Identification of cellular promoters by using a retrovirus promoter trap.
A retrovirus vector has been developed that selects for instances in which the provirus integrates in close proximity to cellular promoters. Coding sequences for a selectable marker (histidinol dehydrogenase) were inserted into the 3' long terminal repeat (LTR) of an enhancerless Molony murine leukemia virus. Although 1.8 kilobase pairs in size, the elongated LTR did not appear to interfere with virus replication or integration. Thus, when the virus was passaged, the elongated LTRs efficiently duplicated, placing histidinol dehydrogenase-coding sequences in the 5' LTR just 30 nucleotides from the flanking cellular DNA. Selection for histidinol expression generated cell clones in which histidinol gene sequences in the 5' LTR were invariably expressed on transcripts initiating in nearby cellular sequences. The efficiency of transducing histidinol resistance was 2,500-fold lower than the efficiency of transducing neomycin resistance when the neomycin phosphotransferase gene was located within the retrovirus and expressed from an independent promoter. By tagging transcriptionally active sites, the vector provides a means to identify and isolate promoters active in different cell types. Furthermore, the virus may be useful as an insertional mutagen, since selection for cell populations containing proviruses only in expressed sites is expected to reduce the number of intergrants needed to screen for loss of gene function.
von Melchner
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Center for Cancer Research, Massachusetts Institute of Technology, Cambridge 02139.
Ruley
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D000429:Alcohol Oxidoreductases / Q000096:biosynthesis / Q000235:genetics; D015152:Blotting, Northern; D015139:Blotting, Southern; D002460:Cell Line; D005786:Gene Expression Regulation; D005819:Genetic Markers; D005822:Genetic Vectors; D019868:Kanamycin Kinase; D008979:Moloney murine leukemia virus / Q000235:genetics*; D009693:Nucleic Acid Hybridization; D010770:Phosphotransferases / Q000235:genetics; D011401:Promoter Regions, Genetic; D011533:Proviruses / Q000235:genetics*; D012091:Repetitive Sequences, Nucleic Acid; D015183:Restriction Mapping; D012190:Retroviridae / Q000235:genetics*; D014158:Transcription, Genetic
D005819:Genetic Markers; D000429:Alcohol Oxidoreductases; C021358:histidinol dehydrogenase; D010770:Phosphotransferases; D019868:Kanamycin Kinase