Articles
CHARACTERIZATION OF SX/2, AN APPLE CHLOROTIC LEAF SPOT VIRUS ISOLATE SHOWING UNUSUAL COAT PROTEIN PROPERTIES
Article number
472_1
Pages
43 – 50
Language
Abstract
The plum isolate SX/2 of apple chlorotic leaf spot virus (ACLSV) showed properties of coat protein different from those of other tested isolates.
It migrated faster in SDS-PAGE and did not react (or reacted weakly) with some monoclonal and polyclonal antibodies prepared against ACLSV isolates from apple and cherry.
The PCR amplification of the 358 bp fragment of the CP gene with primers A52 and A53 did not occur for ACLSV SX/2, at conditions suitable for the other isolates.
RNA was isolated from purified virus particles and was used as template for RT-PCR, using a lower annealing temperature.
PCR products were cloned into the pCR-Script SK+ vector.
Several clones containing cDNA covering the coat protein gene and other parts of the genome were selected and sequenced.
Several mismatches were found between the obtained sequence and primers A52 and A53 which must have been responsible for the problems with PCR amplification.
Modified primers were synthesized and initiated properly the amplification of a specific product.
The coat protein gene of SX/2 showed 84% homology of nucleotide sequence with ACLSV-P863 and ACLSV-P205. Amino acids homology was higher: 93% and 91% respectively.
Three amino acids were identified as possibly important for serological properties of SX/2 isolate.
It migrated faster in SDS-PAGE and did not react (or reacted weakly) with some monoclonal and polyclonal antibodies prepared against ACLSV isolates from apple and cherry.
The PCR amplification of the 358 bp fragment of the CP gene with primers A52 and A53 did not occur for ACLSV SX/2, at conditions suitable for the other isolates.
RNA was isolated from purified virus particles and was used as template for RT-PCR, using a lower annealing temperature.
PCR products were cloned into the pCR-Script SK+ vector.
Several clones containing cDNA covering the coat protein gene and other parts of the genome were selected and sequenced.
Several mismatches were found between the obtained sequence and primers A52 and A53 which must have been responsible for the problems with PCR amplification.
Modified primers were synthesized and initiated properly the amplification of a specific product.
The coat protein gene of SX/2 showed 84% homology of nucleotide sequence with ACLSV-P863 and ACLSV-P205. Amino acids homology was higher: 93% and 91% respectively.
Three amino acids were identified as possibly important for serological properties of SX/2 isolate.
Authors
T. Malinowski, B. Komorowska, M. Cieslinska, B. Zawadzka, T. Candresse
Keywords
ACLSV, PCR, RT-PCR, primers, homology, epitopes
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