RESUMEN
Genetic testing for non-specific intellectual disability (ID) presents challenges in daily clinical practice. Historically, the focus of the genetic elucidation of non-specific ID has been on genes on the X chromosome, and recent research has brought attention to the growing contribution of autosomal genes. In addition, next-generation sequencing (NGS) has greatly improved the ability to simultaneously analyze multiple genetic loci, making large panel testing a practical approach to testing for non-specific ID. We performed NGS analysis of a total of 90 genes implicated in non-specific ID. The 90 genes included 56 X-linked genes and 34 autosomal genes. Pathogenic variants were identified in 11 of 52 (21%) patient samples. Nine of the eleven cases harbored mutations in autosomal genes including AP4B1, STXB1, SYNGAP1, TCF4 and UBE3A. Our mutation-positive cases provide further evidence supporting the prevalence of autosomal mutations in patients referred for non-specific ID testing and the utility of their inclusion in multi-gene panel analysis.
RESUMEN
Storage of dried blood spots (DBS) on high-density FTA(®) plates could constitute an appealing alternative to frozen storage. However, it remains controversial whether DBS are suitable for high-resolution sequencing of human leukocyte antigen (HLA) alleles. Therefore, we extracted DNA from DBS that had been stored for up to 4 years, using six different methods. We identified those extraction methods that recovered sufficient high-quality DNA for reliable high-resolution HLA sequencing. Further, we confirmed that frozen whole blood samples that had been stored for several years can be transferred to filter paper without compromising HLA genotyping upon extraction. Concluding, DNA derived from high-density FTA(®) plates is suitable for high-resolution HLA sequencing, provided that appropriate extraction protocols are employed.
Asunto(s)
ADN/aislamiento & purificación , Pruebas con Sangre Seca/métodos , Antígenos HLA/química , Prueba de Histocompatibilidad/normas , Alelos , ADN/normas , Pruebas con Sangre Seca/instrumentación , Antígenos HLA/genética , Prueba de Histocompatibilidad/instrumentación , Prueba de Histocompatibilidad/métodos , Humanos , Control de Calidad , Análisis de Secuencia de ADN , Manejo de EspecímenesRESUMEN
Primary Autosomal Recessive Microcephaly (MCPH) is characterized by congenital microcephaly usually without additional clinical findings. The most common gene implicated in MCPH is ASPM and a large percentage of mutations described have been homozygous and in consanguineous families primarily of East Asian and Middle Eastern origin. ASPM sequencing was performed on 400 patients between the years 2009 and 2012. Seventy of the patient samples were also analyzed for copy number changes in the ASPM gene. Forty protein truncating mutations, including 29 novel mutations, were identified in 39 patients with MCPH. Approximately one third of patients were compound heterozygotes, indicative of non-consanguinity in these patients. In addition, 46 non-synonymous variants were identified and interpreted as variants of uncertain significance. No deletion/duplication in ASPM was identified in the patients analyzed. A wide ethnic distribution was observed, including the first reported patients with ASPM-related MCPH of Hispanic descent. Clinical information was collected for 26 of the ASPM-positive patients and 41 of the ASPM-negative patients. As more individuals are identified with MCPH, we anticipate that we will continue to identify ASPM mutation-positive patients from all ethnic origins supporting the occurrence of this genetic condition beyond that of consanguineous families of certain ethnic populations.
Asunto(s)
Microcefalia/genética , Mutación , Proteínas del Tejido Nervioso/genética , Patología Molecular , Preescolar , Consanguinidad , Etnicidad/genética , Genes Recesivos , Heterocigoto , Homocigoto , Humanos , Microcefalia/etiologíaRESUMEN
OBJECTIVE: The purpose of this study was to evaluate the effectiveness of glazing, two zirconia, and two lithium disilicate polishing systems on surface roughness of a CAD/CAM lithium disilicate and virgilite ceramic with atomic force microscopy (AFM) and visual assessment performed by dental students and faculty. METHODS AND MATERIALS: Sixty maxillary right central incisor crowns made of a novel chairside CAD/CAM lithium disilicate and virgilite (CEREC Tessera) were milled for glazing and polishing. The crowns were divided into six groups: no polishing/glazing provided (NoP/G); glazed (GZ); glazed and polished with Brasseler Dialite LD Lithium Disilicate (DiLD); glazed and polished with Meisinger Luster Lithium Disilicate (LuLD); glazed and polished with Brasseler Dialite ZR Zirconia (DiZR); and glazed and polished with Meisinger Luster Zirconia (LuZR). Surfaces were scanned with AFM to measure roughness (Ra) and root mean square roughness (Rq) and generate micrographs. Crowns were visually assessed by 10 dental students and 10 dental school faculty members to determine clinical acceptableness. RESULTS: Glazing and all polishing kits significantly reduced Ra and Rq compared to no polishing/glazing. No significant Ra differences were found between glazing and all polishing kits (p>0.05). DiZR significantly reduced Rq compared to other groups (p<0.05). Visual assessment showed that GZ, LuLD, and DiZR were the most clinically acceptable crowns. CONCLUSION: Polishing and glazing considerably improve the surface smoothness of maxillary central incisor crowns fabricated out of a chairside CAD/CAM lithium disilicate and virgilite ceramic. Altogether, zirconia polishing systems provided smoother and more clinically acceptable surfaces than the lithium disilicate kits.
Asunto(s)
Pulido Dental , Porcelana Dental , Humanos , Ensayo de Materiales , Pulido Dental/métodos , Propiedades de Superficie , Cerámica , Coronas , Diseño Asistido por Computadora , Microscopía Electrónica de RastreoRESUMEN
Staphylococcal colonization of implants is a serious complication of orthopaedic surgery. Anti-infectious modification of implant surfaces may serve to prevent bacterial colonization. The authors set out to develop an in vitro test system for the analysis of prevention of biofilm formation by Staphylococcus epidermidis and Staphylococcus aureus on implant materials. Biofilm growth was monitored over 10 days on titanium disks in order to develop appropriate test parameters. Bacterial cell counts following ultrasonic treatment of the colonized samples were compared with scanning electron microscope images of the specimens. Copper ion containing surfaces (ie copper [Cu] and inter-metallic Ti-Cu films) were used for growth inhibition assays: copper ion releasing specimens led to reduced bacterial numbers in biofilms and decreased bacterial persistence in the model used. The assay used represents an inexpensive and quick in vitro screen for the antibacterial effects of novel implant surface materials.
Asunto(s)
Antibacterianos/farmacología , Biopelículas/efectos de los fármacos , Cobre/farmacología , Prótesis e Implantes/microbiología , Infecciones Relacionadas con Prótesis/prevención & control , Staphylococcus aureus/efectos de los fármacos , Staphylococcus epidermidis/efectos de los fármacos , Titanio/farmacología , Biopelículas/crecimiento & desarrollo , Materiales Biocompatibles Revestidos/farmacología , Medios de Cultivo , Pruebas de Sensibilidad Microbiana/métodos , Infecciones Relacionadas con Prótesis/microbiología , Staphylococcus aureus/crecimiento & desarrollo , Staphylococcus epidermidis/crecimiento & desarrolloRESUMEN
Implant infections remain feared and severe complications after total hip replacement. An even higher rate of periprosthetic infections can be observed after revision surgery in comparison to primary total hip replacement. An additional threat for patients with artificial joints arises from the fact that bacteria resistant to a multitude of antibiotics are encountered with increasing frequency in the hospital setting.Among these the enterobacteria producing extended spectrum ß-lactamases (ESBL) are the second most frequent group of multiresistant pathogens. ESBLs are enzymes which possess the ability to hydrolyse third and fourth generation cephalosporins resulting in a distinctive resistance against these antibiotics. Even though ESBLs were first described in the early 1980's and now represent pathogens of utmost importance in intensive care units, they have been hardly considered in orthopedic and trauma surgery.In the present manuscript we provide an overview of the epidemiology and diagnostics of ESBL-expressing bacteria and demonstrate the difficulties in managing implant-associated infections with resistant bacteria. Furthermore, we emphasize the importance of recognizing ESBL-positive bacteria as increasingly important pathogens which require special precautions and treatment. Clinical evaluations suggest that ESBLs in orthopedic and trauma surgery are not a rare phenomenon any more.
Asunto(s)
Artroplastia de Reemplazo de Cadera/efectos adversos , Infecciones por Enterobacteriaceae/etiología , Infecciones por Enterobacteriaceae/prevención & control , Prótesis de Cadera/efectos adversos , Infecciones por Enterobacteriaceae/metabolismo , Humanos , beta-Lactamasas/biosíntesisRESUMEN
BACKGROUND: Erectile dysfunction is a condition that shows a continuously growing prevalence in the male population. The penis prosthesis implant (PPI) qualifies as an effective form of therapy. OBJECTIVES: The aim of this study was to analyze the sexual satisfaction rate and quality of life in patients who had suffered from erectile dysfunction and who were treated with a penile prosthesis. The patient's partners were also surveyed. METHODS: We collected data from patients who underwent surgery in the Center of Excellence for Penile Implants, University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany. Questionnaires with validated scores (EDITS, EDITS Partner) were sent to all patients and their partners via mail. RESULTS: The satisfaction rate in this study was high which shows that the patients and partners are pleased, and the high sexual satisfaction rate led to a higher quality of life. CONCLUSION: The penile prosthesis implantation as a last option of therapy for erectile dysfunction is useful and brings more than adequate results.
Asunto(s)
Disfunción Eréctil , Prótesis de Pene , Disfunción Eréctil/epidemiología , Alemania , Humanos , Masculino , Orgasmo , Satisfacción del Paciente , Calidad de Vida , Encuestas y CuestionariosRESUMEN
Batterham et al. report that the gut peptide hormone PYY3-36 decreases food intake and body-weight gain in rodents, a discovery that has been heralded as potentially offering a new therapy for obesity. However, we have been unable to replicate their results. Although the reasons for this discrepancy remain undetermined, an effective anti-obesity drug ultimately must produce its effects across a range of situations. The fact that the findings of Batterham et al. cannot easily be replicated calls into question the potential value of an anti-obesity approach that is based on administration of PYY3-36.
Asunto(s)
Depresores del Apetito/farmacología , Regulación del Apetito/efectos de los fármacos , Conducta Alimentaria/efectos de los fármacos , Péptido YY/farmacología , Animales , Animales Endogámicos , Apetito/efectos de los fármacos , Apetito/fisiología , Depresores del Apetito/uso terapéutico , Conducta Animal/efectos de los fármacos , Peso Corporal/efectos de los fármacos , Ambiente , Humanos , Metaanálisis como Asunto , Ratones , Obesidad/tratamiento farmacológico , Fragmentos de Péptidos , Péptido YY/administración & dosificación , Péptido YY/sangre , Péptido YY/uso terapéutico , Ratas , Reproducibilidad de los Resultados , Estrés Fisiológico/complicaciones , Estrés Fisiológico/fisiopatologíaRESUMEN
We have evaluated the data of patients who underwent ectopic high submuscular reservoir placement during implantation of inflatable penile prostheses and compared them to those of patients who underwent to traditional reservoir placement in the space of Retzius (SR). The main focus was on evaluating complications and patient satisfaction rates in both methods of RP. One hundred and forty-two patients underwent implantation of the Coloplast Titan OTR prosthesis with exclusive use of the "Clover Leaf" reservoir. We performed a retrospective evaluation, analyzing the treatment-associated complications by means of the Clavien-Dindo classification. All patients as well as their partners received questionnaires with validated scores. Group I: 70 (49.3%) patients who underwent HSM RP. Group II: 72 (50.7%) patients who underwent SR RP. Neither grade IV nor grade V Clavien-Dindo complications were documented. In total, we observed 4 (3.3%) cases grade III b complications, which resulted in revision. Distribution was as follows: infected device (n = 4), scrotal hematoma (n = 2), scrotal seroma (n = 1), and scrotal skin fistula (n = 1). 88% of the patients with ectopic HSM RP and 81% with traditional SR RP were satisfied with their implant. Of the patients with HSM RP, 64.3% (n = 45; BMI range: 18.5-28.8) reported that they were able to feel their reservoir by palpation immediately after surgery. Palpability disappeared in 80% of the patients in this group (BMI > 26.5) after capsule formation at 3 months post-surgery. Only one patient (1.4%; BMI 20.5) reported that he was able to see the reservoir. Our findings suggest that the novel reservoir placement is safe, efficient and results in excellent patient satisfaction, even if the reservoir is initially palpable or visible.
Asunto(s)
Disfunción Eréctil/cirugía , Satisfacción del Paciente/estadística & datos numéricos , Implantación de Pene/métodos , Prótesis de Pene , Escroto/cirugía , Pared Abdominal/cirugía , Adulto , Anciano , Anciano de 80 o más Años , Índice de Masa Corporal , Humanos , Masculino , Persona de Mediana Edad , Diseño de Prótesis , Estudios Retrospectivos , Encuestas y CuestionariosRESUMEN
The Saccharomyces cerevisiae SIS1 gene was identified as a high copy number suppressor of the slow growth phenotype of strains containing mutations in the SIT4 gene, which encodes a predicted serine/threonine protein phosphatase. The SIS1 protein is similar to bacterial dnaJ proteins in the amino-terminal third and carboxyl-terminal third of the proteins. In contrast, the middle third of SIS1 is not similar to dnaJ proteins. This region of SIS1 contains a glycine/methionine-rich region which, along with more amino-terminal sequences, is required for SIS1 to associate with a protein of apparent molecular mass of 40 kD. The SIS1 gene is essential. Strains limited for the SIS1 protein accumulate cells that appear blocked for migration of the nucleus from the mother cell into the daughter cell. In addition, many of the cells become very large and contain a large vacuole. The SIS1 protein is localized throughout the cell but is more concentrated at the nucleus. About one-fourth of the SIS1 protein is released from a nuclear fraction upon treatment with RNase. We also show that overexpression of YDJ1, another yeast protein with similarity to bacterial dnaJ proteins, can not substitute for SIS1.
Asunto(s)
Proteínas Bacterianas/genética , Escherichia coli/genética , Proteínas Fúngicas/genética , Genes Bacterianos , Genes Fúngicos , Genes Supresores , Proteínas de Choque Térmico/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Secuencia de Bases , Deleción Cromosómica , ADN de Hongos/genética , ADN de Hongos/aislamiento & purificación , Epítopos/análisis , Proteínas de Escherichia coli , Técnica del Anticuerpo Fluorescente , Proteínas Fúngicas/análisis , Proteínas Fúngicas/inmunología , Genotipo , Proteínas del Choque Térmico HSP40 , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fenotipo , Saccharomyces cerevisiae/crecimiento & desarrollo , Homología de Secuencia de Ácido Nucleico , Fracciones Subcelulares/ultraestructuraRESUMEN
Classical cadherins form parallel cis-dimers that emanate from a single cell surface. It is thought that the cis-dimeric form is active in cell-cell adhesion, whereas cadherin monomers are likely to be inactive. Currently, cis-dimers have been shown to exist only between cadherins of the same type. Here, we show the specific formation of cis-heterodimers between N- and R-cadherins. E-cadherin cannot participate in these complexes. Cells coexpressing N- and R-cadherins show homophilic adhesion in which these proteins coassociate at cell-cell interfaces. We performed site- directed mutagenesis studies, the results of which support the strand dimer model for cis-dimerization. Furthermore, we show that when N- and R-cadherins are coexpressed in neurons in vitro, the two cadherins colocalize at certain neural synapses, implying biological relevance for these complexes. The present study provides a novel paradigm for cadherin interaction whereby selective cis-heterodimer formation may generate new functional units to mediate cell-cell adhesion.
Asunto(s)
Cadherinas/metabolismo , Animales , Sitios de Unión , Cadherinas/química , Cadherinas/genética , Adhesión Celular , Comunicación Celular , Dimerización , Expresión Génica , Cobayas , Uniones Intercelulares , Células L , Ratones , Conejos , Ratas , Sinapsis , Triptófano/metabolismoRESUMEN
Gene expression is dependent on the interaction of DNA binding factors with distinct promoter control elements to activate RNA synthesis. The expression of the HIS4 gene in yeast is under two different control systems. One of these, general amino acid control, involves a DNA binding protein, GCN4, that stimulates transcription in response to amino acid starvation by binding to 5'-TGACTC-3' sequences in the HIS4 promoter region. A second system, the basal level control, stimulates HIS4 transcription in the absence of amino acid starvation. The basal level transcription of the HIS4 gene is under the control of two genes, BAS1 and BAS2, which are also required for the control of purine biosynthesis. In addition, BAS2 is required for the utilization of organic phosphates in the growth medium. Genetic mapping and DNA sequence analysis show that BAS2 is PHO2, a gene previously identified as a regulator of phosphate metabolism. Direct biochemical analysis shows that the BAS2 gene encodes a protein that binds to both the HIS4 and PHO5 promoters. The involvement of a single DNA binding protein in the regulation of histidine, adenine, and phosphate metabolism suggests that yeast may use a few key DNA binding proteins to coordinate the regulation of diverse metabolic pathways.
Asunto(s)
Fosfatasa Ácida/genética , Histidina/genética , Saccharomyces cerevisiae/genética , Mapeo Cromosómico , Regulación de la Expresión Génica , Genes Fúngicos , Mutación , Fosfatos/fisiología , Regiones Promotoras Genéticas , Factores de Transcripción/fisiología , Transcripción GenéticaRESUMEN
The BAS1 and BAS2 proteins are both required for activation of GCN4-independent (basal) HIS4 transcription in yeast. BAS1 has an NH2-terminal region similar to those of the myb proto-oncogene family. BAS1 and BAS2, which contains a homeo box, bound to adjacent sites on the HIS4 promoter. The joint requirement of BAS1 and BAS2 for activation is probably not due to cooperative binding or the transcriptional control of one of the genes by the other. Although BAS1 and BAS2 were both required for activation of HIS4 transcription, BAS1 was not required for BAS2-dependent expression of the secreted acid phosphatases. The transcriptional activators of HIS4 have DNA binding domains that are conserved in evolution (BAS1 = Myb, BAS2 = homeo box, GCN4 = Jun). Their interactions, therefore, may be relevant to the control of gene expression in more complex systems.
Asunto(s)
Proteínas Fúngicas/genética , Regulación de la Expresión Génica , Genes Fúngicos , Proteínas Proto-Oncogénicas/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Transactivadores , Transcripción Genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Datos de Secuencia Molecular , Proteínas Proto-Oncogénicas c-myb , Homología de Secuencia de Ácido NucleicoRESUMEN
The relationship between adhesive interactions across the synaptic cleft and synaptic function has remained elusive. At certain CNS synapses, pre- to postsynaptic adhesion is mediated at least in part by neural (N-) cadherin. Here, we demonstrate that upon depolarization of hippocampal neurons in culture by K+ treatment, or application of NMDA or alpha-latrotoxin, synaptic N-cadherin dimerizes and becomes markedly protease resistant. These properties are indices of strong, stable, enhanced cadherin-mediated intercellular adhesion. N-cadherin retained protease resistance for at least 2 hr after recovery, while other surface molecules, including other cadherins, were completely degraded. The acquisition of protease resistance and dimerization of N-cadherin is not dependent on new protein synthesis, nor is it accompanied by internalization of N-cadherin. By immunocytochemistry, we found that high K+ selectively induces surface dispersion of N-cadherin, which, after recovery, returns to synaptic puncta. N-cadherin dispersion under K+ treatment parallels the rapid expansion of the presynaptic membrane consequent to the massive vesicle fusion that occurs with this type of depolarization. In contrast, with NMDA application, N-cadherin does not disperse but does acquire enhanced protease resistance and dimerizes. Our data strongly suggest that synaptic adhesion is dynamically and locally controlled, and modulated by synaptic activity.
Asunto(s)
Cadherinas/metabolismo , Neuronas/metabolismo , Transmisión Sináptica/fisiología , Vesículas Sinápticas/metabolismo , Transactivadores , 2-Amino-5-fosfonovalerato/farmacología , Animales , Biomarcadores , Cadherinas/análisis , Cadherinas/química , Células Cultivadas , Proteínas del Citoesqueleto/análisis , Proteínas del Citoesqueleto/metabolismo , Dimerización , Endopeptidasas/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Técnica del Anticuerpo Fluorescente , Cobayas , Hipocampo/citología , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Ratones , Proteínas del Tejido Nervioso/análisis , Proteínas del Tejido Nervioso/metabolismo , Moléculas de Adhesión de Célula Nerviosa/análisis , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Neuronas/química , Neuronas/citología , Fragmentos de Péptidos/análisis , Potasio/farmacología , Conformación Proteica , Conejos , Ratas , Ratas Sprague-Dawley , Receptores AMPA/análisis , Receptores AMPA/metabolismo , Receptores de N-Metil-D-Aspartato/análisis , Receptores de N-Metil-D-Aspartato/fisiología , Transmisión Sináptica/efectos de los fármacos , Vesículas Sinápticas/química , Sinaptofisina/análisis , Sinaptofisina/metabolismo , beta CateninaRESUMEN
We report the purification of a presynaptic "particle web" consisting of approximately 50 nm pyramidally shaped particles interconnected by approximately 100 nm spaced fibrils. This is the "presynaptic grid" described in early EM studies. It is completely soluble above pH 8, but reconstitutes after dialysis against pH 6. Interestingly, reconstituted particles orient and bind PSDs asymmetrically. Mass spectrometry of purified web components reveals major proteins involved in the exocytosis of synaptic vesicles and in membrane retrieval. Our data support the idea that the CNS synaptic junction is organized by transmembrane adhesion molecules interlinked in the synaptic cleft, connected via their intracytoplasmic domains to the presynaptic web on one side and to the postsynaptic density on the other. The CNS synaptic junction may therefore be conceptualized as a complicated macromolecular scaffold that isostatically bridges two closely aligned plasma membranes.
Asunto(s)
Terminales Presinápticos/química , Terminales Presinápticos/ultraestructura , Vesículas Sinápticas/química , Vesículas Sinápticas/ultraestructura , Proteínas de Transporte Vesicular , Animales , Anticuerpos , Cadherinas/análisis , Cadherinas/inmunología , Membrana Celular/química , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Clatrina/análisis , Clatrina/inmunología , Cadenas Pesadas de Clatrina , Dinaminas , GTP Fosfohidrolasas/análisis , GTP Fosfohidrolasas/inmunología , Proteínas HSP70 de Choque Térmico/análisis , Proteínas HSP70 de Choque Térmico/inmunología , Masculino , Proteínas de la Membrana/análisis , Proteínas de la Membrana/inmunología , Microscopía Inmunoelectrónica , Proteínas Munc18 , Cadenas Pesadas de Miosina/análisis , Cadenas Pesadas de Miosina/inmunología , Proteínas del Tejido Nervioso/análisis , Proteínas del Tejido Nervioso/inmunología , Proteínas de Neurofilamentos/análisis , Proteínas de Neurofilamentos/inmunología , Terminales Presinápticos/metabolismo , Proteínas Qa-SNARE , Conejos , Ratas , Espectrina/análisis , Espectrina/inmunología , Sinapsinas/análisis , Sinapsinas/inmunología , Vesículas Sinápticas/metabolismo , Proteína 25 Asociada a SinaptosomasRESUMEN
Saccharomyces cerevisiae strains containing temperature-sensitive mutations in the SIT4 protein phosphatase arrest in late G1 at the nonpermissive temperature. Order-of-function analysis shows that SIT4 is required in late G1 for progression into S phase. While the levels of SIT4 do not change in the cell cycle, SIT4 associates with two high-molecular-weight phosphoproteins in a cell-cycle-dependent fashion. In addition, we have identified a polymorphic gene, SSD1, that in some versions can suppress the lethality due to a deletion of SIT4 and can also partially suppress the phenotypic defects due to a null mutation in BCY1. The SSD1 protein is implicated in G1 control and has a region of similarity to the dis3 protein of Schizosaccharomyces pombe. We have also identified a gene, PPH2alpha, that in high copy number can partially suppress the growth defect of sit4 strains. The PPH2 alpha gene encodes a predicted protein that is 80% identical to the catalytic domain of mammalian type 2A protein phosphatases but also has an acidic amino-terminal extension not present in other phosphatases.
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Fase G1 , Fosfoproteínas Fosfatasas/genética , Fase S , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Secuencia de Bases , Citoplasma/enzimología , Técnica del Anticuerpo Fluorescente , Genes , Datos de Secuencia Molecular , Peso Molecular , Mutación , Fenotipo , Fosfoproteínas Fosfatasas/metabolismo , Polimorfismo Genético , Pruebas de Precipitina , Proteína Fosfatasa 2 , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae , Homología de Secuencia de Ácido Nucleico , TemperaturaRESUMEN
Interaction of the TATA box-binding protein (TBP) with promoters of RNA polymerase II-transcribed genes is an early and essential step in mRNA synthesis. Previous studies have demonstrated that the rate-limiting binding of TBP to a TATA element can be influenced by transcriptional regulatory proteins. To identify additional factors that may regulate DNA binding by TBP in vivo, we performed a genetic selection for extragenic suppressors of a yeast TBP mutant that exhibits altered and relaxed DNA binding specificity. This analysis has led to the discovery of a previously unidentified gene, RTF1. The original rtf1 suppressor mutation, which encodes a single amino acid change in Rtf1, and an rtf1 null allele suppress the effects of the TBP specificity mutant by altering transcription initiation. Differences in the patterns of transcription initiation in these strains strongly suggest that the rtf1 missense mutation is distinct from a simple loss-of-function allele. The results of genetic crosses indicate that suppression of TBP mutants by mutations in RTF1 occurs in an allele-specific fashion. In a strain containing wild-type TBP, the rtf1 null mutation suppresses the transcriptional effects of a Ty delta insertion mutation in the promoter of the HIS4 gene, a phenotype also conferred by the TBP altered-specificity mutant. Finally, as shown by indirect immunofluorescence experiments, Rtf1 is a nuclear protein. Taken together, our findings suggest that Rtf1 either directly or indirectly regulates the DNA binding properties of TBP and, consequently, the relative activities of different TATA elements in vivo.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas Fúngicas/genética , Genes Fúngicos/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , TATA Box/genética , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Núcleo Celular/química , Clonación Molecular , Cruzamientos Genéticos , ADN de Hongos/metabolismo , Proteínas de Unión al ADN/genética , Proteínas Fúngicas/análisis , Proteínas Fúngicas/química , Datos de Secuencia Molecular , Peso Molecular , Fenotipo , ARN de Hongos/análisis , ARN Mensajero/análisis , Secuencias Repetitivas de Ácidos Nucleicos/genética , Retroelementos/genética , Análisis de Secuencia de ADN , Supresión Genética , Proteína de Unión a TATA-Box , Factores de Transcripción/genética , Transcripción Genética/genéticaRESUMEN
The Saccharomyces cerevisiae CLN3 protein, a G1 cyclin, positively regulates the expression of CLN1 and CLN2, two additional G1 cyclins whose expression during late G1 is activated, in part, by the transcription factors SWI4 and SWI6. We isolated 12 complementation groups of mutants that require CLN3. The members of one of these complementation groups have mutations in the BCK2 gene. In a wild-type CLN3 genetic background, bck2 mutants have a normal growth rate but have a larger cell size, are more sensitive to alpha-factor, and have a modest defect in the accumulation of CLN1 and CLN2 RNA. In the absence of CLN3, bck2 mutations cause an extremely slow growth rate: the cells accumulate in late G1 with very low levels of CLN1 and CLN2 RNA. The slow growth rate and long G1 delay of bck2 cln3 mutants are cured by heterologous expression of CLN2. Moreover, overexpression of BCK2 induces very high levels of CLN1, CLN2, and HCS26 RNAs. The results suggest that BCK2 and CLN3 provide parallel activation pathways for the expression of CLN1 and CLN2 during late G1.
Asunto(s)
Ciclinas/biosíntesis , Proteínas Fúngicas/genética , Fase G1/genética , Regulación Fúngica de la Expresión Génica , Fosfoproteínas/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Proteínas Fúngicas/biosíntesis , Genes Fúngicos/genética , Prueba de Complementación Genética , Péptidos y Proteínas de Señalización Intracelular , Mutación , Fosfoproteínas Fosfatasas/metabolismo , Fosfoproteínas/biosíntesis , Regiones Promotoras Genéticas/genética , Proteína Quinasa C/genética , Proteína Fosfatasa 2 , Proteínas Serina-Treonina Quinasas/genética , ARN Mensajero/biosíntesis , Proteínas Recombinantes/biosíntesis , Transcripción GenéticaRESUMEN
A mutation in the gene that encodes Saccharomyces cerevisiae TFIID (SPT15), which was isolated in a selection for mutations that alter transcription in vivo, changes a single amino acid in a highly conserved region of the second direct repeat in TFIID. Among eight independent spt15 mutations, seven cause this same amino acid change, Leu-205 to Phe. The mutant TFIID protein (L205F) binds with greater affinity than that of wild-type TFIID to at least two nonconsensus TATA sites in vitro, showing that the mutant protein has altered DNA binding specificity. Site-directed mutations that change Leu-205 to five different amino acids cause five different phenotypes, demonstrating the importance of this amino acid in vivo. Virtually identical phenotypes were observed when the same amino acid changes were made at the analogous position, Leu-114, in the first repeat of TFIID. Analysis of these mutations and additional mutations in the most conserved regions of the repeats, in conjunction with our DNA binding results, suggests that these regions of the repeats play equivalent roles in TFIID function, possibly in TATA box recognition.
Asunto(s)
Genes Fúngicos , Mutación , Saccharomyces cerevisiae/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción Genética , Secuencia de Aminoácidos , Secuencia de Bases , Northern Blotting , Escherichia coli/genética , Genotipo , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fenotipo , Plásmidos , ARN de Hongos/genética , ARN de Hongos/aislamiento & purificación , Mapeo Restrictivo , Saccharomyces cerevisiae/metabolismo , TATA Box , Factor de Transcripción TFIIDRESUMEN
The major in vitro binding activity to the Saccharomyces cerevisiae HIS4 promoter is due to the RAP1 protein. In the absence of GCN4, BAS1, and BAS2, the RAP1 protein binds to the HIS4 promoter in vivo but cannot efficiently stimulate HIS4 transcription. RAP1, which binds adjacently to BAS2 on the HIS4 promoter, is required for BAS1/BAS2-dependent activation of HIS4 basal-level transcription. In addition, the RAP1-binding site overlaps with the single high-affinity HIS4 GCN4-binding site. Even though RAP1 and GCN4 bind competitively in vitro, RAP1 is required in vivo for (i) the normal steady-state levels of GCN4-dependent HIS4 transcription under nonstarvation conditions and (ii) the rapid increase in GCN4-dependent steady-state HIS4 mRNA levels following amino acid starvation. The presence of the RAP1-binding site in the HIS4 promoter causes a dramatic increase in the micrococcal nuclease sensitivity of two adjacent regions within HIS4 chromatin: one region contains the high-affinity GCN4-binding site, and the other region contains the BAS1- and BAS2-binding sites. These results suggest that RAP1 functions at HIS4 by increasing the accessibility of GCN4, BAS1, and BAS2 to their respective binding sites when these sites are present within chromatin.