Homozygous Rabbits Research Articles

Isolation and characterizatiopn of light chains from allotype suppressed b9 homozygous rabbits

Light chains have been isolated following sulfitolysis and gel filtration of immunoglobulin G from allotype-suppressed b9 homozygous rabbits. Chemically and immunologically all light chains belong to the λ type, exist only in the form with two disulfide infrachain bridges, and seem to be very similar to the b negative, λ-type light chains which all allotype synthesize, in very low amount, under normal circumstances.

Deletion of b5 immunoglobulin-bearing lymphocytes in allotype-suppressed rabbits.

The proportion of peripheral blood lymphocytes with cell surface b5 immunoglobulin (Ig) was compared with serum b5 Ig levels in allotype-suppressed and recovering b5 homozygous rabbits in order to establish the cellular defect in suppression and to determine the relation between lymphocyte membrane Ig and secreted Ig of the same allotype. In rabbits fully suppressed for b5, the absence of circulating b5 Ig is reflected in a complete deletion of b5-bearing lymphocytes. During spontaneous escape from suppression, the appearance of b5-bearing lymphocytes precedes the appearance of detectable serum b5. During recovery from suppression b5-bearing lymphocytes recover rapidly toward normal levels, but circulating b5 levels remain chronically and disproportionately depressed.

Comparative studies of Aal allotypic specificity in IgG and IgM of rabbits. II. Phenotypic expression of allotypic specificities in rabbits exposed in neonatal life to antibodies directed against Aal allotypic determinants specific for IgG.

Neonatal rabbits were treated with antibodies directed against Aal allotypic determinants specific for IgG and its effects on development of allotypically determined immunoglobulins were analysed in sera of- the treated animals. The treatment of Aa1Aa3 heterozygous rabbits resulted in a delayed appearance in serum of all or some Aa1+ immunoglobulins: In some animals neither Aal+ IgG nor Aal+ IgM were detected for 9 weeks after birth and in others no Aal+ IgG only Aal+ IgM was found up to 5 weeks after birth. In Aa1Aa1 homozygous rabbits, no similar effect was observed. Another effect of the treatment was manifested as supernormal levels of serum IgG and IgM with a maximum at 9 weeks. This effect was more remarkable in homozygotes than in heterozygotes in which phenotypic expression of some Aal allotypic patterns was suppressed. In other heterozygotes in which phenotypic expression of all Aal allotypic patterns was suppressed, IgG levels were lower than controls, suggesting that the suppressed phenotypic expression was not compensated. Quantitative analyses of IgG molecules carrying Aal allotypic specificity have revealed that approx. only 40% of IgG molecules carried the Aal allotypic determinants specific for IgG in an IgG preparation from an Aa1Aa1 homozygote in which more than 60% of the molecules possessed Aal allotypic specificity.

A 50, A New Allotypic Specificity Found on Rabbit IgG‐Immunoglobulin1

Abstract. Isoimmunization of a normal rabbit with a locus negative (a –) IgG‐immunoglobulin molecules obtained from an a2‐suppressed a2a2 homozygous rabbit, resulted in the production of an antiserum that identified a new genetically determined marker ‘A 50’. A large number of animals have been classified for the presence or absence of the allotypic marker.

Presence of the Fc-Fragment Allotypic Determinant, A15, on IgG from an Allotype-Suppressed Aa2 Homozygous Rabbit Lacking the Aa2 Determinants of the Fd Fragment

Abstract Approximately 10% to 30% of normal rabbit IgG molecules lack the allotypic specificities, Aa1, Aa2 or Aa3, of the Aa heavy chain locus (1, 2). Suppression of the Aa locus determinants has been observed in Aa locus homozygous rabbits following exposure to anti-allotype antisera in utero and neonatally (3, 4). Approximately 95% of the IgG obtained from an Aa2Aa2 homozygous suppressed rabbit lacked the Aa2 allotypic specificity (5). Recent data (5) on these Aa-negative molecules have shown that: a) Aa-negative IgG molecules carry a “new” set of allotypic determinants, A31, which are present on the Fab portion of the molecule; b) by peptide mapping, the Fab portion of Aa-negative molecules differs from the Fab portion of Aa-positive molecules; and c) by peptide mapping, the Fc portion of Aa-negative molecules is indistinguishable from the Fc portion of Aa-positive molecules.

Conversion of Non-Immune Rabbit Spleen Cells by Ribonucleic Acid of Lymphoid Cells from an Immunized Rabbit to Produce IgM and IgG Antibody of Foreign Heavy-Chain Allotype

Abstract Spleen cells (SpC) from non-immunized rabbits homozygous for one allotype at the a heavy-chain locus of immunoglobulins were incubated with RNA extracts of lymph nodes from immunized homozygous rabbits of a different genotype to yield IgM “direct” plaque-forming cells (PFC) or IgG “indirect” PFC. The heavy-chain allotype of the antibody in the plaques was identified by radioautography and by inhibition of plaque formation using anti-allotype antibodies. The allotype of the RNA donor rabbit was identified in 75% to 86% of the IgM “direct” plaques and in 43% to 62% of the IgG “indirect” plaques. The allotype of the SpC donor rabbit was found in 2% to 12% of the IgM “direct” plaques and in 24% to 39% of the IgG “indirect” plaques. The lysates of “RNA-converted” SpC were analyzed for immunoglobulins of each heavy-chain allotype by immunodiffusion. Allotypes of both RNA and SpC donor rabbits were found in the lysates, whereas only that of the SpC donor was found in the lysates of untreated SpC, and neither allotype was found in the RNA extracts. These results were similar to those reported previously for light-chain allotypes and indicated that a component of the RNA extract provided information for synthesis of at least part of the heavy chains as well as the light chains of the IgM and IgG antibody molecules.

Structural basis of the A14 and A15 allotypic specificities in rabbit immunoglobulin G.

The structural basis for the A14 and A15 allotypic specificities of rabbit immunoglobulin G was investigated with Fe fragments prepared from homozygous rabbits. The presence of threonine in the Cgamma2 region (position 309: Eu numbering) correlates with the A14 allotype; the presence of alanine at this same position correlates with the A15. Now that the sequence of the Cgamma2 region of rabbit IgG has been completely elucidated, a high degree of homology of the constant region domains of rabbit and human gamma chains is apparent (about 60-80%).

Eression of allelic immunoglobulin in homozygous rabbits injected with RNA extract.

Ribonucleic acid extracts obtained from lymph nodes of immunized rabbits homozygous for the b(4)or b(5)allele of light chain immunoglobulin allotypes were injected intravenously into nonimmunized rabbits homozygous for the alternate allele. Approximately 30 percent of the plaque-forming cells in the spleen yielded plaques with immunoglobulin M antibody possessing the allotype of the RNA donor. The allotype of the RNA donor was also found in the IgG immunoglobulin of lymphoid cell lysates as well as in the IgG isolated from the serum. These results suggest that the injected RNA has an informational role in the in vivo synthesis of immunoglobulins by host lymphoid cells.

Expression of allelic and non-allelic genes in immunoglobulin light chains from homozygous rabbits

Expression of allelic and non-allelic genes in immunoglobulin light chains from homozygous rabbits

Identification and Genetic Control of the Mt-3 and Mt-4 Allotypes of Rabbit Serum α2-Macroglobulin

Abstract Two rabbit serum α2-macroglobulin (α2M) allotypes, Mt-3 and Mt-4, which are distinct from the previously described Mtz1 and Mtz2 allelic allotypes of α2M, were identified. The Mt-3 and Mt-4 allotypes are controlled by a pair of alleles at an autosomal locus that appears to be closely linked to the Mtz locus and not closely linked to the Aa, Ab, or Af immunoglobulin loci, or to the Lpq lipoprotein locus. Sera from Mt-3Mt-4 heterozygous rabbits do not precipitate in agar gel double diffusion plates with either anti-Mt-3 or anti-Mt-4 antisera, but do inhibit the precipitation of each antiserum with serum from the corresponding homozygous rabbits. Serum from a heterozygous rabbit precipitates with a mixture of the two antisera, indicating that both Mt-3 and Mt-4 determinants are present on the same α2M molecule and suggesting that Mt-3 and Mt-4 determinants are each present in a smaller number than that found on molecules from homozygous rabbits. Allelic exclusion does not occur at the molecular level in the Mt-3Mt-4 heterozygous rabbit; in this respect, the Mt-3/Mt-4 system resembles the Mtz1/Mtz2 system, but is dissimilar from all other known serum allotype systems. Individual α2M molecules possess allotypic specificities of both the Mtz1/Mtz2 and Mt-3/Mt-4 systems.

Allotype Suppression in a2a2 Homozygous Rabbits Fostered In Uteri of a2-Immunized a1a1 Homozygous Mothers and Injected at Birth with Anti-a2 Antiserum

Abstract By allotype suppression, rabbits were made agammaglobulinemic with respect to a locus immunoglobulins. This was accomplished by neonatal administration of anti-a2 antiserum to a2a2 homozygous rabbits which had been fostered in uteri of a2-immunized a1a1 homozygous mothers. The sera of these a2-suppressed a2a2 homozygotes were analyzed for a1γG, a2γG and total γG-immunoglobulin concentrations by radial diffusion analysis and by precipitation of 125I-labeled γG-immunoglobulins with anti-allotype antisera. At 14 weeks of age, the a2-suppressed offspring had no detectable a2γG molecules, but instead had compensatory levels of a locus-negative γG molecules. This suggests that another locus is involved in the synthesis of γG heavy chains and that the a+ and a- molecules represent subclasses of γG.

Contribution of the b and c Light Chain Loci to the Composition of Rabbit γG-Immunoglobulins

Abstract The contribution of the b5, c7 and c21 genes to the formation of a single γG-immunoglobulin molecule has been analyzed in partially b5 suppressed b5b5 homozygous rabbits of phenotype c7c21. This was done by means of direct precipitation of 125I-labeled γG preparations with anti-b5, anti-c7 or anti-c21 as well as by indirect precipitation of these allotypes with anti-b4. No two of the allotypic specificities, b5, c7 or c21 were found on the same molecule. This indicates that neither the two unlinked light chain loci b and c, nor the allelic genes c7 and c21 contribute to the formation of the same molecule. Two light polypeptide chain peaks were obtained after partial reduction and alkylation of the γG-immunoglobulins. Quantitative allotype analysis revealed that b5, c7 and c21 light chains accounted for all of the molecules in the first light chain peak, and only b5 light chains accounted for all of the molecules of the second peak. The c21 allotypic specificity is on the light chains as previously found for the b5 and c7 specificities.

Allelic Exclusion and Phenogroup Expression in Individual Molecules of Rabbit Low-Density Lipoprotein Allotypes

Abstract A method for quantitative analysis of lipoprotein allotypes is presented, which is based on indirect precipitation of LDL-125I by an initial reaction with anti-allotype antisera followed by precipitation with goat anti-rabbit γG-Fc fragment. Essentially all LDL molecules prepared from homozygous rabbits have two allotypic specificities of the Lpq locus—q1 and q3, q2 and q3 or q2 and q4—depending on the genotype of the rabbit. In heterozygotes, only one of the two allelic genes contributes to the formation of the same molecule; approximately 50% of the molecules have two specificities controlled by one allele, and the other 50% have two specificities controlled by the alternate allele.

Suppression of heavy and light chain allotypic expression in homozygous rabbits through embryo transfer.

Homozygous suppression of allotypic specificities on both the heavy and light chains of rabbit immunoglobulin has been achieved by two methods involving embryo transfer. In the first method, young were transplanted into does producing antibody against an allotypic specificity homozygously present in the genome of the embryo. In this case, the suppression is caused by antibody transmitted in utero. In the second method, young from a nonimmunized surrogate mother lacking their allotypic genotype are injected neonatally with the antiallotype serum. Release from homozygous suppression appears to be achieved more readily than from heterozygous suppression.

Zygote transfer to facilitate altered expression of immunoglobulin light chain phenotypes in homozygous rabbits.

SummaryBy the techniques of zygote transfer and allotype suppression, b5 homozygous rabbits were produced with an altered expression of immunoglobulin light chain allotypes; i.e., at 24 weeks of age, only 27% of the total γG was b5γG compared to 90% in nonsuppressed homozygotes. Of the 73% b-negative molecules, 44% were c7γG and 29% were both b- and c-negative. These results suggest that there may be at least three “loci” which control the synthesis of light chains.

An evaluation of the number of b4 allotypic determinants on rabbit γG-immunoglobulin using double-labeled, soluble complexes with univalent antibody fragments

Univalent anti-b4 Fab-fragments which react with γG-immunoglobulin from b4 homozygous rabbits were used to form soluble antibody-antigen complexes under conditions of antibody excess. Antibody-antigen molar ratios in the soluble complexes were derived from velocity ultracentrifugation date, molecular weight determinations and analyses of Fab-Ag complexes isolated by gel filtration and specifically labeled with 125I and 131I. At least some of the γG-immunoglobulin molecules were found to have a minimum of two or three b4 determinants per light polypeptide chain. Confirmatory quantitative evidence is presented for the lack of b4 specificity of some γG-immunoglubulin molecules. The heterogeneity of the system is recognized and the resultant limitations upon interpretation of the data are discussed.

Differences in amino acid composition of rabbit γG-immunoglobulin light polypeptide chains controlled by allelic genes

The light polypeptide chains (∼22,000 mol. wt.) of γG-immunoglobulin from b4 and b5 homozygous rabbits were analyzed for their amino acid composition. These b4 and b5 light chains, controlled by allelic genes, differed by an average of fifteen residues per chain; two of these, one aspartic acid and one lysine, were charged amino acids. The difference involved at least seven amino acids (aspartic acid, leucine, lysine, proline, serine, threonine and valine) and probably another (alanine). No significant differences were found for the remaining amino acids. Cystine analyses indicated that b4 or b5 light chains each have three intrachain disulfide bonds. The b4 and b5 light chains were not distinguishable by the disc electrophoresis in polyacrylamide gel although each preparation consisted of at least seven discrete electrophoretic light chain components. The a locus does not contribute to the amino acid composition of the light chains and the allotypic specificity controlled by the a locus was not detectable on the light chain.