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In vitro expression of a full‐length DNA copy of cowpea mosaic virus B RNA: identification of the B RNA encoded 24‐kd protein as a viral protease
Author(s) -
Verver Jan,
Goldbach Rob,
Garcia Juan A.,
Vos Pieter
Publication year - 1987
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.1002/j.1460-2075.1987.tb04789.x
Subject(s) - biology , garcia , rna , microbiology and biotechnology , humanities , genetics , gene , art
Double‐stranded cDNA was synthesized from B component RNA of cowpea mosaic virus and cloned into appropriate vectors. Using four clones, together representing the entire B RNA sequence, a full‐length DNA copy was constructed and subsequently positioned downstream of a phage SP6 or T7 promoter. RNA molecules transcribed from this full‐size DNA copy using SP6 or T7 RNA polymerase were efficiently translated in rabbit reticulocyte lysates into a 200‐kd polypeptide similar to RNA isolated from viral B components. Moreover this polypeptide was rapidly cleaved into 32‐kd and 170‐kd polypeptides, exactly like the 200‐kd polypeptide encoded by viral B RNA. In vitro transcription and translation of a DNA copy in which an 87‐bp‐long deletion in the coding sequence for the 24‐kd polypeptide was introduced revealed that the 24‐kd polypeptide bears the proteolytic activity involved in the primary cleavage of the B RNA‐encoded polyprotein.