RESUMO
The filter paper disc method for cloning inducible lymphocytes was used to census the splenic B cell population of C57BL/6 and BALB/c mice for the expression of three VH gene-families, VH X-24, -Q52, and -J558. B cell colonies, arising from single founder lymphocytes, were identified by in situ hybridization with VH family- and C mu-specific cDNA probes. Some 6.7 X 10(4) C mu+ colonies were screened. Among C57BL/6- or BALB/c-derived colonies, approximately 3% were VH X-24+, approximately 19% were VH Q52+, and approximately 54% were VH J558+. These frequencies are consistent with a process of equiprobable expression for individual VH segments, and provide direct evidence that normal splenic B lymphocytes use a process of random genetic combinatorics to generate the antibody repertoire.
Assuntos
Linfócitos B/imunologia , Cadeias Pesadas de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Animais , Diversidade de Anticorpos , Divisão Celular , Linhagem Celular , DNA/genética , Variação Genética , Genótipo , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Hibridização de Ácido Nucleico , Baço/citologiaRESUMO
V kappa gene family expression among LPS-reactive murine B lymphocytes, unlike that of VH gene families, is not proportional to genomic complexity, i.e., nonstoichiometric. Furthermore, no positional bias for the overexpression of J-proximal V kappa genes (V kappa 21) is observed among neonatal B lymphocytes. Yet, the V kappa 1 and V kappa 9 families located in the center of V kappa locus are preferentially used by neonatal B splenocytes. Thus, the mechanisms of V kappa gene rearrangement and expression appear to differ significantly from those controlling the VH locus.
Assuntos
Regulação da Expressão Gênica , Genes de Imunoglobulinas , Região Variável de Imunoglobulina/genética , Cadeias kappa de Imunoglobulina/genética , Animais , Animais Recém-Nascidos , Sondas de DNA , Cadeias Pesadas de Imunoglobulinas/genética , Camundongos , Camundongos Endogâmicos C57BL , Hibridização de Ácido NucleicoRESUMO
The mechanisms responsible for the periodic accumulation and decay of histone mRNA in the mammalian cell cycle were investigated in mouse erythroleukemia cells, using a cloned mouse H3 histone gene probe that hybridizes with most or all H3 transcripts. Exponentially growing cells were fractionated into cell cycle-specific stages by centrifugal elutriation, a method for purifying cells at each stage of the cycle without the use of treatments that arrest growth. Measurements of H3 histone mRNA content throughout the cell cycle show that the mRNA accumulates gradually during S phase, achieving its highest value in mid-S phase when DNA synthesis is maximal. The mRNA content then decreases as cells approach G2. These results demonstrate that the periodic synthesis of histones during S phase is due to changes in the steady-state level of histone mRNA. They are consistent with the conventional view in which histone synthesis is regulated coordinately with DNA synthesis in the cell cycle. The periodic accumulation and decay of H3 histone mRNA appear to be controlled primarily by changes in the rate of appearance of newly synthesized mRNA in the cytoplasm, determined by pulse-labeling whole cells with [3H]uridine. Measurements of H3 mRNA turnover by pulse-chase experiments with cells in S and G2 did not provide evidence for changes in the cytoplasmic stability of the mRNA during the period of its decay in late S and G2. Furthermore, transcription measurements carried out by brief pulse-labeling in vivo and by in vitro transcription in isolated nuclei indicate that the rate of H3 gene transcription changes to a much smaller extent than the steady-state levels of the mRNA or the appearance of newly synthesized mRNA in the cytoplasm. The results suggest that post-transcriptional processes make an important contribution to the periodic accumulation and decay of histone mRNA and that these processes may operate within the nucleus.
Assuntos
Histonas/metabolismo , RNA Mensageiro/metabolismo , Animais , Ciclo Celular , Células Clonais/metabolismo , Regulação da Expressão Gênica , Histonas/genética , Leucemia Eritroblástica Aguda/metabolismo , Camundongos , Hibridização de Ácido Nucleico , RNA Mensageiro/genéticaRESUMO
Genetic interaction as a mechanism for the generation of mutations is suggested by recurrent, multiple nucleotide substitutions that are identical to nucleotide sequences elsewhere in the genome. We have sequenced the mutant K gene from the bm6 mouse, which is one of a series of eight closely related, yet independently occurring mutants known collectively as the "bg series." Two changes from the Kb gene are found, positioned 15 nucleotides apart: an A-to-T change and a T-to-C change in the codons corresponding to amino acids 116 and 121, resulting in Tyr-to-Phe and Cys-to-Arg substitutions, respectively. Hybridization analysis with an oligonucleotide specific for the altered Kbm6 sequence identifies one donor gene, Q4, located in the Qa region of the H-2 complex. The two altered nucleotides that differentiate Kbm6 and Kb are present in Q4 in a region where Kb and Q4 are otherwise identical for 95 nucleotides, delineating the maximum genetic transfer between the two genes. Because the Kbm6 mutation arose in an homozygous mouse these data indicate that the Q4 gene contains the only donor sequence and demonstrates that Q-region gene sequences can interact with the Kb gene to generate variant K molecules.
Assuntos
Genes , Antígenos de Histocompatibilidade , Complexo Principal de Histocompatibilidade , Mutação , Animais , Sequência de Bases , Enzimas de Restrição do DNA , Antígenos de Histocompatibilidade/análise , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Hibridização de Ácido NucleicoRESUMO
The H-2K(b) gene is a member of the large major histocompatibility complex class I gene family. Since many members of this family cross-hybridize with class I cDNA probes, the cloned H-2K(b) gene was identified by hybridization with specific oligonucleotide probes. This clone was definitively shown to encode the H-2K(b) polypeptide by partial DNA sequencing and by serological and tryptic peptide analyses of the expressed product.
Assuntos
Antígenos H-2/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Genes , Hibridização de Ácido Nucleico , Oligodesoxirribonucleotídeos/síntese químicaRESUMO
DNA hybridization was used to establish DNA sequence relationships among seven Drosophila species. Single-copy DNA was isolated from four species within the Drosophila mulleri complex, D. mojavensis, D. arizonensis, D. ritae and D. starmeri. These single-copy DNAs were used as tracers to be hybridized with each other and one additional member of the mulleri complex, D. aldrichi, a member of a closely related complex, D. hydei, and a distantly related species, D. melanogaster. Two methods have been used to determine the relatedness between these species: (1) the extent of duplex formed as measured by binding to hydroxyapatite and (2) the thermal stability of the duplexed DNA. Moderately repetitive DNA was purified from these species and used similarly to determine the divergence of this family of sequences. The rate of nucleotide substitution was estimated to be 0.2 +/-, 0.1% base pair change per million years for both single-copy and middle-repetitive DNAs. The size of the D. arizonensis genome, a representative of the mulleri complex, was calculated to be 2.2 X 10(8) base pairs from its kinetic complexity similar to that of D. hydei. The relative amounts (18%) and average reiteration frequency (100 copies) of the middle-repetitive DNA are similar for all Drosophila species studied. Finally, the data are presented in a phylogenetic tree.
Assuntos
DNA/genética , Drosophila/genética , Animais , Desnaturação de Ácido Nucleico , Hibridização de Ácido Nucleico , Sequências Repetitivas de Ácido Nucleico , Homologia de Sequência do Ácido Nucleico , Especificidade da EspécieRESUMO
The gene for H-2K class I major histocompatibility antigen on the bm8 variant was cloned and the DNA sequence compared with the parental gene. Sequence analysis demonstrated that seven nucleotides were changed with respect to the parental gene sequence spanning 24 nucleotides. These changes represent an alteration of four amino acids from the parent protein. As this mutation occurred in a single generation, a potential donor gene for such a complex mutation was suggested and identified. The Q4 gene class I-like molecule has a stretch of 95 nucleotides of identity in the region of the bm8 mutation. Genomic Southern analysis of the mutant and parental DNA with a gene-specific oligonucleotide demonstrated that the potential donor gene Q4 is a likely candidate sequence for such an event. The amino acid alterations for the H-2Kbm8 mutation are discussed in consideration of hte three-dimensional structure of the characterized human class I glycoprotein.
Assuntos
Antígenos H-2/genética , Mutação , Animais , Sequência de Bases , Southern Blotting , Clonagem Molecular , DNA , Antígenos H-2/imunologia , Antígenos de Histocompatibilidade Classe I/genética , Camundongos , Camundongos Mutantes , Dados de Sequência Molecular , Relação Estrutura-AtividadeRESUMO
Although inhibition of the Na+/Ca2+ exchanger normally increases [Ca2+]i in neonatal cardiac myocytes, application of the inhibitor Ni2+ appears to reduce [Ca2+] measured by fluo-3. To investigate how the apparent reduction in [Ca2+]i occurs we examined Ca2+ transport by the human Na+/Ca2+ exchanger expressed in Sf9 cells. Transport of Ca2+ by the Na+/Ca2+ exchanger was examined using a laser-scanning confocal microscope and the fluorescent Ca2+ indicator fluo-3, and the electrogenic function was determined by measuring the Na+/Ca2+ exchange current (INaCa) using patch clamp methods. INaCa was elicited with voltage-clamp steps or flash photolysis of caged Ca2+. We show significant expression of Na+/Ca2+ exchanger function in Sf9 cells infected with a recombinant Baculovirus carrying the Na+/Ca2+ exchanger. In addition to measurements of INaCa, characterization includes Ca2+ transport via the Na+/Ca2+ exchanger and the voltage dependence of Ca2+ transport. Application of Ni2+ blocked INaCa but, contrary to expectation, decreased fluo-3 fluorescence. Experiments with infected Sf9 cells suggested that Ni2+ was transported via the Na+/Ca2+ exchanger at a rate comparable to the Ca2+ transport. Once inside the cells, Ni2+ reduced fluorescence, presumably by quenching fluo-3. We conclude that Ni2+ does indeed block INaCa, but is also rapidly translocated across the cell membrane by the Na+/Ca2+ exchanger itself, most likely via an electroneutral partial reaction of the exchange cycle.
Assuntos
Níquel/metabolismo , Trocador de Sódio e Cálcio/biossíntese , Animais , Transporte Biológico , Cálcio/metabolismo , Linhagem Celular , Células Cultivadas , Humanos , Miocárdio/metabolismo , Ratos , Ratos Wistar , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Trocador de Sódio e Cálcio/genética , Spodoptera , Fatores de TempoRESUMO
A sensitive, highly resolvable, and quantitative method was designed to analyse the diversity of polymerase chain reaction (PCR)-amplified transcripts which possess length polymorphism. A reverse transcriptase-PCR technique was used to amplify rearranged T cell antigen receptor (TCR) transcripts isolated from human blood. Oligonucleotide primers specific for conserved TCR V and C region sequences were used in PCR, with one of the primers end-labeled with 32P. Amplified cDNA products were analysed by polyacrylamide sequencing gel electrophoresis with an M13mp18 sequencing ladder as a size marker. 32P-labeled products were detected by either autoradiography or PhosphorImager. The method allowed determination of the sizes of PCR products with the precision of one nucleotide. The resolution using this technique was much higher than by electrophoresis in agarose gel with ethidium bromide staining. The sizes of PCR products determined by sequencing gel electrophoresis were consistent with the lengths of nucleotide sequences obtained after subcloning PCR products in competent bacterial cells. Analysis of PCR products by sequencing gel electrophoresis was more rapid and as accurate as nucleotide sequence analysis in determining the relative ratios of TCR mRNA in mixtures of T cell clones. The method is applicable for analysis of both rearranged TCR and immunoglobulin genes.
Assuntos
Variação Genética , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Sequência de Bases , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Células Clonais , Eletroforese/métodos , Expressão Gênica , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , Receptores de Antígenos de Linfócitos T alfa-beta/análiseRESUMO
We describe the use of an oligonucleotide construction as a hybridization probe to detect short noncontiguous regions of sequence identity. Oligonucleotides complementary to various portions of the conserved heptamer and nonamer sequences flanking immunoglobulin variable region genes at the 3' end were used in this model system. We show that short oligonucleotides alone (7 bp or 9 bp) cannot be used as hybridization probes, but a construction containing both conserved sequences linked by a bridge will hybridize. The bridge is formed by degenerate bases (any base potentially at each position) and serves to maintain the spacing originally present between heptamer and nonamer. We show that such a bridging oligonucleotide probe can be used for hybridization analysis both on Southern blots and in bacterial screening.
Assuntos
Hibridização de Ácido Nucleico , Oligonucleotídeos , Sequência de Bases , Southern Blotting , DNA/análise , Biblioteca Gênica , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , Polimorfismo de Fragmento de RestriçãoRESUMO
The causes of observed deficiencies to the humoral immune response in aged humans are unknown. Since a major source of antibody diversity is generated at the VH-D-JH junctional regions of the immunoglobulin heavy chain, we determined whether differences in junctional diversity are manifested with aging. We compared the CDR3 regions of IgM heavy chain transcripts isolated from young adult and aged humans. A PCR assay that measures CDR3 length in the majority of mu-heavy chains showed the same average size and normal range of CDR3 length in aged individuals as observed in young adults. To characterize the features of junctional diversity of aged adults in more detail, we determined the CDR3 sequences of a subset of the mu-heavy chain repertoire that utilizes members of the VH 5 family. In general CDR3 length, D family usage, and JH gene usage were similar in aged compared to young adults. Thus, in contrast to dramatic changes in heavy chain junctional diversity associated with fetal to adult development, no major differences were found between young and aged adults. Since the CDR3 repertoire generated in aged individuals appears to be as diverse as that observed in younger adults, the decline in humoral immunocompetence with aging cannot be attributed to a restriction in heavy chain junctional diversification processes.
Assuntos
Idoso/fisiologia , Cadeias Pesadas de Imunoglobulinas/genética , Região de Junção de Imunoglobulinas/genética , Imunoglobulina M/genética , Imunoglobulina M/imunologia , Região Variável de Imunoglobulina/genética , RNA/análise , Adulto , Humanos , Reação em Cadeia da Polimerase , Polimorfismo Genético , RNA/genética , Alinhamento de Sequência , Análise de Sequência de RNA , Deleção de Sequência , Transcrição GênicaRESUMO
Alternative splicing of RNA transcripts is a general characteristic for NCX genes in mammals, mollusks, and arthropods. Among the family of three NCX genes in mammals, the NCX1 gene contains six exons, namely, A, B, C, D, E, and F, that make up the alternatively spliced region. Studies of the NCX1 gene transcripts suggested that 16 distinct gene products can be produced from the NCX1 gene. The exons A and B are mutually exclusive when expressed. Generally, exon A-containing transcripts are predominantly found in excitable cells like cardiomyoctes and neurons, whereas exon B-containing transcripts are mostly found in nonexcitable cells like astrocytes and kidney cells. Other alternatively spliced exons (C-F) appear to be cassette-type exons and are found in various combinations. Interestingly, exon D is present in all characterized transcripts. The alternatively spliced isoforms of NCX1 show tissue-specific expression patterns, suggesting functional adaptation to tissues. To investigate functional differences among alternatively spliced isoforms of NCX1, we expressed an exon A-containing transcript present in cardiac tissue (NCX1.1) and an exon B-containing transcript found in the kidney (NCX1.3) in Xenopus oocytes. We demonstrated that the Na(+)/Ca(2+) exchangers expressed by exon A- and exon B-containing transcripts display differences in activation by PKA and by [Ca(2+)](i). We also observed that these two isoforms show differences in voltage dependence. Surprisingly, the alternatively spliced isoforms of NCX1 display greater functional differences among themselves than the products of different gene loci, NCX1, NCX2, and NCX3.
Assuntos
Processamento Alternativo , Regulação da Expressão Gênica , Proteínas de Membrana Transportadoras , Trocador de Sódio e Cálcio/genética , Transcrição Gênica , Sequência de Aminoácidos , Animais , Cálcio/fisiologia , Éxons , Humanos , Mamíferos , Potenciais da Membrana , Dados de Sequência Molecular , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiologia , Trocador de Sódio e Cálcio/fisiologiaRESUMO
We describe an analysis of the NCX1 gene and show that various tissues express different alternatively spliced forms of the gene. Alternative splicing has been confirmed by the genomic analysis of the Na(+)-Ca2+ exchanger gene. We also describe the Drosophila Na(+)-Ca2+ exchanger as having many of the same structural characteristics of the mammalian exchangers and this locus as possibly undergoing alternative splicing in the same region that has been described in the NCX1 gene. The general structure of the exchangers is similar to that of the alpha-subunit of the (Na(+)+ K+)-A Pase. Finally, sequence comparison of the various molecules demonstrates that structural characteristics of these molecules are more strongly conserved than the primary sequence of these products.
Assuntos
Processamento Alternativo , Proteínas de Transporte/genética , Sequência de Aminoácidos , Animais , Cálcio/metabolismo , Humanos , Dados de Sequência Molecular , Homologia de Sequência , Sódio/metabolismo , Trocador de Sódio e Cálcio , TransfecçãoAssuntos
Escherichia coli/metabolismo , Proteoma , Trocador de Sódio e Cálcio/química , Sequência de Aminoácidos , Animais , Transporte Biológico , Cálcio/metabolismo , Membrana Celular/metabolismo , Sequência Conservada , Feminino , Dados de Sequência Molecular , Oócitos/fisiologia , Células Procarióticas , XenopusAssuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Animais , Clonagem Molecular , Drosophila/fisiologia , Oócitos/fisiologia , Fosforilação , Processamento de Proteína Pós-Traducional , Ratos , Proteínas Recombinantes/metabolismo , Trocador de Sódio e Cálcio/genética , Transfecção , XenopusAssuntos
Odorantes , Neurônios Receptores Olfatórios/fisiologia , Trocador de Sódio e Cálcio/fisiologia , Animais , Cálcio/fisiologia , Cílios/fisiologia , Proteínas de Ligação ao GTP/fisiologia , Reação em Cadeia da Polimerase , Olfato/fisiologia , Trocador de Sódio e Cálcio/genética , Transcrição GênicaAssuntos
Proteínas de Transporte/genética , Proteínas de Transporte/fisiologia , Coração/fisiologia , Linfócitos/fisiologia , Músculo Liso/fisiologia , Fenômenos Fisiológicos do Sistema Nervoso , Animais , Cálcio/metabolismo , Humanos , Rim/fisiologia , Neuroglia/fisiologia , Neurônios/fisiologia , Sódio/metabolismo , Trocador de Sódio e CálcioRESUMO
Short DNA sequence motifs have been demonstrated to interact with DNA binding proteins and regulate flanking genes. The short nature and the lack of continuity of many of these DNA binding sites make it difficult to develop an approach to characterize genes that have the same flanking sequences. We tested various oligonucleotide combinations using an immunoglobulin variable region gene family as a model amplification system. One successful amplification strategy used an oligonucleotide containing two known noncontiguous short sequences connected by random insertion of all four bases to maintain the appropriate spacing. A second approach used an oligonucleotide having a single short homologous sequence with the addition of all four bases randomly placed at the 5' end to increase the extent of homology. Both strategies will permit the priming of members of a specific gene family, with the two short sequences bridged by all four bases randomly added being more efficient in the amplification process.