Analysis of variability of P1 gene region of N strain of potato virus Y using temperature-gradient gel electrophoresis and DNA heteroduplex analysis.
Reaction conditions specific for reverse taranscription-polymerase chain reaction (RT-PCR) of potato virus Y strain NTN (PVYNTN) were used to amplify a 394 bp fragment of the P1 gene from selected PVY isolates with the aim to study the PVY variability within this genomic region. The P1 gene fragment from the Nicola isolate (Nicola P1/1 clone) was sequenced and characterized by temperature-gradient gel electrophoresis (TGGE). The Nicola P1/1 clone differed from that from the Hungarian isolate by double point mutation resulting in two changes at the deduced amino acid level. The clone showed simple transition from double-stranded to single-stranded form with two characteristic melting end points of about 41 degrees C and 48 degrees C. A more complicated TGGE pattern was, however, found for the whole P1 cDNA library of the Nicola isolate, suggesting accumulation of some minor sequence variants of PVY in this isolate. Based on the TGGE pattern, 46 degrees C was selected as the standard temperature for electrophoretic analysis of heteroduplex DNAs formed with the Nicola P1/1 DNA as reference. More than 40 other PVY isolates from PVYN group were analysed using this method. In most cases only minor fractions of electrophoretically distinguishable DNA heteroduplexes were found, however, in most isolates of PVYN-Wilga type, mixtures of the major sequence variants were observed. Two of these variants from the hybrid 220-5 (Czech Republic) were sequenced. Both of them differed from the Nicola P1/1 clone by 6 point mutations, which led to several changes at the amino acid level.
Matousek
J
J
Department of Molecular Genetics, Czech Academy of Sciences, Ceské Budĕjovice, Czech Republic. jmat@umbr.cas.cz
Ptácek
J
J
Dĕdic
P
P
Schubert
J
J
D000595:Amino Acid Sequence; D018076:DNA, Complementary / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D014644:Genetic Variation; D016679:Genome, Viral; D020180:Heteroduplex Analysis; D006358:Hot Temperature; D008969:Molecular Sequence Data; D017800:Potyvirus / Q000235:genetics*; D012367:RNA, Viral / Q000032:analysis / Q000235:genetics; D016415:Sequence Alignment; D011198:Solanum tuberosum / Q000821:virology
D018076:DNA, Complementary; D012367:RNA, Viral