Open AccessIsolation of cellular promoters by using a retrovirus promoter trap.
Author(s) -
Harald von Melchner,
S Reddy,
H E Ruley
Publication year - 1990
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.87.10.3733
Subject(s) - promoter , biology , retrovirus , gene , microbiology and biotechnology , long terminal repeat , genetics , reporter gene , dna , selectable marker , gene expression , plasmid
A retrovirus vector has been used to isolate transcriptional promoters from mammalian cells. The virus contains a selectable gene encoding histidinol dehydrogenase (his) in the U3 region of the 3' long terminal repeat (LTR). When the virus is passaged, duplication of LTRs places his sequences just 30 nucleotides from the adjacent cellular DNA. As a result, selection for histidinol resistance generates cell clones in which his is expressed on transcripts initiating in the flanking cellular DNA. Upstream cellular sequences, cloned after amplification by polymerase chain reaction, hybridized to RNA from uninfected cells, indicating that the adjacent promoters were transcriptionally active prior to virus integration. Two cloned transcribed flanking sequences also contained highly active transcriptional promoters, as estimated by their ability to activate expression of a linked reporter gene. Thus, U3His vectors provide a rapid and efficient means to isolate promoters active in different cell types. Moreover, by selecting for cell clones containing proviruses integrated in expressed genes, the virus may make an effective insertional mutagen.