Basic Immunogenetics.

Size polymorphism of chicken major histocompatibility complex-encoded B-G molecules is due to length variation in the cytoplasmic heptad repeat region.

“…Recent isolation of partial cDNA clones for two homologous B-G molecules (represented by the clones G3/G7 and G8/G9) revealed the presence of long regions of 21-nucleotide (nt) repeats in the 3' coding region (12). While the nucleotide and amino acid sequences in these regions are poorly conserved, the first (and fourth or fifth, depending on the register) residue of the seven-amino acid repeat is almost always hydrophobic, and the others are often highly charged (Fig.…”

“…Chicken strains from the Gipf-Oberfrick farm of the Basel Institute for Immunology, mAbs and rabbit antisera to B-G molecules, the isolation and characterization of purified B-G molecules, and B-G cDNA clones have been described in detail (11,12). Isolation of chicken blood cells, cell surface iodination using lactoperoxidase and glucose oxidase, detergent lysis with phenylmethylsulfonyl fluoride as protease inhibitor, immunoprecipitation using protein A-Sepharose, sodium dodecyl sulfate (SDS)/polyacrylamide gel electrophoresis (with or without 2 mM dithiothreitol as reductant), and fluorography using phosphotungstate screens were performed essentially as described (12,13).…”

“…Isolation of chicken blood cells, cell surface iodination using lactoperoxidase and glucose oxidase, detergent lysis with phenylmethylsulfonyl fluoride as protease inhibitor, immunoprecipitation using protein A-Sepharose, sodium dodecyl sulfate (SDS)/polyacrylamide gel electrophoresis (with or without 2 mM dithiothreitol as reductant), and fluorography using phosphotungstate screens were performed essentially as described (12,13). lodination of purified B-G molecules by the Iodo-Gen method and the protease inhibitor cocktail used in the experiment for Fig.…”

“…For reduction and alkylation under nondenaturing conditions, immunoprecipitates in wash buffer containing Nonidet P-40 (12,13) were incubated at room temperature with 2 mM dithiothreitol for 30 min and then with 6 mM iodoacetamide in the dark for 30 min. Then the samples were boiled in sample buffer containing 2% SDS with or without 10 mM dithiothreitol before analysis by SDS/gel electrophoresis.…”

“…A B-G molecule isolated from GB-1 chicken strain (B13 haplotype) with a monoclonal antibody (mAb) was shown to be a disufide-linked multimer that bore no demonstrable N-linked or 0-linked carbohydrate and that required detergent for solubilization (11). Using rabbit antisera derived from this purified molecule, we isolated B-G19 cDNA clones with a number of distinctive features, including multiple small repeats and short "introns" that may code for protein when not spliced out (12). In this article, we use these rabbit antisera to show that there is considerable size polymorphism for B-G molecules within and between MHC haplotypes, but that these molecules all have the canonical features found for the B-G13 molecule, along with intrachain disulfide bonds in similar-size extracellular regions of at least 30 kDa.…”