AU707704C - Regulated expression of heterologous genes in plants and transgenic fruit with a modified ripening phenotype - Google Patents

Description

REGULATED EXPRESSION OF HETEROLOGOUS GENES IN PLANTS

AND TRANSGENIC FRUIT WITH A MODIFIED RIPENING PHENOTYPE

FIELD OF THE INVENTION The present invention relates the regulated expression of heterologous genes in transgenic fruit- bearing plants, where fruit produced by the these plants has a modified ripening phenotype. The invention also relates to the fruit produced by such transgenic plants, to methods of producing the transgenic plants and to the transgenic plants themselves.

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AND IDENTIFICATION OF ETHYLENE (Hillman, J. , Ed.) Cambridge University Press, Cambridge, MA, pp. 135-151 (1978) .

BACKGROUND OF THE INVENTION Ethylene is a plant hormone which is a powerful regulator of plant metabolism, acting, and interacting with other plant hormones in trace amounts. Ethylene is a gas under normal physiological conditions. Even at low concentrations, ethylene has profound hormonal effects on plants.

The effects of ethylene, whether produced by the plant itself or applied exogenously, are numerous, dramatic, and of considerable commercial importance. Among the diverse physiological effects are the following: leaf abscission; fading in flowers; flower wilting; leaf yellowing; leaf epinasty; and stimulation of ripening in fruits and vegetables. Ethylene promotes senescence in plants, both in selected groups of cells and in whole organs, such as, fruits, leaves, or flowers. Senescence is the natural, genetically controlled degenerative process which usually leads to death in plants.

Normally, ethylene production from plant tissue is low. Large quantities of ethylene, however, are produced during ripening and senescence processes. A large amount of ethylene is also produced following trauma caused by chemicals, temperature extremes, water stress, ultraviolet light, insect damage, disease, or mechanical wounding. Ethylene produced by plants under such trauma conditions is referred to as "wound ethylene" or "stress ethylene". In fruits and vegetables, the stimulation of ethylene production by cuts or bruises may be very large and bear considerably on storage effectiveness. Ethylene-induced leaf browning is a common basis for loss in many plants, including lettuce and tobacco. In some tissues, exposure to only a small amount of ethylene may cause an avalanche of ethylene production in adjacent plants or plant tissues such as fresh produce. This autocatalytic effect can be very pronounced and lead to loss of fruit quality during transportation and storage.

Current technologies that specifically address post-harvest storage life have been in existence for decades and are hampered by such problems as high cost, side effects, and an inability to completely shut off ethylene production. Included in this group are controlled atmosphere (CA) storage, chemical treatment, packaging, and irradiation. CA facilities slow ethylene biosynthesis through: (i) low temperature, (ii) reducing the oxygen level below 3%, and (iii) elevating the carbon dioxide level in the storage area to the 3%-5% range. Expensive scrubbers are sometimes added which reduce ethylene already respired to the atmosphere. Drawbacks are that

CA facilities are expensive to construct, have a high utility cost, and are unable to completely eliminate ethylene production and side effects. Also, CA storage techniques can only control external ethylene and not that which resides inside the plant tissue. CA storage can also lead to undesirable side effects: injury can result from high C0₂ levels, low 0₂ levels, or low temperature.

Another treatment is to limit the ethylene biosynthesis in the plant tissue through chemical treatment. Aminoethoxyinylglycine (AVG) , an analog of the antibiotic rhizobitoxine, is one such inhibitor. However, AVG cannot be used as a chemical additive in foods due to its high toxicity. Silver thiosulfate (STS) is also effective in slowing fruit ripening and flower fading, but is also toxic and cannot be used on foods. Further, STS only works with certain flowers and often causes black spotting.

Recently, molecular genetic approaches leading to transgenic plants with impaired biosynthesis of ethylene have been reported. Hamilton, et al . , identified a cDNA clone for tomato EFE (pTOM13) by inhibiting ethylene synthesis with an antisense gene expressed in transgenic plants. Oeller, et al . , showed that expression of antisense RNA to the rate-limiting enzyme in the biosynthetic pathway of ethylene, 1- aminocyclopropane-1-carboxylate synthase, inhibits fruit ripening in tomato plants. Klee, et al . , cloned the gene encoding ACC deaminase, from soil bacteria, and introduced it into tomato plants. Reduction in ethylene synthesis in transgenic plants did not cause any apparent vegetative phenotypic abnormalities. However, fruits from these plants exhibited significant delays in ripening, and the mature fruits remained firm for at least 6 weeks longer than the non-transgenic control fruit.

SUMMARY OF THE INVENTION

The present invention relates to plant transformation vectors, chimeric genes and related DNA constructs. In one embodiment the invention relates to a transgenic fruit-bearing plant having (i) a DNA sequence encoding a product that is effective to reduce ethylene biosynthesis in fruit from the plant, and (ii) a promoter whose expression is induced during fruit ripening, by a plant cell cytokine, or by ethylene synthesis by the fruit. The DNA sequence is heterologous to the promoter and is operably linked to the promoter to enable expression of said product. The DNA sequence may encode any of the following products: S-adenosylmethionine hydrolase, aminocyclopropane-1- carboxylic acid (ACC) deaminase, ACC oxidase antisense molecule, ACC synthase antisense molecule, ACC oxidase cosuppression molecule, and ACC synthase cosuppression molecule.

Promoters useful in the present invention may be selected from a variety of plant sources, including, but not limited to, genes homologous to a tomato E4 or E8 genes, including tomato and raspberry E4 and E8 genes. Exemplary other promoters may be obtained from the avocado cellulase gene, tomato ACC oxidase gene, tomato polygalacturonase gene, or homologs of any of these genes in other plants.

The present invention also includes a method for modifying ripening fruit of a fruit bearing plant. In the method, the transgenic plants described above are grown to produce a transgenic plant bearing fruit. Fruit produced by the plant has an initial burst of ethylene production, followed by a reduction in the level of ethylene synthesis by the fruit, resulting in a fruit having a modified ripening phenotype. Such phenotypes include the delay and/or suspension of fruit ripening, typically relative to wild-type (i.e., non- transgenic) fruit. In another aspect, the present invention includes the above described transgenic fruit and fruit cells.

Further, the invention includes a method for producing a transgenic fruit-bearing plant, where fruit produced by the plant has a modified ripening phenotype. In this method, the following chimeric gene is introduced (e.g., by transformation) into progenitor cells of the plant: (i) a DNA sequence encoding a product effective to reduce ethylene biosynthesis in fruit from the plant, and (ii) a promoter whose expression is induced during fruit ripening, by a plant cytokine, or by ethylene synthesis by the fruit. As above, the DNA sequence is heterologous to the promoter and is operably linked to said promoter to enable expression of the product. The transformed progenitor are grown cells to produce a transgenic plant bearing fruit. The method further includes transforming progenitor cells of the plant with a selectable vector containing chimeric gene. The DNA sequences and promoters may be as described above.

In another aspect, this method includes isolating useful promoters, typically, employing the following steps: (i) selecting a probe DNA molecule containing a sequence homologous to a region of the promoter of interest, such as from the tomato E4 or E8 genes;

(ii) contacting the probe with a plurality of target DNA molecules derived from the genome of a selected fruit-bearing plant under conditions favoring specific hybridization between the probe molecule and a target molecule homologous to the probe molecule;

(iii) identifying a target molecule having a DNA sequence homologous to the probe; and

(iv) isolating promoter sequences associated with the target molecule. The invention also includes expression vectors containing the chimeric genes described above. These vectors are useful for transformation of plant cells and may be included in commercial kits. Another embodiment of the present invention is a polypeptide in a plant having (i) a DNA sequence encoding a product that is effective to reduce ethylene biosynthesis in fruit from the plant, and (ii) a promoter whose expression is induced during fruit ripening or by ethylene synthesis by said fruit, where said DNA sequence is heterologous to said promoter and said DNA sequence is operably linked to said promoter to enable expression of said product. In another aspect, the invention includes plant and fruit cells containing such chimeric genes, as well as vectors containing such genes.

One embodiment of the invention is a method for delaying wound-induced ripening of fruit of a fruit- bearing plant. This method includes transforming progenitor cells of the plant with a selectable vector containing (i) a promoter that has the sequence of the tomato E4 gene promoter or the sequence of a promoter for a gene homologous to the tomato E4 gene, and (ii) a heterologous gene, whose product is effective to reduce ethylene biosynthesis in fruit from the plant, which is under the control of the promoter, growing the transformed progenitor cells to produce a transgenic plant bearing fruit, and subjecting the fruit to a wound. The method also includes subjecting the fruit to a wound by picking the fruit from a transgenic plant. In the method, an exemplary heterologous gene is S-adenosylmethionine hydrolase.

The transgenic plant may be selected from the group consisting of, but not limited, to tomato, raspberry, strawberry and melon. The promoter may be selected from the same group. Further plant species are described in the specification. In one embodiment the promotor is a tomato E4 promoter.

In another aspect of the invention, the promoter is isolated by the steps of (i) selecting a probe DNA molecule containing a sequence homologous to a region of tomato E4 gene DNA, (ii) contacting the probe with a plurality of target DNA molecules derived from the genome of a selected fruit-bearing plant under condi- tions favoring specific hybridization between the probe molecule and a target molecule homologous to the probe molecule, (iii) identifying a target molecule having a DNA sequence homologous to tomato E4 gene, and (iv) isolating promoter sequences associated with the target molecule.

The probe molecule may carry a reporter moiety; the identifying step then includes detecting the moiety.

The method also describes isolating the promotor by (i) selecting first and second oligonucleotide primers corresponding to an upstream and a downstream region, respectively, of an E4 gene, (ii) amplifying a region of the E4 gene DNA between the first and second primers to generate probe molecules, (iii) contacting the probe molecules with a plurality of target DNA molecules derived from the genome of a selected fruit- bearing plant under conditions favoring specific hybri¬ dization between the probe molecule and a target mole¬ cule homologous to the probe molecule, (iv) identifying a target molecule having a DNA sequence homologous to tomato E4 gene, and (v) isolating promoter sequences associated with the target molecule.

The invention also describes a delayed-ripening fruit, containing (i) a heterologous S-adenosylmeth- ionine hydrolase gene, and (ii) a promoter effective to produce transient expression of the gene when the fruit is picked.

A method is described for inducing transient, wound-induced expression of a heterologous gene in fruit of a fruit-bearing plant, comprising transforming progenitor cells of the plant with a selectable vector containing (i) a promoter that has the sequence of the tomato E4 gene promoter or the sequence of a promoter for a gene homologous to the tomato E4 gene, and (ii) a heterologous gene which is under the control of the promoter, growing the transformed progenitor cells to produce a transgenic plant bearing fruit, and subject¬ ing the fruit to a wound. In this method, expression of the heterologous gene typically reduces ethylene biosynthesis. In one embodiment, the heterologous gene encodes S-adenosylmethionine hydrolase.

In one embodiment, the present invention includes, a method for delaying ripening of the fruit of a fruit- bearing plant. The method includes transforming plant progenitor cells, or host cells, with a selectable vector containing a plant E4 gene promoter and a heterologous gene, such as S-adenosylmethionine hydrolase, whose product reduces ethylene biosynthesis in the plant. The transformed cells are grown to produce a transgenic plant bearing fruit. In a related embodiment, the method is used, essentially as above, to delay senescence in flowers and vegetables. The E4 promoter, and/or the transformed plant, may be selected from a variety of plants, including fruit-bearing plants, such as tomato, eggplant, legumes, raspberry, strawberry, melon, avocado, cherry, apricot, citrus fruits, etc.; flowers, such as roses and carnations; and vegetables, such as cauliflower, and lettuce.

In another aspect, the invention includes a method of isolating E4 promoters from plants. The method includes selecting an E4 probe, hybridizing it with genomic DNA from selected plant species, identifying DNA or clones from positively-hybridizing targets, and isolating promoter sequences associated with the positive target molecule. Positively-hybridizing targets can be identified by either primary or secondary detection of reporter moieties attached to the probe. The probe can be obtained by a number of methods, including primer-based DNA amplification or isolation of restriction digest fragments.

In another embodiment, the invention includes a delayed-ripening fruit, containing a heterologous gene, such as the S-adenosylmethionine hydrolase gene, and a promoter, such as the E4 promoter, effective to produce transient expression of the heterologous gene when the fruit is picked. The promoter may be obtained from a variety of plants, such as outlined above, using methods of the present invention.

Also included in the present invention is an E4 promoter molecule and a DNA construct containing the promoter molecule operably linked to a heterologous gene, where expression of the heterologous gene is under the control of the promoter DNA molecule. These and other objects and features of the invention will be more fully appreciated when the following detailed description of the invention is read in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE FIGURES Figure 1A schematically illustrates the metabolic reactions for the synthesis of ethylene from methionine under both normal and stress conditions. Figure IB schematically illustrates the effect of the enzyme SAMase (AdoMetase) on ethylene biosynthesis. Figure 2 schematically illustrates the steps described for the genetic engineering of the AdoMetase- encoding gene in vector pAG-111.

Figure 3 illustrates the elements of the tomato E8 promoter and the primers used to amplify and isolate the promoter sequences.

Figure 4 outlines the steps involved in the construction of pGA-SESKN from pGA-ESKN and shows the elements of the E8 gene adjacent the AdoMetase (SAMase) coding sequences which are followed by nosT transcription termination sequences.

Figure 5A schematically represents the structure of the pGA-ESKN vector. Figure 5B schematically represents the structure of the pGA-SESKN vector. Figure 6 shows the photograph of an autoradiogram which demonstrates the AdoMetase mRNA levels in fruit derived from two different transgenic plants.

Figure 7 shows a quantitation of the results presented in Figure 6. These results illustrate the effect of variations of the E8 promoter on AdoMetase mRNA levels in ripening tomatoes.

Figure 8 is a graph representing the relative levels of AdoMetase activity in ripening tomatoes at different stages. Figures 9A to 9D presents the data for ethylene production in the fruit of 4 different transgenic plants (Figure 9A, ES 19-2; Figure 9B, LS 4-2; Figure 9C, ES 35-1; and Figure 9D, ES22 A-l) over a ten day period after entry of the fruit into breaker stage. Figure 10 illustrates the post-harvest shelf life of tomatoes obtained from SESKN transgenic plants. Figure 11 presents the sequence of the SAM-K modification of the AdoMetase gene derived from bacteriophage T3. Figure 12A shows a diagram of the steps followed in constructing vector pAG-114 (pESKN) .

Figure 12B shows a diagram of the steps followed in constructing vector pAG-5321 (pGA-ESKN) . Figure 13 presents the sequence of the upstream minus 2216 base pair region of the tomato E8 gene (SEQ ID NO:24) .

Figure 14 shows a photograph of an autoradiogram of a Southern blot of tomato, raspberry, strawberry, melon, carnation and cauliflower DNA probed with a fragment containing the coding sequence from the tomato E4 gene.

Figure 15 shows a DNA sequence of an isolated E4 gene from a raspberry genomic DNA library. Figure 16 shows a schematic of the tomato E4 gene, and the primers used to isolate a 1.18 kb tomato E4 promoter fragment.

Figures 17A-D show a diagram of the steps followed in constructing vectors pAG-lll (Figures 17A-17B) , pAG- 117 (Figures 17B-17C) and pAG-5520 (Figures 17C-17D) .

Figures 18A and 18B show a the results of an RNAse protection assay to detect SAMase mRNA in fresh and wounded leaves of normal and E4/SAMase transgenic tomato plants. Figure 19 shows a bar graph of ethylene production in wounded leaves of normal and E4/SAMase transgenic tomato plants.

Figures 20 and 21 show the results of an RNAse protection assay to detect SAMase mRNA in E4/SAMase transgenic tomato ripening fruit at four stages of ripening.

Figure 22 shows the results of a Western blot, of E4/SAMase transgenic tomatoes at four stages of ripening, probed with an antibody to SAMase. Figure 23 shows a chart of ethylene production in normal and E4/SAMase transgenic tomatoes as a function of time after harvest.

Figure 24 shows the DNA sequence of the tomato E4 gene.

Figure 25 shows the DNA sequence of a Hindlll/Kpnl fragment containing the tomato E4 promoter and the SAMase gene, and the translated amino acid sequence of the SAMase gene.

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

"Homologous" DNA refers to DNA not introduced into a host organism by recombinant means. "Heterologous" DNA refers to DNA which has been transfected into a host organism. Typically, heterolo¬ gous DNA refers to DNA that is not originally derived from the transfected or transformed cells' genomic DNA (e . g . , CAT and _/S-galactosidase gene sequences) . However, any DNA introduced into an organism by recombinant means is referred to as heterologous DNA (e . g . , introduction into a tomato of a vector carrying the tomato E4 promoter) .

A "chimeric gene," in the context of the present invention, typically comprises a promoter sequence operably linked to non-homologous DNA sequences that encode a gene product (e.g., a tomato E8 promoter adjacent DNA sequences encoding S-adenosylmethionine cleaving enzyme) . Two nucleotide sequences are considered to be

"functionally homologous" if they hybridize with one another under moderately stringent conditions, i.e. 0.1% SSC at room temperature. Examples of such hybridization conditions are given in Examples 6 and 7. Typically, two homologous nucleotide sequences are greater than or equal to about 60% identical when optimally aligned using the ALIGN program (Dayhoff) . Two amino acid sequences are considered "homologous" if their amino acids are greater than or equal to about 60% identical when optimally aligned using the ALIGN program mentioned above.

A "modified ripening" phenotype typically refers to an alteration of the rate of ripening of a transgenic fruit relative to corresponding wild-type fruit. For example, delayed ripening fruit (i.e., ripening takes .longer than corresponding wild-type fruit) or suspension of the fruit's ability to complete the ripening process. A "product" encoded by a DNA molecule includes, for example, RNA molecules and polypeptides.

II . Regulated Promoters for Expression of Genes in Plants. The present invention provides a method to regulate plant cell expression of any gene in a tissue or development stage-specific manner, in particular, genes whose products reduce ethylene synthesis in plant cells. In one embodiment, the invention teaches the use of tissue/stage-specific promoters whose expression is induced during fruit ripening or by the presence of ethylene. To regulate cellular production of ethylene, a gene whose product results in a reduction of ethylene synthesis is operably linked to such promoter (creating a chimeric gene) . In one aspect of the invention, an initial burst of ethylene synthesis occurs, expression of the chimeric gene is induced and production of ethylene by the cell is subsequently reduced. When the chimeric gene is present in fruit cells, the result is fruit having a modified ripening phenotype relative to wild-type (non-transgenic) fruit.

The initial burst of ethylene allows fruit to initiate ripening, but the subsequent down regulation of ethylene production delays the course of fruit ripening, i.e., the fruit are suspended in their ability to complete the ripening process. Climateric or non-climateric fruit may be produced by this method. Several examples of regulated promoters are described below, including two embodiments of the tomato E8 promoter, the tomato E4 promoter and the raspberry E4 promoter. Further useful promoters include, but are not limited to, the avocado cellulase promoter (Casε, et al . ) , the tomato polygalacturonase promoter (Bird, et al . ) , and the tomato ACC oxidase

(ACCO) promoter, as well as promoters from homologous genes obtained from other plants.

Exemplary gene products that result in reduction of ethylene synthesis include, but are not limited to the following: S-adenosylmethionine hydrolase; 1- aminocyclopropane-1-carboxylate deaminase (Klee, et al . ; Sheehy, et al . ) ; the ACC synthase gene in an antisense or cosuppression configuration (Oeller, et al . ; Van der Straeten, et al . ) ; and the ACC oxidase gene in either an antisense or cosuppression configuration (Hamilton, et al . ; Holdsworth, et al . ) . Cosuppression has been described by Jorgensen, et al . (1991, 1993) , both herein incorporated by reference. Other gene products that may be useful in the reduction of ethylene biosynthesis include catalytic antibodies and ribozyme molecules.

The present invention provides, in one aspect, nucleic acid constructs suitable for transforming plants with heterologous genes under the control of an E4 promoter- (originally isolated from tomato; Cordes, et al . ) or an E8 promoter. In one embodiment, the plant is a fruit-bearing plant, and the heterologous gene is a gene effective to reduce ethylene biosynthesis in fruit from the plant. In another embodiment, the plant is a flowering plant, and the heterologous gene is a gene effective to reduce ethylene biosynthesis in flowers of the plant. In still another embodiment, the plant is a vegetable, and the heterologous gene is a gene effective to reduce ethylene biosynthesis in the vegetable.

Experiments performed in support of the present invention demonstrate that an exemplary heterologous gene effective to reduce ethylene biosynthesis in tissues of a plant is the AdoMetase gene, isolated from bacteriophage T3 (Hughes, et al . , 1987a) . These experiments show that ethylene production in ripe transgenic tomatoes containing the AdoMetase gene under the control of a tissue/stage specific promoter is significantly lower. In addition, the tomatoes develop color to a light red stage and then cease further color development, and remain firm longer than control tomatoes. Further, the experiments show that the E4 promoter coupled to a heterologous gene can be activated by wounding plant tissue containing the construct, and that the activation of the promoter is typically transient in nature.

A. Tomato E4 Promoter The tomato E4 promoter is both stage and tissue specific (Cordes, et al . ) . Typically, E4 mRNA is abundant in ripening fruit and is not detected in leaf, root, stem, or unripe fruit. E4 gene expression can, however, be activated by ethylene, and the ethylene- induced expression can be detected in a variety of plant tissues (Lincoln, et al . , Lincoln and Fischer) . Further, the rin (ripening inhibited) mutation, that blocks many aspects of ripening, including softening, ethylene production, and color development (Giovannoni, et al . ) , reduces the concentration of E4 mRNA by greater than 10-fold. The sequence of the E4 promoter has been published (Cordes, et al . ) and the DNA sequence of the minus 1173 base pair region is presented in the first portion of Figure 25. The tomato E4 promoter may be employed in vector constructs used to produce transgenic plants, such as transgenic tomatoes. For example, a vector engineered according to methods of the present invention (detailed below) , containing the tomato E4 promoter connected to the AdoMetase gene (e.g. vector pAG-5520) , may be used to produce transgenic raspberries, strawberries, melons, carnations, cauliflower, and the like. The AdoMetase gene will be expressed in the fruit of these transgenic plants will delay ripening. An advantage of this method is a savings of time and resources involved in vector construction, since the same vector can be used to transform many different plant types. Experiments performed in support of the present invention have demonstrated the ability to the tomato E4 promoter to facilitate DNA coding sequence expression in raspberries.

Alternatively, E4 promoter sequences may be isolated from the same type of plant that is to be transformed, and incorporated into the vector constructs used to perform the transformations. For example, a raspberry E4 promoter may be connected to a heterologous gene, such as the AdoMetase gene, and used to transform raspberries. This method is typically preferable, because a promoter from the same type of plant as is-transformed is more likely to contain all of the regulatory elements required for appropriate stage and tissue specificity of expression. For example, isolation of a genomic copy of a raspberry E4 gene with 5' regulatory sequences is described below. E4 promoters may isolated from other plants using a number of methods, including those described below.

B. Identification of Plant E4 Promoters

The present invention provides for the use E4 promoters from species other than tomato in vector constructs containing heterologous genes. Southern blot experiments performed in support of the present invention demonstrate the presence of DNA molecules having high sequence homology with the tomato E4 gene in raspberry, strawberry, melon, carnation and cauliflower. Similar Southern blot analyses may be performed on other fruit-bearing plants to identify additional E4 genes.

A Southern blot analysis used herein is detailed in Example 6. E4 homologues are identified in a

Southern blot of the genomic DNA of the plants listed above probed with a labelled DNA fragment containing the coding sequence of the tomato E4 gene.

The probe is selected to contain the coding sequence of tomato E4, rather than the promoter sequence, because coding sequences are typically more conserved from species to species than are promoter sequences. In the experiments detailed in Example 6, probe molecules are generated from tomato genomic DNA using primer-specific amplification (Mullis; Mullis, et al . ) . The oligonucleotide primers are selected such that the amplified region included the entire coding sequence of the tomato E4 gene. Primers may also be selected to amplify only a selected region of the E4 gene. Alternatively, a probe can be made by isolating restriction-digest fragments containing the sequence of interest from plasmid DNA.

The probe is labeled with a detectable moiety to enable subsequent identification of homologous target molecules. Exemplary labeling moieties include radioactive nucleotides, such as ³²P-labeled nucleotides, digoxygenin-labeled nucleotides, biotinylated nucleotides, and the like, available from commercial sources.

In the case of primer-amplified probe, labeled nucleotides may be directly incorporated into the probe during the amplification process. Probe molecules derived from DNA that has already been isolated, such as restriction-digest fragments from plasmid DNA, are typically end-labeled (Ausubel, et al . ) .

Target molecules, such as Hindlll DNA fragments from the genomes of the above-listed plants, are electrophoresed on a gel, blotted, and immobilized onto a nylon or nitrocellulose filter. Labeled probe molecules are then contacted with the target molecules under conditions favoring specific hybridization between the probe molecules and target molecules homologous to the probe molecules. Conditions favoring specific hybridization are referred to as moderately to highly stringent, and are affected primarily by the salt concentration and temperature of the wash buffer (Ausubel, et al . , Sambrook, et al . ) . Conditions such as those used in the final wash in Example 6 are typically classified as moderately stringent, due to the low salt concentration, and are expected to preserve only specific hybridization interactions, allowing the identification and isolation of homologous genes in different plant species. Following contacting, hybridization, and washing, target molecules with sequences homologous to the probe are identified by detecting the label on the probe. The label may be detected directly, for example, as in radioactive label detected on autoradiograms, or it may be detected with a secondary moiety, for example, fluorescently-labeled streptavidin binding to a biotinylated probe.

C. Isolation of Other E4 Promoters

Following the identification of plants containing E4 genes, the DNA encoding the genes, including the promoter regions, may be isolated from the respective species, by, for example, screening a genomic DNA library. Experiments performed in support of the present invention, detailed in Example 7, demonstrate the isolation of a genomic copy of a raspberry E4 gene from a raspberry genomic DNA library.

The library of interest is screened with a probe containing sequences corresponding to the coding sequence of a known E4 gene, such as the tomato E4 gene. The screening is done using known methods (Ausubel, et al . , Sambrook, et al . ) , essentially as described above. Positive plaques or colonies are isolated, and the insert DNA is sequenced and compared to known E4 sequences. Clones containing inserts with sequences corresponding to genes homologous to tomato E4 are identified and, if necessary, used to obtain additional clones until the promoter region of interest is isolated. The sequence of the raspberry E4 gene is presented in Figure 15 (SEQ ID NO:26) . III. Heterologous Genes

According to methods of the present invention, heterologous genes are linked to the promoters of the present invention. Exemplary heterologous gene for the transformation of plants include genes whose products are effective to reduce ethylene biosynthesis in specific tissues of those plants, e.g. the fruits, flowers or leaves. One of these genes, AdoMetase, is discussed in detail below.

A. Ethylene Synthesis

The amino acid methionine has been shown to be a precursor of ethylene (C₂H₄) in plant tissues (reviewed by Imaseki) . Methionine, however, is not the immediate precursor but first must be converted to the sulfonium compound S-adenosylmethionine (SAM) and, subsequently, aminocyclopropane-1-carboxylic acid (ACC) prior to conversion to ethylene. The metabolic reactions for the synthesis of ethylene from methionine under both normal and stress conditions are presented in Figure

1A, and summarized as follows:

Methionine → SAM → ACC → Ethylene

ACC synthase catalyzes the degradation of SAM to ACC and 5'-methylthioadenosine (MTA) . This enzymatic reaction appears to be the rate limiting step in ethylene formation. For example, the natural plant hormone indoleacetic acid (IAA or auxin) stimulates ethylene production by inducing the synthesis of ACC synthase. Conversely, the synthesis of SAM from methionine and the production of ethylene from ACC do not require auxin induction.

In addition, wounding and fruit ripening induces the formation of ACC synthase and, therefore, the conversion of SAM to ACC. The other product of the ACC synthase reaction, MTA, must be recycled back into methionine so as to provide an adequate supply of methionine for continual ethylene production. This recycling pathway from MTA to methionine, also presented in Figure 1A, has been shown to exist in plant tissue (Adams, et al . ; Kushad, et al . ) . The degradation of MTA has added significance in light of the finding that MTA is a potent inhibitor of ACC synthase. The importance of the degradation and recycling of MTA in normal plant tissues is, therefore, twofold: 1) to prevent the direct inhibition of ethylene synthesis by MTA, and 2) to provide adequate methionine for continual ethylene synthesis. The first step in the degradation of MTA in plant tissue is the hydrolysis of this nucleoside to 5- methylthioribose (MTR) by a specific MTA nucleosidase. MTR not only provides its methylthio moiety for the formation of methionine, but also contributes four carbons from its ribose towards the synthesis of this amino acid. Therefore, the methylthio group is conserved by recycling. It should be noted that this pathway merely maintains a methionine supply for ethylene biosynthesis, but does not result in a net increase in methionine synthesis.

1. AdoMet hydrolase

One approach to reduce ethylene biosynthesis in plants reported here utilizes a gene that encodes the enzyme S-adenosylmethionine hydrolase. This approach has been described in the PCT International Application US90/07175. This enzyme, encoded by the E . coli bacteriophage T3 , hydrolyses AdoMet to homoserine and MTA. The enzyme is known as its recommended name, AdoMet hydrolase (AdoMetase) , or by its other name, S- adenosyl ethionine cleaving enzyme (SAMase) (Studier, et al . ) . Both products of the reaction (i.e., homoserine and MTA) are recycled to methionine; MTA as previously shown (Figure 1A) and homoserine via a metabolism pathway known to exist in plant tissues.

The AdoMetase gene has been identified, isolated, cloned, and sequenced (Hughes, et al . , 1987a; Hughes, et al . , 1987b) . The gene contains two in-frame reading sequences that specify polypeptides of 17105 and 13978 daltonε. Both polypeptides terminate at the same ochre codon. This results in the 14 kd polypeptide being identical to 82% of the 17kd polypeptide starting at the carboxyl end of the longer polypeptide. Both polypeptides are present in partially purified cells and from E . coli expressing the cloned gene (Hughes, et al . , 1987b; Studier, et al . , 1976) . Other bacteriophages that encode the AdoMetase or SAMase genes are coliphage BA14, Klebsiella phage Kll, and Serratia phage IV (Mertens, et al . ; Horsten, et al . ) . The effect AdoMetase expression in plant cells has on the plant methionine recycling pathway is shown schematically in Figure IB. Experiments performed in support of the present invention, using transgenic tomatoes expressing an AdoMetase gene and monitoring ethylene production, have demonstrated that the effect of AdoMetase on the pathway is to "short circuit" the branch that produces ethylene: ethylene production is reduced in such transgenic plants.

Different bacteriophages may be expected to contain AdoMetase genes with variations in their DNA sequences. The isolation of AdoMetase coding sequences from bacteriophage coding sequences can be accomplished as previously described for AdoMetase from bacteriophage T3. Alternatively, degenerative hybridization probes for AdoMetase coding sequences can be generated and used to screen plasmids carrying fragments of a selected bacteriophage's genome for the presence of homologous sequences. AdoMetase enzymatic activity can be evaluated by standard biochemical tests (see for example, Example 13) .

Furthermore, the amino acid sequence of AdoMetase may be modified by genetic techniques to produce enzymes with altered biological activities (see below) . An increase in the biological activity could permit the use of lower amounts of the enzyme to control ethylene biosynthesis in plants.

IV. Vector Construction

Plant transformation vectors are constructed according to methods known in the art (see, for example, Houck, et al . , and Becker, et al . )

A series of recombinant DNA manipulations are performed in the AdoMetase gene prior to placement in an Agrobacterium expression vector. Initially, a Maelll to Ba HI fragment from M13HB1 (Hughes, et al . , 1987a) is subcloned into the pUC19 plasmid vector to produce pAG-110 (Example 1, pUC19-SAMase Figure 2) . To increase the translational efficiency of the AdoMetase gene in plants, site directed mutagenesis of the nucleic acid sequences surrounding the ATG start codon is performed. A synthetic double stranded 39 base pair oligonucleotide is synthesized and substituted for the BamHI to Xmnl fragment at the 5' end of the gene (Figure 2) . The net effect of this substitution is to change the CACCAAATGA (SEQ ID NO: 14) in the native T3 sequence to GCCACCATGG (SEQ ID NO: 15) which is an optimal eukaryotic translational initiation sequence (Kozak, et al . ; Lutcke, et al . ) .

The change also introduces an NcoJ site (CCATGG) at the AdoMetase start codon which facilitates fusions to different promoters. The only alteration to the AdoMetase coding sequence is the amino acid at amino acid position two which is changed from isoleucine to valine: this is a highly conservative amino acid change. The altered form of AdoMetase was named SAM-K (Figure 11) .

A recombinant vaccinia vector with SAM-K (w:SAM- K) was constructed. Expression of this vector in African green monkey cells or T3-infected bacterial cells was compared with the gene to the native T3 gene when expressed in the same cells. The specific activity of AdoMetase was higher in the vv:SAM-K infected cells than in the T3 infected bacterial cells demonstrating that SAM-K encodes a fully functional version of AdoMetase.

Experiments performed in support of the present invention have demonstrated constitutive expression of AdoMetase in transgenic plants. In these plants there is a significant reduction in the ability of these plants to synthesize ethylene.

Using the sequence shown in Figure 24 primers are prepared for use in the polymerase chain reaction (PCR) to amplify a 1177 base pair region of the E4 promoter from tomato genomic DNA (Example 8) . The primers are designed with unique restriction sites at each end and were used to place the promoter in the proper orientation 5' of the SAMase gene in pAG-lll (Figures 17A, 17B) . The 5' end of the promoter fragment has a Hindi I I site, while the 3' end has an Ncol site (CCATGG) placed such that the ATG start codon of the E8 gene product is used as the ATG in the Ncol site. This allows precise placement of the entire E4 promoter directly in front of the SAMase amino acid coding sequences with no intervening sequences (Example 8) . A selectable vector expressing AdoMetase under the control of the E4 promoter (pAG-5520) is constructed as detailed in Example 8 and schematized in Figures 17A- 17D. For selection, the vector contains the neomycin phosphotransferase II gene, providing aminoglycoside antibiotic (e.g. kanamycin) resistance.

V. Plant Transformation

A. Methods of Transforming Plants pAG-5520 is transferred to tomato plants (Example 9) to generate transgenic plants expressing AdoMetase. Tomato progenitor cells (tomato cotyledon tissue explantε) are transformed with EHA101 bacteria containing pAG-5520 and grown in tissue culture in the presence of kanamycin for 8 to 10 weeks to produce plants.

A number of methods, in addition to Agrobacterium- based methods, may be employed to elicit transformation of plant progenitor cells, such as electroporation, microinjection, and microprojectile bombardment. These methods are well known in the art (Comai, et al . , Klein, et al . ; Miki, et al . ; Bellini, et al . ) and provide the means to introduce selected DNA into plant genomes: such DNA may include a DNA cassette which consists of the E4 gene promoter functionally adjacent, for example, AdoMetase coding sequences.

B. Identifying and Evaluating Transformants Several transgenic plants are assayed for their ability to synthesize AdoMetase mRNA, AdoMetase protein, and their ability to inhibit the biosynthesis of ethylene. The assays are performed after the plant tissue being assayed has been subjected to a wound, and are carried out both on leaves of the plant, as well as the fruit. -Leaf wounds are typically cuts on the leaf, performed either with a dull knife or with a circular bore. Fruit "wounds" can be as mild as picking the fruit from the plant.

Leaf-based assays can be informative if the promoter driving the heterologous gene (transgene) is at least somewhat active in leaf tissue, as is the case for the E4 promoter. In such cases, leaf-based assays are useful for initial screens of the expression level of a transgene, since they can be performed much earlier than fruit-based assays. Fruit-based assays, on the other hand, provide more accurate data on transgene expression in the target tissue itself (fruit) . The results of both types of assays are detailed in Example 13. AdoMetase mRNA levels are determined using, for example, an RNAase protection assay (RPA) (Example 10) . Figures 18A and 18B show the results of an RPA using fresh or wounded leaves from normal and transformed tomato plants. Plants showing detectable transgene mRNA expression in leaves are grown to produce fruit, and the fruit is then tested mRNA expression. Figures 20 and 21 shows the results of an RPA using the fruit from one transgenic plant at different stages of fruit ripening. While the absolute level of expression varies considerably among different transformed lines, the relative level of expression as a function of ripening stage is consistently transient, typically peaking at the orange stage, with expression at low levels in fully ripe fruit in all but one case. Ethylene biosynthesis is measured in leaves and fruit of transgenic tomatoes. Figure 19 shows the level of ethylene synthesis in wounded leaves from normal (M) and four transgenic tomato plants. The experiments are performed as detailed in Example 11. Some of lines show a reduction in ethylene biosynthesis, but the values are not well correlated with those obtained in the RNAse assay presented in Figures 18A and 18B. The assay is nevertheless useful for screening, as suggested above, since plants negative for AdoMetase expression in leaf wound assays are also negative for AdoMetase expression in fruit.

Ethylene biosynthesis in transgenic fruit, is typically reduced relative to control fruit, as is shown in Figure 23. Whereas control tomatoes show an increasing rate of ethylene synthesis during the 5 days following harvest, fruit from transgenic line E4-05 shows a decreasing rate of synthesis, which bottoms out approximately one week post-harvest. This result demonstrates that constructs of the present invention are effective at reducing the level of ethylene biosynthesis in the fruit of fruit-bearing plants.

After these time-points, the rates of ethylene synthesis reverse directions for both normal and transgenic plants. This characteristic demonstrates the physiological effects of the transient nature of heterologous gene expression under control of an E4 promoter, and that activation of the E4 promoter, for example, by wounding, can transiently inhibit ethylene biosynthesis. To correlate the effects of AdoMetase mRNA expression, Western Blots were performed,as detailed in Examples 12 and 13. The results are shown in Figure 22. These results demonstrate that transformation of tomato progenitor cells with a construct containing the E4 promoter coupled to the AdoMetase gene is effective to reduce ethylene biosynthesis in the fruit of the transgenic plants, and further, that this reduction can have a transient time-course.

The biological effects of AdoMetase gene expression include a cessation of color development beyond the light red stage, and tomatoes that remain firm much longer than untransformed controls.

VI. E8 Promoter Regulated Gene Expression. Regulatable promoters have been employed in the method of the present invention. One exemplary regulatable promoter is the tomato E8 gene promoter. Expression of the E8 gene has been shown to be induced (i) at the onset of ripening, and (ii) by treatment of tomatoes with ethylene (Deikman, et al . , 1988; Lincoln, et al . ; Giovannoni, et al . ) . The sequence of the E8 promoter has been published (Deikman, et al . , 1988; Deikman, et al . , 1992) and the DNA sequence of the minus 2216 base pair region is presented in Figure 13. Using the sequence shown in Figure 13 primers were prepared for use in the polymerase chain reaction (PCR) to amplify the 1124 base pair promoter from tomato genomic DNA (Example 1) . The primers were designed with unique restriction sites at each end and were used to place the promoter in the proper orientation 5' of the SAM-K gene in pUC19 (Figure 3) . The 3' end of the promoter fragment had an Ncol site (CCATGG) placed such that the ATG start codon of the E8 gene product was used as the ATG in the Ncol site. This allowed precise placement of the entire E8 promoter directly in front of the SAM-K amino acid coding sequences with no intervening sequences (Example 1, Figure 12A) .

Two AdoMetase expressing vectors were constructed (Example 1) , the pAG-5321 (pGA-ESKN) vector (Figures 12A, 12B and Figure 5A) and the pGA-SESKN vector

(Figure 4 and Figure 5B) . The pAG-5321 vector contains a portion of the E8 promoter (Figure 4, lower E8 promoter) adjacent the AdoMetase coding sequences. A lambda EMBL-3 clone containing genomic tomato sequences that hybridize to the -1124 E8 region was isolated and used as the source for a region upstream of the -1124 E8 (lower E8) promoter. Restriction mapping analysis and subcloning allowed identification of an approximately 1200 bp Hindlll to Xbal fragment as the region immediately upstream of the original -1124 bp E8 promoter (Figure 4) . This region was added to the pAG- 5321 construct to yield pGA-SESKN, which contained the approximately -2254 bp E8 promoter fused to the AdoMetase gene (Figure 4, SE8) . Both of these vectors were transferred to tomato plants (Example 2) to generate transgenic plants expressing AdoMetase. A number of methods, in addition to Agrobacterium-based methods, may be employed to elicit transformation of the plant host, such as electroporation, microinjection, and microprojectile bombardment. These methods are well known in the art (Klein, et al . ; Miki, et a2. ; Bellini, et al . ) . Further, these methods provide the means to introduce selected DNA into plant genomes: such DNA may include a DNA cassette which consisting of the E8 gene promoter functionally adjacent AdoMetase coding sequences.

Several transgenic plants were assayed for their ability to synthesize AdoMetase mRNA using a sensitive RNAase protection assay (RPA) (Example 3) . Figures 6 and 7 show the results of an RPA using the fruit from two transgenic plants (ESKN, transformed with pAG-5321, and SESKN, transformed with pGA-SESKN) at different stages of fruit ripening. Other tissues from these plants including immature and mature leaves, flowers and stems were negative for the presence of AdoMetase

RNA. Although the expression of AdoMetase in ESKN transgenic plants was regulated to the post mature green fruit, it was repeatedly observed (as shown in Figures 6 and 7) that the expression of AdoMetase turned off in the fully ripe fruit. On the other hand, the SESKN transgenic fruit maintained AdoMetase mRNA expression in ripe fruit.

To determine whether the presence of AdoMetase enzyme activity correlated with the level of AdoMetase mRNA, an AdoMetase assay was performed using extracts from four fruit obtained at different stages from an ESKN transgenic plant (Example 5) . Figure 8 shows the level of AdoMetase activity in mature green, breaker, orange, and ripe fruit from a single pAG-5321 transgenic plant. These data demonstrate that

AdoMetase activity follows roughly the same expression pattern in ripening fruit as the AdoMetase mRNA levels. The data presented above suggest that inclusion of the upstream region of the native E8 promoter in a chimeric gene construct enhances long-lived expression of the chimeric gene in ripening transgenic tomatoes. Figure 6 shows the RPA results from pGA-SESKN line 22A- 1 and from pAG-5321 line 18. ESKN line 18 had one of the highest levels of AdoMetase expression of all the ESKN transgenic lines. Quantitative measurement of

AdoMetase mRNA is shown in Figure 7. The results show that the -2254 bp E8 promoter expression is maintained through the fully ripe stage of fruit development. This expression pattern is in sharp contrast to the - 1124 bp E8 promoter (ESKN) mRNA levels also shown in Figure 6.

Ethylene evolution measurements from fruit picked at breaker and analyzed daily are shown in Figure 9. The rate at which fruit from SESKN lines 22A and 35-1 produced lycopene was reduced as evidenced by the time required for orange fruit development. Furthermore, the total amount of ethylene produced from these tomatoes was reduced by approximately 80%. The expression of AdoMetase and a reduction in ethylene biosynthesis was strictly correlated in the 25 SESKN transgenic plants analyzed.

The SESKN tomatoes that synthesized less ethylene were assessed for their shelf life properties when stored at room temperature (22°C) (Example 5) . Three fruit each from SESKN lines 22A-1 and 35-1 were compared with untransformed normal tomatoes. Senescence was determined by visually observing contraction and wrinkles on the tomato skin. Firmness was not measured but was noted to be much greater in the transgenic lines. The results of these senescence assessments are shown in Figure 10. Even at 55 days post-breaker, the 22A-1 tomatoes remained firm and appeared to be suffering more from dehydration than from the softening-induced senescence of the normal tomatoes.

These results demonstrate the ability to provide tissue specific regulation to DNA sequences encoding a gene product, e.g., AdoMetase enzyme, in transgenic plants. In addition, the results obtained with the two different E8 promoters (lower E8 and SE8) suggest the use of these promoters for similar tissue specific expression of any desired gene product. Further, the two regions of the E8 promoter (lower E8 and upper E8, Figure 4) can be used as hybridization probes against libraries of DNA representative of the genomes of other plant species. Homologous sequences to the E8 promoter are then tested for tissue specific expression in the plant species from which they were isolated. Such promoters, as well as the E8 promoter itself, can be tested for regulatable expression in heterologous plant systems using the methods described herein. A reporter gene, such as GUS (?-glucuronidase) , can be used to test tissue specific regulatable expression from these promoters. Expression of GUS protein can be easily measured by fluorometric, spectrophotometric or histochemical assays (Jefferson, 1987) .

Variants of the E8 promoter may be isolated from different tomato cultivars or other plant species by standard recombinant manipulations such as primer specific amplification (Mullis; Mullis, et al . ) or oligonucleotide hybridization (Ausubel, et al . ; Sambrook, et al . ) .

VII. Advantages of the Invention

These results demonstrate the ability to provide tissue and stage specific regulation of gene expression in transgenic plants. A tissue or stage specific promoter is a region of DNA that regulates transcription of the immediately adjacent (downstream) gene to a specific plant tissue or developmental stage of the plant or plant tissue. Other gene products which may be useful to express using these promoters include genes encoding (i) flavor (e.g., thaumatin) or color modification (e.g., products that modify lycopene synthesis) , (ii) enzymes or other catalytic products (such as, ribozymes or catalytic antibodies) that modify plant cell processes, and (iii) gene products that affect ethylene production, such as antisense molecules, enzymes that degrade precursors of ethylene biosynthesis, catalytic products or cosuppression molecules. Further, it is useful to restrict expression of some genes to specific tissues, such as the fruit — for example, any gene that would be deleterious to the plant if it were expressed constitutively. Such genes would include genes which encoded degradative enzymes that deplete necessary metabolites. As can be seen from the results described above, derivatives of the E8 and E4 promoter regions can be used as on/off switches for the tissue and stage specific expression of genes whose expression is under their control.

The constructs and methods of the present invention are applicable to all higher plants including, but not limited to, the following:

Solanaceae. Lycopersicon (tomato) and Capsicum (peppers) ;

Cucurbitaceae. Cucurbita (squashes) , Cucumis (melons, cantaloupe) or Citrulluε (watermelon) ;

Rosaceae. Malus (apple) , Prunus (peaches, plums, nectarines) , Rubus (raspberry) , Fragaria (strawberry) , and Pyrus (pears) ;

Annonaceae. Annona (sweetsop, cherimoya) ; Musaceae. Muεa (banana) ; Lauraceae. Persea (avocado) ; Saxifragaceae. Ribes (currents) ; Ebenaceae. Diospyros (persimmon) ; Caricaceae. Carica (papaya) ; Anacardiaceae. Mangifera (mango) ; Myrtaceae. Psidium (guava) ; Actinidiaceae. Actinidia (kiwifruit) . Variants of the E8 and E4 promoter may be isolated from different tomato cultivars and from other plants by the methods described above. A reporter gene, such as GUS ( ?-glucuronidase) , can be used to test tissue specific regulatable expression from such promoters. Expression of GUS protein can be easily measured by fluorometric, spectrophotometric or histochemical assays (Jefferson, 1987) .

A further advantage of the present invention is the ability to produce fruit that initiate ripening, but the subsequent down regulation of ethylene production delays the overall time course of fruit ripening, i.e., the fruit are suspended or delayed in their ability to complete the ripening process.

VIII. Utility

A. The Vectors of the Present Invention.

The present invention provides vectors suitable for the transformation of plants. The vectors, chimeric genes and DNA constructs of the present invention are also useful for the expression of heterologous genes. Transgenic plants, and their fruit products, carrying the chimeric genes of the present invention, may be a useful source of recombinantly- expressed material.

In one embodiment, the chimeric genes of the present invention have two components: (i) a DNA sequence encoding a product that is effective to reduce ethylene biosynthesis in fruit from the plant, and (ii) a promoter whose expression is induced during fruit ripening or in response to ethylene.

The vectors of the present invention may be constructed to carry an expression cassette containing and insertion site for DNA coding sequences of interest. The transcription of such inserted DNA is then under the control of a suitable promoter (i.e., a promoter whose expression is induced during fruit ripening, in response to ethylene, or in response to a plant cytokine) . Exemplary of such promoters are promoters obtained from tomato E4 or E8 genes, or homologs thereof (e.g., raspberry E4) , and avocado cellulase gene or tomato polygalacturonase gene.

Such expression cassettes may have single or multiple transcription termination signals at the coding-3'-end of the DNA sequence being expressed. The expression cassette may also include, for example, DNA sequences encoding (i) a leader sequence (e.g., to allow secretion or vacuolar targeting) , and (ii) translation termination signals. Further, the vectors of the present invention may include selectable markers for use in plant cells (such as, the nptll kanamycin resistance gene) . The vectors may also include sequences that allow their selection and propagation in a secondary host, such as, sequences containing an origin of replication and a selectable marker. Typical secondary hosts include bacteria and yeast. In one embodiment, the secondary host is Escherichia coli , the origin of replication is a colEl- type, and the selectable marker is a gene encoding ampicillin resistance. Such sequences are well known in the art and are commercially available as well (e.g., Clontech, Palo Alto, CA; Stratagene, La Jolla, CA) .

The vectors of the present invention may also be modified to intermediate plant transformation plasmids that contain a region of homology to an Agrobacterium tumefacienε vector, a T-DNA border region from Agrobacterium tumefaciens , and chimeric genes or expression cassettes (described above) . Further, the vectors of the invention may comprise a disarmed plant tumor inducing plasmid of Agrobacterium tumefaciens . The vectors of the present invention are useful for stage and/or tissue specific expression of nucleic acid coding sequences in plant cells. For example, a selected peptide or polypeptide coding sequence can be inserted in an expression cassette of a vector of the present invention. The vector transformed into host cells, the host cells cultured under conditions to allow the expression of the protein coding sequences, and the expressed peptide or polypeptide isolated from the cells. Transformed progenitor cells can also be used to produce transgenic plants bearing fruit.

In one aspect of the invention, fruit produced by such transgenic plants has an initial burst of ethylene production, followed by a reduction in the level of ethylene synthesis by the fruit. The fruit then demonstrates a modified ripening phenotype.

The vectors, chimeric genes and DNA constructs of the present invention can be sold individually or in kits for use in plant cell transformation and the subsequent generation of transgenic plants.

B. Transgenic Plants and Fruit.

Experiments performed in support of the present invention demonstrate that plants carrying chimeric genes of the present invention (comprising, a DNA sequence encoding a product that is effective to reduce ethylene biosynthesis in fruit from the plant and a promoter whose expression is induced during fruit ripening or in response to ethylene) exhibit significantly lower levels of ethylene production following harvest. Examples of this result have been described herein, for example, using expression of the AdoMetase gene under the control of a E4 and E8 promoters. Due to the deleterious effects of ethylene biosynthesis on the handling and storage of commercially-important plants and plant products (such as fruits, vegetables and flowers) plants in which ethylene synthesis is reduced are of substantial value. Similarly, flowering plants containing heterologous genes effective to reduce ethylene biosynthesis will retain a fresh appearance longer than untransformed counterparts. Reduced ethylene biosynthesis in leafy vegetables, such as lettuce, would reduce leaf browning and lead to a longer shelf- life. The transgenic tomatoes described herein are illustrative of present invention. These transgenic tomatoes remain firm much longer after harvest than normal tomatoes, such transgenic tomatoes may be harvested at a later, vine-ripened, stage and still retain the transportability previously associated with, for example, green tomatoes.

Further, the expression constructs of the present invention allows an initial burst of ethylene synthesis to begin the ripening process. The promoters of the present invention can be manipulated to suspend further ripening or to increase the time-course of the ripening process (relative to wild-type fruit) . The constructs of the present invention demonstrate the first example of tissue-type or developmental stage-specific control over ethylene production.

C. Expression in Heterologous Plant Systems

Experiments performed in support of the present invention demonstrate the versatility of the chimeric gene constructs of the invention. The vector constructs of the present invention can be used for transformation and expression of heterologous sequences in transgenic plants independent of the original plant source for the promoter sequence. For example, the tomato E4-Adometase chimeric gene was introduced into raspberries. The transgenic raspberries were propagated under green house conditions. Proteins were prepared from the transgenic raspberry fruit, the proteins size-fractionated and transferred to nylon membranes. The bound proteins were then probed with a monoclonal antibody specifically immunoreactive with the Adometase protein. Results from these experiments demonstrate the expression of Adometase in the raspberry fruit. These data suggest that the tomato E4 promoter is useful for the promotion of gene expression in tomato and heterologous systems, i.e., plant cells other than tomato. Further, the expression mediated by the promoter appears to be tissue/developmental-stage specific even in heterologous plants. These findings support the usefulness of the vectors, chimeric genes and DNA constructs of the present invention for transformation of species of fruit-bearing plants, where such plants are different species than the plant source of the promoter sequences.

The following examples illustrate, but in no way are intended to limit the present invention.

Materials and Methods Tomato seed (Lycopersicon esculentum Mill. var. cerasi forme (Dunal) Alef. cv. Large Red Cherry) were obtained from Peto Seed, Inc. (Saticoy, CA) and were grown under standard greenhouse conditions. Harvested fruit were stored at room temperature (22°C) .

Standard recombinant DNA techniques were employed in all constructions (Adams, et al . ; Ausubel, et al . , Sambrook, et al . ) .

EXAMPLE 1 Cloning of the AdoMetase Gene A. Isolation of the AdoMetase Gene. The AdoMetase gene was identified on an Alul- Haelll restriction fragment from purified T3 DNA

(Hughes, et al . , 1987a) . Bacteriophage T3 is available under ATCC No. 11303-B3 (American Type Culture Collection, 12301 Parklawn Dr., Rockville MD 20852). The DNA fragment was first cloned into the bacteriophage M13 MP8 vector (Pharmacia LKB Biotechnology, Inc., Pistcataway, NJ) . A Maelll to BamHI fragment was subcloned into the pUC19 plasmid vector (Pharmacia) to produce pUC19-AdoMetase (pAGHO or pUC19-SAMase; Figure 2) . This vector was transformed into E . coli and used as a source of DNA for further construction experiments and for DNA sequence determination.

B. Modification of the Amino-Terminal Sequence of the Cloned AdoMetase Gene.

The cloned AdoMetase gene was further engineered to contain a consensus eukaryotic translation initiation site (Kozak; Lutcke, et al . ) by altering the nucleotide sequence surrounding the SAMase ATG start- codon using a synthetic double-stranded oligonucleotide.

The plasmid pUC19-AdoMetase (pAGHO) was digested with Xmal and BamHI and the 1.9 kb and 1.3 kb fragments were purified by electro-elution after agarose gel electrophoresis. A double stranded synthetic oligonucleotide linker having the sequence indicated in Figure 3 was ligated to the 1.9 kb fragment and this ligated DNA subjected to Xmal digestion to remove excess linkers. The linkered 1.9 kg fragment was then re-purified by electrophoresis on low melting temperature agarose and ligated to the 1.3 kb fragment to form the plasmid pUC19-SAM-K (pAG-lll) . The altered gene region was subjected to DNA sequence analysis. The gene sequence is given in Figure 11. This gene was designated SAM-K and used to construct the following plant expression vectors. This plasmid DNA can also be used to directly transform the plant host via electroporation, microinjection, or microprojectile bombardment. C. Vector Constructions using the Tomato E8 Promoter.

Two different forms of the E8 promoter were used to construct SAM-K-containing vectors. The first (- 1124 bp) was isolated from tomato (Lycopersicon esculentum var. cerasiform) DNA using polymerase chain reaction (PCR) (Mullis; Mullis, et al . ; Perkin-Elmer

Cetus, Norwalk CT) . The primers used in the PCR reaction were based on the sequence described by Deikman, et al . (1988) . The sequences of the oligonucleotide primers are given in Figure 3. The oligonucleotides were designed to incorporate restriction endonuclease sites (Xbal and Ncol) at the 5' and 3' ends, respectively, of the amplified E8- promoter fragment. These restriction endonuclease cleavage sites facilitated subcloning into the pUC19- SAM-K vector (see Figure 2) : an Ncol site is present near the ATG start codon region in the synthetic oligonucleotide. Figure 12A outlines the generation of the vector pAG-5321 (pGA-ESKN) starting from vector pNCN (Pharmacia, Inc., Piscataway, NJ) and pUC-SAM-K (described above) . The sequence of the E8 promoter (the lower E8 promoter) is similar to the sequence presented as bases 1189 to 2214 in Figure 13.

Figure 12B outlines one approach to the generation of Agrobacterium vectors for use in the present invention. However, the E8/AdoMetase cassette, present in, for example, pAG-5321, can be incorporated in a number of vectors useful for plant transformation.

Agrobacterium binary vectors were developed from pGA482 (An, et al . , 1985) , a pBIN19 derivative (Clontech Laboratories) containing the neomycin phosphotransferase II gene fused to the nopaline synthesis gene promoter (An, et al . , 1988) . The resulting vector, designated pAG-5321 is shown in Figure 5A.

The second E8 promoter (-2254 bp) was isolated from a lambda EMBL-3 clone that contained the entire E8 gene. The E8 gene clone was selected from a tomato (Lycopersicon esculentum var. VFN8) genomic library obtained from Clontech Laboratories (Palo Alto, CA) using the PCR-derived E8 promoter fragment (described above) as a hybridization probe in plaque-lift filter hybridizations. The lambda clone carrying the E8 gene was identified by a positive hybridization signal. The E8-bearing phage was plaque purified and the lambda phage DNA isolated.

The lambda E8 genomic clone was used as a source of the Hindlll to Xbal fragment that is the approximately -2254 to -1124 bp upstream region of the E8 promoter. This fragment was inserted 5' of the approximately -1124 bp E8 promoter in pAG-5321 at the Hindi I I and Xbal sites (Figure 4) . The resulting plasmid was named pGA-SESKN. Figure 13 shows the nucleotide sequence of the -2216 bp region from one cultivar (Deikman, et al . , 1988, 1992) . The Hindi I I to Xbal fragment (used for construction of the approximately -2254 promoter) contains additional sequences 5' to the end of this -2216 bp sequence. Figure 4 shows the relationship of the two portions of the E8 promoter that are present in pGA- SESKN.

Standard recombinant DNA techniques were employed in all constructions (Adams, et al . ; Ausubel, et al . ) . Another lambda vector, pGEM7Zf(+)SAM-K, was constructed by cloning the BamHI to Kpnl AdoMetase fragment from pUC19-SAM-K into the same sites of pGEM7Xf(+) (Promega, Inc., Madison, WI) . Other plant cloning vectors, such as pBI121 (Clontech Laboratories, Inc. , Palo Alto, CA) , can also be used to practice the present invention. The plant promoter upstream of the AdoMetase gene sequence can be varied to obtain tissue specific expression, temperature dependent expression, or light dependent expression in the transgenic plants. Another useful plant promoter, in addition to the E8 promoter described above, is the constitutive Cauliflower Mosaic Virus (CaMV) promoter (Pharmacia) .

EXAMPLE 2 Plant Transformation The pAG-5321 and pGA-SESKN AdoMetase plasmids were separately introduced into Agrobacterium using a direct transformation method.

Agrobacterium tumefaciens strain EHA101 (Hood, et al . ) , a disarmed derivative of Agrobacterium tumefaciens strain C58, was used to introduce coding sequences into plants. This strain contains a T-DNA- less Ti plasmid. The pAG-5321 and pGA-SESKN AdoMetase plasmids were transferred into EHA101 using electroporation essentially as described by Nagel, et al . Briefly, an Agrobacterium tumefaciens culture was grown to mid-log phase (OD 600 0.5 to 1.0) in YEP media (10 g yeast extract, 10 g peptone, and 5 g NaCI per liter) . After chilling on ice, 50 is of these cells were pelleted, resuspended in 1 ml of ice cold 20 mM CaCl₂ and split into 1 ml aliquots. Typically, one μg of plasmid DNA was added to an aliquots and incubated on ice for 30 minutes. The aliquot was then frozen in liquid nitrogen and thawed at 37°C for 5 minutes. One ml of YEP media was added and incubated at 28°C for 2 hours. The cells were pelleted, r.esuspended in 50 μl of YEP, and plated on YEP agar plates containing 20 μg/ml kanamycin. Kanamycin-resistant transformed colonies appear within 2 days.

Tomato cotyledon tissue explants were excised from both the tip and base of the cotyledon. Cotyledon explants were pre-conditioned for 2 days on tobacco feeder plates (Fillatti, et al . ) . The pre-conditioned explants were inoculated with EHA101 containing the pAG-5321 or pGA-SESKN AdoMetase plasmid of interest and finally placed in a 10 ml overnight culture of

EHA101/ [pAG-5321 or pGA-SESKN] for 5 minutes. The explants were then co-cultivated with the EHA101 strains for 2 days on tobacco feeder plates as described by Fillatti, et al . The explants were grown in tissue culture media (Fillatti, et al . ) containing 2Z media, MS salts, Nitsch and Nitsch vitamins, 3% sucrose, 2 mg/1 seatin, 500 mg/1 carbenicillin, 100 mg/1 kanamycin and 0.7% agar. The explants were grown in tissue culture for 8 to 10 weeks. The carbenicillin treatments were kept in place for 2 to 3 months in all media. The explants and plants were kept on carbenicillin until they were potted in soil as a counter-selection to rid the plants of viable Agrobacterium tumefaciens cells.

EXAMPLE 3

RNAase Protection Assays for the Detection of SAMase mRNA

Tomato fruits at various stages of development from transgenic plants and wild-type plants were used as mRNA sources. mRNA was extracted from tomato cells and purified using the "QUICK PREP RNA" kit from Pharmacia, Inc. RNAse Protection Assays (RPA) were performed following the manufacturer's instructions using an "RPAII" kit from Ambion, Inc. (Hialeah, FL) . This method has been previously described by Lee, et al . pGEM7Zf(+) SAM-K was used to generate ³²P-UTP- labeled RNA probe using bacteriophage T7 RNA polymerase as contained in the "RIBOPROBE IT T7 RNA POLYMERASE

SYSTEM" from Promega, Inc. The radiolabeled probe was purified on a preparative polyacrylamide gel and used for up to one week.

One microgram of isolated mRNA was hybridized to approximately 10,000 CPM of the RNA probe and further processed as per the instructions in the "RPA II" kit. Briefly, one microgram of the purified mRNA was mixed with 10,000 CPM of the RNA probe in a total volume of 15 μl . 20 μl of a hybridization buffer that allows hybridization of complementary sequences (Ausubel, et al . ; Maniatis, et ai . ; Sambrook, et al . ) is then added. The hybridization solution is provided in the "RPAII" kit from Ambion. The solution was heated to 90°C for 3-4 minutes to denature all the RNA and incubated at 45°C overnight to allow hybridization of complementary sequences. The solution was cooled to 37°C and RNase (provided in the Ambion kit) was added which degrades all un-hybridized probe.

Protected probe was resolved on a denaturing polyacrylamide gel, dried, and exposed to film for up to 16 hours. Quantitative analysis of the RPA signals was accomplished by excising each band from the gel, dissolving the band in a liquid fluor, and determining the radioactivity present in the sample using liquid scintillation counting. A standard curve was generated using various amounts of unlabeled RNA synthesized from a AdoMetase fragment cloned into pGEM5Z(+) in the sense orientation. The linear range of the assay was dependent on the amount of input P-labeled RNA probe in the RNAase protection assay but typically ranged from 10 pg to 1 ng of mRNA.

EXAMPLE 4 Ethylene Measurements

The assay for tomato ethylene evolution is performed over a 0.5 to 1.0 hour period by sealing glass jars containing individual fruit and sampling 2 ml aliquots for gas chromatographic analysis. A Hewlett Packard 5890 (Palo Alto, CA) gas chromatograph with a flame ionization detector and a 6ft Porapak N column was used for ethylene measurements (Adams, et al . ) . This system combined with an HP Vectra computer and the current version of "CHEMSTATION" (Hewlett Packard) allows measurement of ethylene concentrations as low as 0.2 nl of ethylene in a 2 ml sample (0.1 ppm) . After measurement of the ethylene in the headspace, the values are converted to nl of ethylene per gram of tissue per hour.

EXAMPLE 5

Characterization of Transgenic Tomatoes

A. Promoter Effect on SAMase mRNA Levels in Ripening Transgenic Fruit. Transgenic fruit were selected from two transgenic plants, ESKN #18 and SESKN #22A, at three stages of ripening, breaker (Br) , Orange (Or) and Ripe (Ri) . Transgenic plant ESKN #18 contained the lower E8 promoter (Figure 4) adjacent the Sam-K AdoMetase gene. Transgenic plant SESKN #22A contained the entire SE8 promoter (Figure 4) adjacent the Sam-K AdoMetase gene. The AdoMetase mRNA level in ripening transgenic fruit was determined as described in Example 3.

The products of the RNA protection assay were resolved on polyacrylamide gels and exposed to X-ray film. A representative autoradiogram of the RNA protection assay is presented in Figure 6. As can be seen in the figure, AdoMetase mRNA was present in both transgenic plants at the breaker stage of fruit ripening. However, the levels of AdoMetase mRNA drop in the ESKN transgenic plant, relative to the SESKN transgenic plant, at the orange and ripe stages of fruit ripening.

The level of AdoMetase mRNA was quantitated as described in Example 3 by liquid scintillation counting and determination of mRNA concentrations relative to a standard curve. Figure 7 presents the results of this analysis. The results are consistent with those shown in Figure 6. AdoMetase mRNA was present in both transgenic plants at the breaker stage of fruit ripening with the concentrations lower in ESKN #18. At the orange and ripe stages of fruit ripening the levels of AdoMetase mRNA drop in the ESKN transgenic plant, relative to the level at breaker stage and the levels in the fruit from the SESKN transgenic plant. The

AdoMetase mRNA levels stay relatively constant in the SESKN transgenic plant.

B. Relative Levels of SAMase Activity in Ripening Transgenic Tomatoes.

To determine whether the presence of AdoMetase enzyme activity correlated with the level of AdoMetase mRNA, a 14C-SAM-based AdoMetase assay was performed using extracts from four different fruit stages from a single pAG-5321 transgenic plant (ESKN) .

Plant tissues to be assayed for AdoMetase enzyme activity were frozen and ground to a powder in liquid nitrogen. The ground tissue was then suspended •in 1.5 volumes of 200 mM Tris-HCl (pH 7.5) , 10 mM DTT, and 10 mM EDTA. The suspension was vortexed vigorously then subjected to centrifugation at 40,000 x g at 4°C for 20 minutes. The following was added to 50 μl of extract: 5 μl of ¹⁴C-SAM (DuPont-New England Nuclear, NEC-363) at 20 μCi/ml and a specific activity of 58.0 mCi/mmol. The reaction was incubated at 37°C for 1 hour then 40 μl of the reaction was spotted on a cellulose think layer chromatography (TLC) plate (J.T. Baker, Inc., Phillipsburg, N.J., Baker-Flex Cellulose F) and resolved for 3 hours in 70:70:20:40, butanol:acetone:acetic acid:water. The MTA and MTR spots were identified using autoradiography, excised, and counted using liquid scintillation.

Figure 8 shows the level of AdoMetase activity in mature green, breaker, orange, and ripe fruit. The level of AdoMetase activity is defined as the percent conversion of SAM (S-adenosylmethionine) to MTA (5'- Methylthioadenosine) and MTR (5'-Methylthioribose) . The decreasing level of AdoMetase activity from breaker to ripe fruit in the ESKN transgenic plant is consistent with the AdoMetase mRNA levels shown in

Figure 7.

Untransformed tomato fruit extracts do not degrade

SAM to MTA or MTR at any stage of ripening when used in this assay.

C. Ethylene Production in Ripening Transgenic Fruit.

Ethylene produced from transgenic tomatoes carrying the AdoMetase gene under the regulation of the SE8 promoter (Figure 4) was determined as described in

Example 4. Greenhouse grown tomatoes from 4 transgenic lines were tested. The results of the analysis are presented in Figures 9A to 9D. Each of the four graphs shown in Figure 9 represent the comparison of fruit from one pGA-SESKN transgenic line (Es 19-2, LS 4-2, ES 35-1 and ES 22A-1) with the fruit from untransformed controls. The control values (open squares) are the same in each of the four graphs and represent the average of six fruit from two different plants. The values from each transgenic line (closed symbols) are the average of ethylene determinations for three fruit. Error bars represent one standard deviation of the data.

The data represent a time period of ten days after the breaker stage of fruit ripening (post-breaker) . These data demonstrate a reduction in the amount of ethylene production in transgenic tomatoes versus normal fruit over the ten day period.

D. Post-Harvest Shelf-life of SESKN Tomatoes. Tomatoes from the SESKN transgenic plants that synthesized less ethylene were assessed for their shelf life properties when stored at 22°C. Three fruit from each from SESKN lines 35-1, 22A-1 and LS4-2 were compared with tomatoes from two untransformed, normal plants (M16 and M15) . Senescence was determined each day by visual examination of the fruit for the occurrence of contraction and wrinkles on the tomato skin. The results of these senescence assessments are shown in Figure 10.

As can be seen from the results in the figure, the bar graph shows the time for the fruit to achieve each stage: all fruit were picked at the breaker stage. For instance, line 35-1 took 18 days to ripen (Ripe stage) but then senescence developed at day 27. Line 22A-1 took 7 days to turn orange, 13 days to turn red, then 52 days to senescence. Even at 55 days post- breaker, the 22A-1 tomatoes remained firm and appeared to be suffering more from dehydration than from the softening-induced senescence of the normal tomatoes. Firmness was not measured for the tomatoes from the five plants described above, however, the firmness was noted to be much greater in the fruit from the transgenic lines.

EXAMPLE 6

Southern Blot Analysis of E4 Homologues in Several

Species of Plants

A Southern blot analysis was conducted to determine if sequences homologous to the tomato E4 gene were present in other plant species. The blot consisted of Hindlll digests of six genomic plant DNAs: tomato, raspberry, strawberry, melon, carnation and cauliflower, along with size standards. This blot was hybridized with a probe following standard methods (Maniatis, et al . ) . The probe was a -740 bp polymerase chain reaction (PCR; Mullis, Mullis., et al . ) product amplified from genomic tomato DNA using PCR primers flanking the coding sequence of the E4 gene. The probe was labeled by incorporating ³²P-labeled nucleotides into the PCR reaction.

The primers were designed according to Cordes, et al . (1989). The 5' primer sequence, corresponding to the region between nucleotides 1439 and 1452 of the E4 gene (SEQ ID NO:8) , is represented as SEQ ID NO: 6 (ACG CAT GGA GGG TAA CAA) . Positions 5-7 of this primer correspond to the ATG start codon of the E4 gene. The 3' primer sequence, corresponding to the region between nucleotides 2160 and 2177 of the E4 gene (SEQ ID NO:8), is represented as SEQ ID NO:7 (GAA GCA AGA CAG CAA ATG) .

An autoradiograph of the blot is shown in Figure 14. Several bands are apparent in each lane, with the lane corresponding to tomato DNA showing the strongest signal.

EXAMPLE 7 Isolation of DNA Fragments Homologous to Tomato E4 from a Raspberry Genomic Library

A. Screening of the Library.

A raspberry genomic library in lambda GEM-ll was- obtained from Novagen (Madison, WI) and screened by standard methods with the tomato E4 gene probe described above. Three lambda clones which hybridized to the probe were identified. The clones were purified by 3 rounds of plaque purification. One of the clones was selected for further analysis.

B. Analysis of a Positive Clone.

The clone was digested with several enzymes (Apa I, Bam HI, Eco RI, Hind III, Nco I, Sac I, and Sal I) , run on a gel, and transferred to a "SUREBLOT" nylon membrane (Oncor, Gaithersburg, MD) . The blot was hybridized overnight at 42°C with the tomato E4 probe in "HYBRISOL I" hybridization cocktail (Oncor, Gaithersburg, MD) . The final (most stringent) wash was 0.1% SSC, 0.1% SDS for 30 minutes at room temperature

(22°C) .

A 1.6kb Sac I fragment which hybridized to the probe was subcloned into pGEMSZf (+) (Promega, Madison, WI) and further characterized. A 225 bp region in that fragment was found to be highly homologous to the tomato E4 gene at both the DNA level (74%) and the amino acid level (80%) . The sequence of this region (SEQ ID NO:12) was compared to the sequence of a portion of the tomato E4 gene (SEQ ID NO:8). Additional raspberry E4 gene sequences were obtained by further hybridization screening of raspberry genomic library clones. The sequence of a genomic copy of a raspberry E4 gene is presented in Figure 15 (nucleotide sequence: SEQ ID NO:25; polypeptide sequence: SEQ ID NO:26).

EXAMPLE 8 Cloning of the AdoMetase Gene

A. Isolation of the AdoMetase Gene.

The AdoMetase (SAMase) gene was identified on an Alul-Haelll restriction fragment from purified T3 DNA

(Hughes, et al . , 1987a) . Bacteriophage T3 is available under ATCC No. 11303-B3 (American Type Culture Collection, 12301 Parklawn Dr., Rockville MD 20852) . The DNA fragment was first cloned into the bacteriophage M13 MP8 vector (Pharmacia LKB

Biotechnology, Inc. , Pistcataway, NJ) . A Maelll to BamHI fragment was subcloned into the pUC19 plasmid vector (Pharmacia) to produce pAG-110 (pUC19-SAMase, Figure 2) . This vector was transformed into E . coli and used as a source of DNA for further construction experiments, detailed below.

B. Modification of the Amino-Terminal Sequence of the Cloned AdoMetase Gene. The cloned AdoMetase gene was further engineered to contain a consensus eukaryotic translation initiation site (Kozak; Lutcke, et al . ) by altering the nucleotide sequence surrounding the AdoMetase ATG start-codon using a synthetic double-stranded oligonucleotide.

Plasmid pAG-110 was digested with Xmnl and BamHI and the 1.9 kb and 1.3 kb fragments were purified by electro-elution after agarose gel electrophoresis. A double stranded synthetic oligonucleotide linker formed by annealing oligonucleotides represented by SEQ ID N0:1 and SEQ ID NO:3 (Figure 2) was ligated to the 1.9 kb fragment. This ligated DNA was subjected to Xmnl digestion to remove excess linkers.

The linkered 1.9 kb fragment was then re-purified by electrophoresis on low melting temperature agarose and ligated to the 1.3 kb fragment to form the plasmid pAG-lll. The altered gene region was sequenced to confirm its identity. pAG-lll was used in subsequent recombinant DNA manipulations, including the construction of plant expression vectors, detailed below. This plasmid DNA can also be used to directly transform the plant host via electroporation, microinjection, or microprojectile bombardment.

C. Vector Constructions using the Tomato E4 Promoter.

1. PAG-110, PAG-lll, pAG-117

A 1.18kb E4 promoter was isolated from tomato

(Lycopersicon esculentum var. cerasiform) DNA using the polymerase chain reaction (PCR; Mullis; Mullis, et al . ; Perkin-Elmer Cetus, Norwalk CT) . The primers used in the PCR reaction were based on the sequence described by Cordeε, et al . The sequences of the 5' and 3' oligonucleotide primers, shown in Figure 16, are represented as SEQ ID NO:4 and SEQ ID NO: 5, respectively. The oligonucleotides were designed to incorporate restriction endonuclease sites (Hindlll and -VcoT) at the 5' and 3' ends, respectively, of the amplified E4-promoter fragment. These restriction endonuclease cleavage sites were used to subclone the E4-promoter fragment (Figure 17A) into the pAG-lll vector (Figure 17B) , which contains an NcoJ site at the ATG start codon in the region modified by the synthetic oligonucleotide (see Figure 2) . The resulting vector, containing an E4:SAMase chimeric construct in the region between the Hindlll /Kpnl sites, was termed pAG- 117.

2. PAG-5321 Agrobacterium binary vectors were developed from pGA482 (An, et al . , 1985) , a pBIN19 derivative (Clontech Laboratories) containing the neomycin phosphotransferase II gene (providing kanamycin resiεtance) fused to the nopaline synthesis (NOS) gene promoter (An, et al . , 1988) .

Figure 12B outlines the generation of vector pAG- 5321 starting from vectors pGA482 and pAG-114. Figure 12A outlines the generation of the vector pAG-114 from vectors pAG-lll (described above) and pNCN (Pharmacia, Inc., Piscataway, NJ) .

3. PAG-5520

The E4:SAMase chimera was excised from pAG-117 with a Hindlll /Kpnl digest. The resulting 1.7 kb fragment was purified as above, and cloned upstream of the nopaline synthase polyA addition site in pAG-5321, resulting in pAG-5520 (Figure 17D) . The identity of the Hindlll /Kpnl insert waε confirmed by DNA εequence analysis. This vector was used to generate the transgenic plants described herein.

Figures 17A-D and 12B-B outline one approach to the generation of Agrobacterium vectors for use in the present invention. However, the E4/SAMase cassette, present in, for example, pAG-117, can be incorporated in a number of vectors useful for plant transformation, such as pBI121 (Clontech Laboratories, Inc., Palo Alto, CA) . 4. PAG-924

Vector pAG-924 was constructed by cloning the BamHI /Kpnl SAMase fragment from pAG-lll into the same sites of pGEM7Zf(+) (Promega, Inc., Madison, WI) . This plasmid was used to make the RNA probe for RNase protection assays, described in Example 8.

5. Other Constructs

DNA constructs may be made using genes other than the AdoMetase gene under the control of an E4 promoter, for example, other genes effective to reduce ethylene biosynthesis. Preferably, the E4 promoter is isolated from the εame species of plant into which the construct is being introduced. For example, a tomato E4 promoter may be used to direct the expression of a heterologous gene, such as AdoMetase, in tomatoes, while a raspberry E4 promoter may be used to direct the expression of a heterologous gene in raspberries.

EXAMPLE 9

Plant Transformation Agrobacterium tumefaciens strain EHA101 (Hood, et al . ) , a disarmed derivative of Agrobacterium tumefaciens strain C58, was used to introduce coding sequences into plants. This strain contains a T-DNA- lesε Ti plasmid. The pAG-5520 construct was transferred into EHA101 using electroporation esεentially aε described by Nagel, et al . Briefly, an Agrobacterium tumefaciens culture was grown to mid-log phase (OD 600 0.5 to 1.0) in MG/L media (5 gm tryptone, 2.5 g yeast extract, 5 gm NaCI, 5 gm mannitol, 1.17 gm sodium glutamate, 0.25 gm K₂HP0₄, 0.1 g MgS0₄, 2 μg biotin per liter, pH adjusted to 7.2 with NaOH) . After chilling on ice 250 ml of the culture were pelleted, resuεpended in sterile, chilled 1 mM Hepes/KOH pH 7.0, pelleted and resuεpended as before, pelleted again, resuspended in sterile, chilled 10% glycerol, pelleted again, resuspended in 500 μl sterile, chilled 10% glycerol and split into 80 μl aliquots which were frozen on dry ice/ethanol and stored at -80°C.

Typically, 0.1-1 μg of plasmid DNA was added to a 40 μl aliquot of cells and incubated on ice 30-60 secondε. The mix waε then transferred to a 0.1 cm gap electroporation cuvette (Invitrogen) and pulsed at 1.25 kV (BioRad Gene Pulser at 25 μF, BioRad Pulse controller at 200 Ω) . One ml of MG/L media was added quickly after the pulse. The mixture was transferred to a microfuge tube and allowed to incubate at 28°C for

1 hour. The cells were diluted 1:100, and 10 and 100 μl were plated on two MG/L plates containing 20 μg/ml kanamycin, respectively. Kanamycin-resistant transformed colonies appeared within 2 days.

Seven to eight day-old tomato cotyledon tissue explants were excised from both the tip and base of the cotyledon. Cotyledon explants were pre-conditioned for

2 days on tobacco feeder plates (Fillatti, et al . ) . The pre-conditioned explants were inoculated with EHA101 containing the pAG-5520 plaεmid and placed in a 10 ml overnight culture of EHA101/pAG-5520 for 5 minuteε. The explantε were then co-cultivated with the EHA101 strain for 2 days on tobacco feeder plates as described by Fillatti, et al .

The explants were grown in tissue culture media (Fillatti, et al . ) containing 2Z media, MS salts, Nitsch and Nitsch vitamins, 3% sucrose, 2 mg/1 seatin, 500 mg/1 carbenicillin, 100 mg/1 kanamycin and 0.7% agar for 8 to 10 weeks. Carbenicillin was used for 2 to 3 months (until the plants were potted in soil) in all media as a counter-selection to rid the plantε of viable Agrobacterium tumefaciens cells. EXAMPLE 10

RNAase Protection Assays for the Detection of AdoMetase mRNA Tomato fruits at various stages of development from transgenic plants and wild-type plants were used as mRNA sources. mRNA was extracted from tomato cells and purified using the "QUICK PREP RNA" kit from Pharmacia, Inc (Piscataway, NJ) . Alternatively, total RNA was isolated using a LiCl precipitation procedure. Tissues were frozen in liquid nitrogen and ground to a fine powder. 550 μl phenol/buffer (1:1, Tris-saturated phenol, pH 6.9 : Extraction buffer (100 mM LiCl, 100 M Tris-HCl pH 8.0, 10 mM EDTA, 1% SDS)) at 80°C was added for every 100 mg of the powder. The mixture was vortexed for 60 seconds, 250 μl chloroform was added, the mixture vortexed for another 30 seconds, and spun to separate the phases. The aqueous phase was removed to a new tube, an equal volume of 4 M LiCl was added and mixed, and the sample waε placed at -20°C for 2 to 24 hourε. The RNA waε pelleted, washed and resuεpended in water.

RNAεe Protection Aεεayε (RPA) were performed following the manufacturer's instructions using an "RPAII" kit from Ambion, Inc. (Hialeah, FL) , as previously described by Lee, et al .

Plasmid pAG-924 was used to generate ³P-UTP- labeled RNA probe using bacteriophage T7 RNA polymerase as contained in the "RIBOPROBE II T7 RNA POLYMERASE SYSTEM" from Pro ega, Inc. The radiolabeled probe waε purified on a preparative polyacrylamide gel and uεed for up to one week.

One microgram of isolated mRNA was hybridized to approximately 10,000 CPM of the RNA probe and further processed as per the instructions in the "RPA II" kit. Briefly, between 0.7 and 1.0 nanogram of the purified mRNA waε mixed with 10,000 CPM of the ³²P-RNA probe in a total volume of 15 μl. 20 μl of a hybridization buffer that allows hybridization of complementary sequences (Ausubel, et al . ; Maniatis, et al . ; Sambrook, et al . ) is then added. The hybridization solution is provided in the "RPAII" kit from Ambion (Hialeah, FL) . The solution was heated to 90°C for 3-4 minutes to denature all the RNA and incubated at 45°C overnight to allow hybridization of complementary sequences. The solution was cooled to 37°C and RNase (provided in the Ambion kit) , which degrades all un-hybridized probe, was added.

Protected probe was resolved on a denaturing polyacrylamide gel, dried, and exposed to film for up to 3 hours. Quantitative analysiε of the RPA signals was accomplished by excising each band from the gel, dissolving the band in a liquid fluor, and determining the radioactivity present in the sample using liquid scintillation counting. A standard curve was generated using various amounts of unlabeled RNA synthesized from a AdoMetase fragment cloned into pGEM5Z(+) in the senεe orientation. The linear range of the aεεay waε dependent on the amount of input ³²P-labeled RNA probe in the RNAaεe protection aεεay but typically ranged from 10 pg to 1 ng of mRNA.

EXAMPLE 11 Ethylene Measurements A. Leaf disks Measurement of ethylene from leaf discs was performed by excising five one-centimeter leaf discε from mature tomato leaves and placing them in a 25 ml Erlenmeyer flask on top of filter paper saturated with Murishige and Skoog (MS) medium or MS medium supplemented with 10 μM of the auxin naphthalene acetic acid (NAA) .

B. Fruit The assay for tomato ethylene evolution was performed by sealing glass jars containing individual fruit for a 0.5 to 1.0 hour period and sampling 2ml aliquots for gas chromatographic analysis.

B. Ethylene measurements

Ethylene evolution was measured by gas chromatography/flame ionization after 20 hours and recorded as nanoliters of ethylene/gram fresh weight/hour. A Hewlett Packard 5890 (Palo Alto, CA) gas chromatograph with a flame ionization detector and a 6ft Porapak N column was used for all ethylene meaεurementε (Adamε, et al . , Ward, et al . ) . Thiε εystem combined with an HP Vectra computer and the current version of "CHEMSTATION" (Hewlett Packard) allows measurement of ethylene concentrations as low as

0.2 nl of ethylene in a 2ml sample (O.lppm) .

Following measurement of the ethylene in the headspace, the values were converted to nanoliters of ethylene per gram of tisεue per hour.

EXAMPLE 12 Weεtern Blot Analyεiε Frozen tomato tissueε were ground in liquid nitrogen, extracted directly into Lammeli sample buffer (50mM Tris,pH 6.8, 1% SDS, 5% betamecaptoethaon, 10% glycerol, and .005% bromophenol blue), heated to 95°C for 5 minutes and centrifuged to remove debris. Total soluble protein in the supernatants was measured using the Coomassie Plus protein assay (Pierce, Rockford, IL) . Eight- icrograms of soluble protein from each sample, or known quantities of purified AdoMetase (positive control) were resolved on a polyacrylamide gel and electrophoretically transferred to Immobilon-P membrane using standard procedures. The blot was incubated with 2μg/ml of the SAM10-9A3.1.3 monoclonal antibody to SAMase (Goding) in PBS-Tween (phosphate- buffered saline, 0.05% Tween 20), 1% bovine εerum albumin (BSA) buffer for 60 inuteε at 25°C. The blot waε then waεhed 4 times in PBS-Tween buffer and incubated for 60 minutes with a goat anti ouse HRP- conjugate εuεpended in PBS-Tween, 1% BSA buffer (Kirkegaard and Perry Laboratorieε, Inc., Gaitherεburg, MD) . Bound antibody waε detected using the Renaissance chemiluminescence reagent (DuPont NEN, Boston, MA) according to the manufacturer's instructionε.

EXAMPLE 13 Characterization of Transgenic Tomato Plants

A. Promoter Effect on AdoMetase mRNA Levels in Wounded Leaves.

Fresh and wounded leaves from six independent transgenic plants, E4-1, E4-2, E4-3, E4-4, E4-5 and E4- 6, were aεεayed for AdoMetase mRNA levels. All transgenic lines contained the E4 promoter adjacent the AdoMetase gene.

Single, freshly detached leaves were wounded by cutting 6-7 times with a dull knife. Two hours after wounding the total RNA was extracted, reacted and analyzed as described in Example 10. Fresh leaves were dropped directly into liquid nitrogen immediately after picking to halt RNAse activity, and procesεed aε above.

The products of the RNA protection asεay were resolved on polyacrylamide gels and exposed to X-ray film. A representative autoradiogram of the RNA protection asεay is presented in Figure 18A. As can be seen in the figure, expression of SAMase is silent in normal tomato plant leaves (fresh and wounded) and in fresh leaves isolated from transgenic plants. Expression of AdoMetase RNA is clearly evident, however, in wounded leaves from four of the six transgenic lines.

The level of AdoMetase mRNA was quantitated as described in Example 10 by liquid scintillation counting. Figure 18B presents the results of this analysiε, εhown aε a ratio of wound inducability, for four of the lineε εhown in Figure 18A (E4-2, E4-4, E4-5 and E4-6) , along with four other lineε (E4-8, E4-10, E4-12 and E4-13) . The reεults are consiεtent with those shown in Figure 18A, and indicate that a transgene driven by an E4 promoter can be activated in plant tissues by the wounding of those tissueε, in other words, that the E4 promoter is wound-inducible.

B. Promoter Effect on Ethylene Production in Wounded Leaves.

A leaf disc ethylene production assay was conducted, as detailed in Example 11, to measure the impact of wound-induced AdoMetase expreεεion on wound- induced ethylene εyntheεis expected from these tissues. Leaves from a normal (M) and four transgenic (E4-2, E4- 4, E4-5 and E4-12) tomato plants were cut with a cork bore into one cm discε that were meaεured for their ability to release ethylene.

Table 1 summarizeε ethylene εyntheεiε in leaveε from control and four tranεgenic lineε 4.5 and 19 hours after wounding. Leaves from two of the pAG-5520 tranεgenic lineε were εignificantly reduced in their ability to produce ethylene. Lineε E4-4 and E4-5 produced 69.6% and 84.8%, respectively, of the ethylene produced by the control plants during the first 4.5 hourε. The ethylene reduction in thoεe two lineε were greater from 4.5 to 19 hours during which they produced 54.4% and 45.6%, respectively, of the controls. The data in this table are also presented in Figure 19 in graphical form.

TABLE 1

WOUND-INDUCED ETHYLENE SYNTHESIS IN PAG-5520 TRANSGENIC PLANTS

Plant 4.5 hr. 4.5 to 19 hr. ID (nl/g/hr) (nl/g/hr)

Control 17.1 ± 2.7 5.7 ± 1.6

E4-2 20.2 ± 0.42 5.1 ± 1.5

E4-4 11.9 ± 1.3 3.1 ± 0.6

E4-5 14.5 ± 0.5 2.6 ± 0.4

E4-12 21.1 ± 5.1 5.4 ± 1.4

Error values are one standard deviation of the data (n=3) .

C. Adometase mRNA Expreεεion in Ripening Transgenic Fruit.

Expresεion of AdoMetase in ripening pAG-5520 tomato fruit was measured using an RNAεe protection aεεay (RPA), aε detailed in Example 10. Ripening fruit were harveεted at four different εtageε, mature green (MG) , breaker (Br) , orange (Or) and ripe (Ri) .

Figure 20 diεplayε the AdoMetase RNA expresεion level at each of theεe εtages. Figure 21 is a graphical representation of the same data, quantitated as described in Example 10 by liquid scintillation counting. Two of the transgenic lines asεayed εhowed little or no expreεεion of AdoMetase. Six other lines showed significant AdoMetase RNA expression, with the orange stage being predominant. In five of the six lines expressing AdoMetase, the expression level at the ripe stage was substantially diminished, demonstrating the transient nature of the E4-directed expresεion.

D. Weεtern Blot Analvεis of AdoMetase Expression.

Western blot analysiε waε carried out on protein extracts from E4-5 transgenic ripening tomatoes as detailed in Example 12. The reεultε are εhown in Figure 22.

Figure 22 εhows the level of AdoMetase at four stages of fruit ripening. The pattern of expression matches that of AdoMetase transcription, including a decline in AdoMetase at the ripe stage. Known quantities of purified recombinant AdoMetase were run in the control lanes and used to establish a εtandard curve baεed on εignal intenεity. This allowed estimation of the relative amount of AdoMetase in these tomatoes and was calculated to be approximately 0.05% of the total soluble protein at the orange stage of ripening.

E. Ethylene Production in Ripening Transgenic Fruit.

Daily ethylene production by control and pAG-5520 transgenic fruit picked at the breaker stage was measured by sealing glass jars containing individual fruit and sampling 2 ml aliquots for gas chromatographic analysiε, aε detailed in Example 11. Measurements were made over a period of 15 days post- harvest.

The results of the analysiε are presented in Figure 23, which showε a comparison of fruit from one pAG-5520 transgenic line (E4-05) with the fruit from untranεformed controls. The control values are represented aε open εquareε, whereas the values from the transgenic line are represented as diamonds. The values are the average of ethylene determinations for three fruit. Error bars repreεent one standard deviation of the data.

In the transgenic line, the rate of ethylene production declines steeply immediately after harvest, reaching a minimum of approximately 1.0 nl/g/h at 7 days post-harvest. After this point, the rate increaεeε to a level of approximately 2 to 3-fold the minimum and remains relatively constant. The kinetics of ethylene production from these transgenic tomatoeε correlateε with the observed AdoMetase RNA transcription and AdoMetase accumulation in the corresponding fruit. When AdoMetaεe expreεεion iε high the level of ethylene production iε low, as expected. The data represent a time period of fifteen days after the breaker stage of fruit ripening (post- breaker) , and demonstrate a reduction in the amount of ethylene production in transgenic tomatoes versus normal fruit over the fifteen day period. Further, the data graphically illustrate the biological consequence of the transient nature of E4-driven Adometase expreεεion described above.

The effect of AdoMetase gene expression on ripening has several dimensionε, including (i) pAG-5520 tomatoeε (tranεgenic line E4-12-D) develop color to a light red stage and then cease further color development, and (ii) the transgenic tomatoes remain firm for much longer than controls.

While the invention has been described with reference to specific methods and embodiments, it will be appreciated that various modifications and changes may be made without departing from the invention.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: Epitope, Inc.

(C) CITY: Beaverton

(D) STATE: OR

(E) COUNTRY: USA (F) POSTAL CODE: 97005

(ii) TITLE OF INVENTION: Regulated Expression of Heterologous

Genes in Plants and Transgenic Fruit with a Modified Ripening Phenotype

(iii) NUMBER OF SEQUENCES: 26

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Dehlinger & Associates (B) STREET: 350 Cambridge Avenue, Suite 250

(C) CITY: Palo Alto

(D) STATE: CA

(E) COUNTRY: USA

(F) ZIP: 94306

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentln Release #1.0, Version #1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE: (C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 08/261,677

(B) FILING DATE: 17-JUN-1994

(viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Fabian, Gary R.

(B) REGISTRATION NUMBER: 33,875

(C) REFERENCE/DOCKET NUMBER: 4257-0011.41

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (415) 324-0880

(B) TELEFAX: (415) 324-0960

(2) INFORMATION FOR SEQ ID Nθ:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 39 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(C) INDIVIDUAL ISOLATE: Figure 2 - top strand of synthetic oligo

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 12..38

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

GATCCGCCAC C ATG GTT TTC ACT AAA GAG CCT GCG AAC G 39

Met Val Phe Thr Lys Glu Pro Ala Asn 1 5

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Met Val Phe Thr Lys Glu Pro Ala Asn 1 5

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 35 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: Figure 2, bottom strand of synthetic oligo

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

CGTTCGCAGG CTCTTTAGTG AAAACCATGG TGGCG 35

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(C) INDIVIDUAL ISOLATE: Figure 16 - E4 promoter, 5' primer

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

AGCCCATTGA AGCTTAAAGT AAACTT 26

(2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(iii) HYPOTHETICAL: NO

( iv ) ANTI-SENSE : NO

(vi) ORIGINAL SOURCE:

(C) INDIVIDUAL ISOLATE: Figure 16 - E4 promoter, 3' primer

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

TACCCTCCAT GGCTCAATCT CT 22

(2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(C) INDIVIDUAL ISOLATE: E4 gene 5'primer

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

ACGCATGGAG GGTAACAA 18

(2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(iii) HYPOTHETICAL: NO

( iv ) ANTI -SENSE : NO

(vi) ORIGINAL SOURCE:

(C) INDIVIDUAL ISOLATE: tomato E4 gene 3' primer

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:

GAAGCAAGAC AGCAAATG 18

(2) INFORMATION FOR SEQ ID NO:8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2796 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(C) INDIVIDUAL ISOLATE: Figure 24 - E4 tomato gene DNA

(ix) FEATURE:

(A) NAME/KEY: CDS (B) LOCATION: 1439..1774

(ix) FEATURE:

(A) NAME/KEY: exon

(B) LOCATION: 1439..1774

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1859..2113

(ix) FEATURE:

(A) NAME/KEY: exon

(B) LOCATION: 1859..2113

(i ) FEATURE: (A) NAME/KEY: intron

(B) LOCATION: 1775..1858

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:

GAATTCTCAA TTGAGCCCAA TTCAATCTCC AATTTCAACC CGTTTTAAAA CTTTTTATTA 60

AGATATGTTT CTATATTGAA AGTATGAATT ATTATCTATT TAACATCTTT TAGGATTTAT 120

CTATCCATTT GCTACTTTTT TAACAAAAAA TTCTTGAGTG AAAATTCAAA TTGTGATTAT 180

AAAAGTTAAA TATCAATATG TTAAATTATT AAGATTAATC GGGTCAAATT GGCGGGTCAA 240

GGCCCAATTC TTTTTTAGCC CATTTAAGCT CAAAGTAAAC TTGGGTGGGT CAAGACCCAA 300

CTCGATTTCT GTTCAACCCA TTTTAATATT TCTATTTTCA ACCTAACCCG CTCATTTGAT 360 ACCCCTACAA ATATCATATT TGTGTGTGAA ATATTTTTTG GGCTGGAGAG AGAGGCCCCG 420

AGGGGAGTGG AGGGGTGGGG TGGGGAGAGA GAGCGAGAAA GAGTGGAGAG AGAAATTTGA 480

TATGAAATCC TACATATATT ACAGATTGTA ATGTTCTAAA CTATAACGAT TTGTCATAAA 540

CACATATCAT GGATTTGTCT TTTTGTGTAA TTTTCCCAAT TGTAAATAGG ACTTCGTTAT 600

TTGAAACTTG AAAGTGAAGT CACATAGATT AAGTACAAAC ATTAATTAAA GACCGTGGTG 660

GAATGATAAA TATTTATTTA TCTTTAATTA GTTATTTTTT TGGGAGCTCT TTATTCCAAT 720

GTGAGACTTT TGCGACATAT ATTCAAATTT AATCGAATCA CAATATGTAT TAGATTGATA 780

AAAAAATAAT TTTTTTACAA TGTTAGTTGA GACTCATAAC TTACTGCCTA TTGGTAATCT 840

ATGACTCCTA ATTCCTTAAT TATTTAAATA TATCATCTTG ATCGTTAACA AAGTAATTTC 900

GAAAGACCAC GAGTAAGAAG ACAAACGAGA ATACCAAAAA ATTCAAAAAT TTAATGTGAT 960

TTGGTCAATC GATCTACGTC CATAAAGGAG ATGAGTAATC TACTATAAAT ATGAGAGTAC 1020

AAAATACAGA GAGAAACAAC CTCAACTAAT TCACTCGGAA TACATGAGAA GTTCACACAA 1080

GTGATAACGT ATCAAACTTG TGACCCACAC TTTTCCCTCT AACCAAAGCT CTTAAAACTA 1140

TATTGTGAAT GCTGATTAAG TTAAACGAAA CAGTCCTAAA TCTTTTCCGT CCTATGAGAA 1200

ACAAGATTAA TCAATTCACA ATTTTTTTAA AAAGAAAAAC CTGTAAGAAA TTTAGGCAAA 1260

CAAAACCTAA CACAAGTTTG TTTTTGTTTT TACTACCAAC AAGAAATTCA AATGGCAAAT 1320

GTATAACGCA TCTTAGCTAA TTATATGACC AGATTCAGAT TAATATACAT CTTCACCCAT 1380

GCAATCCATT TCTATATAAA GAAACATACA CGAACTTGAT ATTATTAGAG ATTGAGCA 1438

ATG GAG GGT AAC AAC AGC AGT AGC AAG TCA ACC ACC AAT CCA GCA TTG 1486 Met Glu Gly Asn Asn Ser Ser Ser Lys Ser Thr Thr Asn Pro Ala Leu 1 5 10 15

GAT CCG GAT CTG GAC AGC CCG GAT CAG CCG GGT CTG GAG TTT GCC CAA 1534 Asp Pro Asp Leu Asp Ser Pro Asp Gin Pro Gly Leu Glu Phe Ala Gin 20 25 30

TTT GCT GCC GGC TGC TTT TGG GGA GTC GAA TTG GCT TTC CAG AGG GTT 1582 Phe Ala Ala Gly Cys Phe Trp Gly Val Glu Leu Ala Phe Gin Arg Val 35 40 45

GGA GGA GTA GTG AAG ACG GAG GTT GGG TAC TCT CAG GGG AAT GTC CAT 1630 Gly Gly Val Val Lys Thr Glu Val Gly Tyr Ser Gin Gly Asn Val His 50 55 60

GAC CCG AAC TAC AAG CTT ATT TGC TCC GGA ACA ACC GAA CAT GCC GAG 1678

Asp Pro Asn Tyr Lys Leu He Cys Ser Gly Thr Thr Glu His Ala Glu

65 70 75 80

GCC ATT CGG ATC CAG TTT GAC CCG AAT GTC TGC CCG TAT TCC AAT CTC 1726

Ala He Arg He Gin Phe Asp Pro Asn Val Cys Pro Tyr Ser Asn Leu

85 90 95

CTT TCT CTA TTT TGG AGT CGC CAT GAC CCG ACC ACT CTA AAT CGC CAG 1774

Leu Ser Leu Phe Trp Ser Arg His Asp Pro Thr Thr Leu Asn Arg Gin 100 105 110

GTATCAAATT CCTTTGGTGT TTCATTTTAT GTGATTAATA TTAAAAATTT TTTATATAAA 1834

TGTCATGATG ATGGTTGTTG CTAG GGT AAT GAT GTG GGA AAG CAA TAC CGC 1885

Gly Asn Asp Val Gly Lys Gin Tyr Arg 1 5

TCA GGA ATA TAT TAC TAT AAT GAT GCT CAG GCT CAA CTG GCA AGG GAG 1933

Ser Gly He Tyr Tyr Tyr Asn Asp Ala Gin Ala Gin Leu Ala Arg Glu 10 15 20 25

TCG TTA GAA GCT AAG CAG AAG GAA TTT ATG GAT AAG AAA ATT GTC ACT 1981 Ser Leu Glu Ala Lys Gin Lys Glu Phe Met Asp Lys Lys He Val Thr

30 35 40

GAA ATT CTT CCT GCT AAG AGA TTT TAT AGA GCT GAA GAG TAT CAC CAG 2029 Glu He Leu Pro Ala Lys Arg Phe Tyr Arg Ala Glu Glu Tyr His Gin 45 50 55 CAA TAT CTA GAG AAG GGT GGG GGC AGA GGT TGT AAG CAG TCG GCT GCA 2077

Gin Tyr Leu Glu Lys Gly Gly Gly Arg Gly Cys Lys Gin Ser Ala Ala 60 65 70

AAG GGC TGC AAT GAC CCA ATA AGG TGC TAC GGT TGACAGCAGA TCTTTGAATG 2130

Lys Gly Cys Asn Asp Pro He Arg Cys Tyr Gly

75 80 85

TCATAGCAAC TACAAAAGAA CTTGTTAGAC ATTTGCTGTC TTGCTTCTTT AAATTTGAAT 2190

AAACATGACA ATGATTCTTA TAACTACTTG CTCTCTTGGA TGGAATAACT AGTTGTCGTA 2250

AAGTATTCTC CTCTTGCTAA TTATTATCTC TCTTTATATG GTACCTGCAA TTTGTTGCTT 2310

TAGTTACAGA ATAATGGACG TCAATTCTAT ATCTTAATTT GTTTTAAGTC TTAAATGAGG 2370

TGGTTTGTGT TTGAAAGCAA TATCAAGCAT AGTAATACCA ATGATTTAGT AGATGAACTT 2430

AATCAAATCA AATTCCAAAA TGCAGTCTAC AAATTGACAA CATGAAGTTA AGTGTATCTT 2490

ATGTAAATTG ACATCTTTCC TAGTAGATGC CTAATACTTT TGTAAAGACT AAAATAAGCA 2550

CAGATGAGGC TTGTGCATTT AACTTAGAGT TCATCCTTAG GTGTGGCTGC AGGAGACCCT 2610

GTAGGGTTGC TTGAAGTCTT GATGGGGTAG GAGGGTTGCA TTGCTATACC ACACAACCCC 2670

TCTTCAGCGT CAACCTTGCG CTGCATTCTA ATGTATCCTT TTTCTCCCCA TTCAGCTCCC 2730

CATGAGTTCT TCACAATCCA GTATTTGGTT CCATCGACGG TTGTGCCATA CCCCACAATA 2790

GCCACA 2796

(2) INFORMATION FOR SEQ ID NO:9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 196 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:

Met Glu Gly Asn Asn Ser Ser Ser Lys Ser Thr Thr Asn Pro Ala Leu 1 5 10 15

Asp Pro Asp Leu Asp Ser Pro Asp Gin Pro Gly Leu Glu Phe Ala Gin 20 25 30

Phe Ala Ala Gly Cys Phe Trp Gly Val Glu Leu Ala Phe Gin Arg Val 35 40 45

Gly Gly Val Val Lys Thr Glu Val Gly Tyr Ser Gin Gly Asn Val His 50 55 60

Asp Pro Asn Tyr Lys Leu He Cys Ser Gly Thr Thr Glu His Ala Glu 65 70 75 80

Ala He Arg He Gin Phe Asp Pro Asn Val Cys Pro Tyr Ser Asn Leu 85 90 95

Leu Ser Leu Phe Trp Ser Arg His Asp Pro Thr Thr Leu Asn Arg Gin 100 105 110

Gly Asn Asp Val Gly Lys Gin Tyr Arg Ser Gly He Tyr Tyr Tyr Asn 115 120 125

Asp Ala Gin Ala Gin Leu Ala Arg Glu Ser Leu Glu Ala Lys Gin Lys 130 135 140

Glu Phe Met Asp Lys Lys He Val Thr Glu He Leu Pro Ala Lys Arg 145 150 155 160

Phe Tyr Arg Ala Glu Glu Tyr His Gin Gin Tyr Leu Glu Lys Gly Gly 165 170 175

Gly Arg Gly Cys Lys Gin Ser Ala Ala Lys Gly Cys Asn Asp Pro He 180 185 190

Arg Cys Tyr Gly 195 (2) INFORMATION FOR SEQ ID NO: 10:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1678 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(C) INDIVIDUAL ISOLATE: Figure 25 - E4 tomato promoter / AdoMetase gene DNA

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1174..1629

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:

AAGCTTAAAG TAAACTTGGG TGGGTCAAGA CCCAACTCGA TTTCTGTTCA ACCCATTTTA 60

ATATTTCTAT TTTCAACCTA ACCCGCTCAT TTGATACCCC TACAAATATC ATATTTGTGT 120

GTGAAATATT TTTTGGGCTG GAGAGAGAGG CCCCGAGGGG AGTGGAGGGG TGGGGTGGGG 180

AGAGAGAGCG AGAAAGAGTG GAGAGAGAAA TTTGATATGA AATCCTACAT ATATTACAGA 240

TTGTAATGTT CTAAACTATA ACGATTTGTC ATAAACACAT ATCATGGATT TGTCTTTTTG 300

TGTAATTTTC CCAATTGTAA ATAGGACTTC GTTATTTGAA ACTTGAAAGT GAAGTCACAT 360

AGATTAAGTA CAAACATTAA TTAAAGACCG TGGTGGAATG ATAAATATTT ATTTATCTTT 420

AATTAGTTAT TTTTTTGGGA GCTCTTTATT CCAATGTGAG ACTTTTGCGA CATATATTCA 480 AATTTAATCG AATCACAATA TGTATTAGAT TGATAAAAAA ATAATTTTTT TACAATGTTA 540

GTTGAGACTC ATAACTTACT GCCTATTGGT AATCTATGAC TCCTAATTCC TTAATTATTT 600

AAATATATCA TCTTGATCGT TAACAAAGTA ATTTCGAAAG ACCACGAGTA AGAAGACAAA 660

CGAGAATACC AAAAAATTCA AAAATTTAAT GTGATTTGGT CAATCGATCT ACGTCCATAA 720

AGGAGATGAG TAATCTACTA TAAATATGAG AGTACAAAAT ACAGAGAGAA ACAACCTCAA 780

CTAATTCACT CGGAATACAT GAGAAGTTCA CACAAGTGAT AACGTATCAA ACTTGTGACC 840

CACACTTTTC CCTCTAACCA AAGCTCTTAA AACTATATTG TGAATGCTGA TTAAGTTAAA 900

CGAAACAGTC CTAAATCTTT TCCGTCCTAT GAGAAACAAG ATTAATCAAT TCACAATTTT 960

TTTAAAAAGA AAAACCTGTA AGAAATTTAG GCAAACAAAA CCTAACACAA GTTTGTTTTT 1020

GTTTTTACTA CCAACAAGAA ATTCAAATGG CAAATGTATA ACGCATCTTA GCTAATTATA 1080

TGACCAGATT CAGATTAATA TACATCTTCA CCCATGCAAT CCATTTCTAT ATAAAGAAAC 1140

ATACACGAAC TTGATATTAT TAGAGATTGA GCC ATG GTT TTC ACT AAA GAG CCT 1194

Met Val Phe Thr Lys Glu Pro 1 5

GCG AAC GTC TTC TAT GTA CTG GTT TCC GCT TTC CGT TCT AAC CTC TGC 1242

Ala Asn Val Phe Tyr Val Leu Val Ser Ala Phe Arg Ser Asn Leu Cys

10 15 20

GAT GAG GTG AAT ATG AGC AGA CAC CGC CAC ATG GTA AGC ACT TTA CGT 1290

Asp Glu Val Asn Met Ser Arg His Arg His Met Val Ser Thr Leu Arg

25 30 35

GCC GCA CCG GGT CTT TAT GGC TCC GTT GAG TCA ACC GAT TTG ACC GGG 1338

Ala Ala Pro Gly Leu Tyr Gly Ser Val Glu Ser Thr Asp Leu Thr Gly

40 45 50 55

TGC TAT CGT GAG GCA ATC TCA AGC GCA CCA ACT GAG GAA AAA ACT GTT 1386 Cys Tyr Arg Glu Ala He Ser Ser Ala Pro Thr Glu Glu Lys Thr Val

60 65 70 CGT GTA CGC TAC AAG GAC AAA GCG CAG CCA CTC AAT GTT GCA CGC CTA 1434

Arg Val Arg Tyr Lys Asp Lys Ala Gin Pro Leu Asn Val Ala Arg Leu

75 80 85

GCT TCT AAT GAG TGG GAG CAA GAT TGC GTA CTG GTA TAC AAA TCA CAG 1482

Ala Ser Asn Glu Trp Glu Gin Asp Cys Val Leu Val Tyr Lys Ser Gin

90 95 100

ACT CAC ACG GCT GGT CTG GTG TAC GCT AAA GGT ATC GAC GGG TAT AAG 1530 Thr His Thr Ala Gly Leu Val Tyr Ala Lys Gly He Asp Gly Tyr Lys

105 110 115

GCT GAA CGT CTG CCG GGT AGT TTC CAA GAG GTT CCT AAA GGC GCA CCG 1578

Ala Glu Arg Leu Pro Gly Ser Phe Gin Glu Val Pro Lys Gly Ala Pro 120 125 130 135

CTG CAA GGC TGC TTC ACT ATT GAT GAG TTC GGT CGC CGC TGG CAA GTA 1626

Leu Gin Gly Cys Phe Thr He Asp Glu Phe Gly Arg Arg Trp Gin Val

140 145 150

CAA TAACGTGTTA AACTCAAGGT CATGCACGAT GCGTGGCGGA TCGGGTACC 1678

Gin

(2) INFORMATION FOR SEQ ID NO:11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 152 amino acids (B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:H:

Met Val Phe Thr Lys Glu Pro Ala Asn Val Phe Tyr Val Leu Val Ser 1 5 10 15

Ala Phe Arg Ser Asn Leu Cys Asp Glu Val Asn Met Ser Arg His Arg

20 25 30 His Met Val Ser Thr Leu Arg Ala Ala Pro Gly Leu Tyr Gly Ser Val 35 40 45

Glu Ser Thr Asp Leu Thr Gly Cys Tyr Arg Glu Ala He Ser Ser Ala 50 55 60

Pro Thr Glu Glu Lys Thr Val Arg Val Arg Tyr Lys Asp Lys Ala Gin 65 70 75 80

Pro Leu Asn Val Ala Arg Leu Ala Ser Asn Glu Trp Glu Gin Asp Cys

85 90 95

Val Leu Val Tyr Lys Ser Gin Thr His Thr Ala Gly Leu Val Tyr Ala 100 105 110

Lys Gly He Asp Gly Tyr Lys Ala Glu Arg Leu Pro Gly Ser Phe Gin 115 120 125

Glu Val Pro Lys Gly Ala Pro Leu Gin Gly Cys Phe Thr He Asp Glu 130 135 140

Phe Gly Arg Arg Trp Gin Val Gin 145 150

(2) INFORMATION FOR SEQ ID NO:12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 225 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(iϋ) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: Figure 25 - raspberry E4 gene DNA (ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..213

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:

GAG CTC AGG TTT CAG CGA GTG GCC GGT GTG GTC AAG ACC GAG GTT GGG 48 Glu Leu Arg Phe Gin Arg Val Ala Gly Val Val Lys Thr Glu Val Gly 1 5 10 15

TAC TCC CAG GGC CAC GTC CAC GAT CCG AAT TAC AAA CTG GTC TGC TCC 96 Tyr Ser Gin Gly His Val His Asp Pro Asn Tyr Lys Leu Val Cys Ser 20 ^* 25 30

GGA ACT ACC AAC CAT TCG GAG GTC GTT CGG GTC CAG TTC GAC CCG CAA 144 Gly Thr Thr Asn His Ser Glu Val Val Arg Val Gin Phe Asp Pro Gin 35 40 45

GTC TAC CCA TAC TCG GAC CTG CTT TCC GTC TTT TGG TCT CGT CAT GAT 192 Val Tyr Pro Tyr Ser Asp Leu Leu Ser Val Phe Trp Ser Arg His Asp 50 55 60

CCA ACG ACT GTC AAT CGC CAG GTATGGGGAT TG 225

Pro Thr Thr Val Asn Arg Gin 65 70

(2) INFORMATION FOR SEQ ID NO:13:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 71 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:

Glu Leu Arg Phe Gin Arg Val Ala Gly Val Val Lys Thr Glu Val Gly 1 5 10 15

Tyr Ser Gin Gly His Val His Asp Pro Asn Tyr Lys Leu Val Cys Ser 20 25 30

Gly Thr Thr Asn His Ser Glu Val Val Arg Val Gin Phe Asp Pro Gin 35 40 45

Val Tyr Pro Tyr Ser Asp Leu Leu Ser Val Phe Trp Ser Arg His Asp 50 55 60

Pro Thr Thr Val Asn Arg Gin 65 70

(2) INFORMATION FOR SEQ ID NO:14:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 10 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(iii) HYPOTHETICAL: NO

( iv ) ANTI -SENSE : NO

(vi) ORIGINAL SOURCE:

(C) INDIVIDUAL ISOLATE: original T3 sequence

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:

CACCAAATGA 10

(2) INFORMATION FOR SEQ ID NO:15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

( ii ) MOLECULE TYPE : DNA (iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(C) INDIVIDUAL ISOLATE: Kozak sequence

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:

GCCACCATGG 10

(2) INFORMATION FOR SEQ ID NO:16:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 81 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA

(iii) HYPOTHETICAL: NO

( iv ) ANTI -SENSE : NO

(vi) ORIGINAL SOURCE:

(C) INDIVIDUAL ISOLATE: Fig. 2, first DNA sequence

(ix) FEATURE: (A) NAME/KEY: CDS

(B) LOCATION: 49..81

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:

AAGCTTGCAT GCCTGCAGGT CGACTCTAGA GGATCCCCGT AACACCAA ATG ATT TTC 57

Met He Phe

1

ACT AAA GAG CCT GCG AAC GTC TTC 81

Thr Lys Glu Pro Ala Asn Val Phe 5 10

(2) INFORMATION FOR SEQ ID NO:17:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 11 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:

Met He Phe Thr Lys Glu Pro Ala Asn Val Phe 1 5 10

(2) INFORMATION FOR SEQ ID NO:18:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(C) INDIVIDUAL ISOLATE: Fig. 2, T3 SAMase

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:

CACCAAATGA TT 12

(2) INFORMATION FOR SEQ ID NO:19: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(C) INDIVIDUAL ISOLATE: Fig. 2, SAM-K

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:

GCCACCATGG TT 12

(2) INFORMATION FOR SEQ ID NO:20:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(C) INDIVIDUAL ISOLATE: Fig. 3, E8 5' primer

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:

GGTCTAGAAG GAATTTCACG 20 (2) INFORMATION FOR SEQ ID NO:21:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(C) INDIVIDUAL ISOLATE: Fig. 3, E8 3' primer

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:

ATTCACAGTG CAAAAGACCA TGGAA 25

(2) INFORMATION FOR SEQ ID NO:22:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 586 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA

(iϋ) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: Fig. 11, pUC19-SAM-K ( ix ) FEATURE :

(A) NAME/KEY: CDS

(B) LOCATION: 66..521

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:

ACAGCTATGA CCATGATTAC GCCAAGCTTG CATGCCTGCA GGTCGACTCT AGAGGATCCG 60

CCACC ATG GTT TTC ACT AAA GAG CCT GCG AAC GTC TTC TAT GTA CTG 107

Met Val Phe Thr Lys Glu Pro Ala Asn Val Phe Tyr Val Leu 1 5 10

GTT TCC GCT TTC CGT TCT AAC CTC TGC GAT GAG GTG AAT ATG AGC AGA 155 Val Ser Ala Phe Arg Ser Asn Leu Cys Asp Glu Val Asn Met Ser Arg 15 20 25 30

CAC CGC CAC ATG GTA AGC ACT TTA CGT GCC GCA CCG GGT CTT TAT GGC 203 His Arg His Met Val Ser Thr Leu Arg Ala Ala Pro Gly Leu Tyr Gly 35 40 45

TCC GTT GAG TCA ACC GAT TTG ACC GGG TGC TAT CGT GAG GCA ATC TCA 251

Ser Val Glu Ser Thr Asp Leu Thr Gly Cys Tyr Arg Glu Ala He Ser

50 55 60

AGC GCA CCA ACT GAG GAA AAA ACT GTT CGT GTA CGC TAC AAG GAC AAA 299

Ser Ala Pro Thr Glu Glu Lys Thr Val Arg Val Arg Tyr Lys Asp Lys

65 70 75

GCG CAG GCA CTC AAT GTT GCA CGC CTA GCT TGT AAT GAG TGG GAG CAA 347 Ala Gin Ala Leu Asn Val Ala Arg Leu Ala Cys Asn Glu Trp Glu Gin 80 85 90

GAT TGC GTA CTG GTA TAC AAA TCA CAG ACT CAC ACG GCT GGT CTG GTG 395 Asp Cys Val Leu Val Tyr Lys Ser Gin Thr His Thr Ala Gly Leu Val 95 100 105 110

TAC GCT AAA GGT ATC GAC GGG TAT AAG GCT GAA CGT CTG CCG GGT AGT 443 Tyr Ala Lys Gly He Asp Gly Tyr Lys Ala Glu Arg Leu Pro Gly Ser 115 120 125 TTC CAA GAG GTT CCT AAA GGC GCA CCG CTG CAA GGC TGC TTC ACT ATT 491 Phe Gin Glu Val Pro Lys Gly Ala Pro Leu Gin Gly Cys Phe Thr He 130 135 140

GAT GAG TTC GGT CGC CGC TGG CAA GTA CAA TAAGTGTTAA ACTCAAGGTC 541

Asp Glu Phe Gly Arg Arg Trp Gin Val Gin 145 150

ATGCACGATG CGTGGCGGAT CGGGTACCGA GCTCGAATTC ACTGG 586

(2) INFORMATION FOR SEQ ID NO:23:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 152 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:

Met Val Phe Thr Lys Glu Pro Ala Asn Val Phe Tyr Val Leu Val Ser 1 5 10 15

Ala Phe Arg Ser Asn Leu Cys Asp Glu Val Asn Met Ser Arg His Arg 20 25 30

His Met Val Ser Thr Leu Arg Ala Ala Pro Gly Leu Tyr Gly Ser Val 35 40 45

Glu Ser Thr Asp Leu Thr Gly Cys Tyr Arg Glu Ala He Ser Ser Ala 50 55 60

Pro Thr Glu Glu Lys Thr Val Arg Val Arg Tyr Lys Asp Lys Ala Gin 65 70 75 80

Ala Leu Asn Val Ala Arg Leu Ala Cys Asn Glu Trp Glu Gin Asp Cys

85 90 95

Val Leu Val Tyr Lys Ser Gin Thr His Thr Ala Gly Leu Val Tyr Ala 100 105 110

Lys Gly He Asp Gly Tyr Lys Ala Glu Arg Leu Pro Gly Ser Phe Gin 115 120 125

Glu Val Pro Lys Gly Ala Pro Leu Gin Gly Cys Phe Thr He Asp Glu 130 135 140

Phe Gly Arg Arg Trp Gin Val Gin 145 150

(2) INFORMATION FOR SEQ ID NO:24:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2216 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(C) INDIVIDUAL ISOLATE: Fig. 13, E8 promoter

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:

GAATTCATTT TTGACATCCC TAATGATATT GTTCACGTAA TTAAGTTTTG TGGAAGTGAG 60

AGAGTCCAAT TTTGATAAGA AAAGAGTCAG AAAACGTAAT ATTTTAAAAG TCTAAATCTT 120

TCTACAAATA AGAGCAAATT TATTTATTTT TTAATCCAAT AAATATTAAT GGAGGACAAA 180

TTCAATTCAC TTGGTTGTAA AATAAACTTA AACCAATAAC CAAAGANCTA ATAAATCTGA 240

AGTGGAATTA TTAAGGATAA TGTACATAGA CAATGAAGAA ATAATAGGTT CGATGAATTA 300 ATAATAATTA AGGATGTTAC AATCATCATG TGCCAAGTAT ATACACAATA TTCTATGGGA 360

TTTATAATTT CGTTACTTCA CTTAACTTTT GCGTAAATAA AACGAATTAT CTGATATTTT 420

ATAATAAAAC AGTTAATTAA GAACCATCAT TTTTAACAAC ATAGATATAT TATTTCTAAT 480

AGTTTAATGA TACTTTTAAA TCTTTTAAAT TTTATGTTTC TTTTAGAAAA TAAAAATTCA 540

AAAAAATTAA ATATATTTAC AAAAACTACA ATCAAACACA ACTTCATATA TTAAAAGCAA 600

AATATATTTT GAAAATTTCA AGTGTCCTAA CAAATAAGAC AAGAGGAAAA TGTACGATGA 660

GAGACATAAA GAGAACTAAT AATTGAGGAG TCCTATAATA TATAATAAAG TTTATTAGTA 720

AACTTAATTA TTAAGGACTC CTAAAATATA TGATAGGAGA AAATGAATGG TGAGAGATAT 780

TGGAAAACTT AATAATTAAG GATNTTAAAA TATATGGTAA AAGATAGGCA AAGTATCCAT 840

TATCCCCTTT TAACTTGAAG TCTACCTAGG CGCATGTGAA AGGTTGATTT TTTGTCACGT 900

CATATAGCTA TAACGTAAAA AAAGAAAGTA AAATTTTTAA TTTTTTTTAA TATATGACAT 960

ATTTTAAACG AAATATAGGA CAAAATGTAA ATGAATAGTA AAGGAAACAA AGATTAATAC 1020

TTACTTTGTA AGAATTTAAG ATAAATTTAA AATTTAATAG ATCAACTTTA CGTCTAGAAA 1080

GACCCATATC TAGAAGGAAT TTCACGAAAT CGGCCCTTAT TCAAAAATAA CTTTTAAATA 1140

ATGAATTTTA AATTTTAAGA AATAATATCC AATGAATAAA TGACATGTAG CATTTTACCT 1200

AAATATTTCA ACTATTTTAA TCCAATATTA ATTTGTTTTA TTCCCAACAA TAGAAAGTCT 1260

TGTGCAGACA TTTAATCTGA CTTTTCCAGT ACTAAATATT AATTTTCTGA AGATTTTCGG 1320

GTTTAGTCCA CAAGTTTTAG TGAGAAGTTT TGCTCAAAAT TTTAGGTGAG AAGGTTTGAT 1380

ATTTATCTTT TGTTAAATTA ATTTATCTAG GTGACTATTA TTTATTTAAG TAGAAATTCA 1440

TATCATTACT TTTGCCAACT TGTAGTCATA ATAGGAGTAG GTGTATATGA TGAAGGAATA 1500

AACAAGTTCA GTGAAGTGAT TAAAATAAAA TATAATTTAG GTGTACATCA AATAAAAACC 1560 TTAAAGTTTA GAAAGGCACC GAATAATTTT GCATAGAAGA TATTAGTAAA TTTATAAAAA 1620

TAAAAGAAAT GTAGTTGTCA AGTTGTCTTC TTTTTTTTGG ATAAAAATAG CAGTTGGCTT 1680

ATGTCATTCT TTTACAACCT CCATGCCACT TGTCCAATTG TTGACACTTA ACTAATTAGT 1740

TTGATTCATG TATGAATACT AAATAATTTT TTAGGACTGA CTCAAATATT TTTATATTAT 1800

CATAGTAATA TTTATCTAAT TTTTAGGACC ACTTATTACT AAATAATAAA TTAACTACTA 1860

CTATATTATT GTTGTGAAAC AACAACGTTT TGGTTGTTAT GATGAAACGT ACACTATATC 1920

AGTATGAAAA ATTCAAAACG ATTAGTATAA ATTATATTGA AAATTTGATA TTTTTCTATT 1980

CTTAATCAGA CGTATTGGGT TTCATATTTT AAAAAGGGAC TAAACTTAGA AGAGAAGTTT 2040

GTTTGAAACT ACTTTTGTCT CTTTCTTGTT CCCATTTCTC TCTTAGATTT CAAAAAGTGA 2100

ACTACTTTAT CTCTTTCTTT GTTCACATTT TATTTTATTC TATTATAAAT ATGGCATCCT 2160

CATATTGAGA TTTTTAGAAA TTATTCTAAT CATTCACAGT GCAAAAGACC ATGGAA 2216

(2) INFORMATION FOR SEQ ID NO:25:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2708 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(C) INDIVIDUAL ISOLATE: RASPBERRY E4 GENE

(ix) FEATURE:

(A) NAME/KEY: isc feature (B) LOCATION: 1468..1469

(D) OTHER INFORMATION: /note= "small sequencing gap of unknown size"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:

AAGCTTAATT GAGATGATTA GCCCAGACCC AGCAGGATTA GGCTTAATGG TGGTCCATTT 60

GAGAAAAAGA TTAAAAATGA TGTCATAAAA AAACNTGGTC GBCAGGATTC NAACCTGCGC 120

GGGCAAAGCC ACATGATTTC TAGTCATGCC CGATAACCAC TCCGGCACGA CCACAATGAT 180

GCTACAATTG CTTTGTTGTA ATCATTAACT TATGGTTGAG TTTGATGCTG ATTAATACTA 240

TTATGTTTCC ATTAACTACT TTTGAAGTAT ACAAAATTAC GAATTTATAA CCAAATTTGA 300

GGTATAATAT GCGAGAGCTA CCTAAATTTT TCTTACTTAA TTTTAAAGTA CATTCAAATT 360

CTGAATTTAT ATTGTGTATA GTCAGAAAAC AATCTACATA TTTAAACACA TAAATTTCTC 420

ACGTTTATAA TCAATTTTGT CGGTTCCTGT AATTTTTCTA AAATAAAAAG CAACCAAAAT 480

TGTGCATCAA CTTATTACAT ACCATGGGAA ATGCAAACTT CAAAACTTAT GGACTCAAAG 540

GGTACATATC TAAACTACAT ATTGTCAGAT TCTTCACTCT TATTTCTTGA GGGCCTCGAG 600

GCATTACCAA CCAAATCCAA AAATTGCTTT CGAATCTCAA TAAAAAGGAT AACCCCATGA 660

AAAAGACGTG GACGGCAGGA TTCGAACCTG CGCGCAGAGC CCACATGATT TCTAGTCATG 720

CCCGATAACC ACTCCGGCAC GTCCACTTCA CTGTTAACGT TTACAGTAAC AAGTCACTAA 780

CTACTAATCA ACATTAGCTC AGGAAATCAA AACTAGATTA TTTACATTTA CAACGACATG 840

TCGTTCGAAG TAGTTGGTCT GTATCTGAGT AGCTTTGGCG GGTAGATTCA ATCGCATTTC 900

TGCATATAAA ACTGATCCTC CCTCTATCGC CAAAGTCAAA CTGAAAATGG CTTCCACCAC 960

CACCAACAAC CCAGCTCTAG ACCCAGATTC GGACACTCCG GATAATCCGG GTCACGAGTT 1020 TGCTCAATTC GGATCCGGGT GCTTCTGGGG AGCCGAGCTC AGGTTTCAGC GAGTGGCCGG 1080

TGTGGTCAAG ACCGAGGTTG GGTACTCCCA GGGCCACGTC CACGATCCGA ATTACAAACT 1140

GGTCTGCTCC GGAACTACCA ACCATTCGGA GGTCGTTCGG GTCCAGTTCG ACCCGCAAGT 1200

CTACCCATAC TCGGACCTGC TTTCCGTCTT TTGGTCTCGT CATGATCCAA CGACTGTCAA 1260

TCGCCAGGTA TGGGGATTGG GGACTTCTGT TTTCATTTGA ATTTTGATGC TAAAAAATTT 1320

CTTGCTTTTT CATACTACAC AGTACACACA AAAAGTTGTG TTTTTTTTCA TTCTTTTAAA 1380

TAGTAGTTGG AAAAGTGCTC TTGGAGTTGA AGAGTACTTC AGTATTGCAT ATGGTCTCAG 1440

TGAAATGATA GTGATTATCA TAAGGAGTTT AAAGGCAGGA TGCATTTTGT GTATGANTGA 1500

TTTTGGGTAG AATATTTTTG GAACAGTTAA AATTTATGGG CTGCTGCACA CTGGCTATGA 1560

ACAAATGTAT AGCATTAAAG TGCTTATGAC AAATTCACAA TTGTATATTA GCAGCAGAGA 1620

CATTAAAGTT TCTAAATGCC TTTTAAGTAG ATTGGAAAAA AGTGCTTTTT TTGGTTGAAG 1680

AAGCACATTC ACTATTTGCC TGTTAATGGA ATTGGTAATG ATGAATCACA AGGATATTTG 1740

TGAATACAAG CAGGATGCTT TTAGTGTGCA AGTGATCTTT CGGAACATTT AAAATCGTCA 1800

TAACAAAGGT GTAACATAAG AAGGCTTTGA AATATTCTCA ATTTCTCATT GATTGAATGA 1860

ATTATGTGTT AGGGTGGAGA TGTGGGTACT CAATATCGAT CTGGAATATA CTACTACAAC 1920

GAAACGCAGG CCCGTCTAGC ACAGGAATCA AAGGAAGCAA AGCAACTGGA GTTTAAGGAT 1980

AAGAAGGTGG TGACAGAGAT TCTTCCAGCA AAGAGGTTTT ACAGGGCAGA GGAGTACCAT 2040

CAGCAATATC TCGCAAAGGG AGGAGGTAAT GGCAACAAAC AATCTGCTGA AAAAGGTTGC 2100

AATGATCCTA TTCGATGCTA TGGTTGAGAA ACTAATGCAT TATGCCATTA TTAAAACTCT 2160

ACTGGTTTAC TATGCAGAAA CACCTATGTC AGTTCAATTA TACTGAAGGC ACCAAAGTGT 2220

CATCTTAAAT TATATGGCAA TGTTTTACTC GTTATGAATA AAGGAGGTCC AAGTCGACCA 2280 GATATGAACA AATGAAATAT TGCCATGTTA ATTGGAATCC AGTAGTAATT AGGATTTGTT 2340

TTGGTGTATG TACTCCGATA TCAAGATATG CAAATGATGC ATTGTGTTTT TATATATTGA 2400

CAAGTTCCAA ATTATAGTAC TTCGTATGTG TTATGCGGTT TAATTAGTGT TGCTTACTTG 2460

AATGGTATAT TACTATTATG CTTAGTAGGA ACTAGGAACT AGGGAATATG TTGTGATAGA 2520

GTTGTCCAAC GAAATTTTTG ACCAAAGTTA TTTCATTGAA TAAAAACTAC AGTCTTAGAG 2580

ATACATCCAA TTCTATAAAG TGAAAGAAGC AAATATTATT TGTTCATGAG GCTATGAGTC 2640

ATGAACTTTA TGCTATAACC GAAGCAACCT CAGAAAAGTC GAAGTAAATT GTGTATTGTT 2700

TAGAGCTC 2708

(2) INFORMATION FOR SEQ ID NO:26:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 191 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(C) INDIVIDUAL ISOLATE: RASPBERRY E4 PROTEIN

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:

Met Ala Ser Thr Thr Thr Asn Asn Pro Ala Leu Asp Pro Asp Ser Asp 1 5 10 15

Thr Pro Asp Asn Pro Gly His Glu Phe Ala Gin Phe Gly Ser Gly Cys

20 25 30 Phe Trp Gly Ala Glu Leu Arg Phe Gin Arg Val Ala Gly Val Val Lys 35 40 45

Thr Glu Val Gly Tyr Ser Gin Gly His Val His Asp Pro Asn Tyr Lys 50 55 60

Leu Val Cys Ser Gly Thr Thr Asn His Ser Glu Val Val Arg Val Gin 65 70 75 80

Phe Asp Pro Gin Val Tyr Pro Tyr Ser Asp Leu Leu Ser Val Phe Trp

85 90 95

Ser Arg His Asp Pro Thr Thr Val Asn Arg Gin Gly Gly Asp Val Gly 100 105 110

Thr Gin Tyr Arg Ser Gly He Tyr Tyr Tyr Asn Glu Thr Gin Ala Arg 115 120 125

Leu Ala Gin Glu Ser Lys Glu Ala Lys Gin Leu Glu Phe Lys Asp Lys 130 135 140

Lys Val Val Thr Glu He Leu Pro Ala Lys Arg Phe Tyr Arg Ala Glu 145 150 155 160

Glu Tyr His Gin Gin Tyr Leu Ala Lys Gly Gly Gly Asn Gly Asn Lys 165 170 175

Gin Ser Ala Glu Lys Gly Cys Asn Asp Pro He Arg Cys Tyr Gly 180 185 190

Claims (34)

IT IS CLAIMED:

1. A tranεgenic fruit-bearing plant, compriεing (i) a DNA sequence encoding a product that is effective to reduce ethylene biosynthesis in fruit from the plant, and (ii) a promoter whose expression is induced during fruit ripening or by ethylene synthesiε by εaid fruit, where εaid DNA εequence iε heterologous to said promoter and said DNA sequence is operably linked to said promoter to enable expresεion of εaid product.

2. A tranεgenic plant of claim 1, wherein εaid DNA εequence encodeε S-adenosylmethionine hydrolase.

3. A transgenic plant of claim 1, wherein said DNA sequence encodeε a product selected from the group consisting of aminocyclopropane-1-carboxylic acid (ACC) deaminase, ACC oxidase antiεense molecule, ACC synthase antisense molecule, ACC oxidase cosuppression molecule, and ACC synthase cosuppression molecule.

4. A transgenic plant of claim 1, wherein the promoter is obtained from a gene homologous to a tomato E4 or E8 gene.

5. A transgenic plant of claim 4, wherein the promoter is from a tomato E4 or E8 gene.

6. A transgenic plant of claim 4, wherein the promoter is from a raspberry E4 gene.

7. A transgenic plant of claim 1, wherein the_. promoter is obtained from a gene selected from the group consisting of avocado cellulase gene and tomato polygalacturonase gene.

8. A transgenic plant of claim 1, wherein the promoter is obtained from a gene homologous to a gene εelected from the group consisting of avocado cellulase gene and tomato polygalacturonase gene.

9. A method for modifying ripening fruit of a fruit bearing plant, compriεing, growing the plant of claim 1, to produce a transgenic plant bearing fruit, wherein fruit produced by said plant has an initial burst of ethylene production, followed by a reduction in the level of ethylene synthesiε by said fruit, resulting in a fruit having a modified ripening phenotype.

10. A fruit produced by the plant of claim 1.

11. A method for producing a transgenic fruit- bearing plant, where fruit produced by said plant has a modified ripening phenotype, compriεing introducing into progenitor cellε of the plant (i) a DNA εequence encoding a product effective to reduce ethylene bioεynthesis in fruit from the plant, and (ii) a promoter whoεe expreεsion is induced during fruit ripening or by ethylene syntheεiε by εaid fruit, where εaid DNA sequence is heterologous to said promoter and said DNA sequence is operably linked to said promoter to enable expresεion of εaid product, and growing the transformed progenitor cells to produce a transgenic plant bearing fruit, wherein fruit produced by said plant has an initial burst of ethylene production, followed by a reduction in the level of ethylene syntheεis by said fruit, resulting in fruit having a modified ripening phenotype.

12. A method of claim 11, where said introducing includes transforming progenitor cells of the plant with a selectable vector containing said DNA sequence and said promoter.

13. A method of claim 11, wherein said heterologous DNA sequence encodes S-adenosylmethionine hydrolase.

14. A method of claim 11, wherein said DNA sequence encodes a product selected from the group consisting of aminocyclopropane-1-carboxylic acid (ACC) deaminase, ACC oxidase antisense molecule, ACC synthase antisense molecule, ACC oxidase cosuppression molecule, and ACC synthase cosuppression molecule.

15. A method of claim 11, wherein the promoter is obtained from a gene homologous to a tomato E4 or E8 gene.

16. A method of claim 15, wherein the promoter is from a tomato E4 or E8 gene.

17. A method of claim 15, wherein the promoter is from a raspberry E4 gene.

18. A method of claim 11, wherein the promoter is obtained from a gene selected from the group consiεting of avocado cellulase gene and tomato polygalacturonase gene.

19. A method of claim 11, wherein the promoter is obtained from a gene homologous to a gene selected from the group consisting of avocado cellulase gene and tomato polygalacturonase gene.

20. A method of claim 11, wherein the promoter is isolated by the steps of:

(i) selecting a probe DNA molecule containing a sequence homologous to a region of tomato E4 gene DNA, (ii) contacting the probe with a plurality of target DNA molecules derived from the genome of a selected fruit-bearing plant under conditions favoring specific hybridization between the probe molecule and a target molecule homologous to the probe molecule, (iϋ) identifying a target molecule having a DNA sequence homologous to tomato E4 gene, and

(iv) isolating promoter sequences asεociated with the target molecule.

21. A method of claim 11, wherein the promoter is isolated by the steps of:

(i) selecting a probe DNA molecule containing a εequence homologouε to a region of tomato E8 gene DNA,

(ii) contacting the probe with a plurality of target DNA moleculeε derived from the genome of a εelected fruit-bearing plant under conditionε favoring εpecific hybridization between the probe molecule and a target molecule homologouε to the probe molecule,

(iii) identifying a target molecule having a DNA εequence homologouε to tomato E4 gene, and

(iv) iεolating promoter εequenceε aεεociated with the target molecule.

22. An expression vector for use in transforming plant cells, compriεing

(i) a DNA sequence encoding a product that is effective to reduce ethylene biosynthesis in fruit from a plant, and

(ii) a promoter whose expresεion is induced during fruit ripening or by ethylene synthesiε by said fruit, where said DNA sequence is heterologous to εaid promoter and said DNA sequence is operably linked to said promoter to enable expression of said product.

23. A plant cell expresεion vector of claim 22, wherein said DNA sequence encodes S-adenosylmethionine hydrolase.

24. A plant cell expreεεion vector of claim 22, wherein said DNA sequence encodes a product selected from the group consisting of aminocyclopropane-1-carboxylic acid (ACC) deaminase, ACC oxidase antisense molecule, ACC synthase antisense molecule, ACC oxidase cosuppression molecule, and ACC synthase cosuppression molecule.

25. A plant cell expression vector of claim 22, wherein the promoter is obtained from a gene homologous to a tomato E4 or E8 gene.

26. A plant cell expression vector of claim 25, wherein the promoter is from a tomato E4 or E8 gene.

27. A plant cell expression vector of claim 25, wherein the promoter is from a raεpberry E4 gene.

28. A plant cell expreεεion vector of claim 22, wherein the promoter iε obtained from a gene selected from the group consisting of avocado cellulase gene and tomato polygalacturonase gene.

29. A plant cell expresεion vector of claim 22, wherein the promoter iε obtained from a gene homologous to a gene selected from the group consisting of avocado cellulaεe gene and tomato polygalacturonase gene.

30. A kit for use in plant transformation, comprising the vector of claim 22.

31. A kit of claim 28, where the plant is a fruit bearing plant.

32. A chimeric gene capable of expresεing a polypeptide in a plant, compriεing

(i) a DNA sequence encoding a product that is effective to reduce ethylene biosyntheεiε in fruit from the plant, and (ii) a promoter whose expression is induced during fruit ripening or by ethylene synthesiε by said fruit, where said DNA sequence is heterologous to said promoter and said DNA sequence is operably linked to said promoter to enable expression of said product.

33. A plant cell containing the chimeric gene of claim 32.

34. A plant tranεformation vector containing the chimeric gene of claim 32.