Simplified plasmid rescue of host sequences adjacent to integrated proviruses.
We have previously described a Moloney murine leukemia retroviral (MoMLV) vector useful for the generation of anchored long-range maps of complex mammalian genomes. We now report the development of a modified vector carrying the ColE1 origin of replication and the chloramphenicol-resistance (CmR) gene to facilitate the recovery of genomic sequences adjacent to integrated proviruses. We demonstrate the utility of this new vector for the rescue in plasmid form of a 6-kb human fragment containing portions of an Alu element adjacent to the proviral 3'-LTR from an infected human primary fibroblast clone. We generated a sequence-tagged site (STS) from the derived sequence outside the Alu element, and used a somatic cell hybrid mapping panel to assign this STS to human chromosome 10 by a polymerase chain reaction-based assay. We suggest that this new CmR vector will be useful for recovering sequences of genes interrupted by proviral insertion, to generate directional maps with respect to the inserted provirus using the single cleavage sites within the vector, and to generate site-specific STS for defining and mapping the integration site.
Kurdi-Haidar
B
B
Department of Pediatrics, UCSD School of Medicine, La Jolla, CA 92093-0634, USA. bhaidar@ucsd.edu
Friedmann
T
T
D000818:Animals; D001483:Base Sequence; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis; D002874:Chromosome Mapping; D002879:Chromosomes, Human, Pair 10; D002999:Clone Cells; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli; D005822:Genetic Vectors; D015894:Genome, Human; D006801:Humans; D006822:Hybrid Cells; D019868:Kanamycin Kinase; D008156:Luciferases / Q000096:biosynthesis; D008322:Mammals; D008969:Molecular Sequence Data; D008979:Moloney murine leukemia virus / Q000235:genetics*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000096:biosynthesis; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011533:Proviruses / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis; D012091:Repetitive Sequences, Nucleic Acid; D018741:Replication Origin; D016324:Sequence Tagged Sites; D014162:Transfection; D016662:Virus Integration
D017931:DNA Primers; D011994:Recombinant Proteins; D008156:Luciferases; D015500:Chloramphenicol O-Acetyltransferase; D017853:Phosphotransferases (Alcohol Group Acceptor); D019868:Kanamycin Kinase