RESUMO
Blimp-1 is a transcriptional repressor that is both required and sufficient to trigger terminal differentiation of B lymphocytes and monocyte/macrophages. Here we report the organization of the mouse Blimp-1 gene, an analysis of Blimp-1 homologs in different species, the characterization of Blimp-1 mRNA isoforms and initial studies on the transcription of Blimp-1. The murine Blimp-1 gene covers approximately 23 kb and contains eight exons. There are Blimp-1 homologs in species evolutionarily distant from mouse (Caenorhabditis elegans and Drosophila melanogaster) but no homolog was found in the unicellular yeast Saccharomyces cerevisiae. The three major Blimp-1 mRNA isoforms result from the use of different polyadenylation sites and do not encode different proteins. Run-on transcription analyses were used to show that the developmentally regulated expression of Blimp-1 mRNA in B cells is determined by transcription initiation. Multiple Blimp-1 transcription initiates sites were mapped near an initiator element and a region conferring basal promoter activity has been identified.
Assuntos
Processamento Alternativo/genética , Regiões Promotoras Genéticas/genética , Proteínas Repressoras , Fatores de Transcrição/genética , Sequência de Aminoácidos , Animais , Linfócitos B/citologia , Linfócitos B/metabolismo , Sequência de Bases , Diferenciação Celular , Linhagem Celular , Clonagem Molecular , RNA Polimerases Dirigidas por DNA/metabolismo , Evolução Molecular , Éxons/genética , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter/genética , Humanos , Camundongos , Dados de Sequência Molecular , Ensaios de Proteção de Nucleases , Poli A/genética , Fator 1 de Ligação ao Domínio I Regulador Positivo , Isoformas de Proteínas/genética , RNA Mensageiro/análise , RNA Mensageiro/genética , TATA Box/genética , Fatores de Transcrição/química , Transcrição Gênica/genética , TransfecçãoRESUMO
VDJ genes are assembled in two sequential DNA recombination steps, rearrangement of D and J(H) gene segments followed by rearrangement of a V(H) gene segment. In mouse and human, VDJ gene rearrangements occur in a regulated step in which D rearranges to J(H) on both alleles before V(H) rearranges to DJ. To determine how VDJ gene rearrangements are regulated in the rabbit, we studied the IgH gene rearrangements in rabbit B cells by examining the gene rearrangement on the unexpressed allele in rabbit hybridomas. By Southern blot analysis with probes from the J(H) and 3'V(H)1 regions, we found that approximately 50% of the hybridomas had DJ gene rearrangements on the unexpressed allele, whereas the remaining 50% did not rearrange either J(H) or V(H) genes. Unexpectedly, we found a VD gene rearrangement on the unexpressed allele of a rabbit B cell line, so we PCR-amplified the VD gene rearrangements from splenocyte DNA and found a few VD gene rearrangements in normal B cells as well. The data taken together show that IgH gene rearrangements in rabbit B cells are regulated differently than those in the mouse, and we hypothesize that in some B cells, VDJ gene rearrangements proceed through a VD intermediate rather than through a DJ intermediate.
Assuntos
Linfócitos B/fisiologia , Rearranjo Gênico de Cadeia Pesada de Linfócito B , Coelhos/imunologia , Alelos , Animais , Sequência de Bases , Expressão Gênica , Humanos , Hibridomas , Camundongos , Dados de Sequência Molecular , Coelhos/genética , Mapeamento por RestriçãoRESUMO
B cells in rabbit preferentially utilize the 3'-most VH gene, VH1, in VDJ genes. To determine whether the preferential utilization of VH1 results from preferential rearrangement, we examined VH gene usage in nonproductive VDJ gene rearrangements, genes that would not be influenced by antigen selection. Since nonproductive VDJ gene rearrangements are found infrequently in neonatal and adult rabbits, we examined the VDJ gene rearrangements during early fetal development. According to nested-primer polymerase chain reaction analyses, VDJ genes in fetal liver appeared around day 14 of gestation and nucleotide sequence analyses of VDJ genes cloned from 14- to 28-day-old fetuses showed that many of the genes were nonproductive. We found that most of these nonproductive genes had utilized VH1 indicating to us that the preferential utilization of VH1 is due to preferential rearrangement. We compared the nucleotide sequence of the promoter region of VH1 with sequences of several other unutilized and infrequently utilized VH genes and found that the core transcriptional factor motifs were similar in all VH promoters examined. We suggest that transcriptional activity of the VH1 promoter region does not explain the preferential rearrangement of VH1.
Assuntos
Rearranjo Gênico , Cadeias Pesadas de Imunoglobulinas/imunologia , Cadeias J de Imunoglobulina/imunologia , Fígado/imunologia , Animais , Linfócitos B/imunologia , Sequência de Bases , Desenvolvimento Embrionário e Fetal/genética , Desenvolvimento Embrionário e Fetal/imunologia , Fígado/embriologia , Dados de Sequência Molecular , Coelhos , Alinhamento de SequênciaRESUMO
We developed transgenic rabbits with a DNA construct containing the proto-oncogene c-myc conjugated to the Ig kappa-chain enhancer gene, E kappa. One of four transgenic rabbits was mated to a normal rabbit and we used the offspring to develop a colony of rabbits carrying the E kappa-myc transgene in their germline. Of a total of 19 E kappa-myc transgenic rabbits, eight developed tumors. The tumors were characterized histologically and four were diagnosed as lymphoma, and one each was diagnosed as embryonic carcinoma, hepatoma, ovarian carcinoma and basal cell carcinoma. By Southern analysis, we showed the four lymphomas were of B-lymphoid lineage and by nucleotide sequence analysis we found three of them most likely used VH1 in their VDJ gene rearrangements. Cells from the embryonic carcinoma, the hepatoma and two of the B-lymphomas were adapted to tissue culture. We discuss the possibility that tumors of non-lymphoid origin develop in the E kappa-myc transgenic rabbits because of the potential for NF-kappa B to activate the kappa-enhancer in cells other than B-lymphoid lineage cells.
Assuntos
Elementos Facilitadores Genéticos , Genes myc , Cadeias kappa de Imunoglobulina/genética , Linfoma de Células B/genética , NF-kappa B/genética , Neoplasias Experimentais/genética , Proteínas Proto-Oncogênicas c-myc/genética , Animais , Animais Geneticamente Modificados , Sequência de Bases , Northern Blotting , Southern Blotting , Carcinoma Embrionário/genética , Feminino , Rearranjo Gênico de Cadeia Pesada de Linfócito B , Neoplasias Hepáticas Experimentais/genética , Masculino , Dados de Sequência Molecular , Linhagem , CoelhosRESUMO
The neonatal antibody repertoire in both mouse and humans differs from that of the adult repertoire in that the neonatal repertoire uses a limited set of JH-proximal VH genes but the adult repertoires use many different VH genes. Rabbits are unusual in that adults use only three or four VH genes, with approximately 80% of the B cells using VH1, the 3'-most VH gene. To investigate whether the repertoire of neonatal rabbits differs from that of adults, we analyzed VH, D, and JH gene usage in B cells of neonatal rabbits. A total of 68 rearranged VDJ genes was cloned from mRNA and genomic DNA isolated from lymphoid tissues of newborn to 10-day-old rabbits. We found that 74% of the VDJ gene rearrangements utilized VH1 and 15% utilized the genes that we designated VHx or VHy. From the remaining VDJ genes we identified seven novel VH genes, one, VHz, which was found in mRNA. We conclude that the repertoire of utilized VH genes in neonates is limited and is similar to that of adult rabbits. We also found the D1, D2a, D2b, and JH4 gene segments preferentially rearranged. We suggest that the preferential usage of VH, D, and JH gene segments in VDJ genes is caused by preferential rearrangement rather than by selective expansion of B cells that utilize the gene segments.
