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Southern blot analysis of DNA polymorphism among HLA-DR5-associated Dw subtypes
Abstracts
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which is present in normal relatives of the patients in addition to the diabetics themselves.
EVIDENCE FOR GENE DUPLICATION WITHIN THE HLA CLASS II REGION DETECTED BY SEROLOGICAL, IMMUNOCHEMICAL, AND DNA ANALYSES. Avraham Amar, Erik Mickelson, Gayle Baldwin, Brenda Nisperos, John A. Hansen, and Gerald T. Nepom; Genetic Systems Corporation, Puget Sound Blood Center, and the University of Washington School of Medicine. Seattle, WA Two-dimensional electrophoresis and restriction fragment length polymorphism (RFLP) analysis were performed on a cell line established from an individual homozygous for an H L A - D specificity related to HLA-Dw8 (MN75). Immunoprecipitated H L A - D R antigens from MN75 reveal the existence of two sets of/3 chain patterns. These patterns correlate with two serologically defined specificities assigned to this cell line, DR5 and DRw8. In addition, Southern blots of D N A extracted from this cell line, hybridized with a DR/3 c D N A probe, identify RFLP corresponding to DR5-1ike and DR8-1ike genes, correlating with the serological and biochemical data. Analysis of an individual heterozygous for M N 7 5 and another HLA specificity, Dw4, demonstrated that DR5-1ike and DR8-1ike genes are related in a Cis configuration on a single haplotype. It is suggested that M N 7 5 has undergone a gene duplication within HLA class II genes, and carries two sets of expressed D R genes on the same haplotype. This haplotype, designated as D R [5 + 8], is composed of both DR5 and DRw8 molecules similar to those seen in the homozygous cell lines SWEIG and OLG, respectively.
SOUTHERN BLOT ANALYSIS OF DNA POLYMORPHISM AMONG HLA-DR5-ASSOCIATED Dw SUBTYPES. Avraham Amar, Erik Mickelson, Brenda Nisperos, John A. Hansen, and Gerald T. Nepom; Genetic Systems Corp., Puget Sound Blood Center. and the University of Washington School of Medicine. Seattle, WA Class II variation among a panel of ten HLA-DR5 homozygous typing cells was investigated by analysis of restriction fragment length polymorphisms in genomic DNA. Very little polymorphism was found using restriction enzyme fragment analysis by Southern blotting among eight DR5-Dw5 homozygous individuals using DR/3, DQ~, or DQ/3 probes. However, restriction fragment polymorphism was easily detected among HLA DR5- Dw-(blank) homozygous individuals, using the above mentioned probes, and their patterns were distinct from those of the DR5-Dw5 cluster. Probes were hybridized to cellular D N A digested with several restriction enzymes including Hind III, Bgl II, Taq I, and Bam HI. The general patterns of polymorphism are consistent with the serological pattern and MLC reactivity used to characterize the DR5 panel of cells.
A STRAIGHTFORWARD APPROACH TO TYPE FOR c~ AND /3 ALLELES AT THE HLADQ AND DX LOCI. Massimo Trucco, Sharon Rosenshine, Isabella Cascino, Adriana Zeevi, and Rene Duquesnoy; The Wistar Institute of Anatomy and Biology, Philadelphia, PA Today's most intriguing aspect of the study of H L A - D region products is the location of determinants recognized by alloantisera and monoclonal antibodies showing the so-called "supertypic" specificities like those of the MT and MB series. We have been looking for the most informative and the simplest (i.e., not requiring statistical tests) approach to find the link between patterns at the D N A
191
which is present in normal relatives of the patients in addition to the diabetics themselves.
EVIDENCE FOR GENE DUPLICATION WITHIN THE HLA CLASS II REGION DETECTED BY SEROLOGICAL, IMMUNOCHEMICAL, AND DNA ANALYSES. Avraham Amar, Erik Mickelson, Gayle Baldwin, Brenda Nisperos, John A. Hansen, and Gerald T. Nepom; Genetic Systems Corporation, Puget Sound Blood Center, and the University of Washington School of Medicine. Seattle, WA Two-dimensional electrophoresis and restriction fragment length polymorphism (RFLP) analysis were performed on a cell line established from an individual homozygous for an H L A - D specificity related to HLA-Dw8 (MN75). Immunoprecipitated H L A - D R antigens from MN75 reveal the existence of two sets of/3 chain patterns. These patterns correlate with two serologically defined specificities assigned to this cell line, DR5 and DRw8. In addition, Southern blots of D N A extracted from this cell line, hybridized with a DR/3 c D N A probe, identify RFLP corresponding to DR5-1ike and DR8-1ike genes, correlating with the serological and biochemical data. Analysis of an individual heterozygous for M N 7 5 and another HLA specificity, Dw4, demonstrated that DR5-1ike and DR8-1ike genes are related in a Cis configuration on a single haplotype. It is suggested that M N 7 5 has undergone a gene duplication within HLA class II genes, and carries two sets of expressed D R genes on the same haplotype. This haplotype, designated as D R [5 + 8], is composed of both DR5 and DRw8 molecules similar to those seen in the homozygous cell lines SWEIG and OLG, respectively.
SOUTHERN BLOT ANALYSIS OF DNA POLYMORPHISM AMONG HLA-DR5-ASSOCIATED Dw SUBTYPES. Avraham Amar, Erik Mickelson, Brenda Nisperos, John A. Hansen, and Gerald T. Nepom; Genetic Systems Corp., Puget Sound Blood Center. and the University of Washington School of Medicine. Seattle, WA Class II variation among a panel of ten HLA-DR5 homozygous typing cells was investigated by analysis of restriction fragment length polymorphisms in genomic DNA. Very little polymorphism was found using restriction enzyme fragment analysis by Southern blotting among eight DR5-Dw5 homozygous individuals using DR/3, DQ~, or DQ/3 probes. However, restriction fragment polymorphism was easily detected among HLA DR5- Dw-(blank) homozygous individuals, using the above mentioned probes, and their patterns were distinct from those of the DR5-Dw5 cluster. Probes were hybridized to cellular D N A digested with several restriction enzymes including Hind III, Bgl II, Taq I, and Bam HI. The general patterns of polymorphism are consistent with the serological pattern and MLC reactivity used to characterize the DR5 panel of cells.
A STRAIGHTFORWARD APPROACH TO TYPE FOR c~ AND /3 ALLELES AT THE HLADQ AND DX LOCI. Massimo Trucco, Sharon Rosenshine, Isabella Cascino, Adriana Zeevi, and Rene Duquesnoy; The Wistar Institute of Anatomy and Biology, Philadelphia, PA Today's most intriguing aspect of the study of H L A - D region products is the location of determinants recognized by alloantisera and monoclonal antibodies showing the so-called "supertypic" specificities like those of the MT and MB series. We have been looking for the most informative and the simplest (i.e., not requiring statistical tests) approach to find the link between patterns at the D N A