Characterization of the Mpv 20 proviral insertion site and of the Unp gene.

Abstract

Mpv 20 is a transgenic mouse line generated by retroviral infection of embryos in the laboratory of R. Jaenisch. Of the seventy lines generated, only four displayed a recessive lethal phenotype. The fourth line Mpv 17 (Weiher et al. 1990) displays an adult lethal phenotype. The proviral integration site was cloned and a series of genomic clones were isolated from DNA libraries generated from both heterozygote and wild type animals. Using a combination of exon trapping and genomic sequencing of a 3.5 kb region surrounding the proviral integration site, it was determined that the virus integrated into an intron of the murine homologue of the human Npat (Nuclear Protein at the AT locus) gene (accession D83243, U58852, X97186). Northern analysis using a partial murine lung Npat cDNA, generated by RT-PCR, demonstrated on average a reduction of 1.6 fold Npat message in RNA obtained from heterozygote tissues relative to a wild type littermate control. A 5$\sp\prime$ RACE protocol was used to isolate a longer cDNA which was used on Southern blots to examine the possibility of methylation differences between the different animals. Under the experimental conditions utilized, no observable differences were detected between wild type and heterozygote animals. RACE product did however detect a truncated message on a Northern blot present in heterozygote animals but not in the wild type controls. During a search for candidate genes present at the Mpv 20 locus, a cDNA named Unp was isolated (Gupta et al. 1993). The second part of the thesis dealt with the determination of the genomic structure of the Unp gene. The Unp gene was found to be present in 22 exons spanning 47.3 kb and has been assigned the Genbank accession AF026469. All exons, with the exception of exon 1 which contains the transcriptional start site and exon 22 which contains the complete untranslated region, were flanked by the consensus splice acceptor/donor dinucleotides AG/GT (Mount 1982). Compilation of exonic sequences identified the presence of sequencing errors present in the initially published cDNA sequence (Gupta et al. 1993). A 1.1 kb Eco RI/Sac I region upstream of the transcriptional start site was examined for promoter activity using a CAT reporter construct. Low activity was observed in this construct. Surprisingly, a substantial increase in CAT activity was observed when a 556 bp segment at the 5$\sp\prime$ end was deleted from the reporter construct leading us to believe that some repressor element must be present in the deleted region. The promoter fragment lacking the repressor area was found to display activity in both orientations in our experimental assay. (Abstract shortened by UMI.)