Articles
MOLECULAR CLONING OF THE DOUBLE-STRANDED RNAS ASSOCIATED WITH STRAWBERRY MILD YELLOW-EDGE VIRUS
Article number
236_14
Pages
111 – 116
Language
Abstract
For many virus diseases of woody hosts it has not been possible to purify virus particles for the production of antisera or the extraction of genomic viral RNA for the production of cDNA probes.
Sap of strawberry leaf tissues is very viscous and difficult to work with, and the successful transmission of strawberry mild yellow-edge virus (SMYEV) to Rubus rosaefolius has allowed us to purify microgram quantities of SMYEV dsRNA. The dsRNA was gel-purified and then denatured with methylmercuric hydroxide.
First-strand cDNA synthesis was initiated with random primers and second-strand synthesis carried out by standard methods.
The double-stranded DNA was size-fractionated through a CL-4B Sepharose column and larger cDNA recovered by ethanol precipitation.
After C-tailing, the DNA was annealed into G-tailed pUC9 and transformed into Escherichia coli. About 800 transformants were obtained and 110 colonies selected on the basis of colony hybridization for plasmid isolation and characterization.
The hybridization of 32P-dATP oligo-labeled cDNA clones to Northern blots of SMYEV dsRNA from strawberry demonstrates that these cDNA clones are specific for SMYEV.
Sap of strawberry leaf tissues is very viscous and difficult to work with, and the successful transmission of strawberry mild yellow-edge virus (SMYEV) to Rubus rosaefolius has allowed us to purify microgram quantities of SMYEV dsRNA. The dsRNA was gel-purified and then denatured with methylmercuric hydroxide.
First-strand cDNA synthesis was initiated with random primers and second-strand synthesis carried out by standard methods.
The double-stranded DNA was size-fractionated through a CL-4B Sepharose column and larger cDNA recovered by ethanol precipitation.
After C-tailing, the DNA was annealed into G-tailed pUC9 and transformed into Escherichia coli. About 800 transformants were obtained and 110 colonies selected on the basis of colony hybridization for plasmid isolation and characterization.
The hybridization of 32P-dATP oligo-labeled cDNA clones to Northern blots of SMYEV dsRNA from strawberry demonstrates that these cDNA clones are specific for SMYEV.
Authors
R.R. Martin, W. Jelkmann, S. Spiegel, R.H. Converse
Keywords
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