RETROVIRAL INSERTIONAL MUTAGENESIS OF THE BETA(2)-MICROGLOBULIN GENE

Abstract

Retroviral insertions into the host chromosome appear to be random with respect to the nucleotide sequence of the target site, and can theoretically be used as insertional mutagens of any host gene. Somatic cell mutants in the gene encoding the cell-surface antigen {dollar}\beta\sb2{dollar}-microglobulin (B2m {dollar}\sp{lcub}\rm b{rcub}){dollar} were studied. 3 of 22 B2m {dollar}\sp{lcub}\rm b{rcub}{dollar} mutants isolated after infection with Moloney-MuLV contained an insertion in B2m {dollar}\sp{lcub}\rm b{rcub}.{dollar} The B2m {dollar}\sp{lcub}\rm b{rcub}{dollar} gene in mutant i1 harbored an Ab-MuLV provirus in the same transcriptional orientation as the gene, and mutants i7 and i18 each had a Mo-MuLV provirus inserted in the reverse orientation. All three proviruses were clustered within 500 bp of each other in the 5{dollar}\sp\prime{dollar} end of B2m {dollar}\sp{lcub}\rm b{rcub}{dollar} intron I.;The three insertional mutations in B2m {dollar}\sp{lcub}\rm b{rcub}{dollar} were analyzed further to determine effects on gene expression. None of the mutants had wild-type levels of functional B2m {dollar}\sp{lcub}\rm b{rcub}{dollar} mRNA, but mutants i1 and i7 did have small amounts of functional B2m {dollar}\sp{lcub}\rm b{rcub}{dollar} RNA. Moreover, mutant i1 produced some {dollar}\beta\sb2{dollar}-m{dollar}\sp{lcub}\rm b{rcub}{dollar} protein. Mutant i1 also had a non-functional, Ab-MuLV-initiated transcript that spliced into B2m {dollar}\sp{lcub}\rm b{rcub}{dollar} exon II. In contrast, mutant i7 and i18 Mo-MuLV proviruses caused transcription complementary to B2m coding sequence. This readthrough viral transcription may have a role in B2m {dollar}\sp{lcub}\rm b{rcub}{dollar} inactivation, and also reflects the potential for insertional activation of host genes.;45% of spontaneous mutants in this model system (10 of 22 mutants) arose by loss of heterozygosity at B2m and a more distal marker on chromosome 2. It was therefore possible to distinguish non-insertional B2m mutants from potential insertional mutants without probes for B2m, since distal markers could be used instead. This principle may be important in eliminating non-insertional mutants of uncloned genes when attempting insertional mutagenesis. In preliminary attempts at mutagenizing Ly-18.2, which maps to chromosome 12, it was found that many of the spontaneous mutants occurred by recombination in and near the centromeric rDNA cluster of the chromosome. These mutations resulted in the loss of heterozygosity of several distal markers. By this method, up to 90% of spontaneous Ly-18 surface antigen mutants could be eliminated, thus facilitating analysis of those mutants that are potentially insertional.