AU731938B2

AU731938B2 - Polyphenol oxidase genes from banana, tobacco and pineapple

Info

Publication number: AU731938B2
Application number: AU73268/98A
Authority: AU
Inventors: Simon Piers Robinson
Original assignee: Commonwealth Scientific and Industrial Research Organization CSIRO
Current assignee: Commonwealth Scientific and Industrial Research Organization CSIRO
Priority date: 1997-05-19
Filing date: 1998-05-19
Publication date: 2001-04-05
Anticipated expiration: 2018-05-19
Also published as: AU7326898A

Description

i ii WO 98/53080 PCT/AU98/00362 1 POLYPHENOL OXIDASE GENES FROM BANANA, TOBACCO PINEAPPLE The present invention relates to the isolation of genes encoding polyphenol oxidase (PPO) from plants.

Browning of plant tissues often occurs following injury or damage and this generally results in spoilage of fruit and vegetables. Undesirable browning also occurs during processing of plant materials to produce food or other products.

Steps are taken during transport, storage, and processing to prevent these browning reactions. Often this involves the use of chemicals such as sulphur dioxide but the use of these substances is likely to be restricted in the future due to concerns about their safety and consumer acceptance. For example, the US Food and Drug Administration banned the use of sulphite for most fresh fruit and vegetables in 1986. The production of fruit and vegetable varieties with an inherently low susceptibility to brown would remove the need for these chemical treatments.

It will be understood that browning in plants is predominantly catalysed by the enzyme PPO. PPO is localised in the plastids of plant cells whereas the phenolic substrates of the enzyme are stored in the plant cell vacuole. This compartmentation prevents the browning reaction from occurring unless the plant cells are damaged and the enzyme and its substratesare mixed.

The prior art includes International Application PCT/AU92/00356 to the present applicant which describes the cloning of PPO genes from grapevine, broad bean leaf, apple fruit and potato tuber. This application recognises that PPO levels in plants may be manipulated by increasing or decreasing expression of PPO gene. The application also identifies two conserved copper binding sites in PPO genes, designated CuA and CuB. However, the method described in PCT/AU92/00356 which was used to clone the PPO genes from apple and potato involved the use of an oligo dT reverse primer for polymerase chain reaction (PCR). Whilst the method is acceptable, in some tissues, it does not give rise to a strong band of the predicted size or else it gives rise to many additional products making it difficult to resolve the PPO fragment.

Accordingly, it is an object of the present invention to overcome or at least alleviate one or more of the difficulties related to the prior art.

qf r WO 98/53080 PCT/AU98/00362 2 In a first aspect of the present invention there is provided a method for preparing nucleic acid encoding PPO, fragments and derivatives thereof, which method includes providing a source of a polypeptide having PPO activity, a first primer having a sequence corresponding to a first conserved region of PPO, and a second primer having a sequence corresponding to a second conserved region of PPO orientation; isolating RNA from the source of polypeptide having PPO activity; treating the RNA to construct copy DNA (cDNA) therefrom; and amplifying the cDNA so formed using the first and second primers.

Applicant has found that the method of the present invention, which involves the use of a second primer based on PPO, means that there is less likelihood that other (non-PPO) genes are amplified. Furthermore, the method of the present invention dramatically increases the amount of genuine product formed in most cases. Moreover, the added specificity provided by the second PPO-based primer makes it possible to clone PPO more readily from certain plants in which it was difficult to obtain a clone using one primer and oligo-dT.

In a preferred aspect of the present invention there is provided a method for preparing nucleic acid encoding banana, tobacco or pineapple PPO, fragments and derivatives thereof, which method includes providing a source of a polypeptide having banana, tobacco or pineapple PPO activity, a first primer having a sequence corresponding to a first conserved region of banana, tobacco or pineapple PPO, and a second primer having a sequence corresponding to a second conserved region of banana, tobacco or pineapple PPO; isolating RNA from the source of polypeptide having banana, tobacco or pineapple PPO activity; treating the RNA to construct copy DNA (cDNA) therefrom; and WO 98/53080 PCT/AU98/00362 3 amplifying the cDNA so formed using the first and second primers.

The terms "nucleic acid encoding banana/tobacco/pineapple PPO" and "banana/tobacco/pineapple PPO gene" as used herein should be understood to refer to a banana, tobacco and/or pineapple PPO gene or a sequence substantially homologous therewith. For example, these terms include sequences which differ from the specific sequences given in the Examples hereto but which, because of the degeneracy of the genetic code, encode the same protein. Applicants have found that there are families of PPO genes in most plants. Thus, there are likely to be other PPO genes in banana, tobacco and/or pineapple, in addition to those which have been isolated. These could be cloned using the methods of the present invention. Thus, the terms "nucleic acid encoding banana/tobacco/pineapple PPO" and banana/tobacco/pineapple

PPO

gene" should be understood to include banana, tobacco and/or pineapple PPO genes other than those specific genes that have been isolated. The terms may also include presequences such as chloroplast transit sequence as well as sequences encoding mature PPO protein.

The term "derivative" as used herein includes nucleic acids that have been chemically or otherwise modified, for example mutated, or labelled, or nucleic acids incorporating a catalytic cleavage site.

The term "fragment" includes functionally active fragments of a PPO gene which are capable of altering expression of the PPO genes. Examples of alteration of the gene may include up-regulation or down-regulation of the gene, coding of the gene, transcription of the gene, binding of the gene or stability of the gene sequence.

The source of polypeptide having PPO activity is preferably a source of polypeptide having banana or tobacco or pineapple PPO activity. The source of polypeptide having banana PPO activity may be banana peel, preferably young banana peel. More preferably the peel of young banana fruit. The source of polypeptide having tobacco PPO activity may be tobacco leaves, preferably young tobacco leaves. The source of polypeptide having pineapple PPO activity may be pineapple fruit, preferably the flesh of the pineapple fruit, more preferably the flesh of pineapple fruit exhibiting blackheart disorder.

WO 98/53080 PCT/AU98/00362 4 The RNA may be isolated by any suitable method including extraction for example with a detergent such as CTAB, use of an oligo-dT spun column as described in PCT/AU92/00356 and PCT/AU96/00310 the entire disclosure of each application which is incorporated herein by reference, or use of a commercially available kit such as the PolyATtract 1000 system from Promega Corporation.

The step of treating the RNA to construct cDNA according to this aspect of the present invention may include treating the RNA with reverse transcriptase and an adapter primer to form cDNA.

The adapter primer may be an oligonucleotide adapter primer including the following sequence or part thereof: I I Il I I II I I 1 -3' The step of treating the RNA to construct cDNA according to this aspect of the present invention may include treating the RNA with reverse transcriptase and a reverse primer to form cDNA.

The adapter primer may be replaced with a reverse primer having a sequence corresponding to a conserved region of PPO genes including the following sequence or part thereof: REV2 :5'-GCCTGCAGTT[TC]TC[AG]TC[AG]TAGAA-3' The first primer has a sequence corresponding to a first conserved region of PPO. Preferably the first primer has a sequence corresponding to at least a portion of or in close proximity to a first copper binding site of PPO. The second primer has a sequence corresponding to a second conserved region of PPO.

Preferably the second primer has a sequence corresponding to at least a portion of or in close proximity to a second copper binding site of PPO. More preferably the first primer has a sequence corresponding to at least a portion of or in close proximity to one of the CuA or CuB binding sites of PPO, and the second primer has a sequence corresponding to at least a portion of or in close proximity to the other of the CuA or CuB binding sites of PPO.

The first and second primers may be degenerate. The first primer may WO 98/53080 PCT/AU98/00362 include one of the following sequences or part thereof: GEN8 5'-GCGAATTCGATCCIACITT[TC]GC[GT]TTICC-3'.

GEN9 5'GCGAATTCTICA[TC]TG[TC]GCITA[TC]TG-3'.

5'GCGAATTCTTICCIT[TA][TCITGGAA[TC]TGGG-3'.

The second primer may include the following sequences or part thereof REV1 5'-GCCTGCAGCCACATIC[TG][AG]TCIAC[AG]TT-3'.

REV2: 5'GCCTGCAGTT[TC]TC[AG]TC[AG]TAGAA-3'.

The cDNA may be amplified using the polymerase chain reaction (PCR).

Those skilled in the art will appreciate that if the Cu binding sites are internal, the nucleic acid isolated will be a fragment of the PPO gene lacking 3' and 5' termini. However, it is possible to determine the complete nucleic acid sequence of the PPO gene and to prepare or isolate nucleic acid encoding such PPO or antisense to such PPO.

Accordingly, in a further aspect of the present invention there is provided a method for preparing nucleic acid encoding the 3' end of PPO, which method includes providing a source of polypeptide having PPO activity a primer in sense orientation; and an adapter primer; isolating RNA from the source of polypeptide having PPO activity; treating the RNA to construct cDNA therefrom; and amplifying the cDNA so formed using the primers.

In a further aspect of the present invention there is provided a method for preparing nucleic acid encoding the 5' end of PPO, which method includes providing a source of polypeptide having PPO activity, an anchor, primers in antisense orientation; and an anchor primer; isolating RNA from the source of polypeptide having PPO activity; treating the RNA to construct cDNA therefrom; WO 98/53080 PCT/AU98/00362 6 attaching the anchor to the 5' end of the cDNA so formed; and amplifying the cDNA using the primers.

The source of polypeptide having PPO activity is preferably a source of polypeptide having banana or tobacco or pineapple PPO activity. The source of polypeptide having banana PPO activity may be banana peel, preferably young banana peel. More preferably the peel of young banana fruit. The source of polypeptide having tobacco PPO activity may be tobacco leaves, preferably young tobacco leaves. The source of polypeptide having pineapple PPO activity may be pineapple fruit, preferably the flesh of the pineapple fruit, most preferably the flesh of pineapple fruit exhibiting blackheart disorder.

The RNA may be isolated by any suitable method including extraction for example with a detergent such as CTAB, use of an oligo-dT spun column as described in PCT/AU92/00356 or PCT/AU96/00310 the entire disclosure of each patent application which is incorporated herein by reference, or use of a commercially available kit such as the PolyATtract 1000 system from Promega Corporation.

The adapter primer may be an oligonucleotide adapter primer including one of the following sequences or part thereof: I Ill I I IIl 1-3' The adapter primer may be replaced with a reverse primer having a sequence corresponding to a conserved region of PPO genes including the following sequence or part thereof: REV2 5'-GCCTGCAGTT[TC]TC[AG]TC[AG]TAGAA-3' The primer in sense orientation may be a banana, tobacco or pineapple PPO specific primer.

The adapter primer may include the following sequence or part thereof: 5'-GACTCGAGTCGACATCGA-3'.

The primers in antisense orientation may be banana, tobacco or pineapple WO 98/53080 PCT/AU98/00362 7 PPO specific primers.

The anchor may be of any suitable type. The anchor may be attached by ligation for example using T4 RNA ligase. The anchor primer should be capable of hybridizing with the anchor.

The cDNA may be amplified using PCR.

Those skilled in the art will appreciate that using the methods of the present invention it is possible to determine the complete nucleic acid sequence of the PPO gene of interest and to prepare or isolate nucleic acid encoding such PPO or antisense to such PPO.

In a further aspect of the present invention, there is provided a nucleic acid encoding banana PPO or antisense to banana PPO, fragments and derivatives thereof. Preferably the nucleic acid has the sequence shown in Fig. 1, 2, 3 or 4, fragments and derivatives thereof, and substantially homologous sequences.

In a further aspect of the present invention, there is provided a nucleic acid encoding tobacco PPO or antisense to tobacco PPO, fragments and derivatives thereof. Preferably the nucleic acid has the sequence shown in Fig. 5, 6 or 7 fragments and derivatives thereof, and substantially homologous sequences.

In a further aspect of the present invention, there is provided a nucleic acid encoding pineapple PPO or antisense to pineapple PPO, fragments and derivatives thereof. Preferably the nucleic acid has the sequence shown in Fig.

8, 9 or 10 fragments and derivatives thereof, and substantially homologous sequences.

The nucleic acid may be prepared by a method as hereinbefore described.

The nucleic acid may be modified, for example by inclusion of a catalytic cleavage site.

In a further aspect of the present invention there is provided a method for preparing a recombinant vector including a nucleic acid encoding banana PPO or antisense to banana PPO, fragments and derivatives thereof, which method includes providing nucleic acid encoding banana PPO or antisense to banana PPO, fragments and derivatives thereof; and WO 98/53080 PCT/AU98/00362 8 a vector; and reacting the nucleic acid and the vector to deploy the nucleic acid within the vector.

In a further aspect of the present invention there is provided a method for preparing a recombinant vector including a nucleic acid encoding tobacco PPO or antisense to tobacco PPO, fragments and derivatives thereof, which method includes providing nucleic acid encoding tobacco PPO or antisense to tobacco PPO, fragments and derivatives thereof; and a vector; and reacting the nucleic acid and the vector to deploy the nucleic acid within the vector.

In a further aspect of the present invention there is provided a method for preparing a recombinant vector including a nucleic acid encoding pineapple PPO or antisense to pineapple PPO, fragments and derivatives thereof, which method includes providing nucleic acid encoding pineapple PPO or antisense to pineapple PPO, fragments and derivatives thereof; and a vector; and reacting the nucleic acid and the vector to deploy the nucleic acid within the vector.

The nucleic acid may be prepared by a method as hereinbefore described.

The vector may be a plasmid expression vector. For example Bluescript SK has been found to be suitable. Alternatively, the vector may be a binary vector. The recombinant vector may contain a promoter, preferably a constitutive promoter upstream of the nucleic acid.

The cloning step may take any suitable form. A preferred form may include WO 98/53080 PCT/AU98/00362 9 fractionating the cDNA, for example on a column or a gel; isolating a fragment of the expected size, for example from the column or gel; and ligating said fragment into a suitable restriction enzyme site of the vector, for example the EcoRV site of a Bluescript SK vector.

In order to test the clones so formed, a suitable microorganism may be transformed with the vector, the microorganism cultured and the polypeptide encoded therein expressed. The microorganism may be a strain of Escherichia coli, for example E.coli DH5 has been found to be suitable. Alternatively, appropriate vectors may be used to transform plants.

In a further aspect of the present invention there is provided a recombinant vector including a nucleic acid encoding banana PPO or antisense to banana PPO, fragments and derivatives thereof, which vector is capable of being replicated, transcribed and translated in a unicellular organism or alternatively in a plant.

In a further aspect of the present invention there is provided a recombinant vector including a nucleic acid encoding tobacco PPO or antisense to tobacco PPO, fragments and derivatives thereof, which vector is capable of being replicated, transcribed and translated in a unicellular organism or alternatively in a plant.

In a further aspect of the present invention there is provided a recombinant vector including a nucleic acid encoding pineapple PPO or antisense to pineapple PPO, fragments and derivatives thereof, which vector is capable of being replicated, transcribed and translated in a unicellular organism or alternatively in a plant.

The nucleic acid may be prepared by a method as hereinbefore described.

The vector may be a plasmid expression vector. For example Bluescript SK* has been found to be suitable. Alternatively, the vector may be a binary vector. The recombinant vector may contain a promoter, preferably a constitutive promoter upstream of the nucleic acid encoding banana, tobacco or pineapple WO 98/53080 PCT/AU98/00362 PPO or antisense to banana, tobacco or pineapple PPO, fragments and derivatives thereof.

The microorganism may be a strain of Escherichia coli, for example E.coli has been found to be suitable.

In a further aspect of the present invention there is provided a method of decreasing the level of PPO activity in a plant tissue, which method includes providing a nucleic acid encoding banana PPO, a modified nucleic acid encoding banana PPO, or a nucleic acid antisense to banana PPO, fragments and derivatives thereof; and a plant sample; and introducing said nucleic acid into said plant sample to produce a transgenic plant.

In a further aspect of the present invention there is provided a method of decreasing the level of PPO activity in a plant tissue, which method includes providing a nucleic acid encoding tobacco PPO, a modified nucleic acid encoding tobacco PPO, or a nucleic acid antisense to tobacco PPO, fragments and derivatives thereof; and a plant sample; and introducing said nucleic acid into said plant sample to produce a transgenic plant.

In a further aspect of the present invention there is provided a method of decreasing the level of PPO activity in a plant tissue, which method includes providing a nucleic acid encoding pineapple PPO, a modified nucleic acid encoding pineapple PPO, or a nucleic acid antisense to pineapple PPO, fragments and derivatives thereof; and a plant sample; and introducing said nucleic acid into said plant sample to produce a transgenic plant.

PPO activity may be decreased by the use of sense constructs WO 98/53080 PCT/AU98/00362 11 (cosuppression). Alternatively the nucleic acid may include a sequence encoding antisense mRNA to banana or tobacco or pineapple PPO or a functionally active fragment thereof. Alternatively the nucleic acid may encode banana or tobacco or pineapple PPO or a functionally active fragment thereof and incorporate a catalytic cleavage site (ribozyme). The nucleic acid may be included in a recombinant vector as hereinbefore described. In a preferred aspect, the nucleic acid may be included in a binary vector. In a further preferred aspect, the introduction of a binary vector into the plant may be by infection of the plant with an Aqrobacterium containing the binary vector or by bombardment with nucleic acid coated microprojectiles. Methods for transforming banana, tobacco or pineapple with Agrobacterium are known to those skilled in the art and are described in, for example, May et al., Bio/technology (1995) 13:486-492; Michelmore et al., Plant Cell Reports (1987) 6:439-442, and Curtis et al., Journal of Experimental Botany (1994) 45:1141-1149, the entire disclosures of which one incorporated herein by reference. Methods for transforming banana, tobacco or pineapple by bombardment with DNA coated microprojectiles are known to those skilled in the art and are described in, for example, Sagi et al., Bio/technology (1995) 13:481-485, the entire disclosure of which is incorporated herein by reference.

In a further aspect of the present invention there is provided a method of increasing the level of PPO activity in a plant tissue, which method includes providing a nucleic acid encoding banana PPO or a fragment thereof; and a plant sample; and introducing said nucleic acid into said plant sample to produce a transgenic plant.

In a further aspect of the present invention there is provided a method of increasing the level of PPO activity in a plant tissue, which method includes providing a nucleic acid encoding tobacco PPO or a fragment thereof; and a plant sample; and introducing said nucleic acid into said plant sample to produce a WO 98/53080 PCT/AU98/00362 12 transgenic plant.

In a further aspect of the present invention there is provided a method of increasing the level of PPO activity in a plant tissue, which method includes providing a nucleic acid encoding pineapple PPO or a fragment thereof; and a plant sample; and introducing said nucleic acid into said plant sample to produce a transgenic plant.

The nucleic acid may be included in a recombinant vector as hereinbefore described. In a preferred aspect, the nucleic acid may be included in a binary vector. In a further preferred aspect, the introduction of the binary vector into the plant may be by infection of the plant with an Aqrobacterium containing the binary vector or by bombardment with nucleic acid coated microprojectiles.

The plant may be of any suitable type. However the method is particularly applicable to banana, tobacco or pineapple.

In a further aspect of the present invention there is provided a transgenic plant, which plant contains nucleic acid capable of modifying expression of the normal banana PPO gene.

The plant may be of any suitable type. Preferably, the plant is banana.

In a further aspect of the present invention there is provided a transgenic plant, which plant contains nucleic acid capable of modifying expression of the normal tobacco PPO gene.

The plant may be of any suitable type. Preferably, the plant is tobacco.

In a further aspect of the present invention there is provided a transgenic plant, which plant contains nucleic acid capable of modifying expression of the normal pineapple PPO gene.

The plant may be of any suitable type. Preferably, the plant is pineapple.

The nucleic acid may be as hereinbefore described.

In a still further aspect of the present invention there is provided a plant vaccine including nucleic acid encoding banana PPO or antisense to banana PPO, fragments and derivatives thereof.

In a still further aspect of the present invention there is provided a plant PCT/AU98/00362 Received 19 May 1999 P:\OPER\MRO\98-00362.PCT 18/5/99 -13 vaccine including nucleic acid encoding tobacco PPO or antisense to tobacco PPO, fragments and derivatives thereof.

In a still further aspect of the present invention there is provided a plant vaccine including nucleic acid encoding pineapple PPO or antisense to pineapple PPO, fragments and derivatives thereof.

The present invention will now be more fully described with reference to the accompanying Examples. It should be understood, however, that the description following is illustrative only and should not be taken in any way as a restriction on the generality of the invention described above.

IN THE FIGURES: FIGURE 1: The BPPO2 cDNA nucleotide sequence (SEQ ID NO: 1) and derived protein sequence (SEQ ID NO: 2) encoding part of a banana PPO protein.

FIGURE 2: The BPPO8 cDNA nucleotide sequence (SEQ ID NO:3) and derived protein sequence (SEQ ID NO: 4) encoding part of a banana PPO protein.

FIGURE 3: The BANPPO34 cDNA nucleotide sequence (SEQ ID NO:5) and derived protein sequence (SEQ ID NO: 6) encoding part of a banana PPO protein.

FIGURE 4: The BANPPO35 cDNA nucleotide sequence (SEQ ID NO:7) and derived protein sequence (SEQ ID NO:8) encoding part of a banana PPO protein.

FIGURE 5: The TOBPPO6 cDNA nucleotide sequence (SEQ ID NO: 9) and derived protein sequence (SEQ ID NO:10) encoding part of a tobacco PPO protein.

FIGURE 6 The TOBPPO25 cDNA nucleotide sequence (SEQ ID NO: 11) and derived protein sequence (SEQ ID NO: 12) encoding part of a tobacco PPO protein.

FIGURE 7: The TOBPPO26 cDNA nucleotide sequence (SEQ ID NO:13) and derived 7 A---30 protein sequence (SEQ ID NO: 14) encoding part of a tobacco PPO protein.

AMENDED SHEIt'Paticle 34) (IPEA/AU) PCT/AU98/00362 Received 19 May 1999 P:\OPER\MRO\98-00362.PCT 18/5/99 -14- FIGURE 8: The PINPPO20 cDNA nucleotide sequence (SEQ ID NO: 15) and derived protein sequence (SEQ ID NO: 16) encoding part of a pineapple PPO protein.

FIGURE 9: The PINPPO2 cDNA nucleotide sequence (SEQ ID NO: 17) and derived protein sequence (SEQ ID NO: 18) encoding part of a pineapple PPO protein.

FIGURE 10: The PINPPOFL cDNA nucleotide sequence (SEQ ID NO:19) and derived protein sequence (SEQ ID NO:20) encoding a pineapple PPO protein.

EXAMPLE 1: Cloning Banana Peel PPO genes Total RNA was isolated from the peel of young banana fruit. Fruit tissue (3g) was frozen and ground to a fine powder in liquid nitrogen with a coffee grinder then added to ml of extraction buffer hexadecyltrimethylammonium bromide (CTAB), 2% polyvinyl pyrolidone, 100 mM Tris-CHI, pH 8.0, 25 mM EDTA, 2 M NaCI, 0.05% spermidine, 2% P-mercaptoethanol) at 65°C. The extract was mixed with 20 ml of chloroform IAA then centrifuged for 20 minutes at 5,000 RPM and the aqueous phase was re-extracted with chloroform IAA. The aqueous phase was filtered through Miracloth and 0.25 volumes of M LiCl were added then the sample was incubated overnight at 4 0 C before centrifuging for 20 minutes at 8,000 RPM. The supematant was removed and the pellet was resuspended in 0.5 ml of 1 M NaCI, 0.5% SDS, 10 mM Tris, pH 8.0, ImM EDTA. The RNA was extracted once with an equal volume of chloroform IAA and 2 volumes of ethanol was added. After incubation for 40 mins at -70 0 C the solution was centrifuged for 15 minutes at 10,000 RPM. The supernatant was removed and the pellet was rinsed with 80% ethanol, drained and dried. The pellet was resuspended in 50 /L of sterile water.

First strand cDNA was synthesised from 10/g total RNA with reverse transcriptase as described in Ref 2, utilising an oligosaccharide-dT primer adapter (Ref 1): B26 (SEQ ID NO:32): (5'GACTCGAGTCGACATCGATT TTTTTTTTITTTTT-3').

AMENDED SHEET (Article 34) (IPEA/AU) PCT/AU98/00362 Received 19 May 1999 P:\OPER\MRO\9S-00362.PCT 18/5/99 Oligonucleotide primers were designed based on known plant PPO DNA sequences.

Comparison of a number of PPO sequences from a range of different plants allowed identification of the conserved regions of the gene, which are mostly in or near the regions which encode the two copper binding sites, CuA and CuB Forward primers designed around the CuA site (GEN8, GEN9 and GEN 10) and reverse primers designed around the CuB site (REV1 and REV2) were synthesised: GEN8 (SEQ ID NO:22): (5'-GCGAATTCGATCCIACITT[TC]GC[GT]TTICC-3') GEN9 (SEQ ID NO:23): (5'-GCGAATTCTICA[TC]TG[TC]GCITA[TC]TG-3') (SEQ ID NO:24):(5'-GCGAATTCTTICCIT[TA][TC]TGGAA[TC]TGGG-3') REV1 (SEQ ID NO:25):(5'-GCCTGCAGCCACATIC[TG][AG]TCIAC[AG]TT-3') REV2 (SEQ ID NO:26):(5'-GCCTGCAGTT[TC]TC[AG]TC[AG]TAGAA-3') The first strand reaction was amplified by the polymerase chain reaction (PCR) essentially according to the method of Frohman using GEN and REV primers, each at a final concentration of 1IM Amplification involved an initial program of 2 cycles of denaturation at 94°C for 1 min, annealing at 37°C for 2 min, a slow ramp to 72 0 C for 3 min, followed by 33 cycles of denaturation at 94 0 C for 1 min, annealing at 55°C for 1 min, and elongation at 72°C for 3 min. A sample of the amplified DNA was run on an agarose gel and stained with ethidium bromide to determine the size of the PCR products. The remainder was run on a low melting point agarose gel and the bands of interest were excised. DNA was purified from the agarose with a QIAquick PCR Purification kit (Qiagen).

The purified DNA was cloned into Eco -RV-cut Bluescript SK vector (Stratagene) which had been T-tailed with Taq Polymerase and the ligated DNA was introduced into E. coli by electroporation. Recombinant clones which had an insert of the predicted size were selected and their DNA sequence was determined by automated sequencing. Two putative banana PPO clones (BPPO2 and BPPO8) were identified based on their homology to known plant PPO genes.

AMENDED SHEET (Article 34) (IPEA/AU) PCT/AU98/00362 Received 19 May 1999 P:\OPER\MRO\98-00362.PCT 18/5/99 -16- The 3'-end of BPPO2 was cloned using a primer designed to the sequence of BPPO2: BAN8F (SEQ ID NO:27): (5'-GTTGCTCTTCTTAGGCTCGGCTTAC-3') at a final concentration of 1l M and a B25 adaptor primer: ID NO:28):(5'GACTCGAGTCGACATCGA-3') at a final concentration of lpM (ref Amplification involved 35 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 1 min, and elongation at 72*C for 3 min. A sample of the amplified DNA was run on an agarose gel and stained with ethidium bromide to determine the size of the PCR products. The remainder was run on a low melting point agarose gel and the bands of interest were excised. DNA was purified from the agarose with a QIAquick PCR Purification kit (Qiagen).

The purified DNA was cloned into Eco RV-cut Bluescript SK vector (Stratagene) which has been T-tailed with Taq Polymerase and the ligated DNA was introduced into E.coli by electroporation. Recombinant clones which had an insert of the predicted size (1150 bp) were selected and their DNA sequence was determined by automated sequencing. Two putative banana PPO clones (BANPPO34 and BANPPO35) were identified based on their homology to know plant PPO genes. The sequence of BANPPO34 was identical to that of BPPO2.

EXAMPLE 2: Cloning Tobacco Leaf PPO genes Total RNA was isolated from young leaves (1-3cm long) of glasshouse grown plants.

Approximately 2 g of frozen leaf material was ground to a fine powder in liquid nitrogen then extracted in 15 ml of extraction buffer (50 mM Tris-HCl, pH 9.0, 150 mM LiCI, EDTA, 5% SDS and 0.6% P-mercaptoethanol) by shaking vigorously in a 50 ml screw cap tube for 1-2 minutes.

Approximately 15 ml of phenol chloroform IAA (25:24:1) was added and the homogenate AMENDED SHEET (Article 34) (IPEA/AU) WO 98/53080 PCT/AU98/00362 17 mixed then centrifuged for 15 minutes at 5,000 RPM, 4 0 C. The upper aqueous phase was removed and re-extracted twice with phenol chloroform IAA and then once with chloroform IAA and then centrifuged for 10 minutes at 5,000 RPM, 4°C. The supernatant was removed, LiCI was added to a final concentration of 2 M and the mixture was incubated overnight at 4 0 C. After centrifuging for 10 minutes at 8,000 RPM, 4 0 C the supernatant was removed and the pellet was resuspended in 6 ml of 0.4 M LiCI then 2 ml of 8M LiCI was added and the mixture was incubated overnight at 4 0 C. The mixture was centrifuged for minutes at 8,000 RPM, 4 0 C, the supernatant was removed and the pellet was resuspended in 0.5 ml of sterile water and centrifuged briefly to remove any insoluble material.

mRNA was isolated from the total RNA using a PolyATtract kit (Promega). First strand cDNA was synthesised from 10 pg total RNA or 2 gg mRNA with reverse transcriptase as described in Ref 2, utilising an oligo-dT primer adapter (Ref 1): B26: (5'-GACTCGAGTCGACATCGAI I I I I I II I I I I I I I The first strand reaction was amplified by the polymerase chain reaction (PCR) essentially according to the method of Frohman using GEN and REV primers described in Example 1, each at a final concentration of 1 iM Amplification involved an initial program of 2 cycles of denaturation at 940 C for 1 min, annealing at 370 C for 2 min, a slow ramp to 720 C over 2 min and elongation at 72° C for 3 min, followed by 28 cycles of denaturation at 940 C for 1 min, annealing at 550 C for 1 min, and elongation at 720 C for 3 min. A sample of the amplified DNA was run on an agarose gel and stained with ethidium bromide to determine the size of the PCR products. The remainder was run on a low melting point agarose gel and the bands of interest were excised. DNA was purified from the agarose with a QIAquick PCR Purification kit (Qiagen).

The purified DNA was cloned into Eco RV-cut Bluescript SK vector (Stratagene) which had been T-tailed with Taq Polymerase and the ligated DNA was introduced into E. coli DH5a by electroporation. Recombinant clones which had an insert of the predicted size were selected and their DNA sequence was WO 98/53080 PCT/AU98/00362 18 determined by automated sequencing. Three putative tobacco PPO clones (TOBPPO6, TOBPPO25 and TOBPP026) were identified based on their homology to known plant PPO genes.

EXAMPLE 3: Cloning Pineapple PPO genes Mature pineapple fruit were treated to induce blackheart disorder by holding the fruit for 17 days at 120C then for 4 days at 250C. Flesh showing blackheart symptoms was dissected from the fruit, frozen in liquid nitrogen and ground to a fine powder in a pre-cooled coffee grinder. To isolate total RNA 10 g of the powder was ground in a mortar and pestle then extracted with 30 ml of homogenisation buffer (100mM Tris-HCI, pH9.0, 200mM NaCI, 15 mM EDTA, sarkosyl and 1% p-mercaptoethanol), 30 mi of phenol and 6 ml of chloroform IAA. The mixture was stirred in a beaker, 2.1 ml of 3M NaAc (pH 5.2) was added and the mixture was kept on ice for 15 minutes then centrifuged for 15 minutes at 8,000 RPM, 40C. The upper aqueous phase was removed and an equal volume of isopropanol was added. The mixture was incubated for minutes at -700C then centrifuged for 20 minutes at 8,000 RPM, 4 0 C in Corex tubes. The supernatant was removed and the pellet was rinsed with ethanol and centrifuged for 5 minutes at 8,000 RPM, 4°C. The ethanol was removed and the pellet was air dried then resuspended in 0.75 ml sterile water and centrifuged to remove any insoluble material. LiCI was added to a final concentration of 3 M and the mixture was incubated overnight at -200C then centrifuged for 30 minutes at 15,000 RPM, 4°C. The pellet was rinsed with ethanol, centrifuged briefly, drained and air dried. The pellet was resuspended in 75 0l sterile water and centrifuged to remove any insoluble material.

Oligonucleotide primers were designed based on known plant PPO DNA sequences. Comparison of a number of PPO sequences from a range of different plants allowed identification of the conserved regions of the gene, which are mostly in or near the regions which encode the two copper binding sites, CuA and CuB. Forward primers designed around the CuA site (GEN8, GEN9 and WO 98/53080 PCT/AU98/00362 19 GEN 10) and reverse primers designed around the CuB site (REV1 and REV2) were synthesised GEN8: (5'-GCGAATTCGATCCIACITT[TC]GC[GT]TTICC-3') GEN9: (5'-GCGAATTCTICA[TC]TG[TC]GCITA[TC]TG-3') GEN10: (5'-GCGAATTCTTICCIT[TA][TC]TGGAA[TC]TGGG-3') REV1 (5'-GCCTGCAGCCACATIC[TG][AG]TCIAC[AG]TT-3') REV2: (5'-GCCTGCAGTT[TC]TC[AG]TC[AG]TAGAA-3') First strand cDNA was synthesised from 10 pg total RNA with reverse transcriptase as described in Ref 2, utilising the REV2 primer: REV2: (5'-GCCTGCAGTT[TC]TC[AG]TC[AG]TAGAA-3') The first strand reaction was amplified by the polymerase chain reaction (PCR) essentially according to the method of Frohman using the GEN and REV primers described in Example 1, each at a final concentration of 1 iM Amplification involved an initial program of 2 cycles of denaturation at 940 C for 1 min, annealing at 370 C for 2 min, a slow ramp to 720 C over 2 min and elongation at 720 C for 3 min, followed by 33 cycles of denaturation at 940 C for 1 min, annealing at 550 C for 1 min, and elongation at 720 C for 3 min.

A sample of the amplified DNA was run on an agarose gel and stained with ethidium bromide to determine the size of the PCR products. The remainder was run on a low melting point agarose gel and the bands of interest were excised.

DNA was purified from the agarose with a QIAquick PCR Purification kit (Qiagen).

The purified DNA was cloned into Eco RV-cut Bluescript SK vector (Stratagene) which had been T-tailed with Taq Polymerase and the ligated DNA was introduced into E. coli DH5a by electroporation. Recombinant clones which had an insert of the predicted size were selected and their DNA sequence was determined by automated sequencing. A putative pineapple PPO clone (PINPPO20) was identified based on its homology to known plant PPO genes.

WO 98/53080 PCT/AU98/00362 First strand cDNA was also synthesised from 10 [pg total RNA with reverse transcriptase as described in Dry, I.B. and Robinson, S. P (1994), utilising an oligo-dT primer adapter (Ref 1): B26: (5'-GACTCGAGTCGACATCGA IIIT I I I I I I I I-3') This first strand reaction was amplified by the polymerase chain reaction (PCR) essentially according to the method of Frohman, M.A. (1990) using GEN9 and primers GEN9 (5'-GCGAATTCTICA[TC]TG[TC]GCITA[TC]TG-3') (5'-GCGAATTCTTICCIT[TA][TC]TGGAA[TC]TGGG-3') at a final concentration of 1 pM and a B25 adaptor primer: (5'-GACTCGAGTCGACATCGA-3') at a final concentration of 0.1 lM (Frohman, M.A. (1990); Dry, I.B. and Robinson, S.P. (1994)) Amplification involved a program of 33 cycles of denaturation at 940 C for 1 min, annealing at 550 C for 1 min, and elongation at 720 C for 3 min.

The purified DNA was cloned into Eco RV-cut Bluescript SK vector (Stratagene) which had been T-tailed with Taq Polymerase and the ligated DNA was introduced into E. coli DH5a by electroporation. Recombinant clones which had an insert of the predicted size were selected and their DNA sequence was determined by automated sequencing. Two putative pineapple PPO clones (PINPPO1 and PINPPO2) were identified based on their homology to known plant PPO genes. The sequence of PINPPO1 was found to be nearly identical to that of The 5'-end of PINPPO1 was obtained using a 5'-RACE system for rapid amplification of cDNA ends, Version 2.0, from GIBCO-BRL, according to the PCT/AU98/00362 Received 19 May 1999 P:\OPER\MRO\98-00362.PCT 185/99 -21 manufacturer's instructions. Specific oligonucleotide primers based on the sequences of PINPPO1 and PINPPO2 were used: PINE 1: (SEQ ID NO:29): 5-'ATATCACCTGTCGGTACATGACGGC-3' PINE 2: (SEQ ID NO:30):5'- GTGCCATTGTAGTCGAGGTCAATCA-3' A number of clones were sequenced and one, 5PINA, was found to be nearly identical to PINPPO1 in the overlapping regions.

A full-length pineapple cDNA clone was isolated using a primer designed to the sequence of 5PIN1: (SEQ ID NO:31):(5'-CCAGTGCCTGGTTTAGGTGTATTCAC-3') are used with the B25 adaptor primer as described above to amplify cDNA prepared from blackheart-induced pineapple fruit RNA. Amplification involved a program of 33 cycles of denaturation at 94 C for 1 min, annealing at 55 C for 1 min, and elongation at 72°C for 3 min.

A sample of the amplified DNA was run on an agarose gel and stained with ethidium bromide to determine the size of the PCR products. The remainder was run on a low melting point agarose gen and the bands of interest were excised. DNA was purified from the agarose with a QIAquick PCR Purification kit (Qiagen).

The purified DNA was cloned into Eco RV-cut Bluescript SK vector (Stratagene) which had been T-tailed with Taq Polymerase and the ligated DNA was introduced into E.coli by electroporation. Recombinant clones which had an insert of the predicted size (2.2kbp) were selected and their DNA sequence was determined by automated sequencing. A pineapple PPO clone (PINPPOFL) was identified based on its homology to the PINPPO1 and 5 PINA clones. The sequence of PINPPOFL was found to be nearly identical to that of PINPPO20, PINPPO1 and 5PINA in the overlapping regions.

AMENDED SHEET (Article 34) (IPEA/AU) PCT/AU98/00362 Received 19 May 1999 P:\OPER\MRO\98-00362.PCT 18/5/99 -22-

GENERAL

Those skilled in the art will be aware that the invention described herein is subject to variations and modifications other than those specifically described. It is to be understood that the invention described herein includes all such variations and modifications. The invention also includes all such steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

Throughout this specification, unless the context requires otherwise the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

REFERENCES

1. Frohman, MA (1990) in "PCR Protocols: A guide to Methods and Applications" (MA Innis, DH Gelfrand, JJ Sninsky and TJ White, eds) Academic Press, New York, pp 28-38.

2. Dry, IB and Robinson, SP (1994) "Molecular cloning and characterisation of grape berry polyphenol oxidase". Plant Mol. Biol. 26, 495-502.

AMENDED SHEET (Article 34) (IPEA/AU) EDITORIAL NOTE: CASE FILE NO.: 73268/98 THE FOLLOWING SEQUENCE LISTING PAGES 1 27 IS PART OF THE DESCRIPTION.

PCT/AU98/00362 Received 19 May 1999 A:\2144866.SEQ- 18/5/99 1- SEQUENCE LISTING <110> COMMONWEALTH SCIENTIFIC AND INDUSTRIAL RESEARCH ORGANISATION <120> Polyphenol oxidase genes from banana, tobacco and pineapple <130> PCT/AU98/00362 <140> PCT/AU98/00362 <141> 1998-05-19 <150> AU PP6849 <151> 1997-05-19 <160> 33 <170> PatentIn Ver. <210> 1 <211> 582 <212> DNA <213> banana <220> <221> CDS <222> <400> 1 cac tgt gcg tat tgt gat ggc gcc tac gac cag ate ggc ttc ccc aac 48 His Cys Ala Tyr Cys Asp Gly Ala Tyr Asp Gin Ile Gly Phe Pro Asn 1 5 10 ctc gag etc caa gtc cac aac tcc tgg ctc ttc ttc cct tgg cac cgc 96 Leu Glu Leu Gin Val His Asn Ser Trp Leu Phe Phe Pro Trp His Arg 25 ttc tac etc tac ttc cac gag agg atc etc gga aag ctc ata ggc gac 144 Phe Tyr Leu Tyr Phe His Glu Arg Ile Leu Gly Lys Leu Ile Gly Asp 40 gac act ttc gcc ctc cct ttc tgg aac tgg gac gcg ccc ggc ggc atg 192 Asp Thr Phe Ala Leu Pro Phe Trp Asn Trp Asp Ala Pro Gly Gly Met 55 aag ctg ccg tcg atc tac gcc gac cct tcg tcc tcg ctc tat gac aag 240 Lys Leu Pro Ser Ile Tyr Ala Asp Pro Ser Ser Ser Leu Tyr Asp Lys 70 75 ttt cgc gac gcc aag cac cag ccg cca gtc ctc gtc gac ctc gac tac 288- Phe Arg Asp Ala Lys His Gin Pro Pro Val Leu Val Asp Leu Asp Tyr 90 aac gga acc gac cct agt ttc acc gac gca gag cag atc gat cag aac 336 Asn Gly Thr Asp Pro Ser Phe Thr Asp Ala Glu Gin Ile Asp Gin Asn 100 105 110 ctc aag atc atg tac egg cag gtg ate tec aac ggc aag acg ccg ttg 384 Leu Lys Ile Met Tyr Arg Gin Val Ile Ser Asn Gly Lys Thr Pro Leu ,A 115 120 125 AMENDED SHEET (Article 34) (IPEA/AU) PCT/AU98/00362 Received 19 May 1999 A:\2144866.SEQ 18/5/99 -2ctc ttc tta ggc tcg get tac cgt gcc ggc gac aac cca aac ccc ggc 432 Leu Phe Leu Gly Ser Ala Tyr Arg Ala Gly Asp Asn Pro Asn Pro Gly 130 135 140 gcg ggc tcg etc gag aac ata cca cac ggc ccc gtc cac ggg tgg act 480 Ala Gly Ser Leu Glu Asn Ile Pro His Gly Pro Val His Gly Trp Thr 145 150 155 160 ggc gac aga age caa ccc aat ctc gag gac atg ggc aac ttc tac tcc 528 Gly Asp Arg Ser Gin Pro Asn Leu Glu Asp Met Gly Asn Phe Tyr Ser 165 170 175 gcg ggg cgc gac cct ate ttc ttc gcc cac cat tea aat gtc gat cgc 576 Ala Gly Arg Asp Pro Ile Phe Phe Ala His His Ser Asn Val Asp Arg 180 185 190 atg tgg 582 Met Trp <210> 2 <211> 194 <212> PRT <213> banana <400> 2 His Cys Ala Tyr Cys Asp Gly Ala Tyr Asp Gin Ile Gly Phe Pro Asn 1 5 10 Leu Glu Leu Gin Val His Asn Ser Trp Leu Phe Phe Pro Trp His Arg 25 Phe Tyr Leu Tyr Phe His Glu Arg Ile Leu Gly Lys Leu Ile Gly Asp 40 Asp Thr Phe Ala Leu Pro Phe Trp Asn Trp Asp Ala Pro Gly Gly Met 55 Lys Leu Pro Ser Ile Tyr Ala Asp Pro Ser Ser Ser Leu Tyr Asp Lys 70 75 Phe Arg Asp Ala Lys His Gin Pro Pro Val Leu Val Asp Leu Asp Tyr 90 Asn Gly Thr Asp Pro Ser Phe Thr Asp Ala Glu Gin Ile Asp Gin Asn 100 105 110 Leu Lys Ile Met Tyr Arg Gin Val Ile Ser Asn Gly Lys Thr Pro Leu 115 120 125 Leu Phe Leu Gly Ser Ala Tyr Arg Ala Gly Asp Asn Pro Asn Pro Gly 130 135 140 Ala Gly Ser Leu Glu Asn Ile Pro His Gly Pro Val His Gly Trp Thr 145 150 155 160 Gly Asp Arg Ser Gin Pro Asn Leu Glu Asp Met Gly Asn Phe Tyr Ser 165 170 175 Ala Gly Arg Asp Pro Ile Phe Phe Ala His His Ser Asn Val Asp Arg 180 185 190 AMENDED SHEET (Article 34) (IPEA/AU) PCT/AU98/00362 Received 19 May 1999 A:\21448. SEQ 18/5,99 -3- Met Trp <210> 3 <211> 426 <212> DNA <213> banana <220> <221> CDS <222> (426) <400> 3 ttg ccg ttt tgg aat tgg gac gcg Asp Ala Leu 1 ate Ile aag Lys ect Pro tac Tyr tcg Ser gag Glu caa Gin Pro tac Tyr eac His agt Ser egg Arg gct Ala aae Asn cCC Pro Phe gee Ala eag Gin tte Phe eag Gin tae Tyr ata Ile aat Asn 115 Trp gae Asp ccg Pro ace Thr gtg Va1 egt Arg cca Pro 100 etc Leu Asn Trp 5 ect tcg Pro Ser ccg gte Pro Val gac gca Asp Ala ate tee Ile Ser 70 gee gge Ala Gly eac gge His Gly gag gac Glu Asp ccc gge gge atg aag etg ceg tcg Pro Gly Gly Met Lys Leu Pro Ser 10 tee Ser etc Leu gag Glu 55 aae Asn gac Asp ccc Pro atg Met tcg Ser gte Va1 40 cag Gin ggc Gly aac Asn gte Va1 ggC Gly 120 etc Leu 25 gac Asp ate Ile aag Lys eca Pro eac His 105 aac Asn tat Tyr etc Leu gat Asp acg Thr aac Asn 90 ggg Gly tte Phe gae Asp gac Asp eag Gin ccg Pro 75 ccc Pro tgg Trp tac Tyr aag Lys tac Tyr aae Asn ttg Leu ggc Gly act Thr tee Ser ttt Phe aae Asn etc Leu etc Leu gcg Ala gge Gly gcg Ala 125 ege Arg gga Gly aag Lys tte Phe gge Gly gac Asp 110 ggg Gly gac gee Asp Ala ace gac Thr Asp ate atg Ile Met tta ggc Leu Gly tcg etc Ser Leu aga age Arg Ser cgc gac Arg Asp 48 96 144 192 240 288 336 384 426 ect ate tte ttc Pro Ile Phe Phe 130 gee Ala eac eat tea aat His His Ser Asn gte gat Val Asp age atg tgg Ser Met Trp 140 <210> 4 <211> 142 <212> PRT <213> banana <400> 4 Leu Pro Phe Trp Asn Tip Asp Ala Pro Gly Gly Met Lys Leu Pro Ser 1 5 10 AMENDED SHEET (Article 34) (IPEA/AU) PCT/AU98/00362 SA:\21866.SEQ 18/5/99 Received 19 May 1999 -4- Ile Tyr Ala Asp Pro Ser Ser Ser Leu. Tyr Asp Lys Phe Arg Asp Ala 25 Lys His Gin Pro Pro Val Leu Val Asp Leu Asp Tyr Asn Gly Thr Asp 40 Pro Ser Phe Thr Asp Ala Glu Gin Ile Asp Gin Asn Leu Lys Ile Met 55 Tyr Arg Gin Val Ile Ser Asn Gly Lys Thr Pro Leu Leu Phe Leu Gly 70 75 Ser Ala Tyr Arg Ala Gly Asp Asn Pro Asn Pro Gly Ala Gly Ser Leu 90 Glu Asn Ile Pro His Gly Pro Val His Gly Trp Thr Gly Asp Arg Ser 100 105 110 Gin Pro Asn Leu Glu Asp Met Gly Asn Phe Tyr Ser Ala Gly Arg Asp 115 120 125 Pro Ile Phe Phe Ala His His Ser Asn Val Asp Ser Met Trp 130 135 140 <210> <211> 925 <212> DNA <213> banana <220> <221> CDS <222> <400> g ttg ctc ttc tta ggc tcg get tac cgt gcc ggc gac aac cca aac ccc 49 Leu Leu Phe Leu Gly Ser Ala Tyr Arg Ala Gly Asp Asn Pro Asn Pro 1 5 10 ggc gcg ggc tcg etc gag aac ata cca cac ggc ccc gtc cac ggg tgg 97 Gly Ala Gly Ser Leu Glu Asn Ile Pro His Gly Pro Val His Gly Trp 25 act ggc gac aga aac caa ccc aat ctc gag gac atg ggc aac ttc tac 145 Thr Gly Asp Arg Asn Gin Pro Asn Leu Glu Asp Met Gly Asn Phe Tyr 40 tec gcg ggg cgc gac cct ate ttc ttc gcc cac cat tea aac gtc gac 193 Ser Ala Gly Arg Asp Pro Ile Phe Phe Ala His His Ser Asn Val Asp 55 cgc atg tgg tac ttg tgg aag aag etc ggc ggg aag cat cag gac ttt 241 Arg Met Trp Tyr Leu Trp Lys Lys Leu Gly Gly Lys His Gin Asp Phe 70 75 aac gat aag gac tgg etc aac ace ace ttc etc tte tac gac gag aat 289 Asn Asp Lys Asp Trp Leu Asn Thr Thr Phe Leu Phe Tyr Asp Glu Asn 90 get gac tta gtt cga gtc ace etc aag gac tgc ttg cag ccg gag tgg 337 Ala Asp Leu Val Arg Val Thr Leu Lys Asp Cys Leu Gin Pro Glu Trp UnJ AMENDED SHEET (Article 34) (IPEA/AU) PCT/AU98/00362 Received 19 May 1999 A:\'144866.SEQ 18/5/99 ctt Leu ccg Pro aaa Lys 145 age Ser gaa Glu gae Asp gge Gly eeg Pro 225 ctc Leu gae Asp gtg Val1 egt Arg act Thr 130 get Ala acg Thr gag Glu tac Tyr atc Ile 210 eac His tge Cys gac Asp t eg Ser tae Tyr 115 ec Pro aca Thr a eg Thr gaa Giu ttc Phe 195 aeg Thr aag Lys ctg Leu age Ser gte Val1 100 gat Asp aaa Lys gea Ala gtg Val1 gag Glu 180 ata Ile eeg Pro eac His ggg Gly gtg Val1 260 gee Ala tae Tyr gee Ala gag Glu agg Arg 165 gag Giu aag Lys gge Gly aag Lys ate Ile 245 et e Leu gge Gly eaa Gin t tg Leu a eg Thr 150 agg Arg gte Val1 tte Phe gee Ala eae His 230 aet Thr gte Val1 ete gae Asp aag Lys 135 ceg Pro eee Pro ete Leu gae Asp age Ser 215 age Ser gae Asp aee Thr ege gte Val 120 geg Ala tte Phe aag Lys ate Ile gte Val1 200 gag Glu aag Lys etg Leu ate Ile ate 105 gag Giu eag Gin eeg Pro gta Val1 gtg Val1 185 tte Phe tte Phe aag Lys ete Leu gte Val1 265 gat Asp ate Ile aaa Lys gtg Val1 tcg Ser 170 gag Giu gtg Val1 geg Ala gag Giu gag Glu 250 ecg Pro tte Phe ccg tgg Pro Trp aee gea Thr Ala 140 aeg etg Thr Leu 155 agg age Arg Ser ggg ate Gly Ilie aac gee Asn Ala ggc age Gly Ser 220 aag aag Lys Lys 235 gac ate Asp Ilie aaa gee Lys Ala cea. aat Pro Asn etg Leu 125 geg Ala eaa Gin Gly gag Glu aee Thr 205 t te Phe etg Leu ggg Gly gga Gly 110 aag Lys aaa Lys tee Ser aag Lys tte Phe 190 gag Giu gte Val1 aag Lys geg Ala aag Lys 270 aee Thr aea Thr geg Al a gag Glu 175 ga e Asp ggt Gly aae Asn aeg Thr gag Giu 255 gge Gly egg Arg etg Leu gtg Val1 160 aag Lys ege Arg gag Giu gte Val1 agg Arg 240 ga e Asp aag Lys 385 433 481 529 577 625 673 721 769 817 863 923 925 tgaagtaata Leu Arg Ile 275 280 etatatattt etaetaeeta teaaggaaaa taaaageegc aeeategtaa eaaaaaaaa aa <210> 6 <211> 284 <212> PRT <213> banana <400> 6 Leu Leu Phe Leu Gly Ser Ala Tyr Arg Ala Gly Asp Asn Pro Asn Pro 1 5 10 Gly Ala Gly Ser Leu Glu Asn Ile Pro His Gly Pro Val His Gly Trp AMIENDED SHEET (Article 34) (EPEA/AU) a PCT/AU98/00362 Received 19 May 1999 A:\2144866.SEQ 18/5199 Thr Ser Arg Asn Ala Leu Pro Lys 145 Ser Giu Asp Gly Pro 225 Leu Asp Val1 Gly Ala Met Asp Asp Arg Thr 130 Ala Thr Glu Tyr Ile 210 His Cys Asp Ser Asp Gly Trp Lys Leu Tyr 115 Pro Thr Thr Giu Phe 195 Thr Lys Leu Ser Val1 275 Arg Arg Tyr Asp Val1 100 Asp Lys Ala Val1 Giu 180 Ile Pro His Gly Val1 260 Asn Asp Leu Trp Arg Tyr Ala Giu Arg 165 Giu Lys Gly Lys Ile 245 Leu Gin Pro Trp, 70 Leu Val1 Gin Leu Thr 150 Arg Val1 Phe Al a His 230 Thr Val1 Pro Ile 55 Lys Asn Thr Asp Lys 135 Pro Pro Leu Asp Ser 215 Ser Asp Thr Asn Leu 40 Phe Phe Lys Leu Thr Thr Leu Lys 105 Val Giu 120 Ala Gin Phe Pro Lys Val Ile Vai 185 Val Phe 200 Giu Phe Lys Lys Leu Leu Ile Val 265 Glu Asp Met Gly Asn Phe Tyr Ala Gly Phe 90 Asp Ile Lys Val1 Ser 170 Giu Val1 Al a Giu Giu 250 Pro His Gly 75 Leu Cys Pro Thr Thr 155 Arg Gly Asn Gly Lys 235 Asp Lys His Lys Phe Leu Trp Ala 140 Leu Ser Ile Ala Ser 220 Lys Ile Ala Ser His Tyr Gin Leu 125 Ala Gin Gly Giu Thr 205 Phe Leu Gly Gly Asn Gin Asp Pro 110 Lys Lys Ser Lys Phe 190 Giu Val1 Lys Ala Lys 270 Val1 Asp Giu Giu Thr Thr Ala Giu 175 Asp Gly Asn Thr Giu 255 Gly Asp Phe Asn Trp Arg Leu Val1 160 Lys Arg Glu Val1 Arg 240 Asp Lys Ala Gly Leu Arg Ile Asp Phe Pro Asn <210> 7 <211> 960 <212> DN~A <213> banana <220> <221> CDS <222> <400> 7 AMENDED SHEET (Article 34) (I]PEA/AU) PCT/AU98/00362 Received 19 May 1999 A.\2-144W6.SEQ 18/5/99 -7g ttg etc ttc tta ggc tcg got tac cgt gec ggt gac cag ect aac ccc 49 Leu Leu Phe Leu Gly Ser Ala Tyr Arg Ala Gly Asp Gin Pro Asn Pro 1 5 10 ggc Gly acc Thr gcg Ala cga Arg aat Asn gce Al a etg Leu ceg Pro aaa Lys 145 aaa Lys gaa Glu ga e Asp ggo Gly cog Pro 225 gcg Ala ggc Gly gcg Ala atg Met gac Asp ga c Asp cgc Arg act Thr 130 gcc Ala gtg Val1 ga t Asp tac Tyr ate Ile 210 eac His gga Gly gao Asp geg Ala tgg Trp tog Ser tta Leu tao Tyr 115 ccc Pro ace Thr aeg Thr gag Glu tto Phe 195 aeg Thr aag Lys tee ate gag Ser Ile Glu ego ace cag Arg Thr Gin ego gao ccc Arg Asp Pro tao otg tgg Tyr Leu Trp 70 gao tgg etc Asp Trp Leu gtt egg gte Val Arg Val 100 aeg tao eaa Thr Tyr Gin aag etc gee Lys Leu Ala gog gag gtg Ala Glu Val 150 gtg aag agg Val Lys Arg 165 gag gag ata Giu Glu Ile 180 ate aag tto Ilie Lys Phe gee ggg gee Ala Gly Ala eac aag eac His Lys His 230 aae atg Asn Met ccc aae Pro Asn 40 ate tto Ile Phe 55 aag aag Lys Lys aaa get Lys Ala aeg gte Thr Val gao gtg Asp Val 120 aag geg Lys Ala 135 eag tto Gin Phe ccc aag Pro Lys etc ata Leu Ile gao gte Asp Val 200 agt gag Ser Giu 215 ego aag Arg Lys cog cac aac aae Pro 25 tto Phe tte Phe etc Leu tee Ser aag Lys 105 aag Lys agg Arg cot Pro gtg Val1 gtg Val1 185 tte Phe tto Phe ga t Asp His gag Giu gee Ala age Ser tto Phe 90 gao Asp ate Ilie aaa Lys gtg Val1 ggg Gly 170 gag Glu gtg Val1 gee Ala gag Giu Asn aac Asn eac His agg Arg 75 etc Leu tge Cys eca Pro gee Ala aeg Thr 155 agg Arg ggg Gly aao Asn ggc Gly aat Asn 235 Asn atg MIet cac His aag Lys tte Phe ttg Leu tgg Trp ggc Gly 140 otg Leu age Ser ate Ile geg Ala age Ser 220 aag Lys gtg Val1 ggc Gly gee Al a ca e His tao Tyr gag Giu geg Ala 125 age Ser gaa Giu ggc Gly gag Giu aeg Thr 205 tto Phe otg Leu eac ttg tgg His Leu Trp ace tto tao Thr Phe Tyr aac ate gao Asn Ile Asp eag gao tto Gin Asp Phe gao gag aac Asp Giu Asn ace gag tgg Thr Giu Trp 110 aac ace oga Asn Thr Arg aga tog etg Arg Ser Leu tee cog gte Ser Pro Val 160 aag gag aag Lys Glu Lys 175 tte gao ego Phe Asp Arg 190 gag ggc gao Giu Gly Asp gtg aac gte Val Asn Val aag aeg agg Lys Thr Arg 240 97 145 193 241 289 337 385 433 481 529 577 625 673 721 769 otg tgt ctg gga ate aoe gao etg etc gag gao ate ggc geg gag gao AMENDED SHEET (Article 34) (EPEA/AU) PCT/AU98/00362 Received 19 May 1999 A:\2-144866.SEQ 18/5/99 -8 Leu Cys Leu Gly Ilie Thr Asp Leu Leu Glu Asp Ile Gly Ala Glu Asp 245 250 255 gac gac agc gtg ctc gtc acc atc gtg ccg aag gca ggc aaa gga aag 817 Asp Asp Ser Val Leu Val Thr Ilie Val Pro Lys Ala Gly Lys Gly Lys 260 265 270 gtg tcc gtc ggc ggt ctt cgg att gac ttt tcc aag tgaggaaata 863 Val Ser Val Gly Gly Leu Arg Ile Asp Phe Ser Lys 275 280 aaagaattca cgtgccgtgc ctgctttcaa tgtacgaata aaataagagt gcatcatcac 923 cgaccatggt tctactttaa aaaaaaaaaa aaaaaaa 960 <210> 8 <211> 284 <212> PRT <213> banana <400> 8 Leu Gly Thr Ala Arg Asn Ala Leu Pro Lys 145 Lys Glu Asp Leu Ala Gly Ala Met Asp Asp Arg Thr 130 Ala Val1 Asp Tyr Phe Gly Asp Ala Trp Ser Leu Tyr 115 Pro Thr Thr Glu Phe 195 Leu Gly Ser Ile Arg Thr Arg Asp Tyr Leu Asp Trp Val Arg 100 Thr Tyr Lys Leu Ala Glu Val Lys 165 Giu Glu 180 Ile Lys Ser Ala Tyr Arg Ala Gly Asp Gin Pro Asn Pro 10 Giu Gin Pro Trp 70 Leu Val1 Gin Ala Val1 150 Arg Ile Phe Asn Pro Ile 55 Lys Lys Thr Asp Lys 135 Gin Pro Leu Asp Met Asn 40 Phe Lys Al a Val1 Val1 120 Ala Phe Lys Ilie Val1 200 Pro His 25 Phe Giu Phe Ala Leu Ser Ser Phe 90 Lys Asp 105 Lys Ile Arg Lys Pro Val Val Giy 170 Val Giu 185 Phe Val Asn Asn Asn Met His His Arg Lys 75 Leu Phe Cys Leu Pro Trp Ala Gly 140 Thr Leu 155 Arg Ser Gly Ile Asn Ala Val His Gly Thr Ala Asn His Gin Tyr Asp Giu Thr 110 Ala Asn 125 Ser Arg Giu Ser Gly Lys Glu Phe 190 Thr Glu 205 Leu Trp, Phe Tyr Ile Asp Asp Phe Glu Asn Glu Trp Thr Arg Ser Leu Pro Val 160 Glu Lys 175 Asp Arg Gly Asp ,zz,

LL~

0 C AMENDED SHEET (Article 34) (LPEAIAU) PCT/AU98/003 62 Received 19 May 1999 A:\-'144866.SEQ 18/5/99 Gly Ile Thr 210 Pro His Lys 225 Leu Cys Leu Asp Asp Ser Val Ser Val 275 Ala Gly Ala Ser Giu 215 His Lys His Arg Lys 230 Gly Ile Thr Asp Leu 245 Vai Leu Val Thr Ile 260 Gly Gly Leu Arg Ile 280 -9- Phe Asp Leu Va 1 265 Asp Ala Gly Ser Phe Val Asn Vai 220 Giu Asn Lys Leu Lys Thr Arg 235 240 Giu Asp Ilie Gly Ala Giu Asp 250 255 Pro Lys Ala Gly Lys Gly Lys 270 Phe Ser Lys <210> 9 <211> 545 <212> DNA <213> tobacco <220> <221> CDS <222> <400> 9 gat ccg acg ttt gcg ttg cca Asp 1 atg Met tac Tyr ct t Leu agc Ser tgt Cys aaa Lys att Ile ga t Pro cgt Arg ga t Asp ggt Giy aat Asn c ca Pro cca Pro tgg Trp atg Thr ttg Leu cca Pro cat His aac Asn caa Gin ggg Gly gtt Val1 115 gga.

Phe Ala Leu Pro tat tgg aac tgg gat cat cca aag ggc Tyr Trp Asn Trp Asp His Pro Lys Gly 5 10 cca Pro aga Arg ttt Phe ctt Leu ctc Leu cag Gin 100 ggt Gly aat cac His cgt Arg ggt Giy act Thr ttt Phe gga Gly agt Ser ttc atg ttt Met Phe aac caa Asn Gin caa gac Gin Asp 55 cta atg Leu Met 70 ttc ggt Phe Gly gct att Ala Ile aag cct Lys Pro tat tca ga t Asp gaa.

Giu 40 gtg Val1 tat Tyr aag Lys gaa Giu aat Asn 120 gct Ala caa cca aac Gin Pro Asn 25 cac cgc ggt His Arg Gly aaa gga act Lys Gly Thr cgt caa at~g Arg Gin Met 75 cca tat tgt Pro Tyr Cys 90 aac atc cct Asn Ile Pro 105 gag aat aac Giu Asn Asn ggt aag gat Gly Lys Asp gtg Val1 tct.

Ser ga c Asp att, Ilie acg Thr cat His tgt Cys cct.

Pro 140 tac Tyr gta Val ttg Leu acc Thr gaa Giu act Thr aaa Lys 125 gct Ala cct Pro atc Ile caa Gin aat Asn gtt Val1 cct Pro 110 aac Asn ttc Phe gat Asp atg Met atg Met t ca Ser gga Gly gtc Val1 ggt Gly tat Tyr ctt Leu gac Asp atg Met cca Pro ccc Pro cac His gaa Giu agt Ser 96 144 192 240 288 336 384 432 Asp Met Gly Asn Phe Tyr Ser 130 135 AMENDED SHEET (Article 34) (1PEA/AU) AA\214486SEQ. -18/5/99 Rec 10 cac cat gca aat gta gat cgc atg tgg aca ata tgg aaa aca tta gga His His Ala Asn Val Asp Arg Met Trp Thr Ile Trp, Lys Thr Leu Gly 145 150 155 160 gga aaa cgc aag gac atc aac aag cca gat tat ttg aac act gag ttc Gly Lys Arg Lys Asp Ilie Asn Lys Pro Asp Tyr Leu Asn Thr Giu Phe 165 170 175 ttt ttc tac gac gaa aa Phe Phe Tyr Asp Giu 180 <210> <211> 181 <212> PRT <213> tobacco PCT/AU98/00362 ~eived 19 May 1999 480 528 545 <400> Asp Pro Thr Phe Ala Leu Pro Tyr Trp Asn Trp Asp His Pro Lys Gly 1 Met Tyr Leu Ser Cys Lys Ile Asp His 145 Gly Phe Arg Asp Gly Asn Pro Pro Trp Met 130 His Lys Phe Leu Pro His Asn Gin Gly Val1 115 Gly Ala Arg Tyr 5 Pro Arg Phe Leu Leu Gin 100 Gly Asn Asn Lys Asp 180 His Arg Gly Thr Phe Gly Ser Phe Val1 Met Asn Gin Leu 70 Phe Ala Lys Tyr Asp 150 Phe Gin Asp 55 Met Gly Ilie Pro Ser 135 Arg Asp Giu 40 Val1 Tyr Lys Glu Asn 120 Ala Met Gin 25 His Lys Arg Pro Asn 105 Giu Gly Trp Pro Arg Gly Gin Tyr 90 Ile Asn Lys Thr Asn Gly Thr Met 75 Cys Pro Asn Asp Ilie 155 Val1 Ser Asp Ilie Thr His Cys Pro 140 Trp Tyr Val1 Leu Thr Glu Thr Lys 125 Ala Lys Pro Ile Gin Asn Val1 Pro 110 Asn Phe Thr Asp Met Met Ser Gly Val1 Gly Tyr Leu Leu Asp Met Pro Pro His Glu Ser Gly 160 Asp 165 Giu Ilie Asn Lys Pro Asp Tyr Leu Asn Thr Giu Phe 170 175 <210> 11 <211> 673 <212> DNA <213> tobacco AMENDED SHEET (Article 34) (IPEA/AU) PCT/AU98/00362 Received 19 May 1999 A:\2 144S66SEQ 18/5/99 -1I1I- <220> <221> CDS <222> (671) <400> 11 tg cac tgt gcg tat tgc aac ggt gct tac aaa att ggt ggc aaa gag His Cys Ala Tyr Cys Asn Gly Ala Tyr Lys Ile Gly Gly Lys Giu 1 5 10 tta caa gtc cat ttc tcg tgg ctt ttt ttc cct ttt cat aga tgg tac Leu t tg Leu ttt Phe cct Pro aac Asn caa Gin ata Ile ttt Phe act Thr agg Arg 160 tac Tyr gac Asp ct c Leu Gin tac Tyr ggt Gly ccc Pro caa Gin gaa Giu atg Met ggt Gly att Ilie 145 ctt Leu t cg Ser cgg Arg acg Thr Val1 ttc Phe ttg Leu 50 atg Met agt Ser gtc Vali tat Tyr cag Gin 130 gaa Glu ga t Asp gcc Ala atg Met cac His 210 His Phe tat gaa Tyr Giu 35 cca tat Pro Tyr ttc gat Phe Asp cac cgt His Arg caa aca Gin Thr 100 cgt caa Arg Gin 115 cct tac Pro Tyr aac atc Asn Ile gag aat Glu Asn ggt tta Gly Leu 180 tgg tcc Trp Ser 195 aaa gat Lys Asp Ser aga Arg tgg Trp cgt Arg aat Asn 85 act Thr atg Met cct Pro cct Pro aat Asn 165 gac Asp gag Giu tgg Trp atc Ile aac Asn gaa Glu 70 gga Gly caa Gin ata Ile cta Leu cat His 150 acg Thr ccg Pro tgg Tr~p t tg Leu ttg Leu tgg Trp 55 ggg Gly acc Thr ctg Leu act Thr gga Giy 135 act Thr aaa Lys ctt Leu aaa Lys aac Phe ggc Gly 40 ga c Asp tct Ser ata Ile cag Gin aat Asn 120 act Thr cct Pro cac His ttc Phe gcc Ala 200 tcc Ser Phe 25 tct.

Ser cat His tcC Ser att Ilie cag Gin 105 gc t Ala ga t Asp gtc Val1 ggt Gly tat Tyr 185 tta Leu gag Glu Pro tta Leu cca Pro Ctt Leu ga t Asp 90 atg Met Cct Pro ccc Pro cac His gag Giu 170 tcc Ser gga Gly ttc Phe Phe att Ilie aag Lys tac Tyr ctt Leu act Thr tgc Cys agt Ser att Ilie 155 ga t Asp cat *His ggg *Gly ttt Phe His aa t Pisn Giy gac Asp ggt Gly aat Asn ccc Pro cca Pro 140 tgg Trp atg Met cac His aaa Lys ttc Phe 220 Arg gat Asp atg Met gaa Giu cat His aac Asn ttg Leu 125 ggg Gly gt t Val1 ggt Gly gcc Ala aga Arg 205 tac Tyr rrp cct Pro cgt A.rg aaa Lys ttc Phe t ta Leu 110 ctc Leu atg Met ggt Gly aat Asn aat Asn 190 agS ArS gat Asj Tyr act Thr ata Ile cgt Arg ggt Gly act Thr ttc Phe ggc Gly agt Ser ttt Phe 175 gtg Val gat Asp gaa Glu 143 191 239 287 335 383 431 479 527 575 623 671 Trp Leu Asn 215 AMENDED SHEET (Article 34) (IPEA/AU) PCT/AU98/003 62 Received 19 May 1999 A:\2144W6.SEQ 18/5/99 12 aa <210> 12 <211> 223 <212> PRT <213> tobacco <400> 12 His 1 Gin Tyr Gly Pro Gin Giu Met Gly Ile 145 Leu Ser Arg Thr Cys Val1 Phe Leu Met Ser Val1 Tyr Gin 130 Giu Asp Ala Met His 210 Aia His Tyr Pro Phe His Gin Arg 115 Pro Asn Giu Gly Trp 195 Lys Tyr Phe Giu Tyr Asp Arg Thr 100 Gin Tyr Ilie Asn Leu 180 Ser Cys Asn 5 Ser Trp Arg Ile Trp Asn Arg Glu 70 Asn Gly Thr Gin Met Ilie Pro Leu Pro His 150 Asn Thr 165 Asp Pro Giu Trp Gly Ala Leu Phe Leu Gly 40 Trp Asp 55 Gly Ser Thr Ile Leu Gin Thr Asn 120 Gly Thr 135 Thr Pro Lys His Leu Phe Lys Ala 200 Tyr Phe 25 Ser His Ser Ile Gin 105 Ala Asp Val1 Gly Tyr 185 Leu Lys 10 Pro Leu Pro Leu Asp 90 Met Pro Pro His Giu 170 Ser Gly Ilie Phe Ilie Lys Tyr 75 Leu Thr Cys Ser Ile 155 Asp His Gly Gly His Asn Gly Asp Gly Asn Pro Pro 140 Trp Met His Lys Gly Arg Asp Met Giu His Asn Leu 125 Gly Val1 Gly Ala Arg 205 Lys Trp Pro Arg Lys Phe Leu 110 Leu Met Gly Asn Asn 190 Arg Giu Tyr Thr Ile Arg.

Gly Thr Phe Gly Ser Phe 175 Val1 Asp Leu Leu Phe Pro pisn Gin Ile Phe Thr Arg 160 Tyr Asp Leu Asp Trp Leu Asn Ser Glu Phe Phe Phe Tyr Asp Giu 215 220 <210> 13 <211> 685 <212> DNA <213> tobacco <220> <221> CDS <222> AMENDED SHEET (Article 34) (IPEA/AU) PCT/AU98/00362 Received 19 May 1999 AA' 144866,SEQ. -I18//99 13 <400> 13 tg cat tgt geg tat tgc aac gat get tae aca atg ggt gac caa aag His Cys Ala Tyr Cys Asn Asp Ala Tyr Thr Met Gly Asp Gin Lys tta Leu ttg Leu ttt Phe eet Pro aat Asn ga t Asp eta Leu ttc Phe ace Thr gtg Val1 160 atg Met ea e His aaa Lys tte Phe eaa Gin tae Tyr get Al a get Ala eea Pro gaa Giu atg Met gga Gly att Ile 145 egg Arg ggt Gly gee Al a aga Arg tac Tyr 225 gt t Val1 tte Phe etg Leu atg Met eat His gte Val1 tat Tyr gag Giu 130 gaa Giu tgt Cys aat Asn aat Asn agg Arg 210 gae Asp eac His tae Tyr eea Pro tte Phe gte Val1 aaa Lys egt Arg 115 ect Pro aae Asn aeg Thr tte Phe gtg Val1 195 gat Asp gaa Glu eaa teg tgg ett tte tte ceg ttt eat aga tgg tae Gin gag Giu tat Tyr ga t Asp egt Arg act Thr 100 eaa Gin tac Tyr att Ile ga t Asp tae Tyr 180 ga e Asp etc Leu aa Ser aga Arg tgg Trp gte Vali aat Asn 85 aat Asn atg Met aga Arg cet Pro ttg Leu 165 tea Ser ege Arg aca Thr Trp ate Ile aac Asn gaa Glu 70 gga Gly gaa Giu ata Ile ttC Phe cat His 150 ggt Gly get Aia atg Met gae Asp Leu ttg Leu tgg Trp 55 ggt Gly ace Thr ata Ile act Thr gga Gly 135 aet Thr aat Asn ggt Gly tgg Trp aat Asn 215 Phe gge Gly 40 ga e Asp tet Ser ata Ile eag Gin aat Asn 120 tet Ser ceg Pro tgt Cys tta Leu aat Asn 200 ga t Asp Phe 25 tee Ser eat His tee Ser atc Ile atg Met 105 get Al a aaa Lys gtt Val1 gtg Val1 ga e Asp 185 gaa Glu tgg Trp Pro ctc Leu cca Pro cte Leu ga t Asp 90 ata Ilie eea Pro Ccc Pro cac His cea Pro 170 cca Pro t9g Trp tta Leu Phe ate Ile age Ser tac Tyr et t Leu act Thr tgC Cys aa t Asn att Ilie 155 tea Ser gt t Val1 aaa Lys aac Asn His gat Asp ggC Gly ga t Asp ggt Gly aac Asn ceg Pro ceg Pro 140 tgg Trp tae Tyr ttt Phe gea Ala t eg Ser 220 Arg ga t Asp atg Met gea Al a ttt Phe aae Asn ctg Leu 125 ggg Gly act Thr ggt Gly tac Tyr eta Leu 205 gag Giu Trp eca Pro cgt Arg aga Arg tte Phe tta Leu 110 ttg Leu cag Gln ggt Gly gag Glu age Ser 190 gga Gly ttC Phe Tyr act Thr t tg Leu cgt Arg ggt Giy att Ile ttc Phe gga Gly aet Thr ga t Asp 175 cac His ggg Gly ttt Phe 47 143 191 239 287 335 383 431 479 527 575 623 671 685 AMENDED SHEET (Article 34) (I]PEA/AU) PCT/AU98/00362 Received 19 May 1999 AA\2144866.SEQ 18/5/99 14- <210> 14 <211> 227 <212> PRT <213> tobacco <400> 14 His Cys Ala Tyr Cys Asn Asp Ala Tyr Thr Met Gly Asp Gin Lys Leu 1 Gin Tyr Ala Ala Pro Giu Met Gly Ile 145 Arg Gly Ala Arg Tyr 225 Val1 Phe Leu Met His Val1 Tyr Glu 130 Giu Cys Asn Asn Arg 210 Asp 5 His Gin Ser Tyr Giu Arg Pro Tyr Trp Phe Asp Val Val Arg Asn Lys Thr Asn 100 Arg Gin Met 115 Pro Tyr Arg Asn Ilie Pro Thr Asp Leu 165 Phe Tyr Ser 180 Val Asp Arg 195 Asp Leu Thr Glu Trp Ile Asn Giu 70 Gly Giu Ile Phe His 150 Gly Al a Met Leu Leu Trp 55 Gly Thr Ile Thr Gly 135 Thr Asn Gly Trp Phe Gly 40 Asp Ser Ile Gin Asn 120 Ser Pro Cys Leu Asn 200 Phe 25 Ser His Ser Ile Met 105 Ala Lys Val1 Va. 1 Asp 185 Giu 10 Pro Leu Pro Leu Asp 90 Ile Pro Pro His Pro 170 Pro Trp Phe Ile Ser Tyr 75 Leu Thr Cys Asn Ilie 155 Ser Val1 Lys His Asp Gly Asp Gly Asn Pro Pro 140 Trp Tyr Phe Ala Arg Asp Met Ala Phe Asn Leu 125 Gly Thr Gly Tyr Leu 205 Trp Tyr Pro Thr Arg Leu Arg Arg Phe Giy Leu Ile 110 Leu Phe Gin Gly Gly Thr Giu Asp 175 Ser His 190 Gly Gly Leu Phe Pro Asn Asp Leu Phe Thr Val1 160 Met His Lys Asp Asn Asp Trp Leu Asn Ser Giu Phe Phe Phe <210> <211> 670 <212> DNA <213> pineapple <220> <221> CDS AMEND)ED SIHEET (Article 34) (EPEA/AU) PCT/AU98/00362 Received 19 May 1999 A:QX 14486SEQ- 18/5/99 <222> -(668) <400> tg cat tgt gcg tac tge gac ggc gcg tat gac caa atc ggc ttc ccc 47 His Cys Ala Tyr Cys Asp Gly Ala Tyr Asp Gln Ile Gly Phe Pro 1 5 10 gat etc gag atc cag ate cac aac tcg tgg etc ttc ttt cct tgg cac Asp Leu Giu Ile Gin Ile His Asn Ser Trp Leu Phe Phe Pro Trp His 25 cgg ttc tac etc tac ttc aae gag cgc ata etc ggg aaa ett ate gge 143 Arg Phe Tyr Leu Tyr Phe Asn Giu Arg Ile Leu Gly Lys Leu Ile Gly 40 gac gac acg tte geg etg ect tte tgg aac tgg gac gcg ceg ggg gge 191 Asp Asp Thr Phe Ala Leu Pro Phe Trp Asn Trp Asp Ala Pro Gly Gly 55 atg cag ttc ceg tet ate tae aeg gae cet tea tee teg eta tat gae 239 Met Gin Phe Pro Ser Ile Tyr Thr Asp Pro Ser Ser Ser Leu Tyr Asp 70 aag ctg egt gat geg aag eac eag ceg ceg act ttg att gac etc gac 287 Lys Leu Arg Asp Ala Lys His Gin Pro Pro Thr Leu Ile Asp Leu Asp 85 90 tac aat ggc ace gat ect ace tte tee ect gaa gaa eag att aac cac 335 Tyr Asn Gly Thr Asp Pro Thr Phe Ser Pro Giu Giu Gin Ile Asn His 100 105 110 aae etc gee gte atg tac ega eag gtg ata tee agt gga aag aca eca 383 Asn Leu Aia Vai Met Tyr Arg Gin Val Ile Ser Ser Gly Lys Thr Pro 115 120 125 gag ctg ttt atg ggc tea geg tac cgc gee ggt gac cag ect gac ccc 431 Glu Leu Phe Met Giy Ser Ala Tyr Arg Ala Gly Asp Gin Pro Asp Pro 130 135 140 gge gca ggt tet gta gag eag aag ceg eac gge ccg gtg cat gtg tgg 479 Gly Ala Gly Ser Vai Glu Gin Lys Pro His Gly Pro Val His Val Trp 145 150 155 aca ggt gat cgc aac eag ccc aat ege gaa gac atg ggc aeg etc tac 527 Thr Gly Asp Arg Asn Gin Pro Asn Arg Giu Asp Met Gly Thr Leu Tyr 160 165 170 175 tcg geg geg tgg gac ccc gtt ttt ttc gca eac cac gge aac ate gac 575 Ser Ala Ala Trp Asp Pro Val Phe Phe Ala His His Gly Asn Ile Asp 180 185 190 ege atg tgg tac gtg tgg agg aac ett ggc gge aag cac cge aac ttc 623 Arg Met Trp Tyr Val Trp Arg Asn Leu Giy Gly Lys His Arg Asn Phe 195 200 205 ace gac ccc gac tgg etc aae geg tee ttc ctg ttc tac gac gaa aa 670 Thr Asp Pro Asp Trp Leu Asn Ala Ser Phe Leu Phe Tyr Asp Glu 210 215 220 <210> 16 AMENDED SHEET (Article 34) (1PEA/AU) PCT/AU98/00362 Received 19 May 1999 A\2 144866SEQ. -8/5/99 <211> 222 <212> PRT <213> pineapple 16- <400> 16 His Leu Phe Asp Gin Leu Asn Leu Leu Ala 145 Gly Ala Met Asp Cys Glu Tyr Thr Phe Arg Gly Ala Phe 130 Gly Asp Ala Trp, Pro 210 Ala Ile Leu Phe Pro Asp Thr Val1 115 Met Ser Arg Trp Tyr 195 Asp Tyr Gin Tyr Ala Ser Al a Asp 100 Met Gly Val1 Asn Asp 180 Val1 Cys Asp Gly Ala Tyr Asp Gin Ilie Gly Phe Pro Asp 10 Ile Phe Leu Ile Lys Pro Tyr Ser Giu Gin 165 Pro Trp His Asn Pro Tyr 70 His Thr Arg Ala Gin 150 Pro Val1 Arg Asn Glu Phe 55 Thr Gin Phe Gin Tyr 135 Lys Asn Phe Asn Ser Arg 40 Trp Asp Pro Ser Val1 120 Arg Pro Arg Phe Leu 200 Trp 25 Ile Asn Pro Pro Pro 105 Ile Al a His Glu Ala 185 Gly Leu Leu Trp Ser Thr 90 Glu Ser Gly Gly Asp 170 His Gly Phe Gly Asp Ser 75 Leu Glu Ser Asp Pro 155 Met His Lys Phe Lys Al a Ser Ile Gin Gly Gin 140 Val1 Gly Gly His Pro Leu Pro Leu Asp Ile Lys 125 Pro His Thr Asn Arg 205 Trp Ile Gly Tyr Leu Asn 110 Thr Asp Val1 Leu Ile 190 Asn His Gly Gly Asp Asp His Pro Pro Trp Tyr 175 Asp Phe Arg Asp Met Lys Tyr Asn Giu Gly Thr 160 Ser Arg Thr Trp Leu Asn Ala Ser Phe Leu Phe Tyr Asp Glu 215 220 <210> 17 <211> 1319 <212> DNA <213> pineapple <220> <221> CDS <222> (1)..(1053) <400> 17 ttg ccg ttt tgg aat tgg gac gcg ccg ggg ggc atg cag atc ccg gcc 48 Leu Pro Phe Trp Asn Trp Asp Ala Pro Gly Gly Met Gin Ile Pro Ala 1 5 10 AMENDED SHEET (Article 34) (IPEA/AU) PCT/AU98/00362 Received 19 May 1999 A:\21144866.SEQ 18/5/99 17ate tac gcc gae gct Ile Tyr Ala Asp Ala tcg tee ccg Ser Ser Pro aag Lys ceg Pro tac Tyr gcg Ala gag Glu caa Gin ec Pro tgg Trp 145 etc Leu gtc Val1 cag Gin aca Thr gtg Val1 225 cac His ace Thr ega Arg gcg Ala etc Leu ec Pro atc Ile 130 cgg Arg aac Asn aaa Lys ga c Asp cg Pro 210 ctg Leu eag Gin tte Phe cag Gin tac Tyr gtg Val1 aac Asn 115 tte Phe aaa Lys geg Al a gta Val1 gte Val1 195 ggg Gly ga t Asp ceg Pro ace Thr gtg Val1 ege Arg eeg Pro 100 gae Asp tte Phe etc Leu tee Ser aag Lys 180 ga e Asp gge Gly aag Lys eeg Pro eet Pro ata Ilie geg Ala eae His gaa Giu gee Ala ggg Gly tte Phe 165 gae Asp ate Ile get Ala eeg Pro aet Thr gag Giu tee Ser 70 gge Gly aae Asn gae Asp eae His gge Gly 150 et e Leu tge Cys eeg Pro geg Ala gtg Vali 230 ttg Leu eag Gin 55 gge Gly gac Asp aeg Thr atg Met eae His 135 a eg Thr tte Phe ttg Leu tgg Trp eet Pro 215 age Ser gag Glu gt e Val 40 cag Gin 9gg Sly geg Al a atg Met gge Gly 120 gge Gly eae His tae Tyr age Ser ate Ilie 200 tee Ser tet Ser gtg Val1 etc Leu gae Asp ate Ile aag Lys eca Pro eat His 105 a eg Thr aae Asn ege Arg gac Asp gee Ala 185 agt S er a eg Thr acg Thr t tg Lei.

tac gac a Tyr Asp I etc gac t Leu Asp gee eac Ala His I aeg eeg Thr Pro 75 gac ceg Asp Pro 90 ttg tgg Leu Trp tte tac Phe Tyr gte gao Val Asp gat ttc Asp Phe 155 gag aac Glu Asn 170 gac gcg Asp Ala geg aag *Ala Lys aea gag *Thr Glu gtg gcg *Val Ala 235 gtg gtg Val Val 250 bag :ac Pyr iae 3ag ,lu acc 1'hr gg Al a ege Arg 140 ace Thr geg Al a etg Leu ceg Pro get Ala 220 agg Arc gac

GIL

ctg c Leu A aae g Asn C etc a Leu 'I ttgt Leu gea Ala gge Gly geg Ala 125 atg Met gae Asp eag Gin Arg aeg Thr 205 *ata Slie ceg Pro gga 1Gly 'ge ~rg ~ge rhr :tt ?he ,ly 3ae %sp 1.10 gg kl a tgg Trp cee Pro ete Leu tae Tyr 190 ceg Pro ttt Phe aag Lys atc Ile aat Asn aee Thr ate Ile atg Met act Thr ccc Pro egg Arg tao Tyr ga e Asp gte Val 175 a eg Thr aag Lys ocs Prc aec Thi ga~ G1i gog Al a gao Asp atg Met gge Giy eta Leu aae Asn gao Asp gtg Val1 tgg Trp 160 oge Arg tae Tyr aaa Lys gtg Val ggg Gly 240 1 etg a Leu 96 144 192 240 288 336 384 432 480 528 576 624 672 720 768 agg agt aet ggg gag gag Arg Ser Thr Giy Giu Giu 245 AMIENDED SHEET (Article 34) (I]PEA/AU) PCT/AU98/00362 Received 19 May 1999 A:\'-)144866.SEQ 18/5/99 18gac aag gac gtg gccc Asp Lys Asp Val Ala 260 aae gaa ggg gtg ggg Asn Giu Gly Val Gly 275 cag gtg ccg cac aag Gin Val Pro His Lys 290 aaa aeg aeg qtc agg Lys Thr Thr Leu Arg 305 gcc gag gac gac gag Ala Glu Asp Asp Glu 325 gag ggg ttg gte aag Glu Gly Leu Val Lys 340 tgatcagcag caaattaacl aagagaataa atgcgtatg ctgaatgaga gttgeatgc gegtggatac gtataacgt aaaaaaaaaa aaaaaaaaa <210> 18 <211> 351 <212> PRT <213> pineapple <400> 18 Leu Pro Phe Trp Asn 1 5 Ile Tyr Ala Asp Ala Lys His Gin Pro Pro Pro Thr Phe Thr Pro Tyr Arg Gin Val Ile Ala Ala Tyr Arg Ala uLeu Val Pro His 100 jtg aag ttc g Jal Lys Phe A 2 7cg gag geg a ?ro Glu Ala S 280 cac aag aag g -us Lys Lys G 295 cte ggg ata a Ueu Gly Ile 1 310 age gtg etcc Ser Val Leu N 9tt ggt ggg c Val Gly GlyI t atacatgaaa t, aatctgeeee tgcgcgeagc egtatgcatg a aaaaaa ac gtg tat .sp Val Tyr 65 .gc gag tte er Glu Phe ~gg aag aag 1y Lys Lys Leg gac etg 'hr Asp Leu 315 ~tc acg etc Tal Thr Leu 330 :ta agg atc jeu Arg Ie 345 gtaaaaaaaa atttgtcaet cataatgcet tataaggaat ata aac gcg Ie Asn Ala 270 gca ggg agc Ala Gly Ser 285 gag aag geg Glu Lys Ala 300 ctc gag gac Leu Glu Asp gtg ceg agS Val Pro Arc gat ttc tec Asp Phe Sex ttgeatttae tttaatttct ggtatagtgt aatgatgagt ccg gac 816 Pro Asp ttc gte 864 Phe Val agg att 912 Arg Ile ate ggc 960 SIle Giy 320 ata gge 1008 f le Gly 335 *aag 1053 *Lys etaeetatag 1113 egagegtgtt 1173 agtagtttag 1233 ttaetatgea 1293 1319 Trp Asp Ala Pro Gly Gly Met Gln 10 Ser Ser Pro Leu Tyr Asp Lys Leu 25 Thr Leu Val Asp Leu Asp Tyr Asn 40 Glu Gin Gin Ile Ala His Asn Leu 55 Ser Gly Gly Lys Thr Pro Glu Leu 70 75 Gly Asp Ala Pro Asp Pro Gly Ala 90 Asn Thr Met His Leu Trp Thr Gly 105 AMENDED SHEET (Airticle 34) ([PEA/AU) Ile Arg Gly Thr Phe Gly Asp 110 Pro Ala Asn Ala Thr Asp Ile Met Met Gly Thr Leu Pro Asri PCT/AU98/00362 Received 19 May 1999 A:\'214.4866.SEQ. -8/5/99 19 Gin Pro Asn Asp Giu Asp Met 115 Pro Trp 145 Leu Val1 Gin Thr Val1 225 Arg Asp Asn Gin Lys 305 Ala Giu Ilie 130 Arg Asn Lys Asp Pro 210 Leu Ser Lys Giu Val1 290 Thr Glu Gly Phe Lys Ala Val1 Val1 195 Gly Asp Thr Asp Gly 275 Pro Thr Asp Leu Phe Leu Ser Lys 180 Asp Gly Lys Gly Val1 260 Val1 His Leu Asp Ala His Gly Gly 150 Phe Leu 165 Asp Cys Ile Pro Ala Ala Pro Val 230 Glu Glu 245 Ala Val Gly Pro Lys His Arg Leu 310 Glu Ser 325 His 135 Thr Phe Leu Trp Pro 215 Ser Glu Lys Glu Lys 295 Gly Val1 Gly Thr 120 Gly Asn His Arg Tyr Asp Ser Ala 185 Ile Ser 200 Ser Thr Ser Thr Val Leu Phe Asp 265 Ala Ser 280 Lys Gly Ile Thr Leu Val Phe Tyr Ala Ala Ala 125 Val Asp Arg Met Trp 140 Asp Phe Thr Asp Pro 155 Glu Asn Ala Gin Leu 170 Asp Ala Leu Arg Tyr 190 Ala Lys Pro Thr Pro 205 Thr Glu Ala Ile Phe 220 Val Ala Arg Pro Lys 235 Val Val Glu Gly Ilie 250 Val Tyr Ile Asn Ala 270 Glu Phe Ala Gly Ser 285 Lys Lys Glu Lys Ala 300 Asp Leu Leu Giu Asp 315 Thr Leu Val Pro Arc 330 Arg Asp Tyr Asp Val1 175 Thr Lys Pro Thr Glu 255 Pro Phe Arg Ile Val1 Trp 160 Arg Tyr Lys Val1 Gly 240 Leu Asp Val1 Ile Gly 320 Ile Gly 335 Val Lys Val Gly Gly Leu Arg Ile Asp Phe Ser Lys 350 <210> 19 <211> 2181 <212> DNA <213> pineapple <220> <221> CDS <222> (2)..(1858) <400> 19 c ggt atc gat aag ctt gat cca gtg cct ggt tta ggt gta ttc act atg 49 Gly Ile Asp Lys Leu Asp Pro Val Pro Gly Leu Gly Val Phe Thr Met 1 5 10 AMENDED SHEET (Article 34) (IPEA/AU) PCT/AU98/003 62 Received 19 May 1999 44866.SEQ~ -8/5/99 gcc acc ctc tct aaa cta gct Ala Thr Leu Ser Lys Leu Ala ccg Pro acc Thr cat His gca Ala gcc Ala ctg Leu t cc Ser gac Asp 145 gcc Ala gag Glu caa Gln ggc Gly cct Pro 225 ctt Leu ctc Leu ttc Phe gca Ala t cg Ser acc Thr gct Ala gcc Al a 130 ttc Phe ca c His Ct C Leu gcg Ala ttc Phe 210 tgg Trp atc Ile cct Pro ctc Leu aat Asn ccc Pro act Thr ccc Pro 115 cga Arg aag Lys ttg Leu a tg Met aaa Lys 195 ccc Pro cac His ggc Gly cct Pro t cc Ser cta Leu gcc Ala ggg Gly 100 gac Asp ccg Pro ctc Leu gtt Val1 agg Arg 180 gtg Val1 gat Asp cgg Arg gac Asp ttg Leu cct Pro agg Arg gcg Al a ct c Leu ctc Leu aca Thr ccc Pro gac Asp 165 gcc Ala cac His ctc Leu ttc Phe gac Asp 245 cat His gta Val agc Ser 70 acc Thr ggc Gly t ca Ser gt t Val1 ccg Pro 150 gcc Ala ctg Leu tgt Cys gag Glu tac Tyr 230 acg Thr 3ct 55 ~aac PAsn tac T'yr ctc Leu act Thr tgc Cys 135 cga Arg ga c Asp ccg Pro gcg Ala atc Ile 215 ctc Lel2 ttc Phe tcc caa Ser Gln 25 cct tct Pro Ser 40 gtc cca Val Pro aag aga Lys Arg tcc tgg Ser Trp aac cgt Asn Arg 105 tgt 9g9g Cys Gly 120 tgc ccg Cys Pro tct gct Ser Ala tac ctg Tyr Leu gcc gac Ala Asp 185 tat tgc Tyr Cys 200 cag atc Gln Ile tac tcc Tyr Ser gcg ctg Ala Leu mca ata aca cct cca ctc tcc Pro Ile Thr Pro Pro Leu Ser :tc Leu aac Pksn atg M4et gcc Al1a 90 cga PArg ccg Pro cca Pro ccg Pro gcc Ala 170 ga c Asp gac Asp ca c His aac Asn cct pro 250 acc Thr ca c His ccg Pro 75 Ctc Leu gcg Al a cct, Pro tac Tyr ctt Leu 155 .aag Lys ccg Pro ggc Gly.

aa c Asr *gaS 23E *tt( Ph~ aaa E Lys cccc Pro aca Thr ggc Gly gcc Ala gcc Ala caa Gln 140 cgc Arg tat Tyr *cgc *Arg gcg Ala tcg 1Ser 220 Scgc 1Arg tgg STrp igc ~er ~tc Ia1 agc Ser 399 ,ly gcc Al1a gac A~sp 125 tcc Ser gtc Val1 aag Lys aac Asn tat Tyr 205 tgS Trj atz Il~ aac Asi ttc a Phe 9) ata E Ile IZ ctg Leu ctt Leu gcc Ala 110 ctc Leu acc Thr cgg Arg aag Lys ttc Phe 190 gac Asp ctc Leu ictc Leu Stgg 'i Trp ~cc 'hr ~ga.

~rg :gg krg :ac ['yr cct Pro cct Pro atc Ile cct Pro gcg Ala 175 gta Val1 caa Glri ttc Phe 999 Gl gac Asy 255 acc Thr tct Ser gcc Ala ggt Gly atc Ile gcc Al a atc Ile gcg Ala 160 gt c Val1 cag Gln atc Ile ttt Phe aaa Lys 240 gcg Ala 97 145 193 241 289 337 385 433 481 529 577 625 673 721 769 817 ccg ggg ggC atg cag ttc ccg tct atc tac aca gac cct tca tcc tcg AMENDED SHEET (Article 34) (IPEA/ALT) v PCT/AU9 8/0 0362 Received 19 May 1999 AA2144866.SEQ 8/5/99 -21 Pro Gly Gly Met Gin Phe Pro Ser 260 cta Leu gac Asp att Ile 305 aag Lys cct Pro cat His acg Thr aac Asn 385 cgc Arg ga t Asp gcc Aia aaa Lys gta Val1 465 gag Giu tat Tyr ctc Leu 290 aac 1Asn acg Thr ga c Asp gtg Val1 etc Leu 370 atc Ilie aac Asn gag Giu ga c Asp gca Aia 450 t cg Ser act Thr ga c Asp 275 ga c Asp ca c His cca Pro ccc Pro tgg Trp 355 tac Tyr ga c Asp ttc Phe aat Asn gca Aia 435 aag Lys a cg Thr aca Thr aag Lys tac Tyr aac Asn gag Giu Giy 340 aca Thr t cg Ser cgc Arg acc Thr gcg Al a 420 atg Met ccg Pro ctg Leu ttt Phe ctg Leu2 aat Asn etc Leu2 ctg Leu 325 gca Ala ggt Giy gcg Aia.

atg Met gac Asp 405 cag Gin Arg aeg Thr aag Lys ceg Pro 485 :gt krg ,iy 3c c ki a 310 ttt Phe 9gC Giy ga t Asp geg Al a tgg Trp 390 ccc Pro etc Leu tac Tyr eca Pro gca Aila 470 gtg Val1 gat Asp ace Thr2 295 gte Vail atg Met tet Ser ege Arg.

tgg Trp 375 tac Tyr gac Asp gte Vali aca Thr aag *Lys 455 *aca *Thr gtg *Val 3eg 280 gat ksp atg Mlet Giy gta Vali aae Asn 360 gac Asp gtg Vali tgg Trp egt Arg tac Tyr 440 agc Ser cca Prc etc Le.

Ile 265 aag Lys I ectE Pro tac Tyr tea Ser2 gag Giu 345 eag Gin ccc Pro tgg Trp etc Leu gt t Vali 425 cag *Gin gee *Aia agg Arg ;gat iAsp 7 r ~ac *is ~cc Chr :ga krg 330 cag Gln ccc Pro gte Vali agg Arg aa e Asn 410 aaa Lys ga t Asr ctz Let.

gg9 G1~ aa.

Ly 49' Thr I cag Gin ttc Phe cag Gin 315 tac Tyr aag Lys aat Asn tte Phe aae Asn 395 gcg *Aia *gta.

Val gta Val cag 1Gin 3 acg {Thr 475 3 ceg sPro 0 ~sp ecg ?ro :cc 3er 300 jtg Vali :gc krg ceg Pro ege Arg tte Phe 380 ctt Leu tcc Ser aaa Lys gag Giu aaS Lys 460 aec Thi gt~ Va] Pro ceg Pro 285 cet Pro ata Ile gcc Aila eac His gaa Giu 365 gca Ala ggc Gi y ttc Phe gac Asp atc IiE 445 Satz Il ;act ;agi L Se Ser 270 act Thr gaa Giu tee Ser ggt Giy ggc Giy 350 ga e Asp ca c His ggC Giy ctg Leu tge ~Cys 430 cg Prc Saac SLys taec r Th2 tgc~ r Al~ Ser S ttg a Leu I gaa c Giu C agt Ser C gac c Asp C 335 ceg Pro atg Met cac His aag Lys tte Phe 415 tta Leu tgg Trp age Ser aca 7Thr i aca a Thr 495 er *tt ie :ag ;in ;ga ily :ag ln 3tg la i Mly Giy cac His 400 tat Tyr gag Giu etc Leu aag Lys gea Aila 480 gtg Val1 865 913 961 1009 1057 1105 1153 1201 1249 1297 1345 1393 1441 1489 1537 get aga ecg aag gee agg agg agt Aia Arg Pro Lys Aia Arg Arg Ser ggg aag gag Giy Lys Giu aag gaa gaa gag gag Lys Giu Giu Giu Giu AMENDED SHEET (Article 34) (IPEA/AU) PCT/AU98/00362 A:\244W.EQ 13//99Received 19 May 1999 22 505 gag Glu gag gtg ttg Giu Val Leu 515 aag ttt gat Lys Phe Asp gtg gag gga Val Glu Gly atc Ile 520 tcg Ser ttg gag aag Leu Glu Lys 510 gtg ttC gtg Val Phe Val gtg ggg ccg Val Gly Pro gtg tat ata Val Tyr Ile 530 gag gcg Glu Ala aac Asn 535 ggg Gly ccg gag cac Pro Glu His gaa Glu 540 agt gag ttc Ser Glu Phe 545 aag Lys goa Ala 550 ggg Gly ago ttc gto Ser Phe Val gtg cca cac aag Val Pro His Lys cac His 560 1585 1633 1681 1729 1777 1825 aag gog aag Lys Ala Lys aag Lys 565 acg Thr aag gag atg Lys Glu Met gc Ala 570 gao Asp atg aac aca Met Asn Thr agg ctt Arg Leu 575 aag oto ggg Lys Leu Gly gag ago gtg Giu Ser Val 595 aag gtt gga Lys Val Gly 610 gao ctg otc Asp Leu Leu gag Giu 585 000 Pro atc ggc got Ilie Gly Ala gag gao gac Giu Asp Asp 590 gga atg gtg Gly Met Val ato acg oto Ile Thr Leu agg ago ggc aag Arg Ser Gly Lys ggg ota agg att Gly Leu Arg Ile 615 ttc too aag Phe Ser Lys tgatgagoat attgtgaaga 1878 gaaaatttgo ttttttatto ttgttgtagt aattattaoa atattoaaoa ga t atttaoogoo ttoaagogta ogatatgtgg ototgagaat ttgtttoota otatagaato ttoagaataa ggtatgtttg aaattagaga tacatctttt gaaaaattgo gagttgogtg gatoagggat, gtttattatg tttggaagaa gtatatgtoo oatgoaogoa aatgatgtga oaagttgott aaaaaaaaaa oattattgtt tgoagooatg a ott tgaat t ggtgtaa tag aaaaaaaatc 1938 1998 2058 2118 2178 2181 <210> <211> 619 <212> PRT <213> pineapple <400> Gly Ilie Asp Lys Leu Asp Pro 1 5 Ala Thr Leu Ser Lys Leu Ala Pro Leu Pro Pro Leu His Ala Thr Phe Leu Ser Pro Val Gly 55 Val Pro Gly 10 Pro Leu Gly Val Phe Thr Met Ser Pro Gin 25 Ser 11ie Thr Pro Leu Thr Lys Pro Leu Ser Phe Thr Thr Ile Arg Ser Val Pro Asn His Pro AMIENDED SHEET (Article 34) (EPEA/AU) PCT/AU98/003 62 Received 19 May 1999 A:\1144866.SEQ- 18/5/99 23 His Ala Ala Leu Ser Asp 145 Ala Glu Gin Gly Pro 225 Leu Pro Leu Asp Ilie 305 Lys Pro His Thr Ala Ser Thr Ala Ala 130 Phe His Leu Ala Phe 210 Trp Ilie Gly Tyr Leu 290 Asn Thr Asp Val1 Leu 370 Asn Pro Thr Pro 115 Arg Lys Leu Met Lys 195 Pro His Gly Gly Asp 275 Asp His Pro Pro Trp 355 Tyr Leu Ala Gly 100 Asp Pro Leu Val1 Arg 180 Val1 Asp Arg Asp Met 260 Lys Tyr Asn Glu Gly 340 Thr Ser Arg Ala Leu Leu Thr Pro Asp 165 Al a His Leu Phe Asp 245 Gin Leu Asn Leu Leu 325 Ala Gly Ala Ser 70 Thr Gly Ser Val1 Pro 150 Ala Leu Cys Glu Tyr 230 Thr Phe Arg Gly Ala 310 Phe Gly Asp Al a Asn Tyr Leu Thr Cys' 135 Arg Asp Pro Ala Ilie 215 Leu Phe Pro Asp Thr 295 Val1 Met Ser Arg Trp 375 Ser ksn Cys 120 Cys Ser Tyr Al a Tyr 200 Gin Tyr Ala Ser Ala 280 Asp Met Gly Val1 Asn 360 Asp A~rg Trp Arg 105 Gly Pro Ala Leu Asp 185 Cys Ile Ser Leu Ile 265 Lys Pro Tyr Ser Giu 345 Gin Pro Met Al a 90 A~rg Pro Pro Pro Al a 170 Asp Asp His Asn Pro 250 Tyr His Thr Arg Ala 330 Gir Pro Val Pro 75 Leu Ala Pro Tyr Leu 155 Lys Pro Gly Asn Giu 235 Phe Thr Gin Phe 315 Ly~ Asi *PhE Thr E Gly C Ala1 Ala Gin 140 Arg Tyr Arg Ala Ser 220 Arg Trp Asp Pro Ser 300 Val Arg SPro iArg E! Phe 380 ,er fly kla ksp 12 Ser Val1 Lys Asn Tyr 205 Ti-p Ile Asn Pro Pro 285 Pro Ile Ala His Git 365 Ala Iieu ,eu kla 110 Leu Thr Arg Tyr Pro Pro Ile

C

Arg Pro Lys Ala 175 Phe Val 190 Asp Gin Leu Phe Leu Gly Ti-p Asp 255 Ser Ser 270 Thr Leu Giu Giu Ser Ser Gly Asp 335 Gly Pro 350 Asp Met His His ~la ;iy Ile kla Ile Dd a 160 Val1 Gin Ilie Phe Lys 240 Ala Ser Ile Gin Gly 320 Gin Vali Gly Gly Asn Ilie Asp Arg Met Ti-p Tyr Val Ti-p Arg Asn Leu Gly Gly Lys His AMENDED SHEET (Article 34) (IPEA/AU) PCT/AU98/00362 Received 19 May 1999 AA2144W6.SEQ. -8/5/99 24 385 Arg Asp Ala Lys Val1 465 Glu Ala Glu Lys Glu 545 Lys Lys Glu Lys Asn Phe Thr Glu Asp Ala 450 Ser Thr Arg Val1 Phe 530 Ala Lys Leu Ser Val1 610 Asn Ala 435 Lys Thr Thr Pro Leu 515 Asp Ser Ala Gly Val1 595 Gly Ala 420 Met Pro Leu Phe Lys 500 Val1 Val1 Giu Lys Ile 580 Leu Asp 405 Gin Arg Thr Lys Pro 485 Al a Val1 Tyr Phe Lys 565 Thr Ile Pro Asp Trp Leu Asn Ala Ser Phe Leu Phe Tyr Leu Tyr.

Pro Ala 470.

Val1 Arg Giu Ile Ala 550 Gly Asp Thr Val1 Thr Lys 455 Thr Val1 Arg Gly Asn 535 Gly Lys Leu Leu Arg Tyr 440 Ser Pro Leu S er Ile 520 Ser Ser Glu Leu Val1 600 Val1 425 Gin Ala Arg Asp Gly 505 Giu Pro Phe Met Giu 585 Pro 410 Lys Asp Leu Gly Lys 490 Lys Leu Glu Val1 Ala 570 Asp Arg Val1 Val1 Gin Thr 475 Pro Giu.

Glu His His 555 Arg Ile Lys Giu Lys 460 Thr Val1 Lys Lys Giu 540 Val1 Met Gly Asp Ile 445 Ile Thr Ser Giu Asp 525 Gly Pro Asn Ala Cys 430 Pro Lys Thr Ala Giu 510 Val1 Val1 His Thr Giu 590 415 Leu Trp Ser Thr Thr 495 Glu Phe Gly Lys Arg 575 Asp Giu Leu Lys Ala 480 Val1 Giu Val1 Pro His 560 Leu Asp Ser Gly Lys 605 Gly Met Val Gly Leu Arg Ilie Asp Phe Ser Lys 615 <210> 21 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 21 gcctgcagtt ytcrtcrtag aa <210> 22 <211> 28 <212> DNA <213> Artificial Sequence ANMNDED SHEET (Article 34) (IPEA/AU) PCT/AU98/00362 S .,Received 19 May 1999 A:\2144866.SEQ- 18/5/99 <220> <223> Description of Artificial Sequence: primer <400> 22 gcgaattcga tccnacntty gckttncc 28 <210> 23 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 23 gcgaattctn caytgygcnt aytg 24 <210> 24 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 24 gcgaattctt nccntwytgg aaytggg 27 <210> <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> gcctgcagcc acatnckrtc nacrtt 26 <210> 26 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 26 gcctgcagtt ytcrtcrtag aa 22 <210> 27 <211> <212> DNA 1kA^^/ <213> Artificial Sequence AMENDED SHEET (Article 34) (IPEA/AU) '9 PCT/AU98/00362 Received 19 May 1999 A:\2144866.SEQ- 18/5/99 -26- <220> <223> Description of Artificial Sequence: primer <400> 27 gttgctcttc ttaggctcgg cttac <210> 28 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 28 gactcgagtc gacatcga 18 <210> 29 <211> <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 29 atatcacctg tcggtacatg acggc <210> <211> <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> gtgccattgt agtcgaggtc aatca <210> 31 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 31 ccagtgcctg gtttaggtgt attcac 26 <210> 32 <211> <212> DNA <213> Artificial Sequence <220> AMENDED SHEET (Article 34) (IPEA/AU) A -PCT/AU98/00362 AA A\144366.SEQ 18/5/99 Received 19 May 1999 27 <223> Description of Artificial Sequence: primer <400> 32 gactcgagtc gacatcgatt tttttttttt ttttt <210> 33 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 33 gactcgagtc gacatcga 18 AMENDED SHEET (Article 34) (IPEA/AU)

Claims

1. An isolated nucleic acid molecule that comprises a nucleotide sequence which encodes or is complementary to a nucleotide sequence which encodes a PPO polypeptide of banana, tobacco or pineapple having an amino acid sequence set forth in any one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18 or 20 or comprising the copper-binding site of any one of said amino acid sequences.

2. An isolated nucleic acid molecule that encodes a PPO polypeptide of banana, tobacco or pineapple wherein said nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of: a nucleotide sequence set forth in any one of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17 or 19; (ii) a fragment of comprising a nucleotide sequence that encodes the copper- binding site of a PPO polypeptide; (iii) a degenerate nucleotide sequence of or and (iv) a nucleotide sequence that is complementary to or (ii) or (iii).

3. The isolated nucleotide sequence according to claims 1 or 2 wherein the copper- binding site is the CuA binding site of said banana, tobacco or pineapple PPO polypeptide.

4. The isolated nucleotide sequence according to claims 1 or 2 wherein the copper- binding site is the CuB binding site of said banana, tobacco or pineapple PPO polypeptide.

5. The isolated nucleic acid molecule according to any one of claims 1 to 4, wherein the PPO polypeptide of banana is at least expressed in banana peel.

6. The isolated nucleic acid molecule according to any one of claims 1 to 4, wherein the PPO polypeptide of tobacco is at least expressed in tobacco leaves. AMENDED SHEET (Article 34) (IPEA/AU) PCT/AU98/00362 P:\OPER\MRO\98-00362. PCT 18/5/99 Received 19 May 1999 -24-

7. The isolated nucleic acid molecule according to any one of claims 1 to 4, wherein the PPO polypeptide of pineapple is at least expressed in pineapple fruit.

8. A recombinant vector comprising the isolated nucleic acid molecule according to any one of claims 1 to 7 inserted within a vector molecule.

9. The recombinant vector according to claim 8 wherein the vector is a plasmid expression vector.

10. The recombinant vector according to claim 9 wherein the plasmid expression vector is Bluescript SK+.

11. The recombinant vector according to claim 8 wherein the vector is a binary vector suitable for introducing into a plant cell, tissue or organ.

12. The recombinant vector according to any one of claims 8 to 11 wherein the vector is capable of being replicated and the PPO-encoding nucleotide sequence is capable of being transcribed and translated in a unicellular organism or in a plant.

13. A method of increasing the level of banana, pineapple or tobacco PPO activity in a plant or a cell, tissue or organ thereof, said method comprising: introducing a nucleotide sequence to said plant or a cell, tissue or organ thereof which sequence encodes a PPO polypeptide of banana, tobacco or pineapple having an amino acid sequence set forth in any one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18 or 20 or an enzymatically-active PPO polypeptide comprising the copper- binding site of any one of said amino acid sequences; and (ii) expressing said nucleotide sequence to produce an enzymatically-active PPO polypeptide.

14. A method of increasing the level of banana, pineapple or tobacco PPO activity in a AMENDED SHEET (Article 34) (IPEA/AU) PCT/AU98/00362 Received 19 May 1999 P:\OPER\MRO\98-00362.PCT 18/5/99 plant or a cell, tissue or organ thereof, said method comprising: introducing a nucleic acid molecule to said plant or a cell, tissue or organ thereof which nucleic acid molecule comprises the nucleotide sequence set forth in any one of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17 or 19 or a degenerate nucleotide sequence thereof; and (ii) expressing said nucleic acid molecule to produce an enzymatically-active PPO polypeptide.

A method of decreasing the level of PPO activity in a plant or a cell, tissue or organ thereof, said method comprising introducing a nucleic acid molecule to said plant or a cell, tissue or organ thereof which comprises a nucleotide sequence selected from the group consisting of: a nucleotide sequence which encodes a PPO polypeptide of banana, tobacco or pineapple having an amino acid sequence set forth in any one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18 or 20 or the copper-binding site of any one of said amino acid sequences; (ii) a nucleotide sequence set forth in any one of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17 or 19; (iii) a fragment of (ii) comprising a nucleotide sequence that encodes the copper- binding site of a PPO polypeptide; and (iv) a nucleotide sequence that is complementary to or (ii) or (iii).

16. The method according to claim 15 further comprising expressing the introduced nucleic acid molecule to produce sense or antisense RNA therefrom.

17. The method according to claims 15 or 16 wherein the PPO activity is decreased by co- suppression of the endogenous PPO-encoding genes that would otherwise be expressed in the plant or a cell, tissue or organ thereof. AL, O

18. The method according to claims 15 or 16, wherein the PPO activity is decreased by AMENDED SHEET (Article 34) (IPEA/AU) PCT/AU98/00362 Received 19 May 1999 P:\OPER\MRO\98-00362.PCT- 18/5/99 -26- the expression of antisense RNA that is complementary to RNA encoded by an endogenous PPO-encoding gene that would otherwise be expressed in the plant or a cell, tissue or organ thereof.

19. The method according to any one of claims 13 to 18 wherein the nucleic acid molecule is introduced into the plant or a cell, tissue or organ thereof by means of Agrobacterium- mediated transformation.

The method according to any one of claims 13 to 18 wherein the nucleic acid molecule is introduced into the plant or a cell, tissue or organ thereof by means of microparticle bombardment using a nucleic acid-coated microprojectile.

21. A transformed plant comprising the isolated nucleic acid molecule according to any one of claims 1 to 7 or a plant part, progeny or propagule thereof that also comprises said nucleic acid molecule.

22. A transformed plant comprising the recombinant vector according to any one of claims 8, 9, 11 or 12 or a plant part, progeny or propagule thereof that also comprises said nucleic acid molecule. AMENDED SHEET (Article 34) (IPEA/AU)