RESUMO
Human terminal deoxynucleotidyl transferase (hTdT) is a DNA polymerase that functions to generate diversity in the adaptive immune system. Here, we focus on the function of naturally occurring single-nucleotide polymorphisms (SNPs) of hTdT to evaluate their role in genetic-generated immune variation. The data demonstrate that the genetic variations generated by the hTdT SNPs will vary the human immune repertoire and thus its responses. Human TdT catalyzes template-independent addition of nucleotides (N-additions) during coding joint formation in V(D)J recombination. Its activity is crucial to the diversity of the antigen receptors of B and T lymphocytes. We used in vitro polymerase assays and in vivo human cell V(D)J recombination assays to evaluate the activity and the N-addition levels of six natural (SNP) variants of hTdT. In vitro, the variants differed from wild-type hTdT in polymerization ability with four having significantly lower activity. In vivo, the presence of TdT varied both the efficiency of recombination and N-addition, with two variants generating coding joints with significantly fewer N-additions. Although likely heterozygous, individuals possessing these genetic changes may have less diverse B- and T-cell receptors that would particularly effect individuals prone to adaptive immune disorders, including autoimmunity.
Assuntos
DNA Nucleotidilexotransferase/genética , Imunidade Adaptativa/genética , DNA Nucleotidilexotransferase/química , DNA Nucleotidilexotransferase/metabolismo , Humanos , Região Variável de Imunoglobulina , Células Jurkat , Modelos Moleculares , Nucleotídeos , Polimorfismo de Nucleotídeo Único , Receptores de Antígenos de Linfócitos T/genética , Recombinação V(D)JRESUMO
We have used the RNA colony blot method to examine VH usage in colonies derived from primary splenic B cells. We found that there are strain-specific differences in the pattern of VH usage. Using parental F1, congenic, and recombinant inbred strains we demonstrate that the genetic element that causes the observed phenotype is: (a) stably expressed; (b) not due to maternal influence; (c) not due to dominate diffusible factors; (d) not linked to cloning efficiency; and (e) outside the Igh locus.
Assuntos
Genes de Imunoglobulinas , Cadeias Pesadas de Imunoglobulinas/imunologia , Região Variável de Imunoglobulina/genética , Animais , Linfócitos B/análise , Cruzamentos Genéticos , Ligação Genética , Camundongos , Camundongos Endogâmicos BALB C/genética , Camundongos Endogâmicos C57BL/genética , Fenótipo , RNA/análise , Baço/citologiaRESUMO
The severe combined immunodeficiency (scid) mouse has a defective V(D)J recombinase activity that results in arrested lymphoid development at the pro-B cell stage in the B lineage. The defect is not absolute and scid mice do attempt gene rearrangement. Indeed, approximately 15% of all scid mice develop detectable levels of oligoclonal serum immunoglobulin and T cell activity. To gain more insight into the scid defect and its effect on V(D)J rearrangement, we analyzed DJH recombination in scid bone marrow. We determined that DJH structures are present in scid bone marrow and occur at a frequency only 10-100 times less than C.B-17+/+. The scid DJH repertoire is limited and resembles fetal liver DJH junctions, with few N insertions and predominant usage of reading frame 1. Moreover, 70% of the DJH structures were potentially productive, indicating that normal V(D)J recombinants should be arising continually.
Assuntos
Linfócitos B/fisiologia , Rearranjo Gênico de Cadeia Pesada de Linfócito B , Genes de Imunoglobulinas , Camundongos SCID/genética , Animais , Sequência de Bases , Células da Medula Óssea , Fígado/embriologia , Camundongos , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos/química , Baço/citologiaRESUMO
Antigen-binding diversity is generated by site-specific V(D)J recombination of the T cell receptor (TCR) and immunoglobulin loci in lymphocyte precursors. Coordinate expression of two structurally distinct recombinase activating genes, RAG-1 and RAG-2, is necessary for activation of site-specific V(D)J recombination. In mice bearing targeted disruptions of either the RAG-1 or RAG-2 genes, T and B lymphocyte development is arrested at the CD4-8- double negative (DN) thymocyte or B220+/CD43+ pro-B cell stage. Development of CD4+CD8+ double positive (DP) thymocytes is restored by expression of a functionally rearranged TCR beta transgene, suggesting that TCR beta expression is critical for this developmental transition. We have found that treatment of adult or newborn RAG-deficient mice with a single sublethal dose of gamma-irradiation rescues the DN to DP transition in early thymocytes, and this is accompanied by a dramatic increase in thymus cellularity. In contrast to the observed induction of thymocyte maturation, there was no phenotypic or functional evidence of coincident B lymphocyte development in irradiated RAG-deficient mice. Interestingly, maturation of DP thymocytes occurred without expression of TCR beta protein in the cytoplasm or on the cell surface. These results suggest an in vivo pathway for DP thymocyte development which is TCR beta chain independent.
Assuntos
Antígenos CD4/análise , Antígenos CD8/análise , Proteínas de Ligação a DNA , Proteínas de Homeodomínio , Proteínas/genética , Receptores de Antígenos de Linfócitos T alfa-beta/fisiologia , Linfócitos T/fisiologia , Animais , Linfócitos B/efeitos da radiação , Sequência de Bases , Células Cultivadas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos SCID , Dados de Sequência Molecular , Proteínas/análise , Linfócitos T/imunologia , Linfócitos T/efeitos da radiaçãoRESUMO
Hematopoietic cell phosphatase (HCP), encoded by the hcph gene, (also called PTP1C, SHP, SH-PTP1, and PTPN6) is deficient in motheaten (me/me), and the allelic viable motheaten (me(v)/me(v)) mice. Since HCP is expressed in many cell types and protein phosphorylation is a major mechanism of regulating protein function, it is not surprising that the motheaten phenotype is pleiotropic. It is commonly thought that immune system involvement causes this disease. If so, the motheaten disease ought to be alleviated when the recombination activation gene-1 (RAG-1) is disrupted because there will be no V(D)J rearrangement and thus impaired development of B and T cells. We bred homozygous, double-mutant me(v)/me(v) x RAG 1 -/- mice and found that, in fact, inflamed paws, and splenomegaly with elevated myelopoiesis. Thus, except for autoantibodies, the motheaten phenotype does not depend on the presence of B and T cells. This observation cautions the use of motheaten mice as a model of autoimmune disease.
Assuntos
Doenças Autoimunes/genética , Proteínas de Homeodomínio , Linfócitos , Mutação , Proteínas Tirosina Fosfatases/genética , Proteínas/genética , Animais , Linfócitos B , Dermatite/patologia , Modelos Animais de Doenças , Genótipo , Peptídeos e Proteínas de Sinalização Intracelular , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Fenótipo , Proteína Tirosina Fosfatase não Receptora Tipo 6 , Baço/patologia , Análise de Sobrevida , Linfócitos TRESUMO
Diversity in immunoglobulin antigen receptors is generated in part by V(D)J recombination. In this process, different combinations of gene elements are joined in various configurations. Products of V(D)J recombination are coding joints, signal joints, and hybrid junctions, which are generated by deletion or inversion. To determine their role in the generation of diversity, we have examined two sorts of recombination products, coding joints and hybrid junctions, that have formed by inversion at the mouse immunoglobulin heavy-chain locus. We developed a PCR assay for quantification and characterization of inverted rearrangements of DH and JH gene elements. In primary cells from adult mice, inverted DJH rearrangements are detectable but they are rare. There were approximately 1,100 to 2,200 inverted DJH coding joints and inverted DJH hybrid junctions in the marrow of one adult mouse femur. On day 16 of gestation, inverted DJH rearrangements are more abundant. There are approximately 20,000 inverted DJH coding joints and inverted DJH hybrid junctions per day 16 fetal liver. In fetal liver cells, the number of inverted DJH rearrangements remains relatively constant from day 14 to day 16 of gestation. Inverted DJH rearrangements to JH4, the most 3' JH element, are more frequently detected than inverted DJH rearrangements to other JH elements. We compare the frequencies of inverted DJH rearrangements to previously determined frequencies of uninverted DJH rearrangements (DJH rearrangements formed by deletion). We suggest that inverted DJH rearrangements are influenced by V(D)J recombination mechanistic constraints and cellular selection.
Assuntos
Linfócitos B/imunologia , Inversão Cromossômica , Rearranjo Gênico , Genes de Imunoglobulinas , Cadeias Pesadas de Imunoglobulinas/genética , Animais , Sequência de Bases , Medula Óssea/imunologia , Células Cultivadas , Mapeamento Cromossômico , Clonagem Molecular , DNA Nucleotidiltransferases/metabolismo , Primers do DNA , Feto , Fígado/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Mutagênese Insercional , Sondas de Oligonucleotídeos , Reação em Cadeia da Polimerase , Baço/imunologia , VDJ RecombinasesRESUMO
Recombination activating genes RAG1 and RAG2 are essential components of V(D)J recombination, a process that generates the specific antigen receptors in lymphocytes. To understand the mechanisms underlying the lineage and developmental regulation of transcription of RAG2, we have characterized the human RAG2 exon 1A promoter. In this study, a series of deletion constructs were used to isolate the promoter while a linker scanning approach was taken to assess functionally relevant cis elements within the promoter. Two regulatory domains were identified. The -140 to -123 region is critical for promoter activity in all cell lines tested. Mutations to the putative Ets (-122 to -118) or to the C/EBP (-137 to -129) consensus core sequences did abrogate promoter activity, although specific DNA/protein interactions remained, as determined by EMSA. The -69 to -48 region demonstrates lineage specific promoter activity. Mutations to an overlapping, BSAP-myb-Ikaros-myb site (-65 to -39) resulted in differential promoter activity in human B and T cells. EMSA analysis of this region showed a B cell specific protein complex. Transfection of BSAP into cell lines trans-activates the human RAG2 promoter. We conclude that transcriptional regulation of the human RAG2 gene is complex, involving both tissue specific and ubiquitous factors, and both proximal and distal regulatory elements.
Assuntos
Proteínas de Ligação a DNA/genética , Regiões Promotoras Genéticas , Sequência de Bases , Éxons , Células HeLa , Humanos , Células Jurkat , Células K562 , Dados de Sequência Molecular , Mutagênese , Proteínas Nucleares , Células Tumorais CultivadasRESUMO
A procedure is described for the routine detection of RNA transcripts in small numbers of hematopoietic cells growing in semi-solid agar. It is suggested that hybridization depends upon RNA expression and that as few as 2500 mRNA molecules per colony are easily detected. Applications of this technique are described in three diverse experimental systems; immunoglobulin gene expression in B cell colonies; neo expression in normal and transformed B cell clones derived from multipotent stem cells infected with a neo-containing retrovirus; and c-myc expression in factor-dependent myeloid colonies.
Assuntos
Linfócitos B/análise , Células-Tronco Hematopoéticas/análise , RNA/análise , Transcrição Gênica , Medula Óssea/análise , Células Cultivadas , Elementos de DNA Transponíveis , Genes Bacterianos , Imunoglobulinas/análise , Hibridização de Ácido Nucleico , Proto-OncogenesRESUMO
A sensitive dot blot procedure for detecting RNA transcripts with radiolabelled cDNA probes in small numbers of cells in suspension culture is described. For a frequent mRNA species such as that for immunoglobulin in an IgM secretor hybridoma, as few as 30 cells can be detected using a C mu probe; for a less frequent mRNA species such as transcripts coding for alpha, beta or gamma genes of the T cell receptor in T cell tumors or CTL clones, as few as 3 X 10(3) to 10(4) cells can be detected using C alpha, C beta or C gamma probes. High sensitivity is achieved by confining the cells to a very small area on the filter on which they are probed, and by treating the filter with formaldehyde.
Assuntos
Linfócitos/imunologia , RNA Mensageiro/genética , Animais , Linhagem Celular , Células Cultivadas , DNA/genética , Genes , Genes de Imunoglobulinas , Camundongos , Camundongos Endogâmicos , Camundongos Nus , Hibridização de Ácido Nucleico , RNA Mensageiro/análise , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T/imunologia , Transcrição GênicaAssuntos
Rearranjo Gênico , Genes de Imunoglobulinas , Região de Junção de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Cadeias delta de Imunoglobulina/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Reação em Cadeia da PolimeraseAssuntos
Linfócitos B/citologia , Ágar , Animais , Formação de Anticorpos , Linfócitos B/imunologia , Divisão Celular , Meios de Cultura , Técnicas de Cultura/métodos , Substâncias de Crescimento , Imunoglobulinas/biossíntese , Imunoglobulinas/genética , Indicadores e Reagentes , Mitógenos , Transcrição GênicaAssuntos
Linfócitos B/imunologia , Rearranjo Gênico de Cadeia Pesada de Linfócito B , Genes de Imunoglobulinas , Células-Tronco Hematopoéticas/imunologia , Animais , Linfócitos B/citologia , Células Cultivadas , Análise Mutacional de DNA , Idade Gestacional , Células-Tronco Hematopoéticas/efeitos dos fármacos , Cadeias Pesadas de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Interleucina-7/farmacologia , Fígado/citologia , Fígado/embriologia , CamundongosAssuntos
Aniseiconia/etiologia , Astenopia/etiologia , Erros de Refração/complicações , Adolescente , Adulto , Criança , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Antigen receptor gene rearrangement is regulated by many factors in B and T lymphocytes. The sequences of the gene segments themselves, their associated recombination signal sequences (RSS), expression of the RAG genes and the chromatin accessibility of the particular gene segments to be rearranged all influence the outcome of recombination and thus antigen receptor diversity. In the present study, we have evaluated the effect of variations in RAG activity level on the junctional diversity of coding joint sequences. Using the pre-B-like 204-1-8 and the mature B DR3 cell lines under different transfection conditions, we were able to investigate recombination activity levels that varied 100-fold. We evaluated the sequences of the coding joints for junctional diversity resulting from nucleotide addition or deletion. Surprisingly, we found that the sequence of coding joints of these recombinants did not exhibit significant variation despite the large difference in recombination frequency. Our results indicate that the fidelity of the joining phase of V(D)J recombination is not jeopardized by varying RAG activity.
Assuntos
Linfócitos B/imunologia , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Rearranjo Gênico do Linfócito B , Animais , Linfócitos B/citologia , Linfócitos B/efeitos dos fármacos , Cafeína/farmacologia , Linhagem Celular , Rearranjo Gênico do Linfócito B/efeitos dos fármacos , Genes RAG-1 , Camundongos , Inibidores de Fosfodiesterase/farmacologiaRESUMO
The immunoglobulin heavy chain variable region (VH) genes of the mouse have been categorized into families based upon sequence homology. Utilizing the RNA colony blot assay we have determined the expression of eight of these families in B cell colonies derived from either surface immunoglobulin positive (sIg+) adult spleen B cells or sIg- fetal liver pre-B cells. We demonstrate, based upon the analysis of greater than 6000 individual colonies, that VH gene usage is a characteristic of the mouse strain studied. C57BL/6 mice most frequently (45%) utilize family VHJ558, the largest VH family, whereas BALB/c mice most frequently (22%) utilize family VH7183, the most JH proximal family in BALB/c mice. Moreover, colonies derived from sIg- fetal liver derived precursors show similar patterns, suggesting that selection based on exogenous antigen is not an important parameter in determining VH gene family usage.
Assuntos
Linfócitos B/imunologia , Genes , Região Variável de Imunoglobulina/genética , RNA/genética , Animais , Linfócitos B/citologia , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Fígado/imunologia , Camundongos , Camundongos Endogâmicos , Hibridização de Ácido Nucleico , Baço/imunologiaRESUMO
Somatic point mutations are usually found in the coding and flanking regions of functionally and aberrantly rearranged immunoglobulin variable region gene segments. Mutations in the unrearranged V gene segments of myelomas or hybridomas have not been described so far. We have cloned and sequenced unrearranged V lambda gene segments from several cell lines. There were no nucleotide changes in four unrearranged V lambda segments: one V lambda 1 from a lambda 3-producing hybridoma and one V lambda 2 from a lambda 1-producing myeloma (J558) and two V lambda 2 from a kappa-producing myeloma (P3X63). However, we found somatic mutations in the unrearranged V lambda segments from the lambda 2-producing myeloma MOPC315. The unrearranged V lambda 1 gene segment had two mutations in the coding region and the unrearranged V lambda 2 had one mutation in the 3' flanking region. We also cloned and sequenced the unrearranged J lambda and C lambda gene segments of MOPC315 and found no sequence alterations. This is consistent with the notion that the overall mutation rate is not higher in this cell line. Therefore, we suggest that the somatic hypermutation system can use unrearranged V gene segments as substrates. The extensive sequencing required for this work revealed a number of errors in the reported nucleotide sequences of the Ig lambda locus in BALB/c mice.