AU696417B2 - Regulation of senescence - Google Patents

Description

I I O95091 P 1/611911),/01990J~l This application relates to novel DNA constructs, plant cells containing such constructs and plants derived therefrom. In particular, it relates to the modification of the senescence process in plants.

The process of plant senescence has been well-studied (see for example, Plant Senescence: Its Biochemistry and Physiology, eds. Thompson et al, 1987). Senescence is a controlled series of biochemical and physiological events comprising the final stage of development. The changes taking place in senescence form a genetically programmed sequence, with close co-ordination at the cell and tissue level. Cells remain viable and show tight metabolic regulation until the end of senescence.

Senescence may be caused by a variety of external factors (including light, temperature, water/minerals, pathogens) or internal factors (including space, light, nutrients, flowering/pollination, growth substances). A genetic switch is triggered which modifies gene expression at the transcriptional and/or post-transcriptional level and induces a change in cell/tissue function resulting in senescence. For example, during leaf senescence, the photosynthetic apparatus is dismantled and leaf function changes from carbon assimilation to nitrogen/phosphorus mobilisation. Senescence involves pigment degradation, proteolysis and nucleic acid breakdown leading to nitrogen redistribution and phosphorus remobilisation to other plant parts. It also involves the respiration of lipids and carbohydrates.

SUBSTITUTE SHEET (RULE 26) [I II I WO 91;/079J( 11(;19 /111 11 In an agricultural context, leaf expansior and senescence are quantified as leaf urea index and leaf area duration, and these factors are known to be major determinants of yield in many crops (Thomas, 1992 in Crop Photosynthesis: Spatial and Temporal Determinants, Baker and Thomas (eds), Elsevier, pages 11-41). The importance of delayed senescence in increasing the yield of determinate crops has been confirmed by studies on several species (for example, Tollenaar and Daynard, 1978, Plant Sci, 78:199-206; Thomas, 1987 in Developmental Mutants in Higher Plants, Thomas and Grierson (eds), Cambridge Univ Press, pages 245-265).

Genetic variation exists for senescence and has, usually incidentally or empirically, been exploited for crop improvement by traditional plant breeding. Variants showing delayed or inoperative senescence are known in a range of species such as maize, sorghum, oats, rice, wheat, soybean, faba bean, fescue, fruit crops and trees. For example, genetic variation exists for symptomF of leaf senescence and genotypes with leaves nich remain green for longer than normal are termed "stay-green" varieties. Stay-green variants are known in cereals, legumes, grasses and fruit species (Thomas and Smart, 1993, Ann Appl Biol, 123:193-219). During senescence some of these variants retain both chlorophyll and photosynthetic competence ("functional" stay-greens). Others keep their chlorophyll but degrade some other components of the photosynthetic apparatus as normal so they cannot utilize the light energy they have harvested ("non-functional" stay greens). Thus measurement SUBSTITUTE SHEET (RULE 26) ~~II II1 ls ,P -a rms~rmnrra~; I WO 95107993 I'CTIG 1i941019901) 3 of greenness in general is not always sufficient to determine the progression of leaf senescence.

Healthy stay-green plants produce a higher yield.

Such plants may also have increased resistance to disease and drought and rossess leaves with higher nutritional quality and attractiveness to grazing animals. Their retention of chlorophyll makes then an ideal source of this pigment for the food industry and ensures that amenity and ornamental plants remain attractive over an e%:ended period.

Although these naturally-occuring genetic variants with contrasting senescence phenotypes exist (differing in tie timing and rate of senescence), the use of such variants in the breeding of improved crop varieties is not entir-ely satisfactory. For example, research has shown that superficially similar stay-greens differ greatly in metabolism and genetic make-up. It is insufficient simply to select stay-greens by eye: potential disadvantages as well as benefits must be considered in crop management and breeding programmes. Normally, loss of green coloration (chlorophyll) directly reflects cessation of photosynthesis. In some stay-greens both processes are simply delayed; in others they occur at the normal time, but more slowly than usual. But in others, chlorophyll is retained despite loss of photosynthetic activity (although leaves retain more protein and lipid). Thus the stay-green character in one genetic line may have only a superficial resemblance to the character in another and may arise from quite different underlying physiological and biochemical modifications.

It is an object of the present invention to SUBSTITUTE SHEET (RULE 26) I WO 95/07993 'CT/GB.19/01990 4 provide a means tc modify senescence at the level of gene expression.

The modification of plant gene expression has been achieved by several methods. The molecular biologist can choose from a range of known methods to decrease or increase gene expression or to a'ter the spatial or temporal expression of a particular gene. For example, the expression of either specific antisense RNA or partial sense RNA has been utilised to reduce the expzession of various target genes in plants (as reviewed by Bird and Ray, 1991, Biotechnology and Genetic Engineering Reviews 9:207-227). These techniques involve the incorporation into the genome of the plant of a synthetic gene designed to express either antisense or sense RNA. They have been successfully used to down-regulate the expression of a range of individual genes, for example those involved in the development and ripening of fruit (Gray et al, 1992, Plant Molecular Biology, 19:69-87). Methods to increase the expression of a target gene have also been developed. For example, additional genes designed to express RNA containing the complete coding region of che target gene may be incorporated into the genome of the plant to "over-express" the gene product. Various other methods to modify gene expression are known; for example, the use of alternative regulatory sequences.

Senescence-related genes having a function in foliar senescence may be classified according to their patterns of expression during leaf development (Smart, 1994, New Phytol, 126:419-448; Thomas, 1994, Reviews in Clinical Gerontology, SUBSTITUTE SHEET (RULE 26) I WO 95/07993 PCT/G94/0I1990 4:5-20). Six broad categories may be recognised: "Housekeeping" genes controlling the primary metabolic activities of viable cells( such as respiration, ribosomal RNA and protein synthesis); Genes expressed early, whose effects become apparent later (for example, homeotic genes and genes encoding mRNAs or proteins such as vacuolar enzymes or zymogens that become active later in the life of the leaf); Genes which encode growth or carbon assimilation components and contribute to the progress of senescence by switching off (such as nuclear and plastid genes for Calvin cycle enzymes and thylakoid proteins); Regulatory genes that are expressed at the initiation of senescence and control its timing and rate of progress; Genes encoding RNAs or proteins induced de novo or showing enhanced expression during senescence (such as enzymes of pigment breakdown); Genes encoding proteins which remobilise storage products (such as enzymes of gluconeogenesis).

According to the present invention there is provided a method of inhibiting or accelerating plant senescence by modifying the expression of at least one senescence-related gene.

A senescence-related gene is a gene which has a role in senescence. The senescence-related gene may be activated during senescence, or may be down-regulated during senescence, or may show an unchanged level of expression during senescence.

SUBSTITUTE SHEET (RULE 26) E SWO 95/07993 IPCTI/G 1941() 1990) 6 Senescence may be inhibited by inhibiting a gene which is normally activated during senescence.

Additionally or alternatively, senescence may be inhibited by increasirng the expression of a gene which is normally down-regulated during senescence.

Senescence may also be delayed or slowed by transforming a plant with a construct in which a promoter from a senescence-activated gene drives expression of a strong senescence antagonist.

Senescence may be accelerated by inhibiting a gene which is normally down-regulated during senescence. Additionally or alternatively, senescence may be accelerated by increasing the expresjion of a gene which is normally activated during senescence.

Additionally or alternatively, senescence may be iihibited or accelerated by respectively inhibiting or increasing the expression of a gene which normally shows an unchanged level of expression during senescence.

The method of the present invention can be applied to any plant, including tomato, lettuce, broccoli, cabbage, carrot, beet, melon, banana, strawberry, wheat, maize rice, canola, rape, sunflower, soybean. Selected plants will show modified senescence phenotypes, which may include one or more of the following characteristics: Prolonged life of the plant or plant part due to delayed or slowed senescence.

Increased yield due to delayed senescence of the leaf and resulting prolonged SUBSTITUTE SHEET (RULE 26) WO 95/0i7993 B94/019901 7 photosynthetic activity.

Increased protein content of fruits and vegetables due to reduced rate of protein breakdown.

Improved quality of leafy vegetables due to the reduced rate of senescence.

Improved disease tolerance due to the presence of less senescing tissue.

Improved tolerance of drought and other stresses.

Improved storage life of the harvested plant or plant part.

Highly-adapted crop in which the senescence process is under external control: onset and rate of senescence can be adapted to the specific environment or other requirements by producing crops with particular characteristics or by inducing those characteristics when needed.

Increased senescence breaking down unwanted plant material more rapidly (for example, avoiding the use of desiccants on crops such as rape) For example, delaying senescence in grain maize, sorghum, wheat or barley can have various beneficial effects on crop phenotype. The resulting increase in leaf area (increased duration of green leaf area) and rise in photosynthetic capacity during grain-filling results in increased yield. Improved stalk integrity (die to delayed senescence in stalk tissue) incredses resistance to pathogenic organisms and allows improved harvesting; yield is again increased. Delaying senescence in early-maturing silage maize or sorghum will maintain leaf integrity and greenness SUBSTITUTE SHEET (RULE 26) i I SWO 95107993 PC'IAI 1191401990 8 throughout the continuing growing season to increase the crop's overall biomass.

Naturally-occurring stay-green variants can be used as parents in crop breeding programmes.

However, the existence of visually identical but physiologically different "functional" and "non-functional" stay-greens complicates their use and the desired effect on crop phenotype is not easily achieved. Also it is uncertain how the stay-green character will behave when crossed into a different genetic background. In contrast, the genetic modification of single genes is a simpler process allowing a higher degree of control of genotype and phenotype. The present invention provides a means of transferring the "modified senescence" trait into elite lines without a prolonged breeding programme which might alter other beneficial traits at the same time. In addition, the generation of improved crop varieties is not limited by the naturally-available genotypes: it is possible to genera-e a different range of allelic forms (for example, those having greatly inhibited or greatly accelerated senescence). Genetic modification of single genes will probably result in a dominant "modified senescence" phenotype, which can be more easily incorporated into traditional breeding programmes.

In one embodiment of the invention, stay-green plant lines could be produced by inhibiting the chlorophyll degradation pathway or by preventing lipid degradation. For example, stay-green silage maize could be produced with improved quality and palatability and with post-harvest stability. The putative pathway of chlorophyll breakdown in SUBSTITUTE SHEET (RULE 26)

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n SWO 95/07993 PCT/G 194/01990 9 senescence involves various enzymes including chlorophyllase, magnesium dechelatase, dioxygenase, proteases and ferredoxin (Matile, 1992). The putative pathway of galactolipid breakdown in senescence involves gluconeogenesis and hence the enzyme pyruvate, orthophosphate dikinase which converts pyruvate to PEP (Matile, 1992).

In another embodiment, low proteolysis plant lines could be produced by down-regulating physiological protein mobilisation or by preventing autolytic degradation. For example, low proteolysis maize lines would have high silage quality and allow improved nitrogen intake and retention by the animal, with a consequent reduction in slurry nitrogen.

Geneti- nodification of single senescence-related genes may also be used in combination with a "gene switch" allowing control of the senescence process according to circumstances. If the relevant senescence-related genes could be switched on or off at will, the timing and progress of senescence and the phenotype of the crop could be directly controlled. For example, induction of senescence ("switching on" senescence) can allow early harvest of the crop.

Modifying the expression of senescence-related genes using a gene switch may also be used to improve the quality of the crop. For example, induction of senescence in late-maturing silage maize or sorghum can result in better quality silage and better harvest timing. Silage consists of about 50% grain and 50% leaf/stem material. For late-maturing silage varieties, there is little dry matter accumulation at the end of the growing SUBSTITUTE SHEET (RULE 26) season as the weather becomes colder. However, the moisture content increases while the crop is left in the field which reduces the quality of the silage. The ability to induce senescence at the time when maximum dry matter has been accumulated can reduce the final moisture content by around concentrating the carbohydrate material and improving silage quality.

According to a further aspect of the present invention, there is provided a method for producing plants having modified senescence characteristics which comprises transformation of plants with a DNA construct adapted to modify the expression of at least one senescence-related gene and subsequent selection of plants in which the senescence process is either inhibited or accelerated.

The expression of the or each senescence-related gene may be either reduced or increased depending on the characteristics desired for the modified plant. "Antisense" or "partial sense" or other techniques may be used to reduce gene expression or expression may be increased, for example, by incorporation ooo of additional senescence-related genes. The additional genes may be designed to give either the same or different spatial and temporal patterns of expression in the plant.

In one aspect the present invention provides a DNA construct adapted to modify the expression of at least one senescence-related gene comprising a Io DNA sequence of at least 20 bases in length occurring in any one of the .•.sequences shown as SEQ ID NO 1 to SEQ ID NO 39 preceded by a 25 transcriptional initiation region operative in plants so that the construct can 0**0 generate RNA in plant cells.

Throughout the description and claims of the specification the word "comprise" and variations of the word, such as "comprising" and "comprises", is not intended to exclude other additives, components, integers or steps.

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A DNA construct according to the invention may be an "antisense" construct generating "antisense" RNA or a "sense" construct (encoding at least part of the functional gene product) generating "sense" RNA. "Antisense RNA" is an RNA sequence which is complementary to a sequence of bases in the corresponding mRNA: complementary in the sense that each base (or the najority of bases) in the antisense sequence (read in the 3' to 5' sense) is capable of pairing with the corresponding base (G with C, A with U) in the mRNA sequence read in the to 3' sense. Such antisense RNA may be produced in the cell by transformation with an appropriate DNA construct arranged to generate a transcript with at least part of its sequence complementary to at least part of the coding strand of the relevant gene (or of a DNA sequence showing substantial homology therewith). "Sense RNA" is an RNA sequence which is substantially homologous to at least part of the corresponding mRNA sequence.

Such sense RNA may be produced in the cell by transformation with an appropriate DNA construct arranged in the normal orientation so as to generate a transcript with a sequence identical to at least part of the coding strand of the relevant gene (or of a DNA sequence showing substantial homology therewith). Suitable sense constructs may b( used to inhibit gene expression (as described in International Patent Publication W091/08299) or to over-express the enzyme.

The constructs of the invention may be inserted into any plant to regulate the expression 1 TI I II SWO 95/07993 P 11T 9-1/019901 12 of one or more senescence-related genes. The constructs may be transformed into any dicotyledonous or monocotyledonous plant.

Depending on the nature of the construct, expression of the senescence-related gene may be increased or reduced, either throughout or at particular stages in the life of the plant.

Generally, as would be expected, gene expression is enhanced only by full-length sense constructs which express RNA homologous to the substantially complete coding region of the gene. Constructs containing an incomplete DNA sequence shorter than that corresponding to the complete gene generally inhibit the expression of the gene, whether they are arranged to express sense or antisense RNA.

Full-length antisense constructs also inhibit gene expression.

In a DNA construct according to the invention, the transcriptional initiation region may be derived from any plant-operative promoter. The transcriptional initiation region may be positioned for transcription of a DNA sequence encoding RNA which is complementary to a substantial run of bases in a senescence-related mPRNA (making the DNA construct a full or partial antisense construct) DNA constructs according to the invention may comprise a DNA sequence at least 10 bases (preferably at least 20 bases) in length for transcription into sense or antisense RNA. There is no theoretical upper limit to the base sequence it may be as long as the relevant mRNA produced by the cell but for convenience it will generally be found suitable to use sequences between 100 and 1000 bases in length. The preparation of such SUBSTITUTE SHEET (RULE 26) II WO 95/07993 /G 194/(01990 13 constructs is described in more detail below.

The sequence of any senescence-related gene may be used in the DNA constructs as suitable genes may be isolated from any plant species. For example, in work leading to this invention we have identified a range of senescence-related genes.

The cDNA of these genes has been cloned and characterised, and may be used to modify the senescence process of plants, including foliar senescence, fruit senescence and senescence of other plant parts. The genes in question are encoded in the following clones: pSENUl, pSENU2, pSENU3, pSENU4, pSENU5, pSEND31, pSEND32, pSEND33, pSEND34, pSEND35, pSENE71 (all isolated from tomato: the pSENU clones correspond to genes which are up-regulated during senescence; the pSEND clones correspond to genes which are down-regulated during senescence; the pSENE clone corresponds to a gene which exhibits even expression during senescence); SEE1, SEE2, SEE3, SEE4, SEE5, SEE6, SEE7, SEES, SEE9, SEC1, SEC2, SED1, SED2 (all isolated from maize; the SEE clones correspond to genes whose expression is enhanced during senescence; the SED clones correspond to genes whose expression is diminished during senescence; the SEC clones correspond to genes whose expression is constant during senescence).

The DNA sequence in a construct according to the invention may be derived from cDNA, genomic DNA or synthesised ab initio.

It is convenient to obtain or derive the DNA sequence from the sequence of any one of the cDNA SUBSTITUTE SHEET (RULE 26) nil I Iq--l- e-"cl I-- I I I Wo '5/07993 PC( .HTI(II1/0199(1 14 clones pSENU1, pSENU2, pSENU3, pSENU4, pSEND31, pSEND32, pSEND33, pSEND34, pSENE71, SEE1, SEE2, SEE3, SEE4, SEE5, SEE6, SEE7, SEE8, SEE9, SEC1, SEC2, SED1, SED2. Full and partial base sequences of some of these clones are set out in SEQ ID NOs 1 to 39. The following clones were deposited at The National Collections of Industrial and Marine Bacteria (23 St Machar Drive, Aberdeen, Scotland, AB2 1RY) under the terms of the Budapest Treaty on 13 July 1393 under the following accession numbers: pSENUl NCIMB 40571 pSENU2 NCIMB 40572 pSENU3 NCIMB 40573 pSENU4 NCIMB 40574 NCIMB 40575 pSEND31 NCIMB 40576 pSEND32 NCIMB 40577 pSEND33 NCIMB 40578 pSEND34 NCIMB 40579 NCIMB 40580 pSENE71 NCIMB 40581 SEE1 NCIMB 40582 SEE2 NCIMB 40584 SEE3 NCIMB 40570 SEE4 NCIMB 40583 cDNA clones similar to pSENUl-5 or pSEND31-35 or pSENE71 may be obtained from the mRNA of senescent tomatoes leaves. cDNA clones similar to SEE1-4 may be obtained from the mRNA of senescent maize leaves. In this way may be obtained sequences coding for the whole, or substantially the whole, of the mRNA produced by the cDNA clones.

An alternative source of DNA for the base sequence for transcription is a suitable genomic SUBSTITUTE SHEET (RULE 26)

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WO 95107993 I' I094/o 1990 sequence encoding a senescence-related mRNA. This gene may differ from the cDNA in that introns may be present. The introns are not transcribed into mRNA (or, if so transcribed, are subsequently cut out). When using such a gene as the source of the base sequence for transcription it is possible to use either intron or exon regions both are useful in modifying the expression of the gene according to the invention. Oligonucleotide probes or the cDNA clone may be used to isolate the actual senescence-related gene(s) by screening genomic DNA libraries. Such genomic clones may include control sequences operating in the plant genome. Thus it is also possible to isolate promoter sequences which may be used to drive expression of the senescence-related protein or any other protein.

These promoters may be particularly responsive to senescence-related events and conditions.

Senescence-related promoters may be used to drive expression of any target gene.

A further way of obtaining a suitable DNA base sequence for transcription is to synthesise it ab initio from the appropriate bases, for example using the sequences in SEQ ID NOs 1 to 39The proposed full sequence of SENU2 by comparison with clone C14 is shown as SEQ ID NO 5 as a guide.

Recombinant DNA constructs according to the present invention may be made using standard techniques. For example, the DNA sequence for transcription may be obtained by treating a vector containing said sequence with restriction enzymes to cut out the appropriate segment. The DNA sequence for transcription may also be generated by annealing and ligating synthetic oligonucleotides SUBSTITUTE SHEET (RULE 26) PII II- WO 95/07993 I'"I'IG;941/19910 16 or by using synthetic oligonucleotides in a polymerase chain reaction (PCR) to give suitable restriction sites at each end. The DNA sequence is then cloned into a vector containing upstream promoter and downstream terminator sequences. If antisense DNA is required, the cloning is carried out so t'.it the cut DNA sequence is inverted with respect to its orientation in the strand from which it was cut.

In a construct expressing antisense RNA, the strand that was formerly the template strand becomes the coding strand, and vice versa. The construct will thus encode RNA in a base sequence which is complementary to some or all of the sequence of the senescence-related mRNA. Thus the two RNA strands are complementary not only in their base sequence but also in their orientations to In a construct expressing sense RNA, the template and coding strands retain the assignments and orientations of the original plant gene.

Constructs expressing sense RNA encode RNA with a base sequence which is homologous to part or all of the sequence of the mRNA. In constructs which express the functional senescence-related gene product, the whole of the coding region of the gene is linked to transcriptional control sequences capable of expression in plants.

For example, constructs according to the present invention may be made as follows. A suitable vector containing the desired base sequence for transcription (such as pSEND35) is treated with restriction enzymes to cut the SUBSTITUTE SHEET (RULE 26) I I WO 95/07993 IT(;IG94/01991 17 sequence out. The DNA strand so obtained is cloned (if desired, in reverse orientation) into a second vector containing the desired promoter sequence for example cauliflower mosaic virus 35S RNA promoter or the promoter of the pSENU1 gene or other genes which are switched on at the onset of senescence) and the desired terminator sequence (for example the 3' of the Agrobacterium tumelaciens nopaline synihase gene, the nos 3' end).

The transcriptional initiation region (or promoter) operative in plants may be a constitutive promoter (such as the 35S cauliflower mosaic virus promoter) or an inducible or developmentally regulated promoter (such as fruit-specific promoters), as circumstances require. Use of a constitutive promoter will tend to affect the senescence process in all parts of the plant, while use of a tissue specific promoter allows more selective control of gene expression and affected functions as the antisense or sense RNA is only produced in the organ in which its action is required. For example, fruit development and/or ripening-specific promoters that could be used include the ripening-enhanced polygalacturonase promoter (International Patent Publication Number W092/08798), the E8 promoter (Diekman Fischer, 1988, EMBO, 7:3315-3320) and the fruit specific 2A11 promoter (Pear et al, 1989, Plant Molecular Biology, 13:639-651). Inducible promoters may also be used. According to a further aspect of the invention, the transcriptional initiation region in the DNA construct comprises a "gene switch".

Several gene promoter sequences are known SUBSTITUTE SHEET (RULE 26) I r r WO 95/07993 I'CT/G 1i94/1990 18 which are responsive to an applied exogenous chemical inducer. This enables external control of expression of the gene controlled by the inducible promoter. For example, European patent application publication number EPA 332104 (published 18th September 1989) describes chemically regulatable DNA sequences isolated from the pathogenesis-related (PR) protein gene; International patent application publication numbers W090/08826 (published 9 August 1990) and W093/01294 (published 21 January 1993) describe a chemically inducible gene promoter sequence isolated from a 27kd subunit of the maize glutathione-S-transferase gene (GST II-27); International patent application number GB93/00764 describes a chemically-inducible gene expression cassette including a regulator protein (such as the Aspergillus nidulans alcR protein) and an inducible promoter (such as the A nidulans alcA promoter).

Such chemically-inducible promoter sequences may be used in "gene switches" to regulate transcription of an associated DNA sequence (or "target gene") in plants or plant tissue.

The gene switch may be a positive switch, where the inducible promoter directly controls the target gene. In the presence of the chemical inducer, the target gene is switched on and the encoded protein is expressed.

For example, the inducible GST II-27 promoter can be operatively linked to one or more target genes to give a chemically switchable construct: expression of the target gene(s) is controlled by application of an effective exogenous inducer. The SUBSTITUTE SHEET (RULE 26)

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IIIIB~*lp~"'"' SWO 95107993 PCT/G1194/01990 19 gene switch construct may be inserted into a plant by transformation. The inducible GST II-27 promoter is functional in both monocotyledons and dicotyledons, and in a variety of tissues including roots, leaves, stems and reproductive tissues.

Effective inducers for use with the GST II-27 promoter include N,N-diallyl-2,2-dichloroacetamide (common name: dichloramid); benzyl-2-chloro-4-(trifluoromethyl) (common name: flurazole); naphth .lene-1,8-dicarboxylic anhydride; 2-dichloromethyl-2-methyl-1,3-dioxolane and several others as described in International patent application publication numbers W090/08826 and W093/01294. The contents of the said applications are incorporated herein by reference.

Alternatively, the gene switch may be a negative switch, where the inducible promoter indirectly controls the target gene via a repressor/operator system. In the presence of the chemical inducer, the target gene is switche. off and the encoded protein is not expressed.

For example, negative gene switches are described in International patent application publication numbers W090/08829, W090/08827 and W090/08830 (all published 9 August 1990). The contents of the said applications are incorporated herein by reference. The switch comprises a chemically-inducible promoter driving expression of a repressor gene encoding a repressor protein, and a promoter containing an operator sequence and driving expression of a target gene.

(The operator region may be introduced into promoter(B) by biotechnological techniques). If SUBSTITUTE SHEET (RULE 26)

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I L WO 95/07993 PCTG1194101990 present, the repressor protein binds to the operator sequence, preventing expression of the target gene. In the absence of inducer, promoter is not active and the repressor protein is not expressed: hence the target gene is expressed. In the presence of the chemical inducer, the repressor protein prevents expression of the target gene.

Promoter may be GST II-27 or any other chemically-inducible promoter sequence. The repressor gene/operator sequences may be taken from the E coli lac operon.

Senescence-related gene expression (and hence senescence characteristics) may be modified to a greater or lesser extent by controlling the degree of sense or antisense mRNA production in the plant cells. This may be done by suitable choice of promoter sequences, or by selecting the number of copies or the site of integration of the DNA sequences that are introduced into the plant genome. For example, the DNA construct may include more than one DNA sequence encoding a senescence-related gene or more than one recombinant construct may be transformed into each plant cell.

It is also possible to modify the activity of the senescence-related gene while also modifying the activity of one or more other plant genes. For example, a first plant may be individually transformed with a senescence-related DNA construct and then crossed with a second plant which has been individually transformed with a construct adapted to modify the expression of another gene. As a further example, single plants may be either consecutively or co-transformed with SUBSTITUTE SHEET (RULE 26) WO 95107993 PCT/GB94/01990 21 senescence-related DNA constructs and with appropriate constructs for modification of the other gene(s). An alternative example is plant transformation with a senescence-related DNA construct which itself contains an additional gene for modification of the activity of the orher gene(s). The senescence-related DNA constructs may contain sequences of DNA for regulation of the expression of the other gene(s) locted adjacent to the senescence-related sequences. These additional sequences may be in either sense or antisense orientation as described in International patent application publication number W093/23551 (single construct having distinct DNA regions homologous to different target genes). By using such methods, the benefits of modifying the activity of the senescence-related gene may be combined with the benefits of modifying the activity of other genes.

The senescence characteristics of plants may be modified by transformation with a DNA construct according to the invention. The inventicon further provides plant cells containing constructs of the invention; plants derived therefrom showing modified senescence characteristics; and seeds of such plants.

A DNA construct of the invention is transformed into a target plant cell. The target plant cell may be part of a whole plant or may be an isolated cell or part of a tissue which may be regenerated into a whole plant. The target plant cell may be selected from any monocotyledonous or dicotyledonous plant species. Suitable plants include any fruit-bearing plant (such as tomatoes, mangoes, peaches, apples, pears, strawberries, SUBSTITUTE SHEET (RULE 26) -r I LP -a I ~I WO 95107993 1191/019911 22 bananas and melons) and other important crops such as maize, rice, wheat, barley, sorghum, sugar beet, canola, rape, soybean. For any particular plant cell, the DNA sequence used in the transformation construct may be derived from the same plant species, or may be derived from any other plant species (as there will be sufficient ,equence similarity to allow modification of related enzyme gene expression).

Constructs according to the invention may be used to transform any plant using any suitable transformation technique to make plants according to the invention. Both monocotyledonous and dicotyledonous plant cells may be transformed in various ways known to the art. In many cases such plant cells (particularly when they are cells of dicotyledonous plants) may be cultured to regenerate whole plants which subsequently reproduce to give successive generations of genetically modified plants. Any suitable method of plant tranE ir-nation may be used. For example, dicotyledonous p. ants such as tomato and melon may be transformed by Agrobacterium Ti plasmid technology, such as described by Bevan (1984, Nucleic Acid Research, 12:8711-8721) or Fillatti et al (Biotechnology, July 1987, 5:726-730). Such transformed plants may be reproduced sexually, or by cell or tissue culture.

The invention will now be described by way of example only, with reference to the SEQUENCE LISTING and to the drawings in which: Figure 1 is a diagram illustrating the construction of a pSENUl sense construct.

Figure 2 is a diagram illustrating the SUBSTITUTE SHEET (RULE 26) ~Crr~~ WO 95/07993 'CT/G 1194/01990 23 construction of a pSENU1 antisense construct.

Figure 3 is a diagram illustrating the construction of a pSENUS sense construct.

Figure 4 is a diagram illustrating the construction of a pSENU5 antisense construct.

EXAMPLE 1 Characterisation of tomato senescence-related clones RNA was extracted from tomato plants at four stages of senescence: mature green (no yellowing), onset (first visible sign of colour loss), mid (about 30% colour loss) and advanced (completely yellow). A tomato leaf senescence cDNA library was constructed using polyA RNA from the onset and mid stages of senescence in the ration 4:1. A Stratagene lambda Uni-ZAP XR cDNA library was generated with 1x10 6 pfu, average insert size 0.9kB. Amplification gave 500,000 clones which were differentially screened (Hodge et al, 1992, Plant Journal, 2:257-260) using mature green and onset/mid polyA single-stranded cDNA. Genes differentially expressed during leaf senescence in tomato were isolated and characterised. Details of the cDNA clones are given below.

pSENU1 pSENU1 (also known as clone 4S1) is a cDNA of approximately 1.0kB, encoding a mRNA of approximately 1.4 kB. The mRNA encoded by pSENU1 is expressed during the onset of senescence in tomato leaves.

SUBSTITUTE SHEET (RULE 26) I SWO 95/07993 PCTI/194/01990 24 pSENU1 encodes a protein of unknown function.

The pSENU1 DNA sequence does not show any significant homology to sequences in publicly-available sequence databases.

The sequence of pSENU1 is shown as SEQ ID NO 1. The clone was deposited at The National Collections of Industrial and Marine Bacteria (Scotland) on 13 July 1993 under the accession number NCIMB 40571.

pSENU2 pSENU2 (also known as clone 8S1) is a cDNA of approximately 1.2kB, encoding a mRNA of approximately 1.4 kB. The mRNA encoded by pSENU2 is expressed during the onset of senescence in tomato leaves.

The pSENU2 sequence exhibits 100% homology with C14 cDNA induced in unripe tomato fruit in response to low temperature. DNA sequence analysis indicated that C14 mRNA encodes a polypeptide with a region that is homologous to the plant thiol proteases actinidin and papain and to animal thiol protease cathepsin H (Schaffer and Fischer, 1988, Plant Physiol, 87:431-436). A similar thiol protease gene is also expressed in pea ovaries during senescence (Granell et al, 1992, Plant J, 2:907-915). We have now found that this kind of protease is also expressed during leaf senescence.

This protease may play an important role in the degradation of peptides at the onset and during foliar senescence.

Partial sequences of pSENU2 are shown as SEQ SUBSTITUTE SHEET (RULE 26) ii i- I- I~ I I 1 I- li- WO95/07993 PCTI/(19./01990 ID NO 2 end) to SEQ ID NO 4 end). The proposed full sequence of SENU2 by comparison with clone C14 is shown as SEQ ID NO 5. The clone pSENU2 was deposited at The National Collections of Industrial and Marine Bacteria (Scotland) on 13 July 1993 under the accession number NCIMB 40572.

pSENU3 pSENU3 (also known as clone 77S3) is a cDNA of 1.1982kB, encoding a mRNA of approximately 1.4 kB.

The mRNA encoded by pSENU3 is expressed during the onset of senescence in tomato leaves.

The pSENU3 sequence exhibits 70% homology with oryzain gamma, a cysteine proteinase expressed in rice seeds and induced by gibberellin, GA3 (Watanabe et al, 1991, J Biol Chem, 266:16897-16902). We have now identified a related gene (pSENU3) expressed in leaves.

The complete sequence of pSENU3 is shown as SEQ ID NO 6. The clone was deposited at The National Collections of Industrial and Marine Bacteria (Scotland) 1 13 July 1993 under the accession number NC '3 40573.

pSENU4 pSENU4 (also known as clone 73S7) is a cD' 0.525kB, encoding a mRNA of approximately 0.7kr The clone is dc- ie-stranded from base 179 to 323; the rest is single-stranded. The mRNA en.

by pSENU4 is expressed during the onset of senescence in tomato leaves.

SUBSTITUTE SHEET (RULE 26) I 9 B WO 95/07993 PC'/17GB94/01990 26 The predicted amino acid sequence encoded by pSENU4 matches that originally determined by Lucas et al (1985, EMBO J, 4:2745-2749) for the tomato extracellular pathogenesis related protein P14.

The P14 protein (PR14_LYCES) contains two isomers, P4 and P6 (van Kan et al, 1992, Plant Mol Biol, 20:513-527). P6 is 15.5kD and is serologically related to the PR1 protein family of tobacco, but has not been assigned a function. The pSENU4 sequence is homologous to the cDNA encoding P6 (clone LEPRP6) which has been isolated from Cladosporium fulvum infected tomato, but pSENU4 lacks 160bp at the 5'end of the P6 cDNA. An identical cDNA (clone LEP1P14A) has also been isolated from ethylene treated tissue (Vera P and Tonero P, unpublised data) which encodes a protein named Pl(Pl4a).

The complete sequence of pSENU4 is shown as SEQ ID NO 7. The clone was deposited at The National Collections of Industrial and Marine Bacteria (Scotland) on 13 July 1993 under the accession number NCIMB 40574.

(also known as clone 72S3) is a cDNA of 0.847kB, encoding a mRNA of approximately The mRNA encoded by pSENU5 is expressed during the onset of senescence in tomato leaves.

encodes a protein of unknown function.

The pSENU5 DNA sequence does not show any significant homology to sequences in publicly-available sequence databases.

SUBSTITUTE SHEET (RULE 26) WO 95/(,,993 t990 27 The complete sequence of pSENU5 is shown as SEQ ID NO 8. The clone was deposited at The National Collections of Industrial and Marine Bacteria (Scotland) on 13 July 1993 under the accession number NCIMB 40575.

pSEND31 pSEND31 (also known as clone 1M4) is a cDNA of approximately 0.9kB, encoding a mRNA of approximately 1.0kB. The mRNA encoded by pSEND31 is expressed in green leaves of tomatoes plants but at the onset of senescence its expression is switched off.

The pSEND31 sequence exhibits 100% homology to the tomato cDNA clone TAS14 which is inducible by salt stress and ABA in tomato seedlings (Godoy et al, 1990, Plant Mol Biol, 15:695-705). Southern analysis suggests that there is one gene per haploid genome. We now show this gene is specifically reduced during tomato leaf senescence.

Partial sequences of pSEND31 are shown as SEQ ID NO 9 end) to SEQ ID NO 10 (towards the 3' end). The proposed full sequence of SEND31 by comparison with clone TAS14 is shown as SEQ ID NO 11. The clone pSEND31 was deposited at The National Collectioins of Industrial and Marine Bacteria (Scotland) on 13 July 1993 under the accession number NCIMB 40576.

pSEND32 pSEND32 (also known as clone 8M2) is a cDNA of 0.8kB, encoding a mRNA of approximately 0.6kB. The SUBSTITUTE SHEET (RULE 26) I C WO 95107993 PCT/G 94/019!)90 28 mRNA encoded by pSEND32 is expressed in green leaves of tomatoes plants but at the onset of senescence its expression is switched off.

pSEND32 encodes a protein of unknown function.

The pSEND32 DNA sequence does not show any significant homology to sequences in publicly-available sequence databases.

The sequence of pSEND32 is shown as SEQ ID NO 12. The clone was deposited at The National Collections of Industrial and Marine Bacteria (Scotland) on 13 July 1993 under the accession number NCIMB 40577.

pSEND33 pSEND33 (also known as clone 59S3) is a cDNA of approximately 0.8kB, encoding a mRNA of approximately 0.6kB. The mRNA encoded by pSEND33 is expressed in green leaves of tomatoes plants but at the onset of senescence its expression is switched off.

The pSEND33 sequence exhibits 60% homology to ferredoxin-1 of pea and spinach. The amino acid sequence of plant ferredoxins is highly conserved.

Plant leaves contain at least two distinct forms of chloroplast-type ferredoxins. Ferredoxin-1 appears more closely related to other angiosperm ferredoxins. Ferredoxin-1 is encoded by a single gene in pea (Elliott et al, 1989, Plant Cell, 1:681-690).

pSEND33 may encode ferredoxin. We have now shown that expression of this gene is specifically SUBSTITUTE SHEET (RULE 26) I 'I C .41'C* I P WO 95/07993 IT/l'(71194/0)1990 29 reduced during tomato leaf senescence. The down-regulation of ferredoxin during senescence may be counteracted by overexpression of the pSEND33 gene. This may lead to prolonged photosynthesis and increased yield of plants.

The sequence of pSEND33 is shown as SEQ ID NO 13. The clone was deposited at The National Collections of Industrial and Marine Bacteria (Scotland) on 13 July 1993 under the accession number NCIMB 40578.

pSEND34 pSEND34 (also known as clone 91S3) is a cDNA of 0.558kB, encoding a mRNA of approximately 0.6kB.

The mRNA encoded by pSEND34 is expressed in green leaves of tomatoes plants but at the onset of senescence its expression is switched off.

The pSEND34 sequence exhibits 90% homology to the potato photosystem II 10kD polypeptide at both the nucleotide and the predicted amino acid level (Eckes et al, 1986, Mol Gen Genet, 205:14-22).

Homology with the same protein from spinach and Arabidopsis is about 80% (Lautner et al, 1988, J Biol Chem, 263:10077-10081; Gil-Gomez et al, 1991, Plant Mol Biol, 17:517-522). We have now shown that this gene is specifically reduced during tomato leaf senescence.

The sequence of pSEND34 is shown as SEQ ID NO 14. The clone was deposited at The National Collections of Industrial and Marine Bacteria (Scotland) on 13 July 1993 under the accession number NCIMB 40579.

SUBSTITUTE SHEET (RULE 26) -L I I r- WO 95/07993 PCIT/; B94/01990 (also known as clone 72S6) is a cDNA of approximately 0.7kB. The mRNA encoded by is expressed in green leaves of tomatoes plants but at the onset of senescence its expression is switched off.

encodes a protein of unknown function.

The pSEND35 DNA sequence does not show any significant homology to sequences in publicly-available sequence databases.

The sequence of pSEND35 is shown as SEQ ID NO The clone was deposited at The National Collections of Industrial and Marine Bacteria (Scotland) on 13 July 1993 under the accession number NCIMB 40580.

(11) pSENE71 pSENE71 (also known as clone 75S3) is a cDNA of 0.6kB. The mRNA encoded by pSENE71 is expressed in green leaves of tomatoes plants and during senescence.

pSENE71 encodes a protein of unknown function.

The pSENE71 DNA sequence does not show any significant homology to sequences in publicly-available sequence databases.

The 5' sequence of pSENE71 is shown as SEQ ID NO 16. The clone was deposited at The National Collections of Industrial and Marine Bacteria (Scotland) on 13 July 1993 under the accession SUBSTITUTE SHEET (RULE 26) ey II~1-" WO 95/07993 ICT/'GB94/019 31 number NCIMB 40581.

EXAMPLE 2 Characterisation of maize senescence-related clones

SUMMARY

Leaf senescence after pollen shed was studied in two maize lines. Relati;e chlorophyll content, photosystem II efficiency (determined by analysis of chlorophyll fluorescence) and photosynthetic CO 2 fixation (measured by infrared gas analyser) declined during senescence. Statistical analysis of the fitted curves revealed that yellowing in the first line was significantly delayed compared with the second line, but the decline in photosynthesis occurred simultaneously in the two lines. Western blotting detected a transition point du- 7ng senescence when pronounced quantitative and qualitative changes occurred in a number of leaf proteins. This point, coinciding with the onset of visible senescence, was delayed in the first line.

Changes in the complement of translatable mRNAs were apparent earlier chan alterations in pigment or protein levels. A cDNA library was constructed from the poly(A) RNA of leaves judged to be in the early stages of senescence and differential screening was employed to isolate senescencerelated clones, which were investigated further by northern analysis. Partial sequencing, followed by comparison with all known sequences in the GenEMBL database, indicated that a number of cDNAs were related to genes of known identity, including oryzain, pyruvate, orthophosphate dikinase and ferredoxin I, while others showed similarity to SUBSTITUTE SHEET (RULE 26) I I SWO 95/07993 PCT/CBg4/019901 32 cDNAs of unknown function, or did not exhibit any significant homology.

EXPERIMENTAL DETAILS Non-destructive measurements of chlorophyll fluorescence and the level of greenness were made on ear leaves from two maize genotypes (X and Ear leaves were harvested at intervals from days before pollen shed until 35 days after pollen shed. The data indicated a significant decrease in the chlorophyll fluorescence parameter and greenness between 20 and 25 days after pollen shed (daps) for Y, and between 25 and 30 daps for X.

The decrease in the photosynthetic rate with increasing leaf age is reflected in the increase in the sub-stomatal CO 2 concentration with age in both genotypes. Western analysis using pziteins extracted from the same leaves gave a pattern of bands obtained with different antibodies which confirmed that a change occurs between 20 and daps for Y and between 25 and 30 daps for X.

Preliminary results of cell-free translation using total RNA extracted from the same leaves suggested that subtle increases in the intensity of certain bands are detectable 10 aays after pollen shed, although more obvious changes are not seen until 20-25 days after pollen shed.

The evidence from the physiological and molecular data suggested that protein and RNA changes occur before visible senescence takes place. Two cDNA libraries were therefore made: one from RNA extracted from leaves 10-20 days after pollen shed (initiation/early senescence) and the other from RNA extracted from leaves 25 days a-ter pollen shed (mid senescence) SUBSTITUTE SHEET (RULE 26) ralII I I i WO 95107993 lPCT/GB94/01990 33 Total RNA was extracted from leaves at the appropriate stages, polyA RNA was purified and cDNA made using a Pharmacia cDNA Synthesis Kit.

Two cDNA libraries (early and mid senescence) were constructed in lambda gtlO (average insert size 1.3 kB). Senescence-related cDNA clones were identified by differential screening, using RNA from leaves at pollen shed and RNA from the same stages as were selected to make the library to provide the necessary probes.

Clones showing stronger hybridisation with a senescing leaf cDNA probe than with a mature green leaf cDNA probe were further characterised. Both lambda minipreps and PCR were utilised to isolate and determine the size of cDNA inserts. Duplicate cDNA clones were identified by Southern analysis.

A number of independent cDNA clones were used as probes on Northern analysis to investigate the level of mRNA in different ages of leaves. The level of mRNA increases with leaf age in genotype Y but not in X for a number of cDNA clones (including SEE4). This s, ggests that these messages become more abundant in normal senescence but that their expression is blocked in some way in the stay-green genotype X. Other messages, such as those hybridising to the cDNAs SEE1 and SEE2, increase with leaf age in both genotypes. These mRNAs may form part of the senescence syndrome which occurs on schedule in both genotypes.

SENESCENCE-RELATED GENES Details of some of the cDNAs (including their approximate sizes and sizes of the corresponding wridrAs) are given below. End-sequencing of the SUBSTITUTE SHEET (RULE 26)

I

I I SWO 95/07993 PCT/GB94/01990 34 cDNAs allowed comparison with known sequences in the GenEMBL database. Some homologies were found.

The cDNA SEE1 shows homology to genes for two thiol proteases, oryzain gamma from rice and aleurain from barley. The cDNA SEE2 shows some homology to a castor bean vacuolar processing enzyme. The cDNA SEE3 is identical to part of the maize pyruvate, orthophosphate dikinase mRNA while the cDNA SEE4 shows homology to maize and Silene ferredoxin mRNAs.

Proteases and ferredoxin are thought to play a part in chlorophyll breakdown during senescence, while pyruvate, orthophosphate dikinase has a role in gluconeogenesis, which has been suggested to occur during galactolipid breakdown in senescence.

Hence the maize cDNAs represent senescence-related genes and may be used to modify the senescence process.

SEE1 (also known as clone p16.4) SEE1 is a senescence-enhanced cDNA clone of approximately 1.7kB, encoding a mRNA of approximately 1.2kB. The mRNA encoded by SEE1 increases in abundance during maize leaf senescence.

SEE1 shows homology to genes for two thiol proteases: oryzain gamma from rice (GenEMBL ID code OSOZC; 77.6% identity in a 304bp overlap) and aleurain from barley (GenEMBL ID code HVLEU; 77.9% identity in a 222bp overlap). A more detailed comparison of the SEE1 sequence with the rice oryzain gamma DNA sequence (Watanabe et al, 1991) SUBSTITUTE SHEET (RULE 26) r WO 95/07993 PCT/GB94/01990 shows a 78.9% identity over 1417 base pairs, with homology over 607 base pairs at the 5' end and 74% homology over 665 base pairs at the 3' end.

The clone SEE1 may thus encode a protease.

This is supported by the finding that a 1kB DNA fragment is amplified by PCR when one of the primers used is derived from a region which is conserved in a range of thiol proteases.

The sequence of SEE1 is shown as SEQ ID NO 17.

It is 1442 base pairs in length with a single long open reading frame between bases 78 to 1160. The predicted amino acid sequence suggests that the encoded protein is 360 amino acids long with a molecular weight of 39 kDa. There are conserved motifs in the protein sequence: a putative vacuolar signal, a Cys active site, a His active site and an Asn active site. The predominantly hydrophilic protein sequence suggests that the protein is soluble. At the protein level, the SEE1 sequence shows 85.6% identity over 355 amino acids to the thiol protease aleurain precursor and 83.7% identity over 363 amino acids to the oryzain gamma precursor.

The SEE1 clone was deposited at The National Collections of Industrial and Marine Bacteria (Scotland) on 13 July 1993 under the accession number NCIMB 40582.

SEE2 (also known as clone D20.2) SEE2 is a senescence-enhanced cDNA clone of approximately 1.3kB, encoding a mRNA of approximately 1.8kB. The mRNA encoded by SEE2 SUBSTITUTE SHEET (RULE 26) lll IIII -lC

I

SWO 95/07993 PCT/G B94/01)90 36 increases in abundance during maize leaf senescence.

SEE2 shows homology to a castor bean vacuolar processing enzyme (Hara-Nishimura et al, 1993): 67% homology over 374 base pairs at the 5' end (and at the protein level, 75% homology over 130 amino acids at the 5' end) but no homology over 431 base pairs at the 3' end.

Partial sequences of SEE2 are shown as SEQ ID NO 18 (isolated using a T7 primer) and SEQ ID NO 19 (isolated using a T3 primer). The clone SEE2 was deposited at The National Collections of Industrial and Marine Bacteria (Scotland) on 13 July 1993 under the accession number NCIMB 40584.

SEE3 (also known as clone pSEl) SEE3 is a senescence-enhanced cDNA clone of approximately 2.5kB, encoding a mRNA of approximately 3.4kB. The mRNA encoded by SEE3 increases in abundance during maize leaf senescence.

The SEE3 sequence exhibits 100% identity to the maize pyruvate, orthophosphate dikinase mRNA (GenEMBL ID code ZMPOD) in a 141bp overlap. A more detailed comparison to the dikinase (Matsuoka et al, 1988) shows a 91% homology over 132 base pairs at the 5' end and an 89% homology over 262 base pairs at the 3' end. SEE3 also exhibits 97.7% identity to the maize pyruvate, orthophosphate dikinase gene, exons 2-19 (GenEMBL TD code ZMPPDK2) in a 128 bp overlap.

SUBSTITUTE SHEET (RULE 26) I I WOg 95/079)93 PICT/GB94/01990 37 The sequence of SEE3 has an internal EcoRI site, and partial sequences of SEE3 are shown as SEQ ID NOs 20 to 23 (SEQ ID NO 20 being the most and SEQ ID NO 23 being the 3' end). The clone SEE3 was deposited at The National Collections of Industrial and Marine Bacteria (Scotland) on 13 July 1993 under the accession number NCIMB 40570.

SEE4 (also known as clone p20.0) SEE4 is a senescence-enhanced cDNA clone of approximately 0.9kB, encoding a mRNA of approximately 1.2kB. The mRNA encoded by SEE4 increases in abundance during maize leaf senescence.

The SEE4 sequence exhibits homology to the following ferredoxin mRNAs: maize ferredoxin I isoprotein mRNA, pFD1 (GenEMBL ID code ZMFD1; 80.6% identity in a 170bp overlap); maize ferredoxin I isoprotein mRNA, pFD1' (GenEMBL ID code ZMFD1P; 80.6% identity in a 170bp overlap); maize ferredoxin isoprotein mRNA, pFD5 (GenEMBL ID code 81.8% identity in a 159bp overlap); maize ferredoxin III isoprotein mRNA (GenEMBL ID code ZMFD3; 67.9% identity in a 140bp overlap); Silene pratensis mRNA for ferredoxin precursor (GenEMBL ID code SPFER1; 67.5% identity in a 120bp overlap). A more detailed comparison of the SEE4 sequence with the ferredoxin I sequence (Hase et al, 1991) shows no homology over 261 base pairs at the 5' end and 79% homology over 247 base pairs at the 3' end (with 67% homology at the protein level over 52 amino acids at the 3' end).

The 5' sequence of SEE4 is shown as SEQ ID NO SUBSTITUTE SHEET (RULE 26) 38 24 and the 3' sequence as SEQ ID NO 25. The clone was deposited at The National Collections of Industrial and Marine Bacteria (Scotland) on 13 July 1993 under the accession number NCIMB 40583.

OTHER CLONES (pl6.1) shows homology to a maize catalase (Abler and Scandalios, unpublished): 87% over 79 base pairs at the 3' end. The 3' sequence of SEE5 is shown as SEQ ID NO 26.

SEE6 (p16.3) shows homology to a maize cab-1 (Sullivan et al, 1989): 97% over 270 base pairs at the 5' end and 98% over 349 base pairs at the 3'end. The 5' sequence of SEE6 is shown as SEQ ID NO 27 and the 3' sequence as SEQ ID NO 28.

SEE7 (p16.5) shows homology to a maize GRP (Didierjean et al, 1992): 61% over 198 base pairs at the 5' end but no homology over 450 base pairs at the 3' end. The 5' sequence of SEE7 is shown as SEQ ID NO 29 and the 3' sequence as SEQ ID NO SEE8 (p16.6) shows no homology to known sequences over 388 base pairs at the 5' end. The partial sequence of SEE8 is shown as SEQ ID NO 31 (isolated with the T3 primer).

SSEE9 (p16.11) shows no homology to known sequences over 209 base pairs at the 5' end and over 390 base pairs at the 3' end. Partial sequences of SEE9 are is shown as SEQ ID NO 32 (isolated with the T7 primer) and SEQ ID NO 33 (isolated with the T3 primer).

r WO 95/07993 P('T/G1194/01990 39 SEC1 (p16.7) shows homology to an Arabidopsis thaliana PS1 typ; III cab (Wang, Zhang and Goodman, unpublished): 79% homology over 136 base pairs at the 5' end. The partial sequence of SEC1 is shown as SEQ ID NO 34 (isolated with the T3 primer).

SEC2 (p16.9) shows no homology to known sequences over 151 base pairs at the 5' end and over 385 base pairs at the 3' end. The 5' sequence of SEC2 is shown as SEQ ID NO 35 and the 3' sequence as SEQ ID NO 36.

SED1 (p16.13) shows homology to an Arabidopsis thaliana AP52 ATP sulfurylase (Leustek, unpublished): 62% homology over 282 base pairs at the 5' end but no homology over 444 base pairs at the 3' end. The 5' sequence of SED1 is shown as SEQ ID NO 37 and the 3' sequence as SEQ ID NO 38.

SED2 (p20.3) shows homology to an Arabidopsis thaliana cDNA 20D6T7 (Newman, unpublished): 68% over 131 base pairs at the 3' end (and at the protein level, 85% over 26 amino acids at the 3' end). The partial sequence of SED2 is shown as SEQ ID NO 39 (isolated with the T7 primer).

EXAMPLE 3 Construction of antisense RNA vectors with the CaMV 35S promoter.

A vector is constructed using sequences corresponding to a restriction fragment obtained from a senescence-related cDNA clone described in Example 1 or 2 and is cloned into the vectors GA643 SUBSTITUTE SHEET (RULE 26) I WO 95/07993 '(CT/G194/01990 (An et al, 1988, Plant Molecular Biology Manual A3: 1-19) or pDH51 (Pietrzak et al, 1986, Nucleic Acids Research, 14:5875-5869) which has previously been cut with a compatible restriction enzyme(s). A restriction fragment from the senescence/pDH51 clone containing the promoter, the senescence-related clone fragment and other pDH51 sequence is cloned into SLJ44026B or SLJ44024B (Jones et al, 1990, Transgenic Research, 1) or a Binl9 (Bevan, 1984, Nucleic Acids Research, 12:8711-8721) which permits the expression of the antisense RNA under control of the CaMV promoter.

After synthesis of the vector, the structure and orientation of the sequences are confirmed by DNA sequence analysis.

EXAMPLE 4 Construction of antisense RNA vectors with the polygalacturonase promoter.

The fragment of the senescence-related cDNA described in Example 3 is also cloned into the vector pJR3. pJR3 is a Binl9 based vector, which permits the expression of the antisense RNA under the control of the tomato polygalacturonase promoter. This vector includes approximacely 5 kb of promoter sequence and 1.8 kb of 3' sequence from the PG promoter separated by a multiple cloning site.

After synthesis, vectors with the correct orientation of senescence-related sequences are identified by DNA sequence analysis.

SUBSTITUTE SHEET (RULE 26) -r I I WO 95/07993 PICT/Gi94/01990 41 EXAMPLE Construction of sense RNA vectors with the CaMV 35S promoter.

The fragment of senescence-related cDNA described in Example 3 is also cloned into the vectors described in Example 3 in the sense orientation.

After synthesis, the vectors with the sense orientation of senescence-related sequence are identified by DNA sequence analysis.

EXAMPLE 6 Construction of sense RNA vectors with the polygalacturonase promoter.

The fragment of senescence-related cDNA that was described in Example 3 is also cloned into the vector pJR3 in the sense orientation.

After synthesis, the vectors with the sense orientation of senescence-related sequence are identified by DNA sequence analysis.

EXAMPLE 7 Construction of an over-expression vector using the CaMV35S promoter.

The complete sequence of the senescence-related cDNA clone is inserted into the vectors described in Example 3.

SUBSTITUTE SHEET (RULE 26) =I I- II- WO 95/07993 P'CI/G; 94/01990 42 EXAMPLE 8 Construction of an over-expression vector using the polygalacturonase promoter.

The complete sequence of the senescence-related cDNA clone is inserted into pJR3.

EXAMPLE 9 Generation of transformed plants Vectors are transferred to Agrobacterium tumefaciens LBA4404 (a micro-organism widely available to plant biotechnologists) and are used to transform tomato plants.

Transformation of tomato cotyledons follows standard protocols Bird et al Plant Molecular Biology 11, 651-662, 1988). Transformed plants are identified by their ability to grow on media containing the antibiotic kanamycin. Plants are regenerated and grown to maturity.

Plants are analysed for modifications to their senescence characteristics.

SUBSTITUTE SHEET (RULE 26) C I I L_ _I_ IWO 5107993 ("I(IG 194/0199() 43 EXAMPLE Construction of further vectors Part of the senescence-related sequence encoded by SEE1 is incorporated into DNA constructs in the sense orientation and under the control of the ubiquitin promoter and the GST II promoter. Similar constructs are made using the SEE2 sequence. These constructs are used to transform maize.

SUBSTITUTE SHEET (RULE 26)

I---II-

WO 95/07993 1194/01990 44 SEQUENCE LISTING GENERAL INFORMATION:

APPLICANT:

NAME: ZENECA LIMITED STREET: 15 Stanhope Gate CITY: London STATE: England COUNTRY: UK POSTAL CODE (ZIP): W1Y 6LN (ii) TITLE OF INVENTION: Regulation of Senescence (iii) NUMBER OF SEQUENCES: 39 (iv) COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.25 (EPO) (vi) PRIOR APPLICATION DATA: APPLICATION NUMBER: GB 9318927.2 FILING DATE: 13-SEP-1993 INFORMATION FOR SEQ ID NO: 1: SEQUENCE CHARACTERISTICS: LENGTH: 660 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: ORGANISM: SENU1 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: GTTGTGTAGC TACTACAAAC ATCATCAATA TTCAATAATG GTTCTCCAAA ATCAACTTGG TCATCATCAA TCATTCTCTC ATGAACACAA CCACTATTCC GCGGAGAGCA ACCATGGACA 120 AATGATGAGG CCTTTCGCCA TGCCATTTTC TCAGTCTTCT CACAATAATC ACATGATGGT 180 CAGCCAACAC AGTGGCGGAT ATTATGGAAG CCATGGCCAT GGCCATAGCA ACTATGGAAG 240 TCATGGCCAT ATGCCACATG AATCAACCAA CTTCTCAAGT AGCACAAGCA TGGTCCATAG 300 SUBSTITUTE SHEET (RULE 26) WO 95107993 WO 9/0793 PtIcr/B94O I990

TGATGGTGGT

CATGGGACAT

CCACCAAAAT

L AAATTTTA

TGTGGTACT

CTACTGCTAT

TATGGTAGTG GAATGAACCA CATGGAAGTC ATGGTCATGG AGCTACAGTC AGAGCCAGAA CACACACATA TATATAATAT ACTTTGGCTA TATGTAGTAC CTTAATTTAA ATTATCTATG

ATCATCCCAC

TCATGGACAT

GGTCAACTGG

ATGTGTGGTG

AAAGGTTTGC

TATCTGCTTT

GCCCATATGT

GGACTAGGTT

GCTCTCAAGA

AAAGTAATAT

TACCTAAATA

ATCATTGACA

CGTCCATGGC

TTGGTGGAAG

ATTTGGATGA

TATGTGTGTT

AGTAAACAAT

AATGATGAAT

INFORMATION FOR SEQ ID NO: 2: Wi SEQUENCE CHARACTERISTICS: LENGTH: 193 base pairs TYPE: nucleic acid STRANDEDNESS: single VD TOPOLOGY: linear (ii) MOLECULE TYPE. cDNA (vi) ORIGINAL SOURCE: ORGANISM: SENJ2 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: AGATGTTCCT GTTAATAACG AAAAGGCGTT GCAAAAGGCT GTTGCACATC AACCTGTGAG CATTGCACTT GAAGCTGGTG GCAGAGACTT CCANCACTAC AAATCTGGTA TCTTCACTGG AAAATGTGGT ACTGCAGTGG ATCA'L3GTGT AGTTATTGCT GGATATGGTA CTGAGAATNN CATGGATTAT TGG INFORMATION FOR SEQ ID NO: 3: Wi SEQUENCE CHARACTERISTICS: LEFGTH: 179 base pairs TYPE: nucleic acid STRANflEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: ORGANISM: SENU2 MIDDLE (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: SUBSTITUTE SHEET (RULE 26) r~C11~ WO 9(107993 PCT/GB94/011990 46 TACAAGCACC AATACTGCGG AATGGGGTGA AAAAGCAGGG ACAACAGAGG ACGTGAATCG ATACATTGAT TAACAAAACC TCATTTTTCC AGCAGAAAGG TGTAGAAACA GATGAATGCA 120 TTATCAAGAT TACAAGATGT TGATTAATGA TTTGTATATA TTGATATGTC ACTTGCTAT 179 INFORMATION FOR SEQ ID NO: 4: SEQUENCE CHARAC"Et'STICS: LENGTH: 200 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: ORGANISM: SENU2 3' (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: AGATGTTGAT TAATGATTTG TATATATTGG ATTATTGTTC AACTTTGTCT AATTATTCAG TACATATTCC TTGTTTCTTA TTATGAAGAC TCCAAGTAAT GCTTTTTAGT CTTCCATCTG 120 TACTTGGTTT CAACATTAAT TAAAAAAAAG GACTATCTTC TGTACCTTTC .iTTAAAAAA 180 AAAAAAAAAA AAAAAAAAAA 200 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 965 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: ORGANISM: SENU2 PROPOSED (xi) SEQUENCE DESCRIPTION: SEQ ID NO: AGATGTTCCT GTTAATAACG AAAAGGCGTT GCAAAAGGCT GTTGCACATC AACCTGTGAG CATTGCACTT GAAGCTGGTG GCAGAGACTT CCANCACTAC AAATCTGGTA TCTTCACTGG 120 AAAATGTGGT ACTGCAGTGG ATCATGGTGT AGTTATTGCT GGATATGGTA CTGAGAATNN 180 CATGGATTAT TGGATCGTTA GGAACTCATG GGGAGCTAAC TGCCGAGAGA ACGGCTACCT 240 SUBSTITUTE SHEET (RULE 2t) I WO 95/07993 WO 95/07993 Cf/GB94/01990

CAGAGTCCAG

ATATCCAGTA

CAAGCCACCT

TATCCTTCAG

TTrGCTGTGAA

AGGAACATGC

TGCACAACCT

AGCACCAATA

ATTGATTAAC

CAAGATTACA

AATTATTCAG

CTTCCATCTG

ATTTC

CGTAACGTTT

AAAACAGGAC

ACAGAGTGTG

TTCCGTAGGT

GACCACTACA

TCAATGAGCA

ATTGGGGCCT

CTGCGGAATG

AAAACCTCAT

AGATGTTGAT

TACATATTCC

TACTTGGTTT

CCAGCTCTAG

CAAATCCTCC

ATGAATATTC

CTTGCTTCTC

GTTGCTGCCC

AGGGCAACCC

TCGGAAATGG

GGGTGAAAAA

TTTTCCAGCA.

TAATGATTTG

TTGTTTCTTA

CAACATTAAT

TGGCTTGTGT

TAPACCCGCT

TCAATGCGCT

TTGGGGATGC

ACACGACTAT

ACTGGGAGTG

AGGAAAOX-*-AG

GCAGGGACAA

GAAAGGTGTA

TATATATTGG

TTATGAAGAC

TAAAAAAAAG

GGTTTAGCCA

CCATCTCCTC

GTCGGCACCA

TGCCCACTTG

CCTATCTGCA

AAGGCAATGA

AGCAGTTCTT

CAGAGGACGT

GAAACAGATG

ATTATTGTTC

TCCAAGTAAT

GACTATCTTC

TAGAGCCTTC

CATCTCCGGT

CTTGCTGCTG

AAGGAGCCAC

ATGTTCGTCA

AGCGCATTCT

GAATTCTACA

GAATCGATAC

AATGCATTAT

AACTTTGTCT

GCTTTTTAGT

TGTACCTTTC

INFORMATION F0'. SEQ ID NO: 6: Wi SEQUENCE CHARACTERISTICS: LENGTH: 1388 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: ORGANISM: SENU3 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: CATTACAATA ATTACAATGT CACGTCTCTC GCTCGTATTG ATTCTCGTCG CGCTACTGCA CTTGCCGGTC CGGCGACCTT CGCTGATAAG AATCCGATCA ATTTCCCGAT GAGCTGGAGA ACGGGATTCT TCAAGTCGTC GGCCAGACTC CTCCTTCGCT CGCTTTGCTA TCAGGCATCG GAAAAGGTAT GACTCCGTTG GCAAAGGTTT GAGATATTTT TGGACAATCT GAAGATGATC CGATCGCATA ACTATCATAC AAACTCGGTA TCAATGAGTT TACCGACCTA ACATGGGATG

CCGGCCTTTT

GGCAAGTCGT

GCAGTGCTCT

AAGAGATCAA

ACAGAAAAGG

AGTTCCGTAA

SUBSTITUTE SHEET (RULE 26) .WO 95/07993 C/B/090 IICT/GB94/01990 ACACAAGTTG GGGGCATCTC AAAACTGTTC TGCCACCACA AAGGGAAATC

TAACGTCGTT

GGCACAGGGC

CTATGCCCAA

TGGAGCTTTT

TAAATTCAAT

ATGTJA.ATTC

GGGTGCTGAA

TGAGGTGGTA

CGACACTCCC

TACTCCCTAC

CAAAATGGAG

TGCCTAAGCT

GATTCAGCAG

TTATACATAT

TAGTTCACAG

TGAAATGGAA

CTGCCAGAGA

AAGTGCGGAT

GCATTTGGGA

AATAACTTTG

GGTGGTCTTG

TCACAAGCAA

TATGAACTGA

AAAGGGTTCA

ATGCArCG"TA.A

TGGCTCATAA

ATGGGAAAGA

TTGGAGTTTT

CAGCATTCAG

AAAAATGGTT

AGTTTATTAT

GAAGAATTTC

CGAAAGACTG

CTTGCTGGAC

AGGGAATCTC

GTTGCAATGG

ACACTGAAGA

ATATTGGTGT

AATACGCAGT

AACAGTACAA

ACCATGCTGT

AGAACTCATG

ATATGTGTGG

GTGAAAAAA

GCTCCATTCT

TAGGCTACTT

GCAGGAAAAA

TTATTGTAAA

GAGGAAAGAT

ATTCAGCACT

TCTGTCAGAG

AGGGTTGCCT

AGCATATCCA

CAAAGTCATC

TGCATTGGTT

GAGCGGAGTT

TCTTGCTGTG

GGGAGCAGAT

TGTTGCAACT

TCTGCATAAA

CAGATTTATA

ATATGAAAGA

ACCCTATGTA

CATTGTTAAT

GGTATTGTTA

ACTGGTGCAC

CAGCAGC2TG

TCTCAAGZAT

TACACCGGCA

AGTTCTGTCA

AGGCCTGTTA

TACGCCAGCA

GGTTACGGTG

TGGGGTGAGG

TGTGCATCCT

TCCGTGTTGT

TTCTGAACAT

AACAATAAGA

ATTTATACCA

AAGGTGTTGT

TCAAGCTCAC

GCCCAGTGAA

TAGAGGCAGC

TGGACTGTGC

TTGAGTACAT

AGAATGGCAT

ATATTACCCT

GTGTTGCTTT

CTGAATGTLG

TTGAAAATGG

ATGGATACTT

ACCCAATCGT

CCCAGTTAAT

GTATGGATTG

TCAAAATATT

TTATAAGTAA

CCTTAGTTCG

420 480 540 600 660 720 780 900 960 1020 1080 1140 1200 1260 1320 1380 1388 TGTCTTTT INFORMATION FOR SEQ ID NO: 7: SEQtENCE CHARACTERISTICS: LENGTH: 523 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: ORGANISM: SENU4 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: GGCATCCCGA GCACAAAACT ATGCCAACTC AAGAGCTGGT GATTGTAACT TGATTCATTC SUBSTITUTE SHEET (RULE 26) 1PCT/G1194/0 1990 .WO 95/07993 TGGTGCTGGG GAGAATCTTG CCAAGGGTGG TGGTGACTTC ATTGTGGGTG TCCGAGAGGC CAAGCTATAA CTACGCTACC AAAGTGTAGA CATTATACTC AAGTAGTCTG GCGCAACTCA GGCACGTTGC AACAACGGAT GGTGGTTCAT TTCTTGCAAC GATCGGACAA CGTCCTTACT AAAATGATCT ATACTTATGA AAATTCTCAT ATGAGACGTC GAGAAGTTAA AATTTAAGTT ACTCCTATCT AAAATATTAA GGGATTAAAT ATTGAACATC TTTTGATGTT GCTAATATGA ATAATTCCAC ATACCATATG INFORMATION FOR SEQ ID NO: 8: Wi SEQUENCE CHARACTERISTICS: LENGTH: 847 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: ORGANISM: SENUS

ACGGGGAGGG

AACCAATGTG

GTCCGACTAG

TATGATCCTG

CATGTTGCTA

TGACATATGA

TATAATTATT

TTC

CAGCCGTGCA

TTGGTGGAAA

GTTGTGGTCG

TAGGCAACTG

GTATTAAATA

ATCAAGTCAA

ATTATTTCCC

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: ATTAAATTTA TGGAGAAGGT TAATTTTTTG AAAAATGGTG TATTAAGAAT TACCCCCAGG

TTTTCGATTT

TCTCTTTTCC

GGGATTTGCC

ATCCAAATGG

TTGACAAGCA

AAACACTTGT

ACGAATATCG

TCTGTCGAAT

CACATGATGA

TGAGTTCTCG

CGCTCCCACC

TTTGCCAGCT

AGGTGATATG

GAATAGGTCA

AATCATATTA

CTTCTATAGA

TCTTGCCAAT

TTTCTTGAAA

GGTTAGAAAC

AGATTCTGAG

GATGAAGAAC

TCTATTATCC

GAG CAAGAAA

AATAGAGCAA

AGGGGACGAC

GGAAAATCTC

CTCGAATCTA

AAAAGAGGCA

AGAGAAATTG

GCATTGCTTC

TTGTGGTACA

CTGAAGTTGA

AGTATTTTTT

CAAATTCAGG

AACAACAACA

CACATGGCTG

ACTATCATCC

ATACTAAAAA

ATAAAAACTC

CGCGAATAGT

ATACTTGAAG

AGTTTACAAA

TAGCACTAAG

ATATTGGAAG

ACAATTGATA

TAGGACCAAT

AATTCAGGGA

TAAGGAGGAA

GCCCGTTGTT

GAACTGAAGA

CGTAAAGTCT

TCTGATCCTT

GAAGTGAAGT

GCTACTGGAA

GGATTGAAGA

TGGATTATGC

AATTGGGTTA

AACATGACAA

TCAGTCAAAA

AGAAGGCATC

SUJBSTITUTE SHEET (RULE 26) 111 9 nn WO 95/07993 PCTr/GB1194/01990 CATAATATTT TACGATTTTA TGGGGAGGAA TAATTCGAAT GGAGTTGCAG CTTCAACGTC 720 AAGTAGTGGA ATCACTGATT TGACTACTAC TAATGAAGAA TCTGATGATC ATGAAGAAAG 780 TACTAGTAGT TTTAATAATT TTACTACTTT TAAAAGAAAA ATTAATTAAT TAATCGTCGT 840 TAAAACG 847 INFORMATION FOR SEQ ID NO: 9: SEQUENCE CHARACTERISTICS: LENGTH: 212 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: ORGANISM: SEND31 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: AAAAAAGAAT TTTTGATCAA AGATGGCACA ATACGGCAAT CAAGACCAAA TGCGCAAGAC TGATGAATAT GGAAACCATG TCCAAGAAAC AGGAGTCTAT CAAGGTACCG GTACTGGCGG 120 TATGATGGGG GGCACGGGTA CTGGCGGTAT GATGGGGGGC ACTGGTGGAG AATATGGAAC 180 TCAAGGCATG GGTACTGGTA CTCATCACCA TG 212 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 180 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: ORGANISM: SEND31 3' (xi) SEQUENCE DESCRIPTION: SEQ ID NO: AATCTAATTA CGTACACTCT TGTGTTTAAA GTCGTGTAAA GTGCGGTGAC GCTATATGCA TATATATATA TATTGGCGCC ATGCCCTGCC CCTTCTGTAT TTTTAAAACA AGAATATTGC 120 TTCCATGCTT GGAAAGCAAT GATCATATTG ATGTGAAAAA AAAAAAAAAA AAAAAAAAAA 180 SUBSTITUTE SHEET (RULE 26) II i I WO 95/07993 PCTIGB94/0 1990 51 INFORMATION FOR SEQ ID NO: 11: SEQUENCE CHARACTERISTICS: LENGTH: 721 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: ORGANISM: SEND31 PROPOSED (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: AAAAAAGAAT TTTTGATCAA AGATGGCACA ATACGGCAAT CAAGACCAAA TGCGCAAGAC

TGATGAATAT

TATGATGGGG

TCAAGGCATG

CAGCTCTAGC

GAAGATAATG

AGAGAAGAAA

TTTGTTTTCA

GCACTAATTA

AAAGTCGTGT

TGCCCCTTCT

GTTGATGTGT

GGAAACCATG

GGCACGGGTA

GGTACTGGTA

TCGTCGGAGG

GAGAAGATGC

GGAATGATGG

TCTCCATCTT

GTAGTAATCG

AAAGTGTGCT

GTATTTTTAA

GTTATGGTGT

TCCAAGAAAC

CTGGCGGTAT

CTCATCACCA

ATGATGGAGA

CTGGACAACA

ACAAAATCAA

ATCTTATGAA

TTATGCCAGT

GACGCTATAT

AACAAGAATA

CCCGTGTTTA

AGGAGTCTAT

GATGGGGGGC

TGAGGGGCAA

AGGTGGGAGG

TGAAGGTGAG

GGACAAGATC

TAAATAAGGT

AATTATCTAA

ACATGTGTGT

TTGCTTCCAT

ATGATGTTTT

CAAGGTACCG

ACTGGTGGAG

CAGCAGCTTC

AGAAAGAAGG

TATGGACAAA

CCTGGGATGC

AGTGCTTGAT

TTACGTACAC

GTATATTGGC

GCTTGGAAAG

GGAAATAATA

GTACTGGCGG

AATATGGAAC

GTCGATCCGA

GTTTGAAGGA

CAACAGGTGA

ATTGAACACC

TCTATTTCAT

TCTTGTGTTT

GGCCATGCCC

CAATGATCAT

TAACTTGTGC

INFORMATION FOR SEQ ID NO: 12: SEQUENCE CHARACTERISTICS: LENGTH: 679 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: SUBSTITUTE SHEET (RULE 26) .WO 95/0l7993 CTG14099 PCT GB94/01990 52 ORGANISM: SEND32 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: AAGCAGACCA ATTTTTTCCC CACCAAACAA ATTCACACTC TGCTTTCTAT ATAAGCCTCA

CTCTTCATCC

ACCTTCTCTT

GCCATCTTCT

CAACATTACC

CCAATCTTCC

CACAACTTCC

CACAACTTCC

CAAGCCCTTA

ATGATTCGTT

CTTTCGTATG

ACCAAAGCTC

CCTCGTTGTC

GGACG.'CAACT

TATGCCCAAT

ACTGCCGCAG

AAACCCTCAG

CAAACCTCAG

AGTACTAATG

TGTATCATTA

TAATCTCTTA

AAGAAACAGT

TTGTCATCAA

TTGCCAGATC

CTTCCACTGC

CCTGGACTGC

CTGCCACCTT

CTACCTCTGC

TTCCTTCAAT

TTGTCGTATT

TTAGTAAGTC

CTGCTATA.AC

TCAGGAGCCA

TCCCCATTGG

CGCAGCCTGG

CCACATTACC

TGCCCAAACC

CACAGTTGCC

GATTGGGAAG

TGTGAACCTG

AATTGTTTGT

AATGGCTCAT

TGTAATTCAA

TCTCCCAAAA

ATTGCCCACA

TATGCCTAAT

GGATTTGCCA

AATACCATTG

TGTACTACGT

CTCATGTTAA

CAGTTAACAT

CAGTCTCTTT

GTTGAGGCTC

CCTGAGCTAC

CTACCTATGT

CTTCCTCAGC

TTACCTATGC

CCACCTCTGC

GCTATCCAGG

TGTTGTTAAA

TATTAAATTA

120 180 240 300 360 420 480 540 600 660 CTTCTGTGA.A TGATTCCTC INFORM~ATION FOR SEQ ID NO: 13: Wi SEQUENCE CHARACTERISTICS: LENGTH: 574 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: ORGANISM: SEND33 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: AGAAAAGAAA. AGAATTGTAA AAAATGGCTA GTATTTCTGG TACAATGATT AGCACTTCTT TCCTTCCAAG AAAGCCAGCT GTGACTAGCC TCAAAGCCAT ATCAAATGTT GGTGAAGCTT TGTTTGGTCT TAAATCTGGT AGAAATGGGA CGATTACTTG CATGGCCAGT TACAAAGTGA AGCTTATTAC ACCAGAAGGA CCTATTGAAT TTGAATGCCC AGATGATGTT TACATTCTTG ACCAAGCTGA GGAAGAAGGA CATGACCTTC CTTACTCATG CAGGGCTGGT TCTTGCTCAT SUBSTITUTE SHEET (RULE 26) IR 1, WO095/07993( TICT/GB94/01990 CTTGTGCTGG AAAAGTTACT GCTGGAAGTG AGGACCAAGA AGCTGCTGGA TTTGTGCTTA CCATTGAGAC TCACAAGGAG GAGGAGCTTA TAAAAAACAA CACTTCATTT TGTTTCATGA TTATCACAAA AGTTGCTATC TAGTTATTTG INFORMATION FOR SEQ ID NO: 14 SEQUENCE CHARACTERISTICS LENGTH: 540 base pa TYPE: nucleic acid STRANDEDNESS: singl TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: ORGANISM: SEND34 TTGATCAGTC TGATGGAAAC TTCCTTGATG CTTGTGTTGC TTACCCAAAG GGTGATGTTA CTGCTTAAAT TACAACCATT TCCATTTTAA GCATTTACTT TTCACATTTT CCCTCTATTG

TGAC

360 420 480 540 574 120 180 2.10 300 360 420 480 540 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: ATTGAGCCTA AANCCAACTT TCACTCTGGA GAAAATATCA GTGAAAGGGC TACTAGATCT TCTTCTTCCT TCAAAGTTNT GGCTAGAGGT GTTAAGAAGC CAAACCCTAT GGAATTAATG GAAGCATGGC CTTGAGAGAT GGGGTTGATN GAAGCCCAAG GGAAAGGGTG TGTACCAATA TGTTGACAAA TATGGAGCTA ATACAGTCCC ATCTACAACA CGGATGAATG GTCTCCAAGT GGTGATGTCT TACCACTGGC TTANNCATAT GGGCGGTNAC CTTGCTTGGC ATTCTTGCAG NCTTGTCTAC AACACAAGTN CTTTNNNGCA ATAGATGTTA TCCTTGTATT GTTTAAGTTG TATTCCATGT TATCTTTTCA AAATATTTCT TGGCATCTTC TTCTTGGCAT CTTCATTAAT ATGTAATTGC TGTGATTTTA CTGTGGATAA

TTCCATCACT

TTAAGACTGA

CCTCAGGCAG

ATGTTGATGG

ATGTTGGAGG

GAGGTGCTCT

GTACTATTAA

ATTAAAATAT

TATTACATCT

INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 524 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear SUBSTITUTE SHEET (RULE 26) IWO 95/07993 P('T/(;B94/)1990 (ii) MOLECULE TYPE: CDNA (vi) ORIGINAL SOURCE: ORGANISM: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: AACAAAGTGC AACCATTATC AACAGAGACA CAAAAAGAAA ACACAGAAAT GCAAAAATAC

ACAATCCTAG

CAAAACTATG

GTATCATTAG

ACATCACAAT

NTCCCGTCCC

CTAGAAAATA

GTATTATATG

GAGCCAAGAA

CCTATGCAGT

TTGGTGTTGA

GGGACCCCAA

CTGTTTTGTC

ATTAGATGAC

TAATAATACA

AATGGGAAAC

ATTTTATTGT

TGGNACAAAA

AACATTTGGC

ATTTCCCCCC

ATATGTTTAA

AAGTATGTTG

AACAATGACA

CACAAAACAG

GTTAAGGCTG

TTTTAAAGTT

ACAAGATCAC

TTGTCTTCTA

TGTTTGTCTT

AATCAGTAGT

AAACAACAGA

TGGCTGTTTG

CTTTAAGGTT

ATTGTTTGGG

AAAATTTGGG

ATACTTTTTG

TTGTCACAAA

GTCATACATG

TCACAAAGGC

ACCACCTGGG

TCCCTAAGAA

TTGTTTTTAT

TCTTGTTGTA

TAATCATACT TTACTATACT A.ATAAATAAT ATATGTTCAC ATTT INFORMATION FOR SEQ ID NO: 16: Wi SEQUENCE CHARACTERISTICS: LENGTH: 474 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: ORGANISM: SENE71.

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: CTCGAGGTT2- GATCCTTGTC GTCCTGCTGA GCAGTACCCG ATTCCTTCTT TGGAAGGCGT ACAAATTGTG TCATTCCGAA AGAAAACAAC TTCAAACAGG GTACAGATCA TGGGAACCTG ACAGGCAAGA CAGATACATC AACAAATGGG ATCCGATCCA CGAGTCACTC ATGAGATTCG CAGCATATGG ATATCATACT TGACAAGTCC TGTGGTCAGA AGGTCGCTTC TCGTCTCACT GTGAAGCCTA AATCAATGAA AATAGTTGAA ATGGTTTCAA GCTGCAAATG TTGAAGGACT S24

GTGTCTTGAA

CAGGGGAGAG

TTGAGTCTTT

TGTCTCAGGC

CAATGTGAAA

AATGCAAAAA

SUBSTITUTE SHEET (RULE 26) .WO 95107993 P'/B4019 AACGTCCGCG TTGTGCTATA AACTGTACTT CTTTTTCAAT CGTAATGTTG TATTTTGTAT CGAATTTCGA TGTCTTGTGT TTTTACTATA ATGATGTTGG AACATACAAC TGTG INFORMATION FOR SEQ ID NO: 17: Wi SEQUENCE CHARACTERISTICS: LENGTH: 1442 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: ORGANISM: SEE1 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1' GAATTCGCGG CCGCTCCATT CTTCTCGCCT TACTCCCTCA

CCAGTCCCGC

CGGCCACCGC

CCGACCGCGC

ACGCGCTCCG

AGGTCCACAA

GCAAAGGCCT

TCCGTGCGAC

GGATGCGCGC

TGAGCCCAGT

CACTTGAGGC

TTGTTGACTG

CCTTTGAATA

GTGTCAATGG

TTAACATCAC

TTAGTGTTGC

GCGACCATTG

CGTCGAGATG

CGCCGCGGCC

GCCCCACGCC

AACTCCGGCT

GGCCTCCGCG CTCGAGTCCA CTTCGCACGC TTCGCCGTCA

GCGGTTCAGG

CTCCTACCGC

CCGGCTCGGT

CGCCGCCGTT

GAAAAACCAG

TGCATATACC

TGGTTTTGCA

CATCAAATAC

AATCTCCAAG

CCTGGGTGCT

CTTCGAGGTG

TGGAACTACA

ATCTTCTCCG

CTCGGCATCA

GCAGCCCAGA

GCGCTGCCGG

GGCCACTGTO

CAGGCAACTG

TTCAACAATT

AATGGTGGCC

TTTAAGAATG

GAGGATGAAC

ATCACTGGTT

CCGATGGATG

GCCTGCTCGT

TCGCGGACTC

CGGTCTTCGC

GGTACGGCAA

AGAGCCTCCA

ACCGCTTCGC

ACTGCTCCGC

AGACGAAAGA

GATCATGCTG

GCAAGCCCAT

TCGGATGCAA

TTGACACTGA

AGAATGTTGG

TGAAGGATGC

TCAGGCTGTA

TGAACCACGC

CAGAACCCAG

CCTCGCCGTC

CAACCCGATC

CGCGCTCGGC

GAGCTACGAG

GCTGGTCCGC

GGACATGAGC

CACGCTTACC

CTGGAGGGAG

GACCTTCAGC

CTCTCTCTCT

CGGAGGCCTT

GGAATCTTAC

AGTCAAGGTT

TGTTGGTCTG

CAAGAGCGGA

TGTTCTGGCT

TAAAATATCG

GTCGCCCTCG

CGCCCCGTCA

CGCACCCGCG

AGCGCGGCGG

TCCACCAACC

TGGGAGGAGT

GGCAACCACC

GATGGGATTG

ACTACTGGTG

GAGCAACAGC

CCATCCCAGG

CCTTACCAAG

TTGGACTCGG

GTTCGCCCAG

GTTTACACTA

GTTGGCTACG

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 SUBSTITUTE SHEET (RULE 26) WO 95/07993 PC'r/G 1194/()1990

GTGTCGAAGA

ATGAGGGTTA

CCTACCCTAT

ATGCATGTTT

AGAAA.ATCAT

AGGTAGATTG

TTATGAAACA

TGGTGTACCC

CTTCAAGATG

TGTCGCATGA

AACCTGAGCT

GAAGGGAAGG

TCAGTTGGGG

AAATACGCAT

TACTGGCTCA

GAAATGGGCA

GGCCCTTACG

TGGCGATGGG

AGTTGACCGG

TTCCGAAGTA

TGATCOTTAT

TCAAGAACTC

AGAACATGTG

AAATGTTACA

TTATACAGAA

ATTCCTGCTT

CTCTACTCAT

ATGGGGCGCT

CGGTGTTGCT

TGGTCTGTTT

CGGAAACTCT

GTACGTCTCC

ACGTGTGTAT

GACTGGGGTG

ACGTGTGCAT

GGCATCAATA

GTTTGTGAAT

CCGACTGTGT

GACAGTTTAT

GGCCGCGAAT

1080 1140 1200 1260 1320 1380 1440 1442 GGTCCAAAAA AAAAAAAAGC INFORMATION FOR SEQ ID NO: 18: SEQUENCE CHARACTERISTICS: LENGTH: 423 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: ORGANISM: SEE2 PRIMER) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: GAATTCGCGG GCGCTCCCGA GCCCTCCACC GGAGTATGAC ACTTGCTTGG TAGTGTTGCT TGGATGGAAG ACAGTGATTT CCACAATCTG CGAACTGAAT GCAATACAAC TTGGTCAAGG ATAGGACAGC GGTTCAGGAT ACATTCAGCT TGTGATGCAA TATGGTTCAT TGGAGTTGAA TGTTAAGCAT CTGTTTTCGT AAACCCTGCT AACGATGACA ACACGTCCAT AGAAGACAAC TCGTTGGCCA AGGCTGTTAA TCAGCGCGAC GCTGGACCTT GTCTTCTTCT GGCAGAAGTA GCAGACAGCT CACATGAGAA AAATGAAGCT CGGGGGGTTT GGCTTGAGGT

GGT

INFORMATION FOR SEQ ID NO: 19: SEQUENCE CHARACTERISTICS: LENGTH: 414 base pairs TYPE: nucleic acid STRANDEDNESS: single

GAGACCTGTA

CTCTCAAGCA

ATGGCTCCCA

ACATTGGCAC

TCATTCTCAA

CCGGAAATTG

GATGGCCACA

SUBSTITUTE SHEET (RULE 26) pi L WO 95/07993 I'CT/G B9.1/01990 TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: ORGANISM: SEE2 (T3 PRIMER) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: GAATTCCACC ACGGATAGTC CATTTCCCAA CAAGAATGGG GTAAAGTTTC CAGTCGGCTA CATAGAAATA GCAGTAACGC GTTCTTCATT TTTATGGAAT ACATCTGCTA ACAAACTATA GGGAATATAT ACAGCAACGA CCAATACAAT CTTTCCGACT ATATATGTAC ACCGGAAATT GGGCATAGCT ATGTAATCTG ACAGCTATAG TAGTGGTATA AGTCTGCTTC ACCTATGGTT CAAGAACTAG AGGACTGGGT AATGTAAACC AAGTCCGTTG GGGNGGGAGC GGCTTGAAAT CACCYTATGA TTCTTTAGGG CGCTTAAAAC CCTCGTGGGT AGAACTCCAG INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 181 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: ORGANISM: SEE3

ACATGAGCAT

TACAAACTAC

TGCCAAATAA

GAACGGTGGA

GACCCCCCTG

ATTTCCGCCT

GGGT

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: CGCGGTAGAN GGTATCGATA AGCTTGNATA TCGCAATNCG TCGGACGCTA TCAACCAGCA TCACTGGCCT CAGGGGCACC GCCGAGTAAC GTGCAGTGCC ATGGTTGCTT CGGTCAACAT GGGGNAACAC TTCTGGCACC GGTGTGCT TCACCAGGTA ACCCCAACAC CGGAGTAGAA

G

INFORMATION FOR SEQ ID NO: 21: SEQUENCE CHARACTERISTICS: LENGTH: 422 base pairs TYPE: nucleic acid STRANDEDNESS: single SUBSTITUTE SHEET (RULE 26) -H III I I WO 95107993 11CI/G1194/01990 TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: ORGANISM: SEE3 MIDDLE (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21: GAATTCAGNC TGCTCCGCTA TCTCATCAGC CACCAGAGCT GCCCTGGGGN TTGTTCCAC TTTGTACCCG ATAGTCTTGC CCACATTGGC GAACACTTCC GGCGGATAAG AGTCACTTGA TGCCCCAGTT CCTGTGGTGT TCCAACAAGA TCTCTGGGAA CACTTGNAAC ACCCTGGTTG GTCATTGCTA TAGCAGCTTC CGGGCTTGCA TCTCTGTCAA TTCAGGGTAC GATATNCCAA GCCTGCACCC AGCATCGGGT TTACTTCTGA AAGCTTTTCA ATTCGCGCGA GGGGATCCTC TCCCGTCTTC AGCACATAAT CACTTACAAT GTCCTCGATG TTCCCCTTCC

TC

ATCTCAATCA

TCAGCAACTT

GGAACCATTA

AAAAATGGCC

ACGGAAGCCA

CTGGTTGGGT

T14GAAGGGAC INFORMATION FOR SEQ ID NO: 22: SEQUENCE CHARACTERISTICS: LENGTH: 203 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLL linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: ORGANISM: SEE3 MIDDLE (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22: GAATTCTTCT CCTCCGGAAC GAACGACCTG ACGCAGATGA CCTTTGGGTA CAGCAGGGNA TGATGTGGGA AAGTTCATCC CCGNCTATCT TGCTCAGGGT CATTCTCCAA CATGACCCCT TCGAGGNTCC TGGACCAGAG GGGTAGTGGG CGAGCTGGTG AAGCTTGCTA CAGAGCAGGG GCCGNAAAGC TAGGCCTAAC TTG INFORMATION FOR SEQ ID NO: 23: SEQUENCE CHARACTERISTICS: LENGTH: 262 base pairs SUBSTITUTE SHEET (RULE 26) 'WO 95107993 P(T/(;G94/01990 59 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: ORGANISM: SEE3 3' (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23: TTTAATCGAT AATTGATGAT GTATTGCATG AACAGAGTTT TGCANCCATG GNAGCAGTTT GGCAAGTTTN ACATGCACTG GGGGAAAAAA AAGGACATGG GCATTGTTGC TTTCATGAGC 120 CACTGCCTAC TAGTTAATGT TCACATAGNC ATGGGCTCTG TAATAATACT GTTGCATCAC 180 CACATCCACC AGCAGCAGGC AATCCGGTTG CCAATGTAGG AGGCAGCCCT CCAGACAAGC 240 ACCTGAGCTG CAGCTAGCCT A' 262 INFORMATION FOR SEQ ID NO: 24: SEQUENCE CHARACTERISTICS: LENGTH: 191 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: ORGANISM: SEE4 (xi) SEQUENCE DESCRIPTION: SIQ ID NO: 24: AATTCGCGGC CGCTCGAGAG ACTCCATTAT ATTATATTGC TCGATCTCTC CATCTGAACG TACACACAGT CCAACACACA TGGCCGCCGC CGCCGCCGCC CTGGTGAGCA GCAACAGCCT 120 CCGCGCCCCG GCGGCCTTCT CGTCGTCGT CCGCGCGGGG CTCCCGGNCG GAGGGTGTCG 180 TCGGAGT.GT G 191 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 304 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear SUBSTITUTE SHEET (RULE 26) WO 95/0799J U/o 1(Wav (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: ORGANISM: SEE4 3' (xi) SEQUENCE DESCRIPTION: SEQ ID NO: GAATTCGCGG CCGCTTTTTT TTTTGGTATG CATACGCATG AGGACCATGT ATAGTTAGTT ATTATTACAT GGCGGCGTCG TTATTATTAT TATTACATGC ATGCAATGCA AGGAGAGCGT GCGTACGTGG TGCATTAATT GCTGACTATA CTAGTACGAC TCGACGAGTG CATCCTCCTT GTGCGTCTGG ATGACGAGGT CCGAGGTGGG GTAGGCGATG CAGGTGAGCA CAAAGCCCTC GGCGACCTGG TCGTCGTCGA GGAAGCTCTG GTCGAACTGG TTGACGGAGC CGGAGACGAT

CTTG

INFORMATION FOR SEQ ID NO: 26: SEQUENCE CHARACTERISTICS: LENGTH: 94 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: ORGANISM: SEES 3' (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26: TTTTTTTTTT CACCTAGTAA TAATAATAAT AATACACATC CACTNGGTTT GGNTTGTCTT GGTACAAAGG CGGGCACGGG CACGGGCTAT ACGT INFORMATION FOR SEQ ID NO: 27: SEQUENCE CHARACTERISTICS: LENGTH: 270 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) (vi) MOLECULE TYPE: cDNA ORIGINAL SOURCE: ORGANISM: SEE6 SUBSTITUTE SHEET (RULE 26) WO 95/07993 [ICTIG1194/01990) 61 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27: CAACCGGTCC AGCACTTTCA CGCTCAGCTC CAGCAATGGC TGCCTCCACC ATGGCGATCT CCTCCACGGC GATGGCCGGC ACCCCCATCA AGGTGGGCTC CTTCGGCGAG GGCCGCATCA CCATGCGCAA GACCGTGGGC AAGCCCAAGG TGGCGGNGTC CGGCAGACCC TGGTACG'GC CCGACCGCGT CAAGTACCTC GGNCCCTTCT CCGGCGAGCC CCCGAGCTAC CTCACCGGCG AGTTCCCCGG CGACTACGGN TGGGACACCG INFORMATION FOR SEQ ID NO: 28: SEQUENCE CHARACTERISTICS: LENGTH: 353 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: ORGANISM: SEE6 3' (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28: TTTTTTTAGA AGAAAAACAA ATTAATGGAT ATC CACCACTATT GCACAAACAT GGTTCATCTT AGT TGCCTATGCA ATGCAACGTG AAACAAAAGA CAC CACGTACGTA CACCCTCTCC GATCCGAAAG ACA GCTTAGTTGN CGGGGACGAA GTTGGTGGCG TAG GCGATGTGGG TCAGCGAAGG TCTCGAGCGG GCC INFORMATION FOR SEQ ID NO: 29: SEQUENCE CHARACTERISTICS: LENGTH: 301 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: ORGANISM: SEE7

AACAATT

GTACAAC

ACATGCA

ATGAAGT

GNCCATG

CTTGGCG

AACCAGCAGG

GCCAACTCAT

TCTCGCAGCA

TGCATAGCAT

CGTTGTTGTT

GTGACGATGG

AAACAAAAAC

CATCTCGACT

TCAATCAATC

AGCCGTTGGA

GACTGGGTCA

CCT

SUBSTITUTE SHEET (RULE 26) WO 95/07993 rCTIGB94I01 990 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29: CAACTGCAAA GCAGCAAGCT CTACTCTTCT TCTGTACTGA ACGTGTGACT AGATAACAAT AAGCGTGAAC CATGGCGGAC GAGTACGGCC GCAGCGGCTA CGGCAGGTCC GGCGCCGGCG ACGACTACGA CAGCGGCTAC AACAGNAAGT CCGGCACTGA TGACTACGGC CGTGGCGAAG GTGGCTACAA CAAGTCGGGC GGCGATGACG ACTACGGTCG CAGCGGCGGC GATGGGTACG GCAGGTCCGG CGGCGACGAC TACGGNCGTN GCACCGGCGG CGGTGGCTAC AACAAGTCCC INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 425 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: ORGANISM: SEE7 3' (xi) SEQUENCE DESCRIPTION: SEQ ID NO: TTTTTTTTTA AGAGTGTCGA AATATATAGT GGTTTATTTC TTTCATTAAA ACAGATGAAG AGAAAGCTTT TATTCTCAAA GCGTTGATAA AAAGCCGAGC TGCACACATC GATCACCAAA CACGCGTAGG AAGTACCATG CACACACATC ACACATGATA TACATAGCG GACAATCAAA CCCCCGGCTG ATTCTTTTAT CGGCCTCCAT GCATGCTCCT TCTGGTCCTG GGGAGGAGGC TACGTACGCA TCGAGCAGGA GGCCGATCAG CCGAAGAAGT GGNGTTCTTC TTCCCCTCC GGATTCTCCT CCTCCCCGGG GTCCCCGCGG GTNTTGGGCT CCTCTTGGCG

GGACG

INFORMATION FOR SEQ ID NO: 31: SEQUENCE CHARACTERISTICS: LENGTH: 388 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear

AATAAAATAT

GAAAACCACG

ATCAGTAACT

TGAGTTCGGT

AGTAGATCGA

CGGGGGGGGC

NGGTGGTCGT

SUBSTITUTE SHEET (RULE 26) WO 95/07993 P(17G1194/( 1990 63 (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: ORGANISM: SEE8 (T3 PRIMER) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31: CCCCAGGAAT GGGCAGCGGG CGTCGTCCTC CTTCCTCGGC GGCAAGACGC GGCCGAGGCG GCCGGCGTCG TTCGCCGTGC GCGCCGCGGG GACCCAACAG TCCCCGGCAG CAACCCTCCG CGTGGCTCGA GGCAGCTTCC CGGGATTCGT GGGCTCGGAT CTGACCGGAG AGCTGGGNGG AACGTGAGGG GNGTGGTGCA ATGTCGGGNG GGGATTCACC GAGTTCTGAC AGACGGTCCA NAACCTCTGT AGATCACAAC ACACCCTCAN ACAGTANCCA CGTGGCGGGC GCGTGGTNAT AACACCACTC CAGAAATACG ACAGTGTC INFORMATION FOR SEQ ID NO: 32: SEQUENCE CHARACTERISTICS: LENGTH: 209 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: ORGANISM: SEES (T7 PRIMER)

TGCTGAGACA

GCGATCTGGT

TTGTTCCCTG

TGCGGTGGGG

CACGCGGAAG

AAACCGAGGA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32: CCCAACACTA GTAGCGTAGC TGAGAGCCTG AGACGATGGC CGACTAC:AC CACGGCGGCA GGGNCATGTA CTCCAACACC GACGAGTGCT ACGACGCCGG CAGGCACGGC GGCAGCGTGA GGGAGTACTC GNGCACCGAC GAGTACTACG ACAACGTGGA CGACCGCATG AGGAGGCCCG CGTACGGCGC CGACGACTNC GGCTACGGC INFORMATION FOR SEQ ID NO: 33: SEQUENCE CHARACTERISTICS: LENGTH: 390 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear SUBSTITUTE SHEET (RULE 26) WO 95/07993I 11CflIMc)114/1990I 64 (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: ORGANISM: SEE9 (T3 PRIMER) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33: GAATTCGCGG CCGCTCTTAA TTGCATGCAT GCATCATTAG A' CGTTACACGC TGCAGAAGCA TGCGCTTAGT CGTTATTTGC Al ATGGAGTAGT GATCGAGCAA CACACACGTG ACGACGTTTG C CGCACGTGTA CGGCGGCTAG GGGCCGTATA TATATCGAGT C GATGGGATGA TCAGTCGGTT TCAGTCGCAG TAGTAGGAGT A( CCCGTGCGGC ACCCTGGTGC TCCTGCTGCC TGCTCTCGTA G( GCGGGTGCTC GNGGGTACGC GTTGGCCGCC INFORMATION FOR SEQ ID NO: 34: SEQUENCE CHARACTERISTICS: LENGTH: 347 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: ORGANISM: SECi (T3 PRIMER)

IATAGTAAC

3ACGATAAT

TCTTATTTA

CTGCATGCA

3CCCGNGCC

AGTGATGGAT

AAGTAGATCG

CTTACACGCA

TCTCGGATCG

CGTGCCCGTG

3TGGGCCTC CTCCTTGCTC 120 180 240 300 360 390 120 180 240 300 347 (xi) SEQUENCE DESCRIPTION: SEQ 1L NO: 34: GAATTCGCGG CCGCTTTTTT TTACCATGTG CAATACAGTT TCCGGTCTCA ATAAGATCAA TACTAGTTTG TTGTTACAGG CAGCAGGCTG GCCATGCATG AATCATGACC GATGCAACGC TTCAGGTTCC TTNACAACAC GCATGCATCT AGCTTTCCAC AGCAAACAAC TGGCGGCGCC; GGCAGCAGGT ATGCTGATGC ACCAACGTTC TAGTGGAACT TGAGGCTGGT GAGCACGTTG TTGGTTGGAC GGGGGTCGGC GANGTGGTCG AGCAGGTTCT GGAACGGGCC AACGCCGGTG GACGAGCCCC TGGATGAAGT AACCGAGGAT GGCGAGCATG GNGAGCC INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 151 base pairs SUBSTITUTE SHEET (RULE 26) WO 95/07993 I'CTIGB94/01990 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: ORGANISM: SEC2 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: CTI-CCATGA ACACGTTCGC CAACGATATC ATCAGCCGCG CCGTGTCCGG NAAGTTCTTC CGGGCGGAGG GNCGGAACAA GCTCTTCCGG GAGCTGGTGG AGGCCAACTC TNCTCTGTTC GGAGGGTTCA ACCTGGAAGA CTACTTCCCG G INFORMATION FOR SEQ ID NO: 36: SEQUENCE CHARACTERISTICS: LENGTH: 385 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: ORGANISM: SEC2 3' (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36: TTTTTTTTAC AAAATAGCTT TTTTTATTAC TATATAAGTA AACACACACA ATAGTTTACA TGACCCGTAC GATAGCAACA CTCGGACATC TTTTCCTAGT ATATATATAT ACATGGAATC CAAACTCCAA ACCATCTATA TCTCTGTAGA CTTCACCATT GGCATGTGCA TTGTACGTAC GTACGTGCTC GTCCTCTACA GCAGTTTTCC TTCGATCGTC AGCAGCTTGG AGGATGGAGT TGNCGAGCAG TGCAGCAGTA GCAGGAAGCT TGGGAACAAG CATGAGCTTC TCCTTTGGAT TCCAAACAAC TCCGGCATGT CAACG INFORMATION FOR SEQ ID NO: 37: SEQUENCE CHARACTERISTICS: LENGTH: 375 base pairs TYPE: nucleic acid STRANDEDNESS: single

TAAGATTAAC

ATAATAATAG

GTGTAGTTCA

NCCGCTGCAT

NTGCAGTAGG

GCACCGTGAG

SUBSTITUTE SHEET (RULE 26) W. 91/07993 rIT/G(IO94/0) I 99() TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: ORGANISM: SEDi (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37: GAATTCGCGG NCGCTCGCAG TCAAGATTTT CTGTTCATCT CTGGz 7CAA TATGCCAAAA CTGGAGAGAA TCCTCCAGAC GGTTTCATGT GCCCGGGTGG CTTGTTGACT ACTACAACAG CCTGCAAGCT GAAGAAGCGA CTCCAGTCCC AAGCTACTAT TTGGACTAAG AGATTATTCA GTGGCGGGGG AAACATACAA AGATCATTTG GAGCTNTTTT CCTTATTGAT TCTTTAGGTT AGTAGTTGAT TCAGCATCTT TCNCGGATTG GGTGGTTTGG TCTTAAATTA TGAGACTCTG AAAAACTAAG GATCT INFORMATION FOR SEQ ID NO: 38: i) SEQUENCE CHARACTERISTICS: LENGTH: 388 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear

GATGCGCACT

GTGGAAGGTT

TGTATGAGGC

GCAAGTGATC

TGATATATGA

AAGCATGGNA

(ii) (vi) MOLECULE TYPE: cD.NA ORIGINAL SOURCE: ORGANISM: SEDl 3' (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38: GAATTCGCGG CCGCTTTTTT TTT1TTTTTTT TTTTGTTGTT TTTTTTTTTT TTTTATGNAC CCNTNCATCT TTTATTATTT AGTCCAACCG GNTTCGCTAC CCATGCTGTK KGGGCTCAAC TTGNGCAGAT CTTTAGTTTT CCCATGCTTC ATTTAAGACA AACAAACAAT CGAAAAGATC TTKATCATAT TTCAAATCAA TAAAGRATCA ATAAGGGRAA AAAGSTCAAA ATGATCTKAT CACTTGCTTG CNCCCATTGA ATAATCTCTT AGTCAAATAG GAGGTTGCCT CAATACAGGG GCTTCTTC.AN CTTGGAGGTT GTGGGGGG

KTTTTTTTTT

AGAACAATGG

AGAGTCTCAT

CTACTTAACC

TNTGTTCCCC

AATTGGNGTC

SUBSTITUTE SHEET (RULE 26) W O 9.1/07993 ffI'MRw«/w 1990 67 INFORMATION FOR SEQ ID NO: 39: SEQUENCE CHARACTERISTICS: LENGTH: 296 base pairs TYPE: nucleic acid 2) STRANDEDNESS: single cD) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: ORGANISM: SED2 (T7 PRIMER) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39: GCTAGCGCCT CCCTAGGTCT CGTTGTCAGC AATGGCCACC ACCGCTGCGT CCAGCCTCCT CAAGTCCTCC TTCGCGGGCT CCCGGCTCCC TTCGGCCACG CGCACCACCA CCCCGTCGTC 120 CGTGGNCGTG GNCACCCCGN GCGGCGGGGG GGGGCCCATC CGCGNGTCCA TCTCCTCCCC 180 CAACCCGNCC TACGACCTGA CGTCCTTCTG GTTCAGCCCC ATCAAGGAGT CCATCGTGTC 240 CCGCGAGATG ACCCGGCGCT ACATGANGGA CATGATCACC CACGNCGACA CCGACG 296 SUBSTITUTE SHEET (RULE 26)

Claims (7)

1. A DNA construct adapted to modify the expression of at least one senescence-related gene comprising a DNA sequence of at least 20 bases in length occurring in any one of the sequences shown as SEQ ID NO 1 to SEQ ID NO 39 preceded by a transcriptional initiation region operative in plants so that the construct can generate RNA in plant cells.

2. A DNA construct as claimed in claim 1 which generates antisense RNA.

3. A DNA construct as claimed in claim 1 in which the DNA sequence is a cDNA sequence.

4. A DNA construct according to any one of the preceding claims containing a sequence at least 20 bases in length occurring in any one of the clones deposited at the National Collections of Industrial and Marine Bacteria under the accession numbers NCIMB 40570 to NCIMB 40584. A method for producing plants having modified senescence characteristics which comprises transformation of plants with a DNA construct 20 adapted to modify the expression of at least one senescence-related genc and subsequent selection of plants in which the senescence process is either inhibited or accelerated, wherein the DNA construct is a construct as claimed in any one of claims 1 to 4.

6. A plant cell containing a DNA construct as claimed in any one of claims 1 a ,to 4.

7. A plant having modified senescence characteristics and derived from a cell as claimed in claim 6. C'WINWORD"JENNYMISPECNKI\76194-94.DOC I 69

8. A DNA construct according to claim 1 substantially as hereinbefore described with reference to any of the examples and/or the sequence listings. DATED 29 July, 1998 PHILLIPS ORMONDE FITZPATRICK Attorneys For: ZENECA LIMITED o. Seek a o e' *e