Polymerase chain-reaction-mediated cloning and expression of the coat protein gene of potato virus Y inEscherichia coli

Abstract

Complementary DNAs (cDNAs) to the RNA genome of a necrotic strain of potato virus Y in Japan (Hokkaido Univ. isolate of PVY-T:PVY-TH) were synthesized and cloned into a plasmid pBR322. About 4.3 kbp of the cDNA sequence containing the 3'-poly(A) tract of PVY-TH was inserted into a recombinant plasmid pBRYT88. The coat protein coding region (CP gene) in pBRYT88 was amplified using the polymerase chain reaction (PCR) and subcloned into a plasmid pUC119. The nucleotide sequence of the CP gene was determined from both the PCR-mediated clones and pBRYT88. The CP gene of PVY-TH consisted of 801 nucleotides, corresponding to 267 amino acids of Mr 29,811. The predicted amino acid sequence of the PVY-TH CP gene was different from that of PVYN (1) in only five amino acids and displayed 98.1% sequence homology. This result indicates that PVY-TH is closely related to PVYN (1). The cDNAs of the PVY-TH CP gene containing an additional initiation codon (ATG) at the 5' end and a stop codon at the 3' end were constructed by PCR amplification and subcloned into an E. coli expression vector, pKK223-3. Five transformed E. coli colonies expressing the PVY-TH CP were identified by immunoscreening using both polyclonal rabbit antiserum against PVY-TH and mouse monoclonal antibody (MoAb) specific to PVY-T. The CP of PVY-TH produced in the E. coli colonies had an electrophoretic mobility identical to that of native PVY-TH CP and reacted strongly to a specific MoAb to PVY-T, but did not react to a specific MoAb to an ordinary strain of PVY (PVY-O). The maximum expression of the CP in E. coli was approximately 7% of the total soluble proteins. The result indicates that the CP gene cloned by PCR was functional and the PCR procedure was useful for producing biologically active cDNA clones from a single, long positive-sense RNA genome encoding a single, large polyprotein precursor, such as potyviruses.

The Genetic Structure of Populations of Potato virus Y in Japan; Based on the Analysis of 20 Full Genomic Sequences

“… T 13 was obtained from PVY‐T H isolate collected in Japan in 1986 (Hataya et al. ). This isolate was not examined for inducing potato tuber necrotic ringspot disease. …”

Frequent occurrence of recombinant potyvirus isolates

“…In addition, the sequences of the following isolates, which are not in the databases, were included: PeMV (Dougherty et al, 1985), Is (Rosner & Raccah,I988), Go16 (Wefels et al, 1989), NL1 (van der Vlugt et al, 1993), Th (Hataya et aL, 1990), 02 (Lawson et aL, 1990) and M1, partially sequenced by one of us (Le Romancer, 1993).…”

Molecular cloning, sequencing, and expression inEscherichia coli of the potato virus Y cytoplasmic inclusion gene

“…It was found that amino acid homologies of CI between strains and distinct members of potyviruses were similar to those observed between coat proteins, which means that amino acid homology of the CI proteins may also be applicable to taxonomy of PVY and potyviruses. The length of the coat proteins differs considerably among potyviruses, whereas lengths of the CI proteins were very similar (PVY-O: 634, PVY-T13: 634, PVY~: 634, PPV-NAT: 635, PPV-R: 635, TEV: 633 and TVMV: 635 amino acids) [1,8,13,[17][18][19]23; Inoue et al, in prep.]. CI proteins showed significant differences at both N-and C-terminal regions, with a conserved core sequence.…”