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Enhancer and silencer-like elements in the vicinity of HLA-A11 class I gene
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Abstracts ENHANCER AND SILENCER-LIKE ELEMENTS IN THE VICINITY OF HLA-A11 CLASS I GENE. H Zinszner, O Blancher, JF Bourge, L Degos, and P Paul, INSERM U93, Hopital Saint Louis, 75010 PARIS. Class I MHC antigens are expressed at various levels in most somatic cells. Recent studies suggest that different cis-acting elements may control class I gene expression and tissue specificity. For the human class I genes only the promoter region 500 base pairs upstream of the transcription initiation site has been well described. We have investigated the existence of new DNA regulatory elements involved in the regulation of classical HLA class I gene expression. A genomic DNA fragment consisting of the entire HLA-A11 gene, 9 kilobases upstream and 2.5 kilobases downstream of this gene was isolated. Subfragments of these regions were independently subcloned in the CAT expression vector. CAT activity assays were performed after transfection in various lymphoid and non lympho'fd cell lines. This allowed us to isolate two elements that show respectively enhancer-like activity contained in a 110 base pairs DNA fragment and silencer-like activity contained in a 450 base pairs fragment. The effect of these two regulatory regions are now being tested on the HLA-A promoter itself and their implication in tissue specific expression of class I genes is tested using band-shift assays.
MOLECULAR ANALYSIS OF A RECOMBINATION BREAKPOINT IN THE HLA-DQ REGION, KS Stranick, BK Kienzle, and .RW Knowles. Memorial Sloan-Kettering Cancer Center, New York, NY. Despite extensive linkage disequilibrium in the HLA-DR and DQ regions, several class II haplotypes, including DR7-DQw2, appear to have resulted from recombination events between the DQB1 and DQA1 genes. In order to understand the molecular mechanism of recombination, the genomic region around the breakpoint in the DR7-DQw2 cell line, MANN (DQ~2.7/DQc~-3.7), was compared to the presumed donor haplotypes represented by the DR3-DQw2 cell line, VAVY (DQI3-2.3/DQc~-2.3), and the DR7-DQw9 cell line, DBB (DQ~-3.3/DQc~-3.7). Preliminary mapping of this region, using a genomic probe located 5 kb downstream from the 3' end of DQB1, indicated that the breakpoint was located near the DQB 1 gene. Genomic clones from MANN and DBB were then isolated from ~. libraries and the breakpoint was further localized by fine restriction mapping. Sequence analysis of this apparent recombination breakpoint has identified a repetitive sequence, (7-purine)15, immediately on the 5' side of the breakpoint in MANN, which replaces a pyrimidine-rich unique sequence in DBB. On the 3' side of the breakpoint, another repetitive sequence, (TG)20, was found near a truncated DQB1 pseudogene, that appears to have resulted from a previous unequal crossing over event. This would suggest that at least two recombination events have occurred near this potential Z-DNA structure, (TG)n, that has also been found near several translocation breakpoints in other genes. Analysis of similar breakpoints in the DQ region of other recombinant haplotypes may further define the role of these repetitive sequences in the molecular mechanism of recombination.
Abstracts ENHANCER AND SILENCER-LIKE ELEMENTS IN THE VICINITY OF HLA-A11 CLASS I GENE. H Zinszner, O Blancher, JF Bourge, L Degos, and P Paul, INSERM U93, Hopital Saint Louis, 75010 PARIS. Class I MHC antigens are expressed at various levels in most somatic cells. Recent studies suggest that different cis-acting elements may control class I gene expression and tissue specificity. For the human class I genes only the promoter region 500 base pairs upstream of the transcription initiation site has been well described. We have investigated the existence of new DNA regulatory elements involved in the regulation of classical HLA class I gene expression. A genomic DNA fragment consisting of the entire HLA-A11 gene, 9 kilobases upstream and 2.5 kilobases downstream of this gene was isolated. Subfragments of these regions were independently subcloned in the CAT expression vector. CAT activity assays were performed after transfection in various lymphoid and non lympho'fd cell lines. This allowed us to isolate two elements that show respectively enhancer-like activity contained in a 110 base pairs DNA fragment and silencer-like activity contained in a 450 base pairs fragment. The effect of these two regulatory regions are now being tested on the HLA-A promoter itself and their implication in tissue specific expression of class I genes is tested using band-shift assays.
MOLECULAR ANALYSIS OF A RECOMBINATION BREAKPOINT IN THE HLA-DQ REGION, KS Stranick, BK Kienzle, and .RW Knowles. Memorial Sloan-Kettering Cancer Center, New York, NY. Despite extensive linkage disequilibrium in the HLA-DR and DQ regions, several class II haplotypes, including DR7-DQw2, appear to have resulted from recombination events between the DQB1 and DQA1 genes. In order to understand the molecular mechanism of recombination, the genomic region around the breakpoint in the DR7-DQw2 cell line, MANN (DQ~2.7/DQc~-3.7), was compared to the presumed donor haplotypes represented by the DR3-DQw2 cell line, VAVY (DQI3-2.3/DQc~-2.3), and the DR7-DQw9 cell line, DBB (DQ~-3.3/DQc~-3.7). Preliminary mapping of this region, using a genomic probe located 5 kb downstream from the 3' end of DQB1, indicated that the breakpoint was located near the DQB 1 gene. Genomic clones from MANN and DBB were then isolated from ~. libraries and the breakpoint was further localized by fine restriction mapping. Sequence analysis of this apparent recombination breakpoint has identified a repetitive sequence, (7-purine)15, immediately on the 5' side of the breakpoint in MANN, which replaces a pyrimidine-rich unique sequence in DBB. On the 3' side of the breakpoint, another repetitive sequence, (TG)20, was found near a truncated DQB1 pseudogene, that appears to have resulted from a previous unequal crossing over event. This would suggest that at least two recombination events have occurred near this potential Z-DNA structure, (TG)n, that has also been found near several translocation breakpoints in other genes. Analysis of similar breakpoints in the DQ region of other recombinant haplotypes may further define the role of these repetitive sequences in the molecular mechanism of recombination.