RESUMO
We have performed a systematic structure-function analysis of Saccharomyces cerevisiae TAF25, an evolutionarily conserved, single-copy essential gene which encodes the 206-amino-acid TAF25p protein. TAF25p is an integral subunit of both the 15-subunit general transcription factor TFIID and the multisubunit, chromatin-acetylating transcriptional coactivator SAGA. We used hydroxylamine mutagenesis, targeted deletion, alanine-scanning mutagenesis, high-copy suppression methods, and two-hybrid screening to dissect TAF25. Temperature-sensitive mutant strains generated were used for coimmunoprecipitation and transcription analyses to define the in vivo functions of TAF25p. The results of these analyses show that TAF25p is comprised of multiple mutable elements which contribute importantly to RNA polymerase II-mediated mRNA gene transcription.
Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/fisiologia , Proteínas Fúngicas/química , Proteínas Fúngicas/fisiologia , Regulação Fúngica da Expressão Gênica , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Fatores Associados à Proteína de Ligação a TATA , Fatores de Transcrição TFII/química , Sequência de Aminoácidos , Sequência Conservada , Proteínas de Ligação a DNA/genética , Proteínas Fúngicas/genética , Genes Fúngicos , Substâncias Macromoleculares , Dados de Sequência Molecular , Mutação , Subunidades Proteicas , RNA Polimerase II/fisiologia , RNA Fúngico/biossíntese , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade , Transativadores/química , Transativadores/metabolismo , Fator de Transcrição TFIID , Técnicas do Sistema de Duplo-HíbridoRESUMO
The yeast transcriptional activator ADR1, which is required for ADH2 and other genes' expression, contains four transactivation domains (TADs). While previous studies have shown that these TADs act through GCN5 and ADA2, and presumably TFIIB, other factors are likely to be involved in ADR1 function. In this study, we addressed the question of whether TFIID is also required for ADR1 action. In vitro binding studies indicated that TADI of ADR1 was able to retain TAFII90 from yeast extracts and TADII could retain TBP and TAFII130/145. TADIV, however, was capable of retaining multiple TAFIIs, suggesting that TADIV was binding TFIID from yeast whole-cell extracts. The ability of TADIV truncation derivatives to interact with TFIID correlated with their transcription activation potential in vivo. In addition, the ability of LexA-ADR1-TADIV to activate transcription in vivo was compromised by a mutation in TAFII130/145. ADR1 was found to associate in vivo with TFIID in that immunoprecipitation of either TAFII90 or TBP from yeast whole-cell extracts specifically coimmunoprecipitated ADR1. Most importantly, depletion of TAFII90 from yeast cells dramatically reduced ADH2 derepression. These results indicate that ADR1 physically associates with TFIID and that its ability to activate transcription requires an intact TFIID complex.
Assuntos
Proteínas de Ciclo Celular , Proteínas de Ligação a DNA/metabolismo , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Fatores Associados à Proteína de Ligação a TATA , Fatores de Transcrição TFII/metabolismo , Fatores de Transcrição/metabolismo , Ativação Transcricional , Álcool Desidrogenase/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Proteínas de Ligação a DNA/genética , Proteínas Fúngicas/genética , Testes de Precipitina , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/metabolismo , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Proteína de Ligação a TATA-Box , Fator de Transcrição TFIID , Fatores de Transcrição/genética , Fatores de Transcrição TFII/genéticaRESUMO
The Gcn4p activation domain contains seven clusters of hydrophobic residues that make additive contributions to transcriptional activation in vivo. We observed efficient binding of a glutathione S-transferase (GST)-Gcn4p fusion protein to components of three different coactivator complexes in Saccharomyces cerevisiae cell extracts, including subunits of transcription factor IID (TFIID) (yeast TAFII20 [yTAFII20], yTAFII60, and yTAFII90), the holoenzyme mediator (Srb2p, Srb4p, and Srb7p), and the Adap-Gcn5p complex (Ada2p and Ada3p). The binding to these coactivator subunits was completely dependent on the hydrophobic clusters in the Gcn4p activation domain. Alanine substitutions in single clusters led to moderate reductions in binding, double-cluster substitutions generally led to greater reductions in binding than the corresponding single-cluster mutations, and mutations in four or more clusters reduced binding to all of the coactivator proteins to background levels. The additive effects of these mutations on binding of coactivator proteins correlated with their cumulative effects on transcriptional activation by Gcn4p in vivo, particularly with Ada3p, suggesting that recruitment of these coactivator complexes to the promoter is a cardinal function of the Gcn4p activation domain. As judged by immunoprecipitation analysis, components of the mediator were not associated with constituents of TFIID and Adap-Gcn5p in the extracts, implying that GST-Gcn4p interacted with the mediator independently of these other coactivators. Unexpectedly, a proportion of Ada2p coimmunoprecipitated with yTAFII90, and the yTAFII20, -60, and -90 proteins were coimmunoprecipitated with Ada3p, revealing a stable interaction between components of TFIID and the Adap-Gcn5p complex. Because GST-Gcn4p did not bind specifically to highly purified TFIID, Gcn4p may interact with TFIID via the Adap-Gcn5p complex or some other adapter proteins. The ability of Gcn4p to interact with several distinct coactivator complexes that are physically and genetically linked to TATA box-binding protein can provide an explanation for the observation that yTAFII proteins are dispensable for activation by Gcn4p in vivo.
Assuntos
Coenzimas/metabolismo , Quinases Ciclina-Dependentes , Proteínas de Ligação a DNA , Proteínas Fúngicas/metabolismo , Proteínas Quinases/metabolismo , RNA Polimerase II/metabolismo , Proteínas de Saccharomyces cerevisiae , Transativadores/metabolismo , Fatores de Transcrição TFII/metabolismo , Fatores de Transcrição/metabolismo , Animais , Sítios de Ligação , Extratos Celulares , Coenzimas/genética , Quinase 8 Dependente de Ciclina , Proteínas Fúngicas/genética , Complexo Mediador , Camundongos , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , RNA Polimerase II/genética , Coelhos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transativadores/genética , Fator de Transcrição TFIID , Fatores de Transcrição/genética , Fatores de Transcrição TFII/genéticaRESUMO
Three cats were diagnosed as hyperthyroid based on clinical signs, historical findings, laboratory abnormalities, and basal serum thyroxine (T4) concentrations, and/or nuclear thyroid scans. Additionally, a presumptive diagnosis of thyroid carcinoma with pulmonary metastasis was made in each cat based on radiographic or scintigraphic evaluation. All three cats had solitary pulmonary nodules 1.5 to 2 cm in diameter on survey thoracic radiographs; one cat also had chylous pleural effusion and pulmonary lobar consolidation. Focal pulmonary accumulation of sodium pertechnetate (99mTcO4-) and/or radioiodine (131I) corresponding to radiographic lesions were seen in all cats. Two cats were treated with single ablative doses (1111 to 1480 MBq) of 131I; the remaining cat was euthanatized. One of the treated cats died 8 days later; the other cat was euthanatized 22 weeks following treatment. Histopathologic examination of tissue obtained at necropsy confirmed metastatic thyroid carcinoma in one cat and bronchogenic adenocarcinoma in two cats. Our findings indicate that increased radionuclide uptake in focal pulmonary lesions and cytologic evaluation of tissue obtained by fine-needle aspiration are not specific for thyroid tissue.
Assuntos
Adenocarcinoma/veterinária , Carcinoma/veterinária , Doenças do Gato/diagnóstico , Hipertireoidismo/veterinária , Neoplasias Pulmonares/veterinária , Neoplasias da Glândula Tireoide/veterinária , Adenocarcinoma/diagnóstico , Adenocarcinoma/radioterapia , Adenocarcinoma/secundário , Animais , Carcinoma/diagnóstico , Carcinoma/radioterapia , Carcinoma/secundário , Doenças do Gato/radioterapia , Gatos , Feminino , Hipertireoidismo/diagnóstico , Radioisótopos do Iodo/uso terapêutico , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/radioterapia , Neoplasias Pulmonares/secundário , Pertecnetato Tc 99m de Sódio , Neoplasias da Glândula Tireoide/diagnóstico , Neoplasias da Glândula Tireoide/radioterapiaRESUMO
A polymerase chain reaction (PCR)-based technique is described which allows for the determination of library plasmid insert DNA sequence directly and rapidly from intact yeast cells. Yeast spheroplasts are used to template a PCR reaction to amplify the insert sequence. This PCR product is then purified and its sequence directly determined using thermal cycle sequencing. Readable sequence can reproducibly be obtained from multiple yeast colonies in just two days. Uses of this technique in yeast two-hybrid screening as well as other types of yeast library screens are discussed.
Assuntos
Genes Fúngicos/genética , Biblioteca Genômica , Plasmídeos/genética , Análise de Sequência de DNA/métodos , Leveduras/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Vetores Genéticos/genética , Reação em Cadeia da Polimerase/métodos , Esferoplastos/citologia , Esferoplastos/genética , Moldes Genéticos , Fatores de Tempo , Leveduras/citologia , Leveduras/crescimento & desenvolvimentoRESUMO
We have studied the competitive binding of histones and the Rous sarcoma virus internal enhancer binding factor (IBF) factor (which recent studies indicate is almost certainly cEBP beta). We find that histones and IBF are incapable of forming a ternary complex with a 159-base pair (bp) fragment of DNA containing a single IBF binding site and that histones and factor are mutually exclusive in binding. We have analyzed the various physical parameters of binding, in an attempt to understand how the factor might establish an exclusive binding in the cell. The stability of the nucleosome and the factor-DNA complex have been determined, and in addition a minimum value for the affinity of the histone octamer has been computed. We find that in simple competition the IBF can successfully compete, only if the substrate DNA is shorter than 140 bp. The relevance of these results is discussed in terms of a kinetic model for successful factor competition during the replication of the factor binding site in the cell.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Histonas/metabolismo , Proteínas Nucleares/metabolismo , Nucleossomos/metabolismo , Fatores de Transcrição/metabolismo , Vírus do Sarcoma Aviário , Ligação Competitiva , Proteínas Estimuladoras de Ligação a CCAAT , Linhagem Celular , Desoxirribonucleoproteínas/química , Elementos Facilitadores Genéticos , Humanos , Técnicas In Vitro , Cinética , Proteínas Virais/metabolismoRESUMO
We demonstrate, utilizing a temperature conditional mutant allele of the gene encoding TAF25p, that this non-histone-like TBP-associated factor, which is shared between the TFIID and SAGA complexes, is required for bulk mRNA gene transcription by RNA polymerase II in vivo. Immunoblotting experiments indicate that at the restrictive temperature, inactivation of TAF25p function results in a reduction of the levels of numerous TFIID and SAGA subunits, indicating its loss of function, like the histone-like TAFs, causes degradation of the constituents of these two multisubunit complexes. These data suggest that TAF25p plays a key structural role in maintaining TFIID and SAGA complex integrity. This is the first demonstration that a non-histone-like TAF is required for continuous, high level RNA polymerase II-mediated mRNA gene transcription in living yeast cells.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Proteínas Fúngicas/fisiologia , RNA Mensageiro/genética , Proteínas de Saccharomyces cerevisiae , Fatores Associados à Proteína de Ligação a TATA , Transcrição Gênica , Alelos , Clonagem Molecular , Proteínas de Ligação a DNA/genética , Escherichia coli , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Hidroxilamina/farmacologia , Temperatura , Fator de Transcrição TFIID , Fatores de Transcrição TFII/química , Fatores de Transcrição TFII/metabolismo , Leveduras/genética , Leveduras/metabolismoRESUMO
Effects of retained fetal membranes on subsequent productive and reproductive performance were measured in a single Florida Holstein dairy herd (n = 216 retained fetal membranes and 208 normal cases). Mean performance was peak daily milk yield, 31.9 kg; 305-d milk yield, 6317 kg; days to first service, 60; days open, 115; and services per conception, 2.3. Yield was unaffected by retained fetal membranes; however, retention resulted in an increase of 4 d to first service, 19 d open, and .2 services per conception. Detrimental effects of parturitions in hot weather (July through September) compared with cool (December through February) were peak daily milk yield, 5.5 kg; 305-d milk yield, 512 kg; days to first service, 3; days open 39; and services per conception, .9. Interactions could not be detected between diagnosis (retained or normal) and season for any response. Sex of calf effects could not be detected for any response. Continued research to reduce occurrence of retained fetal membranes or to obviate its effects should lead to improved reproductive performance.
Assuntos
Membranas Extraembrionárias/fisiologia , Leite/metabolismo , Complicações do Trabalho de Parto/veterinária , Prenhez , Animais , Bovinos , Feminino , Lactação , Gravidez , Estações do AnoRESUMO
Conventional hyperthermia treatment of superficial tumors in the oral cavity is troublesome due to difficulty in accessing the lesion. A new hyperthermia technique employing near-infrared radiation delivered through a flexible silica optical fiber is described. The system consisted of an Nd:YAG laser for tissue heating, a He-Ne laser for aiming beam, a computer-controlled optical shutter, an interstitial thermometer, computer, and a printer. A 3-m-long 600-microns silica fiber delivered laser energy to the tumor via surface illumination. Using the aiming beam, the spot size was adjusted to include 5 mm of surrounding normal tissue. A thermocouple implanted in the tumor base provided temperature feedback to maintain desired hyperthermic temperature within the lesion. Three spontaneously occurring canine (two squamous cell carcinomas on the gum, one pigmented melanoma on the hard palate) and one feline tumor (squamous cell carcinoma on the nose) have been treated with Nd:YAG laser hyperthermia. Hyperthermia was delivered at 43.5 degrees C for 1 h. All animals received standard radiation treatment prior to hyperthermia. Nd:YAG laser hyperthermia allowed effective and efficient delivery of heat to veterinary nasal and oral lesions otherwise not treatable with conventional heating techniques.
Assuntos
Doenças do Gato/terapia , Doenças do Cão/terapia , Hipertermia Induzida/métodos , Terapia a Laser , Neoplasias Bucais/veterinária , Doenças Nasais/veterinária , Silicatos de Alumínio , Animais , Carcinoma de Células Escamosas/radioterapia , Carcinoma de Células Escamosas/terapia , Doenças do Gato/radioterapia , Gatos , Terapia Combinada , Doenças do Cão/radioterapia , Cães , Desenho de Equipamento , Neoplasias Gengivais/radioterapia , Neoplasias Gengivais/terapia , Hipertermia Induzida/instrumentação , Melanoma/radioterapia , Melanoma/terapia , Neoplasias Bucais/radioterapia , Neoplasias Bucais/terapia , Neodímio , Doenças Nasais/radioterapia , Doenças Nasais/terapia , Neoplasias Palatinas/radioterapia , Neoplasias Palatinas/terapia , Terapia Assistida por Computador , ÍtrioRESUMO
In this report we describe the cloning and initial characterization of TAF40, a gene that encodes a yeast TATA-binding protein-associated factor (yTAF) of Mr = approximately 40,000. This gene has many similarities to other yTAFs described thus far in that it is present at a single copy per haploid genome, it is essential for viability, and the deduced protein sequence of yTAF40 exhibits similarity to previously described human and Drosophila TAFIIs. Immunological studies confirm that yTAF40 protein is a subunit of a large multiprotein TATA-binding protein-TAF complex that contains a subset of the total number of the yTAFs present in yeast cell extracts. Transcription reactions performed using yeast whole cell extracts reveal that of the three nuclear RNA polymerases only RNA polymerase II function is abrogated when yTAF40 and associated proteins are immunodepleted from solution, indicating that the functionality of the multiprotein complex containing yTAF40 is RNA polymerase II-specific. By these criteria yTAF40 appears to encode a bona fide RNA polymerase II-specific TAF, and thus the protein that it encodes has been termed yTAFII40.
Assuntos
Proteínas de Ligação a DNA/genética , Genes Fúngicos , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Fatores Associados à Proteína de Ligação a TATA , Fatores de Transcrição TFII/genética , Fatores de Transcrição/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Proteínas de Ligação a DNA/química , Drosophila , Humanos , Dados de Sequência Molecular , RNA Polimerase II/metabolismo , Alinhamento de Sequência , Fator de Transcrição TFIID , Fatores de Transcrição/química , Fatores de Transcrição TFII/químicaRESUMO
We have used a combination of fluorescence anisotropy spectroscopy and fluorescence-based native gel electrophoresis methods to examine the effects of the transcription factor IID-specific subunit TAF130p (TAF145p) upon the TATA box DNA binding properties of TATA box-binding protein (TBP). Purified full-length recombinant TAF130p decreases TBP-TATA DNA complex formation at equilibrium by competing directly with DNA for binding to TBP. Interestingly, we have found that full-length TAF130p is capable of binding multiple molecules of TBP with nanomolar binding affinity. The biological implications of these findings are discussed.
Assuntos
Proteínas de Ligação a DNA/metabolismo , TATA Box , Fatores Associados à Proteína de Ligação a TATA , Fator de Transcrição TFIID , Fatores de Transcrição/metabolismo , DNA/metabolismo , Proteínas de Ligação a DNA/isolamento & purificação , Eletroforese/métodos , Polarização de Fluorescência , Substâncias Macromoleculares , Ligação Proteica , Subunidades Proteicas , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Espectrometria de Fluorescência , Proteína de Ligação a TATA-Box , Fatores de Transcrição/isolamento & purificaçãoRESUMO
We describe the cloning and analysis of TAF25, a previously uncharacterized yeast gene that encodes a yeast TATA-binding protein-associated factor or yTAF of Mr = 25,000. The gene encoding yTAF25 is a single copy essential gene, and the protein sequence deduced from TAF25 exhibits sequence similarity to a metazoan hTAFII. The results from immunological studies confirm that yTAF25 is a subunit of a large multiprotein TATA-binding protein-yeast TATA-binding protein-associated factor complex that contains a subset of the total number of the yTAFs present in yeast cell extracts. Both genetic and biochemical analyses demonstrate that yTAF25 can interact directly with itself. Transcriptional data show that the activity of the multiprotein complex containing yTAF25 is RNA polymerase II-specific, thus indicating that TAF25 encodes a bona fide yeast RNA polymerase II TAF. Hence the protein encoded by TAF25 has been termed yTAFII25.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genes Fúngicos , RNA Polimerase II/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Fatores Associados à Proteína de Ligação a TATA , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais , Sequência de Bases , Clonagem Molecular , DNA Fúngico/genética , Proteínas de Ligação a DNA/imunologia , Proteínas Fúngicas/imunologia , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Proteína de Ligação a TATA-Box , Fator de Transcrição TFIIDRESUMO
The heating potential of a closed loop interstitial hyperthermia system employing 1,064 nm laser light in conjunction with a long frosted contact probe was investigated in hind limb muscle of anesthetized dogs. The laser system was an Nd:YAG surgical laser modified with a single channel thermometry unit, a computer, a printer, and a computer-controlled laser exposure shutter. The long frosted laser probe was implanted into the muscle, and 3.12-5.00 Watts of laser power was delivered interstitially. Temperature distribution was measured in three dimensions around the frosted probe. The temperature distributions generated by this technique were satisfactory for producing desired hyperthermia temperatures in an approximately 3.5 cm3 cylindrical tissue volume. A multiple laser delivery system is needed to induce interstitial hyperthermia in large tumors. A significant potential for the long frosted contact probe may be its use in combining interstitial hyperthermia and interstitial photodynamic therapy. Using this technique, both modalities may be delivered while employing the same treatment setup.