DNA sequencer (Perkin Elmer) and processed with the ABI sequence analysis software. Nucleotide and amino acid sequence analysis and comparisons were done using the UWGCG software package. Plasmids: Plasmid pACCRT-EIB for expressing bacterial carotenoid biosynthesis genes in E. coli, was previously described (Cunningham et al., 1993). Plasmid pBESC and pBPENN were constructed by inserting an 1666 bp of cDNA of the tomato B allele (from L. pennellii) or b allele (from L. esculentum), respectively, in the EcoRV site of the plasmid vector pBluescript KS (Stratagene®). Pigment extraction and analysis: For extraction of pigments from E. coli, aliquots of 2 ml were taken from bacterial suspension cultures. The cells were harvested by centrifugation, washed once with water, resuspended in 2 ml of acetone and incubated at 65 °C for 10 minutes in the dark. The samples were centrifuged again at 13,000 g for 5 minutes and the acetone supernatant containing the pigments was placed in a clean tube. More than 99 % of the carotenoids were extracted by this procedure as determined by re-extraction after breaking and grinding the samples. The pigment extract was blown to dryness under a stream of N2 and stored at - 20 °C until required for analysis. Fruit pigments were extracted from 1.0 gram of fresh tissue. The tissue was ground in 2 ml of acetone and incubated at room temperature in the dark for 10 minutes. Then, 2 ml of dichloro-methane were added and the samples were agitated until all pigments were transferred to the supernatant, which was then filtered. To each sample, 4 ml of ether and 0.4 ml of 12 % w/v NaCl/H2θ were added and the mixture was shaken gently until all pigment was transferred to the upper (ether) phase. The ether was collected, and the pigment extract was blown to dryness under a stream of N2 and stored at -20 °C until required for analysis. Carotenoids were separated by reverse phase HPLC using a Spherisorb ODS-2 column (silica 5 mm 3.2 mm x 250 mm, Phenomenex®). Samples of 50 μl of acetone-dissolved pigments were injected to a Waters 600 pump. The mobile phase consisted of acetonitrile:H2θ (9: 1) - solvent A, and 100 % ethyl acetate - solvent B, which were used in a linear gradient between A and B for 30 minutes, at flow of 1 ml per minute. Light absorption peaks were detected in the range of 200-600 nm using a Waters 996 photo diode-array detector. All spectra were recorded in the eluting HPLC solvent, as was the fine absorbance spectral structure. Carotenoids were identified by their characteristic absorption spectra and their typical retention time, which corresponded to standard compounds of lycopene and β-carotene. Peak areas were integrated by the Millennium chromatography software (Waters). EXPERIMENTAL RESULTS The only difference between the high-beta mutant and the wild-type tomato is in the fruit color due to accumulation of β-carotene at the expense of lycopene. Thus, it was logical to assume that this mutation occurred in the gene that encodes lycopene-β-cyclase (CrtL-b). However, the CrtL-b cDNA that was previously cloned from tomato (Pecker et al., 1996) was mapped to 2 loci on chromosomes Nos. 4 and 10, but not on chromosome 6, where the B locus was mapped. Even at very low stringency of hybridization conditions we were unable to detect any hybridization of the tomato CrtL-b like sequences on chromosome 6. Therefore, the only way to clone the gene B, which is responsible for the high-beta phenotype, was to use map-based ("positional") cloning techniques. Fine mapping of the B locus: As a source to the B mutation, the IL- 6-2 or IL-6-3 (BB) (Eshed and Zamir, 1995) tomato lines were employed. Each line was crossed with the cultivated tomato cv. M-82 (bb), and the hybrids were selfed to create an F-2 population that segregated for both the B phenotype and the introgressed DNA segment. 1335 F-2 plants were scored for the RFLP using markers CT-193 and TG-578, (Pnueli et al., 1998; Tanksley et al., 1992) and for the B phenotype, and recombinant plants were collected. The 32 recombinants collected were further screened with all the available RFLP probes surrounding B to accurately map the mutated locus (Figure 2). One RFLP marker, TM-16 (Pnueli et al., 1998), co-segregated with B in less than 0.0375 cM resolution. The tomato genomic library in YACs was screened with the DNA marker TM-16 as a molecular probe. Two YAC clones, designated 271 and 310, were identified by hybridization. DNA sequences from the ends of the inserts in these YACs were amplified by PCR as previously described (Pnueli et al., 1998) and were used as molecular probes to screen the 32 recombinant plants for Restriction Fragment Length Polymorphism (RFLP). The YAC ends were mapped as shown in Figure 2. It was established that YAC 310 overlaps the B locus, thus ensured that the 200 kb insert of YAC 310 contains the B gene. In contrast, recombination between YAC 271 and the B phenotype indicated that this clone did not carry the B locus. Moreover, it established that B was residing in a confined small region of YAC 310 that did not overlap with YAC 271 (Figure 2). The DNA insert of YAC 310 was cut with EcoRI and the resulting fragments were subcloned in the vector λ-gtl l . Two phage clones designated Bl and B3, co-segregated with the B locus and mapped to the end of YAC 310. The nucleotide sequence of the insert of Bl was determined. The B 1 fragment was further used to screen a genomic library of wild-type tomato (cv VF36) in the lambda vector EMBL3, and a cosmid library of L. pennellii. A single positive phage clone and a single positive cosmid clone were isolated, respectively. The Bl fragment was also used to screen 1.5 million plaques of cDNA library from a tomato fruit and 3 identical clones were isolated. The ca. 1300 bp inserts in these clones contained an open reading frame that was lacking the 5' end, as determined by nucleotide sequence analysis. The full- length cDNAs were then obtained using reverse-transcription polymerase chain reaction (RT-PCR) method with RNA isolated from wild-type (VF- 36) and high-beta (IL 6-3) flowers and fruits. For the PCR reaction we used 5' primers based on the genomic sequence taken from the sequence of Bl insert and the 3' primers based on the cloned cDNA. The full coding region of the cDNA of the allele b of wild type tomato (cv. VF-36) and the allele B from L. pennellii were excised in pBluescript KS " vector which were designated pBESC and pBPENN, respectively. DNA sequence comparison between cDNA and genomic sequences revealed no introns interference in the cDNA sequence. Table 1 below summarizes the sequence data with reference to the sequence listing: TABLE 1 cDNA = complementary DNA; gDNA = genomic DNA; bp = base pairs; aa = amino acid. Cloning and sequence analysis of old-gold-crimson (ogC) mutation'. The old-gold and crimson are two names given to a well-known recessive mutation that was found in the Philippines in 1951 (Butler, 1962 and the SolGenes databases: http:// probe.nal.usda.gov:8300/ cgi- in/webace?db = solgenes & class = Locus & object = og; and: http:// probe.nal.usda.gov:8300/ cgi-bin/webace?db = solgenes & class = Image & object = og%2c + old + gold). The ogC locus was mapped to chromosome 6. At least 2000 F-2 progenies of a cross between High-beta (BB) and ogC were screened for B-ogC double mutants and not a single recombinant plant was found. That locates B and ogC less than 0.025 cM apart. The ogC phenotype is characterized by over accumulation of lycopene, both in fruits and flowers, compare to wild type tomatoes and lack of β-carotene in the fruits. Cloning the B locus from ogC mutant plants was done by PCR method on total genomic DNA extracted from ogC plants using primers that were based on the sequence of the b allele described herein. Sequence analysis of the b-homolog revealed a single base deletion, in the coding sequence of b at position 104 from the initiation codon (compare SEQ ID NOs: 13 and 16). This deletion created a frame-shift mutation that shortened the translatable polypeptide to 56 amino acids. This finding indicates that the ogC is a null mutation of the normal function of the b gene. Sequences comparison of alleles in the B locus: Nucleotide sequence analysis of the 1666 bp cDNA revealed an open reading frame of 498 codons, potentially coding for a polypeptide of 498 amino acids with a calculated molecular mass of 56.4 kDa. Nucleotide sequence analysis showed 98% identity between b (from VF-36, SEQ ID NO: 8) and B (from L. pennellii, SEQ ID NO: 10). The amino acid sequences of B and b are 97.4% identical (SEQ ID NOs: 17 and 19). In the 1200 bp sequences upstream to the translated region of B from L. pennellii there are four sequence insertions as compared with the equivalent region in b from VF-36. The inserts are 26, 13, 9, and 8 bp long and start at (5' end) nucleotides 859, 753, 479 and 306, respectively, of SEQ ID NOs: 1 1, 15. They are located upstream to the initiator methionine codon in the B allele are the main difference between the B and b alleles, and are therefore responsible for the differential expression of the B locus in tomato. Their sequences are TGACTTCACCCTTCTTTCTTGTCTTC (SEQ ID NO:21), AGAGTCTGGGTTC (SEQ ID NO:22), CTAGTATCG (SEQ ID NO:23) and CTAAATAT (SEQ ID NO:24). An additional AATTTTCAAA (SEQ ID NO:25) sequence, which is found in upstream regions of ethylene-activated genes such as E4 and E8 (Montgomery et al., 1993), is shared by the upstream regions of the B and b alleles. All other sequences in the promoter and region are 90-94% conserved in the two allele (compare SEQ ID NOs: 9 and 11). The polypeptide products of B and b are β-carotene synthases: The use of E. coli heterologous system for carotenoid biosynthesis has been proven to be a powerful tool for identifying genes associated with carotenoid biosynthesis. E. coli cells of the strain XLI- Blue, carrying the plasmid pACCRT-EIB accumulate lycopene (Cunnungham et al. 1993). Lycopene-accumulating E. coli cells were co-transformed with the plasmid pBESC or pBPENN and selected on LB medium containing both ampicillin and chloramphenicol. Carotenoids from cells carrying pACCRT-EIB alone, or pACCRT-EIB and either pBESC or pBPENN were extracted and analyzed by HPLC. Cells carrying only the pACCRT-EIB plasmid produced lycopene, while cells carrying both pACCRT-EIB and pBPENN accumulate also β- carotene up to 13 % of total carotenoids. Similarly, cells carrying both pACCRT-EIB and pBESC produced β-carotene up to 5 % of total carotenoids (see Table 2 below). These results indicated that the cDNA- products of both the B and b alleles are capable of converting lycopene to β- carotene by the symmetric formation of two β-ionone rings on the linear lycopene molecule. TABLE 2 The B gene product converts lycopene to β-carotene. Accumulation of carotenoids in E. coli cells expressing alleles B or b from tomato (percent of total carotenoids) plasmid lycopene β-carotene pACCRT-EIB 100 pACCRT-EIB + pBESC (b) 87 13 pACCRT-EIB + pBPENN (B) 95 5 Sequence comparison between B and other carotene cyclases: The nucleotide sequences of the coding region of b and the coding region of the cDNA of the previously published lycopene β-cyclase from tomato, CrtL-b (Pecker et al, 1996) , are 59 % identical. The polypeptide products of these genes are only 52 % identical. These data explain why CrtL-b could not hybridize with the sequence of B. Moreover, while the similarity in amino acid sequence between B and CRTLB suggests a common mechanism of lycopene cyclization, it clearly demonstrates that B is a novel lycopene β- cyclase enzyme. There is no similarity (less than 45 % identities) in the non-translated regions of these two genes. Surprisingly, the nucleotide sequence of the cDNA of b is 83% identical with the cDNA of a gene from bell pepper (Capsicum annuum), which catalyzes the conversion of the ubiquitous 5, 6-epoxy carotenoids, antheraxanthin and violaxanthin, into the ketocarotenoids capsanthin and capsorubin, respectively (Bouvier et al., 1994). This enzyme, called also capsanthin-capsorubin synthase (CCS), is synthesized specifically in pepper fruits. There is 85 % identity in the deduced amino acid sequences of B and CCS. Expression ofB gene during fruit ripening in wild-type and High- beta: Previously, it has been shown that the steady-state levels of mRNA of the genes for early enzymes in the carotenoid biosynthesis pathway, phytoene synthase and phytoene desaturase, increase during fruit ripening in tomato (Hirschberg et al., 1997). In the case of Pds it was demonstrated that transcriptional up-regulation is responsible for this increase (reviewed in Hirschberg et al., 1997). Recently, we have determined that the mRNA level of CrtL-b, which encodes lycopene β-cyclase, decreases during tomato fruit ripening (Pecker et al. 1996). To determine the regulation of expression of B gene during fruit development in tomato, we have measured by RT-PCR its mRNA level at different stages of fruit development. As can be seen in Figure 3, mRNA of the b gene is undetected in leaves and during the green stages of fruit ripening of wild-type tomato. However, it is increased at the 'breaker' stage of ripening but then disappears at later stages of ripening. This marked drop of mRNA of B is contrasted by the dramatic increase in mRNA level of Psy at the same stages of fruit ripening. In contrast to the wild-type tomato, the mRNA level of B in the fruit of the High-beta mutant (containing the B allele) increases dramatically at the 'breaker' stage and remains high during all the subsequent ripening stages (Figure 4). These results indicate that the major difference between alleles b and B is in the level of expression at different ripening stages. The results further explain the phenotype of mutant High-beta, carrying the B allele, where a novel type of lycopene cyclase, which is capable of converting lycopene to β-carotene, is highly expressed during fruit ripening. Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims. REFERENCES CITED IN ALPHABETIC ORDER 1. Bouvier, F., Hugueney, P., d'Harlingue, A., Kuntz, M., and Camara, B. (1994). Xanthophyll biosynthesis in chromoplasts: Isolation and molecular cloning of an enzyme catalyzing the conversion of 5,6-epoxycarotenoid into ketocarotenoid. Plant J. 6:45-54. 2. Britton G (1988) Biosynthesis of carotenoids, Plant Pigments. TW Goodwin, ed, Academic Press), London and New York: pp. 133-180. 3. Butler L, (1962) A new fruit color, Rep. Tomato Genetic Cooperation 12: 17-18. 4. Cunningham, F.X.,Jr., Chamovitz, D., Misawa, N., Gantt, E., and Hirschberg, J. (1993). Cloning and functional expression in Escherichia coli of a cyanobacterial gene for lycopene cyclase, the enzyme that catalyzes the biosynthesis of β-carotene. FEBS Lett. 328: 130-138. 5. Cunningham F.X. r. and Gantt, E. (1998) Genes and enzymes of carotenoid biosynthesis in plants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 49:557-583. 6. Cook (1991) Medicinal chemistry of antisense oligonucleotides - future opportunities. Anti-Cancer Drug Design 6:585-606. 7. Eshed, Y. and Zamir, D. (1995). An introgression line population of Lycopersicon pennellii in the cultivated tomato enables the identification and fine mapping of yield-associated QTL. Genetics 141 : 1147-1162. 8. Fraser, P.D., Truesdale, M.R., Bird, C.R., Schuch, W., and Bramley, P.M. (1994). Carotenoid biosynthesis during tomato fruit development. Plant Physiol. 105:405-413. 9. Gillaspy, G., Ben-David, H., and Gruissem, W. (1993). Fruits - a developmental perspective. Plant Cell 5:1439-1451. lO. Grierson, D. and Schuch, W. (1993). Control of ripening. Philos.Trans.R.Soc.Lond.[Biol]. 342:241-250. 1 1. Hirschberg, J., Cohen, M., Harker, M., Lotan, T., Mann, V., and Pecker, I. (1997). Molecular genetics of the carotenoid biosynthesis pathway in plants and algae. Pure Appl.Chem. 69:2152-2158. 12. Laval-Martin, D., Quennement, J., and Moneger, R. (1975). Pigment evolution in Lycopersicon esculentum during growth and ripening. Biochemistry 14:2357-2362. 13. Mann, V., Pecker, I., and Hirschberg, J. (1994). Cloning and characterization of the gene for phytoene desaturase (Pds) from tomato (Lycopersicon esculentum). Plant Mol.Biol. 24:429-434. 14. Montgomery J, Goldman S, Deikman J, Margossian L, Fischer RL, (1993) Identification of an ethylene-responsive region in the promoter of a fruit-ripening gene. Proc. Natl. Acad. Sci. USA 90:5939-5943. 15. Nicholass FJ, Smith CJ, Schuch W, Bird CR, Grierson D. (1995) High levels of ripening-specific reporter gene expression directed by tomato fruit polygalacturonase gene-flanking regions. Plant Mol Biol 1995 28:423-435. 16. Montgomery J, Pollard V, Deikman J, Fischer RL. (1995) Positive and negative regulatory regions control the spatial distribution of polygalacturonase transcription in tomato fruit pericarp. Plant Cell 5: 1049-1062 17. Pecker, I., Chamovitz, D., Linden, H., Sandmann, G., and Hirschberg, J. (1992). A single polypeptide catalyzing the conversion of phytoene to ζ -carotene is transcriptionally regulated during tomato fruit ripening. Proc.Natl.Acad.Sci.USA 89:4962-4966. 18. Pecker, I., Gabbay, R., Cunningham, F.X., and Hirschberg, J. (1996). Cloning and characterization of the cDNA for lycopene β-cyclase from tomato reveals decrease in its expression during fruit ripening. Plant Mol.Biol. 30:807-819. 19. Pnueli, L., Carmel-Goren, L., Hareven, D., Gutfinger, T., Alvarez, J., Ganal, M., Zamir, D., and Lifschitz, E. (1998). The SELF-PRUNING gene of tomato regulates vegetative to reproductive switching of sympodial meristems and is the ortholog of CEN and TFL1. Development 125: 1979-1989. 20. Ray, J.A., Moureau, P., Bird, A.S., Grierson, D., Maunders, M., Truesdale, M., Bramley, P.M., and Schuch, W. (1992). Cloning and characterization of a gene involved in phytoene synthesis from tomato. Plant Mol.Biol. 19:401-404. 21. Sambrook, J., F, Fritch, E., F.„ and Maniatis, T., 1989. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor. Sandmann, G. (1994a). Phytoene desaturase: Genes, enzymes and phylogenetic aspects. J.Plant Physiol. 143:444-447. 22. Sandmann, G. (1994b). Carotenoid biosynthesis in microorganisms and plants. Eur.J.Biochem. 223:7-24. 23. Tanksley, S.D., Ganal, M.W., Prince, J.C., de Vicente, M.C., Bonierabale, M.W., Broun, P., Fulton, T.M., Giovanonni, J.J., Grandillo, S., Martin, G.B., Messeguer, R., Miller, J.C., Miller, L., Paterson, A.H., Pineda, O., Roder, M.S., Wing, R.A., Wu, W., and Young, N.D. (1992). High density molecular linkage maps of the tomato and potato genomes. Genetics 132: 1141-1160. 24. Van Haaren MJ, Houck CM. (1991) Strong negative and positive regulatory elements contribute to the high-level fruit-specific expression of the tomato 2A11 gene. Plant Mol. Biol. 17:615-630 25. Wing, R.A., Zhang, H.B., and Tanksley, S.D. (1994). Map-based cloning in crop plants - tomato as a model system .1. genetic and physical mapping of jointless. Mol.Gen.Genet. 242:681-688. 26. Zhang, H.B., Martin, G.B., Tanksley, S.D., and Wing, R.A. (1994). Map-based cloning in crop plants: Tomato as a model system .2. Isolation and characterization of a set of overlapping yeast artificial chromosomes encompassing the jointless locus. Mol Gen Genet 244:613-621.

Claims: WHAT IS CLAIMED IS: 1. An isolated complementary or genomic DNA segment comprising a nucleotide sequence coding for a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 18 and 19 and functional naturally occurring and man-induced variants thereof, with the provision that said polypeptide has a major lycopene cyclase catalytic activity. 2. The isolated DNA segment of claim 1 , wherein said nucleotide sequence is selected from the group consisting of SEQ ID NOs: 8, 9, 10 and 11 and functional naturally occurring and man-induced variants thereof. 3. The isolated DNA segment of claim 1 , wherein said nucleotide sequence is a cDNA or a genomic DNA isolated form tomato. 4. An isolated complementary or genomic DNA segment comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 8, 9, 10 and 11. 5. A polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 18 and 19 and functional naturally occurring and man-induced variants thereof, said polypeptide having a major lycopene cyclase catalytic activity. 6. A transduced cell overexpressing a polypeptide including an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 18 and 19 and functional naturally occurring and man-induced variants thereof, said polypeptide having a major lycopene cyclase catalytic activity, the cell therefore over producing β-carotene on an expense of lycopene. 7. The transduced cell of claim 6, selected from the group consisting of a prokaryotic cell and a eukaryotic cell. 8. The transduced cell of claim 7, wherein said eukaryotic cell is of a higher plant. 9. The transduced cell of claim 6, wherein the cell forms a part of a transgenic plant. 10. A method of down-regulating production of β-carotene in a cell comprising the step of introducing into the cell at least one anti-sense polynucleotide sequence capable of base pairing with messenger RNA coding for a polypeptide including an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 18 and 19 and functional naturally occurring and man-induced variants thereof, said polypeptide having a major lycopene cyclase catalytic activity, the cell therefore under producing β-carotene from lycopene. 1 1. The method of claim 10, wherein said at least one anti-sense polynucleotide sequence includes a synthetic oligonucleotide. 12. The method of claim 11, wherein said synthetic oligonucleotide includes a man-made modification rendering said synthetic oligonucleotide more stable in cell environment. 13. The method of claim 11, wherein said synthetic oligonucleotide is selected from the group consisting of methylphosphonate oligonucleotide, monothiophosphate oligonucleotide, dithiophosphate oligonucleotide, phosphoramidate oligonucleotide, phosphate ester oligonucleotide, bridged phosphorothioate oligonucleotide, bridged phosphoramidate oligonucleotide, bridged methylenephosphonate oligonucleotide, dephospho internucleotide analogs with siloxane bridges, carbonate bridge oligonucleotide, carboxymethyl ester bridge oligonucleotide, carbonate bridge oligonucleotide, carboxymethyl ester bridge oligonucleotide, acetamide bridge oligonucleotide, carbamate bridge oligonucleotide, thioether bridge oligonucleotide, sulfoxy bridge oligonucleotide, sulfono bridge oligonucleotide and α-anomeric bridge oligonucleotide. 14. The method of claim 10, wherein said at least one anti-sense polynucleotide sequence is encoded by an expression vector. 15. The method of claim 10, wherein said cell is selected from the group consisting of a prokaryotic cell and a eukaryotic cell. 27 16. The method of claim 15, wherein said eukaryotic cell is of a higher plant. 17. The method of claim 15, wherein the cell forms a part of a transgenic plant. 18. An expression construct for directing an expression of a gene in fruit or flower comprising a regulatory sequence selected from the group consisting of an upstream region of a B allele of tomato and an upstream region of a b allele of tomato. 19. The expression construct of claim 18, comprising a functional part of nucleotides 1- 1210 of SEQ ID NO: 14 or nucleotides 1-1600 of SEQ ID NO: 15, or functional naturally occurring and man-induced variants thereof. 20. The expression construct of claim 18, comprising at least one control element having a sequence selected from the group consisting of SEQ ID NOs:21-24, all derived from SEQ ID NO: l l , and functional naturally occurring and man-induced variants thereof. 21. The expression construct of claim 18, wherein the expression construct is selected from the group consisting of plasmid, cosmid, phage, virus, bacmid and artificial chromosome. 22. The expression construct of claim 18, designed to integrate into a genome of a host. 23. A method of isolating a gene encoding a polypeptide having an amino acid sequence homologous to SEQ ID NOs: 17, 18 and 19 and having a major lycopene cyclase catalytic activity from a species, the method comprising the step of screening a complementary or genomic DNA library prepared from isolated RNA or genomic DNA extracted from said species with a probe having a sequence derived from SEQ ID NOs: 8, 9, 10 or 1 1 and isolating clones reacting with said probe. 24. A transduced cell transduced with the expression construct of claim 18. 25. A transgenic plant transduced with the expression construct of claim 18.

Inventors: FRIEDMAN MARK M, HIRSCHBERG JOSEPH, RONEN GIL, ZAMIR DANNY, FRIEDMAN, MARK, M., HIRSCHBERG, JOSEPH, RONEN, GIL, ZAMIR, DANNY
Applicants: FRIEDMAN MARK M, HIRSCHBERG JOSEPH, RONEN GIL, YISSUM RES DEV CO, ZAMIR DANNY, FRIEDMAN, MARK, M., HIRSCHBERG, JOSEPH, RONEN, GIL, YISSUM RESEARCH AND DEVELOPMENT COMPANY OF THE HEBREW UNIVERSITY OF JERUSALEM, ZAMIR, DANNY
Application year: 1999
Main classification: A01H5
IPC codes: C12N1, C12N9, C12N15