caa a22 4500 465795293 CHVBK 20180323112036.0 cr unu---uuuuu 170327e19901201xx s 000 0 eng 10.1007/BF00570028 doi (NATIONALLICENCE)springer-10.1007/BF00570028 Polymerase chain-reaction-mediated cloning and expression of the coat protein gene of potato virus Y in Escherichia coli [Elektronische Daten] [Tatsuji Hataya, Teruo Sano, Kazusato Ohshima, Eishiro Shikata] 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 amplication and subcloned into anE. coli expression vector, pKK223-3. Five transformedE. 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 theE. 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 inE. coli was apapproximately 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 producting biologically active cDNA clones from a single, long positive-sense RNA genome encoding a single, large polyprotein precursor, such as potyviruses. Kluwer Academic Publishers, 1990 PVY nationallicence potyvirus nationallicence coat protein nationallicence cloning nationallicence PCR nationallicence nucleotide sequence nationallicence Hataya Tatsuji Biotechnology Business Division, Nippon Steel Co., Ltd., 100-71, Tokyo, Japan aut Sano Teruo Department of Botany, Faculty of Agriculture, Hokkaido University, 060, Sapporo, Japan aut Ohshima Kazusato Department of Botany, Faculty of Agriculture, Hokkaido University, 060, Sapporo, Japan aut Shikata Eishiro Department of Botany, Faculty of Agriculture, Hokkaido University, 060, Sapporo, Japan aut Virus Genes Kluwer Academic Publishers 4/4(1990-12-01), 339-350 0920-8569 4:4<339 1990 4 11262 https://doi.org/10.1007/BF00570028 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/BF00570028 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Hataya Tatsuji Biotechnology Business Division, Nippon Steel Co., Ltd., 100-71, Tokyo, Japan aut NATIONALLICENCE 700 1- Sano Teruo Department of Botany, Faculty of Agriculture, Hokkaido University, 060, Sapporo, Japan aut NATIONALLICENCE 700 1- Ohshima Kazusato Department of Botany, Faculty of Agriculture, Hokkaido University, 060, Sapporo, Japan aut NATIONALLICENCE 700 1- Shikata Eishiro Department of Botany, Faculty of Agriculture, Hokkaido University, 060, Sapporo, Japan aut NATIONALLICENCE 773 0- Virus Genes Kluwer Academic Publishers 4/4(1990-12-01), 339-350 0920-8569 4:4<339 1990 4 11262 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer