Biomedical subjects

A M Voie

Publications and source records attributed to A M Voie.

3 recordsLinked to original sources

PubMed1997

Structure and organization of the immunoglobulin M heavy chain genes in Atlantic salmon, Salmo salar.

To determine the structure and organization of the germline immunoglobulin M heavy chain (IgH) genes in Atlantic salmon, Salmo salar, relevant clones from a genomic library (of one individual fish) have been characterized. Two closely related IgH constant region genes, CHA and CHB, have been sequenced completely. In addition, an allotypic variant of CHA was identified and partially sequenced. Five joining (JH) elements were found in a distance of 0.5-1.6 kb upstream of the first constant exon (CH1), in both CHA and CHB, substantiating the hypothesis that the entire gene complex is duplicated; possibly a remnant of a tetraploid event in the salmonid ancestor. An octamer motif (ATGTATTT, and its reverse complementary sequence) was found to be dispersed in the JH-CH1 region, but not elsewhere, signifying a role in these loci. Four closely related variable (VH) genes which were subcloned from three distinct lambda clones showed the classical structure of a two exon unit split by a 100 bp intron. The split-intron and a few hundred base pairs of the flanking sequences of the genes were highly similar. Three of the four genes were interrupted by stop codons and/or frame shifts, indicating a high proportion of VH-pseudogenes in this species. Based on the present results, and comparison with sequences of rainbow trout, Oncorhynchus mykiss, it is likely that the IgH loci have remained tetrasomicly inherited throughout the radiation of the genus Salmo and Oncorhynchus, and that the duplicated loci have gone into a disomic inheritance pattern in the comparatively recent past.

Animals↗

PubMed1995

Efficient low redundancy large-scale DNA sequencing at EMBL.

An efficient low redundancy DNA sequencing strategy should allow high accuracy determination of the consensus sequence on both strands of a DNA fragment from a minimal number of sequencing reactions with minimal overlap. At EMBL we developed a directed strategy for cosmid-scale sequencing based on primer walking, whereas most other sequencing projects of this scale rely on the random 'shotgun' strategy. In our strategy, highly accurate raw data are obtained from automated double-stranded Sanger dideoxy sequencing with inexpensive walking primers (8 to 10 $ per primer), T7 DNA polymerase and internal labelling by fluorescein-15- dATP on A.L.F. DNA sequencers (Pharmacia Biotech). The use of 60-cm long glass plates enables reading length of up to 1000 bases. Comparing various random and directed sequencing strategies in the course of the European Community yeast genome sequencing project on cosmids from chromosomes IX, XI and XV, primer walking was found to be the strategy resulting in the lowest possible redundancy of 2.6 to 2.8. Future development of the sequencing strategy is based on the new EMBL 2-dye sequencing device for simultaneous sequencing on both strands, and implementation of an initial limited random sequencing phase to reduce the number of walking primers required by a factor of 3, while still maintaining a low redundancy of approx. 3.

Animals↗

PubMed1992

Cloning and sequence analysis of two isotypic IgM heavy chain genes from Atlantic salmon, Salmo salar L.

A leukocyte cDNA library from Atlantic salmon, based on oligo-dT priming, was constructed in lambda-gt10. Immunoglobulin heavy chain (IgH) cDNA were isolated from the library using a specific probe generated by polymerase chain reaction (PCR) between two conserved areas within the variable region (second and fourth frame region). Two cDNA clones encoding the entire constant region of membrane-bound IgH, and one cDNA encoding the entire constant region of secretory IgH were sequenced, revealing messages from two isotypic IgM genes. Both genes were shown to be present in haploid embryos and have been isolated from a genomic library, the exons and deduced amino acid sequences of which are presented here (salmon CHA and CHB). The splicing of transcripts encoding the membrane-bound IgH excises the whole fourth exon as in other teleosts. The nucleotide and amino acid identity between salmon CHA and CHB are 98.2%, and 96.2%, respectively. Two subfractions of IgM from Atlantic salmon separated by ion chromatography can be explained by a net exchange of basic residues in salmon CHB compared to CHA. The finding of two closely related salmon CH genes is in accordance with the quasi-tetraploid state of the Atlantic salmon genome.

Amino Acid Sequence↗