Rous sarcoma virus contains sequences which permit expression of the gag gene in Escherichia coli.

Abstract

Several aspects of Rous sarcoma virus gene expression, including transcription, translation, and protein processing, can occur within Escherichia coli containing cloned viral DNA. The viral long terminal repeat contains a bacterial promoter, and viral sequences at or near the authentic viral initiation codon permit the initiation of translation. These signals can direct the synthesis in E. coli of the viral gag gene precursor Pr76 or, when fused to a portion of the lacZ gene, a gag-beta-galactosidase fusion protein. Pr76 is processed into gag structural proteins in E. coli in a process which is dependent upon the gag product p15. These observations suggest that E. coli can be used for the introduction and analysis of mutations in sequences relevant to viral gene expression.

Bacterial beta-galactosidase as a marker of Rous sarcoma virus gene expression and replication.

“…This insertion fuses the RSV gag gene to lac'Z in the same reading frame. As expected from previous results (29), an enzymatically active 3-galactosidase protein was synthesized in bacteria under the direction of the pPN-7 viral sequences (data not shown); it was thus asked whether the plasmid could direct the synthesis of a similar active fusion protein in eucaryotic cells. Parallel cultures of avian cells were transfected with 3 ,ug of either pZ-1 or pPN-7 per 35-mm well; 3 days posttransfection, cells were lysed with buffered SDS, and ,-galactosidase activity was assayed colorimetrically by the rate of hydrolysis of the substrate ONPG.…”

“…Lysis of avian cells and bacteria, as well as immunoprecipitation, was essentially as described (8,29). Each immunoprecipitation contained 1 x 106 to 3 x 106 acid-insoluble cpm.…”

“…DNA hybridization and S1 digestion conditions were essentially as described (29), except that S1 digestions were performed at 42°C. Digested samples were ethanol precipitated before electrophoresis under denaturing condi- (29), but the cultures were at a lower cell density (absorbance at 600 nm = 0.3 to 0.5). After labeling, bacteria were chilled, washed with 10 mM Tris-hydrochloride, and stored as pellets at -70°C.…”

“…In vitro cleavage of immunoprecipitates with disrupted avian myeloblastosis virus was as described previously (29). Samples were subjected to discontinuous SDS-polyacrylamide gel electrophoresis (29). I-Galactosidase assay and its properties.…”

“…It has previously been found that RSV sequences can direct the synthesis of 3-galactosidase as a gag-lacZ fusion protein in E. coli (29). gag is the viral structural gene located nearest to the 5' end of the genome (12).…”