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R G Mage

Publications and source records attributed to R G Mage.

At least 73 records · Page 4Linked to original sources
PubMed1984

Kappa-chain allotypes and isotypes in the rabbit: cDNA sequences of clones encoding b9 suggest an evolutionary pathway and possible role of the interdomain disulfide bond in quantitative allotype expression.

The constant regions of rabbit kappa light chains are unusual because the sequences of the allotypic forms can differ more from each other than do some variable regions with which they associate. We report the nucleic acid sequence of a full-length cDNA clone of b9 allotype and show comparisons to available sequences of the rabbit kappa allotypes b4, b5, and bas-N4. Our analyses suggest that the primordial rabbit kappa gene encoded a bas-like sequence. They also reveal a surprising difference in the position of the variable region cysteine that forms the interdomain disulfide bond that is unique to most rabbit kappa chains. One b9 cDNA sequence lacks the usual cysteine-80 and instead encodes cysteine-108, which in three-dimensional models appears capable of forming the interdomain disulfide bond with cysteine-171 in the constant region. A partial sequence of a second b9 clone encodes both cysteine-80 and cysteine-108; the translation product of this clone could have a free reactive sulfhydryl group that might lead to an unstable nonfunctional Ig molecule. The fact that pre-B cells with b9 kappa chains do not differentiate and expand into productive Ig-producing cells with frequencies comparable to the other allotypes may be explained if a substantial proportion of the gene products have a free sulfhydryl group. Our sequence results suggest that in cells differentiating to produce kappa light chains of b9 allotype the number and location of the cysteines influence immunoglobulin expression.

Animals↗
PubMed1984

Complete sequence of a cloned cDNA encoding rabbit secreted mu-chain of VHa2 allotype: comparisons with VHa1 and membrane mu sequences.

The complete sequence of a cDNA clone (pmu3) encoding a secreted IgM heavy chain from a rabbit of VHa2 allotype has been determined. Comparison of the nucleic acid and amino acid sequences with a comparable mouse mu-chain shows unusually high homologies of the sequences of the fourth domains and secretory portions of the molecules (80 to 90% vs 56 to 73% for the other domains). This high degree of homology is also seen with the sequences of the fourth domain and secreted terminus of human mu-chain. The DNA sequence of a second cDNA clone encoding the membrane form of rabbit IgM is approximately 90% homologous with the mouse and human coding sequences for the membrane terminus, and there is 98 and 97% amino acid sequence homology. We found an unusually long (156 base pair) 5' untranslated region in clone pmu3 and discovered that 105 bases at the 5' end were complementary to the terminal portion of the CH4 domain of our mu sequence. Southern blotting analyses and the finding of a 13 base pair segment in rabbit genomic V region DNA that is homologous to the complementary strand of a segment of the CH4 of rabbit mu strongly suggests that this extended sequence resulted from a reverse transcriptase "hooking error". We have also obtained and sequenced another cDNA clone from a VHa1 cDNA library. Comparison of the V region of clone pmu3 from VHa2 with the 117 available positions of a1 DNA sequence shows 25 differences, nine of which are in allotype-associated codons. There is also a two-codon deletion in the VHa1 compared to VHa2 in the third framework region that may contribute to allotypic differences. Development of VHa allotype-specific probes, now in progress, is complicated by the fact that most of the allotype-associated amino acids of a1, a2, and a3 can be derived from each other by single base changes.

Amino Acid Sequence↗
PubMed1983

The sequences of rabbit kappa light chains of b4 and b5 allotypes differ more in their constant regions than in their 3' untranslated regions.

We report the sequence of a cDNA clone encoding the entire variable and constant regions of a rabbit kappa light chain of b5 allotype. The deduced amino acid sequence of the variable region (positions 1-95) is 86% homologous to that of a b4 light chain protein [BS-1) (1) but the b4 and b5 constant regions are only 74% homologous. Comparison of this DNA sequence to that of a cDNA clone encoding a b4 constant region shows that the kappa allotypes b4 and b5 have diverged significantly more in their coding region than in the 3' untranslated regions (86% vs 96% nucleotide sequence homologies). This implies either a function for the 3' untranslated region with evolutionary pressures to conserve or an accelerated divergence of the coding regions.

Amino Acid Sequence↗
PubMed1983

Nucleotide sequence of a rabbit IgG heavy chain from the recombinant F-I haplotype.

We report the sequence of a cDNA encoding a rabbit immunoglobulin gamma heavy chain of d12 and e14 allotypes with high homology to partial cDNA sequences from rabbits of d11 and e15 allotypes. The encoded rabbit protein shows homologies with human (68-70%) and mouse (60-63%) gamma chains. The nucleotide sequence homologies of the CH domains range from 76-84% with human and 64-76% with mouse sequences. Comparison of the portion of VH encoding amino acid positions 34-112 with a previously determined VH sequence of the same allotype shows high conservation of sequences in the second and third framework segments but more marked differences both in length and encoded amino acids of the second and third complementarity-determining regions (CDRs). We also found a high degree of homology with a human genomic V-region, VH26 (77%) and a remarkable similarity between rabbit and human second CDR sequences and human genomic D minigenes. These results provide additional evidence that D minigene sequences share information with the CDR2 portion of VH regions.

Alleles↗
PubMed1983

Analyses of the splenic mRNA expressed by rabbits of different immunoglobulin kappa-light chain allotypes: conserved sequences in the 3' untranslated region and allotype-specific probes.

The allelism of the structural genes for the complex rabbit b allotypes of immunoglobulin kappa-light chains has been questioned because of observations of unexpected phenotypic expression of "latent" allotypes. We find that the coding sequences of the b4 and b5 "alleles" are only 80% homologous for the last 60 nucleotides but there is a high degree of homology (96%) in the 3' untranslated region (3'DT). The high conservation of 3' DT region sequences enabled us to detect kappa-light chain mRNAs from rabbits of different genetic types (b4, b5, b9 and bbas) on northern blots and dot blots. We can distinguish mRNA encoding b9 and b5 allotypes on dot blots with b5 fragment-probes of known sequence and detect mRNA produced by unstimulated cultured splenic lymphocytes. Analyses of mRNA from cultured cells manipulated to enhance mRNA synthesis and production of unexpected or "latent" b allotypes can now be conducted.

Animals↗
PubMed1982

A subpopulation of small pre-B cells in rabbit bone marrow expresses kappa light chains and exhibits allelic exclusion of b locus allotypes.

The expression of immunoglobulin heavy and light chains by pre-B cells and B lymphocytes was examined by two-color immunoflourescence in heterozygous b5b9 rabbits. Allelic exclusion of b5 and b9 kappa light chain allotypes was observed for both surface immunoglobulin-negative pre-B cells and surface immunoglobulin-positive B lymphocytes. In newborn bone marrow, pre-B cells and immature B lymphocytes expressing b9 were as numerous as those expressing b5. In contrast, circulating B cells and bone marrow plasma cells expressing the b5 marker outnumber b9+ cells by 2 to 1 in adult b5b9 animals. Whereas most B lymphocytes expressed kappa light chain b allotypes, approximately 80% of the micro heavy chain-positive pre-B cells did not. The pre-B cells that expressed detectable light chains were relatively small lymphocytes. A model is presented which includes a "transitional" pre-B cell that expresses both micro chains and kappa chains.

Alleles↗
PubMed1982

Rabbit heavy chain haplotypes--allotypic determinants expressed by VH-CH recombinants.

This report summarizes our current understanding of the heavy chain haplotypes found in our laboratories' rabbits. Independently derived data from several laboratories have been synthesizes into a consistent picture of the linked inheritance of allotypic markers found on the different heavy chain classes and subclasses of rabbit immunoglobulins in pedigreed rabbits, including the families of three apparent VH-CH recombinants. In one recombinant, the entire group of CH markers (C mu, C gamma, and C alpha) recombined with the set of VH. Although in the other two recombinants all CH markers may also have recombined as a group, in one of these only IgG and IgA CH genes were informative; in the other recombinant, only the IgG allotypes were informative. Some allotypic determinants found on IgM molecules ("conformational") appear only when a specific variable region allotype (VHa) is combined with a specific mu constant region allotype (C mu). New combinations of VHa and C mu allotypes were generated in two of the genetic recombinants and led to new "conformational" determinants. The gains and losses observed lend support to the hypothesis that the determinants result from conformations generated by the combination of allotype-specific VH and C mu protein sequences. Conceivably, DNA events that join VH to diversity (D)- and joining (J)-coding sequences or mRNA processing events that splice J to C mu could be involved in generating the sequences that form allotype-specific determinants.

Animals↗
PubMed1982

Expression of kappa chain allotypes at the cell surface and in serum immunoglobulins of normal and allotype-suppressed heterozygous rabbits.

The kappa light chain allotypes b4 and b5 were measured in the serum and on the surfaces of peripheral blood lymphocytes of normal and allotype-suppressed heterozygous rabbits. Surface immunoglobulins (sIg) were detected by fluorescence microscopy and additional quantitative data were obtained by flow microfluorometry. Although a few b5-suppressed animals had no b5 in serum or on cell surfaces for years, most b5-suppressed and all b4-suppressed animals studied had some cells with sIg of the suppressed type by 1 year of age. In suppressed animals the level of serum Ig remained depressed throughout life but cells with sIg appeared in disproportionately large numbers. The effect was particularly striking in those animals suppressed for the b4 type.

Age Factors↗
PubMed1981

Immunofluorescence studies on the expression of VH a allotypes by pre-B and B cells of homozygous and heterozygous rabbits.

Fluorochrome-conjugated antibodies specific for C mu determinants and VH a allotypes were used to examine pre-B cells and B lymphocytes for expression of these markers. The majority of mu+ bone marrow pre-B cells were shown to express a allotypic determinants in conjunction with C mu. Both pre-B and B cells from a2 a3 heterozygous rabbits showed allelic exclusion of these allotypes. Pre-B cells expressing the a2 or a3 specificities appeared to be generated in approximately equal numbers in heterozygotes, while B lymphocytes expressing a3 appeared to undergo preferential clonal expansion very early in development. It was also observed that rabbit bone marrow and blood contained a population of myeloid cells which, in a2 a3 heterozygotes, stained for both a2 and a3 determinants. The frequencies of this cell type, which exhibited bright immunofluorescence staining for a allotypes, could not be reduced by prolonged incubation at 37 degrees C but were readily reduced after cell suspensions were treated with low pH buffer. It is concluded that these myeloid cells bear high avidity Fc receptors for serum immunoglobulin and may be the culprits in studies which have found production of two a or b allotypes by individual B lymphocytes.

Animals↗
PubMed1980

Surface immunoglobulin of rabbit peripheral blood lymphocytes: detection of allotypes by fluorescence and rosetting.

Specific anti-allotype reagents were prepared to detect heavy and light chain allotypes on surface immunoglobulin-positive (sIg+) cells from a3a3b4b4, a2a2b5b5 homozygous and a2a2b4b5 heterozygous rabbits. Total sIg+ peripheral blood lymphocytes (PBL) were detected with fluorescein-labeled goat anti-light chain and sheep anti-rabbit Ig reagents. The total sIg+ cells detected with these reagents and the sum of a and b allotypes individually measured with specific fluorescent anti-allotype reagents (a2 + a3 or b4 + b5) were measured more or ess the same when measured by fluorescence-activated cell sorter (FACS) II flow microfluorometry. The data provided no evidence for single cells expressing both allelic allotypes (allelic inclusion). We found that FACS and resetting techniques were generally equally sensitive. However, we detected a greater proportion of total b4+ plus b5+ cells by rosetting than by fluorescence in some PBL preparations from heterozygous b4b5 rabbits. This was not seen with artificial mixtures of b4b4 and b5b5 cells. The nature of these cells is not yet known. Conceivably, they were not scored as lymphocytes by light-scatter analysis on the FACS and hence were not counted, but were indistinguishable from lymphocytes by microscopy.

Animals↗
PubMed1979

Tritium (3H) radiolabeling of protein A and antibody to high specific activity: application to cell surface antigen radioimmunoassays.

Staphylococcal protein A and several different immunoglobulins have been radiolabeled to high specific activities (greater than 10(6) cpm/microgram) by reductive methylation with tritiated (3H) sodium borohydride. The proteins retain excellent functional and antigenic properties. The utility of these reagents in a variety of assays for cell surface antigens is illustrated. The results indicate that this radiolabeling procedure may become the method of choice for many cell surface and solution immunoassays.

Animals↗
PubMed1979

Lactoperoxidase catalyzed radioiodination of cell surface immunoglobulin: incorporated radioactivity may not reflect relative cell surface Ig density.

Rabbit and mouse splenic lymphocytes were radioiodinated by the lactoperoxidase technique, extracted with non-ionic detergent, immunoprecipitated with high titered rabbit anti-kappa antisera, and compared by SDS-PAGE. Mouse sIg peaks were reproducibly larger in size than rabbit sIg peaks (often greater than 10 times). Neither differences in incorporation of label into the rabbit cell surface, nor differences in average sIg density explain this result. Total TCA-precipitable radioactivity was similar in each species. Estimation of the relative amounts of sIg in the mouse and rabbit showed similar average sIg densities. Differences in detergent solubility, proteolytic lability, or antisera used also do not adequately account for this difference. Thus, these data indicate that radioactivity incorporated after lactoperoxidase catalyzed cell surface radioiodination may not reflect cell surface Ig density. Conclusions about cell surface density based upon relative incorporation of radioactivity should be confirmed by other approaches.

Animals↗