Hormone-responsive expression of an endogenous proviral gene of mouse mammary tumor virus after molecular cloning and gene transfer into cultured cells.
A recombinant lambda phage containing mouse mammary tumor virus (MMTV) proviral DNA was isolated from a gene library constructed from GR mouse liver DNA. Restriction enzyme analyses reveal that the cloned molecule contains a copy of one of the GR endogenous MMTV proviruses flanked on both sides by 2--3 kb of mouse genomic DNA. In this report we have examined the expression of the cloned MMTV provirus after cotransfection with the herpes thymidine kinase (TK; ATP:thymidine 5'-phosphotransferase,, EC 2.7.1.21) gene and integration into mouse LTK- cells. Nine individual TK+ transformants were selected, and all were found to contain MMTV-transfected DNA. One of the TK+ transformants was chosen for further study. Total poly(A)-containing RNA was isolated from the cells, and liquid hybridization analyses with MMTV cDNA showed that it contained 0.02% MMTV-specific RNA. The sizes of the MMTV-specific species were determined and found to correspond to the 35S and 24S mRNAs synthesized in MMTV-infected cells. Glucocorticoid hormones have been shown to increase the concentration of MMTV RNA in virus-infected cultured cells. Therefore, we tested the effect of dexamethasone on the concentration of MMTV-specific RNA in cells transfected with the MMTV proviral DNA. The amount of MMTV-specific poly(A)-containing RNA found in the cells grown in the presence of hormone was 0.17%. Therefore, dexamethasone causes an 8-fold increase in the amount of MMTV-specific RNA in mouse cells containing several copies of a cloned and transfected MMTV proviral gene.
D000818:Animals; D010582:Bacteriophage lambda; D002472:Cell Transformation, Viral; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000235:genetics; D003907:Dexamethasone / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects*; D005814:Genes, Viral; D008324:Mammary Tumor Virus, Mouse / Q000235:genetics*; D051379:Mice
D004279:DNA, Viral; D003907:Dexamethasone