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Biomedical subjects

D M Gibson

Publications and source records attributed to D M Gibson.

16 recordsLinked to original sources

L chain isotype regulation in horse. I. Characterization of Ig lambda genes.

Analysis of 10 cDNA encoding lambda L chains of horse Ig indicated that this species may employ a relatively small number of variable region (V lambda) genes in the splenic B cell population. The V lambda sequences of all of the cDNA analyzed were closely related (> 88% identity at the nucleotide level) and were characterized by a deletion of the amino acid residue at position 3 compared with V lambda sequences so far described in other species. The 10 V lambda sequences could be grouped into three groups, V lambda 1 to V lambda 3, on the basis of a number of linked substitutions. Sequences within the groups showed the greatest divergence in the third cdr regions and at the V-J junctions. The junctional variation included amino acid substitutions on both sides of the V-J junction as well as the insertion or deletion of two to four amino acid residues. Four C lambda genes were identified in genomic blots of horse DNA, and three of these were found expressed in splenic cDNA. The fourth C lambda gene may represent a pseudogene, inasmuch as the associated J region possessed a defective heptamer joining sequence. Six of the nine possible V lambda-C lambda combinations were found in the cDNA analyzed, suggesting that genes belonging to groups V lambda 1 through V lambda 3 may rearrange to any one of three J lambda-C lambda genes. One V lambda germline gene was characterized and found to represent a distinct V lambda group (V lambda 4), not represented in the cDNA sequences analyzed. The number of germline V lambda genes was estimated to be 20 to 30, based on analysis of restriction fragments hybridizing with V lambda probes. On the basis of these data, we propose that the V lambda repertoire in horse may consist of relatively limited number of genes, of which only a few may be used at high frequency in the splenic B cell population. The results indicate that predominance of lambda-chains in horse Ig may not simply be due to the presence of a large germline V lambda gene repertoire.

Amino Acid Sequence

Positive identification of a lambda gt11 clone containing a region of fungal phytase gene by immunoprobe and sequence verification.

As the initial step in a project to provide a more cost-effective source of the phytase enzyme, this paper reports on the use of a polyclonal antibody raised to phytase purified from an isolate of Aspergillus niger (A. ficuum) to screen an A. niger lambda gt11 expression library and the use of amino acid sequencing to identify a clone containing part of the fungal phytase gene. The described use of amino acid sequence fragments to verify the cloning of a gene has potential applications in other cloning projects.

6-Phytase

Two closely related kappa variable region pseudogenes pose an evolutionary paradox.

Two pseudogenes belonging to the Igk-V1 variable region group have been isolated from BALB/c mice. The genes share greater than 96.5% identity of nucleotide sequence in a 1800 base pair (bp) region surrounding the coding region, but deletions of 221 bp and 84 bp have removed essential sequences from the two genes. As the deletions are different in the two pseudogenes, they must have occurred independently in each gene during or subsequent to the duplication event which gave rise to the genes from a common ancestral gene. Polymerase chain reaction analysis was used to identify the pseudogenes in inbred strains of mice. BALB/c (Igkc) and AKR (Igka), prototype strains representative of the predominant kappa haplotypes, possess both pseudogenes but no intact copy. Only one of the pseudogenes was present in SJL (Igka). Strains C58, c.C58 (Igkd) and NZB (Igkb) possessed an intact version of the gene. This distribution of haplotypes is consistent with a close linkage of the pseudogenes with other Igk-V1 genes on chromosome 6. The translated amino acid sequence of the pseudogenes indicates that prior to their acquiring deletions they encoded typical Igk-V1 variable regions except for an unusual FR2 region, in which the conserved proline at position 44 is replaced by leucine and the normally hydrophobic position 36 was occupied by histidine. Possible mechanisms to explain the occurrence of deletions in both of the pseudogenes in the recent evolution of BALB/c are discussed. One explanation would be that the two genes were already nonfunctional at the time of the duplication so that the subsequent deletions represent neutral events which became fixed in the inbred strains by a process of genetic drift. Alternatively, if the genes were functional at the time of duplication, their rapid loss due to deletion events suggests that negative selection may have acted to eliminate the genes from the V-region repertoire.

Alleles

Ef2: a new LY-3-linked light-chain marker expressed in normal mouse serum immunoglobulin.

A new light-chain marker has been detected in normal mouse serum immunoglobulin light chains by gel isoelectric focusing. The marker (Ef2) involves the presence of two major and several minor bands in the normal light-chain IF profiles. Strains expressing the marker IF bands are designated Igk-Ef2a, whereas those lacking the bands are Igk-Ef2b. The majority of inbred strains are Igk-Ef2a. Strains found to be Igk-Ef2b are NZB/BlNJ, BDP/J, C58/J, I/LnJ, CE/J, and P/J. The strain distribution of the alleles differs from the distribution of alleles at the Ly-2 and Ly-3 loci, suggesting the new marker may represent a separate locus. Genetic studies have shown that Igk-Ef2 locus is closely linked to Igk-Ef1 and Hd loci on Chromosome 6, indicating that it is also closely linked to Ly-3. The relative importance of the bands controlled by the Igk-Ef2 locus suggests that the entire normal light-chain pool could be controlled by as few as 100 such loci.

Alleles

Evidence for close linkage of a mouse light chain marker with the Ly-2,3 locus.

Light chains associated with normal serum immunoglobulin can be resolved into a finite number of discrete focusing bands by isoelectric focusing. Four distinct light chain patterns can be distinguished among the inbred mouse strains. In the present studies inheritance of the characteristic light chain patterns has been studied in the AKXL recombinant inbred lines (derived from C57L/J and AKR/J parental lines) and in the inbred Ly-2a,3a congenic line B6.PL-Ly-2aLy-3a/Cy as well as in individual backcross animals of an incipient Ly-2a,3a congenic strain. Virtually complete concordance was observed for the expression of light chains characteristic of phenotype B (AKR-J-like) and the expression of the Ly-2a,3a allele. This observation indicates that a locus controlling light chain structure and/or expression is closely linked (less than 2.6 map units) to the Ly-2,3 locus on mouse Chromosome 6. The locus controlling normal light chain IF-patterns has been designated Ef1.

Animals

Species specificity in the isoelectric spectra of immunoglobulin light chains.

Light chains isolated from normal immunoglobulin give rise to a finite number of discrete bands when analyzed by isoelectric focusing in polyacrylamide gel. The positions and relative intensities of the bands were identical in light chains isolated from different individuals of a species, but clear differences were evident when light chains of several mammalian species were compared.

Animals

Taxonomic position and seasonal variations in marine neritic environment of some gram-negative antibiotic-producing bacteria.

Six marine bacteria which synthesize macromolecular antibiotics were isolated from neritic waters on the French Mediterranean coast, and their frequency recorded over two successive years. They appeared in relatively large numbers during the period August to December, and can be identified as marine pseudomonads; however, the low guaninecytosine ratio of their DNA, lack of catalase and specific self-inhibition are not compatible with the characteristics of the genus Pseudomonas. Two produced violacein, usually synthesized by bacteria belonging to the genus Chromobacterium. Their taxonomic position is discussed.

Anti-Bacterial Agents