Mutation affecting the expression of immunoglobulin variable regions in the rabbit.

Abstract

We have found a variant of the allotype allele a2 in the rabbit, which presumably arose by mutation, that segregates as expected for an allele at the a locus. This allele is called "ali" and the corresponding rabbit strain is called "Alicia." In heterozygous animals (al/al and ali/a3) the concentration of a2 molecules is lower by a factor of 1000 than in standard a2/a2 homozygotes. In homozygous ali/al individuals the a2 concentration varies with age-i.e., very low in young rabbits and higher in older ones-but it never reaches normal levels. The low level of a2 is compensated by increased amounts of a-negative molecules. Southern blot analysis did not reveal any gross changes in the intron between JH and CI (oining region of immunoglobulin heavy chain and constant region of immunoglobulin ,u chain) or in the number of VH gene segments encoding a locus specificities. We suggest that the ali phenotype is due to a mutation in a control element.The immunoglobulin (Ig) molecules are made up of heavy (H) and light (L) polypeptide chains. In a few species, the amino acid sequences of these chains and their DNA sequences have been determined (1)(2)(3). H chains can be divided into a variable (VH) region, which contributes to the antibodycombining site, and a constant (CH) region, which determines the class of the Ig. The H chains of the rabbit are encoded by a cluster of closely linked genetic loci: a-ms-d-e-f-g, with a few alleles being known at each locus (2, 4). At the DNA level, the order of gene segments appears to be VH-J-C,-CY--CF-Ca (where J = joining) (5). Allotypic determinants of the a locus are present in VH regions of all Ig classes (2). Some 5-30% of the IgG molecules lack a locus allotypic determinants and are called a-negative. These molecules express other allotypic specificities, encoded by the w, x, and y loci closely linked to the a locus (2, 4), and their N-terminal amino acid sequence differs from that of the a-positive molecules (6).We describe here a variant, presumably a mutant, in which the expression of a-positive molecules is remarkably altered. MATERIALS AND METHODSAnimals. Rabbits were bred at the institute and were identical by descent with respect to the H-chain haplotypes.Igs. Normal serum of allotyped rabbits was run over a protein A-Sepharose 4B (Pharmacia, Uppsala, Sweden) column. After extensive washing the bound material was eluted with 1 M acetic acid, neutralized immediately with Tris buffer, and dialyzed against phosphate-buffered saline (PBS). Standard anti-a2 was purified by affinity chromatography on a2 IgG coupled to CNBr-activated Sepharose 4B according to the procedure suggested by the manufacturer (Pharmacia). Ascites fluid of monoclonal 3.11 anti-a2 was obtained from W. van der Loo and used as such. Commercial anti-rabbit Fc was obtained from Jackson Immunochemical (Avondale, PA) and also used without further purification. Double Diffusion in Agarose Gel. Undiluted serum and antiserum were allowed to diffuse overnight at +40C in 1.5% agarose Indubiose A45 (L'Indus...

Sequence analysis of 0.5 Mb of the rabbit germline immunoglobulin heavy chain locus

“…Ig heavy chain expression in Alicia rabbits, a mutant strain derived from an a2 rabbit of the F-I haplotype (Kelus and Weiss, 1986), is impaired because of a deletion of VH1 and VH2 Allegrucci et al, 1990;Chen et al, 1993). Amplification of a DNA fragment flanking the deleted sequence and subsequent sequencing showed a deletion of about 10,283 around position 92,942 ( F 36 bp) of BAC 225.…”

Rabbit somatic cell cloning: effects of donor cell type, histone acetylation status and chimeric embryo complementation

“…Ali is a rabbit strain, which has a variant of the allotype allele a2 associated with the H chain of immunoglobulin molecules. Bas is another rabbit strain with a mutant gene at the Ab allotypic locus controlling the synthesis of allotypic specificities (Ab4, Ab5, Ab6, Ab9, Ab4v, and Ab95) of k-immunoglobulin light chains (Garcia et al 1982, Kelus & Weiss 1986.…”

Mechanism for Pre-B Cell Loss in VH-Mutant Rabbits

“…at Loyola University Chicago. Mutant ali/ali rabbits were progeny of rabbits originally described by Kelus and Weiss (11). Studies were approved by the Institutional Animal Care and Use Committee of Loyola University Chicago.…”

“…The mutant ali/ali rabbit was discovered 25 y ago through the study of rabbit V H allotypes (11). Serum analysis of young ali/ali rabbits demonstrated marked reductions in the V H a-allotype Ig (V H a-Ig) compared with wild-type (wt) rabbits.…”