Complementary DNA Cloning and analysis of carnation mottle virus RNA

J. C. Carrington, T. J. Morris

Research output: Contribution to journalArticle

36 Citations (Scopus)

Abstract

A 3850-base pair (bp) cDNA fragment, representing most of the carnation mottle virus (CarMV) genome, was molecularly cloned in Escherichia coli using a modification of the plasmid-primer method of Okayama and Berg (H. Okayama and P. Berg (1982), Mol. Cell. Biol. 2, 161-170). Poly(dT)-tailed pUC8 was used to prime cDNA synthesis from polyadenylated CarMV RNA template. Oligo(dC) was added to the cDNA 3′ end, and second-strand synthesis was specifically primed using an oligo(dG)-tailed linker fragment. cDNA inserts of 3250 and 1950 by were identified from a clone bank as apparently overlapping and were fused at a common BglII restriction site to produce pCarMV-1C. Confirmation that this plasmid harbored CarMV cDNA sequences was achieved by Southern blot hybridization with a randomly primed cDNA probe. Nick-translated pCarMV-1C was used to probe virion and total single-stranded RNA extracts from infected Chenopodtium quinoa leaves by "Northern" blot hybridization. Besides the full-length genomic RNA, two additional species of approximately 1600 and 1750 bases were associated with CarMV. Based on a series of Northern blots with nick-translated subclones derived from pCarMV-1C as probes, these subgenomic RNAs were found to originate from the 3′ domain of the virus genome.

Original languageEnglish (US)
Pages (from-to)22-31
Number of pages10
JournalVirology
Volume139
Issue number1
DOIs
StatePublished - Nov 1984

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Carmovirus
Organism Cloning
Complementary DNA
RNA
Northern Blotting
Plasmids
Chenopodium quinoa
Poly T
Genome
RNA Probes
Southern Blotting
Base Pairing
Virion
Clone Cells
Escherichia coli
Viruses

ASJC Scopus subject areas

  • Virology

Cite this

Complementary DNA Cloning and analysis of carnation mottle virus RNA. / Carrington, J. C.; Morris, T. J.

In: Virology, Vol. 139, No. 1, 11.1984, p. 22-31.

Research output: Contribution to journalArticle

Carrington, J. C. ; Morris, T. J. / Complementary DNA Cloning and analysis of carnation mottle virus RNA. In: Virology. 1984 ; Vol. 139, No. 1. pp. 22-31.
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