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R G Mage

Publications and source records attributed to R G Mage.

At least 37 records · Page 2Linked to original sources
PubMed1996

CD5 is a potential selecting ligand for B cell surface immunoglobulin framework region sequences.

In rabbits nearly all B lymphocytes express the glycoprotein CD5, in contrast to mice and humans, where only a small proportion of B cells express this molecule (Raman, C., and K.L. Knight. 1992. J. Immunol. 149:3858-3864). CD5+ B cells appear to develop early in ontogeny and be maintained throughout life by self-renewal. The function of CD5 on B cells is still unknown. We showed earlier that "positive" selection occurs during B lymphocyte development in the rabbit appendix. This selection favors B cell expressing surface immunoglobulins with VHa2 structures in the first and third framework regions (Pospisil, R., G.O. Young-Cooper, and R.G. Mage. 1995. Proc. Natl. Acad. Sci. USA. 92:6961-6965). Here we report that F(ab')2 fragments, especially those bearing VHa2 framework region determinants, specifically interact with the B cell-surface glycoprotein CD5. This interaction can be inhibited by anti-CD5 antibodies. Furthermore, immobilized F(ab')2 fragments selectively bind CD5 molecules in appendix cell lysates. Interactions of VH framework region structures with CD5 may affect maintenance and selective expansion of particular B cells and thus contribute to autostimulatory growth of autoimmune or transformed cells.

Animals↗
PubMed1996

Rabbit DQ52 and DH gene expression in early B-cell development.

Rabbits predominantly rearrange the most 3'VH gene (VH1); thus combinatorial diversity is very limited. In man and mouse, the most 3'DH gene, DQ52, is preferentially rearranged early in B-cell development. To test whether this preference for rearranging a DH gene segment based on 3' end proximity exists in rabbit, we cloned and sequenced the rabbit DQ52 gene. The 11 base pair coding region sequence is identical to a published mouse DQ52, and 81.8% similar to the human sequence. It is localized approximately 805 bp upstream of the JH1 gene. However, the 3' recombination signal sequence has an atypical nonamer. We prepared mRNA from 15- to 28-day fetal rabbits and amplified expressed VDJ sequences of mu mRNA by RT-PCR. The PCR products with VDJ rearrangements were cloned and sequenced. As expected, 44 of 45 VDJ sequences reflected use of the 3' VH1a2 gene, but the DQ52 gene was utilized very infrequently, if at all. We found only one VDJ sequence from 28-day fetal liver B-cells with 8 bp that matched the germline DQ52 sequence. Instead of expressing DQ52, another DH gene, Df was frequently expressed. We cloned the genomic Df gene and localized it about 32 kb upstream of the JH region. Thus, in contrast to man and mouse, rabbits preferentially express a DH gene located in the middle of the DH region early in B cell ontogeny. This may correlate with more frequent initial rearrangement of VH to DH in rabbit B cells.

Amino Acid Sequence↗
PubMed1995

Preferential expansion and survival of B lymphocytes based on VH framework 1 and framework 3 expression: "positive" selection in appendix of normal and VH-mutant rabbits.

B cells with a rearranged heavy-chain variable region VHa allotype-encoding VH1 gene segment predominate throughout the life of normal rabbits and appear to be the source of the majority of serum immunoglobulins, which thus bear VHa allotypes. The functional role(s) of these VH framework region (FR) allotypic structures has not been defined. We show here that B cells expressing surface immunoglobulin with VHa2 allotypic specificities are preferentially expanded and positively selected in the appendix of young rabbits. By flow cytometry, a higher proportion of a2+ B cells were progressing through the cell cycle (S/G2/M) compared to a2- B cells, most of which were in the G1/G0 phase of the cell cycle. The majority of appendix B cells in dark zones of germinal centers of normal 6-week-old rabbits were proliferating and very little apoptosis were observed. In contrast, in 6-week-old VH-mutant ali/ali rabbits, little cell proliferation and extensive apoptosis were observed. Nonetheless even in the absence of VH1, B cells with a2-like surface immunoglobulin had developed and expanded in the appendix of 11-week-old mutants. The numbers and tissue localization of B cells undergoing apoptosis then appeared similar to those found in 6-week-old normal appendix. Thus, B cells with immunoglobulin receptors lacking the VHa2 allotypic structures were less likely to undergo clonal expansion and maturation. These data suggest that "positive" selection of B lymphocytes through FR1 and FR3 VHa allotypic structures occurs during their development in the appendix.

Animals↗
PubMed1995

Characterization of a rabbit germ-line VH gene that is a candidate donor for VH gene conversion in mutant Alicia rabbits.

Normal rabbits preferentially rearrange the 3'-most VH gene, VH1, to encode Igs with VHa allotypes, which constitute the majority of rabbit serum Igs. A gene conversion-like mechanism is employed to diversify the primary Ab repertoire. In mutant Alicia rabbits that derived from a rabbit with VHa2 allotype, the VH1 gene was deleted. Our previous studies showed that the first functional gene (VH4) or VH4-like genes were rearranged in 2- to 8-wk-old homozygous Alicia. The VH1a2-like sequences that were found in splenic mRNA from 6-wk and older Alicia rabbits still had some residues that were typical of VH4. The appearances of sequences resembling that of VH1a2 may have been caused by gene conversions that altered the sequences of the rearranged VH or there may have been rearrangement of upstream VH1a2-like genes later in development. To investigate this further, we constructed a cosmid library and isolated a VH1a2-like gene, VH12-1-6, with a sequence almost identical to VH1a2. This gene had a deleted base in the heptamer of its recombination signal sequence. However, even if this defect diminished or eliminated its ability to rearrange, the a2-like gene could have acted as a donor for gene-conversion-like alteration of rearranged VH genes. Sequence comparisons suggested that this gene or a gene like it could have acted as a donor for gene conversion in mutant Alicia and in normal rabbits.

Amino Acid Sequence↗
PubMed1995

Secondary rearrangements and post-rearrangement selection contribute to restricted immunoglobulin DJH expression in young rabbit bone marrow.

Extrachromosomal circular DNA purified from bone marrow cells of 2-weeks-old rabbits was assayed by polymerase chain reaction to determine the relative rearrangement frequencies of immunoglobulin DH to JH genes in vivo. DH genes rearranged to individual JH genes with different frequencies. This bias did not correlate with potential sequence overlaps in the DH or JH coding sequences. The JH2 and JH4 genes were the preferred targets of recombination in primary rearrangements. Although primary rearrangements to JH6 were relatively infrequent, secondary rearrangements were detected. This assay also revealed previously undescribed JH pseudogenes with functional recombination signal sequences. Analyses of genomic VDJH indicated that B cells expressing VDJH4 heavy chains survived and dominated in the bone marrow environment due to secondary rearrangements and/or post-rearrangement selection.

Amino Acid Sequence↗
PubMed1995

The rabbit B cell antigen receptor is non-covalently associated with unique heteromeric protein complexes: possible insights into the membrane IgM/IgD coexpression paradox.

We describe several proteins that are components of the rabbit B cell receptor complex. Two proteins (37 kDa and 42 kDa) were found in non-covalent association with IgM expressed on B cells from peripheral blood. These proteins were also immunoprecipitated by anti-B29 (Ig-beta) and anti-mb1 (Ig-alpha) monoclonal antibodies. As in the mouse and human, the IgM associated molecules were found as heteromeric structures with non-reduced apparent molecular weights of approximately 70-75 kDa. On rabbit B cells we also found these proteins in a 100-135 kDa complex which may represent trimeric or tetrameric structures. By Western blot, the 37 kDa protein was identified as rabbit Ig-beta (B29), suggesting that the 42 kDa protein is rabbit Ig-alpha. These data suggest that rabbit IgM is associated with both Ig-alpha/beta and Ig-(alpha beta)2 or alpha beta gamma complexes. When similar immunoprecipitation studies were performed on lysates made from B cells isolated from appendix follicles, we found two additional IgM associated protein complexes containing 34 kDa and 36 kDa proteins.

Animals↗
PubMed1994

The appendix functions as a mammalian bursal equivalent in the developing rabbit.

In this paper we present genomic DNA sequence and histological evidence that the appendix is a site of diversification of the rabbit's primary antibody repertoire. By 6 weeks after birth, the B cell follicular regions of the rabbit appendix and the distribution of the resident lymphoid cells bear a strong morphological resemblance to similar regions within two primary lymphoid tissues, the chicken bursa and the sheep ileal Peyer's patch. However, similarities between the rabbit appendix, chicken bursa and sheep ileal Peyer's patch end as these animals reach adulthood. The rabbit appendix undergoes morphological and cellular distribution changes as it matures taking on the appearance of a secondary lymphoid tissue, while the sheep ileal Peyer's patch and the chicken bursa both involute. We determined DNA sequences of PCR amplified rearranged variable region genes from germinal center B cells of 6 week old rabbits isolated from several different appendix dark zones and light zones. There was a trend toward a higher degree of diversification from the germ-line VH gene DNA sequence in dark zones than light zones. It is likely that both gene conversion and somatic hypermutation are responsible for the nucleotide changes we observed. Our findings suggest that the rabbit appendix functions as a mammalian bursal equivalent early in development. As the rabbit matures, the appendix appears to evolve into a secondary lymphoid tissue resembling secondary GALT in appearance and possibly in function.

Amino Acid Sequence↗
PubMed1994

Rabbit IgH sequences in appendix germinal centers: VH diversification by gene conversion-like and hypermutation mechanisms.

Although the rabbit IgH locus contains approximately 100 VH genes, the majority of B cells rearrange VH1. To produce a primary repertoire containing a sufficient number of protective antibodies, rearranged VH1-DH-JH sequences may diversify within rabbit B cells in an organ that functions like a chicken bursa, sheep ileal Peyer's patch, or both. It was suggested many years ago that the rabbit appendix could be a bursal equivalent. To reexamine this possibility, we analyzed rearranged heavy chain variable region sequences in B cells from light and dark zones of appendix germinal centers from 6-week-old rabbits. Our findings indicate that antibody diversification occurs by gene conversion-like and somatic hypermutation mechanisms in appendix germinal centers of young rabbits.

Amino Acid Sequence↗
PubMed1993

VH gene expression and regulation in the mutant Alicia rabbit. Rescue of VHa2 allotype expression.

Rabbits of the Alicia strain, derived from rabbits expressing the VHa2 allotype, have a mutation in the H chain locus that has a cis effect upon the expression of VHa2 and VHa- genes. A small deletion at the most J-proximal (3') end of the VH locus leads to low expression of all the genes on the entire chromosome in heterozygous ali mutants and altered relative expression of VH genes in homozygotes. To study VH gene expression and regulation, we used the polymerase chain reaction to amplify the VH genes expressed in spleens of young and adult wild-type and mutant Alicia rabbits. The cDNA from reverse transcription of splenic mRNA was amplified and polymerase chain reaction libraries were constructed and screened with oligonucleotides from framework regions 1 and 3, as well as JH. Thirty-three VH-positive clones were sequenced and analyzed. We found that in mutant Alicia rabbits, products of the first functional VH gene (VH4a2), (or VH4a2-like genes) were expressed in 2- to 8-wk-olds. Expression of both the VHx and VHy types of VHa- genes was also elevated but the relative proportions of VHx and VHy, especially VHx, decreased whereas the relative levels of expression of VH4a2 or VH4a2-like genes increased with age. Our results suggest that the appearance of sequences resembling that of the VH1a2, which is deleted in the mutant ali rabbits, could be caused by alterations of the sequences of the rearranged VH4a2 genes by gene conversions and/or rearrangement of upstream VH1a2-like genes later in development.

Amino Acid Sequence↗
PubMed1993

Recombination activating genes-1 and -2 of the rabbit: cloning and characterization of germline and expressed genes.

The recombination activating genes RAG-1 and RAG-2 appear to be necessary components of the machinery needed for the Ig or TCR gene rearrangements that occur in developing B and T lymphocytes. In addition RAG-2 has been implicated in the process of V-gene diversification by somatic gene conversion in the chicken. Because gene conversion may be an important mechanism for V-gene diversification in the rabbit, we cloned the rabbit RAG locus and characterized the coding regions of the genomic RAG-1 and RAG-2. In addition, we sequenced cDNAs encompassing the RAG-2 coding region, part of the RAG-2 5' untranslated region and a 967 bp fragment of cDNA from the RAG-1 coding region. Northern analysis revealed a RAG-1 mRNA of 6.6 kb which is similar in size to the RAG-1 mRNA reported previously for other species, and a major species of RAG-2 mRNA of 4.4 kb, which is larger than that from the mouse (2.2 kb). Analysis of the genomic clones showed that, as in other species, the RAG-1 and RAG-2 genes are oriented so as to be convergently transcribed. The DNA sequence analysis showed that the rabbit RAG-1 coding region is 91, 85 and 72% identical to human, mouse and chicken, respectively. The deduced RAG-1 protein sequence for rabbit is 93, 90 and 78% identical to human, mouse and chicken. Comparison of the rabbit RAG-2 coding region revealed 90, 87 and 71% identity to human, mouse and chicken, respectively, at the nucleotide level, and 91, 90 and 72% at the protein level. Although there is considerable conservation of sequence between species, we obtained evidence for allelic forms of the rabbit RAG locus both by Southern analyses and by sequencing. A remarkable degree of polymorphism was found in our rabbit colonies, particularly in the region 3' of the rabbit RAG-2 coding region. A 5' cDNA probe hybridized with one or more additional fragments that are not detected with the coding region probes, suggesting that the 5' cDNA sequence results from splicing of one or more upstream exons.

Amino Acid Sequence↗
PubMed1991

Identification of enhancer sequences 3' of the rabbit Ig kappa L chain loci.

The rabbit is useful for studies of Ig L chain gene expression because of a great disparity in expression of two isotypic forms of the kappa L chain. Normally, K1 is expressed at high levels and K2 is almost silent; expression of K2 increases in mutant or experimentally allotype-suppressed animals. The reasons for the preferential utilization of the K1 isotype have not been fully elucidated. We were interested in looking for second enhancers 3' of the C kappa genes because the absence of a 3' enhancer in the K2 locus could explain the preferential utilization of the K1 isotype. However, we found a strong region of enhancer activity about 7 kb downstream of the C kappa 2 gene. Sequences in this region are highly conserved between rabbit, man, and mouse. There also appears to be a homologous 3' enhancer region in the rabbit K1 locus. We also confirmed earlier reports that the rabbit K1 intron enhancer is inactive in transient transfections into mouse B cells but find that the same construct has low but significant activity in a human B cell line. In a comparable construct the K2 intron enhancer is without activity suggesting possible differential activity of the intronic enhancers.

Animals↗
PubMed1991

Mapping of the duplicated rabbit immunoglobulin kappa light chain locus.

The rabbit has two isotypic forms of the immunoglobulin kappa light chain, K1 and K2, which probably arose by duplication. In the normal rabbit, only traces of K2 light chains are produced. However, K2 levels are elevated in allotype-suppressed rabbits and in the Basilea strain which does not produce K1 because of a K1 mRNA splice site mutation. Previous cloning and sequencing showed that each isotype has its own set of J kappa genes but it was not known whether the two isotypes utilize shared or separate sets of V kappa genes. In addition, although genetic linkage of allotypes associated with the K1 and K2 genes has been demonstrated, physical linkage had not been previously demonstrated by overlapping cosmid or phage clones. We used pulsed field and transverse alternating field electrophoresis to obtain megabase maps and to estimate the size of the duplication of the rabbit kappa light chain locus. We found that the two C kappa genes are about 1 megabase apart. One explanation for the poor expression of K2, could be great physical distance from V kappa genes. However, we found that there are V kappa, J kappa and C kappa 2 genes within a approximately 105-kb fragment. Thus, physical distance of V kappa from C kappa 2 may not be the basis for poor K2 expression.

Animals↗
PubMed1991

Preferrential rearrangement in normal rabbits of the 3' VHa allotype gene that is deleted in Alicia mutants; somatic hypermutation/conversion may play a major role in generating the heterogeneity of rabbit heavy chain variable region sequences.

The rabbit is unique in having well-defined allotypes in the variable region of the heavy chain. Products of the VHa locus, (with alleles a1, a2, and a3), account for the majority of the serum immunoglobulins. A small percentage of the serum immunoglobulins are a-negative. In 1986, Kelus and Weiss described a mutation that depressed the expression of the Ig VH a2 genes in an a1/a2 rabbit. From this animal the Alicia rabbit strain was developed and the mutation was termed ali. We previously showed, using Southern analysis and the transverse alternating field electrophoresis technique, that the difference between the ali rabbit and normal is a relatively small deletion including some of the most 3' VH genes. The most JH proximal 3' VH1 genes in DNA from normal rabbits of a1, a2 and a3 haplotypes encode a1, a2 and a3 molecules respectively, and it has been suggested that these genes are responsible for allelic inheritance of VHa allotypes. The present study suggests that the 3' end of the VH locus probably plays a key role in regulation of VH gene expression in rabbits because VH gene(s) in this region are the target(s) of preferential VDJ rearrangements. This raises the possibility that mechanisms such as somatic gene conversion and hypermutation are at work to generate the antibody repertoire in this species. Our data support the view that the 3' VH1 gene may be the preferred target for rearrangement in normal rabbits, and for the normal chromosome in heterozygous ali animals. However, homozygous ali rabbits with a deletion that removed the a2-encoding VH1 on both chromosomes do survive, rearrange other VH genes and produce normal levels of immunoglobulins as well as a significant percentage of B cells which bear the a2 allotype. This challenges the view that one VH gene, VH1, is solely responsible for the inheritance pattern of VHa allotypes.

Animals↗
PubMed1991

Molecular analysis of recombination sites within the immunoglobulin heavy chain locus of the rabbit.

Previously, recombinations involving genes of the rabbit immunoglobulin heavy chain locus have been documented serologically. These data indicated that the sites at which the causative recombination events occurred could have been anywhere from within the VH gene cluster up to, or 3' of, C mu. Since these sites could not be localized further by serological methods, we attempted to do this using techniques of molecular biology. DNAs from homozygous recombinant rabbits and from the appropriate non-recombinant parental haplotypes were characterized using Southern blots hybridized with a panel of probes derived from cloned regions of the rabbit immunoglobulin heavy chain gene complex. In all three recombinants, the site was downstream of the entire VH cluster and upstream of the JH cluster within an approximately 50 kilobase (kb) region containing expanses of repetitive-sequence DNA as well as DH genes. DH-specific probes further showed that in two of the recombinants, the recombination appears to have occurred within or 5' of DH1 and 5' of DH2 genes; in the third it occurred 3' of the DH2 genes but at least approximately 5 kb 5' of the JH region.

Animals↗
PubMed1991

Evolutionarily conserved organization and sequences of germline diversity and joining regions of the rabbit T-cell receptor beta 2 chain.

We previously reported that there are three different copies of T-cell receptor beta chain constant region (C beta) genes in some rabbits, two of which are present on an approximately 16-kb and one on an approximately 6-kb Eco RI fragment. We also reported that one of the C beta genes on the approximately 16-kb fragment was chimeric, with a 5' cluster of J beta 2 segments and a 3' untranslated region of beta 1 type. Here we report the complete genomic sequences of the D beta 2 and J beta 2 segments associated with the chimeric C beta gene. The rabbit D beta 2 gene segment has very strong similarity to both its human and mouse counterparts. The sequence similarity also extends rather far from the coding region in both 5' and 3' directions. The content and organization of rabbit J beta 2 gene segments is similar to those found in both human and mouse. The rabbit J beta 2 cluster has six functional segments and one pseudogene, as well as a remnant of another pseudogene between J beta 2.2 and J beta 2.3 equivalent to the one found in man in the same location. The J beta 2.5 gene segment of rabbit has lost the splice signal and is a pseudogene unlike its counterparts in man and mouse. Overall analysis of the rabbit D beta 2-J beta 2 region reveals a closer similarity to human than mouse. However, the general organization of the gene segments in the D beta 2-J beta 2 regions of all three species is remarkably conserved over long stretches of DNA sequence.

Amino Acid Sequence↗
PubMed1990

cDNA clones encoding immunoglobulin lambda chains from rabbit expressing the phenotype c7.

A cDNA library derived from spleen cells of an unimmunized rabbit expressing the c7 phenotype of Ig lambda chains (c7+, c21-) was screened with V lambda or C lambda probes of a lambda light chain bearing c21 epitopes. The nucleotide sequences of three hybridizing clones were found to be identical within the V lambda, J lambda and C lambda regions. The V lambda region was 97% similar to that of the functional germ-line gene V lambda 2, and the C lambda region was identical to that of gene C lambda 6, recently identified. Gene C lambda 6 exhibited four codon differences when compared with gene C lambda 5, the latter encoding c21 epitopes. The data presented here and in the accompanying report (Jaton, J.-C. et al., Eur. J. Immunol. 1990, 20:2713) support the view that gene C lambda 6 encodes the C region of c7 lambda chains and that c7 and c21 markers designate two distinct isotypic forms of lambda chains. On the basis of comparative Southern blotting analyses and restriction maps of cloned genomic regions containing V lambda and C lambda genes, a scheme is proposed to account for the c7- and c21- phenotypes.

Amino Acid Sequence↗
PubMed1990

Chemical and immunochemical identification of the second major rabbit immunoglobulin lambda chain polypeptide bearing c7 epitopes.

The purification of lambda chain-containing IgG fraction from pooled sera of Basilea rabbits, which were bred and selected for the expression of a high level of lambda chains positive for c7 but negative for c21, was carried out. On the basis of specific binding to anti-c7 antiserum, the c7 lambda chain fraction in serum IgG was shown to account for up to 70% of the total immunoglobulin light chains, the remaining 30%, bearing the expected k2 bas isotype. Peptide mapping of the mixed light chains (lambda + k2 bas) followed by microsequencing of the constant region fragments indicated that the C lambda region originated from the cDNA sequence derived from a c7+,c21- Basilea rabbit (i.e. identical to gene C lambda 6), as described in the preceding report (Hayzer, D. J. et al., Eur. J. Immunol. 1990. 20: 2707). In addition, a synthetic peptide encompassing residues 139-159 of the constant region derived from the predicted sequence of gene C lambda 6 was shown to partially inhibit the c7-anti-c7 binding reaction in a sensitive enzyme-linked immunosorbent assay. Taken together, the chemical and immunochemical data clearly demonstrate that gene C lambda 6 indeed encodes c7 epitopes.

Amino Acid Sequence↗