Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 74
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Cell Mol Life Sci ; 66(2): 263-74, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18810317

RESUMO

It has now been more than ten years since the discovery of the major apoptotic nuclease, DNA fragmentation factor (DFF), also known as caspase-activated DNase (CAD). Here we review the recent literature that has uncovered new insight into DFF's regulation, and both its positive and negative roles in human disease. Cells from mice deficient in DFF still undergo apoptotic death without significant cell-autonomous DNA degradation. Their corpses' genomes are subsequently degraded by lysosomal DNase II after phagocytosis. However,DFF-deficient mice are more susceptible to cancer. Indeed, several different cancers in humans are associated with defects in DFF expression and it has been proposed that DFF is a p53-independent tumor suppressor. Negative aspects of DFF expression include contributing to susceptibility to acquire systemic lupus erythematosus, to chromosomal translocations that result in mixed lineage leukemias, and in the possible spreading of oncogenes and HIV due to horizontal gene transfer.


Assuntos
Apoptose/fisiologia , Fragmentação do DNA , Desoxirribonucleases/metabolismo , Animais , Desoxirribonucleases/química , Desoxirribonucleases/genética , Ativação Enzimática , Transferência Genética Horizontal , Predisposição Genética para Doença , Humanos , Leucemia Mieloide Aguda/induzido quimicamente , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Lúpus Eritematoso Sistêmico/genética , Mutação , Neoplasias/genética , Neoplasias/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo
2.
Apoptosis ; 10(4): 821-30, 2005 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16133872

RESUMO

Endonuclease G (EndoG) is a mitochondrial enzyme that becomes an apoptotic nuclease when released from the mitochondrial intermembrane space. EndoG will digest either DNA or RNA, but at physiological ionic strength, RNA is a much more favorable substrate as compared to chromatin. This indicates that EndoG's major in vivo function(s) may be: (i) an apoptotic RNase, and/or (ii) an apoptotic DNase in the presence of additional co-activators. In the present study we have searched for factors that modulate the activity of human EndoG on DNA substrates. We demonstrate that EndoG forms complexes with AIF and FEN-1 but not with PCNA. Interestingly, heat shock proteins 70 interact with EndoG and are involved in the regulation of its activity. Purified Hsp70 prevented stimulation of EndoG DNase activity by other nuclear factors in the ATP-dependent manner.


Assuntos
Trifosfato de Adenosina/farmacologia , Apoptose/efeitos dos fármacos , Endodesoxirribonucleases/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Ribonucleases/metabolismo , Fator de Indução de Apoptose/metabolismo , Extratos Celulares , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/enzimologia , DNA/metabolismo , Endonucleases Flap/metabolismo , Células HeLa , Humanos , Proteínas Nucleares/metabolismo , RNA/metabolismo , Especificidade por Substrato/efeitos dos fármacos
3.
J Biol Chem ; 276(51): 48404-9, 2001 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-11606588

RESUMO

Endonuclease G (endoG) is released from mitochondria during apoptosis and is in part responsible for internucleosomal DNA cleavage. Here we report the action of the purified human recombinant form of this endonuclease on naked DNA and chromatin substrates. The addition of the protein to isolated nuclei from non-apoptotic cells first induces higher order chromatin cleavage into DNA fragments > or = 50 kb in length, followed by inter- and intranucleosomal DNA cleavages with products possessing significant internal single-stranded nicks spaced at nucleosomal ( approximately 190 bases) and subnucleosomal ( approximately 10 bases) periodicities. We demonstrate that both exonucleases and DNase I stimulate the ability of endoG to generate double-stranded DNA cleavage products at physiological ionic strengths, suggesting that these activities work in concert with endoG in apoptotic cells to ensure efficient DNA breakdown.


Assuntos
Cromatina/metabolismo , DNA/metabolismo , Desoxirribonuclease I/metabolismo , Endodesoxirribonucleases/metabolismo , Exonucleases/metabolismo , Catálise , Endodesoxirribonucleases/isolamento & purificação , Humanos , Concentração Osmolar , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Especificidade por Substrato
4.
Mol Cell Biochem ; 218(1-2): 125-30, 2001 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-11330826

RESUMO

The endonuclease DFF40/CAD mediates regulated DNA fragmentation and chromatin condensation in cells undergoing apoptosis. Here we report the enzyme's co-factor requirements, and demonstrate that the ionic changes that occur in apoptotic cells maximize DFF40/CAD activity. The nuclease requires Mg2+, exhibits a trace of activity in the presence of Mn2+, is not costimulated by Ca2+, is inhibited by Zn2+ or Cu2+, and has high activity over a rather broad pH range (7.0-8.5). The enzyme is thermally unstable, and is rapidly inactivated at 42 degrees C. Enzyme activity is markedly affected by ionic strength. At the optimal [K+] of 50-125 mM, which is in the range of the cytoplasmic [K+] for cells undergoing apoptosis, the activity of DFF40/CAD for naked DNA cleavage is about 100-fold higher than at 0 or 200 mM [K+]. Although these ranges of ionic strength do not affect DFF40 homo-oligomer formation, at higher ionic strengths the enzyme introduces single-stranded nicks into supercoiled DNA.


Assuntos
Desoxirribonucleases/metabolismo , Íons/metabolismo , Proteínas Recombinantes/metabolismo , Animais , Apoptose/fisiologia , Cálcio/farmacologia , Caspases/metabolismo , Cromatina/metabolismo , Cobre/farmacologia , Cricetinae , Ativação Enzimática , Estabilidade Enzimática , Células HeLa , Temperatura Alta , Humanos , Concentração de Íons de Hidrogênio , Magnésio/farmacologia , Manganês/farmacologia , Concentração Osmolar , Proteínas de Ligação a Poli-ADP-Ribose , Potássio , Zinco/farmacologia
5.
J Biol Chem ; 275(11): 8226-32, 2000 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-10713148

RESUMO

Here we report the co-factor requirements for DNA fragmentation factor (DFF) endonuclease and characterize its cleavage sites on naked DNA and chromatin substrates. The endonuclease exhibits a pH optimum of 7.5, requires Mg(2+), not Ca(2+), and is inhibited by Zn(2+). The enzyme generates blunt ends or ends with 1-base 5'-overhangs possessing 5'-phosphate and 3'-hydroxyl groups and is specific for double- and not single-stranded DNA or RNA. DFF endonuclease has a moderately greater sequence preference than micrococcal nuclease or DNase I, and the sites attacked possess a dyad axis of symmetry with respect to purine and pyrimidine content. Using HeLa cell nuclei or chromatin reconstituted on a 5 S rRNA gene tandem array, we prove that the enzyme attacks chromatin in the internucleosomal linker, generating oligonucleosomal DNA ladders sharper than those created by micrococcal nuclease. Histone H1, high mobility group-1, and topoisomerase II activate DFF endonuclease activity on naked DNA substrates but much less so on chromatin substrates. We conclude that DFF is a useful reagent for chromatin research.


Assuntos
Cromatina/metabolismo , DNA de Neoplasias/metabolismo , Desoxirribonucleases/metabolismo , Apoptose/fisiologia , Caspase 3 , Caspases/metabolismo , Fragmentação do DNA/fisiologia , Desoxirribonucleases/antagonistas & inibidores , Desoxirribonucleases/efeitos dos fármacos , Ativação Enzimática , Células HeLa , Humanos , Magnésio/farmacologia , Proteínas de Ligação a Poli-ADP-Ribose , Especificidade por Substrato
6.
J Immunol ; 164(2): 812-24, 2000 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-10623827

RESUMO

The mouse Ig kappa L chain gene locus has been extensively studied, but to date high-level expression of germline transgenes has not been achieved. Reasoning that each end of the locus may contain regulatory elements because these regions are not deleted upon V kappa-J kappa joining, we used yeast artificial chromosome-based techniques to fuse distal regions of the contig to create transgene miniloci. The largest minilocus (290 kb) possessed all members of the upstream V kappa 2 gene family including their entire 5' and 3' flanking sequences, along with one member of a downstream V kappa 21 gene family. In addition, again using yeast artificial chromosome-based technology, we created Ig kappa miniloci that contained differing lengths of sequences 5' of the most distal V kappa 2 gene family member. In transgenic mice, Ig kappa miniloci exhibited position-independent and copy number-dependent germline transcription. Ig kappa miniloci were rearranged in tissue and developmental stage-specific manners. The levels of rearrangement and transcription of the distal and proximal V kappa gene families were similar to their endogenous counterparts and appeared to be responsive to allelic exclusion, but were differentially sensitive to numerous position effects. The minilocus that contained the longest 5' region exhibited significantly greater recombination of the upstream V kappa 2 genes but not the downstream V kappa 21 gene, providing evidence for a local recombination stimulating element. These results provide evidence that our miniloci contain nearly all regulatory elements required for bona fide Ig kappa gene expression, making them useful substrates for functional analyses of cis-acting sequences in the future.


Assuntos
Cromossomos Artificiais de Levedura/imunologia , Mapeamento de Sequências Contíguas , Rearranjo Gênico de Cadeia Leve de Linfócito B/genética , Cadeias kappa de Imunoglobulina/genética , Transcrição Gênica/imunologia , Transgenes/imunologia , Alelos , Animais , Linfócitos B/citologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Cromossomos Artificiais de Levedura/genética , Cruzamentos Genéticos , Dosagem de Genes , Genes de Imunoglobulinas/genética , Marcadores Genéticos/imunologia , Células Germinativas/imunologia , Células Germinativas/metabolismo , Região de Junção de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Cadeias kappa de Imunoglobulina/química , Camundongos , Camundongos Transgênicos , Família Multigênica/imunologia , Reprodutibilidade dos Testes
7.
J Biol Chem ; 274(23): 16431-6, 1999 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-10347204

RESUMO

The nucleosomal chromatin structure within genes is disrupted upon transcription by RNA polymerase II. To determine whether this disruption is caused by transcription per se as opposed to the RNA polymerase source, we engineered the yeast chromosomal HSP82 gene to be exclusively transcribed by bacteriophage T7 RNA polymerase in vivo. Interestingly, we found that a fraction of the T7-generated transcripts were 3' end processed and polyadenylated at or near the 3' ends of the hsp82 and the immediately downstream CIN2 genes. Surprisingly, the nucleosomal structure of the T7-transcribed hsp82 gene remained intact, in marked contrast to the disrupted structure generated by much weaker, basal level transcription of the wild type gene by RNA polymerase II under non-heat shock conditions. Therefore, disruption of chromatin structure by transcription is dependent on the RNA polymerase source. We propose that the observed RNA polymerase dependence for transcription-induced nucleosome disruption may be related either to the differential recruitment of chromatin remodeling complexes, the rates of histone octamer translocation and nucleosome reformation during polymerase traversal, and/or the degree of transient torsional stress generated by the elongating polymerase.


Assuntos
Cromatina/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Nucleossomos/metabolismo , RNA Polimerase II/metabolismo , Transcrição Gênica , Bacteriófago T7/enzimologia , Cromatina/química , Engenharia Genética , Saccharomyces cerevisiae/genética , Moldes Genéticos , Proteínas Virais
8.
J Immunol ; 162(10): 6029-39, 1999 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-10229843

RESUMO

The Igkappa gene contains an evolutionarily conserved nuclear matrix association region (MAR) adjacent to the intronic enhancer. To test for the function of this MAR, we created mouse lines with a targeted MAR deletion. In MAR knockout animals, the immune system was normal in nearly all respects, including the distributions of various B cell populations and Ab levels. However, in pro-B cells, enhanced rearrangement was noted on the MAR- allele in heterozygotes. In addition, the efficiencies for targeting and generating somatic mutations were reduced on MAR-deleted alleles. These results provide evidence for the MAR negatively regulating the probability of premature rearrangement and positively regulating the probability of somatic hypermutation.


Assuntos
Sequência Conservada , Rearranjo Gênico do Linfócito B , Genes de Imunoglobulinas , Cadeias kappa de Imunoglobulina/genética , Mutagênese , Alelos , Animais , Linfócitos B/imunologia , Sequência de Bases , Metilação de DNA , Células-Tronco Hematopoéticas/imunologia , Região de Junção de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Camundongos , Camundongos Knockout , Probabilidade , Processamento Pós-Transcricional do RNA , RNA Mensageiro/isolamento & purificação , Recombinação Genética
9.
Methods ; 17(2): 95-103, 1999 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-10075888

RESUMO

In the past decade, site-specific chromosomal DNA cleavage mediated by DNA endonucleases has been used to examine diverse aspects of chromosome structure and function in eukaryotes, such as DNA topology, replication, transcription, recombination, and repair. Here we describe a method with which chromosomes can be linearized at any predefined position in vivo. Yeast homothallic switching endonuclease (HO endo), a sequence-specific double-strand nuclease involved in mating-type switching, is employed for targeting DNA cleavage. HO endo contains discrete functional domains: a N-terminal nuclease and a C-terminal DNA-binding domain, thereby allowing construction of a chimeric nuclease with the cutting site distinct from the original HO recognition sequence. The expression of the nuclease is engineered to be controlled by a tightly regulated, inducible promoter. The cut sites recognized by HO endo or its derivatives are introduced specifically at desired positions in the yeast genome by homologous recombination. Here we present experimental procedures and review some applications based on this approach in yeast and other biological systems.


Assuntos
DNA/química , Desoxirribonucleases de Sítio Específico do Tipo II/química , Biologia Molecular/métodos , Southern Blotting , Enzimas de Restrição do DNA , Modelos Biológicos , Plasmídeos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae
10.
Crit Rev Eukaryot Gene Expr ; 9(3-4): 295-310, 1999.
Artigo em Inglês | MEDLINE | ID: mdl-10651246

RESUMO

MARs are cis-acting DNA sequences that function both negatively and positively in conjunction with transcriptional enhancers to regulate antigen receptor and co-receptor genes. Evidence exists that certain tissue-specific nuclear proteins are involved in this regulation, including SATB1, Bright, and Cux/CDP, possibly by modulating intranuclear gene location, histone acetylation, DNA methylation, and/or nucleosome positioning.


Assuntos
Núcleo Celular/genética , DNA/genética , Matriz Nuclear/genética , Receptores de Antígenos de Linfócitos T/genética , Animais , Metilação de DNA , Regulação da Expressão Gênica/imunologia , Humanos , Transcrição Gênica
11.
Mol Immunol ; 35(10): 609-20, 1998 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-9823759

RESUMO

Previous studies employing ectopic integration of reporter genes have shown that the nuclear matrix association region (MAR) adjacent to the intronic enhancer of the mouse kappa immunoglobulin (Ig) gene is required for high level transcription of rearranged genes, demethylation, reduction of position effects and maximal somatic hypermutation in B cells. To test for the function of this MAR in its natural chromosomal environment, we pursued the 'HIT-and-RUN' procedure with the mouse pre-B cell line 103 to create a targeted MAR deletion. We observed a 'HIT' targeting frequency of 1/684 but 0/2100 'RUN' clones maintained the MAR-deleted germline locus because of an unexpected hyper-recombination for Vkappa-Jkappa joining, specifically to the MAR-deleted allele, and primarily at Jkappa4 and Jkappa5. This hyper-recombination was correlated with undermethylation of the Jkappa-Ckappa region but not with the level of local transcription. These results are consistent with the possibility that the MAR and/or DNA methylation negatively regulate(s) Vkappa-Jkappa joining during the pre-B cell stage of development.


Assuntos
Linfócitos B/metabolismo , DNA Nucleotidiltransferases/metabolismo , Marcação de Genes , Genes de Imunoglobulinas , Células-Tronco Hematopoéticas/metabolismo , Cadeias kappa de Imunoglobulina/genética , Matriz Nuclear/metabolismo , Recombinação Genética , Alelos , Animais , Linfócitos B/imunologia , Sítios de Ligação , DNA/metabolismo , Elementos Facilitadores Genéticos , Rearranjo Gênico de Cadeia Leve de Linfócito B , Células-Tronco Hematopoéticas/imunologia , Cadeias J de Imunoglobulina/genética , Região Variável de Imunoglobulina/genética , Camundongos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Deleção de Sequência , VDJ Recombinases
13.
Acta Biochim Pol ; 45(1): 209-19, 1998.
Artigo em Inglês | MEDLINE | ID: mdl-9701513

RESUMO

Packaging of DNA into chromatin adds complexity to the problem of regulation of gene expression. Nucleosomes affect the accessibility of transcription factors to occupy their binding sites in chromatin of eukaryotic cells. The disruption of nucleosome structure within the enhancer/promoter region of the integrated HIV-1 proviral genome is an instructive example of a chromatin remodeling process during transcriptional activation. To investigate the mechanism responsible for generating nuclease hypersensitive sites that exist in vivo in the promoter/enhancer region of the 5'LTR (long terminal repeat) of integrated HIV-1 we have utilized an in vitro chromatin assembly system with Xenopus oocyte extracts. Chromatin assembly in the presence of Sp1 and NFkappaB transcription factors induces DNase I hypersensitive sites on either side of their binding sites and positions the adjacent nucleosomes. This structure can also be formed in a factor-induced, ATP-dependent chromatin remodeling process and closely resembles the in vivo chromatin structure. The DNase I hypersensitive sites that form within the HIV LTR are probably histone-free and remain after removal of transcription factors.


Assuntos
Regulação Viral da Expressão Gênica/fisiologia , Repetição Terminal Longa de HIV , Nucleossomos/fisiologia , Acetilação , Trifosfato de Adenosina/fisiologia , Animais , Cromatina/genética , Genoma Viral , Histonas/metabolismo
14.
Proc Natl Acad Sci U S A ; 95(15): 8461-6, 1998 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-9671700

RESUMO

We report here the reconstitution of a pathway that leads to the apoptotic changes in nuclei by using recombinant DNA fragmentation factor (DFF), a heterodimeric protein of 40 and 45 kDa. Coexpression of DFF40 and DFF45 is required to generate recombinant DFF, which becomes activated when DFF45 is cleaved by caspase-3. The cleaved fragments of DFF45 dissociate from the DFF40, the active component of DFF. Purified DFF40 exhibited an intrinsic DNase activity that was markedly stimulated by chromatin-associated proteins histone H1 and high mobility group proteins. DFF40 also triggered chromatin condensation when incubated with nuclei. These data suggest that DFF40 is sufficient to trigger both DNA fragmentation and chromatin condensation during apoptosis.


Assuntos
Apoptose , Cromatina/metabolismo , Fragmentação do DNA , Desoxirribonucleases/metabolismo , Sequência de Aminoácidos , Proteínas Reguladoras de Apoptose , Desoxirribonucleases/química , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Dados de Sequência Molecular , Proteínas de Ligação a Poli-ADP-Ribose , Proteínas/química , Proteínas/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos
15.
Genomics ; 43(3): 307-15, 1997 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-9268633

RESUMO

We present a method for genome comparisons and high-resolution hybridization analyses using megabase stretches of known DNA sequences as a reference. The method employs two-dimensional gel electrophoresis, separating genomic segments cut with different restriction endonucleases in the first and second dimensions, to generate filters suitable for image analysis and repeated nucleic acid hybridizations. The corresponding two-dimensional pattern is computed from the reference nucleotide sequence and matched to the observed pattern, thereby identifying each fragment on the filter; at the same time the technique uncovers discrepancies from the reference sequence. This permits genome comparisons as well as automated identification and quantification of hybridization patterns with various probes. The technique is illustrated by an analysis of Saccharomyces cerevisiae chromosome IX.


Assuntos
Hibridização de Ácido Nucleico/métodos , Análise de Sequência de DNA/normas , Passeio de Cromossomo/métodos , Cromossomos Fúngicos , Gráficos por Computador , DNA/análise , Enzimas de Restrição do DNA/metabolismo , Eletroforese em Gel Bidimensional , Filtração/instrumentação , Genes Fúngicos , Processamento de Imagem Assistida por Computador , Hibridização In Situ/métodos , Valores de Referência , Projetos de Pesquisa , Saccharomyces cerevisiae/genética
16.
J Biol Chem ; 272(28): 17654-61, 1997 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-9211915

RESUMO

Nuclease hypersensitive sites exist in vivo in the chromatin of the integrated human immunodeficiency virus (HIV)-1 proviral genome, in the 5'-long terminal repeat (LTR) within the promoter/enhancer region near Sp1 and NFkappaB binding sites. Previous studies from the Kadonaga and Jones laboratories have shown that Sp1 and NFkappaB can establish hypersensitive sites in a truncated form of this LTR when added before in vitro chromatin assembly with Drosophila extracts, thus facilitating subsequent transcriptional activation of a linked reporter gene upon the association of additional factors (Pazin, M. J., Sheridan, P. L., Cannon, K., Cao, Z., Keck, J. G., Kadanaga, J. T., and Jones, K. A. (1996) Genes & Dev. 10, 37-49). Here we assess the role of a full-length LTR and 1 kilobase pair of downstream flanking HIV sequences in chromatin remodeling when these transcription factors are added after chromatin assembly. Using Xenopus laevis oocyte extracts to assemble chromatin in vitro, we have confirmed that Sp1 and NFkappaB can indeed induce sites hypersensitive to DNase I, micrococcal nuclease, or restriction enzymes on either side of factor binding sites in chromatin but not naked DNA. We extend these earlier studies by demonstrating that the process is ATP-dependent when the factors are added after chromatin assembly and that histone H1, AP1, TBP, or Tat had no effect on hypersensitive site formation. Furthermore, we have found that nucleosomes upstream of NFkappaB sites are rotationally positioned prior to factor binding and that their translational frame is registered after binding NFkappaB. On the other hand, binding of Sp1 positions adjacent downstream nucleosome(s). We term this polar repositioning because each factor aligns nucleosomes only on one side of its binding sites. Mutational analysis and oligonucleotide competition each demonstrated that this remodeling required Sp1 and NFkappaB binding sites.


Assuntos
Cromatina/virologia , Repetição Terminal Longa de HIV/genética , NF-kappa B/metabolismo , Nucleossomos/metabolismo , Fator de Transcrição Sp1/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Polaridade Celular , Cromatina/metabolismo , DNA/metabolismo , Pegada de DNA , DNA Super-Helicoidal/metabolismo , Técnicas In Vitro , Nuclease do Micrococo/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Oócitos/metabolismo , Xenopus laevis
17.
Mol Cell Biol ; 17(5): 2825-34, 1997 May.
Artigo em Inglês | MEDLINE | ID: mdl-9111354

RESUMO

To address the role of transient torsional stress in transcription, we have utilized the regulated expression of HO endonuclease in yeast to create double-strand breaks in DNA templates in vivo at preselected sites. Linearization of circular minichromosomes, either 2 kb upstream or immediately downstream of a lacZ reporter gene controlled by the yeast metallothionein gene (CUP1) promoter, did not alter the copper induction profile of lacZ RNA transcripts compared to that of nonlinearized controls. Constructs site-specifically integrated into yeast chromosome II gave similar results. In vivo cross-linking with psoralen as a probe for negative DNA supercoiling demonstrated that template linearization efficiently dissipated DNA supercoiling induced by transcription. Therefore, the efficient transcription of linearized, relaxed templates found here demonstrates that transient torsional tension is not required for transcription of chromatin templates in yeast.


Assuntos
Cromatina/metabolismo , DNA Fúngico/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Moldes Genéticos , Transcrição Gênica , Cobre/metabolismo , Dano ao DNA , DNA Super-Helicoidal/metabolismo , Regulação Enzimológica da Expressão Gênica , Genes Reporter , Mapeamento por Restrição , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae
18.
Proc Natl Acad Sci U S A ; 92(26): 12421-5, 1995 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-8618913

RESUMO

The immunoglobulin kappa gene locus encodes 95% of the light chains of murine antibody molecules and is thought to contain up to 300 variable (V kappa)-region genes generally considered to comprise 20 families. To delineate the locus we have isolated 29 yeast artificial chromosome genomic clones that form two contigs, span > 3.5 megabases, and contain two known non-immunoglobulin kappa markers. Using PCR primers specific for 19 V kappa gene families and Southern analysis, we have refined the genetically defined order of these V kappa gene families. Of these, V kappa 2 maps at least 3.0 Mb from the joining (J kappa) region and appears to be the most distal V kappa gene segment.


Assuntos
Mapeamento Cromossômico , Genes de Imunoglobulinas , Região de Junção de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Cadeias kappa de Imunoglobulina/genética , Família Multigênica , Animais , Sequência de Bases , Southern Blotting , Quimera , Cromossomos Artificiais de Levedura , Primers do DNA , Biblioteca Gênica , Íntrons , Camundongos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Mapeamento por Restrição
19.
Proc Natl Acad Sci U S A ; 90(12): 5623-7, 1993 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-8516310

RESUMO

Chromatin becomes reorganized during mitosis each cell cycle. To identify genes potentially involved in these supramolecular events, we have used a colony-color assay to screen temperature-sensitive mutants of Saccharomyces cerevisiae. When a sequence that mediates attachment to the nuclear matrix in vitro was inserted into the GAL1 promoter of a lacZ fusion gene, beta-galactosidase synthesis was inhibited. This observation permitted screening for temperature-sensitive-inducible mutants on 5-bromo-4-chloro-3-indolyl beta-D-galactoside plates. Only 1 of 20 complementation groups of newly isolated mutants exhibited temperature-sensitive inducibility for the matrix association region but not for control CEN3 or STE6 inserts--a cmd1 mutant in which the last 7 amino acids of calmodulin were truncated by an ochre termination codon. Another mutant (smi1) exhibited a rare phenotype at the nonpermissive condition, which included S phase and budding arrest. We cloned and sequenced the SMI1 gene, which encodes a 57-kDa polypeptide with evolutionarily conserved epitope(s) found in mammalian cell nuclei. Thus, we provide evidence for involvement of calmodulin and another conserved protein in the in vivo binding of a matrix association region.


Assuntos
Calmodulina/metabolismo , Núcleo Celular/metabolismo , Proteínas Nucleares/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos , Sequência de Bases , Evolução Biológica , Clonagem Molecular , Sequência Conservada , Genes Fúngicos , Teste de Complementação Genética , Mamíferos , Dados de Sequência Molecular , Mutagênese , Matriz Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Peptídeos/síntese química , Peptídeos/imunologia , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão/biossíntese , Homologia de Sequência de Aminoácidos , Temperatura , Fatores de Transcrição , beta-Galactosidase/biossíntese
20.
J Biol Chem ; 267(33): 23888-93, 1992 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-1429727

RESUMO

Previous studies have located transcriptional enhancer elements within both the intron and 3'-region of the mouse kappa immunoglobulin gene. Here we address the role of these two enhancers in specifying gene activity and specific chromatin structures. MOPC41 kappa gene constructs, either intact or containing deletions of one or both enhancers, were introduced into S194 mouse plasmacytoma cells for transient and stable expression studies. Transient expression assays revealed that the basal level expression exhibited by enhancerless constructs was activated 100-200-fold by the two enhancers together in a synergistic fashion. A similar trend was observed when both enhancers were present in stably integrated constructs, although the synergy was less pronounced. Analysis of DNase I hypersensitive sites in the chromatin revealed that stably integrated constructs established hypersensitive sites about the enhancer sequences. These sites demonstrated the same nuclease susceptibility as those associated with the endogenous gene(s), and their establishment was independent of the presence of the other enhancer. Thus, although both enhancers are required for maximal gene expression, the elements act independently in determining specific chromatin structures.


Assuntos
Cromatina/fisiologia , Elementos Facilitadores Genéticos , Genes de Imunoglobulinas , Cadeias kappa de Imunoglobulina/genética , Animais , Northern Blotting , Southern Blotting , Núcleo Celular/fisiologia , Células Clonais , DNA de Neoplasias/genética , DNA de Neoplasias/isolamento & purificação , Desoxirribonuclease I , Regulação Neoplásica da Expressão Gênica , Íntrons , Camundongos , Plasmocitoma , Mapeamento por Restrição , Deleção de Sequência , Transfecção , Células Tumorais Cultivadas
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...