Identification of Cellular Promoters by Using a Retrovirus Promoter Trap

Overview

Journal J Virol

Publisher American Society for Microbiology

Date 1989 Aug 1

PMID 2545900

Citations 34

Authors

H von Melchner

H E Ruley

Affiliations

Soon will be listed here.

Abstract

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.

Citing Articles

An Promoter-Trap: Augmenting Genome Annotation and Functional Genomics.

Reid W, Pilitt K, Alford R, Cervantes-Medina A, Yu H, Aluvihare C G3 (Bethesda). 2018; 8(10):3119-3130.

PMID: 30135106 PMC: 6169391. DOI: 10.1534/g3.118.200347.

Systems Biology-Based Investigation of Cellular Antiviral Drug Targets Identified by Gene-Trap Insertional Mutagenesis.

Cheng F, Murray J, Zhao J, Sheng J, Zhao Z, Rubin D PLoS Comput Biol. 2016; 12(9):e1005074.

PMID: 27632082 PMC: 5025164. DOI: 10.1371/journal.pcbi.1005074.

Functional Genomic Strategies for Elucidating Human-Virus Interactions: Will CRISPR Knockout RNAi and Haploid Cells?.

Perreira J, Meraner P, Brass A Adv Virus Res. 2016; 94:1-51.

PMID: 26997589 PMC: 7112329. DOI: 10.1016/bs.aivir.2015.11.001.

A role for H/ACA and C/D small nucleolar RNAs in viral replication.

Murray J, Sheng J, Rubin D Mol Biotechnol. 2014; 56(5):429-37.

PMID: 24477674 PMC: 7090452. DOI: 10.1007/s12033-013-9730-0.

LoxP-FRT Trap (LOFT): a simple and flexible system for conventional and reversible gene targeting.

Chaiyachati B, Kaundal R, Zhao J, Wu J, Flavell R, Chi T BMC Biol. 2012; 10:96.

PMID: 23198860 PMC: 3529186. DOI: 10.1186/1741-7007-10-96.

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