Three sets of cosmid clones-containing the HLADRa chain gene and two additional related genes-were isolated from human genomic DNA libraries by using a cDNA probe for the HLA-DRa chain. Southern blot analysis using DNA from somatic cell hybrids indicated that all of the clones mapped to chromosome 6. Partial sequence analysis showed that the two additional related genes were highly homologous to each other, and to the HLA-DRa chain, in parts of the exon that encoded the a2 domain but were more divergent in intron sequences. One of the genes corresponds to the HLA-DR-related DC series. DNA probes made from this gene revealed marked restriction enzyme polymorphism when hybridized to genomic DNA from HLA-DR typed homozygous cell lines. The patterns obtained from a number of homozygous and heterozygous cell lines correlated with the HLA-DR crossreactive serotypes and also indicated that there is a further sequence in the haploid human genome that is closely homologous with the DCa chain sequence. One family was studied and showed the expected HLA-DR-associated inheritance of restriction enzyme patterns. No polymorphism has yet been demonstrated in restriction enzyme fragments that include the other cloned sequence, which may correspond to the SBa chain gene or to a novel HLA-DR-related gene. These experiments indicate that there are at least three sequences in the human genome related, but not identical, to the HLA-DRa chain gene.
SummaryExtra copies of the human tissue-type plasminogen activator (t-PA) gene were introduced into the Bowes melanoma cell line. We obtained a recombinant cell line (TRBM6) which secretes approximately ten-fold more t-PA than the parent cell line. The identity of the plasminogen activator made by the new cell line was confirmed by sizing on sodium dodecyl sulphate polyacrylamide gels and by specific quenching using anti-t-PA antibody. We estimate that the recombinant line produces t-PA at a rate of approximately 3 pg/cell/24 hr and that t-PA accumulates in the harvest medium at a rate of approximately 4000 International t-PA Units/ml/24 hr.
We have isolated a cDNA clone corresponding to a substantial portion of the human tissue-type plasminogen activator (t-PA) protein. It encodes almost all of the protein B chain and part of the 3' untranslated region. We have used this clone to screen bacteriophage lambda and cosmid libraries of human genomic DNA. Several related genomic clones were isolated. One of these, a cosmid clone, carried approx. 40 kb of human DNA. Mapping experiments indicate that the region containing the protein-coding exons is approx. 20 kb in length. The cosmid, containing the t-PA gene and the aminoglycosyl-3'-phosphotransferase dominant-selection marker, was introduced into mouse L cells. Approximately half of the transformants were shown to produce human t-PA. We demonstrated that the fibrinolytic t-PA activity could be specifically quenched by anti-t-PA antibody and that the recombinant t-PA was of similar size (by SDS-polyacrylamide gel electrophoresis) to the t-PA produced by the human Bowes melanoma cell line. Our results suggest that the cosmid clone carries the whole t-PA coding region together with the regulatory elements necessary for its expression.
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