RESUMO
The morphology of axon terminals changes with differentiation into mature synapses. A molecule that might regulate this process was identified by a screen of Drosophila mutants for abnormal motor activities. The still life (sif) gene encodes a protein homologous to guanine nucleotide exchange factors, which convert Rho-like guanosine triphosphatases (GTPases) from a guanosine diphosphate-bound inactive state to a guanosine triphosphate-bound active state. The SIF proteins are found adjacent to the plasma membrane of synaptic terminals. Expression of a truncated SIF protein resulted in defects in neuronal morphology and induced membrane ruffling with altered actin localization in human KB cells. Thus, SIF proteins may regulate synaptic differentiation through the organization of the actin cytoskeleton by activating Rho-like GTPases.
Assuntos
Proteínas de Drosophila , Drosophila/metabolismo , Fatores de Troca do Nucleotídeo Guanina , Terminações Pré-Sinápticas/metabolismo , Proteínas rac de Ligação ao GTP , Actinas/metabolismo , Sequência de Aminoácidos , Animais , Axônios/fisiologia , Membrana Celular/ultraestrutura , Citoesqueleto/fisiologia , Citoesqueleto/ultraestrutura , DNA Complementar/genética , Drosophila/embriologia , Drosophila/genética , Embrião não Mamífero/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Expressão Gênica , Genes de Insetos , Humanos , Hibridização In Situ , Células KB , Dados de Sequência Molecular , Movimento , Mutação , Junção Neuromuscular/metabolismo , Transdução de SinaisRESUMO
To identify the target genes modulated by the myb gene product (Myb), a co-transfection assay with a Myb expression plasmid was performed. Both c-Myb and B-Myb, another member of the myb gene family, trans-activated the human c-myc promoter. DNAase I footprint analysis using the bacterially expressed c-Myb, identified multiple c-Myb binding sites in the c-myc promoter region. Deletion analysis of the c-myc promoter suggested that some number of Myb binding sites, not a specific Myb binding site, is important for the c-Myb-induced trans-activation of the c-myc promoter. Using the c-myc-chloramphenicol acetyltransferase (CAT) construct as a reporter in a co-transfection assay, the domains of c-Myb required for trans-activation were examined. The functional domains of c-Myb identified using the c-myc promoter were almost the same as those identified previously with the artificial target gene containing Myb binding sites, but unlike the case with the artificial target gene the N-terminal half of the previously identified negative regulatory domains and the C-terminal 136 amino acids were required for the maximal trans-activation of the c-myc promoter. These results indicate that there are some differences in the regulation of Myb-dependent trans-activation in different target genes.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Genes myc , Oncogenes , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/metabolismo , Transcrição Gênica , Ativação Transcricional , Animais , Sequência de Bases , Linhagem Celular , Cloranfenicol O-Acetiltransferase/genética , Cloranfenicol O-Acetiltransferase/metabolismo , Deleção Cromossômica , Proteínas de Ligação a DNA/genética , Humanos , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-myb , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência do Ácido Nucleico , TransfecçãoRESUMO
Primary murine fetal hemopoietic cells were transformed with a fusion protein consisting of the ligand-binding domain of the estrogen receptor and a carboxyl-terminally truncated c-Myb protein (ERMYB). The ERMYB-transformed hemopoietic cells exhibit an immature myeloid phenotype when grown in the presence of beta-estradiol. Upon removal of beta-estradiol, the ERMYB cells display increased adherence, decreased clonogenicity and differentiate to cells exhibiting granulocyte or macrophage morphology. The expression of the c-myc, c-kit, cdc2 and bcl-2 genes, which are putatively regulated by Myb, was investigated in ERMYB cells grown in the presence or absence of beta-estradiol. Neither c-myc nor cdc2 expression was down-regulated after removal of beta-estradiol demonstrating that differentiation is not a consequence of decreased transactivation of these genes by ERMYB. While bcl-2 expression was reduced by 50% in ERMYB cells grown in the absence of beta-estradiol, there was no increase in DNA laddering, suggesting that Myb was not protecting ERMYB cells from apoptosis. In contrast, a substantial (200-fold) decrease in c-kit mRNA level was observed following differentiation of ERMYB cells, and c-kit mRNA could be partially re-induced by the re-addition of beta-estradiol. Furthermore, a reporter construct containing the c-kit promoter was activated when cotransfected with a Myb expression vector, providing further evidence of a role for Myb in the regulation of c-kit.
Assuntos
Regulação para Baixo/genética , Genes cdc , Genes myc , Granulócitos/citologia , Macrófagos/citologia , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas/genética , Receptores de Estrogênio/genética , Proteínas Recombinantes de Fusão/metabolismo , Transativadores/genética , Animais , Apoptose , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Divisão Celular/efeitos dos fármacos , Linhagem Celular Transformada , DNA/biossíntese , DNA/química , Estradiol/deficiência , Estradiol/fisiologia , Feto , Vetores Genéticos/síntese química , Células-Tronco Hematopoéticas , Camundongos , Camundongos Endogâmicos CBA , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-myb , RNA Mensageiro/biossíntese , RNA Mensageiro/química , Receptores de Estrogênio/biossíntese , Receptores de Estrogênio/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Retroviridae/genética , Transativadores/biossíntese , Transativadores/metabolismoRESUMO
The myb gene family has three members, c-myb, A-myb, and B-myb. A-myb mRNA is mainly expressed in testis and peripheral blood leukocytes. A-Myb can activate transcription from the promoter containing Myb-binding sites in all cells examined. In addition to the two domains (a DNA-binding domain and a transcriptional activation domain), two negative regulatory domains have been identified in A-Myb. These results indicate that A-Myb functions as a transcriptional activator mainly in testis and peripheral blood cells, and the regulatory mechanism of A-Myb activity is similar to that of c-Myb.
Assuntos
Proteínas de Ciclo Celular , Regulação da Expressão Gênica/genética , Oncogenes/genética , Proteínas Proto-Oncogênicas/fisiologia , Transativadores/fisiologia , Ativação Transcricional/fisiologia , Células Cultivadas , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Masculino , Especificidade de Órgãos , Plasmídeos/metabolismo , Proteínas Proto-Oncogênicas/análise , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-myb , RNA Mensageiro/análise , Deleção de Sequência/fisiologia , Testículo/química , Transativadores/análise , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/genéticaRESUMO
Three prokaryotic termination signals, the Escherichia coli trp attenuator, lambda 4S RNA terminator, and lambda tR1 terminator, were examined as to their effects on transcription in vivo in HeLa cells. The trp attenuator inhibited the expression of downstream genes in an orientation-dependent manner, but both the lambda 4S RNA and lambda tR1 terminators did not. Furthermore, a point mutation of the attenuator that disrupted the secondary structure of mRNA abolished this inhibitory effect. These results suggest that an attenuator-like secondary structure is effective in inhibiting transcriptional elongation by RNA polymerase II.
Assuntos
RNA Polimerase II/genética , Regiões Terminadoras Genéticas , Bacteriófago lambda/genética , Sequência de Bases , Escherichia coli/genética , Células HeLa , Humanos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , RNA Mensageiro/química , RNA Mensageiro/genética , Transcrição GênicaRESUMO
Stabilizing factor, a 9 kDa protein, stabilizes and facilitates formation of the complex between mitochondrial ATP synthase and its intrinsic inhibitor protein. A clone containing the gene encoding the 9 kDa protein was selected from a yeast genomic library to determine the structure of its precursor protein. As deduced from the nucleotide sequence, the precursor of the yeast 9 kDa stabilizing factor contains 86 amino acid residues and has a molecular weight of 10,062. From the predicted sequence we infer that the stabilizing factor precursor contains a presequence of 23 amino acid residues at its amino terminus. We also used S1 mapping to determine the initiation site of transcription under glucose-repressed or derepressed conditions. These experiments suggest that transcription of this gene starts at three different sites and that only one of them is not affected by the presence of glucose.
Assuntos
Clonagem Molecular , Mitocôndrias/enzimologia , Proteínas Mitocondriais , Proteínas/genética , ATPases Translocadoras de Prótons/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Proteínas Adaptadoras de Transdução de Sinal , Genes , Genes Fúngicos/efeitos dos fármacos , Glucose/farmacologia , Conformação Proteica , Saccharomyces cerevisiae/enzimologia , Transcrição GênicaAssuntos
Proteínas Oncogênicas Virais/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Animais , Sequência de Bases , Elementos Facilitadores Genéticos , Escherichia coli/genética , Humanos , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-myb , Fatores de TranscriçãoRESUMO
Three members of the human myb gene family (c-myb, A-myb, and B-myb) encode transcriptional regulators that can bind to specific DNA sequences. High levels of c-myb expression are usually found in immature hemopoietic cells, but the B-myb is more commonly expressed in many types of cells. To understand the regulation of the activity of B-myb gene product (B-Myb), its functional domains were analyzed. Like c-Myb, B-Myb also has a transcriptional activation domain containing a cluster of acidic amino acids in the region downstream of the DNA-binding domain, which consists of three tandem repeats of 51-52 amino acids. In contrast to c-Myb, B-Myb does not contain a negative regulatory domain. Furthermore, the multiple nuclear localization signals are in at least two regions in the COOH-terminal half of B-Myb, and one of them is adjacent to a potential cdc2 kinase site. These results indicate that B-Myb contains DNA-binding and transcriptional activation domains similar to those of c-Myb, but a regulatory mechanism of B-Myb activity is quite different from that for c-Myb.
Assuntos
Proteínas de Ligação a DNA/biossíntese , Genes myc , Oncogenes , Proteínas Proto-Oncogênicas/biossíntese , Actinas/genética , Sequência de Aminoácidos , Animais , Linhagem Celular , Galinhas , Cloranfenicol O-Acetiltransferase/biossíntese , Cloranfenicol O-Acetiltransferase/metabolismo , Chlorocebus aethiops , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Rim , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-myb , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , Sequências Reguladoras de Ácido Nucleico , Sequências Repetitivas de Ácido Nucleico , Deleção de Sequência , Homologia de Sequência de Aminoácidos , TransfecçãoRESUMO
Morphogen gradients of secreted molecules play critical roles in the establishment of the spatial pattern of gene expression. During midgut development in Drosophila, secreted molecules of Decapentaplegic (Dpp) and Wingless (Wg) establish unique transcriptional regulation within target cells to specify the resultant cell types. Here we report the identification of a novel homeobox gene, defective proventriculus (dve), which is required for the midgut specification under the control of Dpp and Wg. In dve mutants, two distinct parts of the midgut, the proventriculus and middle midgut, are abnormally organized. The Wg signal regulates dve expression during proventriculus development. On the other hand, dve is a downstream target of Dpp in the middle midgut and defines the functional specificity of copper cells along with another Dpp target gene, labial. Thus, the dve gene acts under the two distinct extracellular signals at distant parts of the midgut primordia.
Assuntos
Proteínas de Drosophila , Drosophila/embriologia , Drosophila/genética , Regulação da Expressão Gênica no Desenvolvimento , Genes Homeobox , Genes de Insetos , Proteínas de Homeodomínio/genética , Proteínas de Insetos/genética , Proteínas Proto-Oncogênicas/genética , Sequência de Aminoácidos , Animais , Padronização Corporal/genética , Mapeamento Cromossômico , Sistema Digestório/embriologia , Embrião não Mamífero/fisiologia , Elementos Facilitadores Genéticos , Genes Letais , Proteínas de Homeodomínio/biossíntese , Proteínas de Homeodomínio/química , Proteínas de Insetos/biossíntese , Dados de Sequência Molecular , Proteínas Proto-Oncogênicas/biossíntese , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Proteína Wnt1RESUMO
Asymmetric divisions that produce two distinct cells play fundamental roles in generating different cell types during development. In the Drosophila central nervous system, neural stem cells called neuroblasts divide unequally into another neuroblast and a ganglion mother cell which is subsequently cleaved into neurons. Correct gene expression of ganglion mother cells requires the transcription factor Prospero. Here we demonstrate the asymmetric segregation of Prospero on neuroblast division. Prospero synthesized in neuroblasts is retained in the cytoplasm and at mitosis is exclusively partitioned to ganglion mother cells, in which it is translocated to the nucleus. Differential segregation of Prospero was also found in the endoderm. We have identified a region in Prospero that is responsible for this event. The region shares a common motif with Numb, which also shows unequal segregation. We propose that asymmetric segregation of transcription factors is an intrinsic mechanism for establishing asymmetry in gene expression between sibling cells.
Assuntos
Divisão Celular/fisiologia , Proteínas de Drosophila , Drosophila/embriologia , Proteínas de Homeodomínio/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição , Sequência de Aminoácidos , Animais , Sítios de Ligação , Ciclo Celular/fisiologia , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Drosophila/citologia , Escherichia coli/genética , Gânglios dos Invertebrados/citologia , Proteínas de Homeodomínio/genética , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/genética , Neurônios/citologia , Proteínas Nucleares/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , beta-Galactosidase/genéticaRESUMO
We report that the c-myb protein binds to another site, MBS-II, in the SV40 enhancer with low affinity. In co-transfection experiments with a c-myb expression plasmid, tandem repeats of the sequence containing the MBS-II site induced c-myb-dependent transcriptional repression. Results of mutational analyses of the sequence around the MBS-II site suggested that the c-myb protein represses transcription by competing with another trans-activator. These results indicate that c-myb protein can regulate transcription not only positively but also negatively.
Assuntos
Regulação Viral da Expressão Gênica , Proteínas Proto-Oncogênicas/fisiologia , Sequências Reguladoras de Ácido Nucleico , Proteínas Repressoras/fisiologia , Fatores de Transcrição/fisiologia , Mapeamento Cromossômico , Análise Mutacional de DNA , Proteínas de Ligação a DNA/fisiologia , Desoxirribonuclease I , Elementos Facilitadores Genéticos , Técnicas In Vitro , Plasmídeos , Proteínas Proto-Oncogênicas c-myb , Vírus 40 dos Símios/genética , Transcrição GênicaRESUMO
Structure and function of two domains of c-Myb were analyzed. We show that a leucine zipper structure is a component of the negative regulatory domain, because its disruption markedly increases both the transactivating and transforming capacities of c-Myb. Our results suggest that an inhibitor which suppresses transactivation binds to c-Myb through the leucine zipper, and that c-Myb can be oncogenically activated by mis-sense mutation. We also proposed a model, the "tryptophan cluster", for the structure of the Myb DNA-binding domain, in which the three tryptophans form a cluster in the hydrophobic core in each repeat. The results of NMR analysis of repeat 3 revealed that the conserved tryptophans play a key role to make the hydrophobic core.
Assuntos
Proteínas de Ligação a DNA/genética , Zíper de Leucina/genética , Proteínas Oncogênicas de Retroviridae/genética , Transcrição Gênica , Triptofano/análise , Animais , Linhagem Celular , Genes Reguladores , Células HeLa , Humanos , Espectroscopia de Ressonância Magnética , Modelos Químicos , Conformação de Ácido Nucleico , Proteínas Oncogênicas v-myb , Solubilidade , Triptofano/química , Água/químicaRESUMO
The c-myb protooncogene encodes a sequence-specific DNA-binding protein (c-Myb) that induces transcriptional activation or repression. We have identified three functional domains of the mouse c-Myb protein that are responsible for DNA binding, transcriptional activation, and negative regulation, respectively. In addition to the DNA-binding domain, which is located near the N terminus, an adjacent region (the transcriptional activation domain) containing about 80 amino acids was found to be essential for transcriptional activation. Deletion of a region spanning about 175 amino acids of the C-proximal portion increased transcriptional activation markedly, revealing that this domain normally represses activation. Differences between the transcriptional activation and repression functions of c-Myb and v-Myb are discussed in the light of these functional domains. Our results suggest that transcriptional activation may be involved in transformation by myb gene products.
Assuntos
Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica , Proteínas Proto-Oncogênicas/genética , Proto-Oncogenes , Transcrição Gênica , Actinas/genética , Sequência de Aminoácidos , Animais , Galinhas , Dados de Sequência Molecular , Plasmídeos , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-mybRESUMO
We present evidence that the mouse c-myb proto-oncogene encodes a transcriptional trans-activator. Trans-activation was assayed by cotransfection into CV1 monkey kidney cells of a c-myb cDNA expression plasmid together with a reporter plasmid carrying the chloramphenicol acetyltransferase (CAT) gene under the control of a test promoter and enhancer. Cotransfection with the c-myb cDNA plasmid caused a 20-fold stimulation of transcription from the promoter of the mouse alpha 2(I) collagen gene linked to tandem repeats of the simian virus 40 (SV40) enhancer element. Using different promoters in combination with varying numbers of repeats of the SV40 enhancer element, it was shown that tandem repeats of the SV40 enhancer mediated the c-myb-induced activation of transcription. These results show that the mouse c-myb gene product either is itself or induces, an activator of transcription that recognizes specific sequences in the SV40 enhancer.
Assuntos
Regulação da Expressão Gênica , Proto-Oncogenes , Animais , Southern Blotting , Linhagem Celular , Elementos Facilitadores Genéticos , Genes , Camundongos , Plasmídeos , Regiões Promotoras Genéticas , Vírus 40 dos Símios/genética , TransfecçãoRESUMO
The proto-oncogene c-myb encodes a nuclear protein which binds to DNA. Here we find that bacterially synthesized c-myb protein binds to one site of the simian virus 40 enhancer. The c-myb protein purified from the human T-cell line, Molt4, was also shown to recognize the same sequence. In co-transfection experiments with a c-myb expression plasmid, tandem repeats of a c-myb-binding sequence were shown to function as a c-myb-dependent enhancer. These results indicate the c-myb protein is a simian virus 40 enhancer-binding protein that can positively regulate transcription.
Assuntos
Elementos Facilitadores Genéticos , Proteínas Proto-Oncogênicas/metabolismo , Vírus 40 dos Símios/genética , Transcrição Gênica , Animais , Sequência de Bases , Linhagem Celular , Humanos , Camundongos , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Plasmídeos , Proteínas Tirosina Quinases/metabolismo , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas/isolamento & purificação , Proteínas Proto-Oncogênicas c-myb , Proto-Oncogenes , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , TransfecçãoRESUMO
Three members of the myb gene family have been identified in human cDNA libraries c-myb, A-myb, and B-myb. We compared the DNA binding properties of the B-myb and c-myb proteins (B-MYB and c-MYB) using bacterially synthesized B-MYB and c-MYB in DNase I footprinting. B-MYB bound to most of the c-MYB binding sites examined, including the c-MYB binding site, MBS-I, in the simian virus (SV) 40 enhancer, in which the most frequent sequence was CCTAACTG. The MBS-I site was an enhancer element dependent on B-MYB and c-MYB in a co-transfection assay that used the B-myb or c-myb expression plasmid. Some sites in the SV40 genome, including the MBS-BI site, had high affinity with B-MYB but little or no affinity with c-MYB, in which the most frequent sequence was AGAAANPyrG. The MBS-BI site was an enhancer element dependent on B-MYB and a very weakly dependent on c-MYB. Our results showed that B-MYB is a transcriptional activator, like c-MYB, and that although B-MYB and c-MYB have similar sequence specificity for DNA binding some sequences were recognized by B-MYB preferentially.
Assuntos
Proteínas de Ciclo Celular , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transativadores , Fatores de Transcrição/metabolismo , Transcrição Gênica , Sequência de Bases , Sítios de Ligação , Proteínas de Ligação a DNA/genética , Desoxirribonuclease I , Elementos Facilitadores Genéticos/genética , Escherichia coli/genética , Genes Virais , Globinas/genética , Humanos , Dados de Sequência Molecular , Plasmídeos , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-myb , Proteínas Recombinantes/metabolismo , Vírus 40 dos Símios/genética , Fatores de Transcrição/genética , TransfecçãoRESUMO
In the DNA-binding domain of the c-myb protooncogene product (c-Myb) which consists of three repeats of 51-52 amino acids, there are 3 perfectly conserved tryptophans in each repeat. Site-directed mutagenesis of these tryptophans showed that any single or multiple mutations of tryptophan to hydrophilic residues or alanine abolished or greatly reduced the sequence-specific DNA-binding activity, but mutations to hydrophobic amino acids retained considerable activity. Raman spectroscopic study showed that these tryptophans were buried in the protein core. These 3 tryptophans are proposed to form a cluster in the hydrophobic core in each repeat. This hypothetical structure is referred to as the "tryptophan cluster," and it may represent a characteristic property of a group of DNA-binding proteins including the myb- and ets-related proteins.
Assuntos
DNA/metabolismo , Proteínas Proto-Oncogênicas/química , Triptofano , Animais , Sítios de Ligação , Escherichia coli/genética , Camundongos , Estrutura Molecular , Mutagênese Sítio-Dirigida , Fosforilação , Plasmídeos , Conformação Proteica , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-myb , Sequências Repetitivas de Ácido Nucleico , Análise Espectral Raman , Transcrição Gênica , TransfecçãoRESUMO
The wild-type p53 protein suppresses transformation, but certain missense mutants of p53 can transform cells. Although the wild-type p53 protein contains a transcriptional activation domain, no p53-responsive element has been identified. Here, we identified the p53-responsive element within the Tax-responsive element [21-base-pair (bp) enhancer] of human T-cell leukemia virus type I. Mutation analysis of the 21-bp enhancer indicated that the 16-bp sequence containing the cAMP-responsive element and its surrounding sequence was responsible for p53-induced transactivation. This 16-bp sequence was demonstrated to bind specifically to wild-type human p53 protein in vitro. Using a series of deletion mutants of p53, we showed that almost the entire region of p53 is needed for the transactivating capacity. Furthermore, the transforming mutants of p53 were unable to act as transcriptional activators. The p53-responsive element identified here should be useful to analyze the mechanism by which p53 regulates expression of a set of genes with a negative effect on cellular growth.