Derivatives of Moloney murine sarcoma virus capable of being transcribed in embryonal carcinoma stem cells have gained a functional Sp1 binding site.
The long terminal repeat (LTR) sequences of Moloney murine leukemia virus and its closely related derivative Moloney murine sarcoma virus (Mo-MSV) are incapable of directing transcription in embryonal carcinoma (EC) stem cells. The myeloproliferative sarcoma virus, a derivative of Mo-MSV, has several point mutations in the LTR and is transcribed more efficiently to allow productive infection of F9 EC cells. One of these mutations, at -166 with respect to the transcriptional start, creates a consensus binding site for the well-characterized mammalian transcription factor Sp1. We used gel retardation assays to demonstrate that F9 EC cell extracts form several complexes with the myeloproliferative sarcoma virus sequence around -166. One of these complexes involves a murine Sp1-like protein, which has immunoreactivity, DNA binding specificity, and electrophoretic mobility equivalent to those of purified human Sp1 protein. An equivalent complex forms on the corresponding Mo-MSV sequence but with a fivefold-lower affinity. Consistent with these observations, introduction of the single point mutation at -166 into the Mo-MSV LTR, creating a consensus Sp1 binding site, increases expression in F9 EC cells sixfold.
Prince
V E
VE
Laboratory of Eukaryotic Molecular Genetics, National Institute for Medical Research, The Ridgeway, London, England.
Rigby
P W
PW
D001483:Base Sequence; D001665:Binding Sites; D002448:Cell Adhesion; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism; D016384:Consensus Sequence; D004279:DNA, Viral / Q000378:metabolism; D054278:Embryonal Carcinoma Stem Cells; D015870:Gene Expression; D009052:Leukemia Virus, Murine / Q000235:genetics* / Q000276:immunology; D008969:Molecular Sequence Data; D008980:Moloney murine sarcoma virus / Q000235:genetics* / Q000276:immunology; D009154:Mutation; D014411:Neoplastic Stem Cells / Q000382:microbiology*; D012367:RNA, Viral / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D013724:Teratoma / Q000382:microbiology*
D004279:DNA, Viral; D012367:RNA, Viral; D016329:Sp1 Transcription Factor; D015500:Chloramphenicol O-Acetyltransferase