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Biomedical subjects

C Auffray

Publications and source records attributed to C Auffray.

At least 127 records · Page 7Linked to original sources

Exuberant restriction fragment length polymorphism associated with the DQ alpha-chain gene and the DX alpha-chain gene.

Cellular DNAs from individuals of 23 families were digested with five restriction endonucleases (Pvu II, EcoRI, HindIII, BamHI, and EcoRV) and then probed with a DX alpha-chain gene probe. Seventeen allogenotopes were observed, each of which could be assigned to a serologically defined haplotype by noting its segregation in families. Six sets of allogenotopes forming allelic series were noted. In comparison with restriction maps of the DQ alpha and the DX alpha regions, each of these series has been assigned to the DQ alpha or the DX alpha locus. Allogenotopes of the four DQ alpha series constitute three clusters correlating with the supertypic groups of class II histocompatibility antigens DQw1 (DR1, DR2, and DRw6), DRw53 (DR4, DR7, and DR9), and DR3 plus DR5 plus DR8. These 13 DQ alpha fragments constitute 22 different patterns. The two DX alpha series constitute two clusters, one of which is not found to be correlated strongly with DR specificities, whereas the other is correlated loosely (r = 0.45) with DR5 and DR7. This absence of strong linkage disequilibrium between the DX alpha series and the DR series contrasts with the DQ alpha series and suggests a recombination point between DQ alpha and DX alpha loci.

Alleles↗

Sequence analysis of the human major histocompatibility gene SX alpha.

The DP subregion of the human major histocompatibility complex contains two closely linked gene pairs, DP alpha, DP beta and SX alpha, SX beta. The exon-intron organization and the complete DNA sequence of the SX alpha gene are reported here. There are several mutations within the SX alpha gene which strongly suggest that it is a pseudogene. These include two frameshift mutations, one in the alpha 1 domain and the other in the cytoplasmic domain. A 5' splice site mutation at the end of the alpha 1 exon also exists. DNA sequence homology between DP alpha and SX alpha suggests that these genes arose through a gene duplication event.

Base Sequence↗

A program for prediction of protein secondary structure from nucleotide sequence data: application to histocompatibility antigens.

A computer program is described which, given a nucleotide or an amino acid sequence, outputs protein secondary structure prediction curves as well as hydrophobicity and charged-residue profiles. The program allows for cumulative averaging of properties (secondary structure propensities, hydrophobicity and charge profiles) from several homologous primary structures, a novel concept shown to improve the predictive accuracy. The use of the program is demonstrated on a set of nucleotide and amino acid sequences from human and murine histocompatibility antigens of class I and II. The last extracellular domains of both class I and II antigens (alpha 3 of class I, alpha 2 and beta 2 of class II) and the beta 2-microglobulin domain are predicted to consist of seven anti-parallel beta-strands, in accord with previous claims of homology between these domains and the constant domains of immunoglobulin chains. The remaining extracellular domains are all proposed to form an anti-parallel, four-stranded beta-sheet with one of its faces being covered by alpha-helices and/or structureless segments ("open face sandwiches").

Animals↗

Inter-locus and intra-allelic polymorphisms of HLA class I antigen gene mRNA.

We have constructed cDNA clone libraries from two lymphoblastoid cell lines, JY (HLA-A2, B7, C untypeable) and LB (HLA-A28, B40, Cw3), and isolated clones encoding class I HLA antigens. We have characterized short oligonucleotide probes derived from the coding region of the HLA class I antigens which are specific for the HLA-A and -B loci. These probes have been used to subdivide the class I cDNA clones into subclasses. DNA sequencing of several HLA-A and -B related clones has allowed us to extend the primary structural characterization of these cell-surface antigens. This analysis has also detected a sequence polymorphism at the HLA-A locus, indicating that the previously considered homozygous typing cell line LB expresses two alleles of similar, although not identical, serological specificity.

Alleles↗

Sequence of rat intestinal vitamin D-dependent calcium-binding protein derived from a cDNA clone. Evolutionary implications.

We have recently reported molecular cloning of the cDNA synthesized from rat duodenal mRNA-encoding intestinal calcium-binding protein (ICaBP), a vitamin D3-induced protein (Desplan, C., Thomasset, M., and Moukhtar, M. S. (1983) J. Biol. Chem. 258, 2762-2765). Nucleotide sequence analysis of the longest cDNA insert (375 base pairs) permitted the assignment of 207 nucleotides of the coding region and 104 nucleotides of the entire 3'-noncoding region of the mRNA. Although the derived amino acid sequence for rat ICaBP differed from the bovine and porcine sequences by 16 and 14 residues, respectively, all the residues of each calcium-binding site met the proposed requirements of the "EF hand" theory. In contrast, several differences found in the linker regions might explain the absence of cross-immunoreactivity between rat and porcine ICaBPs. Analysis of nucleotide sequence homologies between the coding and noncoding regions showed that the region coding for the two calcium-binding sites (I and II) was immediately followed in the noncoding region by a sequence very similar to the sequence coding for site I. This suggests that rat ICaBP mRNA contains the remains of an untranslated calcium-binding site III-like structure and that low Mr ICaBP could result in early termination of the translation of a larger molecule containing four sites.

Amino Acid Sequence↗

Polymorphism and complexity of the human DC and murine I-A alpha chain genes.

A cDNA clone encoding the human B cell alloantigen DC alpha chain (pDCH1) has been used to analyse the structure of the human and murine major histocompatibility complexes by the DNA filter hybridization technique. The pDCH1 probe hybridizes to a single DNA sequence present on chromosome 17 in the mouse genome. A restriction enzyme polymorphism enables us to map this sequence to the I-A subregion. Extensive restriction enzyme polymorphism detected in HLA-DR homozygous typing cells is reminiscent of the DR/MT linkage disequilibrium groups, suggesting that the pDCH1 probe could be useful for haplotype typing in the human population. The HLA-DR region appears more complex than the I region since a second DC-like hybridizing sequence is detected in the human genome in these experiments.

Animals↗

Analysis of human class II antigen alpha chain genes: a summary.

A cDNA clone corresponding to the HLA-DR alpha chain has been isolated following immunoprecipitation of polysomes with a monoclonal antibody against the denatured DR alpha chain. This cDNA clone has been used to isolate and solve the complete structure of the DR alpha chain gene and a cDNA clone corresponding to the DC alpha chain. The DR alpha and the DC alpha chains bear remarkable homology to each other and have one domain (alpha 2) which is a member of the immunoglobulin superfamily. The DC alpha chain gene has been demonstrated to be the homologue of the murine I-A alpha chain gene, and it has been localized on chromosome 6 using deletion mutants. A restriction enzyme polymorphism of the DC alpha chain gene has been detected in HLA-DR homozygous typing cells. This report is a summary of a presentation made at the HLA-DR meeting held in Marseille, France.

DNA↗

Genetic mapping of a human class II antigen beta-chain cDNA clone to the SB region of the HLA complex.

A class II antigen beta-chain cDNA clone was isolated from a human B-cell cDNA library by using as a probe the murine I-A beta gene. This cDNA clone, pHA beta, was shown to be distinct from the DC beta- and DR beta-related loci by DNA sequence analysis, thus suggesting that it might correspond to a third polymorphic human class II locus, SB, which encodes secondary B-cell antigens. Genetic mapping of this beta-chain cDNA clone to the SB region was performed by the blot hybridization procedure. We showed that (i) within panels of HLA-DR homozygous human B-cell lines and of unrelated individuals who have been typed for HLA antigens, differential mobility of DNA fragments segregated with distinct SB genotypes; (ii) gamma-ray-induced deletion mutants that have lost the expression of DR or DC/MT antigens but maintain SB expression preserved a pattern consistent with (a) their SB phenotype and (b) the genetic independence of the SB locus with respect to DR and DC/MT; and (iii) within an informative family, two siblings differing only for one allele at the SB locus (because of the occurrence of an internal recombination between DR and GLO) and otherwise HLA identical exhibited a restriction enzyme polymorphism linked to the SB locus. Therefore, all available data are compatible with identity between HA beta and SB beta.

Alleles↗

Identification of a cDNA clone coding for the acetylcholine binding subunit of Torpedo marmorata acetylcholine receptor.

A recombinant DNA plasmid has been constructed that contains sequences of the gene coding for the acetylcholine binding subunit (alpha-subunit, 40 000 daltons) of Torpedo marmorata acetylcholine receptor protein (AChR). Polyadenylated RNA purified from Torpedo electric organ was used to construct a cDNA library. The AChR alpha-subunit cDNA clone was then identified by a two-step screening of 700 recombinant clones. As AChR is present in Torpedo electric organ but not in Torpedo liver or spleen, differential screening led to the selection of 12 clones specific for the electric organ. We then tested the ability of cDNA inserts to hybridize alpha-subunit mRNA specifically, as judged by cell-free translation and immunoprecipitation. The insert from one clone, p alpha-1, selectively hybridized with a mRNA species which elicited the synthesis of a 38 000 mol. wt. polypeptide. This polypeptide was precipitated by: (1) a rabbit serum raised against purified denatured alpha-subunit (the pure alpha-subunit displaced the complex); and (2) a rat monoclonal antibody specific for the denatured alpha-subunit. It was thus identified as a precursor of the alpha chain. Blot hybridization analysis of polyadenylated RNA from Torpedo electric organ with the p alpha-1 probe revealed a major species of 2.0 kb, which thus contains approximately 800 non-coding nucleotides.

Animals↗

Rat immunoglobulin delta heavy chain gene: nucleotide sequence derived from cloned cDNA.

Rat immunoglobulin delta heavy-chain mRNA has been isolated. RNA blot analysis revealed that this mRNA with a length of 1.8 kb encodes for the secreted form of IgD. The corresponding cDNA was cloned in plasmid pBR322 and its sequence was determined. The hybrid plasmid contains a 775-bp insert comprising a partial C delta 1 sequence and complete C delta H, C delta 3, C delta DC and 3' untranslated sequences. Rat and mouse IgD amino acid sequences show striking homology in C delta 3 and C delta DC regions.

Amino Acid Sequence↗

The amino acid sequence and gene organization of the heavy chain of the HLA-DR antigen: homology to immunoglobulins.

The amino acid sequence of the heavy chain of HLA-DR antigens has been elucidated from the analysis of a genomic clone coding for this protein. A 3.2-kilobase EcoRI fragment includes four exons containing 227 amino acids out of 229 in the mature HLA-DR heavy chain. One exon (alpha 2) encodes a domain of 94 amino acids with strong sequence homology both to Ig constant region domains and to Ig-like domains in HLA-B7, beta 2-microglobulin, and the HLA-DR light chain. These results support a structure for the HLA-DR antigen heterodimer consisting of four extracellular domains, two of which are Ig-like [one in the heavy chain (alpha 2) and one in the light chain (beta 2)]. The third is the amino-terminal polymorphic domain in the light chain (beta 1), and the fourth is an invariant domain in the heavy chain (alpha 1).

Amino Acid Sequence↗

cDNA clone for the heavy chain of the human B cell alloantigen DC1: strong sequence homology to the HLA-DR heavy chain.

A cDNA library has been constructed from a B cell mRNA fraction enriched for HLA-DR sequences, and cDNA clones corresponding to sequences specifically expressed in B lymphocytes have been isolated by a differential screening procedure. Analysis of these clones with probes specific for the HLA-DR heavy chain gene allowed the characterization of HLA-DR heavy chain-related sequences. One clone, pDCH1, was demonstrated to encode the DC1 heavy chain because the amino acid sequence predicted from its nucleotide sequence matches eight out of nine residues available for comparison in the amino-terminal sequence of the DC1 heavy chain. The heavy chain of the DC1 alloantigen is composed of 232 amino acids and can be divided into two external domains, alpha 1 (amino acids 1-87) and alpha 2 (amino acids 88-181), a connecting peptide (amino acids 182-194), a hydrophobic transmembrane region (amino acids 195-217), and an intracytoplasmic region (amino acids 218-232). Comparison with the HLA-DR heavy chain reveals strong sequence homology in the second external Ig-like domain (alpha 2) and the transmembrane region. In contrast, the first external domain, the connecting peptide, and the intracytoplasmic region are less conserved.

Amino Acid Sequence↗

Mouse heavy chain variable regions: nucleotide sequence of a germ-line VH gene segment.

We have constructed a library of Balb/c mouse embryo DNA in the vector Charon 4A. The library was searched for sequences homologous to the VH region of a cloned cDNA of the UPC10 heavy chain mRNA. In this paper, we describe the structure and the partial nucleotide sequence of one of such clones (VH441). The nucleotide sequence of this germ-line gene indicates that it encodes amino-acids 1-98 of the X44 and J601 galactan-binding VH regions, but that it differs from the UPC10 VH segment by four single base changes. The VH gene appears to contain a 101 bases long intervening sequence within a precursor sequence identical to the precursor sequence of UPC10. The 3' non coding sequence of the V gene contains the two conserved sequences found in embryonic V DNA segments, CACAGTG and ACATGAACC, separated by 23 nucleotides and a sequence CACTGTG separated by 33 nucleotides from the first heptamer.

Animals↗

Mouse immunoglobulin A: nucleotide sequence of the structural gene for the alpha heavy chain derived from cloned cDNAs.

The cDNAs complementary to mouse immunoglobulin alpha heavy chain mRNAs have been cloned into the PstI site of the plasmid vector pBR322. Recombinant plasmids have been identified by hybrid-arrested translation and purification of alpha heavy chain mRNA on DNA-DBM filters. The nucleotide sequence of the inserts encodes the constant and 3' untranslated regions of the alpha heavy chain mRNA. The CH3 domains of human and mouse alpha chains are highly homologous, including a 36 amino acid fragment not reported in the protein sequence (Robinson and Appella, 1980). As in the case of the mu secreted heavy chain, the alpha heavy chain contains a carboxy terminal piece of 20 amino acids.

Animals↗

Comparison of mouse immunoglobulin gamma 2a and gamma 2b chain genes suggests that exons can be exchanged between genes in a multigenic family.

A 23-kilobase EcoRI DNA fragment coding for the BALB/c immunoglobulin gamma 2a chain was cloned from mouse embryo DNA in the cosmid pJC74, and a nucleotide sequence of 1904 bases was determined for the entire constant region (CH1, CH2, and CH3), the three intervening sequences (IVS 1, IVS 2, and IVS 3) and the 5' and 3' flanking sequences. When the gamma 2a chain nucleotide sequence was compared with the gamma 2b chain nucleotide sequence, the percent homology of corresponding segments (excluding deletion and insertion) was 82% for the 5' flanking sequence, 87% for CH1, 84% for IVS 1, 96% for the hinge, 95% for IVS 2, 94.6% for CH2, 86% for IVS 3, 74% for CH3, 89% for the 3' untranslated region, and 92% for the 3' flanking region. These findings show that different domains of gamma 2a and gamma 2b have independent rates of evolution and that some of the noncoding segments of the gene are more conserved than are adjacent coding segments. Hypotheses on the possible role of IVS is gene evolution and expression are discussed.

Animals↗

Nucleotide sequence of constant and 3' untranslated regions of a kappa immunoglobulin light chain mRNA of a homozygous b4 rabbit.

A homozygous a2/a2 and b4/b4 rabbit has been hyperimmunized with Micrococcus lysodeikticus. Poly(A)-containing RNA has been isolated from the spleen and translated in vitro, and translation products have been analyzed by NaDodSO4/polyacrylamide gel electrophoresis. Double-stranded cDNA has been synthesized from poly(A)-containing RNA template and inserted in the Pst I endonuclease site of plasmid pBR322 by using the oligo(dC).oligo(dG) tailing procedure. Tetracycline-resistant ampicillin-sensitive clones containing cDNA complementary to a kappa light chain mRNA have been selected by differential screening and their ability to hybridize to a spleen mRNA having the same size as a mouse kappa light chain mRNA. Two clones, pRk-15 and pRk-32, have been selected to determine the nucleotide sequence of the constant and 3' untranslated regions of kappa light chain mRNA, by the Maxam and Gilbert partial degradation method. Comparison of homologous regions of mouse kappa chain mRNA and b4 rabbit kappa chain mRNA reveals 61% homology in the constant region and 59% homology in the 3' untranslated region.

Animals↗