WO2000017372A2 - Pineapple mealybug-associated wilt virus proteins and their uses - Google Patents

Pineapple mealybug-associated wilt virus proteins and their uses Download PDF

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WO2000017372A2
WO2000017372A2 PCT/US1999/022152 US9922152W WO0017372A2 WO 2000017372 A2 WO2000017372 A2 WO 2000017372A2 US 9922152 W US9922152 W US 9922152W WO 0017372 A2 WO0017372 A2 WO 0017372A2
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protein
polypeptide
dna molecule
seq
val
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WO2000017372A3 (en
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John S. Hu
Alexander V. Karasev
William O. Dawson
Michael Melzer
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University Of Hawaii
University Of Florida
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8283—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for virus resistance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/00022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention relates to pineapple mealybug-associated wilt virus ("PMWaV”) proteins, DNA molecules encoding these proteins, and their uses.
  • PMWaV pineapple mealybug-associated wilt virus
  • Mealybug wilt of pineapple is a disease of pineapple that is associated with the presence of mealybugs on virus-infected pineapple plants. It is a continuing problem limiting profitable pineapple production in many pineapple growing areas worldwide. MWP is one of the most important factors limiting successful commercial production of pineapple in Hawaii. The yearly value of the Hawaiian pineapple industry is estimated at $230 million with a production of
  • Diazinon ® insecticide Unfortunately, such control is difficult and inefficient, because the mealybugs are covered with a waxy coating and tend to feed near the roots where pesticide coverage is poor. In addition, the prolific reproduction rate of mealybugs raises the threat of pesticide resistance developing in mealybugs. Ants, which contribute considerably to the mealybug problem, are currently controlled with the granular bait-insecticide, hydramethylnon (trade name Amdro ® , American Cynamid Co., Wayne, NJ), one of the last remaining ant insecticides available to pineapple growers. Nearly all other insecticides that are effective against ants have been banned over the last decade by the U.S. Environmental Protection Agency (EPA).
  • EPA U.S. Environmental Protection Agency
  • the second approach is to transmit PMWaV to healthy pineapple using mealybugs to reproduce the disease.
  • Three experiments have been conducted. First, a MWP symptom induction experiment was conducted using groups of potted
  • PMWaV-free plants and PMWaV-infected plants were received 20-100 mealybugs/plant at monthly intervals or were kept mealybug-free for the duration of the experiment. After three months, only plants in the PMWaV-infected group exposed to mealybugs expressed typical symptoms of MWP; plants in the other three groups remained symptomless. Second, a radomized complete block design was used to test whether MWP symptoms could be induced under field conditions. Plots consisted of PMWaV-free plants kept mealybug free, PMWaV-free plants receiving monthly applications of mealybugs, PMWaV-infected plants kept mealybug free and PMWaV-infected plants receiving monthly applications of mealybugs. Each plot was replicated four times and contained 120 plants. Symptoms developed only on
  • PMWaV-infected plants in the plots received mealybug applications. Plants in all other treatments remained healthy looking. Third, cultivar susceptibility to mealybug wilt symptom development in the presence of PMWaV and mealybugs was tested. Six commercially grown Ananus comosus Smooth Cayenne cultivars from Hawaii were tested. All were susceptible to mealybug wilt when both PMWaV and mealybugs were present. PMWaV-free plants exposed to mealybugs shows signs of spotting caused by mealybug feeding but did not develop symptoms of mealybug wilt. PMWaV-free and -infected plants kept mealybug free did not develop MWP symptoms.
  • the present invention is directed to overcoming these deficiencies in the art.
  • the present invention relates to an isolated protein or polypeptide of a pineapple mealybug wilt virus.
  • the encoding RNA and DNA molecules in either isolated form or incorporated in an expression system, a host cell, or a transgenic pineapple cultivar are also disclosed.
  • Another aspect of the present invention relates to a method of imparting pineapple mealybug wilt virus resistance to pineapple cultivars by transforming them with a DNA molecule encoding the protein or polypeptide corresponding to a protein or polypeptide of a pineapple mealybug wilt virus.
  • PMWaV resistant transgenic variants of the current commercial pineapple cultivars allows for more complete control of the virus, while retaining the varietal characteristics of specific cultivars. Furthermore, these variants permit control of pineapple mealybug wilt virus transmitted by mealybug vectors (Sether et al., "Transmission of Pineapple Mealybug Wilt Associated Virus by Two Species of Mealybug (Dysmicoccus spp.),” Phvtopath. 88(11):1224-30 (1998), which is hereby incorporated by reference). With respect to the latter mode of transmission, the present invention circumvents increased restriction of pesticide use which has made chemical control of insect infestation increasingly difficult.
  • the present invention relates to isolated DNA molecules encoding for the proteins or polypeptides of a PMWaV.
  • a substantial portion of the genome has been sequenced for Types I and II of pineapple mealybug virus. Within each genome are a plurality of open reading frames ("ORFs"), each containing DNA molecules in accordance with the present invention.
  • ORFs open reading frames
  • the complete nucleotide sequence for pineapple mealybug virus Type I i.e. PMWaV-1) is as follows (SEQ. ID. No. 1):
  • Type 2 (i.e.PMWaV-2) is as follows (SEQ. ID. No. 2):
  • One DNA molecule of the present invention includes nucleotides 1 to 1799 of SEQ. ID. No. 1 and is believed to code for a helicase. This DNA molecule comprises the nucleotide sequence corresponding to SEQ. ID. No. 3 as follows:
  • This protein or polypeptide encoded by the nucleotide sequence of SEQ. ID. No. 3 has an amino acid sequence corresponding to SEQ. ID. No. 4 as follows: Glu He Gly Ser Pro Ser Gly Arg Ser Arg Cys Arg Phe Glu Gly Val 1 5 10 15
  • Another such DNA molecule includes nucleotides 1783 to 3350 of SEQ. ID. No. 1 and codes for a polymerase.
  • This DNA molecule comprises the nucleotide sequence corresponding to SEQ. ID. No. 5 as follows:
  • the protein encoded by the nucleotide sequence of SEQ. ID. No. 5 has an amino acid sequence corresponding to SEQ. ID. No. 6 as follows:
  • Another such DNA molecule includes nucleotides 3344 to 3349 of SEQ. ID. No. 1 and codes for an unknown protein or polypeptide.
  • This DNA molecule comprises the nucleotide sequence corresponding to SEQ. ID. No. 7 as follows:
  • This DNA molecule encodes a protein or polypeptide with an amino acid sequence corresponding to SEQ. ID. No. 8 as follows: Met Leu Arg Val Asp Asn Phe Leu Trp Ala He Tyr Leu He Thr Phe 1 5 10 15
  • Another such DNA molecule includes nucleotides 3483 to 5012 of
  • SEQ. ID. No. 1 encodes for a heat shock 70 protein or polypeptide.
  • This DNA molecule comprises the nucleotide sequence corresponding to SEQ. ID. No. 9 as follows:
  • nucleotide sequence of SEQ. ID. No. 9 encodes an amino acid sequence corresponding to SEQ. ID. No. 10 as follows:
  • Another such DNA molecule includes nucleotides 8490 to 7398 of SEQ. ID. No. 2 and codes for a first coat protein.
  • This DNA molecule comprises a nucleotide sequence corresponding to SEQ. ID. No. 11 as follows:
  • the nucleic acid of SEQ. ID. No. 11 encodes a protein having an amino acid sequence corresponding to SEQ. ID. No. 12 as follows:
  • DNA molecule of the present invention includes nucleotides 7430 to 8905 of SEQ. ID. No. 2 and codes for a second coat protein.
  • This DNA molecule comprises a nucleotide sequence corresponding to SEQ. ID. No. 13 as follows:
  • the DNA molecule of SEQ. ID. No. 13 encodes a protein having an amino acid sequence corresponding to SEQ. ID. No. 14 as follows: Met Glu Phe Gin Arg He Pro Ala Val Glu Gly Ser Thr Phe Arg Leu 1 5 10 15 Ser Asp Lys Leu Asp Asp Lys Arg Lys Tyr He Asp Leu Asp Asn Gly 20 25 30
  • Another such DNA molecule includes nucleotides 8895 to 9410 of SEQ. ID. No. 2 and codes for an unknown protein.
  • This DNA molecule comprises a nucleotide sequence corresponding to SEQ. ID. No. 15 as follows:
  • the DNA molecule of SEQ. ID. No. 15 encodes a protein which has an amino acid sequence corresponding to SEQ. ID. No. 16 as follows:
  • Another such DNA molecule includes nucleotides 5203 to 6414 of SEQ. ID. No. 2 and codes for an unknown protein or polypeptide.
  • This DNA molecule comprises a nucleotide sequence corresponding to SEQ. ID. No. 17 as follows:
  • the DNA molecule of SEQ. ID. No. 17 encodes the protein or polypeptide having a deduced amino acid sequence corresponding to SEQ. ID. No. 18 as follows:
  • DNA molecule includes nucleotides 1 to 931 of SEQ. ID. No. 2 and codes for a helicase protein or polypeptide.
  • This DNA molecule comprises a nucleotide sequence corresponding to SEQ. ID. No. 19 as follows: caacaaaatt gatcaggagt actttaagga tagatctggc agttgtatta tgaccgccaa 60 tcgtggtagc gctatagata tcaatgatac cattgagagt atagacgctg ctaatgcgag 120 caaagccgct tcgaacaacg tcagcggcgt ggaatcgata gataattatg tttgtgctcg 180 tacagttaac tcacaaatta tgaactgcaa aggagtaatg aattatacct gcgcctagt 240 t
  • This DNA molecule encodes a protein or polypeptide with a deduced amino acid sequence corresponding to SEQ. ID. No. 20 as follows:
  • Another DNA molecule of the present invention includes nucleotides 879 to 2558 of SEQ. ID. No. 2 and codes for a polymerase protein or polypeptide.
  • This DNA molecule comprises a nucleotide sequence corresponding to SEQ. ID. No. 21 as follows:
  • This DNA molecule encodes a protein or polypeptide with a deduced amino acid sequence corresponding to SEQ. ID. No. 22 as follows:
  • Another DNA molecule of the present invention includes nucleotides 3173 to 3326 of SEQ. ID. No. 2 and codes for an unknown protein or polypeptide.
  • This DNA molecule comprises a nucleotide sequence corresponding to SEQ. ID. No. 23 as follows:
  • This DNA molecule encodes a protein or polypeptide with a deduced amino acid sequence corresponding to SEQ. ID. No. 24 as follows:
  • Another DNA molecule of the present invention includes nucleotides 3340 to 4965 of SEQ. ID. No. 2 and codes for a heat shock protein 70 protein or polypeptide.
  • the DNA molecule comprises a nucleotide sequence corresponding to SEQ. ID. No. 25 as follows:
  • the DNA molecule of SEQ. ID. No. 25 encodes a protein of polypeptide with a deduced amino acid sequence corresponding to SEQ. ID. No. 26 as follows:
  • Another DNA molecule of the present invention includes nucleotides 9407 to 9991 of SEQ. ID. No. 2 and codes for an unknown protein or polypeptide.
  • This DNA molecule comprises a nucleotide sequence of SEQ. ID. No. 27 as follows:
  • This DNA molecule encodes a protein or polypeptide with a deduced amino acid sequence corresponding to SEQ. ID. No. 28 as follows:
  • fragments of the DNA molecules of the present invention are constructed by using appropriate restriction sites, revealed by inspection of the DNA molecule's sequence, to: (i) insert an interposon (Felley et al., "Interposon Mutagenesis of Soil and Water Bacteria: a Family of DNA Fragments Designed for in vitro Insertion Mutagenesis of Gram-negative Bacteria," Gene.
  • the sequence can be used to amplify any portion of the coding region, such that it can be cloned into a vector supplying both transcription and translation start signals.
  • Suitable DNA molecules are those that hybridize to a DNA molecule comprising a nucleotide sequence of at least 15 continuous bases of SEQ. ID. Nos.
  • hybridization buffer comprising 0.9M sodium citrate (“SSC") buffer at a temperature of 42°C and remaining bound when subject to washing with SSC buffer at 42°C; and preferably in a hybridization buffer comprising 20% formamide in 0.9M saline/0.9M SSC buffer at a temperature of 42°C and remaining bound when subject to washing at 42°C with 0.2x SSC buffer at 42°C.
  • SSC sodium citrate
  • Variants may also (or alternatively) be modified by, for example, the deletion or addition of nucleotides that have minimal influence on the properties, secondary structure and hydropathic nature of the encoded polypeptide.
  • nucleotides encoding a polypeptide may be conjugated to a signal (or leader) sequence at the N-terminal end of the protein which co-translationally or post-translationally directs transfer of the protein.
  • the nucleotide sequence may also be altered so that the encoded polypeptide is conjugated to a linker or other sequence for ease of synthesis, purification, or identification of the polypeptide.
  • the protein or polypeptide of the present invention is preferably produced in purified form (preferably, at least about 80%, more preferably 90%, pure) by conventional techniques.
  • the protein or polypeptide of the present invention is isolated by lysing and sonication. After washing, the lysate pellet is resuspended in buffer containing Tris-HCl. During dialysis, a precipitate forms from this protein solution. The solution is centrifuged, and the pellet is washed and resuspended in the buffer containing Tris-HCl. Proteins are resolved by electrophoresis through an SDS 12% polyacrylamide gel.
  • the DNA molecule encoding the pineapple mealybut wilt virus protein or polypeptide of the present invention can be incorporated in cells using conventional recombinant DNA technology. Generally, this involves inserting the DNA molecule into an expression system to which the DNA molecule is heterologous (i.e. not normally present). The heterologous DNA molecule is inserted into the expression system or vector in proper sense orientation and correct reading frame. The vector contains the necessary elements for the transcription and translation of the inserted protein-coding sequences.
  • Recombinant genes may also be introduced into viruses, such as vaccinia virus.
  • Recombinant viruses can be generated by transfection of plasmids into cells infected with virus.
  • Suitable vectors include, but are not limited to, the following viral vectors such as lambda vector system gtl 1, gt WES.tB, Charon 4, and plasmid vectors such as pBR322, pBR325, pACYC177, pACYC184, pUC8, pUC9, pUC18, pUC19, pLG339, pR290, pKC37, pKClOl, SV 40, pBluescript II SK +/- or KS +/- (see "Stratagene Cloning Systems” Catalog (1993) from Stratagene, La Jolla, Calif, which is hereby incorporated by reference), pQE, pIH821, pGEX, pET series (see Studier et.
  • viral vectors such as lambda vector system gtl 1, gt WES.tB, Charon 4, and plasmid vectors such as pBR322, pBR325, pACYC
  • host- vector systems may be utilized to express the protein-encoding sequence(s). Primarily, the vector system must be compatible with the host cell used.
  • Host- vector systems include but are not limited to the following: bacteria transformed with bacteriophage DNA, plasmid DNA, or cosmid DNA; microorganisms such as yeast containing yeast vectors; mammalian cell systems infected with virus (e.g., vaccinia virus, adenovirus, etc.); insect cell systems infected with virus (e.g., baculovirus); and plant cells infected by bacteria or transformed via particle bombardment (i.e. biolistics).
  • the expression elements of these vectors vary in their strength and specificities. Depending upon the host- vector system utilized, any one of a number of suitable transcription and translation elements can be used. Different genetic signals and processing events control many levels of gene expression (e.g., DNA transcription and messenger RNA ("mRNA”) translation).
  • mRNA messenger RNA
  • Transcription of DNA is dependent upon the presence of a promoter which is a DNA sequence that directs the binding of RNA polymerase and thereby promotes mRNA synthesis.
  • the DNA sequences of eucaryotic promoters differ from those of procaryotic promoters.
  • eucaryotic promoters and accompanying genetic signals may not be recognized in or may not function in a procaryotic system, and, further, procaryotic promoters are not recognized and do not function in eucaryotic cells.
  • translation of mRNA in procaryotes depends upon the presence of the proper procaryotic signals which differ from those of eucaryotes.
  • SD Shine-Dalgarno
  • Promoters vary in their "strength" (i.e. their ability to promote transcription). For the purposes of expressing a cloned gene, it is desirable to use strong promoters in order to obtain a high level of transcription and, hence, expression of the gene. Depending upon the host cell system utilized, any one of a number of suitable promoters may be used. For instance, when cloning in E.
  • promoters such as the T7 phage promoter, lac promoter, trp promoter, recA promoter, ribosomal RNA promoter, the P R and P L promoters of coliphage lambda and others, including but not limited, to / ⁇ cUV5, ompF, bla, Ipp, and the like, may be used to direct high levels of transcription of adjacent DNA segments. Additionally, a hybrid trp-lac ⁇ JY5 (tac) promoter or other E. coli promoters produced by recombinant DNA or other synthetic DNA techniques may be used to provide for transcription of the inserted gene.
  • promoters such as the T7 phage promoter, lac promoter, trp promoter, recA promoter, ribosomal RNA promoter, the P R and P L promoters of coliphage lambda and others, including but not limited, to / ⁇ cUV5, ompF, bla, Ipp, and the like, may be used to direct high levels
  • Bacterial host cell strains and expression vectors may be chosen which inhibit the action of the promoter unless specifically induced.
  • the addition of specific inducers is necessary for efficient transcription of the inserted DNA.
  • the lac operon is induced by the addition of lactose or IPTG (isopropylthio-beta-D-galactoside).
  • IPTG isopropylthio-beta-D-galactoside.
  • Specific initiation signals are also required for efficient gene transcription and translation in procaryotic cells. These transcription and translation initiation signals may vary in "strength” as measured by the quantity of gene specific messenger RNA and protein synthesized, respectively.
  • the DNA expression vector which contains a promoter, may also contain any combination of various "strong" transcription and/or translation initiation signals. For instance, efficient translation in E. coli requires a Shine-Dalgarno ("SD") sequence about 7- 9 bases 5' to the initiation codon ("ATG”) to provide a ribosome binding site. Thus, any SD-ATG combination that can be utilized by host cell ribosomes may be employed.
  • Such combinations include but are not limited to the SD-ATG combination from the cro gene or the N gene of coliphage lambda, or from the E. coli tryptophan ⁇ , D, C, B or A genes. Additionally, any SD-ATG combination produced by recombinant D ⁇ A or other techniques involving incorporation of synthetic nucleotides may be used.
  • D ⁇ A molecules encoding the various pineapple mealybug wilt virus proteins or polypeptides, as described above, have been cloned into an expression system, they are ready to be incorporated into a host cell. Such incorporation can be carried out by the various forms of transformation noted above, depending upon the vector/host cell system.
  • Suitable host cells include, but are not limited to, bacteria, virus, yeast, mammalian cells, insect, plant, and the like.
  • the present invention also relates to RNA molecules which encode the various pineapple mealybug wilt virus proteins or polypeptides described above.
  • the transcripts can be synthesized using the host cells of the present invention by any of the conventional techniques.
  • the mRNA can be translated either in vitro or in vivo.
  • Cell-free systems typically include wheat-germ or reticulocyte extracts. In vivo translation can be effected, for example, by microinjection into frog oocytes.
  • One aspect of the present invention involves using one or more of the above DNA molecules encoding the various proteins or polypeptides of a pineapple mealybug wilt virus to transform pineapple plants in order to impart pineapple mealybug wilt virus resistance to the plants. The mechanism by which resistance is imparted is not known.
  • the transformed plant can express a protein or polypeptide of pineapple mealybug wilt virus, and, when the transformed plant is inoculated by a pineapple mealybug wilt virus, the expressed protein or polypeptide prevents translation of the viral RNA.
  • the subject DNA molecule incorporated in the plant can be constitutively expressed.
  • expression can be regulated by a promoter which is activated by the presence of pineapple mealybug wilt virus. Suitable promoters for these purposes include those from genes expressed in response to pineapple mealybug wilt virus infiltration.
  • the isolated DNA molecules of the present invention can be utilized to impart pineapple mealybug wilt virus resistance for a wide variety of pineapple plants.
  • the term "pineapple” refers to a member of the genera Ananas and Pseudoananas of the Bromeliaceae family.
  • the genus Pseudoananas is monotypic, i.e., consists of P.bianarius.
  • the genus Ananas consists of five species, namely, A. bracteatus, A. ftitzmuelleri, A. comosus, A. erectif ⁇ lius, and A. ananassoides.
  • Plant tissue suitable for transformation include leaf tissue, root tissue, meristems, zygotic and somatic embryos, and anthers.
  • the expression system of the present invention can be used to transform virtually any plant tissue under suitable conditions.
  • Tissue cells transformed in accordance with the present invention can be grown in vitro in a suitable medium to impart pineapple mealybug wilt virus resistance.
  • Transformed cells can be regenerated into whole plants such that the protein or polypeptide imparts resistance to pineapple mealybug wilt virus in the intact transgenic plants.
  • the plant cells transformed with the recombinant DNA expression system of the present invention are grown and caused to express that DNA molecule to produce one of the above-described pineapple mealybug wilt virus proteins or polypeptides and, thus, impart pineapple mealybug wilt virus resistance.
  • the DNA construct in a vector described above can be microinjected directly into plant cells by use of micropipettes to transfer mechanically the recombinant DNA.
  • the genetic material may also be transferred into the plant cell using polyethylene glycol. Krens, et al., Nature, 296:72-74 (1982), which is hereby inco ⁇ orated by reference.
  • One technique of. transforming plants with the DNA molecules in accordance with the present invention is by contacting the tissue of such plants with an inoculum of a bacteria transformed with a vector comprising a gene in accordance with the present invention which imparts pineapple mealybug wilt virus resistance.
  • this procedure involves inoculating the plant tissue with a suspension of bacteria and incubating the tissue for 48 to 72 hours on regeneration medium without antibiotics at 25-28°C.
  • Bacteria from the genus Agrobacterium can be utilized to transform plant cells. Suitable species of such bacterium include Agrobacterium tumefaciens and Agrobacterium rhizogenes. Agrobacterium tumefaciens (e.g., strains C58, LBA4404, or EHA105) is particularly useful due to its well-known ability to transform plants.
  • Heterologous genetic sequences can be introduced into appropriate plant cells, by means of the Ti plasmid of A. tumefaciens or the Ri plasmid of A. rhizogenes.
  • the Ti or Ri plasmid is transmitted to plant cells on infection by Agrobacterium and is stably integrated into the plant genome. J. Schell, Science, 237:1176-83 (1987), which is hereby inco ⁇ orated by reference.
  • Regeneration generally involves providing a suspension of transformed protoplasts or a petri plate containing explants.
  • Callus tissue is formed and shoots may be induced from callus and subsequently rooted. Alternatively, embryo formation can be induced in the callus tissue. These embryos germinate as natural embryos to form plants.
  • the culture media will generally contain various amino acids and hormones, such as auxin and cytokinins. It is also advantageous to add glutamic acid and proline to the medium. Efficient regeneration will depend on the medium, on the genotype, and on the history of the culture. If these three variables are controlled, then regeneration is usually reproducible and repeatable.
  • transgenic plants of this type are produced, the plants themselves can be propagated vegetatively by tissue culture so that the DNA construct is present in the resulting plants.
  • particle bombardment also known as biolistic transformation
  • particle bombardment also known as biolistic transformation
  • This technique is disclosed in U.S. Patent Nos. 4,945,050, 5,036,006, and 5,100,792, all to Sanford et al., and in Emerschad et al., "Somatic Embryogenesis and Plant Development from Immature Zygotic Embryos of Seedless Grapes (Vitis vinifera) Plant Cell Reports. 14:6-12 (1995) (“Emerschad (1995)”), which are hereby inco ⁇ orated by reference.
  • this procedure involves propelling inert or biologically active particles at the cells under conditions effective to penetrate the outer surface of the cell and to be inco ⁇ orated within the interior thereof.
  • the vector can be introduced into the cell by coating the particles with the vector containing the heterologous DNA.
  • the target cell can be surrounded by the vector so that the vector is carried into the cell by the wake of the particle.
  • Biologically active particles e.g., dried bacterial cells containing the vector and heterologous DNA
  • RNA-mediated resistance RNA-mediated resistance
  • the DNA molecule When pineapple is transformed with such a DNA molecule, the DNA molecule can be transcribed under conditions effective to maintain the messenger RNA in the plant cell at low level density readings.
  • a pineapple mealybug wilt virus Type I or II As a result, translation of the encoded protein is repressed.
  • Pineapple mealybug wilt virus can be detected in a sample using a nucleotide sequence of the DNA molecule, or a fragment thereof, encoding for a protein or polypeptide of the present invention.
  • the nucleotide sequence is provided as a probe in a nucleic acid hybridization assay or a gene amplification detection procedure (e.g., using a polymerase chain reaction procedure).
  • the nucleic acid probes of the present invention may be used in any nucleic acid hybridization assay system known in the art, including, but not limited to, Southern blots (Southern, E.M., "Detection of Specific Sequences Among DNA Fragments Separated by Gel
  • the probes can be used in a gene amplification detection procedure (e.g., a polymerase chain reaction). Erlich, H.A., et. al., "Recent Advances in the Polymerase Chain Reaction," Science 252:1643-51 (1991), which is hereby inco ⁇ orated by reference. Any reaction with the probe is detected so that the presence of a pineapple mealybug wilt virus in the sample is indicated. Such detection is facilitated by providing the probe of the present invention with a label. Suitable labels include a radioactive compound, a fluorescent compound, a chemiluminescent compound, an enzymatic compound, or other equivalent nucleic acid labels.
  • Nucleic acid (DNA or RNA) probes of the present invention will hybridize to complementary pineapple mealybug wilt virus nucleic acids under stringent conditions.
  • stringent conditions are selected to be about 50°C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH.
  • T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.
  • the T m is dependent upon the solution conditions and the base composition of the probe, and may be calculated using the following equation:
  • T m 79.8°C + (18.5 x Log[Na+]) + (58.4°C x %[G+C])
  • Nonspecific binding may also be controlled using any one of a number of known techniques such as, for example, blocking the membrane with protein-containing solutions, addition of heterologous RNA, DNA, and SDS to the hybridization buffer, and treatment with RNase. Wash conditions are typically performed at or below stringency. Generally, suitable stringent conditions for nucleic acid hybridization assays or gene amplification detection procedures are set forth above. More or less stringent conditions may also be selected.
  • Membranes were gently agitated in PBS buffer (140 mM NaCl, 10 mM Na 2 HPO 4 , 3 mM KCl, 2 mM KH 2 PO 4> pH 7.4) with 2% of powdered milk (w/v) being added for 60 min at room temperature. Membranes were transferred to TBS buffer (50 mM Tris-HCl, 50 mM NaCl, pH 7.5) containing 1 ⁇ g of PMWaV monoclonal IgG antibody (Hu et al., "Pineapple Closterovirus in Mealybug Wilt of Pineapple," In: Abstracts of the Xth International Congress of Virology. Jerusalem, Israel, Aug.
  • Membranes were washed as described above, placed in a hybridization bottle with Sigma Fast BCIP/NBT alkaline phosphatase substrate (Sigma), and rotated in a hybridization oven for up to 60 min. Membranes were then rinsed with distilled water and allowed to air dry. When dry, the extent of substrate precipitation for each leaf cross-section was determined under a dissecting scope.
  • Greenhouse-grown pineapple plants were screened for the presence of PMWaV using a monoclonal antibody in a TBIA. Of the thirty-eight plants assayed, twenty-four were positive for the virus (63 %). All substrate precipitate was found in discrete spots corresponding to vascular bundles in the leaf cross-section. The degree of precipitation varied from plant to plant, and was assumed to be an indication of virus titre (Hu et al., "Use of a Tissue Blotting Immunoassay to Examine the Distribution of Pineapple Closterovirus in Hawaii," Plant Pis. 81 :1150-1154 (1997), which is hereby inco ⁇ orated by reference). Of the twenty-four PMWaV-positive plants, thirteen plants with the strongest positive signals had tissue harvested for dsRNA extraction.
  • Double-stranded RNA was extracted from PMWaV-infected pineapple plants using a protocol similar to that of Morris et al., "Isolation and Analysis of Double-stranded RNA From Virus-infected Plant and Fungal Tissue," Phvtopathologv 69:854-85 (1979) and Dale et al., "Double-stranded RNA in Banana Plants with Bunchy Top Disease,” J. Gen. Virol. 67:371-375 (1986), which is hereby inco ⁇ orated by reference).
  • Leaf and stem tissue from TBIA-positive plants was frozen in liquid nitrogen then finely ground with a Bunn model G3 coffee grinder (Bunn-O-Matic; Springfield, IL) and stored at -20 °C. Allotments (50 g) of frozen tissue were added to flasks containing 90 mL STE (100 mM NaCl, 50 mM Tris-HCl, 1 mM EDTA, pH 8.0), 40 mL water-saturated phenol containing 0.1 % ⁇ - mercaptoethanol (v/v) and 0.1 % 8-hydroxyquinoline (w/v), 30 mL 10 % SDS (w/v), 2 mL ⁇ -mercaptoethanol, 0.2 mL NH 4 OH, and 64 mg bentonite.
  • Chloroform (40 mL) was then added, and the sample was stirred for 45 min at 4 °C.
  • the mixture was centrifuged at 7000 g for 10 min at 4 °C, the upper phase from two samples was collected and combined, then adjusted to 16.5 % EtOH.
  • 2 g CF-11 cellulose (Whatman; Maidstone, England)
  • the sample was shaken at room temperature overnight.
  • the sample was then passed through a vertically clamped 60 cc syringe barrel containing a miracloth filter (Calbiochem; La Jolla, CA).
  • the cellulose was washed with approximately 100 mL of STE containing 16.5 % EtOH, and purged of all liquid using the syringe plunger.
  • dsRNA was eluted from the cellulose with 25 mL of STE, and the plunger was used to elute any remaining liquid. The sample was again adjusted to 16.5 % EtOH, and the process was repeated using 1.5 g of cellulose. The final elution of dsRNA from the cellulose was done with three 3 mL aliquots of STE collected in a 15 mL centrifuge tube, with a purge using the syringe barrel following each aliquot.
  • any contaminating cellulose was pelleted by a brief centrifugation, and the supernatant was transferred to a 30 mL centrifuge tube where it was precipitated with 0.1 volume of 3 M NaAc (pH 5.2) and 2 volumes of EtOH (95 %) overnight at -20 °C. Samples were then centrifuged at 12 000 g for 15 min at 4 °C, the supernatant discarded, and the pellet gently resuspended in 0.5 mL H 2 O. The dsRNA was again precipitated and centrifuged as described in a 1.5 mL microfuge tube.
  • the supernatant was discarded, and the pellet washed with 1 mL of cold 70 % EtOH, and centrifuged at 12 000 g for 5 min. The supernatant was discarded and the pellet, representing dsRNA from 100 g of tissue, was resuspended in 10 ⁇ L of H 2 O and stored at -20 °C. Approximately 3 kg of PMWaV-infected tissue was processed using this protocol.
  • Clones 12-1 (1274 bp), 12-2 (1632 bp), and 12-3 (1386 bp) were generated by the step-by-step walking procedure which span nearly the entire 3' end of the viral genome.
  • the 20 - 30 base overlap between each clone was 100% identical in sequence, validating their position relative to each other in the genome.
  • the PMWaV-2-specific primer (#200) used to prime cDNA synthesis for clone 12-1 also annealed downstream on this cDNA strand, allowing its exclusive use in priming subsequent PCR reactions. At first, it was believed there was an inversion in this portion of the PMWaV-2 genome.
  • Virus-specific dsRNAs were isolated from infection pineapple tissue by two cycles of CF-11 cellulose column chromatography. After denaturation with 20 mM methylmercury hydroxide, first strand cDNA was synthesized as previously described (Karasev, et al., "Screening of the Closterovirus Genome by Degenerate Primer-Mediated Polymerase Chain Reaction," J. Gen. Virol. 75:1415-1422 (1994), which is hereby inco ⁇ orated by reference), except that SUPERSCRIPT II reverse transcriptase (BRL) was used instead of the Moloney murine leukemia virus enzyme.
  • SUPERSCRIPT II reverse transcriptase SUPERSCRIPT II reverse transcriptase
  • dsRNA template was denatured at 95 °C for 9 min in an 8 ⁇ L volume containing 3 pmol of PMWaV-2- specific primer and quickly chilled on ice.
  • first strand buffer [375 mM KCl, 250 mM Tris-HCl (pH 8.3), 15 mM MgCl 2 ], 2 ⁇ L of 0.1 M DTT, and 5 ⁇ L of dNTPs (2 mM each)
  • the mixture was incubated at 48 °C for 3 min, and 1 ⁇ L (200 U / ⁇ L) of Superscript II reverse transcriptase (GibcoBRL; Gaithersburg, MD) was added.
  • the reaction was then incubated at 48 °C for 60 min, and terminated by a 15 min incubation at 70 °C.
  • PCR reactions were set up using 1 - 2 ⁇ L of a 1 :5 dilution of the first-strand cDNA synthesis reaction as template in a 20 - 25 ⁇ L reaction containing 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl 2 , 10 pmol of PMWaV-2-specific primer, 10 pmol of random primer, 200 ⁇ M of each dNTP, 1 -2 U of AmpliTaq DNA polymerase (PE Applied Biosystems; Foster City, CA) and overlaid with 1 - 2 drops of mineral oil.
  • the amplification protocol was performed in a Model 480 DNA Thermal Cycler (PE Applied Biosystems) and involved: one cycle of 94 °C for 5 min; forty-five cycles of 94 °C for 1 min, 33 °C for 1 min, 72 °C for 2.5 min; and one cycle of 72 °C for 7 min.
  • PCR reactions were examined for the presence of amplicons in 1.5 % agarose gels run in TAE buffer [40 mM Tris-acetate, 1 mM EDTA (pH 8.0)] at 5 - 7 V / cm.
  • Amplicons desired for cloning were excised from the gel with a razor blade and placed in a 0.6 mL centrifuge tube with a pinhole in the bottom and containing a GF/C glass microf ⁇ bre filter (Whatman). This tube was then placed in a 1.5 mL centrifuge tube and centrifuged at 12,000 g for 30 s. The elutant was either precipitated as described above and the pellet resuspended in a lesser volume, or used directly in vector ligation.
  • the gel-excised amplicons were ligated into either pGEM-T Easy (Promega; Madison, WI) or pBlueScript KS- phagemid (Stratagene; La Jolla, CA) contrasted into a T- vector (Marchuk et al., "Construction of T-vectors, a Rapid and General System for Direct Cloning of Unmodified PCR Products," Nucl. Acids Res. 19:1154 (1991), which is hereby inco ⁇ orated by reference).
  • E. coli DH5 cells were transformed with 2 - 5 ⁇ L of the ligation reaction and cells containing recombinant plasmids were selected with either McConkey agar (Sigma) or LB agar topspread with X-gal and IPTG (Sambrook et al., Molecular Cloning: A Laboratory Manual.
  • Plasmid DNA was sequenced using either T3, T7, SP6, or PMWaV-2- specific primers (Table 2) with Taq DyeDeoxy terminator cycle sequencing (PE Applied Biosystems). Sequencing was conducted on automated sequencers at the Guelph Molecular Supercentre (Model ABI377), University of Guelph, Guelph, Canada, the NCSU DNA Sequencing Facility (ABI377), North Carolina State University, Raleigh, NC, or the Biotechnology/Molecular Biology Instrumentation and Training Facility (ABI373/ABI377), University of Hawaii, Honolulu, HI. Nucleotide sequences were analyzed using various computer programs.
  • PCR products obtained were size separated in low- melting temperature 1% agarose gels (BRL), and the selected bands were isolated and cloned into the AT-based vector pCR2.1 (Invitrogen) according to the manufacturer's protocol.
  • Virus-specific inserts were sequenced directly in dsDNA plasmids using the SEQUENASE 2.0 Kit (USB) according to the manufacture's instructions. Universal T3 and T7 primers were used to sequence the ends of inserts and virus-specific primers to sequence the rest of the cDNAs. Both strands were sequenced at least twice.
  • Example 7 Cloning of the PMWaV-1 and PMWaV-2
  • the initial virus-specific DNA fragments were amplified using two degenerate primers, D2056 and D2128, targeting motifs A and C of the HSP70 protein.
  • a weak, but discrete band of the expected 1-kb size was amplified and cloned.
  • the fourth clone, pcl2 also encoded the N-terminal half of the viral HSP70.
  • the viral HSP70 encoded by this clone came from a different closterovirus.
  • PMWaV- 1 anchored to the clone pel 8
  • PMWaV-2 anchored to the clone pcl2
  • the sequenced 5,217 nucleotide (nt) fragment of the PMWaV- 1 genome spans four open reading frames (ORFs) which may encode four protein products.
  • the first ORF termed ORFla according to conventional designation of the clostero viral genes (Dolja, et al, "Molecular Biology and Evolution of Closteroviruses: Sophisticated Build-up of Large RNA Genomes," Ann. Rev. Phytopathol 32:261-285 (1994), which is hereby inco ⁇ orated by reference), encodes a protein with all eight motifs conserved in the so-called viral helicases (HEL) and, thus, was identified as a respective PMWaV- 1 HEL.
  • HEL viral helicases
  • ORF lb The second ORF (ORF lb) partially overlaps ORFla and encodes a protein with eight conserved domains characteristic of closterovirus RNA-dependent RNA polymerases (RdRp); this ORF lb was identified as the respective PMWaV- 1 RdRp.
  • RdRp closterovirus RNA-dependent RNA polymerases
  • the downstream ORF2 encodes a small 6-kDa protein composed of predominantly hydrophobic amino acid residues.
  • an ORF3 which encodes the PMWaV- 1 HSP70 protein.
  • the overall organization of the PMWaV- 1 genome within this sequenced fragment resembles the gene layout of the type member of the group, beet yellows virus (BYV) (Agranovsky, et al., "Beet Yellows Closterovirus: Complete Genome Structure and Identification of a Leader Papain-like Thiol Protease," Virology 198:311-324 (1994), which is hereby inco ⁇ orated by reference).
  • BYV beet yellows virus
  • the sequenced 10,000 -nt fragment of the PMWaV-2 genome spans eight ORFs which may code for four protein products.
  • the overall genome organization of the PMWaV-2 genome in the sequenced fragment resembles most closely the genome of the grapevine leafroll-associated virus 3 (Ling, et al., "The Coat Protein Gene of Grapevine Leafroll Associated Closterovirus-3 : Cloning, Nucleotide Sequencing, and Expression in Transgenic Plants," Arch Virol. 142:1101- 1116 (1997), which is hereby inco ⁇ orated by reference).
  • ORFla encompasses all motifs characteristic of viral helicases and was identified as a PMWaV-2 HEL.
  • the second ORF designated herein as ORF lb, partially overlaps ORFla and encodes the PMWaV-2 RdRp.
  • ORF lb is probably expressed through a +1 ribosomal frameshift.
  • the downstream ORF2 encodes a small hydrophobic 6-kDa protein
  • the further downstream ORF3 encodes the PMWaV-2 HSP70 protein.
  • ORF4 start codon was 238 bp downstream of the HSP70 homolog stop codon.
  • This ORF potentially encodes a 403 amino acid polypeptide with a theoretical molecular mass of 46.4 kilodaltons (kDa).
  • the function of this potential protein is unknown and database searching via BLASTX did not identify similar proteins except its counte ⁇ art (p55) in GRLaV-3 (Table 3).
  • ORF5 start codon was 75 bp downstream of the p46 stop codon. This ORF potentially encodes a 302 amino acid polypeptide with a theoretical molecular mass of 33.8 kDa.
  • ORF5 was identified as the coat protein gene of PMWaV-2 based on the high degree of homology to GRLaV-3 CP (Table 3), and the moderate degree of homolgy to CPs of other closteroviruses.
  • Amino acid sequence alignment of the CP and the CPs from GLRaV-3, BYV, CTV, and LIYV revealed the invariant amino acid residues S, R, and D, which are conserved in the CP of rod- shaped and filamentous RNA plant viruses (Dolja et al., "Phylogeny of Capsid Proteins of Rod-shaped and Filamentous RNA Plant Viruses: Two Families with Distinct Patterns of Sequence and Probably Structure Conservation," Virology 184:79-86 (1991), which is hereby inco ⁇ orated by reference).
  • Residues R and D are believed to be involved in stabilization of molecules by salt bridge formation and proper folding of the CP (Dolja et al., "Phytogeny of Capsid Proteins of Rod-shaped and Filamentous RNA Plant Viruses: Two Families with Distinct Patterns of
  • ORF6 (CPd)
  • the ORF6 start codon was 33 bp downstream of the CP stop codon.
  • This ORF potentially encodes a 491 amino acid polypeptide with a theoretical molecular mass of 55.8 kDa.
  • This protein was identified as a second coat protein (i.e. CPd) based on the high degree of homology with GRLaV-3 CPd (Table 3) and moderate homologies with the CPd of other closteroviruses.
  • CPd second coat protein
  • Table 3 The S, R, and D residues conserved in the CP were also conserved in the CPd.
  • a start codon 8 bp upstream of the CPd stop codon initiates a ORF7 which potentially encodes a 172 amino acid polypeptide with a theoretical molecular mass of 19.7 kDa.
  • BLASTX analysis reveals this potential protein is homologous with p21 in GLRaV-3, with a 26 % identity and 38 % similarity between the amino acid sequences.
  • ORF7 has been tentatively identified as the PMWaV-2 homolog of p21 of GLRaV-3. This is despite the fact that the N-terminus of p21 does not overlap with the C-terminus of CPd in GLRaV-3 as it does in PMWaV-2.
  • ORF8 The start (AUG) codon for ORF8 shares the UG residues of the stop
  • ORF8 potentially encodes a 194 amino acid polypeptide with a theoretical molecular weight of 22.3 kDa.
  • This protein shares no significant homology with any other closterovirus sequence, however, it interestingly has a region of homology 72 amino acids long (29 % identity, 47 % similarity) with a viral capsid associated protein of nuclear polyhedrosis viruses (Lu, et al., "Nucleotide Sequence and Transcriptional Analysis of the p80 Gene of Autographa Californica Nuclear Polyhedrosis Virus: A Homologue of the Orgyia pseudotsugata Nuclear Polyhedrosis Virus Capsid- Associated Gene," Virology 190:201-209 (1992), which is hereby inco ⁇ orated by reference).
  • Sequence data and characterization of the PMWaV genome(s) will serve two pu ⁇ oses. First, from an academic standpoint, such information will be useful in the classification of the closteroviruses. Sequence homology to other closteroviruses and genome organization are essential to the proper classification of these important pathogens. With this data, the evolutionary relationships within the family and to other RNA plant viruses may also be established. These relationships are especially interesting due to the large coding capacity of the closteroviruses and their novel genes.
  • PMW is a serious disease of pineapple, and is mainly controlled by pesticide use. Increasing pressure to control diseases with non-chemical methods has led to the use of disease resistant crops. Crops resistant to viral diseases by transformation with CP or other viral genes have been established and effective, although the mechanism of resistance is still poorly understood (Hackland et al., "Coat-protein Mediated Resistance in Transgenic Plants," Arch. Virol. 139:1-22 (1994); Prins et al., "RNA-mediated Resistance in Transgenic Crops," Arch. Virol. 141 :2259-2276 (1996), which are hereby inco ⁇ orated by reference).
  • PMWaV appears to be an important factor in PMW
  • PMWaV genes such as CP or RdRp will play an essential role in developing transgenic pineapple plants resistant to PMWaV, and ultimately, PMW.
  • Data obtained by this study allows the immediate implementation of this disease resistance strategy.
  • Further sequencing of the PMWaVs genome, especially genes with unknown function, may also provide valuable insights into vector specificity and virus retention, which are two important factors when studying the spread and the epidemiology of PMW and its associated viruses, PMWaV- 1 and PMwaV-2.
  • the concept of virus-derived resistance is one of the possible solutions to the PMW problem. And it might very well be the fastest solution.
  • Two types of the virus-derived genes have been used to regenerate transgenic plants with demonstrated resistance to viruses: the RdRp and coat protein (CP) genes.
  • the RdRp-mediated resistance seems to be more strict although very specific, i.e. working against the exact virus strain which was a source of the transgene.
  • the CP-mediated resistance seems to be much broader, protecting the plant against a range of virus isolates which may differ in the sequence of the transgene up to 8-10%, although it is not as strict as the RdRp-mediated resistance.
  • the complete RdRp's of both PMWaV- 1 and PMWaV-2 can be used for the generation of transgenic pineapple lines.
  • Two custom-made primers complementary to the start and end of the respective ORF are designed to include an appropriate restriction site to be used for cloning into a plant expression vector.
  • Suitable plant expression vectors include, for example, pEPT8 developed in the laboratory of Prof. D. Gonsalves (Cornell University, Geneva, NY). This vector contains 35S promoter of the cauliflower mosaic virus in front of the inserted ORF and a translation-enhancing leader derived from the alfalfa mosaic virus RNA4, and a CaMV terminator downstream of the insert. The orientation of the insert relative to the 35S promotor can be verified by PCR and restriction enzyme digestion.
  • the resulting plant expression cassette can be excised from the pEPT8-based recombinant plasmid using the Hindlll restriction enzyme and cloned into H ct ⁇ I-digested plant transformation binary vector, pGA482.
  • Hindlll restriction enzyme cloned into H ct ⁇ I-digested plant transformation binary vector, pGA482.
  • Constructs can be mobilized into an avirulent Agrobacterium tumefaciens strain LBA4404 via electroporation, and potential transformants identified on selective media containing 75 ug/ml of gentamycin.
  • A. tumefaciens colonies containing desirable inserts can be used to transform Nicotiana benthamiana and Ananas comosus plants using leaf disk procedures.
  • Kanamycin-resistant R 0 plants can be analyzed by PCR for the respective transgene, and the level of expression tested by Northern-blot hybridization.

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Abstract

The present invention relates to isolated proteins or polypeptides of pineapple mealybug wilt virus. The encoding DNA molecules either alone, in isolated form, or in an expression system, a host cell, or a transgenic pineapple plant are also disclosed. Other aspects of the present invention relate to a method of imparting pineapple mealybug wilt virus resistance to pineapple plants by transforming them with the DNA molecules of the present invention and a method of detecting the presence of a pineapple mealybug wilt virus in a sample.

Description

PINEAPPLE MEALYBUG-ASSOCIATED WILT VIRUS PROTEINS
AND THEIR USES
This application claims the benefit of U.S. Provisional Patent Application Serial No. 60/101,461, filed September 23, 1998, which is hereby incorporated by reference.
FIELD OF THE INVENTION
The present invention relates to pineapple mealybug-associated wilt virus ("PMWaV") proteins, DNA molecules encoding these proteins, and their uses.
BACKGROUND OF THE INVENTION
Mealybug wilt of pineapple (MWP) is a disease of pineapple that is associated with the presence of mealybugs on virus-infected pineapple plants. It is a continuing problem limiting profitable pineapple production in many pineapple growing areas worldwide. MWP is one of the most important factors limiting successful commercial production of pineapple in Hawaii. The yearly value of the Hawaiian pineapple industry is estimated at $230 million with a production of
410,000 tons on 24,000 acres. Yield losses, shifts in fruit size distribution, delays in fruit deliveries, and crop failure caused by mealybug wilt result in severe economic losses (crop yield reductions of 12.2 tons per acre equivalent to 11%). Control of mealybug wilt to minimize crop yield losses and disruption in supply is critical to profitable pineapple production in Hawaii. Without effective mealybug wilt control, economic pineapple production in Hawaii would be in serious jeopardy.
Mealybug wilt nearly destroyed the pineapple industry in Hawaii in the early 1900's. It has been controlled through the intensive use of pesticides aimed at mealybugs or the ants which tend and protect mealybugs from their natural enemies. Pineapple growers have attempted to control mealybugs with foliar application of
Diazinon® insecticide. Unfortunately, such control is difficult and inefficient, because the mealybugs are covered with a waxy coating and tend to feed near the roots where pesticide coverage is poor. In addition, the prolific reproduction rate of mealybugs raises the threat of pesticide resistance developing in mealybugs. Ants, which contribute considerably to the mealybug problem, are currently controlled with the granular bait-insecticide, hydramethylnon (trade name Amdro®, American Cynamid Co., Wayne, NJ), one of the last remaining ant insecticides available to pineapple growers. Nearly all other insecticides that are effective against ants have been banned over the last decade by the U.S. Environmental Protection Agency (EPA). The continued use of Amdro®, however, depends on the annual approval of a special use permit by the EPA and the Hawaii Department of Agriculture. Furthermore, the restrictions on application of Diazinon® and Amdro near irrigation ditches and other waterways seriously impacts control measures aimed at mealybugs and ants, thereby allowing the development of population pockets that escape insecticide. Mealybugs are also easily dispersed by wind throughout the field, making blanket pesticide applications necessary. The pineapple industry has a strong desire, motivated both economically and environmentally, to develop alternative methods that directly control mealybug wilt.
In 1989, closterovirus-like particles from diseased pineapple tissue were purified and partially characterized in Hawaii (Gunasinghe et al., "Purification and Partial Characterization of a Virus from Pineapple," Phvtopathologv 79:1337- 1341 (1989) and Ullman et al., "Serology of a Closteroviruslike Particle Associated with Mealybug Wilt of Pineapple," Phvtopathologv 79:1341-1345 (1989)). More recently, serologically related PMWaV was purified from diseased pineapple in Australia (Thompson, et al., "Detecting of Pineapple Bacilliform Virus Using the Polymerase Chain Reaction," Ann. Appl. Biol. 129:57-69 (1996); Wakeman et al., "Presence of a Clostero-like Virus and a Bacilliform Virus in Pineapple Plants in Queensland," Aust. J. Ag. R. 46:847-958 (1995)). High molecular-weight double- stranded dsRNAs were shown to occur in PMWaV-infected plants (Gunasinghe et al., "Purification and Partial Characterization of a Virus from Pineapple," Phvtopathologv 79:1337-1341 (1989)). PMWaV has been proposed as a cause of MWP (Gunasinghe et al, "Purification and Partial Characterization of a Virus from Pineapple," Phytopathology 79:1337-1341 (1989); Ullman et al., "Serology of a Closteroviruslike Particle Associated with Mealybug Wilt of Pineapple," Phvtopathologv 79:1341-1345 (1989); German et al, "Mealybug Wilt of Pineapple," Advances in Disease Vector Research 9:241-259 (1992); Hu et al., "Detection of Pineapple Closterovirus, a Possible Cause of Mealybug Wilt of Pineapple," Acta Horticulturae 334:411-416 (1992); Hu et al., "Detection of Pineapple Closterovirus in Pineapple Plants and Mealybugs Using Monoclonal Antibodies," Plant Pathology 45:829-836 (1996); Wakman et al., "Presence of a Clostero-like Virus and a Bacilliform Virus in Pineapple Plants in Queensland," Aust. J. Ag. R. 46:847-958 (1995)).
Polyclonal antisera produced to PMWaV in Hawaii and Australia against partially purified virions (Ullman et al., "Serology of a Closteroviruslike Particle Associated with Mealybug Wilt of Pineapple," Phvtopathologv 79:1341-1345 (1989); Wakman et al, "Presence of a Clostero-like Virus and a Bacilliform Virus in Pineapple Plants in Queensland," Aust. J. Ag. R. 46:847-958 (1995)) have been used for the serological detection of PMWaV in pineapple. These antisera, however, cross-react with the plant proteins and, thus, are difficult to use in etiology studies. Recently, monoclonal antibodies (Mabs) were produced against partially purified PMWaV and used for systematic surveys of PMWaV in commercial fields in Hawaii and elsewhere (Hu et al., "Detection of Pineapple Closterovirus in Pineapple Plants and Mealybugs Using Monoclonal Antibodies," Plant Path. 45:829-36 (1996)). Field samples showed that PMWaV is distributed throughout the major pineapple- producing areas of the world (Wakman et al., "Presence of a Clostero-like Virus and a Bacilliform Virus in Pineapple Plants in Queensland," Aust. J. Ag. R. 46:847-958 (1995); Hu et al., "Detection of Pineapple Closterovirus in Pineapple Plants and Mealybugs Using Monoclonal Antibodies," Plant Path. 45:829-36 (1996); Hu et al, "Use of a Tissue Blotting Immunoassay to Examine the Distribution of Pineapple Closterovirus in Hawaii," Plant Pis. 81:1150-1154 (1997)). A worldwide survey of PMWaV in pineapple shows that PMWaV is widespread in Hawaii and at least 12 other countries. Serological studies using polyclonal and monoclonal antibodies suggest that there are at least two different PMWaVs present in afflicted pineapple plants (Wakman et al., "Presence of a Clostero-like Virus and a Bacilliform Virus in Pineapple Plants in Queensland," Aust. J. Ag. R. 46:847-958 (1995); Hu et al, "Detection of Pineapple Closterovirus in Pineapple Plants and Mealybugs Using Monoclonal Antibodies," Plant Path. 45:829-36 (1996). Thus, two approaches have been taken to study the role of the virus in MWP. The first is to examine the distribution in pineapple and to associate it with MWP in the field. Field data shows a strong correlation between mealybug wilt symptomatic plants and infection with closterovirus. All symptomatic plants sampled were positive for closterovirus. Closterovirus infection rate was much lower for symptomless plants sampled in areas of the same field but removed from the area developing wilt. Thus, the closterovirus is designated PMWaV.
The second approach is to transmit PMWaV to healthy pineapple using mealybugs to reproduce the disease. Three experiments have been conducted. First, a MWP symptom induction experiment was conducted using groups of potted
PMWaV-free plants and PMWaV-infected plants. The two groups received 20-100 mealybugs/plant at monthly intervals or were kept mealybug-free for the duration of the experiment. After three months, only plants in the PMWaV-infected group exposed to mealybugs expressed typical symptoms of MWP; plants in the other three groups remained symptomless. Second, a radomized complete block design was used to test whether MWP symptoms could be induced under field conditions. Plots consisted of PMWaV-free plants kept mealybug free, PMWaV-free plants receiving monthly applications of mealybugs, PMWaV-infected plants kept mealybug free and PMWaV-infected plants receiving monthly applications of mealybugs. Each plot was replicated four times and contained 120 plants. Symptoms developed only on
PMWaV-infected plants in the plots received mealybug applications. Plants in all other treatments remained healthy looking. Third, cultivar susceptibility to mealybug wilt symptom development in the presence of PMWaV and mealybugs was tested. Six commercially grown Ananus comosus Smooth Cayenne cultivars from Hawaii were tested. All were susceptible to mealybug wilt when both PMWaV and mealybugs were present. PMWaV-free plants exposed to mealybugs shows signs of spotting caused by mealybug feeding but did not develop symptoms of mealybug wilt. PMWaV-free and -infected plants kept mealybug free did not develop MWP symptoms. Results from the two approaches show that there is a clear association between the PMWaV presence and symptoms of MWP for some of the cultivars, and that both PMWaV and mealybugs are essential for MWP. The working hypothesis for the etiology of MWP involves interactions between PMWaV and stress caused by mealybug feeding (i.e. insect toxins).
The present invention is directed to overcoming these deficiencies in the art.
SUMMARY OF INVENTION
The present invention relates to an isolated protein or polypeptide of a pineapple mealybug wilt virus. The encoding RNA and DNA molecules, in either isolated form or incorporated in an expression system, a host cell, or a transgenic pineapple cultivar are also disclosed.
Another aspect of the present invention relates to a method of imparting pineapple mealybug wilt virus resistance to pineapple cultivars by transforming them with a DNA molecule encoding the protein or polypeptide corresponding to a protein or polypeptide of a pineapple mealybug wilt virus. PMWaV resistant transgenic variants of the current commercial pineapple cultivars allows for more complete control of the virus, while retaining the varietal characteristics of specific cultivars. Furthermore, these variants permit control of pineapple mealybug wilt virus transmitted by mealybug vectors (Sether et al., "Transmission of Pineapple Mealybug Wilt Associated Virus by Two Species of Mealybug (Dysmicoccus spp.)," Phvtopath. 88(11):1224-30 (1998), which is hereby incorporated by reference). With respect to the latter mode of transmission, the present invention circumvents increased restriction of pesticide use which has made chemical control of insect infestation increasingly difficult.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to isolated DNA molecules encoding for the proteins or polypeptides of a PMWaV. A substantial portion of the genome has been sequenced for Types I and II of pineapple mealybug virus. Within each genome are a plurality of open reading frames ("ORFs"), each containing DNA molecules in accordance with the present invention. The complete nucleotide sequence for pineapple mealybug virus Type I (i.e. PMWaV-1) is as follows (SEQ. ID. No. 1):
cggaaattgg atcacccagc ggacgttcga gatgtaggtt cgaaggggtt tttaaacccg 60 ctattcccgt tgtcttcggt gagaaagata aaagaccctt tgaagagtcg tctagagaga 120 gtgattccac tctaatgccc gaaagaaatt tagttgtgga acatacgcaa acgttgattt 180 taggcgacga tgtgaggtca gaaggggaaa gcgtagagaa taaccaaacc gtcaacacgg 240 aagtaaacgt ctcaagatgc gtcaataagg tatcatctcc gctgaagata gtaaaccgac 300 cggagcaagg ggttgtcaag caagtgtgta aggtgcagcc ggtgaaccga tttgaagtta 360 tcaagactat cctagccaat actcttggat tttcatttac tggaatgaac agttttctag 420 acatagaaag ttataggaca ctggttagga gagtaagtgg atctgatggg tattcagccg 480 tattggaagc tttatgtcat tcgctgcacg ggttgagggc tgaggttgac gttttggaca 540 aagttgttaa acaggagatc ccgttaatcg gtcacaaaca cgaattgttt tgtaagaacg 600 tacaagagct ggatagagta aagtacaaac cggacgggtt ccattattac aacgtagagc 660 tcagcaacat ttacggttta gtgaacactc acaagttggt atacaacaac gatcctgtgg 720 ttaaagaatc ggaggggata gttttgttag aaccacatga aatctcgttc aatttaatcc 780 gatcgttagc gttaatagac cttctggtga aagttagcgt cgaggaagtt aataaggcga 840 ttgacaatgt gaaatttgtg aacgctgttc caggggcagg gaaaacctac cagatcaaac 900 agagaatgtt acgctggttc gattcggaaa aggatggtag tgcgttgtta gtgttgactt 960 cgagtagaaa ctcagctgat acgctaaaag ctttcgcaca ggagaagagg ttgggcaaga 1020 tgattcagat tttaacggtc gatgcatttt tgttccaagc gagaggaagg aatgtaagac 1080 tttataaaac gttgctgata gacgaatgct acatgacaca cgcgggcata ttgcgaggta 1140 taatcgccgc tgtgaagccg gaagagtgtg tactttacgg tgataggcga caagtgccct 1200 ttatcaacag aatcaaattg ctaaatgaca acaaatcctt tttgaaaccg agtctcggca 1260 attattcaga gatgttgata accaggcggt gtcccgcaga catctgttgg cggatgtcca 1320 atgttaacaa tgggaagaaa ggagatcgtt tgtactcagg accggtaaaa ttgtttacac 1380 agtctaaacc agtcctaaaa agtgtaacat gtaaggcgtt cagtaaaggt gatcacaatc 1440 tattctcaca agtggacagg gtcatgacct ttactcagaa cgagaagaat gaactcatct 1500 cagagtacat gagcagggga atagggacta tacaagatgc gaaaactttg atcggtacgg 1560 ttgctgagtc ccaaggtgaa acttacaaga gagttcattt agtggcattc aaaccaacag 1620 acgatcaagt attttcttcc atgccacata gactagtagc cctatcgaga cacactatca 1680 gcctgcaata cttttgtata cccaataaga tgaacaaggg cataggtgag gatgtacaat 1740 caataatcaa gctggaggaa agagtcgctg cgaattttgt agtccagcag tgcgtttaac 1800 atttatcttc cgtgtaagga tgtggtcgtc ccaataccgt tgggtttggg aagaccccct 1860 aaagctcact tcgaagccat ccaaagtttt ctcgatgacg cactgaacgg agtggggtct 1920 ttattgtttc tgagcacgga agaacagttt caaatgacgg ataccatcac caacatcacg 1980 gatttttcca tgagtgataa cgatattcgc ttcaaaccga tcaagttttt ctctaatcaa 2040 ccgaggatta gatcacagat ggtggtacgc agacagaaca ccctgaaagc aaacattttg 2100 gcttatgaaa agaggaatgc gggtgtagtt aaatctttgt ggcatccaga tgtaacagac 2160 gaagtaatga ctgtggtaga tgactttttc ggatcatatg tagattctcg aaaattgagt 2220 gaagtgttgg tggatcctgt agaaccaaat atatttgatt tagccgattg gttgttgagc 2280 agaactccaa tgggaaagaa agctttaatg atggaattgg aaaatcctgt tgaactaggt 2340 acaaatttga ataggtttaa gctgatggta aaagcggatg taaaatttaa aacagatttg 2400 gagtgtctgt cggaagtacc acctgggcag aatatagttt tccatgacag agcgatatgc 2460 gcacacttct ccgtgtgttt tagagaattg gtcaatagat tgcgcacagt cgtacacaaa 2520 aatattgtac ttttcaacgg attaagtttt gaagaatttg ctgatcagtt gagcgcggcg 2580 cttaacgatg agtcaattga tacgttcaat tgtgacgagg tagacatatc taaatacgac 2640 aaatcccaat ccacgttcac aaaagctgta gaattggaaa tctacaggag gttaggttta 2700 ccagaaaaga ttcttcaaat ctgggcagcc agcgagttct ttggcaaagc tgttactaat 2760 aagaggtcat tttctgcgga ggtatatgcc caacgtcgca caggtgctgc gaatacgtgg 2820 ataggaaaca ctgtaatcaa catgatgttg ctttcacaga gtgtagacgt gacatcattg 2880 aacgcagtct gttttgccgg tgatgactca ctgattctca agaaaggtgt tcctcgtgtc 2940 aatttcgatg tttacgattt gaagtatgag ttcgatgtca aatactttga ttgtgcctcc 3000 aaatattttt gtggtaagtt ttcaattgaa aactcgggaa agattaaagt gatgcctgat 3060 cctttcaaaa tttttgtgaa gttcggtaaa gaacgacctg aaacagataa gattttgctg 3120 gagcagtggc agtccctttt tgatataacc gaagcttaca cttcggacgc gaacatatca 3180 aagttggtat cgagtttcgc aaacaagtgg gtatcatctc cacatgctta cgaaagcttt 3240 tgtaccataa attctttacg cagcaaccca gaacaattta agaggttgtg gttcgatttg 3300 tatgcttaca atcattccag atttgaagca catataaggt ctgatgttaa gagtggataa 3360 ctttctttgg gcaatatact tgataacttt catagcactt tgcgcgatca tcatcgtttt 3420 gatattgttg tttcaaagag tgctttggcc gaacagttta ccaccaaata atccagtacc 3480 tcatggaggt gggtattgat tttggcacta cctattccac tctgtgtttc tctccaggta 3540 aaggaattga tggttgtgtg gtagagagtg acacgatatt tatacctact gtcgttggtt 3600 acaggaagga caacactcac gccataggtt tgggggcact gttggaaaaa gacttagagg 3660 tttatcgtga tataaaaagg tatttcggac tcaacaagtt caacaaagat gtgtatctcg 3720 ataaattgaa acccacaatc gaggtagtga ttgacgactg gggttgtcct ataggaccag 3780 tagacggtgc gcgcgggaaa gccaaatcag ttctcacttt agcctctgat tttataacgg 3840 gattggtaca actagcgatc aagatgacga atcaacaagt atctgtgtct gtttgttcag 3900 taccagcagc ttacaattct tatcaaaggg gttttatttt tgaaagttgt aagttgagct 3960 ctataaatgt gcaggcggta gtaaacgaac cgaccgcagc tggattgagt gctttcataa 4020 ctaccccaaa agcttctgtg aattatttgt tactctacga tttcggagga ggcacttttg 4080 atagttcctt actcgtggtt gggcctgcgt acgtgggagt actggattcg atgggagata 4140 actatctggg aggcagggac gtagataaca gattgcttga agtttgtgcg gaaaggctga 4200 aggtggacaa gaaagaatta gatcagtttt ctatggaagc cctaaaaata gatatcgtag 4260 acaacccagg gaaaagtgtt aggcgtgtgc tacttaaatc aggcaatgta aaatctattc 4320 agctaacttt ccaagatttt tcagctatat gtcgaccttt cgtggaaaga gcgagacagg 4380 tggttttatc gttgatggct ggaagacgct taacgaaatg tgcagctgtg ttaataggag 4440 gatcttcagt cctacccggg gttatagatt cagtagcttc cttacccatg atcacgaaag 4500 tgatatttga taggaaaact tacagagcgg cagttgctct aggtggagcc ttatatgcgc 4560 aaactttctc tggatcttct aggtatagat tgatagattc catttctggg agtttatcag 4620 atgaatttaa aaatttgaag gctgtgtgca tttttcccaa aggtcacccc atcccgtcta 4680 cggttgagag tcgttttacc atgccctcta cagatactgg cgttgttctt atgcagggtg 4740 agagttccat ggctaatatg aacgagatga ccttttcggg gagtgttaaa acctcgactt 4800 atccacctag atccgtagtg agacaaaaga caagaatttt tgaagatgga agagtagagg 4860 tttatttgaa tggtattaag gtggagaact ccgtgaaacc aagaatacct aacaagaccg 4920 aactcagtcc gaaattcgta agtcctgatg acgttagaat aggacctgag gtcgctgaga 4980 ttaaatcgtt ttattccaaa attgttaggt gagacggggt tgttaacgct tggtagacaa 5040 caaagggaaa tcatatataa aagacatggc tttgagagca actagcgatt acgtttcggc 5100 agacgtgaat gattcagggt ttctggactt actacggact ttctatggta aatcggaagt 5160 tgcagtagaa gccgctgaaa tatttagtta tctacgacgc aattacggtg ctatatc 5217
The complete nucleotide sequence for pineapple mealybug virus
Type 2 (i.e.PMWaV-2) is as follows (SEQ. ID. No. 2):
caacaaaatt gatcaggagt actttaagga tagatctggc agttgtatta tgaccgccaa 60 tcgtggtagc gctatagata tcaatgatac cattgagagt atagacgctg ctaatgcgag 120 caaagccgct tcgaacaacg tcagcggcgt ggaatcgata gataattatg tttgtgctcg 180 tacagttaac tcacaaatta tgaactgcaa aggagtaatg aattatacct gcgccctagt 240 tgacgaaatg tatttgatgc acaagggtct gttgatgttg ggggtattca gttctggagc 300 aagaagagct atattctacg gagatatcaa ccagatcccc ttcataaata gagagaagtg 360 tttttactct aaggagggtg tgtactgtcc aggtaaagat gaaattattt acacatcaga 420 gtcttacaga tgtcctgccg atgtttgtat gtggtcaagc tcactcaagg cgcaagctgg 480 gtctaatcgc tacctgaagg gtgtgtcatg caaccagcgt gaagtggtgt tacgcagttt 540 atccaaacgc ccggtagtgt acgcggaaca agtgatacaa ttggaagctg acgcttatat 600 aacattcaag caggagtgta aagaaaaagt cgtgagggcg ctacgagctg taggcaggag 660 ggataaagtg tttacaagcc atgaggcgca aggtatgact tttgggcggg tcgtgttatg 720 tagattaagt gccactgacg attccgtttt ttcttctgag cctcacattt tagttgcact 780 ctccagacat acacaatcct gtgtctatgc cactcttagt agtaagttag ccgacaaggt 840 aggtgcggcc atagactcag ttacgcgtaa ggaggtaagt gatacggtac ttaagacctt 900 tgtggcgtcg gcgttatttc gagctgattg agcgttgctc ctggcaacaa gatctagagc 960 gtgaagtgtt agcacgctgt tccaacagcc acttttacgt ggttaatagt ttcttggagg 1020 agatggtacc ggggagcgtt agtctggatt accgtttttt cgaggatgac tttgagttct 1080 cggaccatga attcttgata aattcgtgca ttttgcgcga caactctgta aataaactga 1140 cgtatcgtga gaactatatt tatagtttta tccgcagtaa tataggtatg ccgaaacgca 1200 atacactaaa gtgtaatttg gtgacttttg agaatcgtaa ctttaacgtc gatagggact 1260 gttatgtcgg ttgtgacgat ttcgtcgccg atgcgttagt tgagaaactt gtgaatcgat 1320 tctttttggg gaaccgatta tttgagctgc agagcgacgt tgtatgcgct aacgctgtag 1380 ccgcgagcaa ttggatcgac agtaggaccc catctgggta taaagcttta ctaagcgcgc 1440 ttggagggta tttttatacc ccagatggca tgtcgaggta taagctaatg gttaaaagcg 1500 atgccaagcc taaattagac gaaacgccgc taatgaaata cgtaactgga cagaatatag 1560 tgtaccatga tcgagctata acgagcatat ttagccagtg ttttgtgcaa atggtagagc 1620 gcttgaaata tgttactgat tcgaaggtta tactctatca cggtatggat ccgtcgaacc 1680 ttgcgaaaag gatacgcgca gacattgggg atattaacaa atactattgc tatgagctgg 1740 atatctctaa gtacgataag tctcaaggtg cacttatgaa agacgtggaa caacgagtac 1800 taaggttgtt gggactacat gaagagataa tcgatatgtt cttctgcggt gaatatgatt 1860 gtttagtttc aatgacgact cgtgagtttg agacttctat aggagcgcag cgtaggagcg 1920 gtggtgctaa tacatggcta ggcaatacta tcgttgttat gacgttattg tctatattgc 1980 tcgaagagtc acatgtagac tatattgttg tttctggcga tgattctttg attttttcca 2040 cggagccttt ggacctggat acacatactt taactcagaa ctatggcttc gattgtaaat 2100 tattgaacat gaccgcacct tatttttgct ctaagttttt agtccaatgt aaagatttat 2160 gttattttgt acctgaccct tttaagttgt ttgtaaagca tggtatctgt aaatctacta 2220 gtgtatctga cttacatgaa aggtttatgt cgttcgtcga cgtaacgaaa gatctagtaa 2280 gtgaagatgt agtcgcagcc gttgcggaat gtgtactatg gaaatatcat cgaactaatt 2340 acacttacgc cgcgatttgt gttatacacg ttttgagagc taactttcgg caatttttgc 2400 gtatgtacta tttatgcacc cctgccttaa gtatagggtg taataatgga atgaattctt 2460 ttgtcttttc taagttgata gctaagcatt ggttgaattt atttttaggt aattataaag 2520 atgtggtacc aatttttgat aaaactcgtg ctgaataggc gagtgtcatt tttaaatatt 2580 tttatttggt ttaagccgat gtgtagctat ttttagagcc aatactttat tcgtttcttt 2640 gcgtgattgc ctgatttgtt acaatgcgca attttgttta gcgtattgtc attttttgat 2700 tctttattgc ttgtaccgtc atgtgaatat tagttgagct tagtttttta gttttaggtt 2760 gtttctaatt aatgttcgtt ttgttttgta cagcttcata cagaccctcc gattatatcg 2820 cttacaaggc ggttagtgcg gggacacggg gggggtctta gtggtgatgt tggcctgaca 2880 cagtaggtct cctcacagca tcacactttt ggttgggaga gtctgcttct tgcatagcgc 2940 atctcctttg tatatatacg cgttatattg ttgttttaat tatagcgttg gtattactat 3000 tataacatat tctcagggtt ttttagttta cgttagttta taattaggtt ttcttacagt 3060 taatataatt ctattttttc acgattaggt attggctcta tcttgcgtag agtggtagta 3120 ttgtatatta tcgattgtag attgtcagta acacgagatg atatgagtgt atacattaac 3180 agatgttaga cgctttcaca gccataacta tcatagcctc tttaatctta gcttttcttt 3240 ttctgttgat actatttata gtggtactag tgtataatta ttattctaga atgcacagtt 3300 cgatgcgatc gtatggagcg gcgtaagtag tcacgacgta tggaagtagg atacgacttt 3360 ggtacaactt attccaccct gtgttattcg gcagagggtg cgtctggatg tgtgtcgttg 3420 ttcgggtcgc cgtacatcga aacgcaagtg ttcatacgcg ctgacggaac ggggtactct 3480 attgtaaaca agccgaaggc gctgtacaat gctaaagtac ctgggcgtct gtacgttaat 3540 ccaaaacggt gggtaggcgt gaatgcatac gaactagact catacgtgct aaaattaaaa 3600 ccagtgcaca gagtggaagt gttcaaggac gggtcggtaa tgctaggggg tattggtgaa 3660 ggccctgata ggacggtctc tgtaacggat atcatatccc ttttttctaa agcacttata 3720 aaggaagcgg aacagtctac tggactacgc gtaacgggtg cggtggtaac ggtaccagcc 3780 gactacaact cttttaaacg tagttttata actaactgca tgaaagactt gggtattcca 3840 gtaagggcta tagtaaatga accgaccccg gcagcgttat attctttatc tatattacaa 3900 gaaaaggatt tatttctgtc ggcttttgac tttggtggag ggacgtttga tgtgtctttt 3960 gttagaaaac tcggagatgt ggtatgcgta ctgcttagcg ttggcgataa ctttttaggg 4020 gcaagggata tcgacagggc ggtagcagct gaggtgaagg caagagtggg cgaatctatc 4080 gatacagcta cattgtcatt atttgcagcg tctattaaag aggaggtaac taatgagccg 4140 agggcaaaga cgcacgtagt aaaattggtg gatggcgtga aacttataac tttcacgtct 4200 gaagacttaa atgatatagt tcgtccgttt gccgctaggg cgctacacat atatgagcag 4260 gcggcgcaac gataccatcc tgaaacgtcg gtggctgtac tgactggtgg atcgtctgcg 4320 ttgcagtgcg ttcaagaagc actcacagct tccaaatacg actctaaagt ggtatttgat 4380 aagggtgact tcagagcctc agatagctat agtgctaaga tatattgtga tatcctagca 4440 ggagcgtcaa aacttcgatt ggtggatacg ttgacgaaca ctttaagcga tgaggtacta 4500 aacttccggc cagtgatagt attctcaaaa ggaagcgtca ttccttctga aagaaccata 4560 acgtttaata ccggcggtag aaagacgatg tatggtgtct acgaggggga ggaagtccgg 4620 tcgtatttga acgcgctaac ttttcgcgga gagtacatat ctaatgttga aggtaataga 4680 acggacagtg ctacattcag cgtatcgtca gatggtattt tgtcggtatc ggtgaatggc 4740 acgttattaa aaaatgatct cgtgccttct ccacctacag tcttttcgaa gaatctagag 4800 tatctttcca atatagagaa agtagcgaat gaaggaatac ctgagtacgc tcgacagttt 4860 atggcattat acgggcagcg aatatctagg gaagaaatat tagctgatgt cggagcattt 4920 aaagagcata aaatcgttga aaattatagt aagagatggc tataggtgtg ttatcgtgtg 4980 ttactggtga agctgatgat gatttggctc tacggcgaat taagcgtcga tacgcgagat 5040 gcacgacaag agcacacaga ttatttcgtt acctagattc gggtccctgc gttttactgt 5100 tgaatctggc atgtaagtgt agtgactcca tcgggcctgg ctagtgatgt aggtaaagcg 5160 atcgcatggg tggtgcttgt taagggtttg gaaaaaactt tgatgcatcg cgagtccgcc 5220 ttgactcaga tcttggaata tagcaacaat ctgttaacag tacagtctct gtttggaagg 5280 aaactgtatg aaattggcga cccaatggca gttctatcga gctcggagaa aagggcgatt 5340 caagcgataa ccgctaatat gactgctgtg gacaaggcta cgtcagaggc tttgagcgtt 5400 tacttaagaa aagcgactag cgtatcggag ataaaaggag attataccgt accagtagtg 5460 aagcatgaga gaatggaaag agttggagct gaggagaagt tgtttccagc ggtgataaag 5520 gcgtttttgg tagacttctc ggaggtgacg aatttcatga cggacacttc attgaatatt 5580 aaaagcgatg tatactcagc gtacgctcaa gatactgaag agtacctgca ggagtcgata 5640 gctaagcgga tagatacaca attttttaaa aaatgggtta atgtgcgatt cttcaaaagt 5700 agaccgttag atatgaccta cgagaataga gtggcttggt actcctttgt atgcgatgat 5760 attaaagtat atttagataa attttttaat tactcattca acccctctgt tcgaagcgtc 5820 gtaccttacg tcagaatcga atcttcggac aatccacacg agctgaagaa ctacttctct 5880 aacgttaact ttaggcatgg cgctcgtagc gcaagctcgt gtagagctcc gtcgatcatg 5940 ttagaaatgg ttgtacttct aatagattcg aatgttgaga ctcggttggc gccagtgcta 6000 gccatggtaa caattctgtt atggtactcc atctatggga ctaataaaac gaggctaaag 6060 aagaggtaca gatactttat aaacttgcga aacccgaaag gaaaaagagt agatatgcag 6120 gctgttgatg attatgctaa tcgtaaatca gtgagcagcg gcgtaaacat agctaggtac 6180 gtgtgtcgat actacagttc tgtaacgttt tatagtcgca aagtgctggg tataacacgt 6240 aataactggc gatcgctcat ggatattgag gggattctag cttatgatac ggtagatgcg 6300 ttaccagtag ctttagttcc taaagactgg ttacgcagct atgcgcgagc ttgcgagtat 6360 atacggttta atagtaatgt cactagaggc ggagagtatg gttcgaatac ttaatcatat 6420 gttaagtgcg agtatatatt gtaatatact agaagttatt agtgagattg atcgtagatt 6480 aaaggcgata tggctcagaa ttacgtagcc gtagtagaag gcactattct cgaaagtttg 6540 acggctccac ctaaacgatt tagagtggcg acgtctgatg tggggaaata ttacgatagt 6600 agcaaatacc gctctgtaac gggcgtagct acagccgaga gggatcggtt accagcgata 6660 gaggaaactg aactattggc aacaatccca acggaagctt caacagataa gggtgttatt 6720 cccgagactg ttaagaggtc gagtaataaa ccagaaatag tagatgatgt atcaacgttg 6780 ctgttaaatc ctagaaagaa cgttgtacta aatattggat cggttaaaac cgtgccaaag 6840 gtagttaatc agccgggttt gatatcccgg gagattgcta tccgtatagg agaggctctg 6900 aaggaacatt gcaaacaagt tatgggttcg gatagtagta cggacttagc tacatacttt 6960 atacatttga ttcaactcgc tattacgttc tctacatcca aaaatagcga atacaaagag 7020 tttgactata tagaaacaga gacgcaaaag aaaatataca tcaaggacgt gagtgaggtg 7080 gttgagagag cggcgatgaa ttcggggtac gaaaacccgt ttaggcaata tatgcgttat 7140 tttacaagct cgagtataac actaacttta aatggtaaaa taacacctaa cgagagaact 7200 atggctcatc acggagtacc caagcagttc tttgcatata cttacgattt tattgacccc 7260 gactatagcc tcatgaatca ttcggcgatt aatgcttaca acttaacgag gattcaagca 7320 tttaagaata agatagcttc agtgaacaat actatgcata acacatacca gctgaaccag 7380 ggagctgttt cagggtagga ggaagaagta gttaatttag tgttgaaaca tggaatttca 7440 gcggatacct gcagtcgagg gcagtacttt ccggttaagt gacaaactag atgataagcg 7500 taaatatatc gacctcgata acggtagttt aaaatgggaa agagtaccac atggcgaacg 7560 atacgatgcg tatgttaggg ctataacagt tgatgactac cctatagaca tgactaagtc 7620 tgatacgatt gagttagata taactgtgtt tcctatccat acatatgaca cgactagcgg 7680 gtatattatc cattttgagt ttattgagac acaagacaag tgcctgagag ttggcttggg 7740 acataacaca cagtatctag gcagacaaca tttttccgtt aaaagcgtgg acgggaatgc 7800 cagcagtgaa acgatgttag actccaattt cgtgtctaat agctggcttc aaagttctta 7860 caaaatactc ttttcgttgg tcgtaggtag aatagttgtc aaaatcgaca attattacgt 7920 atttcgtact ccggccgtac cgcagattaa cccgaagatt cttagaatag tgcggacgct 7980 gagaagtacg aactcaaatt tgaagtatgg gacagtgacg ccggcgcagt ttgcaaaatt 8040 cgggctgaag ttcggggcta acgttactaa tgttgacgaa tcgaaagtac gacatagact 8100 ggtaccacct gattggaggg cgttcatgcc aaaccgccaa tacgttgata tggttccaga 8160 cgagtcgttg gacaaggatg tgccgacacc tataccatcg gtacaaacga atacaaatgc 8220 gacaccatcg gcggaagcta tacaggagct gaataaagta ctgtcggctg acaaaataat 8280 tgaatctcgt ggtaaagcta tattagagtt tttgcccaac gctactcagt tcaacgaagc 8340 cgatatttat gacgaaaggt acctagacga acaactcagt ctgaaagtga ataccgctct 8400 aagcgcactt tgcgtggagt taatgggtga taaatcgagg ggagcgttgg aaacgctaat 8460 aattgccatg attcagttat gcgtgacgta ttctacggtt aagaatatga taatcaagaa 8520 agaatactat gttgagacaa cttataccag aaaattaata tatgtgtctt acttggcgat 8580 tagatcatgc atagacaaag cagtaggttc gagttttgaa ggtaatgctt tgaggcagta 8640 catgagatac ttcacgtata ctacagtgta cgctatgagg gcgggcttgg tcacgccgaa 8700 ttattcggcc gccgctaagc acggagttcc aaagagattc atcaattatt ccttcgacta 8760 ttgtatgttg gatcctaggt attcacaata tgacgaactt aaagctgcgt ctcttgcgca 8820 cgcttacgcg attaagctta aagccacacg tggtagcgac tctgaggtct acaatactta 8880 tagtttagga aataatggag tttagaccga tagaagtgta ctacgaacct gagaacggag 8940 gcaagctatc gtttaatgag ataaattatg agaccacttt agaaacggct gagtactata 9000 ggctatgcgg ttattcgacc ggttttaccg aaggcgacga gtataccacc aacagctggt 9060 tagtgcttaa ccctgctagg tttagagaag aggtgtatga cttgggaatc ggcgtgccct 9120 ttacttttta taataactta cgtgagctgt tggatataat acctaactta cgaacaatca 9180 aatcggttac gcttcgtcgg ctgttttcag acagtggcga agtacgtatt gttttgaagt 9240 tgaatgtgat cgaaaaagga ggactacctg tttctgtaga gatattacct gaagtgaaag 9300 ggtataggaa tatactgaag gtggtctctt gggaaaggga taatacaaga ggaatagtga 9360 aaaaatcgct attagacgca actattctgt tacccaaaat accagtatga gtgaggagat 9420 cctgaagtcg gcagatggaa tgagctgtgt gtatcactgt ttaactctaa tagctctagg 9480 agagaaaatt acgacagagg gtagagtgga actgttgatt aatcgattat ggtttactca 9540 tttatcggac gacgggaaaa tgcgtcatat gtacgacgtg gttgagaata tactcacgtt 9600 tgcacaacag cataggatta ttattccgca gcacacatcg gttttcttaa aatataatgt 9660 tggtaattta ataaacgtag atggatatac atcgttgttg attgccttag aggaatttct 9720 cgcaagaagc gatgaattac gggaacaagc ggtaagcgag ttcggtgacg gtttcggagg 9780 attttatccg gtatcacaag tagtagagtt atatgcaaaa cataactcaa aaattagcga 9840 aactggtgtt agaaggttgt tggaaaagaa gcctttacga gataaagatg tgcgtttctt 9900 tcctaaagaa ccaagcgaac gagaccttct aagtgcattt gtgtgtatta taacagatga 9960 gttatatacc cgaaactgtc gtaagaaatg aacacgaatg 10000
One DNA molecule of the present invention includes nucleotides 1 to 1799 of SEQ. ID. No. 1 and is believed to code for a helicase. This DNA molecule comprises the nucleotide sequence corresponding to SEQ. ID. No. 3 as follows:
cggaaattgg atcacccagc ggacgttcga gatgtaggtt cgaaggggtt tttaaacccg 60 ctattcccgt tgtcttcggt gagaaagata aaagaccctt tgaagagtcg tctagagaga 120 gtgattccac tctaatgccc gaaagaaatt tagttgtgga acatacgcaa acgttgattt 180 taggcgacga tgtgaggtca gaaggggaaa gcgtagagaa taaccaaacc gtcaacacgg 240 aagtaaacgt ctcaagatgc gtcaataagg tatcatctcc gctgaagata gtaaaccgac 300 cggagcaagg ggttgtcaag caagtgtgta aggtgcagcc ggtgaaccga tttgaagtta 360 tcaagactat cctagccaat actcttggat tttcatttac tggaatgaac agttttctag 420 acatagaaag ttataggaca ctggttagga gagtaagtgg atctgatggg tattcagccg 480 tattggaagc tttatgtcat tcgctgcacg ggttgagggc tgaggttgac gttttggaca 540 aagttgttaa acaggagatc ccgttaatcg gtcacaaaca cgaattgttt tgtaagaacg 600 tacaagagct ggatagagta aagtacaaac cggacgggtt ccattattac aacgtagagc 660 tcagcaacat ttacggttta gtgaacactc acaagttggt atacaacaac gatcctgtgg 720 ttaaagaatc ggaggggata gttttgttag aaccacatga aatctcgttc aatttaatcc 780 gatcgttagc gttaatagac cttctggtga aagttagcgt cgaggaagtt aataaggcga 840 ttgacaatgt gaaatttgtg aacgctgttc caggggcagg gaaaacctac cagatcaaac 900 agagaatgtt acgctggttc gattcggaaa aggatggtag tgcgttgtta gtgttgactt 960 cgagtagaaa ctcagctgat acgctaaaag ctttcgcaca ggagaagagg ttgggcaaga 1020 tgattcagat tttaacggtc gatgcatttt tgttccaagc gagaggaagg aatgtaagac 1080 tttataaaac gttgctgata gacgaatgct acatgacaca cgcgggcata ttgcgaggta 1140 taatcgccgc tgtgaagccg gaagagtgtg tactttacgg tgataggcga caagtgccct 1200 ttatcaacag aatcaaattg ctaaatgaca acaaatcctt tttgaaaccg agtctcggca 1260 attattcaga gatgttgata accaggcggt gtcccgcaga catctgttgg cggatgtcca 1320 atgttaacaa tgggaagaaa ggagatcgtt tgtactcagg accggtaaaa ttgtttacac 1380 agtctaaacc agtcctaaaa agtgtaacat gtaaggcgtt cagtaaaggt gatcacaatc 1440 tattctcaca agtggacagg gtcatgacct ttactcagaa cgagaagaat gaactcatct 1500 cagagtacat gagcagggga atagggacta tacaagatgc gaaaactttg atcggtacgg 1560 ttgctgagtc ccaaggtgaa acttacaaga gagttcattt agtggcattc aaaccaacag 1620 acgatcaagt attttcttcc atgccacata gactagtagc cctatcgaga cacactatca 1680 gcctgcaata cttttgtata cccaataaga tgaacaaggg cataggtgag gatgtacaat 1740 caataatcaa gctggaggaa agagtcgctg cgaattttgt agtccagcag tgcgtttaa 1799
This protein or polypeptide encoded by the nucleotide sequence of SEQ. ID. No. 3 has an amino acid sequence corresponding to SEQ. ID. No. 4 as follows: Glu He Gly Ser Pro Ser Gly Arg Ser Arg Cys Arg Phe Glu Gly Val 1 5 10 15
Phe Lys Pro Ala He Pro Val Val Phe Gly Glu Lys Asp Lys Arg Pro 20 25 30
Phe Glu Glu Ser Ser Arg Glu Ser Asp Ser Thr Leu Met Pro Glu Arg 35 40 45
Asn Leu Val Val Glu His Thr Gin Thr Leu He Leu Gly Asp Asp Val 50 55 60
Arg Ser Glu Gly Glu Ser Val Glu Asn Asn Gin Thr Val Asn Thr Glu 65 70 75 80
Val Asn Val Ser Arg Cys Val Asn Lys Val Ser Ser Pro Leu Lys He 85 90 95 Val Asn Arg Pro Glu Gin Gly Val Val Lys Gin Val Cys Lys Val Gin 100 105 110
Pro Val Asn Arg Phe Glu Val He Lys Thr He Leu Ala Asn Thr Leu 115 120 125
Gly Phe Ser Phe Thr Gly Met Asn Ser Phe Leu Asp He Glu Ser Tyr 130 135 140
Arg Thr Leu Val Arg Arg Val Ser Gly Ser Asp Gly Tyr Ser Ala Val 145 150 155 160
Leu Glu Ala Leu Cys His Ser Leu His Gly Leu Arg Ala Glu Val Asp 165 170 175 Val Leu Asp Lys Val Val Lys Gin Glu He Pro Leu He Gly His Lys 180 185 190
His Glu Leu Phe Cys Lys Asn Val Gin Glu Leu Asp Arg Val Lys Tyr 195 200 205
Lys Pro Asp Gly Phe His Tyr Tyr Asn Val Glu Leu Ser Asn He Tyr 210 215 220
Gly Leu Val Asn Thr His Lys Leu Val Tyr Asn Asn Asp Pro Val Val 225 230 235 240
Lys Glu Ser Glu Gly He Val Leu Leu Glu Pro His Glu He Ser Phe 245 250 255 Asn Leu He Arg Ser Leu Ala Leu He Asp Leu Leu Val Lys Val Ser 260 265 270
Val Glu Glu Val Asn Lys Ala He Asp Asn Val Lys Phe Val Asn Ala 275 280 285 Val Pro Gly Ala Gly Lys Thr Tyr Gin He Lys Gin Arg Met Leu Arg 290 295 300
Trp Phe Asp Ser Glu Lys Asp Gly Ser Ala Leu Leu Val Leu Thr Ser 305 310 315 320
Ser Arg Asn Ser Ala Asp Thr Leu Lys Ala Phe Ala Gin Glu Lys Arg 325 330 335 Leu Gly Lys Met He Gin He Leu Thr Val Asp Ala Phe Leu Phe Gin 340 345 350
Ala Arg Gly Arg Asn Val Arg Leu Tyr Lys Thr Leu Leu He Asp Glu 355 360 365
Cys Tyr Met Thr His Ala Gly He Leu Arg Gly He He Ala Ala Val 370 375 380
Lys Pro Glu Glu Cys Val Leu Tyr Gly Asp Arg Arg Gin Val Pro Phe 385 390 395 400
He Asn Arg He Lys Leu Leu Asn Asp Asn Lys Ser Phe Leu Lys Pro 405 410 415 Ser Leu Gly Asn Tyr Ser Glu Met Leu He Thr Arg Arg Cys Pro Ala 420 425 430
Asp He Cys Trp Arg Met Ser Asn Val Asn Asn Gly Lys Lys Gly Asp 435 440 445
Arg Leu Tyr Ser Gly Pro Val Lys Leu Phe Thr Gin Ser Lys Pro Val 450 455 460
Leu Lys Ser Val Thr Cys Lys Ala Phe Ser Lys Gly Asp His Asn Leu 465 470 475 480
Phe Ser Gin Val Asp Arg Val Met Thr Phe Thr Gin Asn Glu Lys Asn 485 490 495 Glu Leu He Ser Glu Tyr Met Ser Arg Gly He Gly Thr He Gin Asp 500 505 510
Ala Lys Thr Leu He Gly Thr Val Ala Glu Ser Gin Gly Glu Thr Tyr 515 520 525
Lys Arg Val His Leu Val Ala Phe Lys Pro Thr Asp Asp Gin Val Phe 530 535 540
Ser Ser Met Pro His Arg Leu Val Ala Leu Ser Arg His Thr He Ser 545 550 555 560
Leu Gin Tyr Phe Cys He Pro Asn Lys Met Asn Lys Gly He Gly Glu 565 570 575 Asp Val Gin Ser He He Lys Leu Glu Glu Arg Val Ala Ala Asn Phe 580 585 590
Val Val Gin Gin Cys Val 595
and has a molecular weight of about 67 kDa.
Another such DNA molecule includes nucleotides 1783 to 3350 of SEQ. ID. No. 1 and codes for a polymerase. This DNA molecule comprises the nucleotide sequence corresponding to SEQ. ID. No. 5 as follows:
tccagcagtg cgtttaacat ttatcttccg tgtaaggatg tggtcgtccc aataccgttg 60 ggtttgggaa gaccccctaa agctcacttc gaagccatcc aaagttttct cgatgacgca 120 ctgaacggag tggggtcttt attgtttctg agcacggaag aacagtttca aatgacggat 180 accatcacca acatcacgga tttttccatg agtgataacg atattcgctt caaaccgatc 240 aagtttttct ctaatcaacc gaggattaga tcacagatgg tggtacgcag acagaacacc 300 ctgaaagcaa acattttggc ttatgaaaag aggaatgcgg gtgtagttaa atctttgtgg 360 catccagatg taacagacga agtaatgact gtggtagatg actttttcgg atcatatgta 420 gattctcgaa aattgagtga agtgttggtg gatcctgtag aaccaaatat atttgattta 480 gccgattggt tgttgagcag aactccaatg ggaaagaaag ctttaatgat ggaattggaa 540 aatcctgttg aactaggtac aaatttgaat aggtttaagc tgatggtaaa agcggatgta 600 aaatttaaaa cagatttgga gtgtctgtcg gaagtaccac ctgggcagaa tatagttttc 660 catgacagag cgatatgcgc acacttctcc gtgtgtttta gagaattggt caatagattg 720 cgcacagtcg tacacaaaaa tattgtactt ttcaacggat taagttttga agaatttgct 780 gatcagttga gcgcggcgct taacgatgag tcaattgata cgttcaattg tgacgaggta 840 gacatatcta aatacgacaa atcccaatcc acgttcacaa aagctgtaga attggaaatc 900 tacaggaggt taggtttacc agaaaagatt cttcaaatct gggcagccag cgagttcttt 960 ggcaaagctg ttactaataa gaggtcattt tctgcggagg tatatgccca acgtcgcaca 1020 ggtgctgcga atacgtggat aggaaacact gtaatcaaca tgatgttgct ttcacagagt 1080 gtagacgtga catcattgaa cgcagtctgt tttgccggtg atgactcact gattctcaag 1140 aaaggtgttc ctcgtgtcaa tttcgatgtt tacgatttga agtatgagtt cgatgtcaaa 1200 tactttgatt gtgcctccaa atatttttgt ggtaagtttt caattgaaaa ctcgggaaag 1260 attaaagtga tgcctgatcc tttcaaaatt tttgtgaagt tcggtaaaga acgacctgaa 1320 acagataaga ttttgctgga gcagtggcag tccctttttg atataaccga agcttacact 1380 tcggacgcga acatatcaaa gttggtatcg agtttcgcaa acaagtgggt atcatctcca 1440 catgcttacg aaagcttttg taccataaat tctttacgca gcaacccaga acaatttaag 1500 aggttgtggt tcgatttgta tgcttacaat cattccagat ttgaagcaca tataaggtct 1560 gatgttaaga gtggataa 1578
The protein encoded by the nucleotide sequence of SEQ. ID. No. 5 has an amino acid sequence corresponding to SEQ. ID. No. 6 as follows:
Ser Ser Ser Ala Phe Asn He Tyr Leu Pro Cys Lys Asp Val Val Val
1 5 10 15
Pro He Pro Leu Gly Leu Gly Arg Pro Pro Lys Ala His Phe Glu Ala 20 25 30
He Gin Ser Phe Leu Asp Asp Ala Leu Asn Gly Val Gly Ser Leu Leu 35 40 45 Phe Leu Ser Thr Glu Glu Gin Phe Gin Met Thr Asp Thr He Thr Asn 50 55 60
He Thr Asp Phe Ser Met Ser Asp Asn Asp He Arg Phe Lys Pro He 65 70 75 80
Lys Phe Phe Ser Asn Gin Pro Arg He Arg Ser Gin Met Val Val Arg 85 90 95 Arg Gin Asn Thr Leu Lys Ala Asn He Leu Ala Tyr Glu Lys Arg Asn 100 105 110
Ala Gly Val Val Lys Ser Leu Trp His Pro Asp Val Thr Asp Glu Val 115 120 125
Met Thr Val Val Asp Asp Phe Phe Gly Ser Tyr Val Asp Ser Arg Lys 130 135 140
Leu Ser Glu Val Leu Val Asp Pro Val Glu Pro Asn He Phe Asp Leu 145 150 155 160
Ala Asp Trp Leu Leu Ser Arg Thr Pro Met Gly Lys Lys Ala Leu Met 165 170 175 Met Glu Leu Glu Asn Pro Val Glu Leu Gly Thr Asn Leu Asn Arg Phe 180 185 190
Lys Leu Met Val Lys Ala Asp Val Lys Phe Lys Thr Asp Leu Glu Cys 195 200 205
Leu Ser Glu Val Pro Pro Gly Gin Asn He Val Phe His Asp Arg Ala 210 215 220
He Cys Ala His Phe Ser Val Cys Phe Arg Glu Leu Val Asn Arg Leu 225 230 235 240
Arg Thr Val Val His Lys Asn He Val Leu Phe Asn Gly Leu Ser Phe 245 250 255 Glu Glu Phe Ala Asp Gin Leu Ser Ala Ala Leu Asn Asp Glu Ser He 260 265 270
Asp Thr Phe Asn Cys Asp Glu Val Asp He Ser Lys Tyr Asp Lys Ser 275 280 285
Gin Ser Thr Phe Thr Lys Ala Val Glu Leu Glu He Tyr Arg Arg Leu 290 295 300
Gly Leu Pro Glu Lys He Leu Gin He Trp Ala Ala Ser Glu Phe Phe 305 310 315 320
Gly Lys Ala Val Thr Asn Lys Arg Ser Phe Ser Ala Glu Val Tyr Ala 325 330 335 Gln Arg Arg Thr Gly Ala Ala Asn Thr Trp He Gly Asn Thr Val He 340 345 350
Asn Met Met Leu Leu Ser Gin Ser Val Asp Val Thr Ser Leu Asn Ala 355 360 365
Val Cys Phe Ala Gly Asp Asp Ser Leu He Leu Lys Lys Gly Val Pro 370 375 380 Arg Val Asn Phe Asp Val Tyr Asp Leu Lys Tyr Glu Phe Asp Val Lys 385 390 395 400
Tyr Phe Asp Cys Ala Ser Lys Tyr Phe Cys Gly Lys Phe Ser He Glu 405 410 415
Asn Ser Gly Lys He Lys Val Met Pro Asp Pro Phe Lys He Phe Val 420 425 430
Lys Phe Gly Lys Glu Arg Pro Glu Thr Asp Lys He Leu Leu Glu Gin 435 440 445
Trp Gin Ser Leu Phe Asp He Thr Glu Ala Tyr Thr Ser Asp Ala Asn 450 455 460 He Ser Lys Leu Val Ser Ser Phe Ala Asn Lys Trp Val Ser Ser Pro 465 470 475 480
His Ala Tyr Glu Ser Phe Cys Thr He Asn Ser Leu Arg Ser Asn Pro 485 490 495
Glu Gin Phe Lys Arg Leu Trp Phe Asp Leu Tyr Ala Tyr Asn His Ser 500 505 510
Arg Phe Glu Ala His He Arg Ser Asp Val Lys Ser Gly 515 520 525
and a molecular weight of about 59 kDa.
Another such DNA molecule includes nucleotides 3344 to 3349 of SEQ. ID. No. 1 and codes for an unknown protein or polypeptide. This DNA molecule comprises the nucleotide sequence corresponding to SEQ. ID. No. 7 as follows:
ctttctttgg gcaatatact tgataacttt catagcactt tgcgcgatca tcatcgtttt 60 gatattgttg tttcaaagag tgctttggcc gaacagttta ccaccaaata atccagtacc 120 tcatggaggt gggtattga 139
This DNA molecule encodes a protein or polypeptide with an amino acid sequence corresponding to SEQ. ID. No. 8 as follows: Met Leu Arg Val Asp Asn Phe Leu Trp Ala He Tyr Leu He Thr Phe 1 5 10 15
He Ala Leu Cys Ala He He He Val Leu He Leu Leu Phe Gin Arg 20 25 30
Val Leu Trp Pro Asn Ser Leu Pro Pro Asn Asn Pro Val Pro His Gly 35 40 45 Gly Gly Tyr 50
and a molecular weight of about 6 kDa. Another such DNA molecule includes nucleotides 3483 to 5012 of
SEQ. ID. No. 1 and encodes for a heat shock 70 protein or polypeptide. This DNA molecule comprises the nucleotide sequence corresponding to SEQ. ID. No. 9 as follows:
atggaggtgg gtattgattt tggcactacc tattccactc tgtgtttctc tccaggtaaa 60 ggaattgatg gttgtgtggt agagagtgac acgatattta tacctactgt cgttggttac 120 aggaaggaca acactcacgc cataggtttg ggggcactgt tggaaaaaga cttagaggtt 180 tatcgtgata taaaaaggta tttcggactc aacaagttca acaaagatgt gtatctcgat 240 aaattgaaac ccacaatcga ggtagtgatt gacgactggg gttgtcctat aggaccagta 300 gacggtgcgc gcgggaaagc caaatcagtt ctcactttag cctctgattt tataacggga 360 ttggtacaac tagcgatcaa gatgacgaat caacaagtat ctgtgtctgt ttgttcagta 420 ccagcagctt acaattctta tcaaaggggt tttatttttg aaagttgtaa gttgagctct 480 ataaatgtgc aggcggtagt aaacgaaccg accgcagctg gattgagtgc tttcataact 540 accccaaaag cttctgtgaa ttatttgtta ctctacgatt tcggaggagg cacttttgat 600 agttccttac tcgtggttgg gcctgcgtac gtgggagtac tggattcgat gggagataac 660 tatctgggag gcagggacgt agataacaga ttgcttgaag tttgtgcgga aaggctgaag 720 gtggacaaga aagaattaga tcagttttct atggaagccc taaaaataga tatcgtagac 780 aacccaggga aaagtgttag gcgtgtgcta cttaaatcag gcaatgtaaa atctattcag 840 ctaactttcc aagatttttc agctatatgt cgacctttcg tggaaagagc gagacaggtg 900 gttttatcgt tgatggctgg aagacgctta acgaaatgtg cagctgtgtt aataggagga 960 tcttcagtcc tacccggggt tatagattca gtagcttcct tacccatgat cacgaaagtg 1020 atatttgata ggaaaactta cagagcggca gttgctctag gtggagcctt atatgcgcaa 1080 actttctctg gatcttctag gtatagattg atagattcca tttctgggag tttatcagat 1140 gaatttaaaa atttgaaggc tgtgtgcatt tttcccaaag gtcaccccat cccgtctacg 1200 gttgagagtc gttttaccat gccctctaca gatactggcg ttgttcttat gcagggtgag 1260 agttccatgg ctaatatgaa cgagatgacc ttttcgggga gtgttaaaac ctcgacttat 1320 ccacctagat ccgtagtgag acaaaagaca agaatttttg aagatggaag agtagaggtt 1380 tatttgaatg gtattaaggt ggagaactcc gtgaaaccaa gaatacctaa caagaccgaa 1440 ctcagtccga aattcgtaag tcctgatgac gttagaatag gacctgaggt cgctgagatt 1500 aaatcgtttt attccaaaat tgttaggtga 1530
The nucleotide sequence of SEQ. ID. No. 9 encodes an amino acid sequence corresponding to SEQ. ID. No. 10 as follows:
Met Glu Val Gly He Asp Phe Gly Thr Thr Tyr Ser Thr Leu Cys Phe 1 5 10 15 Ser Pro Gly Lys Gly He Asp Gly Cys Val Val Glu Ser Asp Thr He 20 25 30
Phe He Pro Thr Val Val Gly Tyr Arg Lys Asp Asn Thr His Ala He 35 40 45
Gly Leu Gly Ala Leu Leu Glu Lys Asp Leu Glu Val Tyr Arg Asp He 50 55 60
Lys Arg Tyr Phe Gly Leu Asn Lys Phe Asn Lys Asp Val Tyr Leu Asp 65 70 75 80
Lys Leu Lys Pro Thr He Glu Val Val He Asp Asp Trp Gly Cys Pro 85 90 95
He Gly Pro Val Asp Gly Ala Arg Gly Lys Ala Lys Ser Val Leu Thr 100 105 110 Leu Ala Ser Asp Phe He Thr Gly Leu Val Gin Leu Ala He Lys Met 115 120 125
Thr Asn Gin Gin Val Ser Val Ser Val Cys Ser Val Pro Ala Ala Tyr 130 135 140
Asn Ser Tyr Gin Arg Gly Phe He Phe Glu Ser Cys Lys Leu Ser Ser 145 150 155 160
He Asn Val Gin Ala Val Val Asn Glu Pro Thr Ala Ala Gly Leu Ser 165 170 175
Ala Phe He Thr Thr Pro Lys Ala Ser Val Asn Tyr Leu Leu Leu Tyr 180 185 190 Asp Phe Gly Gly Gly Thr Phe Asp Ser Ser Leu Leu Val Val Gly Pro 195 200 205
Ala Tyr Val Gly Val Leu Asp Ser Met Gly Asp Asn Tyr Leu Gly Gly 210 215 220
Arg Asp Val Asp Asn Arg Leu Leu Glu Val Cys Ala Glu Arg Leu Lys 225 230 235 240
Val Asp Lys Lys Glu Leu Asp Gin Phe Ser Met Glu Ala Leu Lys He 245 250 255
Asp He Val Asp Asn Pro Gly Lys Ser Val Arg Arg Val Leu Leu Lys 260 265 270 Ser Gly Asn Val Lys Ser He Gin Leu Thr Phe Gin Asp Phe Ser Ala 275 280 285
He Cys Arg Pro Phe Val Glu Arg Ala Arg Gin Val Val Leu Ser Leu 290 295 300 Met Ala Gly Arg Arg Leu Thr Lys Cys Ala Ala Val Leu He Gly Gly 305 310 315 320
Ser Ser Val Leu Pro Gly Val He Asp Ser Val Ala Ser Leu Pro Met 325 330 335
He Thr Lys Val He Phe Asp Arg Lys Thr Tyr Arg Ala Ala Val Ala 340 345 350 Leu Gly Gly Ala Leu Tyr Ala Gin Thr Phe Ser Gly Ser Ser Arg Tyr 355 360 365
Arg Leu He Asp Ser He Ser Gly Ser Leu Ser Asp Glu Phe Lys Asn 370 375 380
Leu Lys Ala Val Cys He Phe Pro Lys Gly His Pro He Pro Ser Thr 385 390 395 400
Val Glu Ser Arg Phe Thr Met Pro Ser Thr Asp Thr Gly Val Val Leu 405 410 415
Met Gin Gly Glu Ser Ser Met Ala Asn Met Asn Glu Met Thr Phe Ser 420 425 430 Gly Ser Val Lys Thr Ser Thr Tyr Pro Pro Arg Ser Val Val Arg Gin 435 440 445
Lys Thr Arg He Phe Glu Asp Gly Arg Val Glu Val Tyr Leu Asn Gly 450 455 460
He Lys Val Glu Asn Ser Val Lys Pro Arg He Pro Asn Lys Thr Glu 465 470 475 480
Leu Ser Pro Lys Phe Val Ser Pro Asp Asp Val Arg He Gly Pro Glu 485 490 495
Val Ala Glu He Lys Ser Phe Tyr Ser Lys He Val Arg 500 505
and a molecular weight of about 55.4 kDa.
Another such DNA molecule includes nucleotides 8490 to 7398 of SEQ. ID. No. 2 and codes for a first coat protein. This DNA molecule comprises a nucleotide sequence corresponding to SEQ. ID. No. 11 as follows:
atggctcaga attacgtagc cgtagtagaa ggcactattc tcgaaagttt gacggctcca 60 cctaaacgat ttagagtggc gacgtctgat gtggggaaat attacgatag tagcaaatac 120 cgctctgtaa cgggcgtagc tacagccgag agggatcggt taccagcgat agaggaaact 180 gaactattgg caacaatccc aacggaagct tcaacagata agggtgttat tcccgagact 240 gttaagaggt cgagtaataa accagaaata gtagatgatg tatcaacgtt gctgttaaat 300 cctagaaaga acgttgtact aaatattgga tcggttaaaa ccgtgccaaa ggtagttaat 360 cagccgggtt tgatatcccg ggagattgct atccgtatag gagaggctct gaaggaacat 420 tgcaaacaag ttatgggttc ggatagtagt acggacttag ctacatactt tatacatttg 480 attcaactcg ctattacgtt ctctacatcc aaaaatagcg aatacaaaga gtttgactat 540 atagaaacag agacgcaaaa gaaaatatac atcaaggacg tgagtgaggt ggttgagaga 600 gcggcgatga attcggggta cgaaaacccg tttaggcaat atatgcgtta ttttacaagc 660 tcgagtataa cactaacttt aaatggtaaa ataacaccta acgagagaac tatggctcat 720 cacggagtac ccaagcagtt ctttgcatat acttacgatt ttattgaccc cgactatagc 780 ctcatgaatc attcggcgat taatgcttac aacttaacga ggattcaagc atttaagaat 840 aagatagctt cagtgaacaa tactatgcat aacacatacc agctgaacca gggagctgtt 900 tcagggtag 909
The nucleic acid of SEQ. ID. No. 11 encodes a protein having an amino acid sequence corresponding to SEQ. ID. No. 12 as follows:
Met Ala Gin Asn Tyr Val Ala Val Val Glu Gly Thr He Leu Glu Ser 1 5 10 15
Leu Thr Ala Pro Pro Lys Arg Phe Arg Val Ala Thr Ser Asp Val Gly 20 25 30
Lys Tyr Tyr Asp Ser Ser Lys Tyr Arg Ser Val Thr Gly Val Ala Thr 35 40 45
Ala Glu Arg Asp Arg Leu Pro Ala He Glu Glu Thr Glu Leu Leu Ala 50 55 60
Thr He Pro Thr Glu Ala Ser Thr Asp Lys Gly Val He Pro Glu Thr 65 70 75 80 Val Lys Arg Ser Ser Asn Lys Pro Glu He Val Asp Asp Val Ser Thr
85 90 95
Leu Leu Leu Asn Pro Arg Lys Asn Val Val Leu Asn He Gly Ser Val 100 105 110
Lys Thr Val Pro Lys Val Val Asn Gin Pro Gly Leu He Ser Arg Glu 115 120 125
He Ala He Arg He Gly Glu Ala Leu Lys Glu His Cys Lys Gin Val 130 135 140
Met Gly Ser Asp Ser Ser Thr Asp Leu Ala Thr Tyr Phe He His Leu 145 150 155 160 He Gin Leu Ala He Thr Phe Ser Thr Ser Lys Asn Ser Glu Tyr Lys
165 170 175
Glu Phe Asp Tyr He Glu Thr Glu Thr Gin Lys Lys He Tyr He Lys 180 185 190
Asp Val Ser Glu Val Val Glu Arg Ala Ala Met Asn Ser Gly Tyr Glu 195 200 205
Asn Pro Phe Arg Gin Tyr Met Arg Tyr Phe Thr Ser Ser Ser He Thr 210 215 220 Leu Thr Leu Asn Gly Lys He Thr Pro Asn Glu Arg Thr Met Ala His 225 230 235 240
His Gly Val Pro Lys Gin Phe Phe Ala Tyr Thr Tyr Asp Phe He Asp 245 250 255
Pro Asp Tyr Ser Leu Met Asn His Ser Ala He Asn Ala Tyr Asn Leu 260 265 270
Thr Arg He Gin Ala Phe Lys Asn Lys He Ala Ser Val Asn Asn Thr 275 280 285
Met His Asn Thr Tyr Gin Leu Asn Gin Gly Ala Val Ser Gly 290 295 300
and a molecular weight of about 33 kDa.
Yet another DNA molecule of the present invention includes nucleotides 7430 to 8905 of SEQ. ID. No. 2 and codes for a second coat protein. This DNA molecule comprises a nucleotide sequence corresponding to SEQ. ID. No. 13 as follows:
atggaatttc agcggatacc tgcagtcgag ggcagtactt tccggttaag tgacaaacta 60 gatgataagc gtaaatatat cgacctcgat aacggtagtt taaaatggga aagagtacca 120 catggcgaac gatacgatgc gtatgttagg gctataacag ttgatgacta ccctatagac 180 atgactaagt ctgatacgat tgagttagat ataactgtgt ttcctatcca tacatatgac 240 acgactagcg ggtatattat ccattttgag tttattgaga cacaagacaa gtgcctgaga 300 gttggcttgg gacataacac acagtatcta ggcagacaac atttttccgt taaaagcgtg 360 gacgggaatg ccagcagtga aacgatgtta gactccaatt tcgtgtctaa tagctggctt 420 caaagttctt acaaaatact cttttcgttg gtcgtaggta gaatagttgt caaaatcgac 480 aattattacg tatttcgtac tccggccgta ccgcagatta acccgaagat tcttagaata 540 gtgcggacgc tgagaagtac gaactcaaat ttgaagtatg ggacagtgac gccggcgcag 600 tttgcaaaat tcgggctgaa gttcggggct aacgttacta atgttgacga atcgaaagta 660 cgacatagac tggtaccacc tgattggagg gcgttcatgc caaaccgcca atacgttgat 720 atggttccag acgagtcgtt ggacaaggat gtgccgacac ctataccatc ggtacaaacg 780 aatacaaatg cgacaccatc ggcggaagct atacaggagc tgaataaagt actgtcggct 840 gacaaaataa ttgaatctcg tggtaaagct atattagagt ttttgcccaa cgctactcag 900 ttcaacgaag ccgatattta tgacgaaagg tacctagacg aacaactcag tctgaaagtg 960 aataccgctc taagcgcact ttgcgtggag ttaatgggtg ataaatcgag gggagcgttg 1020 gaaacgctaa taattgccat gattcagtta tgcgtgacgt attctacggt taagaatatg 1080 ataatcaaga aagaatacta tgttgagaca acttatacca gaaaattaat atatgtgtct 1140 tacttggcga ttagatcatg catagacaaa gcagtaggtt cgagttttga aggtaatgct 1200 ttgaggcagt acatgagata cttcacgtat actacagtgt acgctatgag ggcgggcttg 1260 gtcacgccga attattcggc cgccgctaag cacggagttc caaagagatt catcaattat 1320 tccttcgact attgtatgtt ggatcctagg tattcacaat atgacgaact taaagctgcg 1380 tctcttgcgc acgcttacgc gattaagctt aaagccacac gtggtagcga ctctgaggtc 1440 tacaatactt atagtttagg aaataatgga gtttag 1476
The DNA molecule of SEQ. ID. No. 13 encodes a protein having an amino acid sequence corresponding to SEQ. ID. No. 14 as follows: Met Glu Phe Gin Arg He Pro Ala Val Glu Gly Ser Thr Phe Arg Leu 1 5 10 15 Ser Asp Lys Leu Asp Asp Lys Arg Lys Tyr He Asp Leu Asp Asn Gly 20 25 30
Ser Leu Lys Trp Glu Arg Val Pro His Gly Glu Arg Tyr Asp Ala Tyr 35 40 45
Val Arg Ala He Thr Val Asp Asp Tyr Pro He Asp Met Thr Lys Ser 50 55 60
Asp Thr He Glu Leu Asp He Thr Val Phe Pro He Leu Thr Tyr Asp 65 70 75 80
Thr Thr Ser Gly Tyr He He His Phe Glu Phe He Glu Thr Gin Asp 85 90 95 Lys Cys Leu Arg Val Gly Leu Gly His Asn Thr Gin Tyr Leu Gly Arg 100 105 110
Gin His Phe Ser Val Lys Ser Val Asp Gly Asn Ala Ser Ser Glu Thr 115 120 125
Met Leu Asp Ser Asn Phe Val Ser Asn Ser Trp Leu Gin Ser Ser Tyr 130 135 140
Lys He Leu Phe Ser Leu Val Val Gly Arg He Val Val Lys He Asp 145 150 155 160
Asn Tyr Tyr Val Phe Arg Thr Pro Ala Val Pro Gin He Asn Pro Lys 165 170 175 He Leu Arg He Val Arg Thr Leu Arg Ser Thr Asn Ser Asn Leu Lys 180 185 190
Tyr Gly Thr Val Thr Pro Ala Gin Phe Ala Lys Phe Gly Leu Lys Phe 195 200 205
Gly Ala Asn Val Thr Asn Val Asp Glu Ser Lys Val Arg His Arg Leu 210 215 220
Val Pro Pro Asp Trp Arg Ala Phe Met Pro Asn Arg Gin Tyr Val Asp 225 230 235 240
Met Val Pro Asp Glu Ser Leu Asp Lys Asp Val Pro Thr Pro He Pro 245 250 255 Ser Val Gin Thr Asn Thr Asn Ala Thr Pro Ser Ala Glu Ala He Gin 260 265 270
Glu Leu Asn Lys Val Leu Ser Ala Asp Lys He He Glu Ser Arg Gly 275 280 285 Lys Ala He Leu Glu Phe Leu Pro Asn Ala Thr Gin Phe Asn Glu Ala
290 295 300
Asp He Tyr Asp Glu Arg Tyr Leu Asp Glu Gin Leu Ser Leu Lys Val 305 310 315 320
Asn Thr Ala Leu Ser Ala Leu Cys Val Glu Leu Met Gly Asp Lys Ser 325 330 335 Arg Gly Ala Leu Glu Thr Leu He He Ala Met He Gin Leu Cys Val 340 345 350
Thr Tyr Ser Thr Val Lys Asn Met He He Lys Lys Glu Tyr Tyr Val 355 360 365
Glu Thr Thr Tyr Thr Arg Lys Leu He Tyr Val Ser Tyr Leu Ala He
370 375 380
Arg Ser Cys He Asp Lys Ala Val Gly Ser Ser Phe Glu Gly Asn Ala 385 390 395 400
Leu Arg Gin Tyr Met Arg Tyr Phe Thr Tyr Thr Thr Val Tyr Ala Met 405 410 415 Arg Ala Gly Leu Val Thr Pro Asn Tyr Ser Ala Ala Ala Lys His Gly 420 425 430
Val Pro Lys Arg Phe He Asn Tyr Ser Phe Asp Tyr Cys Met Leu Asp 435 440 445
Pro Arg Tyr Ser Gin Tyr Asp Glu Leu Lys Ala Ala Ser Leu Ala His 450 455 460
Ala Tyr Ala He Lys Leu Lys Ala Thr Arg Gly Ser Asp Ser Glu Val 465 470 475 480
Tyr Asn Thr Tyr Ser Leu Gly Asn Asn Gly Val 485 490
and a molecular weight of about 54 kDa.
Another such DNA molecule includes nucleotides 8895 to 9410 of SEQ. ID. No. 2 and codes for an unknown protein. This DNA molecule comprises a nucleotide sequence corresponding to SEQ. ID. No. 15 as follows:
atggagttta gaccgataga agtgtactac gaacctgaga acggaggcaa gctatcgttt 60 aatgagataa attatgagac cactttagaa acggctgagt actataggct atgcggttat 120 tcgaccggtt ttaccgaagg cgacgagtat accaccaaca gctggttagt gcttaaccct 180 gctaggttta gagaagaggt gtatgacttg ggaatcggcg tgccctttac tttttataat 240 aacttacgtg agctgttgga tataatacct aacttacgaa caatcaaatc ggttacgctt 300 cgtcggctgt tttcagacag tggcgaagta cgtattgttt tgaagttgaa tgtgatcgaa 360 aaaggaggac tacctgtttc tgtagagata ttacctgaag tgaaagggta taggaatata 420 ctgaaggtgg tctcttggga aagggataat acaagaggaa tagtgaaaaa atcgctatta 480 gacgcaacta ttctgttacc caaaatacca gtatga 516
The DNA molecule of SEQ. ID. No. 15 encodes a protein which has an amino acid sequence corresponding to SEQ. ID. No. 16 as follows:
Met Glu Phe Arg Pro He Glu Val Tyr Tyr Glu Pro Glu Asn Gly Gly 1 5 10 15
Lys Leu Ser Phe Asn Glu He Asn Tyr Glu Thr Thr Leu Glu Thr Ala 20 25 30
Glu Tyr Tyr Arg Leu Cys Gly Tyr Ser Thr Gly Phe Thr Glu Gly Asp 35 40 45
Glu Tyr Thr Thr Asn Ser Trp Leu Val Leu Asn Pro Ala Arg Phe Arg 50 55 60 Glu Glu Val Tyr Asp Leu Gly He Gly Val Pro Phe Thr Phe Tyr Asn 65 70 75 80
Asn Leu Arg Glu Leu Leu Asp He He Pro Asn Leu Arg Thr He Lys 85 90 95
Ser Val Thr Leu Arg Arg Leu Phe Ser Asp Ser Gly Glu Val Arg He 100 105 110
Val Leu Lys Leu Asn Val He Glu Lys Gly Gly Leu Pro Val Ser Val 115 120 125
Glu He Leu Pro Glu Val Lys Gly Tyr Arg Asn He Leu Lys Val Val
130 135 140 Ser Trp Glu Arg Asp Asn Thr Arg Gly He Val Lys Lys Ser Leu Leu
145 150 155 160
Asp Ala Thr He Leu Leu Pro Lys He Pro Val 165 170
and a molecular weight of about 19.7 kDa.
Another such DNA molecule includes nucleotides 5203 to 6414 of SEQ. ID. No. 2 and codes for an unknown protein or polypeptide. This DNA molecule comprises a nucleotide sequence corresponding to SEQ. ID. No. 17 as follows:
atgcatcgcg agtccgcctt gactcagatc ttggaatata gcaacaatct gttaacagta 60 cagtctctgt ttggaaggaa actgtatgaa attggcgacc caatggcagt tctatcgagc 120 tcggagaaaa gggcgattca agcgataacc gctaatatga ctgctgtgga caaggctacg 180 tcagaggctt tgagcgttta cttaagaaaa gcgactagcg tatcggagat aaaaggagat 240 tataccgtac cagtagtgaa gcatgagaga atggaaagag ttggagctga ggagaagttg 300 tttccagcgg tgataaaggc gtttttggta gacttctcgg aggtgacgaa tttcatgacg 360 gacacttcat tgaatattaa aagcgatgta tactcagcgt acgctcaaga tactgaagag 420 tacctgcagg agtcgatagc taagcggata gatacacaat tttttaaaaa atgggttaat 480 gtgcgattct tcaaaagtag accgttagat atgacctacg agaatagagt ggcttggtac 540 tcctttgtat gcgatgatat taaagtatat ttagataaat tttttaatta ctcattcaac 600 ccctctgttc gaagcgtcgt accttacgtc agaatcgaat cttcggacaa tccacacgag 660 ctgaagaact acttctctaa cgttaacttt aggcatggcg ctcgtagcgc aagctcgtgt 720 agagctccgt cgatcatgtt agaaatggtt gtacttctaa tagattcgaa tgttgagact 780 cggttggcgc cagtgctagc catggtaaca attctgttat ggtactccat ctatgggact 840 aataaaacga ggctaaagaa gaggtacaga tactttataa acttgcgaaa cccgaaagga 900 aaaagagtag atatgcaggc tgttgatgat tatgctaatc gtaaatcagt gagcagcggc 960 gtaaacatag ctaggtacgt gtgtcgatac tacagttctg taacgtttta tagtcgcaaa 1020 gtgctgggta taacacgtaa taactggcga tcgctcatgg atattgaggg gattctagct 1080 tatgatacgg tagatgcgtt accagtagct ttagttccta aagactggtt acgcagctat 1140 gcgcgagctt gcgagtatat acggtttaat agtaatgtca ctagaggcgg agagtatggt 1200 tcgaatactt aat 1213
The DNA molecule of SEQ. ID. No. 17 encodes the protein or polypeptide having a deduced amino acid sequence corresponding to SEQ. ID. No. 18 as follows:
Met His Arg Glu Ser Ala Leu Thr Gin He Leu Glu Tyr Ser Asn Asn 1 5 10 15
Leu Leu Thr Val Gin Ser Leu Phe Gly Arg Lys Leu Tyr Glu He Gly 20 25 30 Asp Pro Met Ala Val Leu Ser Ser Ser Glu Lys Arg Ala He Gin Ala 35 40 45
He Thr Ala Asn Met Thr Ala Val Asp Lys Ala Thr Ser Glu Ala Leu
50 55 60
Ser Val Tyr Leu Arg Lys Ala Thr Ser Val Ser Glu He Lys Gly Asp
65 70 75 80
Tyr Thr Val Pro Val Val Lys His Glu Arg Met Glu Arg Val Gly Ala 85 90 95
Glu Glu Lys Leu Phe Pro Ala Val He Lys Ala Phe Leu Val Asp Phe 100 105 110 Ser Glu Val Thr Asn Phe Met Thr Asp Thr Ser Leu Asn He Lys Ser 115 120 125
Asp Val Tyr Ser Ala Tyr Ala Gin Asp Thr Glu Glu Tyr Leu Gin Glu 130 135 140
Ser He Ala Lys Arg He Asp Thr Gin Phe Phe Lys Lys Trp Val Asn 145 150 155 160
Val Arg Phe Phe Lys Ser Arg Pro Leu Asp Met Thr Tyr Glu Asn Arg 165 170 175 Val Ala Trp Tyr Ser Phe Val Cys Asp Asp He Lys Val Tyr Leu Asp 180 185 190
Lys Phe Phe Asn Tyr Ser Phe Asn Pro Ser Val Arg Ser Val Val Pro 195 200 205
Tyr Val Arg He Glu Ser Ser Asp Asn Pro His Glu Leu Lys Asn Tyr 210 215 220 Phe Ser Asn Val Asn Phe Arg His Gly Ala Arg Ser Ala Ser Ser Cys 225 230 235 240
Arg Ala Pro Ser He Met Leu Glu Met Val Val Leu Leu He Asp Ser 245 250 255
Asn Val Glu Thr Arg Leu Ala Pro Val Leu Ala Met Val Thr He Leu 260 265 270
Leu Trp Tyr Ser He Tyr Gly Thr Asn Lys Thr Arg Leu Lys Lys Arg 275 280 285
Tyr Arg Tyr Phe He Asn Leu Arg Asn Pro Lys Gly Lys Arg Val Asp 290 295 300 Met Gin Ala Val Asp Asp Tyr Ala Asn Arg Lys Ser Val Ser Ser Gly 305 310 315 320
Val Asn He Ala Arg Tyr Val Cys Arg Tyr Tyr Ser Ser Val Thr Phe 325 330 335
Tyr Ser Arg Lys Val Leu Gly He Thr Arg Asn Asn Trp Arg Ser Leu 340 345 350
Met Asp He Glu Gly He Leu Ala Tyr Asp Thr Val Asp Ala Leu Pro 355 360 365
Val Ala Leu Val Pro Lys Asp Trp Leu Arg Ser Tyr Ala Arg Ala Cys 370 375 380 Glu Tyr He Arg Phe Asn Ser Asn Val Thr Arg Gly Gly Glu Tyr Gly 385 390 395 400
Ser Asn Thr
and a molecular weight of about 46.4 kDa.
Yet another such DNA molecule includes nucleotides 1 to 931 of SEQ. ID. No. 2 and codes for a helicase protein or polypeptide. This DNA molecule comprises a nucleotide sequence corresponding to SEQ. ID. No. 19 as follows: caacaaaatt gatcaggagt actttaagga tagatctggc agttgtatta tgaccgccaa 60 tcgtggtagc gctatagata tcaatgatac cattgagagt atagacgctg ctaatgcgag 120 caaagccgct tcgaacaacg tcagcggcgt ggaatcgata gataattatg tttgtgctcg 180 tacagttaac tcacaaatta tgaactgcaa aggagtaatg aattatacct gcgccctagt 240 tgacgaaatg tatttgatgc acaagggtct gttgatgttg ggggtattca gttctggagc 300 aagaagagct atattctacg gagatatcaa ccagatcccc ttcataaata gagagaagtg 360 tttttactct aaggagggtg tgtactgtcc aggtaaagat gaaattattt acacatcaga 420 gtcttacaga tgtcctgccg atgtttgtat gtggtcaagc tcactcaagg cgcaagctgg 480 gtctaatcgc tacctgaagg gtgtgtcatg caaccagcgt gaagtggtgt tacgcagttt 540 atccaaacgc ccggtagtgt acgcggaaca agtgatacaa ttggaagctg acgcttatat 600 aacattcaag caggagtgta aagaaaaagt cgtgagggcg ctacgagctg taggcaggag 660 ggataaagtg tttacaagcc atgaggcgca aggtatgact tttgggcggg tcgtgttatg 720 tagattaagt gccactgacg attccgtttt ttcttctgag cctcacattt tagttgcact 780 ctccagacat acacaatcct gtgtctatgc cactcttagt agtaagttag ccgacaaggt 840 aggtgcggcc atagactcag ttacgcgtaa ggaggtaagt gatacggtac ttaagacctt 900 tgtggcgtcg gcgttatttc gagctgattg a 931
This DNA molecule encodes a protein or polypeptide with a deduced amino acid sequence corresponding to SEQ. ID. No. 20 as follows:
Asn Lys He Asp Gin Glu Tyr Phe Lys Asp Arg Ser Gly Ser Cys He 1 5 10 15
Met Thr Ala Asn Arg Gly Ser Ala He Asp He Asn Asp Thr He Glu 20 25 30
Ser He Asp Ala Ala Asn Ala Ser Lys Ala Ala Ser Asn Asn Val Ser 35 40 45
Gly Val Glu Ser He Asp Asn Tyr Val Cys Ala Arg Thr Val Asn Ser 50 55 60 Gin He Met Asn Cys Lys Gly Val Met Asn Tyr Thr Cys Ala Leu Val 65 70 75 80
Asp Glu Met Tyr Leu Met His Lys Gly Leu Leu Met Leu Gly Val Phe 85 90 95
Ser Ser Gly Ala Arg Arg Ala He Phe Tyr Gly Asp He Asn Gin He 100 105 110
Pro Phe He Asn Arg Glu Lys Cys Phe Tyr Ser Lys Glu Gly Val Tyr 115 120 125
Cys Pro Gly Lys Asp Glu He He Tyr Thr Ser Glu Ser Tyr Arg Cys
130 135 140 Pro Ala Asp Val Cys Met Trp Ser Ser Ser Leu Lys Ala Gin Ala Gly 145 150 155 160
Ser Asn Arg Tyr Leu Lys Gly Val Ser Cys Asn Gin Arg Glu Val Val 165 170 175 Leu Arg Ser Leu Ser Lys Arg Pro Val Val Tyr Ala Glu Gin Val He 180 185 190
Gin Leu Glu Ala Asp Ala Tyr He Thr Phe Lys Gin Glu Cys Lys Glu 195 200 205
Lys Val Val Arg Ala Leu Arg Ala Val Gly Arg Arg Asp Lys Val Phe 210 215 220 Thr Ser His Glu Ala Gin Gly Met Thr Phe Gly Arg Val Val Leu Cys 225 230 235 240
Arg Leu Ser Ala Thr Asp Asp Ser Val Phe Ser Ser Glu Pro His He 245 250 255
Leu Val Ala Leu Ser Arg His Thr Gin Ser Cys Val Tyr Ala Thr Leu 260 265 270
Ser Ser Lys Leu Ala Asp Lys Val Gly Ala Ala He Asp Ser Val Thr 275 280 285
Arg Lys Glu Val Ser Asp Thr Val Leu Lys Thr Phe Val Ala Ser Ala 290 295 300 Leu Phe Arg Ala Asp 305
and a molecular weight of about 34.1 kDa. Another DNA molecule of the present invention includes nucleotides 879 to 2558 of SEQ. ID. No. 2 and codes for a polymerase protein or polypeptide. This DNA molecule comprises a nucleotide sequence corresponding to SEQ. ID. No. 21 as follows:
gtgatacggt acttaagacc tttgtggcgt cggcgttatt tcgagctgat tgagcgttgc 60 tcctggcaac aagatctaga gcgtgaagtg ttagcacgct gttccaacag ccacttttac 120 gtggttaata gtttcttgga ggagatggta ccggggagcg ttagtctgga ttaccgtttt 180 ttcgaggatg actttgagtt ctcggaccat gaattcttga taaattcgtg cattttgcgc 240 gacaactctg taaataaact gacgtatcgt gagaactata tttatagttt tatccgcagt 300 aatataggta tgccgaaacg caatacacta aagtgtaatt tggtgacttt tgagaatcgt 360 aactttaacg tcgataggga ctgttatgtc ggttgtgacg atttcgtcgc cgatgcgtta 420 gttgagaaac ttgtgaatcg attctttttg gggaaccgat tatttgagct gcagagcgac 480 gttgtatgcg ctaacgctgt agccgcgagc aattggatcg acagtaggac cccatctggg 540 tataaagctt tactaagcgc gcttggaggg tatttttata ccccagatgg catgtcgagg 600 tataagctaa tggttaaaag cgatgccaag cctaaattag acgaaacgcc gctaatgaaa 660 tacgtaactg gacagaatat agtgtaccat gatcgagcta taacgagcat atttagccag 720 tgttttgtgc aaatggtaga gcgcttgaaa tatgttactg attcgaaggt tatactctat 780 cacggtatgg atccgtcgaa ccttgcgaaa aggatacgcg cagacattgg ggatattaac 840 aaatactatt gctatgagct ggatatctct aagtacgata agtctcaagg tgcacttatg 900 aaagacgtgg aacaacgagt actaaggttg ttgggactac atgaagagat aatcgatatg 960 ttcttctgcg gtgaatatga ttgtttagtt tcaatgacga ctcgtgagtt tgagacttct 1020 ataggagcgc agcgtaggag cggtggtgct aatacatggc taggcaatac tatcgttgtt 1080 atgacgttat tgtctatatt gctcgaagag tcacatgtag actatattgt tgtttctggc 1140 gatgattctt tgattttttc cacggagcct ttggacctgg atacacatac tttaactcag 1200 aactatggct tcgattgtaa attattgaac atgaccgcac cttatttttg ctctaagttt 1260 ttagtccaat gtaaagattt atgttatttt gtacctgacc cttttaagtt gtttgtaaag 1320 catggtatct gtaaatctac tagtgtatct gacttacatg aaaggtttat gtcgttcgtc 1380 gacgtaacga aagatctagt aagtgaagat gtagtcgcag ccgttgcgga atgtgtacta 1440 tggaaatatc atcgaactaa ttacacttac gccgcgattt gtgttataca cgttttgaga 1500 gctaactttc ggcaattttt gcgtatgtac tatttatgca cccctgcctt aagtataggg 1560 tgtaataatg gaatgaattc ttttgtcttt tctaagttga tagctaagca ttggttgaat 1620 ttatttttag gtaattataa agatgtggta ccaatttttg ataaaactcg tgctgaatag 1680
This DNA molecule encodes a protein or polypeptide with a deduced amino acid sequence corresponding to SEQ. ID. No. 22 as follows:
Val He Arg Tyr Leu Arg Pro Leu Trp Arg Arg Arg Tyr Phe Glu Leu 1 5 10 15
He Glu Arg Cys Ser Trp Gin Gin Asp Leu Glu Arg Glu Val Leu Ala 20 25 30
Arg Cys Ser Asn Ser His Phe Tyr Val Val Asn Ser Phe Leu Glu Glu 35 40 45
Met Val Pro Gly Ser Val Ser Leu Asp Tyr Arg Phe Phe Glu Asp Asp 50 55 60
Phe Glu Phe Ser Asp His Glu Phe Leu He Asn Ser Cys He Leu Arg 65 70 75 80 Asp Asn Ser Val Asn Lys Leu Thr Tyr Arg Glu Asn Tyr He Tyr Ser
85 90 95
Phe He Arg Ser Asn He Gly Met Pro Lys Arg Asn Thr Leu Lys Cys 100 105 110
Asn Leu Val Thr Phe Glu Asn Arg Asn Phe Asn Val Asp Arg Asp Cys 115 120 125
Tyr Val Gly Cys Asp Asp Phe Val Ala Asp Ala Leu Val Glu Lys Leu 130 135 140
Val Asn Arg Phe Phe Leu Gly Asn Arg Leu Phe Glu Leu Gin Ser Asp 145 150 155 160 Val Val Cys Ala Asn Ala Val Ala Ala Ser Asn Trp He Asp Ser Arg
165 170 175
Thr Pro Ser Gly Tyr Lys Ala Leu Leu Ser Ala Leu Gly Gly Tyr Phe 180 185 190
Tyr Thr Pro Asp Gly Met Ser Arg Tyr Lys Leu Met Val Lys Ser Asp 195 200 205
Ala Lys Pro Lys Leu Asp Glu Thr Pro Leu Met Lys Tyr Val Thr Gly 210 215 220 Gin Asn He Val Tyr His Asp Arg Ala He Thr Ser He Phe Ser Gin 225 230 235 240
Cys Phe Val Gin Met Val Glu Arg Leu Lys Tyr Val Thr Asp Ser Lys 245 250 255
Val He Leu Tyr His Gly Met Asp Pro Ser Asn Leu Ala Lys Arg He 260 265 270
Arg Ala Asp He Gly Asp He Asn Lys Tyr Tyr Cys Tyr Glu Leu Asp 275 280 285
He Ser Lys Tyr Asp Lys Ser Gin Gly Ala Leu Met Lys Asp Val Glu 290 295 300
Gin Arg Val Leu Arg Leu Leu Gly Leu His Glu Glu He He Asp Met 305 310 315 320 Phe Phe Cys Gly Glu Tyr Asp Cys Leu Val Ser Met Thr Thr Arg Glu
325 330 335
Phe Glu Thr Ser He Gly Ala Gin Arg Arg Ser Gly Gly Ala Asn Thr 340 345 350
Trp Leu Gly Asn Thr He Val Val Met Thr Leu Leu Ser He Leu Leu 355 360 365
Glu Glu Ser His Val Asp Tyr He Val Val Ser Gly Asp Asp Ser Leu 370 375 380
He Phe Ser Thr Glu Pro Leu Asp Leu Asp Thr His Thr Leu Thr Gin 385 390 395 400 Asn Tyr Gly Phe Asp Cys Lys Leu Leu Asn Met Thr Ala Pro Tyr Phe
405 410 415
Cys Ser Lys Phe Leu Val Gin Cys Lys Asp Leu Cys Tyr Phe Val Pro 420 425 430
Asp Pro Phe Lys Leu Phe Val Lys His Gly He Cys Lys Ser Thr Ser 435 440 445
Val Ser Asp Leu His Glu Arg Phe Met Ser Phe Val Asp Val Thr Lys 450 455 460
Asp Leu Val Ser Glu Asp Val Val Ala Ala Val Ala Glu Cys Val Leu 465 470 475 480 Trp Lys Tyr His Arg Thr Asn Tyr Thr Tyr Ala Ala He Cys Val He
485 490 495
His Val Leu Arg Ala Asn Phe Arg Gin Phe Leu Arg Met Tyr Tyr Leu 500 505 510 Cys Thr Pro Ala Leu Ser He Gly Cys Asn Asn Gly Met Asn Ser Phe 515 520 525
Val Phe Ser Lys Leu He Ala Lys His Trp Leu Asn Leu Phe Leu Gly 530 535 540
Asn Tyr Lys Asp Val Val Pro He Phe Asp Lys Thr Arg Ala Glu 545 550 555
and has a molecule weight of about 64 kDa.
Another DNA molecule of the present invention includes nucleotides 3173 to 3326 of SEQ. ID. No. 2 and codes for an unknown protein or polypeptide. This DNA molecule comprises a nucleotide sequence corresponding to SEQ. ID. No. 23 as follows:
atgttagacg ctttcacagc cataactatc atagcctctt taatcttagc ttttcttttt 60 ctgttgatac tatttatagt ggtactagtg tataattatt attctagaat gcacagttcg 120 atgcgatcgt atggagcggc gtaa 144
This DNA molecule encodes a protein or polypeptide with a deduced amino acid sequence corresponding to SEQ. ID. No. 24 as follows:
Met Leu Asp Ala Phe Thr Ala He Thr He He Ala Ser Leu He Leu 1 5 10 15
Ala Phe Leu Phe Leu Leu He Leu Phe He Val Val Leu Val Tyr Asn 20 25 30
Tyr Tyr Ser Arg Met His Ser Ser Met Arg Ser Tyr Gly Ala Ala 35 40 45
and has a molecular weight of about 6 kDa.
Another DNA molecule of the present invention includes nucleotides 3340 to 4965 of SEQ. ID. No. 2 and codes for a heat shock protein 70 protein or polypeptide. The DNA molecule comprises a nucleotide sequence corresponding to SEQ. ID. No. 25 as follows:
atggaagtag gatacgactt tggtacaact tattccaccc tgtgttattc ggcagagggt 60 gcgtctggat gtgtgtcgtt gttcgggtcg ccgtacatcg aaacgcaagt gttcatacgc 120 gctgacggaa cggggtactc tattgtaaac aagccgaagg cgctgtacaa tgctaaagta 180 cctgggcgtc tgtacgttaa tccaaaacgg tgggtaggcg tgaatgcata cgaactagac 240 tcatacgtgc taaaattaaa accagtgcac agagtggaag tgttcaagga cgggtcggta 300 atgctagggg gtattggtga aggccctgat aggacggtct ctgtaacgga tatcatatcc 360 cttttttcta aagcacttat aaaggaagcg gaacagtcta ctggactacg cgtaacgggt 420 gcggtggtaa cggtaccagc cgactacaac tcttttaaac gtagttttat aactaactgc 480 atgaaagact tgggtattcc agtaagggct atagtaaatg aaccgacccc ggcagcgtta 540 tattctttat ctatattaca agaaaaggat ttatttctgt cggcttttga ctttggtgga 600 gggacgtttg atgtgtcttt tgttagaaaa ctcggagatg tggtatgcgt actgcttagc 660 gttggcgata actttttagg ggcaagggat atcgacaggg cggtagcagc tgaggtgaag 720 gcaagagtgg gcgaatctat cgatacagct acattgtcat tatttgcagc gtctattaaa 780 gaggaggtaa ctaatgagcc gagggcaaag acgcacgtag taaaattggt ggatggcgtg 840 aaacttataa ctttcacgtc tgaagactta aatgatatag ttcgtccgtt tgccgctagg 900 gcgctacaca tatatgagca ggcggcgcaa cgataccatc ctgaaacgtc ggtggctgta 960 ctgactggtg gatcgtctgc gttgcagtgc gttcaagaag cactcacagc ttccaaatac 1020 gactctaaag tggtatttga taagggtgac ttcagagcct cagatagcta tagtgctaag 1080 atatattgtg atatcctagc aggagcgtca aaacttcgat tggtggatac gttgacgaac 1140 actttaagcg atgaggtact aaacttccgg ccagtgatag tattctcaaa aggaagcgtc 1200 attccttctg aaagaaccat aacgtttaat accggcggta gaaagacgat gtatggtgtc 1260 tacgaggggg aggaagtccg gtcgtatttg aacgcgctaa cttttcgcgg agagtacata 1320 tctaatgttg aaggtaatag aacggacagt gctacattca gcgtatcgtc agatggtatt 1380 ttgtcggtat cggtgaatgg cacgttatta aaaaatgatc tcgtgccttc tccacctaca 1440 gtcttttcga agaatctaga gtatctttcc aatatagaga aagtagcgaa tgaaggaata 1500 cctgagtacg ctcgacagtt tatggcatta tacgggcagc gaatatctag ggaagaaata 1560 ttagctgatg tcggagcatt taaagagcat aaaatcgttg aaaattatag taagagatgg 1620 ctatag 1626
The DNA molecule of SEQ. ID. No. 25 encodes a protein of polypeptide with a deduced amino acid sequence corresponding to SEQ. ID. No. 26 as follows:
Met Glu Val Gly Tyr Asp Phe Gly Thr Thr Tyr Ser Thr Leu Cys Tyr 1 5 10 15 Ser Ala Glu Gly Ala Ser Gly Cys Val Ser Leu Phe Gly Ser Pro Tyr
20 25 30
He Glu Thr Gin Val Phe He Arg Ala Asp Gly Thr Gly Tyr Ser He 35 40 45
Val Asn Lys Pro Lys Ala Leu Tyr Asn Ala Lys Val Pro Gly Arg Leu 50 55 60
Tyr Val Asn Pro Lys Arg Trp Val Gly Val Asn Ala Tyr Glu Leu Asp 65 70 75 80
Ser Tyr Val Leu Lys Leu Lys Pro Val His Arg Val Glu Val Phe Lys 85 90 95 Asp Gly Ser Val Met Leu Gly Gly He Gly Glu Gly Pro Asp Arg Thr 100 105 110
Val Ser Val Thr Asp He He Ser Leu Phe Ser Lys Ala Leu He Lys 115 120 125
Glu Ala Glu Gin Ser Thr Gly Leu Arg Val Thr Gly Ala Val Val Thr 130 135 140
Val Pro Ala Asp Tyr Asn Ser Phe Lys Arg Ser Phe He Thr Asn Cys 145 150 155 160 Met Lys Asp Leu Gly He Pro Val Arg Ala He Val Asn Glu Pro Thr 165 170 175
Pro Ala Ala Leu Tyr Ser Leu Ser He Leu Gin Glu Lys Asp Leu Phe 180 185 190
Leu Ser Ala Phe Asp Phe Gly Gly Gly Thr Phe Asp Val Ser Phe Val 195 200 205 Arg Lys Leu Gly Asp Val Val Cys Val Leu Leu Ser Val Gly Asp Asn 210 215 220
Phe Leu Gly Ala Arg Asp He Asp Arg Ala Val Ala Ala Glu Val Lys 225 230 235 240
Ala Arg Val Gly Glu Ser He Asp Thr Ala Thr Leu Ser Leu Phe Ala 245 250 255
Ala Ser He Lys Glu Glu Val Thr Asn Glu Pro Arg Ala Lys Thr His 260 265 270
Val Val Lys Leu Val Asp Gly Val Lys Leu He Thr Phe Thr Ser Glu 275 280 285 Asp Leu Asn Asp He Val Arg Pro Phe Ala Ala Arg Ala Leu His He 290 295 300
Tyr Glu Gin Ala Ala Gin Arg Tyr His Pro Glu Thr Ser Val Ala Val 305 310 315 320
Leu Thr Gly Gly Ser Ser Ala Leu Gin Cys Val Gin Glu Ala Leu Thr 325 330 335
Ala Ser Lys Tyr Asp Ser Lys Val Val Phe Asp Lys Gly Asp Phe Arg 340 345 350
Ala Ser Asp Ser Tyr Ser Ala Lys He Tyr Cys Asp He Leu Ala Gly 355 360 365 Ala Ser Lys Leu Arg Leu Val Asp Thr Leu Thr Asn Thr Leu Ser Asp 370 375 380
Glu Val Leu Asn Phe Arg Pro Val He Val Phe Ser Lys Gly Ser Val 385 390 395 400
He Pro Ser Glu Arg Thr He Thr Phe Asn Thr Gly Gly Arg Lys Thr 405 410 415
Met Tyr Gly Val Tyr Glu Gly Glu Glu Val Arg Ser Tyr Leu Asn Ala 420 425 430
Leu Thr Phe Arg Gly Glu Tyr He Ser Asn Val Glu Gly Asn Arg Thr 435 440 445 Asp Ser Ala Thr Phe Ser Val Ser Ser Asp Gly He Leu Ser Val Ser 450 455 460
Val Asn Gly Thr Leu Leu Lys Asn Asp Leu Val Pro Ser Pro Pro Thr 465 470 475 480
Val Phe Ser Lys Asn Leu Glu Tyr Leu Ser Asn He Glu Lys Val Ala 485 490 495 Asn Glu Gly He Pro Glu Tyr Ala Arg Gin Phe Met Ala Leu Tyr Gly 500 505 510
Gin Arg He Ser Arg Glu Glu He Leu Ala Asp Val Gly Ala Phe Lys 515 520 525
Glu His Lys He Val Glu Asn Tyr Ser Lys Arg Trp Leu 530 535 540
and has a molecular weight of 59 kDa.
Another DNA molecule of the present invention includes nucleotides 9407 to 9991 of SEQ. ID. No. 2 and codes for an unknown protein or polypeptide. This DNA molecule comprises a nucleotide sequence of SEQ. ID. No. 27 as follows:
atgagtgagg agatcctgaa gtcggcagat ggaatgagct gtgtgtatca ctgtttaact 60 ctaatagctc taggagagaa aattacgaca gagggtagag tggaactgtt gattaatcga 120 ttatggttta ctcatttatc ggacgacggg aaaatgcgtc atatgtacga cgtggttgag 180 aatatactca cgtttgcaca acagcatagg attattattc cgcagcacac atcggttttc 240 ttaaaatata atgttggtaa tttaataaac gtagatggat atacatcgtt gttgattgcc 300 ttagaggaat ttctcgcaag aagcgatgaa ttacgggaac aagcggtaag cgagttcggt 360 gacggtttcg gaggatttta tccggtatca caagtagtag agttatatgc aaaacataac 420 tcaaaaatta gcgaaactgg tgttagaagg ttgttggaaa agaagccttt acgagataaa 480 gatgtgcgtt tctttcctaa agaaccaagc gaacgagacc ttctaagtgc atttgtgtgt 540 attataacag atgagttata tacccgaaac tgtcgtaaga aatga 585
This DNA molecule encodes a protein or polypeptide with a deduced amino acid sequence corresponding to SEQ. ID. No. 28 as follows:
Met Ser Glu Glu He Leu Lys Ser Ala Asp Gly Met Ser Cys Val Tyr 1 5 10 15
His Cys Leu Thr Leu He Ala Leu Gly Glu Lys He Thr Thr Glu Gly 20 25 30
Arg Val Glu Leu Leu He Asn Arg Leu Trp Phe Thr His Leu Ser Asp 35 40 45 Asp Gly Lys Met Arg His Met Tyr Asp Val Val Glu Asn He Leu Thr 50 55 60 Phe Ala Gin Gin His Arg He He He Pro Gin His Thr Ser Val Phe 65 70 75 80
Leu Lys Tyr Asn Val Gly Asn Leu He Asn Val Asp Gly Tyr Thr Ser 85 90 95
Leu Leu He Ala Leu Glu Glu Phe Leu Ala Arg Ser Asp Glu Leu Arg 100 105 110
Glu Gin Ala Val Ser Glu Phe Gly Asp Gly Phe Gly Gly Phe Tyr Pro 115 120 125
Val Ser Gin Val Val Glu Leu Tyr Ala Lys His Asn Ser Lys He Ser 130 135 140
Glu Thr Gly Val Arg Arg Leu Leu Glu Lys Lys Pro Leu Arg Asp Lys
145 150 155 160 Asp Val Arg Phe Phe Pro Lys Glu Pro Ser Glu Arg Asp Leu Leu Ser
165 170 175
Ala Phe Val Cys He He Thr Asp Glu Leu Tyr Thr Arg Asn Cys Arg 180 185 190
Lys Lys
and has a molecular weight of 22.3 kDa.
Also encompassed by the present invention are fragments of the DNA molecules of the present invention. Suitable fragments capable of imparting pineapple mealybug wilt virus resistance to pineapple plants are constructed by using appropriate restriction sites, revealed by inspection of the DNA molecule's sequence, to: (i) insert an interposon (Felley et al., "Interposon Mutagenesis of Soil and Water Bacteria: a Family of DNA Fragments Designed for in vitro Insertion Mutagenesis of Gram-negative Bacteria," Gene. 52:147-15 (1987), which is hereby incorporated by reference) such that truncated forms of the pineapple mealybug wilt virus polypeptides or proteins, that lack various amounts of the C-terminus, can be produced or (ii) delete various internal portions of the protein. Alternatively, the sequence can be used to amplify any portion of the coding region, such that it can be cloned into a vector supplying both transcription and translation start signals. Suitable DNA molecules are those that hybridize to a DNA molecule comprising a nucleotide sequence of at least 15 continuous bases of SEQ. ID. Nos. 1 or 2 (or any of the above recited portions of those sequences) under stringent conditions characterized by a hybridization buffer comprising 0.9M sodium citrate ("SSC") buffer at a temperature of 42°C and remaining bound when subject to washing with SSC buffer at 42°C; and preferably in a hybridization buffer comprising 20% formamide in 0.9M saline/0.9M SSC buffer at a temperature of 42°C and remaining bound when subject to washing at 42°C with 0.2x SSC buffer at 42°C. Variants may also (or alternatively) be modified by, for example, the deletion or addition of nucleotides that have minimal influence on the properties, secondary structure and hydropathic nature of the encoded polypeptide. For example, the nucleotides encoding a polypeptide may be conjugated to a signal (or leader) sequence at the N-terminal end of the protein which co-translationally or post-translationally directs transfer of the protein. The nucleotide sequence may also be altered so that the encoded polypeptide is conjugated to a linker or other sequence for ease of synthesis, purification, or identification of the polypeptide.
The protein or polypeptide of the present invention is preferably produced in purified form (preferably, at least about 80%, more preferably 90%, pure) by conventional techniques. Typically, the protein or polypeptide of the present invention is isolated by lysing and sonication. After washing, the lysate pellet is resuspended in buffer containing Tris-HCl. During dialysis, a precipitate forms from this protein solution. The solution is centrifuged, and the pellet is washed and resuspended in the buffer containing Tris-HCl. Proteins are resolved by electrophoresis through an SDS 12% polyacrylamide gel.
The DNA molecule encoding the pineapple mealybut wilt virus protein or polypeptide of the present invention can be incorporated in cells using conventional recombinant DNA technology. Generally, this involves inserting the DNA molecule into an expression system to which the DNA molecule is heterologous (i.e. not normally present). The heterologous DNA molecule is inserted into the expression system or vector in proper sense orientation and correct reading frame. The vector contains the necessary elements for the transcription and translation of the inserted protein-coding sequences.
U.S. Patent No. 4,237,224 to Cohen and Boyer, which is hereby incorporated by reference, describes the production of expression systems in the form of recombinant plasmids using restriction enzyme cleavage and ligation with DNA ligase. These recombinant plasmids are then introduced by means of transformation and replicated in unicellular cultures including procaryotic organisms and eucaryotic cells grown in tissue culture.
Recombinant genes may also be introduced into viruses, such as vaccinia virus. Recombinant viruses can be generated by transfection of plasmids into cells infected with virus.
Suitable vectors include, but are not limited to, the following viral vectors such as lambda vector system gtl 1, gt WES.tB, Charon 4, and plasmid vectors such as pBR322, pBR325, pACYC177, pACYC184, pUC8, pUC9, pUC18, pUC19, pLG339, pR290, pKC37, pKClOl, SV 40, pBluescript II SK +/- or KS +/- (see "Stratagene Cloning Systems" Catalog (1993) from Stratagene, La Jolla, Calif, which is hereby incorporated by reference), pQE, pIH821, pGEX, pET series (see Studier et. al., "Use of T7 RNA Polymerase to Direct Expression of Cloned Genes," Gene Expression Technology, vol. 185 (1990), which is hereby incorporated by reference), and any derivatives thereof. Recombinant molecules can be introduced into cells via transformation, transduction, conjugation, mobilization, or electroporation. The DNA sequences are cloned into the vector using standard cloning procedures in the art, as described by Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Springs Laboratory, Cold Springs Harbor, New York (1982), which is hereby incorporated by reference.
A variety of host- vector systems may be utilized to express the protein-encoding sequence(s). Primarily, the vector system must be compatible with the host cell used. Host- vector systems include but are not limited to the following: bacteria transformed with bacteriophage DNA, plasmid DNA, or cosmid DNA; microorganisms such as yeast containing yeast vectors; mammalian cell systems infected with virus (e.g., vaccinia virus, adenovirus, etc.); insect cell systems infected with virus (e.g., baculovirus); and plant cells infected by bacteria or transformed via particle bombardment (i.e. biolistics). The expression elements of these vectors vary in their strength and specificities. Depending upon the host- vector system utilized, any one of a number of suitable transcription and translation elements can be used. Different genetic signals and processing events control many levels of gene expression (e.g., DNA transcription and messenger RNA ("mRNA") translation).
Transcription of DNA is dependent upon the presence of a promoter which is a DNA sequence that directs the binding of RNA polymerase and thereby promotes mRNA synthesis. The DNA sequences of eucaryotic promoters differ from those of procaryotic promoters. Furthermore, eucaryotic promoters and accompanying genetic signals may not be recognized in or may not function in a procaryotic system, and, further, procaryotic promoters are not recognized and do not function in eucaryotic cells. Similarly, translation of mRNA in procaryotes depends upon the presence of the proper procaryotic signals which differ from those of eucaryotes. Efficient translation of mRNA in procaryotes requires a ribosome binding site called the Shine-Dalgarno ("SD") sequence on the mRNA. This sequence is a short nucleotide sequence of mRNA that is located before the start codon, usually AUG, which encodes the amino-terminal methionine of the protein. The SD sequences are complementary to the 3 '-end of the 16S rRNA (ribosomal RNA) and probably promote binding of mRNA to ribosomes by duplexing with the rRNA to allow correct positioning of the ribosome. For a review on maximizing gene expression, see Roberts and Lauer, Methods in Enzymology. 68:473 (1979), which is hereby incorporated by reference.
Promoters vary in their "strength" (i.e. their ability to promote transcription). For the purposes of expressing a cloned gene, it is desirable to use strong promoters in order to obtain a high level of transcription and, hence, expression of the gene. Depending upon the host cell system utilized, any one of a number of suitable promoters may be used. For instance, when cloning in E. coli, its bacteriophages, or plasmids, promoters such as the T7 phage promoter, lac promoter, trp promoter, recA promoter, ribosomal RNA promoter, the PR and PL promoters of coliphage lambda and others, including but not limited, to /αcUV5, ompF, bla, Ipp, and the like, may be used to direct high levels of transcription of adjacent DNA segments. Additionally, a hybrid trp-lac\JY5 (tac) promoter or other E. coli promoters produced by recombinant DNA or other synthetic DNA techniques may be used to provide for transcription of the inserted gene.
Bacterial host cell strains and expression vectors may be chosen which inhibit the action of the promoter unless specifically induced. In certain operons, the addition of specific inducers is necessary for efficient transcription of the inserted DNA. For example, the lac operon is induced by the addition of lactose or IPTG (isopropylthio-beta-D-galactoside). A variety of other operons, such as trp, pro, etc., are under different controls.
Specific initiation signals are also required for efficient gene transcription and translation in procaryotic cells. These transcription and translation initiation signals may vary in "strength" as measured by the quantity of gene specific messenger RNA and protein synthesized, respectively. The DNA expression vector, which contains a promoter, may also contain any combination of various "strong" transcription and/or translation initiation signals. For instance, efficient translation in E. coli requires a Shine-Dalgarno ("SD") sequence about 7- 9 bases 5' to the initiation codon ("ATG") to provide a ribosome binding site. Thus, any SD-ATG combination that can be utilized by host cell ribosomes may be employed. Such combinations include but are not limited to the SD-ATG combination from the cro gene or the N gene of coliphage lambda, or from the E. coli tryptophan Ε, D, C, B or A genes. Additionally, any SD-ATG combination produced by recombinant DΝA or other techniques involving incorporation of synthetic nucleotides may be used.
Once the isolated DΝA molecules encoding the various pineapple mealybug wilt virus proteins or polypeptides, as described above, have been cloned into an expression system, they are ready to be incorporated into a host cell. Such incorporation can be carried out by the various forms of transformation noted above, depending upon the vector/host cell system. Suitable host cells include, but are not limited to, bacteria, virus, yeast, mammalian cells, insect, plant, and the like.
The present invention also relates to RNA molecules which encode the various pineapple mealybug wilt virus proteins or polypeptides described above. The transcripts can be synthesized using the host cells of the present invention by any of the conventional techniques. The mRNA can be translated either in vitro or in vivo. Cell-free systems typically include wheat-germ or reticulocyte extracts. In vivo translation can be effected, for example, by microinjection into frog oocytes. One aspect of the present invention involves using one or more of the above DNA molecules encoding the various proteins or polypeptides of a pineapple mealybug wilt virus to transform pineapple plants in order to impart pineapple mealybug wilt virus resistance to the plants. The mechanism by which resistance is imparted is not known. One possibility, however, is that the transformed plant can express a protein or polypeptide of pineapple mealybug wilt virus, and, when the transformed plant is inoculated by a pineapple mealybug wilt virus, the expressed protein or polypeptide prevents translation of the viral RNA. In this aspect of the present invention the subject DNA molecule incorporated in the plant can be constitutively expressed. Alternatively, expression can be regulated by a promoter which is activated by the presence of pineapple mealybug wilt virus. Suitable promoters for these purposes include those from genes expressed in response to pineapple mealybug wilt virus infiltration.
The isolated DNA molecules of the present invention can be utilized to impart pineapple mealybug wilt virus resistance for a wide variety of pineapple plants. The term "pineapple" refers to a member of the genera Ananas and Pseudoananas of the Bromeliaceae family. The genus Pseudoananas is monotypic, i.e., consists of P. sagenarius. The genus Ananas consists of five species, namely, A. bracteatus, A. ftitzmuelleri, A. comosus, A. erectifώlius, and A. ananassoides. Other genera comprised within the Bromeliaceae family include Tillansia, Aechmea, Neoregrelia, etc. Plant tissue suitable for transformation include leaf tissue, root tissue, meristems, zygotic and somatic embryos, and anthers.
The expression system of the present invention can be used to transform virtually any plant tissue under suitable conditions. Tissue cells transformed in accordance with the present invention can be grown in vitro in a suitable medium to impart pineapple mealybug wilt virus resistance. Transformed cells can be regenerated into whole plants such that the protein or polypeptide imparts resistance to pineapple mealybug wilt virus in the intact transgenic plants. In either case, the plant cells transformed with the recombinant DNA expression system of the present invention are grown and caused to express that DNA molecule to produce one of the above-described pineapple mealybug wilt virus proteins or polypeptides and, thus, impart pineapple mealybug wilt virus resistance.
In producing transgenic plants, the DNA construct in a vector described above can be microinjected directly into plant cells by use of micropipettes to transfer mechanically the recombinant DNA. Crossway, Mol. Gen. Genetics,
202:179-85 (1985), which is hereby incorporated by reference. The genetic material may also be transferred into the plant cell using polyethylene glycol. Krens, et al., Nature, 296:72-74 (1982), which is hereby incoφorated by reference.
One technique of. transforming plants with the DNA molecules in accordance with the present invention is by contacting the tissue of such plants with an inoculum of a bacteria transformed with a vector comprising a gene in accordance with the present invention which imparts pineapple mealybug wilt virus resistance. Generally, this procedure involves inoculating the plant tissue with a suspension of bacteria and incubating the tissue for 48 to 72 hours on regeneration medium without antibiotics at 25-28°C.
Bacteria from the genus Agrobacterium can be utilized to transform plant cells. Suitable species of such bacterium include Agrobacterium tumefaciens and Agrobacterium rhizogenes. Agrobacterium tumefaciens (e.g., strains C58, LBA4404, or EHA105) is particularly useful due to its well-known ability to transform plants.
Heterologous genetic sequences can be introduced into appropriate plant cells, by means of the Ti plasmid of A. tumefaciens or the Ri plasmid of A. rhizogenes. The Ti or Ri plasmid is transmitted to plant cells on infection by Agrobacterium and is stably integrated into the plant genome. J. Schell, Science, 237:1176-83 (1987), which is hereby incoφorated by reference.
After transformation, the transformed plant cells must be regenerated. Plant regeneration from cultured protoplasts is described in Evans et al, Handbook of Plant Cell Cultures, Vol. 1: (MacMillan Publishing Co., New York, 1983); and Vasil I.R. (ed.), Cell Culture and Somatic Cell Genetics of Plants. Acad. Press, Orlando, Vol. I, 1984, and Vol. Ill (1986), which are hereby incoφorated by reference. It is known that practically all plants can be regenerated from cultured cells or tissues.
Regeneration generally involves providing a suspension of transformed protoplasts or a petri plate containing explants. Callus tissue is formed and shoots may be induced from callus and subsequently rooted. Alternatively, embryo formation can be induced in the callus tissue. These embryos germinate as natural embryos to form plants. The culture media will generally contain various amino acids and hormones, such as auxin and cytokinins. It is also advantageous to add glutamic acid and proline to the medium. Efficient regeneration will depend on the medium, on the genotype, and on the history of the culture. If these three variables are controlled, then regeneration is usually reproducible and repeatable.
Once transgenic plants of this type are produced, the plants themselves can be propagated vegetatively by tissue culture so that the DNA construct is present in the resulting plants.
Another approach to transforming plant cells with a gene which imparts resistance to pathogens is particle bombardment (also known as biolistic transformation) of the host cell. This can be accomplished in one of several ways. The first involves propelling inert or biologically active particles at cells. This technique is disclosed in U.S. Patent Nos. 4,945,050, 5,036,006, and 5,100,792, all to Sanford et al., and in Emerschad et al., "Somatic Embryogenesis and Plant Development from Immature Zygotic Embryos of Seedless Grapes (Vitis vinifera) Plant Cell Reports. 14:6-12 (1995) ("Emerschad (1995)"), which are hereby incoφorated by reference. Generally, this procedure involves propelling inert or biologically active particles at the cells under conditions effective to penetrate the outer surface of the cell and to be incoφorated within the interior thereof. When inert particles are utilized, the vector can be introduced into the cell by coating the particles with the vector containing the heterologous DNA. Alternatively, the target cell can be surrounded by the vector so that the vector is carried into the cell by the wake of the particle. Biologically active particles (e.g., dried bacterial cells containing the vector and heterologous DNA) can also be propelled into plant cells.
One approach to transforming pineapple plants is disclosed in U.S. Patent Application Serial No. 09/078,862, filed May 14, 1998 which is hereby incoφorated by reference.
Once a pineapple plant tissue is transformed in accordance with the present invention, the transformed tissue is regenerated to form a transgenic plant. Generally, regeneration is accomplished by culturing transformed tissue on medium containing the appropriate growth regulators and nutrients to allow for the initiation of shoot meristems. Appropriate antibiotics are added to the regeneration medium to inhibit the growth of Agrobacterium and to select for the development of transformed cells. Following shoot initiation, shoots are allowed to develop tissue culture and are screened for marker gene activity. The DNA molecules of the present invention can be made capable of transcription to a messenger RNA, which, although encoding for a pineapple mealybug wilt virus protein or polypeptide, does not translate to the protein. This is known as RNA-mediated resistance. When pineapple is transformed with such a DNA molecule, the DNA molecule can be transcribed under conditions effective to maintain the messenger RNA in the plant cell at low level density readings. Alternatively, it may be desirable to transform pineapple with a DNA molecule that encodes an antisense RNA which hybridizes to mRNA molecules transcribed from a pineapple mealybug wilt virus Type I or II. As a result, translation of the encoded protein is repressed. Pineapple mealybug wilt virus can be detected in a sample using a nucleotide sequence of the DNA molecule, or a fragment thereof, encoding for a protein or polypeptide of the present invention. The nucleotide sequence is provided as a probe in a nucleic acid hybridization assay or a gene amplification detection procedure (e.g., using a polymerase chain reaction procedure). The nucleic acid probes of the present invention may be used in any nucleic acid hybridization assay system known in the art, including, but not limited to, Southern blots (Southern, E.M., "Detection of Specific Sequences Among DNA Fragments Separated by Gel
Electrophoresis," J. Mol. Biol.. 98:503-17 (1975), which is hereby incoφorated by reference), Northern blots (Thomas, P.S., "Hybridization of Denatured RNA and Small DNA Fragments Transferred to Nitrocellulose," Proc. Nat'l Acad. Sci. USA. 77:5201-05 (1980), which is hereby incoφorated by reference), and Colony blots (Grunstein, M., et al., "Colony Hybridization: A Method for the Isolation of Cloned cDNAs that Contain a Specific Gene," Proc. Nat'l Acad. Sci. USA. 72:3961-65 (1975), which is hereby incoφorated by reference). Alternatively, the probes can be used in a gene amplification detection procedure (e.g., a polymerase chain reaction). Erlich, H.A., et. al., "Recent Advances in the Polymerase Chain Reaction," Science 252:1643-51 (1991), which is hereby incoφorated by reference. Any reaction with the probe is detected so that the presence of a pineapple mealybug wilt virus in the sample is indicated. Such detection is facilitated by providing the probe of the present invention with a label. Suitable labels include a radioactive compound, a fluorescent compound, a chemiluminescent compound, an enzymatic compound, or other equivalent nucleic acid labels.
Nucleic acid (DNA or RNA) probes of the present invention will hybridize to complementary pineapple mealybug wilt virus nucleic acids under stringent conditions. Generally, stringent conditions are selected to be about 50°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. The Tm is dependent upon the solution conditions and the base composition of the probe, and may be calculated using the following equation:
Tm = 79.8°C + (18.5 x Log[Na+]) + (58.4°C x %[G+C])
(820 / #bp in duplex) (0.5 x % formamide) Nonspecific binding may also be controlled using any one of a number of known techniques such as, for example, blocking the membrane with protein-containing solutions, addition of heterologous RNA, DNA, and SDS to the hybridization buffer, and treatment with RNase. Wash conditions are typically performed at or below stringency. Generally, suitable stringent conditions for nucleic acid hybridization assays or gene amplification detection procedures are set forth above. More or less stringent conditions may also be selected.
EXAMPLES
The following examples are provided to illustrate embodiments of the present invention but are by no means intended to limit its scope.
Example 1 - Identification of PMWaV-infected Plants
Thirty-eight Champaka 153 Smooth Cayenne pineapple plants grown under glasshouse conditions were assayed for the presence of PMWaV using a tissue blot immunoassay (TBIA) described by Hu et al., "Use of a Tissue Blotting Immunoassay to Examine the Distribution of Pineapple Closterovirus in Hawaii," Plant Dis. 81 :1150-1154 (1997), which is hereby incoφorated by reference. A young stem leaf (3 - 4 cm wide, 25 - 30 cm long) was pulled from each plant and a transverse cut was made at the leaf base with a razor blade. The freshly cut cross- sections were immediately pressed onto a 0.45 μm Nitro ME nitrocellulose membrane (MSI; Westboro, MA). Membranes were gently agitated in PBS buffer (140 mM NaCl, 10 mM Na2HPO4, 3 mM KCl, 2 mM KH2PO4> pH 7.4) with 2% of powdered milk (w/v) being added for 60 min at room temperature. Membranes were transferred to TBS buffer (50 mM Tris-HCl, 50 mM NaCl, pH 7.5) containing 1 μg of PMWaV monoclonal IgG antibody (Hu et al., "Pineapple Closterovirus in Mealybug Wilt of Pineapple," In: Abstracts of the Xth International Congress of Virology. Jerusalem, Israel, Aug. 11-16, 1996, Abstract PW61-7 (1996), which is hereby incoφorated by reference) per mL of buffer and gently agitated for 4 h at room temperature, and stored in this solution overnight at 4°C. Following two 10 min washes in PBST [PBS + 0.5% Tween 20 (v/v)], membranes were placed in TBS buffer containing a 1:1000 dilution of goat anti-mouse IgG alkaline phosphatase conjugate (Sigma; St Louis, MO) and gently agitated for 2 h at room temperature. Membranes were washed as described above, placed in a hybridization bottle with Sigma Fast BCIP/NBT alkaline phosphatase substrate (Sigma), and rotated in a hybridization oven for up to 60 min. Membranes were then rinsed with distilled water and allowed to air dry. When dry, the extent of substrate precipitation for each leaf cross-section was determined under a dissecting scope.
Greenhouse-grown pineapple plants were screened for the presence of PMWaV using a monoclonal antibody in a TBIA. Of the thirty-eight plants assayed, twenty-four were positive for the virus (63 %). All substrate precipitate was found in discrete spots corresponding to vascular bundles in the leaf cross-section. The degree of precipitation varied from plant to plant, and was assumed to be an indication of virus titre (Hu et al., "Use of a Tissue Blotting Immunoassay to Examine the Distribution of Pineapple Closterovirus in Hawaii," Plant Pis. 81 :1150-1154 (1997), which is hereby incoφorated by reference). Of the twenty-four PMWaV-positive plants, thirteen plants with the strongest positive signals had tissue harvested for dsRNA extraction.
Example 2 - Extraction of dsRNA
Double-stranded RNA was extracted from PMWaV-infected pineapple plants using a protocol similar to that of Morris et al., "Isolation and Analysis of Double-stranded RNA From Virus-infected Plant and Fungal Tissue," Phvtopathologv 69:854-85 (1979) and Dale et al., "Double-stranded RNA in Banana Plants with Bunchy Top Disease," J. Gen. Virol. 67:371-375 (1986), which is hereby incoφorated by reference). Leaf and stem tissue from TBIA-positive plants was frozen in liquid nitrogen then finely ground with a Bunn model G3 coffee grinder (Bunn-O-Matic; Springfield, IL) and stored at -20 °C. Allotments (50 g) of frozen tissue were added to flasks containing 90 mL STE (100 mM NaCl, 50 mM Tris-HCl, 1 mM EDTA, pH 8.0), 40 mL water-saturated phenol containing 0.1 % β- mercaptoethanol (v/v) and 0.1 % 8-hydroxyquinoline (w/v), 30 mL 10 % SDS (w/v), 2 mL β-mercaptoethanol, 0.2 mL NH4OH, and 64 mg bentonite. Chloroform (40 mL) was then added, and the sample was stirred for 45 min at 4 °C. The mixture was centrifuged at 7000 g for 10 min at 4 °C, the upper phase from two samples was collected and combined, then adjusted to 16.5 % EtOH. Following the addition of 2 g CF-11 cellulose (Whatman; Maidstone, England), the sample was shaken at room temperature overnight. The sample was then passed through a vertically clamped 60 cc syringe barrel containing a miracloth filter (Calbiochem; La Jolla, CA). The cellulose was washed with approximately 100 mL of STE containing 16.5 % EtOH, and purged of all liquid using the syringe plunger. dsRNA was eluted from the cellulose with 25 mL of STE, and the plunger was used to elute any remaining liquid. The sample was again adjusted to 16.5 % EtOH, and the process was repeated using 1.5 g of cellulose. The final elution of dsRNA from the cellulose was done with three 3 mL aliquots of STE collected in a 15 mL centrifuge tube, with a purge using the syringe barrel following each aliquot. Any contaminating cellulose was pelleted by a brief centrifugation, and the supernatant was transferred to a 30 mL centrifuge tube where it was precipitated with 0.1 volume of 3 M NaAc (pH 5.2) and 2 volumes of EtOH (95 %) overnight at -20 °C. Samples were then centrifuged at 12 000 g for 15 min at 4 °C, the supernatant discarded, and the pellet gently resuspended in 0.5 mL H2O. The dsRNA was again precipitated and centrifuged as described in a 1.5 mL microfuge tube. The supernatant was discarded, and the pellet washed with 1 mL of cold 70 % EtOH, and centrifuged at 12 000 g for 5 min. The supernatant was discarded and the pellet, representing dsRNA from 100 g of tissue, was resuspended in 10 μL of H2O and stored at -20 °C. Approximately 3 kg of PMWaV-infected tissue was processed using this protocol.
Approximately 3 kg of PMWaV-infected pineapple tissue underwent dsRNA extraction. From this tissue, only 10 to 15 ng of viral dsRNA was extracted per 100 g fresh weight, suggesting 300 to 450 ng of dsRNA was recovered in total.
Although Gunasinghe et al., "Purification and Partial Characterization of a Virus from Pineapple," Phytopathology 79:1337-1341 (1989), which is hereby incoφorated by reference, did not estimate their recovery of PMWaV dsRNA from diseased pineapple tissue, they also appeared to have low recovery of viral dsRNA. This low recovery rate, related to low virus titre, is also common to GLRaV-2 (Zhu et al., "Nucleotide and Genome Organization of Grapevine Leafroll-associated Virus-2 are Similar to Beet Yellows Virus, the Closterovirus Type Member," J. Gen. Virol. 79:1289-1298 (1998), which is hereby incoφorated by reference) and GLRaV-3 (Ling et al., "Nucleotide Sequence of the 3'-terminal Two-thirds of the Grapevine Leafroll- associated Virus-3 Genome Reveals a Typical Monopartite Closterovirus," J. Gen. Virol. 79:1299-1307 (1998), which is hereby incoφorated by reference). In contrast, Dodds et al., "Double-stranded RNA from Plants Infected with Closteroviruses," Phvtopathologv 73:419-423 (1983), which is hereby incoφorated by reference, were able to extract B YV, cirus tristeza virus (CTV), and carnation necrotic fleck virus (CNFV) dsRNAs for agarose gel analysis from 0.2 to 7.0 g of diseased host tissue with ease. When resolved by agarose gel electrophoresis, two to five distinct bands were present and estimated to be 1.7, 6, 8, 16, and 18 DNA kilobase pairs (kbp) in size. Susceptibility to RNase A but not mung bean nuclease confirmed these bands to be dsRNA. The two highest molecular weight bands were always present, and may represent the replicative form (RF) of two different viral RNAs (Dodds et al., "Double-stranded RNA from Plants Infected with Closteroviruses," Phytopathology 73:419-423 (1983); Gunasinghe et al., "Purification and Partial Characterization of a Virus from Pineapple," Phvtopathologv 79:1337-1341 (1989), which are hereby incoφorated by reference). The presence of the three lower molecular weight bands was variable, and these may represent subgenomic (Hilf et al., "Characterization of Citrus Tristeza Virus Subgenomic RNAs in Infected Tissue," Virology 208:576-582 (1995); Mawassi et al., "Populations of Citrus Tristeza Virus Contain Smaller-than- full-length Particles Which Encapsidate Sub-genomic RNA Molecules," J. Gen. Virol. 76:651-659 (1995); Karasev et al, "Transcriptional Strategy of Closteroviruses: Mapping the 5' Termini of the Citrus Tristeza Virus Subgenomic RNAs," J. Virol. 71 :6233-6236 (1997), which are hereby incoφorated by reference) or defective dsRNAs (Mawassi et al, "Defective RNA Molecules Associated with Citrus Tristeza Virus," Virology 208:383-387 (1995); Mawassi et al, "Multiple Species of Defective RNAs in Plants Infected with Citrus Tristeza Virus," Virology 214:264-268 (1995), which are hereby incoφorated by reference). The second highest molecular weight band (ca. 16 kbp) is believed to be the RF of a second virus; if PMWaV follows the same transcriptional strategy of CTV (Karasev et al., "Transcriptional Strategy of Closteroviruses: Mapping the 5' Termini of the Citrus Tristeza Virus Subgenomic RNAs," J. Virol. 71 :6233-6236 (1997), which is hereby incoφorated by reference), no subgenomic dsRNA would approach this size. Also, no defective dsRNAs of this size have been reported in the closteroviruses. This observational evidence of a second RF would also agree with the two PMWaVs found by sequence analysis. Clones 12-1 (1274 bp), 12-2 (1632 bp), and 12-3 (1386 bp) were generated by the step-by-step walking procedure which span nearly the entire 3' end of the viral genome. The 20 - 30 base overlap between each clone was 100% identical in sequence, validating their position relative to each other in the genome. The PMWaV-2-specific primer (#200) used to prime cDNA synthesis for clone 12-1 also annealed downstream on this cDNA strand, allowing its exclusive use in priming subsequent PCR reactions. At first, it was believed there was an inversion in this portion of the PMWaV-2 genome. Sequence data from the 5' end of clone 12-2 (which contained this downstream annealing site), however, revealed that primer #200 complemented the actual template only at the first five bases of the 5' end of the primer (CCATC), and more importantly, at the final six bases of the 3' end (CTAGAG). In fact, none of the random minus-sense primers used to generate amplicons had 100% homology with the dsRNA template, but they were still able to prime DNA polymerization because of the low annealing temperature (33 °C) used in PCR.
Example 3 - Source of Virus-specific RNAs and First Strand cDNQ Synthesis
Virus-specific dsRNAs were isolated from infection pineapple tissue by two cycles of CF-11 cellulose column chromatography. After denaturation with 20 mM methylmercury hydroxide, first strand cDNA was synthesized as previously described (Karasev, et al., "Screening of the Closterovirus Genome by Degenerate Primer-Mediated Polymerase Chain Reaction," J. Gen. Virol. 75:1415-1422 (1994), which is hereby incoφorated by reference), except that SUPERSCRIPT II reverse transcriptase (BRL) was used instead of the Moloney murine leukemia virus enzyme.
Example 4 - Primers and Cloning Strategy
Primers used for cloning and run-off transcription are listed in Table 1 as follows: Table 1. Primers sued for cloning of the PMWaV- 1 and PMWaV-2 genomes.
Figure imgf000051_0001
'Redundancy code: N=A,G,C,T; Y=T,C; R=A,G; M=A,C; S=C,G; W=A,T.
To clone the fragments of the pineapple mealybug wilt-associated viruses described, two approaches were utilized: targeting the conserved domains A and C of the PMWaV- 1 and PMWaV-2 heat shock (HSP70) protein (see Karasev, et al., "Screening of the Closterovirus Genome by Degenerate Primer-Mediated Polymerase Chain Reaction," J. Gen. Virol. 75 : 1415- 1422 ( 1994), which is hereby incoφorated by reference) or random, step-by-step walking in the 5' and 3' directions from the already sequenced fragment of the virus genome according to previously described procedures (Karasev, et al., "Screening of the Closterovirus Genome by Degenerate Primer-Mediated Polymerase Chain Reaction," J. Gen. Virol. 75:1415- 1422 (1994) and Karasev, et al., "Complete Sequence of the Citrus Tristeza Virus RNA Genome," Virology 208:511-520 (1995), which are hereby incoφorated by reference).
Example 5 - Primers, cDNA Synthesis, and Cloning Strategy for PMWaV-2
A step-by-step walking procedure using RT-PCR was used to clone the 3'-terminal half of the PMWaV genome. The existing sequence of PMWaV-2 allowed the design of a PMWaV-2-specific primer which annealled just downstream of the HSP70 homolog C-terminus. Primers were designed so that a 20 to 30 base overlap would occur between neighboring clones. This verified each clone generated was in fact an extension of the previous one. All PMWaV-2-specific primers employed are described in Table 2
To synthesize a cDNA template for PCR, 5 - 10 ng of dsRNA template was denatured at 95 °C for 9 min in an 8 μL volume containing 3 pmol of PMWaV-2- specific primer and quickly chilled on ice. After the addition of 5 μL of first strand buffer [375 mM KCl, 250 mM Tris-HCl (pH 8.3), 15 mM MgCl2], 2 μL of 0.1 M DTT, and 5 μL of dNTPs (2 mM each), the mixture was incubated at 48 °C for 3 min, and 1 μL (200 U / μL) of Superscript II reverse transcriptase (GibcoBRL; Gaithersburg, MD) was added. The reaction was then incubated at 48 °C for 60 min, and terminated by a 15 min incubation at 70 °C.
Table 2 - Primers used to clone and sequence the 3' region of PMWaV strain 12. PMWaV-2-specific primers are upper case, random primers are lower case.
Plus-sense primers (5' to 3') Minus-sense primers (5' to 3') Clone
#200 (CCATCGGGAATGGCTAGTG) #200 (ccatcgggaatggctagtg)a 12-1
(SEQ. ID. No. 35) (SEQ. ID. No. 35)
#208 (CTCAGCGTACGCTCAAGA) #206 (CATGATCGACGGAGCTCT) 12-1 (seq)b
(SEQ. ID. No. 36) (SEQ. ID. No. 37)
#201 (ACTGGTTACGCAGCTATGC) #203 (ctgtgttaatcggcggcac) (SEQ. 12-2
(SEQ. ID. No. 38) ID. No. 39)
#210 (AAGTTTGACGGCTCCACC) #209 (TCGTTCGCCATGTGGTAC) 12-2 (seq)
(SEQ. ID. No. 40) (SEQ. ID. No. 41)
#227 (GCTTCAACAGATAAGGGTG)
(SEQ. ID. No. 42)
#207 (TCGTTGGTCGTAGGTAGAA) #36 (gcttgtttcgcgcattca) (SEQ. 12-3
(SEQ. ID. No. 43) ID. No. 44)
#215 (CCGTTTCTAAAGTGGTCTC) #214 (GCCGACACCTATACCATC) 12-3 (seq)
(SEQ. ID. No. 45) (SEQ. ID. No. 46) considered a random primer in this application
PMWaV-2-specific primers designed to obtain internal sequence of the respective clone
Approximately twenty PCR reactions were performed simultaneously at each walking step. Each reaction combined the PMWaV-2-specific primer used to prime the first strand cDNA synthesis reaction with a different random primer, using the cDNA as template. Random primers which produced an amplicon with their respective PMWaV-2-specifιc primer are described in Table 2. PCR reactions were set up using 1 - 2 μL of a 1 :5 dilution of the first-strand cDNA synthesis reaction as template in a 20 - 25 μL reaction containing 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl2, 10 pmol of PMWaV-2-specific primer, 10 pmol of random primer, 200 μM of each dNTP, 1 -2 U of AmpliTaq DNA polymerase (PE Applied Biosystems; Foster City, CA) and overlaid with 1 - 2 drops of mineral oil. The amplification protocol was performed in a Model 480 DNA Thermal Cycler (PE Applied Biosystems) and involved: one cycle of 94 °C for 5 min; forty-five cycles of 94 °C for 1 min, 33 °C for 1 min, 72 °C for 2.5 min; and one cycle of 72 °C for 7 min. PCR reactions were examined for the presence of amplicons in 1.5 % agarose gels run in TAE buffer [40 mM Tris-acetate, 1 mM EDTA (pH 8.0)] at 5 - 7 V / cm. Amplicons desired for cloning were excised from the gel with a razor blade and placed in a 0.6 mL centrifuge tube with a pinhole in the bottom and containing a GF/C glass microfϊbre filter (Whatman). This tube was then placed in a 1.5 mL centrifuge tube and centrifuged at 12,000 g for 30 s. The elutant was either precipitated as described above and the pellet resuspended in a lesser volume, or used directly in vector ligation.
The gel-excised amplicons were ligated into either pGEM-T Easy (Promega; Madison, WI) or pBlueScript KS- phagemid (Stratagene; La Jolla, CA) contrasted into a T- vector (Marchuk et al., "Construction of T-vectors, a Rapid and General System for Direct Cloning of Unmodified PCR Products," Nucl. Acids Res. 19:1154 (1991), which is hereby incoφorated by reference). Equimolar amounts of vector and amplicon DNA were ligated in a 10 μL reaction containing 30 mM Tris- HCl (pH 7.8), 10 mM MgCl2, 10 mM DTT, 1 mM ATP, 5% PEG 8000 (v/v), and 3 U T4 DNA ligase (Promega) incubated at 4 °C overnight. E. coli DH5 cells were transformed with 2 - 5 μL of the ligation reaction and cells containing recombinant plasmids were selected with either McConkey agar (Sigma) or LB agar topspread with X-gal and IPTG (Sambrook et al., Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989), which is hereby incoφorated by reference) containing 75 or 50 ppm ampicillin, respectively. White colonies underwent plasmid mini-preps using a Qiaprep Spin Miniprep Kit (Qiagen; Valencia, CA) following manufacturers instructions.
Plasmid DNA was sequenced using either T3, T7, SP6, or PMWaV-2- specific primers (Table 2) with Taq DyeDeoxy terminator cycle sequencing (PE Applied Biosystems). Sequencing was conducted on automated sequencers at the Guelph Molecular Supercentre (Model ABI377), University of Guelph, Guelph, Canada, the NCSU DNA Sequencing Facility (ABI377), North Carolina State University, Raleigh, NC, or the Biotechnology/Molecular Biology Instrumentation and Training Facility (ABI373/ABI377), University of Hawaii, Honolulu, HI. Nucleotide sequences were analyzed using various computer programs. Open reading frames, amino acid translation, and restriction endonuclease sites were determined using the MAP function, and protein comparisons were made using the PRETTY function of the GCG sequence analysis software package (Genetics Computer Group; Madison, WI). PMWaV-2-specific primers were designed using the Design-PCR-Primer function of Primer Designer version 1.01 (Scientific and Educational Software). The program BLASTX, based on the Basic Local Alignment Search Tool (BLAST) algorithm (Altschul et al., "Basic Local Alignment Search Tool," J. Mol. Biol. 215:403-410 (1990), which is hereby incoφorated by reference), was used to translate nucleotide sequences to amino acid sequences in all reading frames and compare these putative translation products with the non-redundant amino acid sequence database on the National Center for Biotechnology Information website (NCBI; National Institute of Health).
Example 6 - Cloning, Sequencing, and Sequence Analysis
In most cases, PCR products obtained were size separated in low- melting temperature 1% agarose gels (BRL), and the selected bands were isolated and cloned into the AT-based vector pCR2.1 (Invitrogen) according to the manufacturer's protocol. Virus-specific inserts were sequenced directly in dsDNA plasmids using the SEQUENASE 2.0 Kit (USB) according to the manufacture's instructions. Universal T3 and T7 primers were used to sequence the ends of inserts and virus-specific primers to sequence the rest of the cDNAs. Both strands were sequenced at least twice. The sequence obtained was conceptually translated and potential expressibility of open reading frames (ORFs) was evaluated by the algorithm of Trifonov, E.N., "Translation Framing Code and Frame-Monitoring Mechanism as Suggested by the Analysis of mRNA and 16S rRNA Nucleotide Sequence," J. Mol. Biol. 194:643-652 (1987), which is hereby incoφorated by reference. Putative translation products were compared with the nonredundant sequence database of the National Center for Biotechnology Information using the BLAST program (Altschul, et al., "Basic Local Alignment Search Tool," J. Mol. Biol. 215:403-410 (1990) and Altschul, et al., "Issues in Searching Molecular Sequence Databases," Nature Genetics 6:119-129 (1994), which are hereby incoφorated by reference).
Example 7 - Cloning of the PMWaV-1 and PMWaV-2 The initial virus-specific DNA fragments were amplified using two degenerate primers, D2056 and D2128, targeting motifs A and C of the HSP70 protein. A weak, but discrete band of the expected 1-kb size was amplified and cloned. Four clones, which were picked at random, were sequenced. Unexpectedly, only three of these clones, pcl5, pcl6, and pcl8, had an identical, ca. 1-kb, insert encoding the N-terminal half of viral HSP70. The fourth clone, pcl2, also encoded the N-terminal half of the viral HSP70. However, based on a preliminary sequence analysis, the viral HSP70 encoded by this clone came from a different closterovirus. Thus, it was determined that there were two different, although related closteroviruses. These two viruses were designated PMWaV- 1 (anchored to the clone pel 8), and PMWaV-2 (anchored to the clone pcl2). An adjustment was made in regard to the cloning strategy: from this point on, different specific primers were used to "walk" along the two genomes of the viruses. Specificity of the sequences obtained was tested with the two pairs of specific primers in PCR amplifications performed on the first-strand cDNA generated from the initial virus-specific dsRNA.
Example 8 - Organization of the PMWaV-1 Genome
The sequenced 5,217 nucleotide (nt) fragment of the PMWaV- 1 genome spans four open reading frames (ORFs) which may encode four protein products. The first ORF, termed ORFla according to conventional designation of the clostero viral genes (Dolja, et al, "Molecular Biology and Evolution of Closteroviruses: Sophisticated Build-up of Large RNA Genomes," Ann. Rev. Phytopathol 32:261-285 (1994), which is hereby incoφorated by reference), encodes a protein with all eight motifs conserved in the so-called viral helicases (HEL) and, thus, was identified as a respective PMWaV- 1 HEL. The second ORF (ORF lb) partially overlaps ORFla and encodes a protein with eight conserved domains characteristic of closterovirus RNA-dependent RNA polymerases (RdRp); this ORF lb was identified as the respective PMWaV- 1 RdRp. The expression of ORFlbv probably occurs through a +1 translational frameshift as large fusion polyprotein la/lb, as seen in other closteroviruses (see Dolja, et al., "Molecular Biology and Evolution of Closteroviruses: Sophisticated Build-up of Large RNA Genomes," Ann- Rev. Phytopathol 32:261-285 (1994), which is hereby incoφorated by reference). The downstream ORF2 encodes a small 6-kDa protein composed of predominantly hydrophobic amino acid residues. Further downstream is an ORF3 which encodes the PMWaV- 1 HSP70 protein. The overall organization of the PMWaV- 1 genome within this sequenced fragment resembles the gene layout of the type member of the group, beet yellows virus (BYV) (Agranovsky, et al., "Beet Yellows Closterovirus: Complete Genome Structure and Identification of a Leader Papain-like Thiol Protease," Virology 198:311-324 (1994), which is hereby incoφorated by reference).
Example 9 - Organization of the PMWaV-2 Genome
The sequenced 10,000 -nt fragment of the PMWaV-2 genome spans eight ORFs which may code for four protein products. The overall genome organization of the PMWaV-2 genome in the sequenced fragment resembles most closely the genome of the grapevine leafroll-associated virus 3 (Ling, et al., "The Coat Protein Gene of Grapevine Leafroll Associated Closterovirus-3 : Cloning, Nucleotide Sequencing, and Expression in Transgenic Plants," Arch Virol. 142:1101- 1116 (1997), which is hereby incoφorated by reference). The first incomplete
ORFla encompasses all motifs characteristic of viral helicases and was identified as a PMWaV-2 HEL. The second ORF, designated herein as ORF lb, partially overlaps ORFla and encodes the PMWaV-2 RdRp. As in PMWaV- 1 and other closteroviruses, ORF lb is probably expressed through a +1 ribosomal frameshift. The downstream ORF2 encodes a small hydrophobic 6-kDa protein, and the further downstream ORF3 encodes the PMWaV-2 HSP70 protein. In contrast to PMWaV- 1, there is an extended intergenic region of about 600 nt between ORFs lb and 2. A total of 10,000 bases of the PMWaV-2 genome were sequenced and characterized. Downstream of the HSP70 homolog and existing sequence data for PMWaV-2, three complete ORFs have been sequenced, as well as the 5' terminus of a fourth ORF. In convention with BYV, the genus Closterovirus type species, these would be ORF4, ORF5, ORF6, ORF7, and ORF8.
ORF4 ψ46
The ORF4 start codon was 238 bp downstream of the HSP70 homolog stop codon. This ORF potentially encodes a 403 amino acid polypeptide with a theoretical molecular mass of 46.4 kilodaltons (kDa). The function of this potential protein is unknown and database searching via BLASTX did not identify similar proteins except its counteφart (p55) in GRLaV-3 (Table 3).
Table 3. Percentage amino acid and nucleotide homology between PMWaV-2 and GLRaV-3 for different genes in the virus genomes.
Amino Acid Gene Nucleotide
Identity Similarity
ORF5 (p46) 31.1 41.8 43.7
ORF6 (CP) 40.3 49.7 49.4
ORF7 (CPd) 32.3 40.0 43.7
ORF5 (CP)
The ORF5 start codon was 75 bp downstream of the p46 stop codon. This ORF potentially encodes a 302 amino acid polypeptide with a theoretical molecular mass of 33.8 kDa. ORF5 was identified as the coat protein gene of PMWaV-2 based on the high degree of homology to GRLaV-3 CP (Table 3), and the moderate degree of homolgy to CPs of other closteroviruses. Amino acid sequence alignment of the CP and the CPs from GLRaV-3, BYV, CTV, and LIYV revealed the invariant amino acid residues S, R, and D, which are conserved in the CP of rod- shaped and filamentous RNA plant viruses (Dolja et al., "Phylogeny of Capsid Proteins of Rod-shaped and Filamentous RNA Plant Viruses: Two Families with Distinct Patterns of Sequence and Probably Structure Conservation," Virology 184:79-86 (1991), which is hereby incoφorated by reference). Residues R and D are believed to be involved in stabilization of molecules by salt bridge formation and proper folding of the CP (Dolja et al., "Phytogeny of Capsid Proteins of Rod-shaped and Filamentous RNA Plant Viruses: Two Families with Distinct Patterns of
Sequence and Probably Structure Conservation," Virology 184:79-86 (1991), which is hereby incoφorated by reference). A large discrepency has been identified between the CP theoretical molecular weight of 33.8 kDa, and the 23.8 kDa observation given by Gunasinghe et al., "Purification and Partial Characterization of a Virus from Pineapple," Phvtopathologv 79:1337-1341 (1989), which is hereby incoφorated by reference, using SDS-PAGE analysis. As two different PMWaVs have been identified by sequencing, and possibly two RFs of differing size have been observed by gel electrophoresis, it is plausible that 33.8 kDa CP of PMWaV-2 belongs to the larger viral RF, while 23.8 kDa protein observed by Gunasinghe et al., "Purification and Partial Characterization of a Virus from Pineapple," Phvtopathologv 79: 1337- 1341 (1989) belongs to the smaller RF.
ORF6 (CPd) The ORF6 start codon was 33 bp downstream of the CP stop codon.
This ORF potentially encodes a 491 amino acid polypeptide with a theoretical molecular mass of 55.8 kDa. This protein was identified as a second coat protein (i.e. CPd) based on the high degree of homology with GRLaV-3 CPd (Table 3) and moderate homologies with the CPd of other closteroviruses. The S, R, and D residues conserved in the CP were also conserved in the CPd.
ORF7
A start codon 8 bp upstream of the CPd stop codon initiates a ORF7 which potentially encodes a 172 amino acid polypeptide with a theoretical molecular mass of 19.7 kDa. BLASTX analysis reveals this potential protein is homologous with p21 in GLRaV-3, with a 26 % identity and 38 % similarity between the amino acid sequences. ORF7 has been tentatively identified as the PMWaV-2 homolog of p21 of GLRaV-3. This is despite the fact that the N-terminus of p21 does not overlap with the C-terminus of CPd in GLRaV-3 as it does in PMWaV-2. The function of p21 to GLRaV-3 is unknown, and it shares no significant sequence homologies with other proteins in current databases (Ling et al., "Nucleotide Sequence of the 3'- terminal Two-thirds of the Grapevine Leafroll-associated Virus-3 Genome Reveals a Typical Monopartite Closterovirus," J. Gen. Virol. 79:1299-1307 (1998), which is hereby incoφorated by reference).
ORF8 The start (AUG) codon for ORF8 shares the UG residues of the stop
(UGA) codon for ORF7. ORF8 potentially encodes a 194 amino acid polypeptide with a theoretical molecular weight of 22.3 kDa. This protein shares no significant homology with any other closterovirus sequence, however, it interestingly has a region of homology 72 amino acids long (29 % identity, 47 % similarity) with a viral capsid associated protein of nuclear polyhedrosis viruses (Lu, et al., "Nucleotide Sequence and Transcriptional Analysis of the p80 Gene of Autographa Californica Nuclear Polyhedrosis Virus: A Homologue of the Orgyia pseudotsugata Nuclear Polyhedrosis Virus Capsid- Associated Gene," Virology 190:201-209 (1992), which is hereby incoφorated by reference). Sequence data and characterization of the PMWaV genome(s) will serve two puφoses. First, from an academic standpoint, such information will be useful in the classification of the closteroviruses. Sequence homology to other closteroviruses and genome organization are essential to the proper classification of these important pathogens. With this data, the evolutionary relationships within the family and to other RNA plant viruses may also be established. These relationships are especially interesting due to the large coding capacity of the closteroviruses and their novel genes.
The second interest in this information is from a more applied standpoint. PMW is a serious disease of pineapple, and is mainly controlled by pesticide use. Increasing pressure to control diseases with non-chemical methods has led to the use of disease resistant crops. Crops resistant to viral diseases by transformation with CP or other viral genes have been established and effective, although the mechanism of resistance is still poorly understood (Hackland et al., "Coat-protein Mediated Resistance in Transgenic Plants," Arch. Virol. 139:1-22 (1994); Prins et al., "RNA-mediated Resistance in Transgenic Crops," Arch. Virol. 141 :2259-2276 (1996), which are hereby incoφorated by reference). As PMWaV appears to be an important factor in PMW, PMWaV genes such as CP or RdRp will play an essential role in developing transgenic pineapple plants resistant to PMWaV, and ultimately, PMW. Data obtained by this study allows the immediate implementation of this disease resistance strategy. Further sequencing of the PMWaVs genome, especially genes with unknown function, may also provide valuable insights into vector specificity and virus retention, which are two important factors when studying the spread and the epidemiology of PMW and its associated viruses, PMWaV- 1 and PMwaV-2.
Example 10 - PMWaV-1 and PMWaV-2 Sequences as Tools for Generation of Virus-Resistant Plants
The most promising long-term strategy to control the disease which is also environmentally friendly, is generation of the resistant plants. The concept of virus-derived resistance is one of the possible solutions to the PMW problem. And it might very well be the fastest solution. Two types of the virus-derived genes have been used to regenerate transgenic plants with demonstrated resistance to viruses: the RdRp and coat protein (CP) genes. The RdRp-mediated resistance seems to be more strict although very specific, i.e. working against the exact virus strain which was a source of the transgene. The CP-mediated resistance seems to be much broader, protecting the plant against a range of virus isolates which may differ in the sequence of the transgene up to 8-10%, although it is not as strict as the RdRp-mediated resistance. The complete RdRp's of both PMWaV- 1 and PMWaV-2 can be used for the generation of transgenic pineapple lines.
The following general strategy can be used to obtain transgenic plants. Two custom-made primers complementary to the start and end of the respective ORF are designed to include an appropriate restriction site to be used for cloning into a plant expression vector. Suitable plant expression vectors include, for example, pEPT8 developed in the laboratory of Prof. D. Gonsalves (Cornell University, Geneva, NY). This vector contains 35S promoter of the cauliflower mosaic virus in front of the inserted ORF and a translation-enhancing leader derived from the alfalfa mosaic virus RNA4, and a CaMV terminator downstream of the insert. The orientation of the insert relative to the 35S promotor can be verified by PCR and restriction enzyme digestion. The resulting plant expression cassette can be excised from the pEPT8-based recombinant plasmid using the Hindlll restriction enzyme and cloned into H ctΗI-digested plant transformation binary vector, pGA482. (An, G., "Binary Ti vectors," Plant Phvsiol. 81:86-91 (1987); Ling, et al., "The Coat Protein Gene of Grapevine Leafroll Associated Closterovirus-3: Cloning, Nucleotide Sequencing, and Expression in Transgenic Plants," Arch Virol. 142:1101-1116 (1997), which are hereby incoφorated by reference). Both sense and anti-sense constructs can be used. Constructs can be mobilized into an avirulent Agrobacterium tumefaciens strain LBA4404 via electroporation, and potential transformants identified on selective media containing 75 ug/ml of gentamycin. A. tumefaciens colonies containing desirable inserts can be used to transform Nicotiana benthamiana and Ananas comosus plants using leaf disk procedures. Kanamycin-resistant R0 plants can be analyzed by PCR for the respective transgene, and the level of expression tested by Northern-blot hybridization.
Although the invention has been described in detail for the puφose of illustration, it is understood that such detail is solely for that puφose, and variations can be made therein by those skilled in the art without departing from the spirit and scope of the invention which is defined by the following claims.

Claims

WHAT IS CLAIMED:
1. An isolated protein or polypeptide of a pineapple mealybug wilt virus.
2. An isolated protein or polypeptide according to claim 1, wherein the protein or polypeptide is a PMWaV Type I protein or polypeptide.
3. An isolated protein or polypeptide according to claim 2, wherein the protein or polypeptide is selected from the group consisting of a helicase, polymerase, and heat shock protein 70.
4. An isolated protein or polypeptide according to claim 3, wherein the protein or polypeptide is a helicase
5. An isolated protein or polypeptide according to claim 4, wherein the protein or polypeptide has an amino acid sequence comprising SEQ. ID. No. 4.
6. An isolated protein or polypeptide according to claim 3, wherein the protein or polypeptide is a polymerase.
7. An isolated protein or polypeptide according to claim 6, wherein the protein or polypeptide has an amino acid sequence comprising SEQ. ID. No. 6.
8. An isolated protein or polypeptide according to claim 3, wherein the protein or polypeptide is a heat shock 70 protein.
9. An isolated protein or polypeptide according to claim 8, wherein the protein or polypeptide has an amino acid sequence comprising SEQ. ID. No. 10. - όl -
10. An isolated protein or polypeptide according to claim 2, wherein the protein or polypeptide has an amino acid sequence comprising SEQ. ID. No. 8.
11. An isolated protein or polypeptide according to claim 1 , wherein the protein or polypeptide is a PMWaV Type II protein or polypeptide.
12. An isolated protein or polypeptide according to claim 11, wherein the protein or polypeptide is selected from the group consisting of a coat protein, polymerase, helicase, and heat shock protein 70.
13. An isolated protein or polypeptide according to claim 12, wherein the protein or polypeptide is a coat protein.
14. An isolated protein or polypeptide according to claim 13, wherein the protein or polypeptide has an amino acid sequence comprising SEQ. ID. No. 12.
15. An isolated protein or polypeptide according to claim 13 , wherein the protein or polypeptide has an amino acid sequence comprising SEQ. ID. No. 14.
16. An isolated protein or polypeptide according to claim 12, wherein the protein or polypeptide is a helicase.
17. An isolated protein or polypeptide according to claim 16, wherein the protein or polypeptide has an amino acid sequence comprising SEQ. ID. No. 20.
18. An isolated protein or polypeptide according to claim 12, wherein the protein or polypeptide is a heat shock protein 70.
19. An isolated protein or polypeptide according to claim 18, wherein the protein or polypeptide has an amino acid sequence comprising SEQ. ID. No. 26.
20. An isolated protein or polypeptide according to claim 12, wherein the protein or polypeptide is a polymerase.
21. An isolated protein or polypeptide according to claim 20, wherein the protein or polypeptide has an amino acid sequence comprising SEQ.
ID. No. 22.
22. An isolated protein or polypeptide according to claim 11, wherein the protein or polypeptide has an amino acid sequence comprising SEQ. ID. No. 16
23. An isolated protein or polypeptide according to claim 11 , wherein the protein or polypeptide has an amino acid sequence comprising SEQ. ID. No. 18.
24. An isolated protein or polypeptide according to claim 11 , wherein the protein or polypeptide has an amino acid sequence comprising SEQ. ID. No. 24.
25. An isolated protein or polypeptide according to claim 11 , wherein the protein or polypeptide has an amino acid sequence comprising SEQ. ID. No. 28.
26. An isolated protein or polypeptide according to claim 1, wherein the protein or polypeptide is purified.
27. An isolated protein or polypeptide according to claim 1 , wherein the protein or polypeptide is recombinant.
28. An isolated RNA molecule encoding a protein or polypeptide according to claim 1.
29. An isolated RNA molecule encoding a protein or polypeptide according to claim 28, wherein the protein or polypeptide is a PMWaV Type I protein or polypeptide.
30. An isolated RNA molecule according to claim 29, wherein the protein or polypeptide is selected from a group consisting of a helicase, polymerase, and heat shock protein 70.
31. An isolated RNA molecule encoding a protein or polypeptide according to claim 28, wherein the protein or polypeptide is PMWaV Type II protein or polypeptide.
32. An isolated RNA molecule according to claim 31 , wherein the protein or polypeptide is selected from the group consisting of a coat protein, polymerase, helicase, and heat shock protein 70.
33. An isolated DNA molecule encoding a protein or polypeptide according to claim 1.
34. An isolated DNA molecule according to claim 33, wherein the protein or polypeptide is a PMWaV Type I protein or polypeptide.
35. An isolated DNA molecule according to claim 34, wherein the protein or polypeptide is selected from a group consisting of a helicase, polymerase, and heat shock protein 70.
36. An isolated DNA molecule according to claim 35, wherein the protein or polypeptide is a helicase.
37. An isolated DNA molecule according to claim 36, wherein the protein or polypeptide has an amino acid sequence comprising SEQ. ID.
No. 4.
38. An isolated DNA molecule according to claim 36, wherein the DNA molecule has a nucleotide sequence comprising SEQ. ID. No. 3 or a nucleotide sequence which hybridizes to SEQ. ID. No. 3 under stringent conditions of 42°C in a 0.9M sodium citrate buffer.
39. An isolated DNA molecule according to claim 35, wherein the protein or polypeptide is a polymerase.
40. An isolated DNA molecule according to claim 39, wherein the protein or polypeptide has an amino acid sequence comprising SEQ. ID.
No. 6.
41. An isolated DNA molecule according to claim 39, wherein the DNA molecule has a nucleotide sequence comprising SEQ. ID. No. 5 or a nucleotide sequence which hybridizes to SEQ. ID. No. 5 under stringent conditions of 42°C in a 0.9M sodium citrate buffer.
42. An isolated DNA molecule according to claim 35, wherein the protein or polypeptide is a heat shock protein 70.
43. An isolated DNA molecule according to claim 42, wherein the protein or polypeptide has an amino acid sequence comprising SEQ. ID. No. 10
44. An isolated DNA molecule according to claim 42, wherein the DNA molecule has a nucleotide sequence comprising SEQ. ID. No. 9 or a nucleotide sequence which hybridizes to SEQ. ID. No. 9 under stringent conditions of 42°C in a 0.9M sodium citrate buffer.
45. An isolated DNA molecule according to claim 34, wherein the protein or polypeptide has an amino acid sequence comprising SEQ. ID.
No. 8.
46. An isolated DNA molecule according to claim 34, wherein the DNA molecule has a nucleotide sequence comprising SEQ. ID. No. 7 or a nucleotide sequence which hybridizes to SEQ. ID. No. 7 under stringent conditions of 42°C in a 0.9M sodium citrate buffer.
47. An isolated DNA molecule according to claim 34, wherein the DNA molecule has a nucleotide sequence comprising SEQ. ID. No. 1 or a nucleotide sequence which hybridizes to SEQ. ID. No. 1 under stringent conditions of 42°C in a 0.9M sodium citrate buffer.
48. An isolated DNA molecule according to claim 33, wherein the protein or polypeptide is a pineapple mealybug wilt virus Type II protein or polypeptide.
49. An isolated DNA molecule according to claim 48, wherein the protein or polypeptide is selected from a group consisting of a coat protein, polymerase, helicase, and heat shock 70 protein.
50. An isolated DNA molecule according to claim 49, wherein the protein or polypeptide is a coat protein.
51. An isolated DNA molecule according to claim 50, wherein the protein or polypeptide has an amino acid sequence comprising SEQ. ID.
No. 12
52. An isolated DNA molecule according to claim 50, wherein the DNA molecule has a nucleotide sequence comprising SEQ. ID. No. 11 or a nucleotide sequence which hybridizes to SEQ. ID. No. 11 under stringent conditions of 42°C in a 0.9M sodium citrate buffer.
53. An isolated DNA molecule according to claim 50, wherein the protein or polypeptide has an amino acid sequence comprising SEQ. ID. No. 14.
54. An isolated DNA molecule according to claim 50, wherein the DNA molecule has a nucleotide sequence comprising SEQ. ID. No. 13 or a nucleotide sequence which hybridizes to SEQ. ID. No. 13 under stringent conditions of 42°C in a 0.9M sodium citrate buffer.
55. An isolated DNA molecule according to claim 49, wherein the protein or polypeptide is a helicase.
56. An isolated DNA molecule according to claim 55, wherein the protein or polypeptide has an amino acid sequence comprising SEQ. ID. No. 20.
57. An isolated DNA molecule according to claim 55, wherein the DNA molecule has a nucleotide sequence comprising SEQ. ID. No. 19 or a nucleotide sequence which hybridizes to SEQ. ID. No. 19 under stringent conditions of 42°C in a 0.9M sodium citrate buffer.
58. An isolated DNA molecule according to claim 49, wherein the protein or polypeptide is a heat shock protein 70.
59. An isolated DNA molecule according to claim 58, wherein the protein or polypeptide has an amino acid sequence comprising SEQ. ID. No. 26
60. An isolated DNA molecule according to claim 58, wherein the DNA molecule has a nucleotide sequence comprising SEQ. ID. No. 25 or a nucleotide sequence which hybridizes to SEQ. ID. No. 25 under stringent conditions of 42°C in a 0.9M sodium citrate buffer.
61. An isolated DNA molecule according to claim 49, wherein the protein or polypeptide is a polymerase.
62. An isolated DNA molecule according to claim 61, wherein the protein or polypeptide has an amino acid sequence comprising SEQ. ID. No. 22.
63. An isolated DNA molecule according to claim 61 , wherein the DNA molecule has a nucleotide sequence comprising SEQ. ID. No. 21 or a nucleotide sequence which hybridizes to SEQ. ID. No. 21 under stringent conditions of 42°C in a 0.9M sodium citrate buffer.
64. An isolated DNA molecule according to claim 48, wherein the protein or polypeptide has an amino acid sequence comprising SEQ. ID.
No. 16
65. An isolated DNA molecule according to claim 48, wherein the DNA molecule has a nucleotide sequence comprising SEQ. ID. No. 15 or a nucleotide sequence which hybridizes to SEQ. ID. No. 15 under stringent conditions of 42°C in a 0.9M sodium citrate buffer.
66. An isolated DNA molecule according to claim 48, wherein the protein or polypeptide has an amino acid sequence comprising SEQ. ID. No. 18.
67. An isolated DNA molecule according to claim 48, wherein the DNA molecule has a nucleotide sequence comprising SEQ. ID. No. 17 or a nucleotide sequence which hybridizes to SEQ. ID. No. 17 under stringent conditions of 42°C in a 0.9M sodium citrate buffer.
68. An isolated DNA molecule according to claim 48, wherein the protein or polypeptide has an amino acid sequence comprising SEQ. ID. No.
24.
69. An isolated DNA molecule according to claim 48, wherein the DNA molecule has a nucleotide sequence comprising SEQ. ID. No. 23 or a nucleotide sequence which hybridizes to SEQ. ID. No. 23 under stringent conditions of 42°C in a 0.9M sodium citrate buffer.
70. An isolated DNA molecule according to claim 48, wherein the protein or polypeptide has an amino acid sequence comprising SEQ. ID.
No. 28.
71. An isolated DNA molecule according to claim 48, wherein the DNA molecule has a nucleotide sequence comprising SEQ. ID. No. 27 or a nucleotide sequence which hybridizes to SEQ. ID. No. 27 under stringent conditions of 42°C in a 0.9 M sodium citrate buffer.
72. An isolated DNA molecule according to claim 48, wherein the DNA molecule has a nucleotide sequence comprising SEQ. ID. No. 2.
73. An expression system comprising a DNA molecule according to claim 33 in a vector heterologous to the DNA molecule.
74. An expression system according to claim 73, wherein the protein or polypeptide is a PMWaV Type I protein or polypeptide.
75. An expression system according to claim 74, wherein the protein or polypeptide is selected from a group consisting of a helicase, polymerase, and heat shock protein 70.
76. An expression system according to claim 73, wherein the protein or polypeptide is a PMWaV Type II protein or polypeptide.
77. An expression system according to claim 76, wherein the protein or polypeptide is selected from a group consisting of a coat protein, polymerase, helicase, and heat shock 70 protein.
78. A host cell transformed with a heterologous DNA molecule according to claim 33.
79. A host cell according to claim 78, wherein the host cell is Agrobacterium tumefaciens.
80. A host cell according to claim 78, wherein the host cell is a pineapple cell.
81. A transgenic plant cultivar comprising the DNA molecule according to claim 33.
82. A transgenic plant cultivar according to claim 81 , wherein the plant cultivar is a pineapple cultivar.
83. A transgenic plant according to claim 82, wherein the protein or polypeptide is a PMWaV Type I protein or polypeptide.
84. A transgenic plant cultivar according to claim 83, wherein the protein or polypeptide is selected from a group consisting of a helicase, polymerase, or heat shock protein 70.
85. A transgenic plant according to claim 82, wherein the protein or polypeptide is a PMWaV Type II protein or polypeptide.
86. A transgenic plant cultivar according to claim 85, wherein the protein or polypeptide is selected from a group consisting of a coat protein, polymerase, helicase, or heat shock protein 70.
87. A method of imparting pineapple mealybug wilt virus resistance to a pineapple cultivar comprising: transforming a pineapple cultivar with a DNA molecule according to claim 33 under conditions effective to impart resistance to pineapple mealybug wilt virus.
88. A method according to claim 87, wherein the protein or polypeptide is a PMWaV Type I protein or polypeptide.
89. A method according to claim 88, wherein the protein or polypeptide is selected from a group consisting of a helicase, polymerase, and heat shock protein 70.
90. A method according to claim 87, wherein the protein or polypeptide is a PMWaV Type II protein or polypeptide.
91. A method according to claim 90, wherein the protein or polypeptide is selected from a group consisting of a coat protein, polymerase, helicase, and heat shock protein 70.
92. A method according to claim 87, wherein said transforming is Agrobacterium-mediated.
93. A method according to claim 87, wherein said transforming comprises: propelling particles at pineapple plant cells under conditions effective for the particles to penetrate into the cell interior and introducing an expression vector comprising the DNA molecule into the cell interior.
94. A method for detection of pineapple mealybug wilt virus in a sample, said method comprising: providing a nucleotide sequence of the DNA molecule according to claim 33 as a probe in a nucleic acid hybridization assay; contacting the sample with the probe; and detecting any reaction which indicates that pineapple mealybug wilt virus is present in the sample.
95. A method according to claim 94, wherein the nucleic acid hybridization assay is selected from a group consisting of dot blot hybridization, tissue printing, southern hybridization, and northern hybridization.
96. A method for detection of pineapple mealybug wilt virus in a sample: providing a nucleotide sequence of the DNA molecule according to claim 33 as a probe in a gene amplification detection procedure; contacting the sample with the probe; and detecting any reaction which indicates that pineapple mealybug wilt virus is present in the sample.
97. A method according to claim 96, wherein the gene amplification detection procedure is selected from a group consisting of polymerase chain reaction and immunocapture polymerase chain reaction.
PCT/US1999/022152 1998-09-23 1999-09-22 Pineapple mealybug-associated wilt virus proteins and their uses WO2000017372A2 (en)

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