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PMID: 6296764 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

A general strategy for cloning double-stranded RNA: nucleotide sequence of the Simian-11 rotavirus gene 8.

Nucleic acids research ·Vol. 10 ·No. 22 ·1982-11-25 ·Pages 7075-88

Both GW, Bellamy AR, Street JE, Siegman LJ

Abstract

Using Simian-11 rotavirus RNA, a strategy has been developed for the production of full length cloned copies of the genes of double-stranded (dsRNA) viruses. Genomic RNA segments were polyadenylated and reverse transcribed to yield a mixture of full length cDNA copies of both possible polarities. The cDNAs were annealed, filled in to complete any partial copies, tailed and inserted into the PstI site of pBR322 using dG/dC tailing. Cloned rotavirus cDNA gene copies were assigned to genomic RNA segments by Northern hybridization. The complete sequence of gene 8 which codes for NCVP3, a non-structural protein of SA11 rotavirus, was determined from a cloned gene copy. It is 1059 bases in length and has an open reading frame which could code for a protein containing 317 amino acids. The apparent 5' and 3' terminal non coding regions are 46 and 59 bases in length, respectively. The sequence ATGTGACCOH at the 3' end of the plus strand is conserved in four of the eleven genes examined. The cloning procedures used should be generally applicable to viruses with segmented dsRNA genomes.

MeSH Terms
Base Sequence Cloning, Molecular DNA/biosynthesis Genes, Viral Plasmids RNA, Double-Stranded/genetics Reoviridae/genetics Rotavirus/genetics
Chemicals
RNA, Double-Stranded DNA
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Both G W
Bellamy A R
Street J E
Siegman L J
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45 references, click to expand
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Article Info
Journal
Nucleic acids research
Abbr.
Nucleic Acids Res
ISSN
0305-1048
DOI
10.1093/nar/10.22.7075
Published
1982-11-25
Pages
7075-88
Language
English
Region
England
NLM ID
0411011
PMCID
PMC326989
Subset
IM
Databases
GENBANK
J02353
External Links
PubMed source DOI: 10.1093/nar/10.22.7075 PMC Full Text View journal details
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