In vitro transcription with K562 cell nuclear extract and globin genes.
Cloned human epsilon-, A/gamma- and beta-globin genes, the insulin gene and the adenovirus 2 major late promoter (Ad2MLP) were employed for transcription in vitro with K562 nuclear extracts. Nuclear extracts could direct accurate initiation of transcription from epsilon-globin without supplement of a whole cell extract. A clear dependence of protein concentration of nuclear extracts on transcriptional enhancement was observed with the epsilon-globin gene. To examine the cis-acting DNA sequences 5' of the promoter region which may be important in specific expression of globin genes, the epsilon-globin template was truncated using restriction enzymes. Transcriptional activity of the epsilon-globin gene varied according to the truncation suggesting possible regions to which nuclear proteins involved in transcription may bind. Fractionation of nuclear extracts by ion exchange chromatography indicated that activity could be recovered in a 175 mM ammonium sulfate fraction while the 50 mM ammonium sulfate fraction decreased transcription activity. A/gamma globin gene and Ad2MLP could be transcribed in nuclear extracts at higher concentrations, however, beta-globin and the insulin gene were not transcribed at any concentration assayed, either from induced or uninduced cells. Transcription of the beta-globin gene was observed in vitro when K562 nuclear extracts were supplemented with HeLa whole cell extracts.
Wada
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Y
Laboratory of Chemical Biology, NIDDK, Bethesda, Maryland 20892.
Noguchi
C T
CT
D002467:Cell Nucleus / Q000032:analysis; D002474:Cell-Free System; D005786:Gene Expression Regulation / Q000187:drug effects*; D005796:Genes; D005814:Genes, Viral; D005914:Globins / Q000096:biosynthesis / Q000235:genetics*; D006367:HeLa Cells / Q000032:analysis; D006801:Humans; D007328:Insulin / Q000235:genetics; D004915:Leukemia, Erythroblastic, Acute / Q000378:metabolism / Q000473:pathology*; D009363:Neoplasm Proteins / Q000302:isolation & purification / Q000494:pharmacology; D009687:Nuclear Proteins / Q000302:isolation & purification / Q000494:pharmacology*; D014158:Transcription, Genetic / Q000187:drug effects*; D014407:Tumor Cells, Cultured / Q000032:analysis
D007328:Insulin; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D005914:Globins