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Bidirectional function of the human hprt promoter
88 From this experiment we cannot say anything about the proportion of viable clonogenic cells after the flow sorting. We have studied time dependence of Rhodamine 123 staining and found that the highest proportion of stained cells is seen 2 days after stimulation. We have some ideas how to improve the resolution in order to sort out single 6-TG cells. We know that not all high-fluorescent cells are T-lymphocytes and we are studying the DNA distribution in different parts of the Rhodamine 123 spectrum. In the future we will also look at mutation frequencies after sorting with Rhodamine 123 to analyze the influence of the dye itself.
15 Johnson, P., and T. Friedmann, Department of Pediatrics and Center for Molecular Genetics, School of Medicine UCSD, La Jolla, CA 92093 (U.S.A.) Bidirectional function of the human hprt promoter The promoter region of the human hprt gene is characterized by its high GC content, the absence of a TATA-like sequence and its heterogeneous start sites for transcription in the vicinity of a number of SP-1 binding site-like ' G C boxes'. As part of our studies on the design and function of efficient transducing vectors, we have begun to study the transcription of several reporter genes including firefly luciferase, E. coli B-galactosidase, the chloramphenicol acetyl transferase gene (CAT) and the Tn 5 phosphotransferase gene that confers
resistance to the neomycin analogue G418 from the hprt promoter in transfected cells. We have prepared vectors expressing a single reporter gene from the hprt promoter in both orientations and other vectors expressing 2 reporter genes simultaneously in divergent directions. We have been able to detect efficient expressions of the luciferase gene from the normal and the inverted orientation of the hprt promoter in both single- and double-gene constructs. Expression of the CAT and neomycin resistance genes has been efficient in the ordinary orientation but much less so from the inverted hprt promoter. To date, we have found no convincing evidence for the existence of a naturally occurring transcript from the opposite strand in HeLa and other human cells. We have been able to demonstrate transcription and gene expression from the reporter genes in single-gene vectors containing the promoter from another human X-linked 'housekeeping' gene, phosphoglycerol kinase (PGK) in ordinary and reverse orientations. We cannot yet explain the poor expression of the neomycin-resistance and CAT genes from the inverted hprt promoter, but it is possible that there is a contribution to the promoter activity from the luciferase gene that is absent from the other reporter genes. The ability, however, of the hprt promoter to function bidirectionally under some circumstances lends some support to previous demonstrations of opposite transcripts from the mouse dhfr and hprt genes, but the possible significance and function for such transcription is not clear.
15 Johnson, P., and T. Friedmann, Department of Pediatrics and Center for Molecular Genetics, School of Medicine UCSD, La Jolla, CA 92093 (U.S.A.) Bidirectional function of the human hprt promoter The promoter region of the human hprt gene is characterized by its high GC content, the absence of a TATA-like sequence and its heterogeneous start sites for transcription in the vicinity of a number of SP-1 binding site-like ' G C boxes'. As part of our studies on the design and function of efficient transducing vectors, we have begun to study the transcription of several reporter genes including firefly luciferase, E. coli B-galactosidase, the chloramphenicol acetyl transferase gene (CAT) and the Tn 5 phosphotransferase gene that confers
resistance to the neomycin analogue G418 from the hprt promoter in transfected cells. We have prepared vectors expressing a single reporter gene from the hprt promoter in both orientations and other vectors expressing 2 reporter genes simultaneously in divergent directions. We have been able to detect efficient expressions of the luciferase gene from the normal and the inverted orientation of the hprt promoter in both single- and double-gene constructs. Expression of the CAT and neomycin resistance genes has been efficient in the ordinary orientation but much less so from the inverted hprt promoter. To date, we have found no convincing evidence for the existence of a naturally occurring transcript from the opposite strand in HeLa and other human cells. We have been able to demonstrate transcription and gene expression from the reporter genes in single-gene vectors containing the promoter from another human X-linked 'housekeeping' gene, phosphoglycerol kinase (PGK) in ordinary and reverse orientations. We cannot yet explain the poor expression of the neomycin-resistance and CAT genes from the inverted hprt promoter, but it is possible that there is a contribution to the promoter activity from the luciferase gene that is absent from the other reporter genes. The ability, however, of the hprt promoter to function bidirectionally under some circumstances lends some support to previous demonstrations of opposite transcripts from the mouse dhfr and hprt genes, but the possible significance and function for such transcription is not clear.