Biomedical subjects
Publications and source records attributed to F Bregegere.
A novel algorithm has been developed to map restriction fragments, starting from experimental size values with realistic error rates. A high performing PASCAL program has been derived from this algorithm to construct linear maps in minimal computation times.
The complete nucleotide sequences of three cDNAs coding for the C-terminal part of mouse histocompatibility (H-2) antigens, and for the 3' non coding regions of these clones have been determined. Comparison of the sequence indicates a large homology throughout the coding and non-coding regions and suggests the existence of a genetic mechanism which homogenizes nucleotide sequences among genes of the H-2 multigene family.
The products of the highly polymorphic genes known as the major histocompatibility complex (MHC) have been shown to play a major part in the control of several aspects of the immune response and of susceptibility to certain diseases. The major transplantation antigens are membrane proteins composed of two noncovalently associated polypeptide chains: a light, non-polymorphic chain of molecular weight 12,000 (MW), called beta 2-microglobulin, and a polymorphic, glycosylated heavy chain of approximately 45,000 MW. The heavy chains in man and mouse are encoded in at least three loci of the MHC named HLA-A, -B and -C, and H-2D, K and L respectively. The available amino acid sequence data indicate extensive homology between human and mouse heavy chains. We therefore used a recently isolated cloned mouse H-2 cDNA probe to screen a human gene library. We have now characterized one of the recombinant phages obtained, lambda HLA-12, and shown that it contains an authentic HLA sequence with evidence of a second one close by.
Poliovirus cDNA.RNA hybrids were prepared from the Mahoney strain of poliovirus type 1 by using reverse transcriptase (RNA-dependent DNA nucleotidyltransferase) and cloned in the Escherichia coli plasmid pBR322. Bacteria colonies carrying recombinant plasmids were selected by in situ hybridization with virus-specific RNase T1-resistant oligonucleotides. Analysis of the cDNA inserts by restriction mapping and electron microscopy showed that the cloned cDNAs, the longest of which was 3.2 kilobase pairs, originated from various parts of the viral RNA, covering at least 99% of the genome length. Due to overlapping of the clones, the restriction map of the poliovirus genome could be reconstructed. The complete 5' end of the genome was successfully cloned in at least one of the recombinant plasmids, pPV1-366.
mRNA coding for mouse major transplantation antigens of the d haplotype was partially purified, copied into double-stranded cDNA, and cloned in Escherichia coli. Clones were selected by their ability to hybridize specifically with mRNA coding for H-2K, D, or L antigens. One of these clones, pH-2d-1, carries a 1200-base-pair insert, comprising the noncoding region, including poly(A) at the 3' end and part of the coding region. A partial sequence of the latter region showed extensive homology with the known amino acid sequences of H-2Kb,Kk, and HLA-B7 antigens. From this comparison, it appears that the coding region extends from amino acid 133 in the second domain, through the third domain, to the cytoplasmic COOH-terminal region. A stretch of 24 hydrophobic or uncharged residues, located 31 amino acids from the COOH-terminal end, could represent the segment that spans the membrane. This is followed on the cytoplasmic side of the membrane by a cluster of basic amino acids and a possible phosphorylation site on a threonine residue.
Poliovirus type 1 RNA was reverse-transcribed into c-DNA and inserted at the Pst I site of the plasmid vector pBR322 of E. coli. Resulting recombinant plasmids were analyzed by hybridization with RNase T1-resistant 32P-labeled oligonucleotides, and by restriction enzyme mapping. All of the genome was cloned in a series overlapping cDNA inserts, the longest of which was 3.2 kb. The restriction map of the poliovirus cDNA is presented.
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