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The tentative amino acid sequence of pooled, papain-solubilized HLA antigen heavy chains has been determined. The amino acid sequence comprises 273 residues. As the structural analyses were performed on HLA antigen heavy chains comprising a mixture of several allelic forms derived from the A, B, and possibly C loci, multiple residues were encountered in several positions. However, a quantitatively dominating residue could always be easily identified. The present data suggest that the amino acid variability of the HLA-A, -B, and -C antigens is found in restricted regions of the molecule. The COOH-terminal third of the HLA antigen heavy chain appears to be less variable than other regions of the molecule. Previous work has shown that the HLA antigen heavy chain contains two immunoglobulin-like disulphide loops. The COOH-terminal third of the heavy chain was shown to be similar in primary structure to β2-microglobulin and the immunoglobulin G constant domains. [ABSTRACT FROM AUTHOR]

Purified intestine epithelial cells express la antigens as determined by immunoprecipitation and sodium dodecyl sulphate polyacrylamide-gel electropheresis. The intestimal la antigens react at least with alloantisera directed against the H-2 I-A subregion. [ABSTRACT FROM AUTHOR]

Deoxycholate-solubilized HLA antigens have been isolated from platelets and comprised a mixture of 43,000- and 39,000-dalton polypeptide chains associated with β2-microglobulin. Limited proteolysis experiments suggested that the 39,000-dalton chain is a fragment of the intact 43,000-dalton chain. Further proteolysis of the 39.000-dalton fragment yields a 33,000- dalton component. The 39,000-dalton molecule is more acidic than both the 43,000- and the 33,000-dalton chains. Differences in the amino acid compositions of the 43,000- and 39,000- dalton species demonstrate that the peptide(s) released on generation of the 39,000-dalton comcomponent are charged. The proteolytic split most probably occurs in the COOH-terminal end, which, owing to its content of charged amino acids, most probably is not integrated into the hydrocarbon matrix of the membrane. The 39,000- and 43,000-dalton components bind detergent in micellar form and can be incorporated into liposomes. The 33,000-dalton fragment has lost the ability to bind detergent micelles and is not incorporated into liposomes. [ABSTRACT FROM AUTHOR]

Papain-solubilized HLA-A,B,C antigen heavy chains have been cleaved by combined acid and CNBr treatment to yield three large fragments. A 14,000-dalton peptide comprises the NH2-terminal portion of the molecule, less a five-membered peptide. The 14,000-dalton fragment is followed in the linear sequence by a 9000-dalton peptide connected through an aspartyl-prolyl bond to the COOH-terminal 11,000-dalton fragment. The 9000- and 11,000-dalton fragments contain disulphide bridges that are immunoglobulin-like inasmuch as they encompass some fifty-five to sixty amino acid residues. The NH2-terminal portion of the HLA antigen heavy chain is devoid of cysteine. NH2-terminal amino acid sequence analyses do not reveal homologies between the 14,000- and 9000-dalton fragments, β2-microglobulin, and the constant immunoglobulin domains. However, the NH2-terminal sequence of the 11,000-dalton fragment is as homologous to β2-microglobulin and the constant immunoglobulin domains as they are to one another. [ABSTRACT FROM AUTHOR]

The genetic basis for the extensive polymorphism of major histocompatibility complex (MHC) class II molecules was investigated by statistical analysis. Nucleotide sequences of human DQA1, DQB1, DRB1, and DRB3 genes and routine Aα, Aβ, and Eβ genes were used. The results show that polymorphism is selected for in the antigen recognition site of class II molecules since replacement substitutions in this region were found to occur at a significantly higher frequency than expected in the absence of selection. In contrast, replacement substitutions are selected against in the remaining part of the first domain exon and in the second domain exon. Furthermore, comparing the sequence variability pattern among different class II α and sequences, using a variability index for each residue, showed that, with few exceptions, highly polymorphic residues occur in the antigen recognition site. There was a strong and highly significant correlation in the variability pattern in the homologous DRB/Eβ sequences but not for DQB/Aβ or DQA/Aα sequences. This difference may be related to the fact that both α and chains of DQ/A molecules are polymorphic, while only β chains of DR/E molecules vary. [ABSTRACT FROM AUTHOR]

A truncated form of the class II antigen DRα chain of the human major histoeompatibility complex was produced in bacteria. A cDNA clone encoding the intact chain was modified so that the segment encoding the signal sequence was replaced by an ATG codon and the 3′ region downstream to the part corresponding to the third exon was replaced by a stop codon. The new construct was put under the control of the Tae promoter in a bacterial expression vector. The distance between the Shine-Delgarno sequence and the initiation codon was randomized so that clones with optimal expression of the truncated DRα chain could be obtained after induced expression and immunoscreening. The truncated DR a chain was subjected to limited proteolysis with chymotrypsin, and the resulting cleavage products were analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Two fragments were visualized by western blotting. Electrophoresis in the absence and presence of reducing agents suggested that one of the proteolytic fragments contained a disulphide bridge. It is concluded that the extracellular portion of the DRα chain is composed of two compactly folded domains connected by an extended stretch of the polypeptide chain. [ABSTRACT FROM AUTHOR]

Thirty-six consecutive paediatric patients (0-16 years old) with recently contracted juvenile diabetes (IDDM) during 1982-84 were included in the study. Sera were assayed for recent or current Coxsackie B virus (CBV) infection using a specific and sensitive IgM RIA, Eighteen patients (50%) had IgM against CBV 1-5, The patients were also assayed for restriction fragment length polymorphism (RFLP) patterns with DNA probes coding for HLA-DR and DQ beta chains. The CBV-positive patients (n=18) had either RFLP patterns associated with HLA-DR 3 or 4 or HLA-DQ patterns III or IV beta. Two of the CBV negative patients had neither HLA-DR 3 nor DR 4 and four of them had neither DQ patterns III nor IV, Eleven out of 18 CBV-positive patients had HLA-DQ III and DR 3 (61%) versus 5 out of 18 (28%) of the CBV-negative patients. All 11 patients with serology positive for CBV 2,3, and 5 had HLA-DR 4 and DQ IV patterns. This was significantly (P<0.01) different from all five CBV 4-positive patients, who in contrast all had HLA-DR 3 or HLA-DQ III patterns, CBV 1-positive patients (n=2) all had HLA-DR 3, 4, and HLA-DQ III, IV patterns. Thus CBV 4 seems to be significantly associated with a different host genetic constitution from at any rate CBV 2,3, and 5, and possibly CBV 1. [ABSTRACT FROM AUTHOR]

The signal sequences of two HLA-DR β chains and the DR α chain were determined. In addition, the major part of a DC β-chain signal sequence was also elucidated. The data were obtained by combining amino acid sequence analyses of isolated α and β chains with nucleotide sequencing of four cDNA clones. All signal sequences comprise 25 amino acids or more. The two HLA-DR β-chain signal sequences are identical and exhibit only marginal homology to the DC β-chain signal sequence. No homology is apparent between α- and β-chain signal sequences. The differences in the signal sequences of the DR and DC β chains suggest that these sequences may be used to assign β chains to different loci of the human major histocompatibility complex. [ABSTRACT FROM AUTHOR]

Membrane-bound mRNA was isolated from Raji cells and enriched for message coding for the HLA-DR transplantation antigen α-chain by sucrose gradient centrifugation. Double-stranded cDNA was constructed from this mRNA fraction, ligated to plasmid pBR322, and cloned into Escherichia coli. By hybrid selection, a plasmid, pDR-α-1, able to hybridize with mRNA coding for the HLA-DR α-chain was identified. From the nucleotide sequence of one end of the insert an amino acid sequence was predicted which is identical to part of the amino-terminal sequence of an HLA-DR α-chain preparation isolated from Raji cells. This clearly shows that pDR-α-1 carries almost the complete message for an HLA-DR α-chain. From the nucleotide sequence of this plasmid it will be possible to predict the primary structure of an HLA-DR α-chain. [ABSTRACT FROM AUTHOR]

cDNA for a Βchain of HLA-DR antigens was cloned and the partial nueleotide sequence was determined. The data suggest that the fl-chain consists of approximately 230 amino acids. ot which about 200 are exposed on the cell surface. The 6-chain appears to be composed of Two exposed disulphide-containing domains. The arrangement of the disulphide loops suggests that the 5-chain is similar in structure to the HLA-A, B, C antigen subunits and the immunoglobulin chains. for the Βchain domain closest to the membrane this similarity was verified at the level of primary structure. The partial amino acid sequence of the NH2>terminal domain did not display any apparent homology to HLA-A, B, C antigens and immunoglobulins. However, the similarity established here between the two types of major histocompatibility antigen subunits and the immunoglobulin chains suggests a common ancestral origin for at least some regions of these molecules. [ABSTRACT FROM AUTHOR]

A nucleoside diphosphate kinase has been highly purified from baker's yeast, and has been obtained in a crystalline form from an ethanol‐containing medium. During short‐time incubation with [32P]ATP, the enzyme was phosphorylated to an extent of about 3–4 phosphoryl groups per mole of enzyme assuming a molecular weight of 105. This indicates an intermediate phosphorylation of the enzyme. 1‐[32P]Phosphohistidine was shown to be the dominating radioactive degradation product in an alkaline hydrolysate of the 32P‐labelled enzyme. In addition, 3‐[32P]‐phosphohistidine and Ne‐[32P]phospholysine were obtained in small amounts. The results indicate that the amino acid sequence around the reactive 1‐phosphohistidine of the yeast enzyme is different from that of human erythrocytic and bovine liver nucleoside diphosphate kinase. [ABSTRACT FROM AUTHOR]

Human filamin A is a 280 kDa protein involved in actin-filament cross-linking. It is structurally divided into an actin-binding headpiece (ABD) and a rod domain containing 24 immunoglobulin-like (Ig) repeats. A fragment of human filamin A (Ig repeats 14-16) was cloned and expressed in Escherichia coli and the purified protein was crystallized in 1.6 M ammonium sulfate, 2% PEG 1000 and 100 mM HEPES pH 7.5. The crystals diffracted to 1.95 A and belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 50.63, b = 52.10, c = 98.46 A, alpha = beta = gamma = 90 degrees.

Class II genes of the bovine major histocompatibility complex (MHC) were investigated by Southern blot analysis using human cDNA probes for DQ alpha, DQ beta, DR alpha and DR beta. The presence of a DQ-like and a DR-like subregion in cattle was clearly indicated. Highly polymorphic restriction fragment patterns were obtained when genomic DNA, digested with any one of the BamHI, EcoRI or PvuII restriction enzymes, was hybridized with the DQ alpha and the DQ beta probe. The polymorphisms were interpreted genetically by analysing five paternal half-sib families of the Swedish Red and White breed. The material comprised, besides the bulls, 28 offspring and their dams. The analysis resolved 9 and 12 allelic variants of DQ alpha and DQ beta respectively. Thus, this investigation establishes a method for routine typing of MHC class II gene polymorphism in cattle. The results were entirely consistent with close linkage of DQ alpha and DQ beta since no recombinant was found and since alleles at these loci occurred in complete linkage disequilibrium in the material investigated. Close linkage between DQ and the blood group locus M, which has previously been found to be closely linked to the serologically defined BoLA-A locus, was also indicated. In this study DNA was isolated from frozen semen samples of dead bulls, which shows that this type of analysis will be useful in genetic investigations in cattle breeds, where artificial insemination is practised.