RESUMEN
BACKGROUND AND PURPOSE: Biomarker changes in cognitively impaired patients with small vessel disease are largely unknown. The rate of amyloid/lacune progression, cortical thinning and cognitive decline were evaluated in subcortical vascular mild cognitive impairment (svMCI) patients. METHODS: Seventy-two svMCI patients were divided into early stage (ES-svMCI, n = 39) and late stage (LS-svMCI, n = 33) according to their Clinical Dementia Rating Sum of Boxes score. Patients were annually followed up with neuropsychological tests and brain magnetic resonance imaging for 3 years, and underwent a second [11 C] Pittsburgh compound B (PiB) positron emission tomography scan within a mean interval of 32.4 months. RESULTS: There was no difference in the rate of increase in PiB uptake or lacune number between the ES-svMCI and LS-svMCI. However, LS-svMCI showed more rapid cortical thinning and cognitive decline than did the ES-svMCI. CONCLUSIONS: We suggest that, whilst the rate of change in pathological burden did not differ between ES-svMCI and LS-svMCI, cortical thinning and cognitive decline progressed more rapidly in the LS-svMCI.
Asunto(s)
Corteza Cerebral/diagnóstico por imagen , Enfermedades de los Pequeños Vasos Cerebrales/diagnóstico por imagen , Disfunción Cognitiva/fisiopatología , Progresión de la Enfermedad , Anciano , Anciano de 80 o más Años , Enfermedades de los Pequeños Vasos Cerebrales/complicaciones , Disfunción Cognitiva/etiología , Femenino , Humanos , Estudios Longitudinales , Imagen por Resonancia Magnética/métodos , Masculino , Pruebas Neuropsicológicas , Tomografía de Emisión de Positrones/métodosRESUMEN
Recent studies reporting trans-differentiation of mononucleated cells derived from human umbilical cord blood into neuronal cells aroused interest among investigators for their clinical implication and significance in regenerative medicine. In the present study, purified populations of hematopoietic stem cells were isolated via magnetic bead sorting and fluorescence-activated cell sorter (FACS) using a specific CD133 antibody, a cell type-specific marker for hematopoietic stem cells, and grown in culture in the presence of retinoic acid (RA). CD133+ hematopoietic stem cells expressed neuronal and glial phenotypes after RA treatment. RT-PCR analysis indicated that the RA treated CD133+ cells expressed mRNA transcripts for ATP-binding cassettes transporter ABCG2 (a universal stem cell marker), nestin (a specific cell type marker for neural stem cells), Musashi1 (a specific marker for neural stem cells) and RA receptors (RAR) including RAR-alpha, RAR-beta, and retinoid X receptor (RXR)-gamma. RA-treated CD133+ cells expressed mRNA transcripts for neuron-specific markers neurofilament proteins (NF-L, -M, -H) and synaptophysin as determined by RT-PCR, structural proteins characteristic of neurons including tubulin beta III and neuron specific enolase (NSE) by Western blot, and neuron-specific markers NeuN and microtubule-associated protein-2 (MAP2) by immunocytochemistry. RA-treated CD133+ cells also expressed the astrocyte-specific marker glial fibrillary acidic protein (GFAP), as demonstrated by RT-PCR, Western blot, and immunocytochemistry. In addition, RA-treated CD133+ cells expressed cell type-specific markers for oligodendrocytes including myelin basic protein (MBP) as shown by RT-PCR, proteolipid protein (PLP) by Western blot analysis, and cyclic nucleotide phosphodiesterase (CNPase) by immunostaining. Upregulated expression of several basic helix-loop-helix (bHLH) transcription factors important for early neurogenesis, including Otx2, Pax6, Wnt1, Olig2, Hash1 and NeuroD1, was also demonstrated in CD133+ cells after RA treatment. These results indicate that human cord blood-derived CD133+ hematopoietic stem cells could trans-differentiate into neural cell types of neuron-like cells, astrocytes, and oligodendrocytes by RA treatment.
Asunto(s)
Sangre Fetal/efectos de los fármacos , Células Madre Hematopoyéticas/efectos de los fármacos , Neuroglía/efectos de los fármacos , Neuronas/efectos de los fármacos , Tretinoina/farmacología , Células Cultivadas , Sangre Fetal/citología , Sangre Fetal/metabolismo , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Humanos , Neuroglía/citología , Neuroglía/metabolismo , Neuronas/citología , Neuronas/metabolismoRESUMEN
The Schizosaccharomyces pombe DNA repair gene rhp51(+) encodes a RecA-like protein with the DNA-dependent ATPase activity required for homologous recombination. The level of the rhp51(+) transcript is increased by a variety of DNA-damaging agents. Its promoter has two cis-acting DNA damage-responsive elements (DREs) responsible for DNA damage inducibility. Here we report identification of Rdp1, which regulates rhp51(+) expression through the DRE of rhp51(+). The protein contains a zinc finger and a polyalanine tract similar to ones previously implicated in DNA binding and transactivation or repression, respectively. In vitro footprinting and competitive binding assays indicate that the core consensus sequences (NGG/TTG/A) of DRE are crucial for the binding of Rdp1. Mutations of both DRE1 and DRE2 affected the damage-induced expression of rhp51(+), indicating that both DREs are required for transcriptional activation. In addition, mutations in the DREs significantly reduced survival rates after exposure to DNA-damaging agents, demonstrating that the damage response of rhp51(+) enhances the cellular repair capacity. Surprisingly, haploid cells containing a complete rdp1 deletion could not be recovered, indicating that rdp1(+) is essential for cell viability and implying the existence of other target genes. Furthermore, the DNA damage-dependent expression of rhp51(+) was significantly reduced in checkpoint mutants, raising the possibility that Rdp1 may mediate damage checkpoint-dependent transcription of rhp51(+).
Asunto(s)
Adenosina Trifosfatasas/aislamiento & purificación , Daño del ADN , Reparación del ADN , Proteínas de Unión al ADN , Proteínas Fúngicas/genética , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces/genética , Adenosina Trifosfatasas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Unión Competitiva , Relación Dosis-Respuesta a Droga , Relación Dosis-Respuesta en la Radiación , Farmacorresistencia Microbiana , Regulación Fúngica de la Expresión Génica , Metilmetanosulfonato/toxicidad , Datos de Secuencia Molecular , Unión Proteica , Recombinasa Rad51 , Homología de Secuencia de Aminoácido , Transcripción Genética , Rayos Ultravioleta/efectos adversos , Dedos de ZincRESUMEN
Exposure of Schizosaccharomyces pombe cells to UV light results in increased uvi15(+) gene expression at both the mRNA and protein levels, leading to elevated cell survival. This UV-induced expression of the uvi15(+) gene was reduced in Deltasty1 and Deltawis1 cells lacking the stress-activated protein kinase pathway, but not in DNA damage checkpoint mutants. To further understand the cellular mechanisms responsible for this UV-induced expression, the transcription rate and mRNA half-life were investigated. Transcription run-on assays revealed that the rate of uvi15(+) transcription was increased 1.8-fold regardless of Sty1 when cells were UV irradiated. The half-life of uvi15(+) mRNA was also increased 1.5-fold after UV irradiation, but it was decreased in the Deltasty1 background for both basal and UV-induced mRNAs, indicating that the stress-activated MAPK cascade can mediate UV-induced gene expression by increasing mRNA half-life. Deletion analyses identified a 54 nt element downstream of the distal poly(A) site, which was involved in the increased half-life of uvi15(+) mRNA. These results suggest that both Sty1 and the 3'-regulatory element regulate UV-induced expression of the uvi15(+) gene at the post-transcriptional level.
Asunto(s)
Regulación Fúngica de la Expresión Génica/efectos de la radiación , Genes Fúngicos/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos/genética , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces/efectos de la radiación , Rayos Ultravioleta , Regiones no Traducidas 3'/genética , Secuencia de Bases , Daño del ADN/genética , Daño del ADN/efectos de la radiación , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Eliminación de Gen , Semivida , Cinética , Sistema de Señalización de MAP Quinasas/efectos de la radiación , Proteínas Quinasas Activadas por Mitógenos/genética , Poli A/genética , Estabilidad del ARN/efectos de la radiación , ARN de Hongos/genética , ARN de Hongos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Schizosaccharomyces/enzimología , Schizosaccharomyces/genética , Transcripción Genética/genética , Transcripción Genética/efectos de la radiación , Regulación hacia Arriba/genética , Regulación hacia Arriba/efectos de la radiaciónRESUMEN
DNA double-strand breaks can be introduced by exogenous agents or during normal cellular processes. Genes belonging to the RAD52 epistasis group are known to repair these breaks in budding yeast. Among these genes, RAD52 plays a central role in homologous recombination and DNA double-strand break repair. Despite its importance, its mechanism of action is not yet clear. It is known, however, that the human homologue of Rad52 is capable of binding to DNA ends in vitro. Herein, we show that Rad22 protein, a Rad52 homologue in the fission yeast Schizosaccharomyces pombe, can similarly bind to DNA ends at double-strand breaks. This end-binding ability was demonstrated in vitro by electron microscopy and by protection from exonuclease attack. We also showed that Rad22 specifically binds near double-strand break associated with mating type switching in vivo by chromatin immunoprecipitation analysis. This is the first evidence that a recombinational protein directly binds to DNA double-strand breaks in vivo.
Asunto(s)
Daño del ADN , Reparación del ADN , Proteínas de Unión al ADN , ADN/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces/química , Cromatina/metabolismo , Reactivos de Enlaces Cruzados/farmacología , ADN/ultraestructura , Relación Dosis-Respuesta a Droga , Escherichia coli/metabolismo , Exonucleasas/metabolismo , Proteínas Fúngicas/genética , Genotipo , Microscopía Electrónica , Plásmidos/metabolismo , Reacción en Cadena de la Polimerasa , Pruebas de Precipitina , Unión Proteica , Schizosaccharomyces/genéticaRESUMEN
Hrp1 of Schizosaccharomyces pombe is a member of the CHD protein family, characterized by a chromodomain, a Myb-like telobox-related DNA-binding domain and a SNF2-related helicase/ATPase domain. CHD proteins are thought to be required for modification of the chromatin structure in transcription, but the exact roles of CHD proteins are not known. Here we examine the sub-cellular localization and biochemical activity of Hrp1 and the phenotypes of hrp1 Delta and Hrp1-overexpressing strains. Fluorescence microscopy revealed that Hrp1 protein is targeted to the nucleus. We found that Hrp1 exhibited DNA-dependent ATPase activity, stimulated by both single- and double-stranded DNA. Overexpression of Hrp1 caused slow cell growth accompanied by defective chromosome condensation in anaphase resulting in a 'cut' (celluntimelytorn) phenotype and chromosome loss. The hrp1 Delta mutation also caused abnormal anaphase and mini-chromosome loss phenotypes. Electron micrographs demonstrated that aberrantly shaped nucleoli appeared in Hrp1-overexpressing cells. Therefore, these results suggest that Hrp1 may play a role in mitotic chromosome segregation and maintenance of chromatin structure by utilizing the energy from ATP hydrolysis.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Cromatina/metabolismo , Segregación Cromosómica , Proteínas de Unión al ARN/metabolismo , Ribonucleoproteínas/metabolismo , Proteínas de Saccharomyces cerevisiae , Schizosaccharomyces/metabolismo , Factores de Escisión y Poliadenilación de ARNm , Adenosina Trifosfato/metabolismo , Anafase , Nucléolo Celular/genética , Nucléolo Celular/metabolismo , Nucléolo Celular/ultraestructura , Núcleo Celular/química , Cromatina/genética , Cromosomas Fúngicos/genética , Cromosomas Fúngicos/metabolismo , ADN/fisiología , Técnica del Anticuerpo Fluorescente Indirecta , Eliminación de Gen , Regulación Fúngica de la Expresión Génica , Hidrólisis , Mitosis , Proteínas de Unión al ARN/genética , Ribonucleoproteínas/genética , Schizosaccharomyces/genética , Schizosaccharomyces/ultraestructuraRESUMEN
The Saccharomyces cerevisiae DNA repair gene PHR1 encodes a photolyase that catalyzes the light-dependent repair of pyrimidine dimers. PHR1 expression is induced at the level of transcription by a variety of DNA-damaging agents. The primary regulator of the PHR1 damage response is a 39-bp sequence called URS(PHR1) which is the binding site for a protein(s) that constitutes the damage-responsive repressor PRP. In this communication, we report the identification of two proteins, Rph1p and Gis1p, that regulate PHR1 expression through URS(PHR1). Both proteins contain two putative zinc fingers that are identical throughout the DNA binding region, and deletion of both RPH1 and GIS1 is required to fully derepress PHR1 in the absence of damage. Derepression of PHR1 increases the rate and extent of photoreactivation in vivo, demonstrating that the damage response of PHR1 enhances cellular repair capacity. In vitro footprinting and binding competition studies indicate that the sequence AG(4) (C(4)T) within URS(PHR1) is the binding site for Rph1p and Gis1p and suggests that at least one additional DNA binding component is present in the PRP complex.
Asunto(s)
Apoenzimas/genética , Reparación del ADN , Desoxirribodipirimidina Fotoliasa/genética , Proteínas Fúngicas , Glicoproteínas de Membrana , Secuencias Reguladoras de Ácidos Nucleicos , Proteínas Represoras/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Unión Competitiva , Daño del ADN , Huella de ADN , Represión Enzimática , Biblioteca Genómica , Histona Demetilasas , Datos de Secuencia Molecular , Unión Proteica , Proteínas Represoras/genética , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/efectos de la radiación , Homología de Secuencia de Aminoácido , Rayos Ultravioleta/efectos adversos , Dedos de Zinc/genéticaRESUMEN
The SNF2/SWI2 ATPase/helicase family comprises proteins from a variety of species, which serve a number of functions, such as transcriptional regulation, maintenance of chromosome stability during mitosis, and various types of DNA repair. Several proteins with unknown functions are also included in this family. The number of genes that belong to this family is rapidly expanding, which makes it easier to analyze the common biological functions of the family members. This study was designed to clone the SNF2/SWI2 helicase-related genes from the fission yeast Schizosaccharomyces pombe in the hope that this would help to elucidate the common functions of the proteins in this family. The hrp1+ (helicase-related gene from S. pombe) gene was initially cloned by PCR amplification using degenerate primers based on conserved SNF2 motifs within the ERCC6 gene, which encodes a protein involved in DNA excision repair. The hrp1+ ORF codes for an 1373-amino acid polypeptide with a molecular mass of 159 kDa. Like other SNF2/SWI2 family proteins, the deduced amino acid sequence of Hrp1 contains DNA-dependent ATPase/7 helicase domains, as well as a chromodomain and a DNA-binding domain. This configuration is similar to that of mCHD1 (mouse chromo-ATPase/helicase-DNA-binding protein 1), suggesting that Hrp1 is a S. pombe homolog of mCHD1, which is thought to function in altering the chromatin structure to facilitate gene expression. Northern blot analysis showed that the hrp1+ gene produces a 4.6-kb transcript, which reaches its maximal level just before the cells enter the exponential growth phase, and then decreases gradually. DNA-damaging agents, such as MMS, MNNG and UV, decrease the rate of transcription of hrp1+. Deletion of the hrp1+ gene resulted in accelerated cell growth. On the other hand, overexpression of Hrp1 caused a reduction in growth rate. These results indicate that hrp1+ may act as a negative regulator of cellular growth.
Asunto(s)
Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/aislamiento & purificación , ADN Helicasas/genética , ADN Helicasas/aislamiento & purificación , Proteínas de Unión al ADN/genética , Proteínas Fúngicas/genética , Genes Fúngicos/genética , Proteínas Nucleares , Schizosaccharomyces/genética , Factores de Transcripción/genética , Secuencia de Aminoácidos , Secuencia de Bases , Codón Iniciador , Proteínas de Unión al ADN/aislamiento & purificación , Proteínas Fúngicas/aislamiento & purificación , Eliminación de Gen , Expresión Génica , Datos de Secuencia Molecular , Proteínas de Schizosaccharomyces pombe , Factores de Transcripción/aislamiento & purificaciónRESUMEN
In Saccharomyces cerevisiae UV radiation and a variety of chemical DNA-damaging agents induce the transcription of specific genes, including several involved in DNA repair. One of the best characterized of these genes is PHR1, which encodes the apoenzyme for DNA photolyase. Basal-level and damage-induced expression of PHR1 require an upstream activation sequence, UAS(PHR1), which has homology with DRC elements found upstream of at least 19 other DNA repair and DNA metabolism genes in yeast. Here we report the identification of the UME6 gene of S. cerevisiae as a regulator of UAS(PHR1) activity. Multiple copies of UME6 stimulate expression from UAS(PHR1) and the intact PHR1 gene. Surprisingly, the effect of deletion of UME6 is growth phase dependent. In wild-type cells PHR1 is induced in late exponential phase, concomitant with the initiation of glycogen accumulation that precedes the diauxic shift. Deletion of UME6 abolishes this induction, decreases the steady-state concentration of photolyase molecules and PHR1 mRNA, and increases the UV sensitivity of a rad2 mutant. Despite the fact that UAS(PHR1) does not contain the URS1 sequence, which has been previously implicated in UME6-mediated transcriptional regulation, we find that Ume6p binds to UAS(PHR1) with an affinity and a specificity similar to those seen for a URS1 site. Similar binding is also seen for DRC elements from RAD2, RAD7, and RAD53, suggesting that UME6 contributes to the regulated expression of a subset of damage-responsive genes in yeast.
Asunto(s)
Apoenzimas/genética , Proteínas de Ciclo Celular , Reparación del ADN/genética , Proteínas de Unión al ADN/metabolismo , Desoxirribodipirimidina Fotoliasa/genética , Endodesoxirribonucleasas , Regulación Fúngica de la Expresión Génica , Glicoproteínas de Membrana , Proteínas Serina-Treonina Quinasas , Proteínas Represoras , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Factores de Transcripción/metabolismo , Apoenzimas/biosíntesis , Sitios de Unión , Quinasa de Punto de Control 2 , Clonación Molecular , Desoxirribodipirimidina Fotoliasa/biosíntesis , Proteínas Fúngicas/genética , Eliminación de Gen , Dosificación de Gen , Genes Fúngicos , Mutagénesis , Unión Proteica , Proteínas Quinasas/genética , Dímeros de Pirimidina/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos , Saccharomyces cerevisiae/efectos de la radiación , Transcripción Genética , Rayos Ultravioleta/efectos adversosRESUMEN
The Schizosaccharomyces pombe rhp51+ gene encodes a recombinational repair protein that shares significant sequence identities with the bacterial RecA and the Saccharomyces cerevisiae RAD51 protein. Levels of rhp51+ mRNA increase following several types of DNA damage or inhibition of DNA synthesis. An rhp51::ura4 fusion gene was used to identify the cis-acting promoter elements involved in regulating rhp51+ expression in response to DNA damage. Two elements, designated DRE1 and DRE2 (for damage-responsive element), match a decamer consensus URS (upstream repressing sequence) found in the promoters of many other DNA repair and metabolism genes from S. cerevisiae. However, our results show that DRE1 and DRE2 each function as a UAS (upstream activating sequence) rather than a URS and are also required for DNA-damage inducibility of the gene. A 20-bp fragment located downstream of both DRE1 and DRE2 is responsible for URS function. The DRE1 and DRE2 elements cross-competed for binding to two proteins of 45 and 59 kDa. DNase I footprint analysis suggests that DRE1 and DRE2 bind to the same DNA-binding proteins. These results suggest that the DRE-binding proteins may play an important role in the DNA-damage inducibility of rhp51+ expression.
Asunto(s)
Reparación del ADN , Proteínas Fúngicas/genética , Regiones Promotoras Genéticas , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces/genética , Secuencia de Bases , Clonación Molecular , Daño del ADN , ADN de Hongos/genética , Proteínas de Unión al ADN/genética , Proteínas Fúngicas/metabolismo , Eliminación de Gen , Regulación Fúngica de la Expresión Génica , Datos de Secuencia Molecular , Unión Proteica , Recombinasa Rad51 , Rec A Recombinasas/genética , Proteínas de Saccharomyces cerevisiae , Schizosaccharomyces/metabolismoRESUMEN
The rhp51+ gene encodes three transcripts of 1.9, 1.6 and 1.3 kb which have at least six polyadenylation sites. Primer-extension analysis revealed that two transcription start points (tsp) at - 166 and - 136 were responsible for the DNA damage inducibility of this gene. Northern blot analyses showed that the three transcripts were expressed differentially in response to a variety of DNA damage. During the mitotic cell cycle, only the largest transcript exhibited periodic expression, reaching the maximal level in front of the cdc22+ transcript which peaks at the G1/S boundary. Unexpectedly, the steady-state levels of the three transcripts were differentially regulated during the growth cycle. The largest and smallest transcripts accumulated in large quantity at the diauxic shift and during the entry into stationary phase, respectively. To localize the regions responsible for the differential expression of rhp51+, we constructed rhp51::ura4 and ura4::rhp51 hybrid genes, and analyzed their expression patterns in response to methyl methanesulfonate (MMS)-induced DNA damage. The results showed that the promoter region and 5' half of rhp51+ are sufficient to confer damage-responsiveness while the 3' end of the gene alone can direct the formation of multiple, discrete 3' ends of the transcripts. From these results, we conclude that this novel one gene-multiple product system is possible through the cooperation of both the promoter and 3' terminal regions.
Asunto(s)
Proteínas Fúngicas/genética , Genes Fúngicos , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces/genética , Secuencia de Aminoácidos , Secuencia de Bases , Daño del ADN , Proteínas de Unión al ADN/genética , Regulación Fúngica de la Expresión Génica , Datos de Secuencia Molecular , Poli A/metabolismo , Regiones Promotoras Genéticas , ARN de Hongos/genética , ARN Mensajero/genética , Recombinasa Rad51 , Rec A Recombinasas/genética , Schizosaccharomyces/crecimiento & desarrolloRESUMEN
To understand the role of the Rhp51 protein in Schizosaccharomyces pombe, we examined the phenotypes of the null mutant for the rhp51+ gene. Unlike Saccharomyces cerevisiae rad51 mutants, S. pombe rhp51 mutants (rhp51delta cells) displayed slow growth and heterogeneity in cell size, indicating perturbation of the cell cycle. Furthermore, many aberrant nuclear structures found in 4',6'-diamidino-2-phenylindole (DAPI)-stained rhp51delta cells and the caffeine hypersensitivity of the mutant cells suggested an involvement of the Rhp51 protein in normal chromosome segregation. These data suggested that the Rhp51 proteins were required for normal cell growth as well as a DNA repair pathway. Moreover, rhp51delta mutants showed a considerable sensitivity to ultraviolet (UV) light-irradiation as well as methyl methanesulfonate (MMS) treatment, indicating that the Rhp51 proteins are involved in both the active excision mechanism of UV-induced DNA damage and recombinational repair in S. pombe. Taken together, we suggest that the role(s) of the Rhp51 protein in S. pombe may be different from those of Rad51 in S. cerevisiae.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas Fúngicas/genética , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces/metabolismo , Daño del ADN , Proteínas de Unión al ADN/metabolismo , Proteínas Fúngicas/metabolismo , Mitosis , Mutación Puntual , Recombinasa Rad51 , Proteínas de Saccharomyces cerevisiae , Schizosaccharomyces/genética , Schizosaccharomyces/crecimiento & desarrolloRESUMEN
A simple and efficient procedure was described for the isolation of total RNA from the fission yeast Schizosaccharomyces pombe. The present study demonstrated that the quality and the quantity of S. pombe RNA were increased by substituting phenol/chloroform mixture for phenol as a deproteinizing agent in the first vortexing step and using an ice bath instead of a dry ice-ethanol bath in the freezing step. Additionally, this protocol had the advantage of extracting total RNA without any degradation of S. pombe cells. Furthermore, the high amounts and quality of RNA extracted by this modified procedure enabled us to perform some experiments such as Northern blot, S1 mapping, primer extension, and reverse transcriptase reaction-polymerase chain reaction (RT-PCR) without further RNA purification. We suggest that this procedure is very useful to analyse primary structures and steady-state levels of RNA from S. pombe.
Asunto(s)
ARN de Hongos/aislamiento & purificación , Schizosaccharomyces/genética , Secuencia de Bases , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Schizosaccharomyces/químicaRESUMEN
The SFP2 gene of Saccharomyces cerevisiae has been characterized. The deduced amino acid sequence contained twelve highly hydrophobic domains and showed 50, 47, 44 and 48% homologies to Neurospora crassa sulfate permease II (CYS14), soybean GMAK170 nodulin, human colon mucosa protein (DRA) and a putative open reading frame (ORF) downstream of Escherichia coli prs (phosphoribosyl pyrophosphatate synthetase) gene, respectively, in the aligned regions. Cells lacking SFP2 were viable and displayed no obvious decrease in their growth rate. Southern blot analysis revealed that SFP2 exists as a single copy in haploid genome. Northern blot analysis showed that SFP2 produced a 2.8-kb transcript which was highly expressed under sulfur derepressing condition. SFP2 mRNA was found to turn over with a half-life of approximately 15 min, which may contribute to the regulation of sulfate permease function, and reached its maximal level in about 22 h after depression.
Asunto(s)
Proteínas de Transporte de Anión , Genes Fúngicos , Proteínas de Transporte de Membrana/genética , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Secuencia de Bases , Northern Blotting , Clonación Molecular , Cartilla de ADN , Escherichia coli/enzimología , Escherichia coli/genética , Humanos , Proteínas de Transporte de Membrana/biosíntesis , Proteínas de Transporte de Membrana/química , Datos de Secuencia Molecular , Mutagénesis , Neurospora crassa/enzimología , Neurospora crassa/genética , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Mapeo Restrictivo , Homología de Secuencia de Aminoácido , Glycine max/genética , Transcripción GenéticaRESUMEN
A homology (rhp51+) of the RAD51 gene in Schizosaccharomyces pombe was cloned by screening a Sz. pombe genomic library using the 3'-end of RAD51 from Saccharomyces cerevisiae as a probe. As in S. cerevisiae, the sequence of rhp51+ showed two MluI cell-cycle boxes and a putative DNA damage-responsive element in its upstream region. The open reading frame codes for a 365-amino-acid (aa) polypeptide with an estimated molecular mass of 40,555 Da. The deduced aa sequence shows 27, 66, 75 and 80% identity with Escherichia coli RecA, S. cerevisiae Rad51 and the Rad51 homologs from chicken and humans, respectively. The aa sequence encoded by rhp51+ contains A- and B-type nucleotide-binding consensus sequences, as found in other RAD51 homologs. Northern blot analysis showed that rhp51+ encodes a 1.7-kb transcript. Methyl methanesulfonate treatment increased the level of this transcript three- to fivefold. Southern hybridization analysis suggests that a single copy of rhp51+ exists in the Sz. pombe genome.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas Fúngicas/genética , Genes Fúngicos/genética , Saccharomyces cerevisiae/genética , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces/genética , Secuencia de Aminoácidos , Animales , Proteínas Aviares , Secuencia de Bases , Clonación Molecular , Daño del ADN , Reparación del ADN/genética , Proteínas de Unión al ADN/química , Proteínas Fúngicas/química , Humanos , Datos de Secuencia Molecular , Sistemas de Lectura Abierta/genética , ARN Mensajero/análisis , Recombinasa Rad51 , Rec A Recombinasas/genética , Mapeo Restrictivo , Proteínas de Saccharomyces cerevisiae , Alineación de Secuencia , Análisis de Secuencia de ADN , Transcripción GenéticaRESUMEN
A DNA polymerase alpha-associated multienzyme complex isolated from mouse LP1-1 cells transfected with the thymidine kinase gene of herpes simplex virus type I (1) showed activities of DNA polymerase alpha, topoisomerase II, and thymidine kinase (TK) in the complex. TK antiserum recognized a 43 kDa polypeptide only in the fraction of the multienzyme complex prepared from the LP1-1 cells but not that from L-M(TK-) cells. In permeabilized cells, hydroxyurea did not show any inhibitory effect on either DNA polymerase or TK, whereas aphidicolin, novobiocin, and TK antiserum inhibited both enzymes. These results provide evidence for the functional association and an allosteric interaction between the viral TK and host DNA polymerase alpha.