The SV40 early transcriptional regulatory element is unable to direct gene expression in pituitary GH-3 cells.
The SV40 early (SV40E) transcriptional regulatory element (TRE) is able to direct heterologous gene expression in a variety of eukaryotic cell lines. This ability is conferred, in part, by the presence of several cis-elements. Transfection studies, mutational analyses, and in vitro DNA binding assays have demonstrated that the SV40E TRE is capable of interacting with several cellular transcription (trans) factors. In the present study, we have investigated the inability of the SV40E TRE to direct gene expression in cultured rat anterior pituitary GH-3 cells. Gel shift analysis demonstrated that nuclear factors within these cells can recognize and specifically bind to DNA containing SV40 enhancer sequences. Surprisingly, we have found that both HeLa and GH-3 cells possess relatively equal quantities of Sp1-specific RNA; however, a dramatic decrease in Sp1 protein was seen in GH-3 cells. Transfection studies utilizing CAT reporter plasmids revealed that the intact SV40E TRE is inactive in these cells, and that subsequent deletion of a region(s) where nuclear factor binding occurs does not result in detectable levels of gene expression. Thus, removal of cis-sites potentially involved in repressor binding does not result in activation of the SV40E TRE in these cells. Subcloning an SV40 enhancer fragment upstream of a heterologous TK promoter yielded chimeric TREs that could direct high levels of gene expression in HeLa but not GH-3 cells. Therefore, the prototypic SV40 enhancer, in the context of GH-3 cells, cannot enhance gene expression.
Coleman
T A
TA
Department of Zoological and Biomedical Sciences, Ohio University, Athens.
Hou
Y T
YT
Kopchick
J J
JJ
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002872:Chromosome Deletion; D004279:DNA, Viral / Q000235:genetics; D004268:DNA-Binding Proteins / Q000502:physiology*; D004742:Enhancer Elements, Genetic / Q000502:physiology; D005786:Gene Expression Regulation / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000502:physiology*; D010903:Pituitary Gland, Anterior / Q000166:cytology; D051381:Rats; D012045:Regulatory Sequences, Nucleic Acid / Q000502:physiology*; D013539:Simian virus 40 / Q000235:genetics*; D016329:Sp1 Transcription Factor / Q000032:analysis; D014158:Transcription, Genetic / Q000502:physiology
D004279:DNA, Viral; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D016329:Sp1 Transcription Factor