CA2262505A1 - Genetic control of fruit ripening - Google Patents
Genetic control of fruit ripening Download PDFInfo
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- CA2262505A1 CA2262505A1 CA002262505A CA2262505A CA2262505A1 CA 2262505 A1 CA2262505 A1 CA 2262505A1 CA 002262505 A CA002262505 A CA 002262505A CA 2262505 A CA2262505 A CA 2262505A CA 2262505 A1 CA2262505 A1 CA 2262505A1
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- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
- C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
- C12N15/8249—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving ethylene biosynthesis, senescence or fruit development, e.g. modified tomato ripening, cut flower shelf-life
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Abstract
A method of modulating the ripening and/or senescence characteristics in plants of the genus Musa comprises transforming plants with one or more sequences obtainable from the deposited cDNA library having the accession number 40184, regenerating said plants and selecting from the population of transformants those plants having modulated and/or tissue senescence characteristics.
Description
GENETIC CONTROL OF FRUIT RIPENING
This invention relates generally to the modification of a plant phenotype bv the regulation of plant ene ~A~les~ion. More specifically it relates to the modulation of the ripening and/or 5 tissue s~-.esc~-.re characteristics and plants derived the.~tL~ . One suitable application of the present invention is the modulation of ripening and/or sen~scçnre processes in plants of the genus Musa (referred to herein as banana).
Two plme"~al methods for the control of t~,es~ion are known, viz.: o~/e,~AI)~s~ion and undc.~Aylcssion. OveleA~,e;,~ion may be achieved by insertion of one or more than one extra o copies of the selected gene. It is, however, not unknown for plants or their progeny.
originallv transforrned with one or more than one extra copy of a nucleotide sequence. to exhibit the effects of unde,eA~-~ssion as well as ov~,~A~ ion.
For undcl~AI.ies~ion. often referred to as "gene silencing", there are two principle methods which are commonly referred to in the art as '~ tispl~e downregulation" and "sense 5 downregulation (also referred to as "cosuppression"). Both of these methods lead to an inhibition of expression of the target gene. Other lesser used methods involve modification of the Penetic control elements, the promoter and control sequences, to achieve greater or lesser expression of an inserted gene.
There is no reason to doubt the operability of these methods: they are well- established, used 20 routinelv in laboratories around the world and products in which the~ have been used are on the market.
Gene control by any of these methods requires the insertion of a selected gene or genes into plant m~teriAl which can be regeneTated into plants. This transformation process can be p~ cd via a number of methods, for example: the Agrobacterium-m~ t~d 25 tran~fu~ Lion method.
In the microparticle bombardment method, microparticles of dense material, usually gold or tungsten. are fired at high velocity at the target cells where they penetrate the cells. opening an aperture in the cell wall through which DNA may enter. The DNA may be coated on to the microparticles or may be added to the culture m~flium.
30 In microinjection, the DNA is inserted by injection into individual cells via an ultrafine hollow needle.
Sl,~;~ ITE SHEET (RU~E 26) Another mPth~--l viz. fibre-me~ ted transformation, applicable to both monocots and dicots, involves creating a suspension of the target cells in a liquid, adding microscopic needle-like material, such as silicon carbide or silicon nitride "whiskers", and ~it~ting so that the cells and whiskers collide and DNA present in the liquid enters the cell.
5 In ~ Aly, then, the re~ui~.nc~ for both sense and ~nti~P.n~e technology are known and the methods by which the required sequences may be introduced are known. What remains, then is to identify genes whose regulation will be exrect~çd to have a desired effect, isolate them or isolate a fragment of sufficiently effective length, construct a chimeric gene in which the effective fragment is inserted between promoter and terrnin~tion signals, and insert the o construct into cells of the target plant species by transformation. Whole plants may then be regenerated from the transformed cells.
R~n~n~ are a globally i~ O~ fruit crop. They are not only a popular dessert fruit, but represent a vital carbohydrate staple in the tropics with as many as 100 million people 5 subsisting on bananas and pl~nt~in~ as their main energy source. The cultivated dessert banana is commonly triploid, parth~noc~rpic and belongs to the Musa AAA genome group, eg. Cavendish subtypes. R~n~n~c are climac~eric fruits and ripening is regulated by ethylene produced by the fruit and involves numerous biochemical changes including the conversion of starch to sugars. cell wall ~ s~n~hly, synthesis of volatile compounds, chan_es in 20 phenolic constituents and degradation of chlorophyll in the peel. The conversion of starch to sugars is particularly striking, where starch accounts for 20-25% of the fresh wei~ht of the unripe fruit and depending on the genetic background, can ~e converted almost entirely to sugars.
The triploid nature of the cultivated dessert banana crop has h~.,p~.~d conventional methods 2s of breeding for improved ch~cl~,.;stics. As a result of this an enormous pool of genetic resources for Pnh~nring postharvest ch~c~listics of the fruit has remained ull~,ued.
According to the present invention there is provided a method of mod~ ting the ripening or tissue s~nPscenre process in plants of the genus Musa colllp,i~ing inserting into plant 30 material at least one polynucleotide sequence selected from the sequences depicted as SEQ
ID-Nos. 1 -57~ regenerating said plant material and selecting from the transformed SUBSTITUTE SHEET (RULE 26) WO 98111228 PCT/&s97tO2424 ley?c~ , plants with mo~ ted ripening or tissue se~-escF.-ce characteristics. The said polynucleotide mav be obtained from the cDNA library having the NCIMB Accession Number 40814.
Further accordil1g to the present invention is a method of modul~ting the ripenin_ or tissue 5 s~ se~-~ce process in plants of the genus Musa colllylisillg inserting into plant m~teri~l at least one polynucleotide sequence or a rlay?llle.l~ thereof, obtainable by hybridisation. from the cDNA library having the NCIMB Accession Number 40814, by the use of at least one of the sequences depicted as SEQ ID Nos 1-~7 as oligonucleotide probes, said hyhri~icz~tion being corlA~lcted at a tell,pc,aLul~ from 60~C to 65~C in 0.3 strength citrate buffered saline o col~t~ 0.1% SDS followed by rinsing at the same te,l,pe~aLule with 0.3 strenPth citrate buffered saline COl1t~i--i,.g 0.1% SDS, regerl~ lg said plant material and seleclin~ from the transformed legene~ , plants with modulated ripening or tissue senescence characteristics.
The invention further provides a method as described above wherein the said polvnucleotide is capable of m~dnl~ting the production of pectate lyase and more specifically the 5 polynucleotide comprises at least one of the sequences depicted in the sequence listings as SEQ-ID-Nos. 13-1 8.
A l~lefelled method for inserting the said polynucleotides into plant material according to the method of the present invention? may be selected from the group comprising the Agrobacterium, microparticle bombardment, fibre m.o~ t~d or direct insertion methods.
20 The invention further provides plants, their progeny and seed and material obtained from said plants, produced according to the method of the present invention. It is prefetred that the said plants, their progeny and seed and material obtained from said plants are derived from plants of the genus Musa.
The present invention also provides a vector functional in plants comprising a promoter 2~ region which is operable in plant cells, at least one of the polynucleotide sequences described above and a transcription t~nnin~tion sequence. The promoter may be constitutive, developm~nt~lly regulated or switchable. In addition to this the promoter may also be tissue specific or organ specific.
Further provided is a banana produced via the preceding method. having altered fruit 30 characteristics when compared with a banana which is not transformed with at least one of the polynucleotide sequences described above.
SUBSTITUTE SHEET (RULE 26) CA 02262~0~ 1999-02-02 The present invention also provides an improved method of controlling plant pathogens comprising the application of an anti-pathogenic agent to plants, characterised in that plants to which the said agent is applied, are plants according to the present invention.
The gene se~uences of the present invention may be synth~cic~-~ ab initio, using the sequence 5 data in the sequence listing provided herewith, or isolated from a library using the standard techniques know ~,vithin the art. The sequences depicted in the sequence listing or parts thereof may also be used to create oligonucleotide probes for the purposes of isolating from the library those polynucleotides which are capable of producing the desired proteins. To assist the isolation of these polynucleotides we have deposited with the National Collection o of Industrial & Marine Bacteria, St. Machar Drive, Aberdeen, UK, a cDNA library of the banana peel ripening related genes. The library was deposited on 9th July 1996 and has the NCIMB Accession Number 40814.
Thus, this invention is based on the identification of genes which encode proteins involved in banana ripening-related processes, specifically within banana pulp. DNA sequences 15 accor.ling to the sequence listing or those sequences obtainable from the deposited library, may be used in the process of modifying the plant ripening characteristics of plants and/or fruit.
By virtue of this invention banana plants can be generated which. amongst other phenotypic modifications, may have one or more of the following fruit characteristics: improved 20 resistance to damage during harvest, pac~ing and transportation due to slowin~ of the ripening and over-ripening processes; longer shelf life and better storage charac~eristics due to reduced activity of degradative pathways (e.g. cell wall hydrolysis); improved processing characteristics due to changed activity of proteins/enzymes contributing to factors such as:
viscosity, solids, pH, elasticity; improved flavour and aroma at the point of sale due to 25 modification of the sugarlacid balance and other flavour and aroma components responsible for characteristics of the ripe fruit; modified colour due to changes in activity of enzymes involved in the pathways of pigment biosynthesis (e.g. Iycopene, ~-carotene~ chalcones and anthocvanins); increased resict~nce to post-harvest pathogens such as fungi.
The activity of the ripening-related proteins may be either increased or reduced depending on 30 the characteristics desired for the modified plant part (fruit, leaf, flower, etc). The levels of protein may be increased; for example, by incorporation of additional genes. The additional - SIJ~S 111 ~ITE SHEET (RULE 26~
CA 02262~0~ 1999-02-02 wo 98111228 pcTlGs97to2424 genes may be ~leciEn~d to give either the same or dirr~.c.~t spatial and temporal patterns of t~pression in the fruit. '~nti~ncel~ or "partial sense" or other techniques may be used to reduce the ~p~ ion of ripening-related protein.
The activity of each ripening-related protein or enzyme may be modified either individually or in combination with mo~lific~tion of the activity of one or more other ripening- related proteinste~. ~ylllcs. In addition, the activities of the ripening-related proteins/enzymes may be m~difi~d in combination with modification of the activity of other enzymes involved in fruit rirçnin~ or related ylocesses~
DNA constructs according to the invention for gene silencing, may comprise a base sequence o at least 10 bases (preferably at least 35 bases) in length for transcription into 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 l 00 and 1000 bases in length. When using genomic DNA as the source of a base sequence for transcription it is possible to use either intron or exon regions or a combination 15 ofboth.
As a source of the DNA base sequence for transcription, a suitable cDNA or genomic DNA
or synthetic polynucleotide may be used. The isolation of suitable ripening-related sequences is described above; it is convenient to use DNA sequences derived from the ripening-related clones deposited at NCIMB in Aberdeen. Sequences coding for the whole, or subst~nti~lly the whole, of the ~,op,late ripening-related protein may thus be obtained.
Suitable lengths of this DNA sequence may be cut out for use by means of restriction enzymes. The prepaldlion of such constructs is described in more detail below.
Constructs suitable for expression of the ~l,.opl.ate ripening-related sequence in banana cells, may be produced using a cDNA sequence selected from the deposited library having the NCIMB Accession Nurnber 40814 or the gene sequence as found in the chromosome of the banana plant. Recombinant DNA constructs may be made using standard techniques.
In this specification modulation means an incre~e or decre~e of the desired effect.
More specifically '~modulation of the ripening or tissue ~en~scence process in plants of the genus Musa'' means an increase or decrease in production of a ripening related protein resulting from the method as described above. For example, where an increased ripening related protein is desired, plants may be transformed according to the method as described SUBSTITUTE SHEET (RULE 26) above and those plants exhibiting the desired effect may be selected from the population of transformants. Fu~lhe~lllore~ it may be desirable to provide a plant with modulated ripening or tissue sen~scenre characteristics by increasing the production of one protein and decreasing the production of another protein in the same plant. For example, a banana fruit, modified using the present method, and having decreased levels of the enzyme pectate Iyase would be beneficial because pulp softening would require a sllbst~nti~lly longer lime when compared with a control. In addition to this and by incle~illg the levels of another ripening related protein such as an antifungal protein in the same fruit using the present method, would complement the extended life of the banana pulp with inereased re~i~t~nce to disease.
o Increase in production of a ripening related protein includes inserting into plant material one or more copies of anv of the polynucleotides described above, wherein the said polynucleotides must be capable of producing a protein and thereby increasing protein levels when compared with a control plant.
Decrease in production of a ripening related protein includes inserting into plant m~tPri~l one 15 or more copies of any of the polynucleotides described above, wherein the said polvnucleotides must be capable of producing an m~NA which is capable of interfering with endogenous mRNA to such a degree that the levels of tr~n~l~t~d protein are reduced when compared with a control plant.
Ripening process of plants means the process of maturing or developing .
20 Senescence means the progressive deterioration in function of cells. tissues, organs etc., related to the period of time since that function commPnse~ .
Control plant means a comparable plant used for the purposes of 11~t~PrTT ining modulation of the ripening or tissue s~nPscenre process effect in plants. Specifically, in plant transform~tion~ the control plant is usually of the same species and variety as the m~tPri~l 2~ used before the l,dllsÇo,,,,ation process and is grown in the same conditions, (usually with the transformant selection step modified in some way on the part of the control plant)~ as the transformed plants. More specifically the control plant may comprise an untransforrned contlol plant or a transformed control plant providing it has not already been transformed with the same polynucleotide sequence as the plant material to be transformed.
30 "Plant material" includes plant cells and any other type of plant Icgcne.able m~tPri7~1.
SlJ~;~ JTE SHEET (RUEE 26) WO 98111228 PCT/Gs97/02424 The following examples further illustrate the invention but are not to be construed to limit the scope thereof:
TABLE 1. Is a list of clones isolated from banana pulp and the co~ .ollding sequence 5 identity number as provided in the sequence listing herein. The table also illustrates the approximate clone size, the perce.,~ge identity and, where relevant, nucleotide similarity with published sequences, based on the results obtained from conlpa,isons with the EMBL
se~uence rl~t~h~ce The table also provides, where relevant, the gene identity of those p~ hPd se~uences and their ~t~ba~e accession numbers.
o FIGURE 1. Plant transformation vector pUN~ col-l;.inil-g the UBI polyubi~uitin promoter.
FIGURE 2. Plant transformation vector pSHYN, co~ ;"i,~g hygromycin resistance gene for selection of ~ sro-,l,cd plants.
FIGURE 3. Plant transformation vector pAN~ cont~ining the banana ACC oxidase promoter.
Construction of a cDNA library of ripening genes 1.1 Isolation of me~ nger RNA
Total RNA was isolated from ripening (24 hours after ethylene treatment) banana pulp (Musa 20 acuminata cv. Grand Nain) as described bv Chang et al. Plant Molecular Biology Reporter.
Vol. 11(2) 113-116 (1993). Messenger RNA was isolated from total RNA bv Oligo(dT)-cellulose chromatography according to Bantle et al., Analytical Biocll~n~ tr-~ 72, 413-427 (1976).
25 1.2 Synthesis of cDNA and Cloning into Vector The first and second strands of the cDNAs were synth~ci~ed from the messenger RNAs using a com~nercial cDNA synthesis kit (Catalog No. 200450, ZAP ExpressrM Gold Cloning kit, Stratagene Ltd, Carnbridge, Cambs, UK). Double stranded cDNAs were cloned into the ZAP Express~M vector, packaged, mixed with plating bacteria to deterrnine titre and for 30 library screening, following instructions of the suppliers protocol.
SlJts:i 111 ulTE SHEFT (RULE 26) 1.3 Screening of the cDNA library from banana pulp.
The url~mplified cDNA library from ripening banana pulp was differentially screened using cDNA from unripe and ripening banana peel tissue. A proportion of the librarv was plated individually at low density and duplicate plaque lifts made onto Hybond N nylon filters 5 (,~m~rsh~m) according to the m~nl~f~ctllrer's instructions. One filter was hybridised to dCTP
radiolabelled cDNA from green fruit and the duplicate filter hybridised to dCTP
radiolabelled cDNA from ripening fruit. Hybridization's were at high stringency. Plaques hybridising ~.efe.~.~Lially with ripening or green radiolabelled cDNA were picked and re-plated for a second round of selection by differential scl~enillg. These clones were numbered lo as ripening up- or down-regulated peel clones. The clones were in-vivo excised from the ZAP expresslM vector into the pBK-CMV phagemid vector using the ExAssistTM
interference-resistant helper phage, following instructions from m~nllf~ctl-rers protocol.
1.4 Characterisation of the ripening pulp cDNA library and the ripening-related clones.
15 The ripening cDNA li~orary from pulp tissue were prepared with an efficienc~ of 3.2 x 10S
plaque-forming units per microgram of cDNA. The sizes of the inserts in the peel library was 0.4 - 6.7 Kb with a mean size insert of 1.47 Kb.
From the 250 plaques used in the first screen, 73 putative ripening-related clones were obtained. These 73 clones were partially sequenced using the ABI PRISMTM Dye 20 Tennin~tor Cycle Sequencing Ready Reaction kit with Ampli~aq~ DNA poivmerise (Applied Biosystems, Warrington, Cheshire, UK) with forward primers specific for the pBK-CMV vector. From these, the following ripening-related clones were selected. Comparisons of these sequences in the EMBL d~t~b~se using GCG ('Wisconsing') software has identified homologies for the clones listed in TABLE 1 below.
Cons~ruction of truncated sense RNA vectors with the maize polyubiquitin promoter.
A vector is constructed using the sequences corresponding to a fraement of the inserts of 30 one of the sequences I to 57. This fragment is synthPcised by polymerase chain reaction using svnthetic primers incorporating BamHI restriction sites suitable for clonin between a S~ 111 ulTE SHEET (RULE 26) rnaize UBI polyubiquitin promoter (Chri~tenc~n et al, 1992, Plant Molecular Biology, 18:675-689) and a nopaline synthase 3'end termination sequences in the vector pUN (Fig.
1.).
The truncated sense expression cassette is excised by digestion with AscI, the ends of the fr~rnPnt are made flush with T4 polymerase and it is cloned into the vector pSHYN (Fig. 2.) which has been cut with KpnI and the ends made flush with Klenow polymerase. pSHYN
col.t;.;.~ hygromycin resi~t~nre gene for selection of transformed plants.
After synthesis of the vector, the structure and orientation of the sequences are confirmed by DNA sequence analysis.
lo Construction of truncated sense RNA vectors with a fruit enh~nced promoter.
The 1386bp HindIII fragment cont~inin~ the banana ACC oxidase promoter (UK.
Application No. 9607700.3) is cloned the HindIII site in pMSC2 (Fig.3.) to give the vector pAN.
A vector is constructed using the sequences corresponding to a fragment of the inserts of one of the sequences I to 57. This fragment is synth~-si~e-l by polymerase chain reaction using synthetic primers incol~u,d~ g BamHI restriction sites suitable for cloning between a 20 maize UBI polyubiquitin promoter (Chri~t~n~en et al, 1992, Plant Molecular Biology, 18:67~-689) and a nopaline synthase 3'end t~ ion sequences in the vector pAN.
The Ll~-c~l~d sense e,.~ ssion cassette is excised by digestion with AscI, the ends of the fragment are made flush with T4 polymerase and it is cloned into the vector pSHYN (Fig. 2.) which has been cut with KpnI and the ends made flush with Klenow polymerase. pSHYN
25 contains hy~lulllycin resict~nre gene for selection of ~ ~lllled plants.
After synthesis of the vector, the structure and orientation of the sequences are confirmed by DNA sequence analysis.
Construction of an over-expression vector with the maize polyubiquitin promoter.
Sllts~ 1 l l ulTE SHEET (RULE 26) The complete sequence of a ripening -related cDNA co.~ lg a ~11 open-reading frame is inserted into the vectors described in EXAMPLE 2.
Construction of an over-~A~lession vector with a fruit enh~nred promoter.
The complete sequence of a ripening -related cDNA cont~ining a full open-reading frame is inserted into the vectors described in EXAMPLE 3.
Generation of transformed Musa plants Transformed Musa plants cont~ining the vectors are produced by the method described in Sagi et al. (1995) Biotechnology Vol. 13 pp 481-485. Regenerated transformed plants are identified by their ability to grow on hygromycin and grown to maturity. Ripening fruit are analysed for a modulation in their ripening related or senescence characteristics.
Other suitable transformation methods for banana are described in Sagi et al. ( 1994) Plant Cell Reports. Vol. 13. pp 262-266. and May et al. (1995) Biotechnology. Vol. 13 pp 486-492.
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SU~ JTE SHEET (RULE 26) SEQUENCE LISTING
~1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: ZENECA LIMITED
(B) STREET: 15 STANHOPE GATE
(C) CITY: LONDON
(D) STATE: LONDON
(E) COUNTRY: UNITED KINGDOM
(F) POSTAL CODE (ZIP): WlY 6LN
(ii) TITLE OF INVENTION: GENETIC CONTROL OF FRUIT RIPENING
(iii) NUMBER OF SEQUENCES: 57 ~iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version ~1.30 (EPO) (v) CURRENT APPLICATION DATA:
APPLICATION NUM8ER: GB PPD
(2) INFORMATION FOR SEO ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 785 base pairs ~B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA
(vii~ IMMEDIATE SOURCE:
(B) CLONE: U-Ug (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 813 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA
(vii) IMMEDIATE SOURCE:
(B) CLONE: U-U17 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 746 base pairs (B~ TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA
(vii) IMMEDIATE SOURCE:
(B) CLONE: U-U66 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
GGTTAGGTAA CCCTACCTTC GCTCTCGTCC GGAGCACCAC CGCCcccGcc GGCCAACCCG 180 CA 02262505 l999-02-02 t2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A~ LENGTH: 795 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA
(vii~ IMMEDIATE SOURCE:
(B) CLONE: U-U104 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
(2) INFORMATION FOR SEQ ID NO: 5:
(i) SEOUENCE CHARACTERISTICS:
(A~ LENGTH: 797 base pairs (B~ TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown ~ii) MOLECULE TYPE: cDNA
(vii~ IMMEDIATE SOURCE:
(B) CLONE: U-U13 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
IS
(2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 792 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D~ TOPOLOGY: unknown (ii~ MOLECULE TYPE: cDNA
(vii) IMMEDIATE SOURCE:
(B) CLONE: U-Ul36 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
AAGCCCTCAG GAACTCCAAC ATCCAAGTCC TGTTGGATGT CccccGATcc GACGTGCAGT 300 WO g8111228 PCT/GB97/02424 CCGGGCGTTT CTTCCTTTGC CAGGGANTNG GGGCGAAT~C CCNNCTTTTC CANAAACCTT 780 (2) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 855 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLEC~LE TYPE: cDNA
(vii) IMMEDIATE SOURCE:
(8) CLONE: U-U2l (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
CGCCACCGGT CGCGAGGGTT GGAGCCATGG AATTCTTGCG GTTTGGGGCT GAGGTGGCGG l20 (2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 722 base pairs (B) TYPE: nucleic acid CA 02262505 l999-02-02 W 0 98111228 PCTIGBg7/02424 (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA
(vii) IMMEDIATE SOURCE:
(B~ CLONE: U-U31 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
TGGCNTTTTC GACTANNAGT TTGACCGATG GCTATGTTCG TTTTGCTTTT CAC~llll~l 480 40 (2) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 793 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TyeE: cDNA
(vii) IMMEDIATE SOURCE:
(B) CLONE: U-U131 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
WO 98111228 rCT/GB97t02424 TTCTTTCCCC CCNCCCANTC NAACNTTCCN ACGAATTCCC GTTTTACCCN AAA~ACCAAN 780 (2) INFORMATION FOR SEQ ID NO: l0:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 847 base pairs (B) TYPE: nucleic acid ~C) STRANDEDNESS: single (D) TOPOLOGY: unknown ~ii) MOLECULE TYPE: cDNA
(vii) IMMEDIATE SOURCE:
(B) CLONE: V-U32 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: l0:
TATTCACTGA CCGCGCCGGC CGCCGCCGTG CCGACTGCCT CTCCTTCGCT GTCGACGACC l80 (2) INFORMATION FOR SEQ ID NO: ll:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 822 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown ~ii) MOLECULE TYPE: cDNA
(vii) IMMEDIATE SOURCE:
(B) CLONE: U-U55 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
CCCCTTCGCC GCAACCCACG GGAAAGGGGA TCTTCGAGAC NACACNCNAA CAGCCGGGGT ' 480 (2) INFOP~MATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 743 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA
(vii) IMMEDIATE SOURCE:
(B) CLONE: U-U72 ~xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
CA 02262505 l999-02-02 (2) INFORMATION FOR SEQ ID NO: 13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 806 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA
(vii) IMMEDIATE SOURCE:
(B) CLONE: U-U68 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:
(2) INFORMATION FOR SEQ ID NO: 14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 687 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: sin~le (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA
(vii) IMMEDIATE SOURCE:
~B) CLONE: U-U69 CA 02262~0~ 1999-02-02 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:
(2~ INFORMATION FOR SEQ ID NO: 15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 741 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA
(vii) IMMEDIATE SOURCE:
(B) CLONE: U-U84 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:
CA 02262~0~ l999-02-02 - 2~ -~2) INFORMATION FOR SEQ ID NO: 16:
~i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 727 base pairs (B) TYPE: nucleic acid ~C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA
(vii) IMMEDIATE SOURCE:
~B) CLONE: U-U89 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:
(2) INFORMATION FOR SEQ ID NO: ~7:
(i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 842 base pairs (B) TYPE: nucleic acid ~C) STRANDEDNESS: single ~D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA
~vii) IMMEDIATE SOURCE:
~B) CLONE: U-U115 ~xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:
AGTAGTACAG GAAGTACAAA GAAGCTTGAA CGTGTCGCGG CGGCGACTGG GCTACTTGTC 1~0 CA 02262505 l999-02-02 WO 98/11228 PCTtGB97/02424 ATCCACGGCG TCCACATCCA CGAATGCAAG CAGGGCGGGA ACGCGTNCGT TcGcNAcTcc 600 (2) INFORMATION FOR SEQ ID NO: 18:
~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 724 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA
(vii) IMMEDIATE SOURCE:
(B) CLONE: U-U117 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:
(2) INFORMATION FOR SEQ ID NO: 19:
~i) SEQ~ENCE CHARACTERISTICS:
(A) LENGTH: 797 base pairs ~B) TYPE: nucleic acid (C) STRANDEDNESS: single CA 02262~0~ 1999-02-02 W 0 98/11228 PCT/GB97r02424 (D) TOPOLOGY: unknown (ii~ MOLECULE TYPE: cDNA
(vii) IMMEDIATE SOURCE:
(B) CLONE: U-V80 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: l9:
CAAAGCTCGG CGGAGGCAAG CTTGTCCTGA AACCACTGGG CTTGGCGTTG CCTCACTATT l80 (2) INFORMATION FOR SEQ ID NO: 20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 742 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA
(vii) IMMEDIATE SOURCE:
(B) CLONE: U-U90 ~5 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:
CTGTTGGAGT TGGAACTGTG GGTTCCTCCA GAAGCAATCA TGcTTGcAGG ACTTGCATTC 420 CA 02262~0~ 1999-02-02 S
~2) INFORMATION FOR SEQ ID NO: 21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 807 base pairs ~B) TYPE: nucleic acid ~C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA
~vii) IMMEDIATE SOURCE:
(B) CLONE: U-U92 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:
(2) INFORMATION FOR SEQ ID NO: 22:
(i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 770 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA
(vii) IMMEDIATE SOURCE:
CA 02262505 l999-02-02 (B) CLONE: U-U91 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:
GACATTGATT TTGAAAAAGA ACCCAATTGG AACAGGGGAA GGATGGGAAA AAACATATAC 5q0 (2) INFORMATION FOR SEQ ID NO: 23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGT~: 810 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA
~vii) IMMEDIATE SOURCE:
(B) CLONE: U-U96 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:
CCAGGATTGA ~l~l.G~l-C TTGTCTACAG ACCCCATCAG ATGTTGATAA GCTTCCNAAA 420 T iTGAAATC AGGGATTTTG ATGCATTTGA TTTGAAGATG ANACTTCCTG CNGTANTTAC 600 CA 02262505 l999-02-02 (2) INFORMATION FOR SEQ ID NO: 24:
(i~ SEQUENCE CHARACTERISTICS:
(A) LENGTH: 765 base paLrs (B) TYPE: nuclelc acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA
(vii) IMMEDIATE SOURCE:
(B) CLONE: U-U103 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:
(2) INEORMATION FOR SEQ ID NO: 25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 774 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA
(vii) IMMEDIATE SOURCE:
(B) CLONE: U-U93 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25:
70 GGCACGAGCG GAGAANGCAA TCTCGCTCTC TCTCTACTGC ATcGcGGcTc TCGTCCTCGA 60 CA 02262505 l999-02-02 W O 98/11228 PCTtGB97/02424 (2) INFORMATION FOR SEQ ID NO: 26:
~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 786 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA
(vii) IMMEDIATE SOU~CE:
(B) CLONE: U-U125 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26:
GGCACGAGCT AGTCTCGAGT llllllllll llllllllAA AATTGAAATT AGATGCGATT 60 (2) INFORMATION FOR SEQ ID NO: 27:
CA 02262505 l999-02-02 WO 98/11228 PCT/GB97tO2424 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 486 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA
(vii) IMMEDIATE SOURCE:
~B) CLONE: U-U105 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:
GGCACGAGCG A~llllllll llllllllll llllilllll TTTTCACAAG GNAACCATAG 60 (2) INFORMATION FOR SEQ ID NO: 28:
(i) SEQUENCE CHARACTERISTICS:
;A~ LENGTH: 791 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single ~D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA
(vii) IMMEDIATE SOURCE:
(B) CLONE: U-U116 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28:
AAAGGAAATN AANCCNNTTG GGTTTTCCCC TTTGGCCTTT lllL~ lG AAAATCCTNT 780 ~lllllGGTG T 791 (2) INFORMATION FOR SEQ ID NO: 29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 814 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA
(vii) IMMEDIATE SOURCE:
(B) CLONE: U-U120 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29:
NCCACCGTCC TTACCCTCCA TCGACGCCAC CCCACCATCC GAAGCACCCG CCTTCTCAGC 5g0 GGG~lllllN TTAANATTTN AANAAANCNA AN1N11 L LCC CCCCCNTNTT GGTTNATGGC 780 55 CTATGGGTTT ~ L L L ~'1 U L lC TTGGCTTTTC CCGG 814 (2) INFORMATION FOR SEQ ID NO: 30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 742 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA
(vii) IMMEDIATE SOURCE:
(B) CLONE: U-U126 CA 02262505 l999-02-02 ~xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30:
~2) INFORMATION FOR SEQ ID NO: 31:
li) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 794 base pairs ~B) TYPE: nucleic acid ~C) STRANDEDNESS: single tD) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA
(vii) IMMEDIATE SOURCE:
~B) CLONE: U-U129 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31:
CA 02262~0~ 1999-02-02 CCNCCCCCCC CAATTAACCC N1lCllGCTC TTCCCCCTTG CCTTCANCCA ACCCGCCCNT 780 (2) INFORMATION FOR SEQ ID NO: 32:
(i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 799 base pairs ~B) TYPE: nucleic acid ~C) STRANDEDNESS: single ~D) TOPOLOGY: unknown ~ii) MOLECULE TYPE: cDNA
~vii) IMMEDIATE SOURCE:
(8) CLONE: U-Ul30 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32:
GCCGGCCAGG ATCATCAGGG AAGCACTGTC GAAGGCGCTG GTGAAGTACT ACCCCTTCGC l20 CGCCCGA~AT CGCCCGTGGC CTGCCNAANC CCACCGTGGA NCCTGCATGG TCCCGGGANG 480 TTTTTTCCTT TCCTTCCCCC CCCAAAANAA CTTCCCCCCA CC~ l TGCNCCCAAG 780 ~2) INFORMATION FOR SEQ ID NO: 33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 759 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single ~D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA
~vii) IMMEDIATE SOURCE:
(B) CLONE: U-U43 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33:
TAATAGCAGT GGCTACTTGG TGCTTCATTG GGGTGCAATT CATAATAGAA GGAATAACTG l20 ATGAATTAAA TAAANGAACN CNCCTNCAAA CTNAGGGAA 75g (2) INFORMATION FOR SEQ ID NO: 34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 785 ~ase pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA
(vii) IMMEDIATE SOURCE:
(B) CLONE: U-U70 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34:
CNTCGGCTGC CCTCTTCCTT GCTCTCAACC TTGTCTTCTT TGCACTCACC AGTGCTTGTG lB0 TlllL~llGAG CATTCCTTTG TGTCTTTACT TCCCTTTGTT TTTGGCTTTT TGATGACGAA 600 (2) INFORMATION FOR SEQ ID NO: 35:
CA 02262~0~ l999-02-02 (i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 784 ~ase pairs ~B) TYPE: nucleic acid ~C) STRANDEDNESS: single ~D) TOPOLOGY: unknown ~ii) MOLECULE TYPE: cDNA
~vii) IMMEDIATE SOURCE:
~B) CLONE: U-U16 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35:
45 (2) INFORMATION FOR SEQ ID NO: 36:
~i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 817 base pairs ~B) TYPE: nucleic acid ~C) STRANDEDNESS: single ~D) TOPOLOGY: unknown ~ii) MOLECULE TYPE: cDNA
(vii) IMMEDIATE SOURCE:
(B) CLONE: U-U30 ~xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36:
CA 02262~0~ 1999-02-02 (2) INFORMATION FOR SEQ ID NO: 37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 837 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA
(vii) IMMEDIATE SOURCE:
(B) CLONE: U-U40 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37:
~ GG GTATCAGGCA AAGATGGATA CTTCCAGGTT TGCTATTTAA 60 (2) INFORMATION FOR SEQ ID NO: 38:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 799 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single CA 02262505 l999-02-02 tD~ TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA
(vii~ IMMEDIATE SOURCE:
(B~ CLONE: U-U108 ~xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38:
(2) INFORMATION FOR SEQ ID NO: 39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 786 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA
(vii) IMMEDIATE SOURCE:
(B~ CLONE: U-U128 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39:
GTGGAAAATG CAGTGTCATA CAGCTCTAGG TCACTGCTTT CAACCGGATC CcTGGGAAAG 300 WO 98tll228 PCT/GB97102424 (2) INFORMATION FOR SEQ ID NO: 40:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 804 baSe PairS
(B) TYPE: nUC1eiC aCid (C) STRANDEDNESS: Sing1e tD) TOPOLOGY: UnknOWn (ii) MOLECULE TYPE: CDNA
(Vii~ IMMEDIATE SOURCE:
(B) CLONE: U-D4 (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 40:
60 TGGGGGAAAN 1L11L11"111 TTTT 804 (2) INFORMATION FOR SEQ ID NO: 41:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 826 baSe PairS
(B) TYPE: nUC1eiC aCi~
(C) STRANDEDNESS: single (D) TOPOLOGY: UnknOWn (ii) MOLECULE TYPE: CDNA
W O 98/11228 rCT/GB97/02424 ~vii) IMMEDIATE SOURCE:
(B) CLONE: U-D10 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41:
AGGACCTTCC TAAACAGAAG GGTTCTACAC TTGA&GAACC AAACCACCGC TTACGAAGGA 180 35 GGCNCCCTGG TCCTCCTNCC TGCTACTTAA AAA~ TCNTNT 826 (2) INFORMATION FOR SEQ ID NO: 42:
(i~ SEQUENCE CHARACTERISTICS:
(A) LENGTH: 813 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown ~ii) MOLECULE TYPE: cDNA
(vii) IMMEDIATE SOURCE:
(B) CLONE: U-Dl3 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42:
GGAGGACTCG GTGATGTTCT TGGAGGACTG CCACCGGGCC ATGGCTGCAA ATGGACACAG g80 WO 98/11228 PCI'/GB97/02424 ANAAGAATTG ACAGGGTTTT CATTGATCAC ~ LCTT GAAAAGGTGT GGGGAAAAAC 660 (2) INFORMATION FOR SEQ ID NO: 43:
~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 805 base pairs ~B) TYPE: nucleic acid ~C) STRANDEDNESS: single ~D) TOPOLOGY: unknown ~ii) MOLECULE TYPE: cDNA
~vii) IMMEDIATE SOURCE:
~B) CLONE: U-D66 ~xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43:
GTGATGACTA TAGCTCCACG CTATGATCAG TACAAGGATG GGTGGGATAC A~ATGTCCTG 480 CCTNCCTACT TA~AAANTTT TTTCC 805 ~2) INFORMATION FOR SEQ ID NO: 44:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 790 base pairs ~B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA
(vii) IMMEDIATE SOURCE:
~B) CLONE: U-D111 CA 0226250~ l999-02-02 ~xi) SEQUENCE DESCRIPTION: SEQ ID NO: 94:
AAC~l..l~l TGCCAGCACT TTGGGAACCN CAAGGATCCT GGATCTTAAC AACNCAANTC 720 (2) INFORMATION FOR SEQ ID NO: 45:
~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 813 base palrs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECVLE TYPE: cDNA
(vii) IMMEDIATE SOURCE:
(B) CLONE: U-Dl12 ~xi) SEQUENCE DESCRIPTION: SEQ ID NO: 45:
~ iG GAGCATAATA GCAATTTATT CAGGAGGATC AAACTCAAAC 60 CTGTGATGCC TTCTATGACA GTGTCAGCAA GTCCACCAGT TGTTGCGCAC ATGGGGGGAA ~20 CA 02262505 l999-02-02 WO 98/11228 PCT/GBg7/02424 (2~ INFORMATION FOR SEQ ID NO: 96:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 808 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA
(vii) IMMEDIATE SOURCE:
tB) CLONE: U-D39 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 46:
GGCACGAGCG A~ l GCTGTGATGA TATTATGCAT CTTATTAGTT 60 ACN~lll;~ll AAAACATCNG TNCCNAAT B08 (21 INFORMATION FOR SEQ ID NO: 47:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 809 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECVLE TYPE: cDNA
(vii~ IMMEDIATE SOURCE:
(B) CLONE: U-D50 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 47:
, CA 02262~0~ l999-02-02 (2) INFORMATION FOR SEQ ID NO: 48:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 809 ~ase pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA
(vii) IMMEDIATE SOURCE:
(B) CLONE: U-D86 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 48:
CA 02262505 l999-02-02 (2) INFORMATION FOR SEQ ID NO: 49:
(i) SEQUENCE CHARACTERISTICS:
tA) LENGTH: 785 base pairs ~B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA
~vii) IMMEDIATE SOURCE:
(B) CLONE: ~-D90 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 49:
(2) INFORMATION FOR SEQ ID NO: 50:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 813 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA
(vii) IMMEDIATE SOURCE:
(B) CLONE: U-D93 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 50:
, CA 02262~0~ l999-02-02 CAAATGCGTT CNACAATTGG ~ CCA NCCTGCNCAA AACTGTTCCT TGGGCTCCCG 780 (2) INFORMATION FOR SEQ ID NO: 51:
(i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 819 base pairs (B~ TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA
(vii) IMMEDIATE SOURCE:
(B) CLONE: U-D61 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 51:
CAACCCGGGA CAGATCTCGC TGCCCTACGC CCTGTTCACG GCCTCCGGCG TCGTCGTGCA 4 e o TTGATCAGGC ATGTTNGGCG G.~AGAACCCC AAGGAAAACC AGGGAAGGAA ATCAAGGCTT 720 (2) INFORMATION FOR SEQ ID NO: 52:
(i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 794 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA
(vii) IMMEDIATE SOURCE:
(B) CLONE: U-D63 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 52:
CGCCGTGCAT ACAATGATAC CTCTGATAGG GTTGAATGCA CAAACCACTC ATCGTGCTAT l80 TTTATTGCTT TGCATATTTT GTGCTGGAAA CCATGCCAAT CCAACTCCTT TGCAAAACTT g20 (2) INFORMATION FOR SEQ ID NO: 53:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 798 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA
(vii) IMMEDIATE SOURCE:
(B) CLONE: U-D65 ~xi) SEQ~ENCE DESCRIPTION: SEQ ID NO: 53:
CACCGCCCAC CCCCGTGGCC AAGCCGCCTT CAGTTGAACC ACCACCCTAC CACGGCCCTC l80 CA 02262505 l999-02-02 WO 98111228 PCTtGB97/02424 (2) INFORMATION FOR SEQ ID NO: 54:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 824 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA
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(2) INFORMATION FOR SEQ ID NO: 55:
~i) SEQUENCE CHARACTERISTICS:
(A) LENGT~: 797 base pairs (B) TYPE: nucleic acid CA 02262~0~ l999-02-02 (C~ STRANDEDNESS: single (D~ TOPOLOGY: unknown (ii~ MOLECULE TYPE: cDNA
(vii~ IMMEDIATE SOURCE:
(B) CLONE: U-D75 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 55:
TTGAANATGA AGAANAATTN TTTGTGCCNC TTTCAAGGTG GAANGATCTG C~ ullGC 720 (2~ INFORMATION FOR SEQ ID NO: 56:
(i~ SEQUENCE CHARACTERISTICS:
(A) LENGTH: 804 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii~ MOLECULE TYPE: cDNA
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~0 AACCTGGGCA GCTTGAGGCA GCAGTATGGG CTTTCCAAGA GTGCCGACNA GGTGGTGCTG 360 CA 02262505 l999-02-02 GTCATCGAAG CCTACAAGGC CCTCCGTGAC CGANCTCCCT ATCCTCCCAG CTTCATGCTC ~20 l5 A,-~ATCCTCT TTAAAANCAT AAAA 804 (2) INFORMATION FOR SEQ ID NO: 57:
(i~ SEQUENCE CHARACTERISTICS:
(A) LENGTH: 803 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D~ TOPOLOGY: unknown (ii~ MOLECULE TYPE: cDNA
(vii~ IMMEDIATE SOURCE:
(~) CLONF: U-D64 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 57:
This invention relates generally to the modification of a plant phenotype bv the regulation of plant ene ~A~les~ion. More specifically it relates to the modulation of the ripening and/or 5 tissue s~-.esc~-.re characteristics and plants derived the.~tL~ . One suitable application of the present invention is the modulation of ripening and/or sen~scçnre processes in plants of the genus Musa (referred to herein as banana).
Two plme"~al methods for the control of t~,es~ion are known, viz.: o~/e,~AI)~s~ion and undc.~Aylcssion. OveleA~,e;,~ion may be achieved by insertion of one or more than one extra o copies of the selected gene. It is, however, not unknown for plants or their progeny.
originallv transforrned with one or more than one extra copy of a nucleotide sequence. to exhibit the effects of unde,eA~-~ssion as well as ov~,~A~ ion.
For undcl~AI.ies~ion. often referred to as "gene silencing", there are two principle methods which are commonly referred to in the art as '~ tispl~e downregulation" and "sense 5 downregulation (also referred to as "cosuppression"). Both of these methods lead to an inhibition of expression of the target gene. Other lesser used methods involve modification of the Penetic control elements, the promoter and control sequences, to achieve greater or lesser expression of an inserted gene.
There is no reason to doubt the operability of these methods: they are well- established, used 20 routinelv in laboratories around the world and products in which the~ have been used are on the market.
Gene control by any of these methods requires the insertion of a selected gene or genes into plant m~teriAl which can be regeneTated into plants. This transformation process can be p~ cd via a number of methods, for example: the Agrobacterium-m~ t~d 25 tran~fu~ Lion method.
In the microparticle bombardment method, microparticles of dense material, usually gold or tungsten. are fired at high velocity at the target cells where they penetrate the cells. opening an aperture in the cell wall through which DNA may enter. The DNA may be coated on to the microparticles or may be added to the culture m~flium.
30 In microinjection, the DNA is inserted by injection into individual cells via an ultrafine hollow needle.
Sl,~;~ ITE SHEET (RU~E 26) Another mPth~--l viz. fibre-me~ ted transformation, applicable to both monocots and dicots, involves creating a suspension of the target cells in a liquid, adding microscopic needle-like material, such as silicon carbide or silicon nitride "whiskers", and ~it~ting so that the cells and whiskers collide and DNA present in the liquid enters the cell.
5 In ~ Aly, then, the re~ui~.nc~ for both sense and ~nti~P.n~e technology are known and the methods by which the required sequences may be introduced are known. What remains, then is to identify genes whose regulation will be exrect~çd to have a desired effect, isolate them or isolate a fragment of sufficiently effective length, construct a chimeric gene in which the effective fragment is inserted between promoter and terrnin~tion signals, and insert the o construct into cells of the target plant species by transformation. Whole plants may then be regenerated from the transformed cells.
R~n~n~ are a globally i~ O~ fruit crop. They are not only a popular dessert fruit, but represent a vital carbohydrate staple in the tropics with as many as 100 million people 5 subsisting on bananas and pl~nt~in~ as their main energy source. The cultivated dessert banana is commonly triploid, parth~noc~rpic and belongs to the Musa AAA genome group, eg. Cavendish subtypes. R~n~n~c are climac~eric fruits and ripening is regulated by ethylene produced by the fruit and involves numerous biochemical changes including the conversion of starch to sugars. cell wall ~ s~n~hly, synthesis of volatile compounds, chan_es in 20 phenolic constituents and degradation of chlorophyll in the peel. The conversion of starch to sugars is particularly striking, where starch accounts for 20-25% of the fresh wei~ht of the unripe fruit and depending on the genetic background, can ~e converted almost entirely to sugars.
The triploid nature of the cultivated dessert banana crop has h~.,p~.~d conventional methods 2s of breeding for improved ch~cl~,.;stics. As a result of this an enormous pool of genetic resources for Pnh~nring postharvest ch~c~listics of the fruit has remained ull~,ued.
According to the present invention there is provided a method of mod~ ting the ripening or tissue s~nPscenre process in plants of the genus Musa colllp,i~ing inserting into plant 30 material at least one polynucleotide sequence selected from the sequences depicted as SEQ
ID-Nos. 1 -57~ regenerating said plant material and selecting from the transformed SUBSTITUTE SHEET (RULE 26) WO 98111228 PCT/&s97tO2424 ley?c~ , plants with mo~ ted ripening or tissue se~-escF.-ce characteristics. The said polynucleotide mav be obtained from the cDNA library having the NCIMB Accession Number 40814.
Further accordil1g to the present invention is a method of modul~ting the ripenin_ or tissue 5 s~ se~-~ce process in plants of the genus Musa colllylisillg inserting into plant m~teri~l at least one polynucleotide sequence or a rlay?llle.l~ thereof, obtainable by hybridisation. from the cDNA library having the NCIMB Accession Number 40814, by the use of at least one of the sequences depicted as SEQ ID Nos 1-~7 as oligonucleotide probes, said hyhri~icz~tion being corlA~lcted at a tell,pc,aLul~ from 60~C to 65~C in 0.3 strength citrate buffered saline o col~t~ 0.1% SDS followed by rinsing at the same te,l,pe~aLule with 0.3 strenPth citrate buffered saline COl1t~i--i,.g 0.1% SDS, regerl~ lg said plant material and seleclin~ from the transformed legene~ , plants with modulated ripening or tissue senescence characteristics.
The invention further provides a method as described above wherein the said polvnucleotide is capable of m~dnl~ting the production of pectate lyase and more specifically the 5 polynucleotide comprises at least one of the sequences depicted in the sequence listings as SEQ-ID-Nos. 13-1 8.
A l~lefelled method for inserting the said polynucleotides into plant material according to the method of the present invention? may be selected from the group comprising the Agrobacterium, microparticle bombardment, fibre m.o~ t~d or direct insertion methods.
20 The invention further provides plants, their progeny and seed and material obtained from said plants, produced according to the method of the present invention. It is prefetred that the said plants, their progeny and seed and material obtained from said plants are derived from plants of the genus Musa.
The present invention also provides a vector functional in plants comprising a promoter 2~ region which is operable in plant cells, at least one of the polynucleotide sequences described above and a transcription t~nnin~tion sequence. The promoter may be constitutive, developm~nt~lly regulated or switchable. In addition to this the promoter may also be tissue specific or organ specific.
Further provided is a banana produced via the preceding method. having altered fruit 30 characteristics when compared with a banana which is not transformed with at least one of the polynucleotide sequences described above.
SUBSTITUTE SHEET (RULE 26) CA 02262~0~ 1999-02-02 The present invention also provides an improved method of controlling plant pathogens comprising the application of an anti-pathogenic agent to plants, characterised in that plants to which the said agent is applied, are plants according to the present invention.
The gene se~uences of the present invention may be synth~cic~-~ ab initio, using the sequence 5 data in the sequence listing provided herewith, or isolated from a library using the standard techniques know ~,vithin the art. The sequences depicted in the sequence listing or parts thereof may also be used to create oligonucleotide probes for the purposes of isolating from the library those polynucleotides which are capable of producing the desired proteins. To assist the isolation of these polynucleotides we have deposited with the National Collection o of Industrial & Marine Bacteria, St. Machar Drive, Aberdeen, UK, a cDNA library of the banana peel ripening related genes. The library was deposited on 9th July 1996 and has the NCIMB Accession Number 40814.
Thus, this invention is based on the identification of genes which encode proteins involved in banana ripening-related processes, specifically within banana pulp. DNA sequences 15 accor.ling to the sequence listing or those sequences obtainable from the deposited library, may be used in the process of modifying the plant ripening characteristics of plants and/or fruit.
By virtue of this invention banana plants can be generated which. amongst other phenotypic modifications, may have one or more of the following fruit characteristics: improved 20 resistance to damage during harvest, pac~ing and transportation due to slowin~ of the ripening and over-ripening processes; longer shelf life and better storage charac~eristics due to reduced activity of degradative pathways (e.g. cell wall hydrolysis); improved processing characteristics due to changed activity of proteins/enzymes contributing to factors such as:
viscosity, solids, pH, elasticity; improved flavour and aroma at the point of sale due to 25 modification of the sugarlacid balance and other flavour and aroma components responsible for characteristics of the ripe fruit; modified colour due to changes in activity of enzymes involved in the pathways of pigment biosynthesis (e.g. Iycopene, ~-carotene~ chalcones and anthocvanins); increased resict~nce to post-harvest pathogens such as fungi.
The activity of the ripening-related proteins may be either increased or reduced depending on 30 the characteristics desired for the modified plant part (fruit, leaf, flower, etc). The levels of protein may be increased; for example, by incorporation of additional genes. The additional - SIJ~S 111 ~ITE SHEET (RULE 26~
CA 02262~0~ 1999-02-02 wo 98111228 pcTlGs97to2424 genes may be ~leciEn~d to give either the same or dirr~.c.~t spatial and temporal patterns of t~pression in the fruit. '~nti~ncel~ or "partial sense" or other techniques may be used to reduce the ~p~ ion of ripening-related protein.
The activity of each ripening-related protein or enzyme may be modified either individually or in combination with mo~lific~tion of the activity of one or more other ripening- related proteinste~. ~ylllcs. In addition, the activities of the ripening-related proteins/enzymes may be m~difi~d in combination with modification of the activity of other enzymes involved in fruit rirçnin~ or related ylocesses~
DNA constructs according to the invention for gene silencing, may comprise a base sequence o at least 10 bases (preferably at least 35 bases) in length for transcription into 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 l 00 and 1000 bases in length. When using genomic DNA as the source of a base sequence for transcription it is possible to use either intron or exon regions or a combination 15 ofboth.
As a source of the DNA base sequence for transcription, a suitable cDNA or genomic DNA
or synthetic polynucleotide may be used. The isolation of suitable ripening-related sequences is described above; it is convenient to use DNA sequences derived from the ripening-related clones deposited at NCIMB in Aberdeen. Sequences coding for the whole, or subst~nti~lly the whole, of the ~,op,late ripening-related protein may thus be obtained.
Suitable lengths of this DNA sequence may be cut out for use by means of restriction enzymes. The prepaldlion of such constructs is described in more detail below.
Constructs suitable for expression of the ~l,.opl.ate ripening-related sequence in banana cells, may be produced using a cDNA sequence selected from the deposited library having the NCIMB Accession Nurnber 40814 or the gene sequence as found in the chromosome of the banana plant. Recombinant DNA constructs may be made using standard techniques.
In this specification modulation means an incre~e or decre~e of the desired effect.
More specifically '~modulation of the ripening or tissue ~en~scence process in plants of the genus Musa'' means an increase or decrease in production of a ripening related protein resulting from the method as described above. For example, where an increased ripening related protein is desired, plants may be transformed according to the method as described SUBSTITUTE SHEET (RULE 26) above and those plants exhibiting the desired effect may be selected from the population of transformants. Fu~lhe~lllore~ it may be desirable to provide a plant with modulated ripening or tissue sen~scenre characteristics by increasing the production of one protein and decreasing the production of another protein in the same plant. For example, a banana fruit, modified using the present method, and having decreased levels of the enzyme pectate Iyase would be beneficial because pulp softening would require a sllbst~nti~lly longer lime when compared with a control. In addition to this and by incle~illg the levels of another ripening related protein such as an antifungal protein in the same fruit using the present method, would complement the extended life of the banana pulp with inereased re~i~t~nce to disease.
o Increase in production of a ripening related protein includes inserting into plant material one or more copies of anv of the polynucleotides described above, wherein the said polynucleotides must be capable of producing a protein and thereby increasing protein levels when compared with a control plant.
Decrease in production of a ripening related protein includes inserting into plant m~tPri~l one 15 or more copies of any of the polynucleotides described above, wherein the said polvnucleotides must be capable of producing an m~NA which is capable of interfering with endogenous mRNA to such a degree that the levels of tr~n~l~t~d protein are reduced when compared with a control plant.
Ripening process of plants means the process of maturing or developing .
20 Senescence means the progressive deterioration in function of cells. tissues, organs etc., related to the period of time since that function commPnse~ .
Control plant means a comparable plant used for the purposes of 11~t~PrTT ining modulation of the ripening or tissue s~nPscenre process effect in plants. Specifically, in plant transform~tion~ the control plant is usually of the same species and variety as the m~tPri~l 2~ used before the l,dllsÇo,,,,ation process and is grown in the same conditions, (usually with the transformant selection step modified in some way on the part of the control plant)~ as the transformed plants. More specifically the control plant may comprise an untransforrned contlol plant or a transformed control plant providing it has not already been transformed with the same polynucleotide sequence as the plant material to be transformed.
30 "Plant material" includes plant cells and any other type of plant Icgcne.able m~tPri7~1.
SlJ~;~ JTE SHEET (RUEE 26) WO 98111228 PCT/Gs97/02424 The following examples further illustrate the invention but are not to be construed to limit the scope thereof:
TABLE 1. Is a list of clones isolated from banana pulp and the co~ .ollding sequence 5 identity number as provided in the sequence listing herein. The table also illustrates the approximate clone size, the perce.,~ge identity and, where relevant, nucleotide similarity with published sequences, based on the results obtained from conlpa,isons with the EMBL
se~uence rl~t~h~ce The table also provides, where relevant, the gene identity of those p~ hPd se~uences and their ~t~ba~e accession numbers.
o FIGURE 1. Plant transformation vector pUN~ col-l;.inil-g the UBI polyubi~uitin promoter.
FIGURE 2. Plant transformation vector pSHYN, co~ ;"i,~g hygromycin resistance gene for selection of ~ sro-,l,cd plants.
FIGURE 3. Plant transformation vector pAN~ cont~ining the banana ACC oxidase promoter.
Construction of a cDNA library of ripening genes 1.1 Isolation of me~ nger RNA
Total RNA was isolated from ripening (24 hours after ethylene treatment) banana pulp (Musa 20 acuminata cv. Grand Nain) as described bv Chang et al. Plant Molecular Biology Reporter.
Vol. 11(2) 113-116 (1993). Messenger RNA was isolated from total RNA bv Oligo(dT)-cellulose chromatography according to Bantle et al., Analytical Biocll~n~ tr-~ 72, 413-427 (1976).
25 1.2 Synthesis of cDNA and Cloning into Vector The first and second strands of the cDNAs were synth~ci~ed from the messenger RNAs using a com~nercial cDNA synthesis kit (Catalog No. 200450, ZAP ExpressrM Gold Cloning kit, Stratagene Ltd, Carnbridge, Cambs, UK). Double stranded cDNAs were cloned into the ZAP Express~M vector, packaged, mixed with plating bacteria to deterrnine titre and for 30 library screening, following instructions of the suppliers protocol.
SlJts:i 111 ulTE SHEFT (RULE 26) 1.3 Screening of the cDNA library from banana pulp.
The url~mplified cDNA library from ripening banana pulp was differentially screened using cDNA from unripe and ripening banana peel tissue. A proportion of the librarv was plated individually at low density and duplicate plaque lifts made onto Hybond N nylon filters 5 (,~m~rsh~m) according to the m~nl~f~ctllrer's instructions. One filter was hybridised to dCTP
radiolabelled cDNA from green fruit and the duplicate filter hybridised to dCTP
radiolabelled cDNA from ripening fruit. Hybridization's were at high stringency. Plaques hybridising ~.efe.~.~Lially with ripening or green radiolabelled cDNA were picked and re-plated for a second round of selection by differential scl~enillg. These clones were numbered lo as ripening up- or down-regulated peel clones. The clones were in-vivo excised from the ZAP expresslM vector into the pBK-CMV phagemid vector using the ExAssistTM
interference-resistant helper phage, following instructions from m~nllf~ctl-rers protocol.
1.4 Characterisation of the ripening pulp cDNA library and the ripening-related clones.
15 The ripening cDNA li~orary from pulp tissue were prepared with an efficienc~ of 3.2 x 10S
plaque-forming units per microgram of cDNA. The sizes of the inserts in the peel library was 0.4 - 6.7 Kb with a mean size insert of 1.47 Kb.
From the 250 plaques used in the first screen, 73 putative ripening-related clones were obtained. These 73 clones were partially sequenced using the ABI PRISMTM Dye 20 Tennin~tor Cycle Sequencing Ready Reaction kit with Ampli~aq~ DNA poivmerise (Applied Biosystems, Warrington, Cheshire, UK) with forward primers specific for the pBK-CMV vector. From these, the following ripening-related clones were selected. Comparisons of these sequences in the EMBL d~t~b~se using GCG ('Wisconsing') software has identified homologies for the clones listed in TABLE 1 below.
Cons~ruction of truncated sense RNA vectors with the maize polyubiquitin promoter.
A vector is constructed using the sequences corresponding to a fraement of the inserts of 30 one of the sequences I to 57. This fragment is synthPcised by polymerase chain reaction using svnthetic primers incorporating BamHI restriction sites suitable for clonin between a S~ 111 ulTE SHEET (RULE 26) rnaize UBI polyubiquitin promoter (Chri~tenc~n et al, 1992, Plant Molecular Biology, 18:675-689) and a nopaline synthase 3'end termination sequences in the vector pUN (Fig.
1.).
The truncated sense expression cassette is excised by digestion with AscI, the ends of the fr~rnPnt are made flush with T4 polymerase and it is cloned into the vector pSHYN (Fig. 2.) which has been cut with KpnI and the ends made flush with Klenow polymerase. pSHYN
col.t;.;.~ hygromycin resi~t~nre gene for selection of transformed plants.
After synthesis of the vector, the structure and orientation of the sequences are confirmed by DNA sequence analysis.
lo Construction of truncated sense RNA vectors with a fruit enh~nced promoter.
The 1386bp HindIII fragment cont~inin~ the banana ACC oxidase promoter (UK.
Application No. 9607700.3) is cloned the HindIII site in pMSC2 (Fig.3.) to give the vector pAN.
A vector is constructed using the sequences corresponding to a fragment of the inserts of one of the sequences I to 57. This fragment is synth~-si~e-l by polymerase chain reaction using synthetic primers incol~u,d~ g BamHI restriction sites suitable for cloning between a 20 maize UBI polyubiquitin promoter (Chri~t~n~en et al, 1992, Plant Molecular Biology, 18:67~-689) and a nopaline synthase 3'end t~ ion sequences in the vector pAN.
The Ll~-c~l~d sense e,.~ ssion cassette is excised by digestion with AscI, the ends of the fragment are made flush with T4 polymerase and it is cloned into the vector pSHYN (Fig. 2.) which has been cut with KpnI and the ends made flush with Klenow polymerase. pSHYN
25 contains hy~lulllycin resict~nre gene for selection of ~ ~lllled plants.
After synthesis of the vector, the structure and orientation of the sequences are confirmed by DNA sequence analysis.
Construction of an over-expression vector with the maize polyubiquitin promoter.
Sllts~ 1 l l ulTE SHEET (RULE 26) The complete sequence of a ripening -related cDNA co.~ lg a ~11 open-reading frame is inserted into the vectors described in EXAMPLE 2.
Construction of an over-~A~lession vector with a fruit enh~nred promoter.
The complete sequence of a ripening -related cDNA cont~ining a full open-reading frame is inserted into the vectors described in EXAMPLE 3.
Generation of transformed Musa plants Transformed Musa plants cont~ining the vectors are produced by the method described in Sagi et al. (1995) Biotechnology Vol. 13 pp 481-485. Regenerated transformed plants are identified by their ability to grow on hygromycin and grown to maturity. Ripening fruit are analysed for a modulation in their ripening related or senescence characteristics.
Other suitable transformation methods for banana are described in Sagi et al. ( 1994) Plant Cell Reports. Vol. 13. pp 262-266. and May et al. (1995) Biotechnology. Vol. 13 pp 486-492.
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SU~ JTE SHEET (RULE 26) SEQUENCE LISTING
~1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: ZENECA LIMITED
(B) STREET: 15 STANHOPE GATE
(C) CITY: LONDON
(D) STATE: LONDON
(E) COUNTRY: UNITED KINGDOM
(F) POSTAL CODE (ZIP): WlY 6LN
(ii) TITLE OF INVENTION: GENETIC CONTROL OF FRUIT RIPENING
(iii) NUMBER OF SEQUENCES: 57 ~iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version ~1.30 (EPO) (v) CURRENT APPLICATION DATA:
APPLICATION NUM8ER: GB PPD
(2) INFORMATION FOR SEO ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 785 base pairs ~B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA
(vii~ IMMEDIATE SOURCE:
(B) CLONE: U-Ug (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 813 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA
(vii) IMMEDIATE SOURCE:
(B) CLONE: U-U17 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 746 base pairs (B~ TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA
(vii) IMMEDIATE SOURCE:
(B) CLONE: U-U66 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
GGTTAGGTAA CCCTACCTTC GCTCTCGTCC GGAGCACCAC CGCCcccGcc GGCCAACCCG 180 CA 02262505 l999-02-02 t2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A~ LENGTH: 795 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA
(vii~ IMMEDIATE SOURCE:
(B) CLONE: U-U104 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
(2) INFORMATION FOR SEQ ID NO: 5:
(i) SEOUENCE CHARACTERISTICS:
(A~ LENGTH: 797 base pairs (B~ TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown ~ii) MOLECULE TYPE: cDNA
(vii~ IMMEDIATE SOURCE:
(B) CLONE: U-U13 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
IS
(2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 792 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D~ TOPOLOGY: unknown (ii~ MOLECULE TYPE: cDNA
(vii) IMMEDIATE SOURCE:
(B) CLONE: U-Ul36 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
AAGCCCTCAG GAACTCCAAC ATCCAAGTCC TGTTGGATGT CccccGATcc GACGTGCAGT 300 WO g8111228 PCT/GB97/02424 CCGGGCGTTT CTTCCTTTGC CAGGGANTNG GGGCGAAT~C CCNNCTTTTC CANAAACCTT 780 (2) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 855 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLEC~LE TYPE: cDNA
(vii) IMMEDIATE SOURCE:
(8) CLONE: U-U2l (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
CGCCACCGGT CGCGAGGGTT GGAGCCATGG AATTCTTGCG GTTTGGGGCT GAGGTGGCGG l20 (2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 722 base pairs (B) TYPE: nucleic acid CA 02262505 l999-02-02 W 0 98111228 PCTIGBg7/02424 (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA
(vii) IMMEDIATE SOURCE:
(B~ CLONE: U-U31 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
TGGCNTTTTC GACTANNAGT TTGACCGATG GCTATGTTCG TTTTGCTTTT CAC~llll~l 480 40 (2) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 793 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TyeE: cDNA
(vii) IMMEDIATE SOURCE:
(B) CLONE: U-U131 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
WO 98111228 rCT/GB97t02424 TTCTTTCCCC CCNCCCANTC NAACNTTCCN ACGAATTCCC GTTTTACCCN AAA~ACCAAN 780 (2) INFORMATION FOR SEQ ID NO: l0:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 847 base pairs (B) TYPE: nucleic acid ~C) STRANDEDNESS: single (D) TOPOLOGY: unknown ~ii) MOLECULE TYPE: cDNA
(vii) IMMEDIATE SOURCE:
(B) CLONE: V-U32 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: l0:
TATTCACTGA CCGCGCCGGC CGCCGCCGTG CCGACTGCCT CTCCTTCGCT GTCGACGACC l80 (2) INFORMATION FOR SEQ ID NO: ll:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 822 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown ~ii) MOLECULE TYPE: cDNA
(vii) IMMEDIATE SOURCE:
(B) CLONE: U-U55 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
CCCCTTCGCC GCAACCCACG GGAAAGGGGA TCTTCGAGAC NACACNCNAA CAGCCGGGGT ' 480 (2) INFOP~MATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 743 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA
(vii) IMMEDIATE SOURCE:
(B) CLONE: U-U72 ~xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
CA 02262505 l999-02-02 (2) INFORMATION FOR SEQ ID NO: 13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 806 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA
(vii) IMMEDIATE SOURCE:
(B) CLONE: U-U68 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:
(2) INFORMATION FOR SEQ ID NO: 14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 687 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: sin~le (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA
(vii) IMMEDIATE SOURCE:
~B) CLONE: U-U69 CA 02262~0~ 1999-02-02 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:
(2~ INFORMATION FOR SEQ ID NO: 15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 741 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA
(vii) IMMEDIATE SOURCE:
(B) CLONE: U-U84 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:
CA 02262~0~ l999-02-02 - 2~ -~2) INFORMATION FOR SEQ ID NO: 16:
~i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 727 base pairs (B) TYPE: nucleic acid ~C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA
(vii) IMMEDIATE SOURCE:
~B) CLONE: U-U89 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:
(2) INFORMATION FOR SEQ ID NO: ~7:
(i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 842 base pairs (B) TYPE: nucleic acid ~C) STRANDEDNESS: single ~D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA
~vii) IMMEDIATE SOURCE:
~B) CLONE: U-U115 ~xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:
AGTAGTACAG GAAGTACAAA GAAGCTTGAA CGTGTCGCGG CGGCGACTGG GCTACTTGTC 1~0 CA 02262505 l999-02-02 WO 98/11228 PCTtGB97/02424 ATCCACGGCG TCCACATCCA CGAATGCAAG CAGGGCGGGA ACGCGTNCGT TcGcNAcTcc 600 (2) INFORMATION FOR SEQ ID NO: 18:
~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 724 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA
(vii) IMMEDIATE SOURCE:
(B) CLONE: U-U117 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:
(2) INFORMATION FOR SEQ ID NO: 19:
~i) SEQ~ENCE CHARACTERISTICS:
(A) LENGTH: 797 base pairs ~B) TYPE: nucleic acid (C) STRANDEDNESS: single CA 02262~0~ 1999-02-02 W 0 98/11228 PCT/GB97r02424 (D) TOPOLOGY: unknown (ii~ MOLECULE TYPE: cDNA
(vii) IMMEDIATE SOURCE:
(B) CLONE: U-V80 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: l9:
CAAAGCTCGG CGGAGGCAAG CTTGTCCTGA AACCACTGGG CTTGGCGTTG CCTCACTATT l80 (2) INFORMATION FOR SEQ ID NO: 20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 742 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA
(vii) IMMEDIATE SOURCE:
(B) CLONE: U-U90 ~5 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:
CTGTTGGAGT TGGAACTGTG GGTTCCTCCA GAAGCAATCA TGcTTGcAGG ACTTGCATTC 420 CA 02262~0~ 1999-02-02 S
~2) INFORMATION FOR SEQ ID NO: 21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 807 base pairs ~B) TYPE: nucleic acid ~C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA
~vii) IMMEDIATE SOURCE:
(B) CLONE: U-U92 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:
(2) INFORMATION FOR SEQ ID NO: 22:
(i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 770 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA
(vii) IMMEDIATE SOURCE:
CA 02262505 l999-02-02 (B) CLONE: U-U91 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:
GACATTGATT TTGAAAAAGA ACCCAATTGG AACAGGGGAA GGATGGGAAA AAACATATAC 5q0 (2) INFORMATION FOR SEQ ID NO: 23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGT~: 810 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA
~vii) IMMEDIATE SOURCE:
(B) CLONE: U-U96 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:
CCAGGATTGA ~l~l.G~l-C TTGTCTACAG ACCCCATCAG ATGTTGATAA GCTTCCNAAA 420 T iTGAAATC AGGGATTTTG ATGCATTTGA TTTGAAGATG ANACTTCCTG CNGTANTTAC 600 CA 02262505 l999-02-02 (2) INFORMATION FOR SEQ ID NO: 24:
(i~ SEQUENCE CHARACTERISTICS:
(A) LENGTH: 765 base paLrs (B) TYPE: nuclelc acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA
(vii) IMMEDIATE SOURCE:
(B) CLONE: U-U103 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:
(2) INEORMATION FOR SEQ ID NO: 25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 774 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA
(vii) IMMEDIATE SOURCE:
(B) CLONE: U-U93 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25:
70 GGCACGAGCG GAGAANGCAA TCTCGCTCTC TCTCTACTGC ATcGcGGcTc TCGTCCTCGA 60 CA 02262505 l999-02-02 W O 98/11228 PCTtGB97/02424 (2) INFORMATION FOR SEQ ID NO: 26:
~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 786 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA
(vii) IMMEDIATE SOU~CE:
(B) CLONE: U-U125 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26:
GGCACGAGCT AGTCTCGAGT llllllllll llllllllAA AATTGAAATT AGATGCGATT 60 (2) INFORMATION FOR SEQ ID NO: 27:
CA 02262505 l999-02-02 WO 98/11228 PCT/GB97tO2424 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 486 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA
(vii) IMMEDIATE SOURCE:
~B) CLONE: U-U105 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:
GGCACGAGCG A~llllllll llllllllll llllilllll TTTTCACAAG GNAACCATAG 60 (2) INFORMATION FOR SEQ ID NO: 28:
(i) SEQUENCE CHARACTERISTICS:
;A~ LENGTH: 791 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single ~D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA
(vii) IMMEDIATE SOURCE:
(B) CLONE: U-U116 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28:
AAAGGAAATN AANCCNNTTG GGTTTTCCCC TTTGGCCTTT lllL~ lG AAAATCCTNT 780 ~lllllGGTG T 791 (2) INFORMATION FOR SEQ ID NO: 29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 814 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA
(vii) IMMEDIATE SOURCE:
(B) CLONE: U-U120 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29:
NCCACCGTCC TTACCCTCCA TCGACGCCAC CCCACCATCC GAAGCACCCG CCTTCTCAGC 5g0 GGG~lllllN TTAANATTTN AANAAANCNA AN1N11 L LCC CCCCCNTNTT GGTTNATGGC 780 55 CTATGGGTTT ~ L L L ~'1 U L lC TTGGCTTTTC CCGG 814 (2) INFORMATION FOR SEQ ID NO: 30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 742 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA
(vii) IMMEDIATE SOURCE:
(B) CLONE: U-U126 CA 02262505 l999-02-02 ~xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30:
~2) INFORMATION FOR SEQ ID NO: 31:
li) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 794 base pairs ~B) TYPE: nucleic acid ~C) STRANDEDNESS: single tD) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA
(vii) IMMEDIATE SOURCE:
~B) CLONE: U-U129 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31:
CA 02262~0~ 1999-02-02 CCNCCCCCCC CAATTAACCC N1lCllGCTC TTCCCCCTTG CCTTCANCCA ACCCGCCCNT 780 (2) INFORMATION FOR SEQ ID NO: 32:
(i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 799 base pairs ~B) TYPE: nucleic acid ~C) STRANDEDNESS: single ~D) TOPOLOGY: unknown ~ii) MOLECULE TYPE: cDNA
~vii) IMMEDIATE SOURCE:
(8) CLONE: U-Ul30 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32:
GCCGGCCAGG ATCATCAGGG AAGCACTGTC GAAGGCGCTG GTGAAGTACT ACCCCTTCGC l20 CGCCCGA~AT CGCCCGTGGC CTGCCNAANC CCACCGTGGA NCCTGCATGG TCCCGGGANG 480 TTTTTTCCTT TCCTTCCCCC CCCAAAANAA CTTCCCCCCA CC~ l TGCNCCCAAG 780 ~2) INFORMATION FOR SEQ ID NO: 33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 759 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single ~D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA
~vii) IMMEDIATE SOURCE:
(B) CLONE: U-U43 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33:
TAATAGCAGT GGCTACTTGG TGCTTCATTG GGGTGCAATT CATAATAGAA GGAATAACTG l20 ATGAATTAAA TAAANGAACN CNCCTNCAAA CTNAGGGAA 75g (2) INFORMATION FOR SEQ ID NO: 34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 785 ~ase pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA
(vii) IMMEDIATE SOURCE:
(B) CLONE: U-U70 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34:
CNTCGGCTGC CCTCTTCCTT GCTCTCAACC TTGTCTTCTT TGCACTCACC AGTGCTTGTG lB0 TlllL~llGAG CATTCCTTTG TGTCTTTACT TCCCTTTGTT TTTGGCTTTT TGATGACGAA 600 (2) INFORMATION FOR SEQ ID NO: 35:
CA 02262~0~ l999-02-02 (i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 784 ~ase pairs ~B) TYPE: nucleic acid ~C) STRANDEDNESS: single ~D) TOPOLOGY: unknown ~ii) MOLECULE TYPE: cDNA
~vii) IMMEDIATE SOURCE:
~B) CLONE: U-U16 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35:
45 (2) INFORMATION FOR SEQ ID NO: 36:
~i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 817 base pairs ~B) TYPE: nucleic acid ~C) STRANDEDNESS: single ~D) TOPOLOGY: unknown ~ii) MOLECULE TYPE: cDNA
(vii) IMMEDIATE SOURCE:
(B) CLONE: U-U30 ~xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36:
CA 02262~0~ 1999-02-02 (2) INFORMATION FOR SEQ ID NO: 37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 837 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA
(vii) IMMEDIATE SOURCE:
(B) CLONE: U-U40 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37:
~ GG GTATCAGGCA AAGATGGATA CTTCCAGGTT TGCTATTTAA 60 (2) INFORMATION FOR SEQ ID NO: 38:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 799 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single CA 02262505 l999-02-02 tD~ TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA
(vii~ IMMEDIATE SOURCE:
(B~ CLONE: U-U108 ~xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38:
(2) INFORMATION FOR SEQ ID NO: 39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 786 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA
(vii) IMMEDIATE SOURCE:
(B~ CLONE: U-U128 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39:
GTGGAAAATG CAGTGTCATA CAGCTCTAGG TCACTGCTTT CAACCGGATC CcTGGGAAAG 300 WO 98tll228 PCT/GB97102424 (2) INFORMATION FOR SEQ ID NO: 40:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 804 baSe PairS
(B) TYPE: nUC1eiC aCid (C) STRANDEDNESS: Sing1e tD) TOPOLOGY: UnknOWn (ii) MOLECULE TYPE: CDNA
(Vii~ IMMEDIATE SOURCE:
(B) CLONE: U-D4 (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 40:
60 TGGGGGAAAN 1L11L11"111 TTTT 804 (2) INFORMATION FOR SEQ ID NO: 41:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 826 baSe PairS
(B) TYPE: nUC1eiC aCi~
(C) STRANDEDNESS: single (D) TOPOLOGY: UnknOWn (ii) MOLECULE TYPE: CDNA
W O 98/11228 rCT/GB97/02424 ~vii) IMMEDIATE SOURCE:
(B) CLONE: U-D10 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41:
AGGACCTTCC TAAACAGAAG GGTTCTACAC TTGA&GAACC AAACCACCGC TTACGAAGGA 180 35 GGCNCCCTGG TCCTCCTNCC TGCTACTTAA AAA~ TCNTNT 826 (2) INFORMATION FOR SEQ ID NO: 42:
(i~ SEQUENCE CHARACTERISTICS:
(A) LENGTH: 813 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown ~ii) MOLECULE TYPE: cDNA
(vii) IMMEDIATE SOURCE:
(B) CLONE: U-Dl3 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42:
GGAGGACTCG GTGATGTTCT TGGAGGACTG CCACCGGGCC ATGGCTGCAA ATGGACACAG g80 WO 98/11228 PCI'/GB97/02424 ANAAGAATTG ACAGGGTTTT CATTGATCAC ~ LCTT GAAAAGGTGT GGGGAAAAAC 660 (2) INFORMATION FOR SEQ ID NO: 43:
~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 805 base pairs ~B) TYPE: nucleic acid ~C) STRANDEDNESS: single ~D) TOPOLOGY: unknown ~ii) MOLECULE TYPE: cDNA
~vii) IMMEDIATE SOURCE:
~B) CLONE: U-D66 ~xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43:
GTGATGACTA TAGCTCCACG CTATGATCAG TACAAGGATG GGTGGGATAC A~ATGTCCTG 480 CCTNCCTACT TA~AAANTTT TTTCC 805 ~2) INFORMATION FOR SEQ ID NO: 44:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 790 base pairs ~B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA
(vii) IMMEDIATE SOURCE:
~B) CLONE: U-D111 CA 0226250~ l999-02-02 ~xi) SEQUENCE DESCRIPTION: SEQ ID NO: 94:
AAC~l..l~l TGCCAGCACT TTGGGAACCN CAAGGATCCT GGATCTTAAC AACNCAANTC 720 (2) INFORMATION FOR SEQ ID NO: 45:
~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 813 base palrs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECVLE TYPE: cDNA
(vii) IMMEDIATE SOURCE:
(B) CLONE: U-Dl12 ~xi) SEQUENCE DESCRIPTION: SEQ ID NO: 45:
~ iG GAGCATAATA GCAATTTATT CAGGAGGATC AAACTCAAAC 60 CTGTGATGCC TTCTATGACA GTGTCAGCAA GTCCACCAGT TGTTGCGCAC ATGGGGGGAA ~20 CA 02262505 l999-02-02 WO 98/11228 PCT/GBg7/02424 (2~ INFORMATION FOR SEQ ID NO: 96:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 808 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA
(vii) IMMEDIATE SOURCE:
tB) CLONE: U-D39 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 46:
GGCACGAGCG A~ l GCTGTGATGA TATTATGCAT CTTATTAGTT 60 ACN~lll;~ll AAAACATCNG TNCCNAAT B08 (21 INFORMATION FOR SEQ ID NO: 47:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 809 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECVLE TYPE: cDNA
(vii~ IMMEDIATE SOURCE:
(B) CLONE: U-D50 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 47:
, CA 02262~0~ l999-02-02 (2) INFORMATION FOR SEQ ID NO: 48:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 809 ~ase pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA
(vii) IMMEDIATE SOURCE:
(B) CLONE: U-D86 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 48:
CA 02262505 l999-02-02 (2) INFORMATION FOR SEQ ID NO: 49:
(i) SEQUENCE CHARACTERISTICS:
tA) LENGTH: 785 base pairs ~B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA
~vii) IMMEDIATE SOURCE:
(B) CLONE: ~-D90 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 49:
(2) INFORMATION FOR SEQ ID NO: 50:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 813 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA
(vii) IMMEDIATE SOURCE:
(B) CLONE: U-D93 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 50:
, CA 02262~0~ l999-02-02 CAAATGCGTT CNACAATTGG ~ CCA NCCTGCNCAA AACTGTTCCT TGGGCTCCCG 780 (2) INFORMATION FOR SEQ ID NO: 51:
(i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 819 base pairs (B~ TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA
(vii) IMMEDIATE SOURCE:
(B) CLONE: U-D61 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 51:
CAACCCGGGA CAGATCTCGC TGCCCTACGC CCTGTTCACG GCCTCCGGCG TCGTCGTGCA 4 e o TTGATCAGGC ATGTTNGGCG G.~AGAACCCC AAGGAAAACC AGGGAAGGAA ATCAAGGCTT 720 (2) INFORMATION FOR SEQ ID NO: 52:
(i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 794 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA
(vii) IMMEDIATE SOURCE:
(B) CLONE: U-D63 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 52:
CGCCGTGCAT ACAATGATAC CTCTGATAGG GTTGAATGCA CAAACCACTC ATCGTGCTAT l80 TTTATTGCTT TGCATATTTT GTGCTGGAAA CCATGCCAAT CCAACTCCTT TGCAAAACTT g20 (2) INFORMATION FOR SEQ ID NO: 53:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 798 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA
(vii) IMMEDIATE SOURCE:
(B) CLONE: U-D65 ~xi) SEQ~ENCE DESCRIPTION: SEQ ID NO: 53:
CACCGCCCAC CCCCGTGGCC AAGCCGCCTT CAGTTGAACC ACCACCCTAC CACGGCCCTC l80 CA 02262505 l999-02-02 WO 98111228 PCTtGB97/02424 (2) INFORMATION FOR SEQ ID NO: 54:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 824 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA
~viij IMMEDIATE SOURCE:
(B) CLONE: U-D8q txi) SEQUENCE DESCRIPTION: SEQ ID NO: 54:
(2) INFORMATION FOR SEQ ID NO: 55:
~i) SEQUENCE CHARACTERISTICS:
(A) LENGT~: 797 base pairs (B) TYPE: nucleic acid CA 02262~0~ l999-02-02 (C~ STRANDEDNESS: single (D~ TOPOLOGY: unknown (ii~ MOLECULE TYPE: cDNA
(vii~ IMMEDIATE SOURCE:
(B) CLONE: U-D75 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 55:
TTGAANATGA AGAANAATTN TTTGTGCCNC TTTCAAGGTG GAANGATCTG C~ ullGC 720 (2~ INFORMATION FOR SEQ ID NO: 56:
(i~ SEQUENCE CHARACTERISTICS:
(A) LENGTH: 804 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii~ MOLECULE TYPE: cDNA
(vii) IMMEDIATE SOURCE:
(B) CLONE: U-D33 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 56:
~0 AACCTGGGCA GCTTGAGGCA GCAGTATGGG CTTTCCAAGA GTGCCGACNA GGTGGTGCTG 360 CA 02262505 l999-02-02 GTCATCGAAG CCTACAAGGC CCTCCGTGAC CGANCTCCCT ATCCTCCCAG CTTCATGCTC ~20 l5 A,-~ATCCTCT TTAAAANCAT AAAA 804 (2) INFORMATION FOR SEQ ID NO: 57:
(i~ SEQUENCE CHARACTERISTICS:
(A) LENGTH: 803 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D~ TOPOLOGY: unknown (ii~ MOLECULE TYPE: cDNA
(vii~ IMMEDIATE SOURCE:
(~) CLONF: U-D64 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 57:
Claims (14)
1. A method of modulating the ripening or tissue senescence process in plants of the genus Musa comprising inserting into plant material at least one polynucleotide sequence selected from the sequences depicted as SEQ ID Nos 1-57, regenerating said plant material and selecting from the transformed regenerants, plants with modulated ripening or tissue senescence characteristics.
2. A method according to claim 1 wherein the polynucleotide is obtained from the cDNA library having the NCIMB Accession Number 40814.
3 A method of modulating the ripening or tissue senescence process in plants of the genus Musa comprising inserting into plant material at least one polynucleotide sequence or a fragment thereof, obtainable by hybridisation, from the cDNA library having the NCIMB Accession Number 40814, by the use of at least one of the sequences depicted as SEQ ID Nos 1-57 as oligonucleotide probes, said hybridisation being conducted at a temperature from 60°C to 65°C in 0.3 strength citrate buffered saline containing 0.1% SDS
followed by rinsing at the same temperature with 0.3 strength citrate buffered saline containing 0.1% SDS, regenerating said plant material and selecting from the transformed regenerants, plants with modulated ripening or tissue senescence characteristics.
followed by rinsing at the same temperature with 0.3 strength citrate buffered saline containing 0.1% SDS, regenerating said plant material and selecting from the transformed regenerants, plants with modulated ripening or tissue senescence characteristics.
4. A method according to either of claims 1 to 3, characterised in that the said polynucleotide modulates the production of pectate lyase.
5. A method according to claim 4 in which the polynucleotide sequence comprises at least one of the sequences depicted in the sequence listings as SEQ ID Nos.
13-18.
13-18.
6. A method according to any preceding claim wherein the plant material is transformed using the Agrobacterium, microparticle bombardment, fibre mediated or direct insertion method.
7. A plants, its progeny and seed and material obtained from said plant, having inserted within its genome at least one polynucleotide selected from the sequences depicted as SEQ ID Nos 1 to 4.
8. A vector functional in plants comprising a promoter region which is operable in plant cells, at least one of the polynucleotide sequences described in claim 1 to 3 and a transcription termination sequence.
9. A vector according to claim 8 wherein the promoter is constitutive, developmentally regulated or switchable.
10. A vector according to claim 9 wherein the promoter is tissue specific or organ specific.
11. A banana produced via the method according to claims 1 to 6 having altered fruit characteristics when compared with a banana which is not transformed with at least one of the polynucleotide sequences described in claims 1 to 3.
12. A method of controlling plant pathogens comprising the application of an anti-pathogenic agent to plants, characterised in that plants to which the said agent is applied are plants according to claim 7.
13. A polynucleotide associated with fruit ripening selected from SEQ ID Nos.
1-57.
1-57.
14. A fruit of a plant of the genus Musa having a retarded ripening phenotype generated by inserting into the genome of the said plant at least one of the polynucleotide(s) as claimed in claim 13.
Applications Claiming Priority (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB9618862.8 | 1996-09-10 | ||
| GBGB9618862.8A GB9618862D0 (en) | 1996-09-10 | 1996-09-10 | Genetic control of fruit ripening |
| GB9708366.1 | 1997-04-25 | ||
| GBGB9708366.1A GB9708366D0 (en) | 1997-04-25 | 1997-04-25 | Genetic control of fruit ripening |
| PCT/GB1997/002424 WO1998011228A2 (en) | 1996-09-10 | 1997-09-08 | Genetic control of fruit ripening |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2262505A1 true CA2262505A1 (en) | 1998-03-19 |
Family
ID=26310006
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002262505A Abandoned CA2262505A1 (en) | 1996-09-10 | 1997-09-08 | Genetic control of fruit ripening |
Country Status (5)
| Country | Link |
|---|---|
| EP (1) | EP0931150A2 (en) |
| JP (1) | JP2000517164A (en) |
| AU (1) | AU4129197A (en) |
| CA (1) | CA2262505A1 (en) |
| WO (1) | WO1998011228A2 (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB9716766D0 (en) * | 1997-08-07 | 1997-10-15 | Innes John Centre | Pectin degrading enzymes |
| EP1017820A2 (en) * | 1997-09-25 | 2000-07-12 | Boyce Thompson Institute For Plant Research, Inc. | Banana proteins, dna, and dna regulatory elements associated with fruit development |
| US6864404B1 (en) * | 1999-10-15 | 2005-03-08 | Carnegie Institution Of Washington | Engineering disease resistance with pectate lyase-like genes |
| US20130036515A1 (en) * | 2011-08-05 | 2013-02-07 | Giovannoni James J | Banana MADS-Box Genes for Banana Ripening Control |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5908973A (en) * | 1993-03-22 | 1999-06-01 | Zeneca Limited | DNA encoding fruit-ripening-related proteins, DNA constructs, cells and plants derived therefrom |
| GB9318927D0 (en) * | 1993-09-13 | 1993-10-27 | Zeneca Ltd | Regulation of senescence |
| US5792935A (en) * | 1993-12-09 | 1998-08-11 | Texas A&M University | Agrobacterium tumefaciens transformation of Musa species |
-
1997
- 1997-09-08 JP JP09533834A patent/JP2000517164A/en not_active Withdrawn
- 1997-09-08 AU AU41291/97A patent/AU4129197A/en not_active Abandoned
- 1997-09-08 EP EP97939069A patent/EP0931150A2/en not_active Withdrawn
- 1997-09-08 CA CA002262505A patent/CA2262505A1/en not_active Abandoned
- 1997-09-08 WO PCT/GB1997/002424 patent/WO1998011228A2/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| JP2000517164A (en) | 2000-12-26 |
| WO1998011228A2 (en) | 1998-03-19 |
| AU4129197A (en) | 1998-04-02 |
| WO1998011228A3 (en) | 1998-04-23 |
| EP0931150A2 (en) | 1999-07-28 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EEER | Examination request | ||
| FZDE | Discontinued |