RESUMEN
Restricting the localization of the histone H3 variant CENP-A (Cse4 in yeast, CID in flies) to centromeres is essential for faithful chromosome segregation. Mislocalization of CENP-A leads to chromosomal instability (CIN) in yeast, fly and human cells. Overexpression and mislocalization of CENP-A has been observed in many cancers and this correlates with increased invasiveness and poor prognosis. Yet genes that regulate CENP-A levels and localization under physiological conditions have not been defined. In this study we used a genome-wide genetic screen to identify essential genes required for Cse4 homeostasis to prevent its mislocalization for chromosomal stability. We show that two Skp, Cullin, F-box (SCF) ubiquitin ligases with the evolutionarily conserved F-box proteins Met30 and Cdc4 interact and cooperatively regulate proteolysis of endogenous Cse4 and prevent its mislocalization for faithful chromosome segregation under physiological conditions. The interaction of Met30 with Cdc4 is independent of the D domain, which is essential for their homodimerization and ubiquitination of other substrates. The requirement for both Cdc4 and Met30 for ubiquitination is specifc for Cse4; and a common substrate for Cdc4 and Met30 has not previously been described. Met30 is necessary for the interaction between Cdc4 and Cse4, and defects in this interaction lead to stabilization and mislocalization of Cse4, which in turn contributes to CIN. We provide the first direct link between Cse4 mislocalization to defects in kinetochore structure and show that SCF-mediated proteolysis of Cse4 is a major mechanism that prevents stable maintenance of Cse4 at non-centromeric regions, thus ensuring faithful chromosome segregation. In summary, we have identified essential pathways that regulate cellular levels of endogenous Cse4 and shown that proteolysis of Cse4 by SCF-Met30/Cdc4 prevents mislocalization and CIN in unperturbed cells.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Inestabilidad Cromosómica , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas F-Box/metabolismo , Proteínas Ligasas SKP Cullina F-box/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Centrómero/metabolismo , Segregación Cromosómica , Dominios Proteicos , Proteolisis , UbiquitinaciónRESUMEN
Centromere identity is specified epigenetically by specialized nucleosomes containing the evolutionarily conserved centromeric histone H3 variant (Cse4 in budding yeast, CENP-A in humans) which is essential for faithful chromosome segregation. However, the mechanisms of epigenetic regulation of Cse4 have not been clearly defined. We have identified two kinases, Cdc5 (Plk1 in humans) and Ipl1 (Aurora B kinase in humans) that phosphorylate Cse4 to prevent chromosomal instability (CIN). Cdc5 associates with Cse4 in mitosis and Cdc5-mediated phosphorylation of Cse4 is coincident with the centromeric enrichment of Cdc5 during metaphase. Defects in Cdc5-mediated Cse4 phosphorylation causes CIN, whereas constitutive association of Cdc5 with Cse4 results in lethality. Cse4 is also a substrate for Ipl1 and phospho-mimetic cse4 mutants suppress growth defects of ipl1 and Ipl1 kinetochore substrate mutants, namely dam1 spc34 and ndc80. Ipl1-mediated phosphorylation of Cse4 regulates kinetochore-microtubule interactions and chromosome biorientation. We propose that collaboration of Cdc5- and Ipl1-mediated phosphorylation of Cse4 modulates kinetochore structure and function, and chromosome biorientation. These findings demonstrate how phosphorylation of Cse4 regulates the integrity of the kinetochore, and acts as an epigenetic marker for mitotic control.
Asunto(s)
Aurora Quinasas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Unión al ADN/metabolismo , Mitosis , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Aurora Quinasas/genética , Proteínas de Ciclo Celular/genética , Centrómero/metabolismo , Proteína A Centromérica/genética , Proteína A Centromérica/metabolismo , Proteínas Cromosómicas no Histona/genética , Segregación Cromosómica/genética , Proteínas de Unión al ADN/genética , Cinetocoros/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
DNA methylation is a common epigenetic signaling mechanism associated with silencing of repeated DNA and transcriptional regulation in eukaryotes. Here we report that DNA methylation in the human fungal pathogen Candida albicans is primarily localized within structural genes and modulates transcriptional activity. Major repeat sequences and multigene families are largely free of DNA methylation. Among the genes subject to DNA methylation are those associated with dimorphic transition between yeast and hyphal forms, switching between white and opaque cells, and iron metabolism. Transcriptionally repressed methylated loci showed increased frequency of C-to-T transitions during asexual growth, an evolutionarily stable pattern of repression associated mutation that could bring about genetic alterations under changing environmental or host conditions. Dynamic differential DNA methylation of structural genes may be one factor contributing to morphological plasticity that is cued by nutrition and host interaction.
Asunto(s)
Candida albicans/genética , Metilación de ADN/genética , Epigénesis Genética/genética , Regulación Fúngica de la Expresión Génica/genética , Genes Fúngicos/genética , Fenotipo , Secuencia de Bases , Northern Blotting , Southern Blotting , Cartilla de ADN/genética , Hierro/metabolismo , Datos de Secuencia Molecular , Mutación/genética , Análisis de Secuencia de ADN , Especificidad de la EspecieRESUMEN
The kinetochore (centromeric DNA and associated proteins) is a key determinant for high fidelity chromosome transmission. Evolutionarily conserved Scm3p is an essential component of centromeric chromatin and is required for assembly and function of kinetochores in humans, fission yeast, and budding yeast. Overexpression of HJURP, the mammalian homolog of budding yeast Scm3p, has been observed in lung and breast cancers and is associated with poor prognosis; however, the physiological relevance of these observations is not well understood. We overexpressed SCM3 and HJURP in Saccharomyces cerevisiae and HJURP in human cells and defined domains within Scm3p that mediate its chromosome loss phenotype. Our results showed that the overexpression of SCM3 (GALSCM3) or HJURP (GALHJURP) caused chromosome loss in a wild-type yeast strain, and overexpression of HJURP led to mitotic defects in human cells. GALSCM3 resulted in reduced viability in kinetochore mutants, premature separation of sister chromatids, and reduction in Cse4p and histone H4 at centromeres. Overexpression of CSE4 or histone H4 suppressed chromosome loss and restored levels of Cse4p at centromeres in GALSCM3 strains. Using mutant alleles of scm3, we identified a domain in the N-terminus of Scm3p that mediates its interaction with CEN DNA and determined that the chromosome loss phenotype of GALSCM3 is due to centromeric association of Scm3p devoid of Cse4p/H4. Furthermore, we determined that similar to other systems the centromeric association of Scm3p is cell cycle regulated. Our results show that altered stoichiometry of Scm3p/HJURP, Cse4p, and histone H4 lead to defects in chromosome segregation. We conclude that stringent regulation of HJURP and SCM3 expression are critical for genome stability.
Asunto(s)
Inestabilidad Cromosómica/genética , Proteínas Cromosómicas no Histona/genética , Proteínas de Unión al ADN/genética , Histonas/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Centrómero/genética , Centrómero/metabolismo , Inmunoprecipitación de Cromatina , Proteínas Cromosómicas no Histona/metabolismo , Segregación Cromosómica/genética , Proteínas de Unión al ADN/metabolismo , Expresión Génica , Histonas/metabolismo , Humanos , Cinetocoros/metabolismo , Saccharomyces cerevisiae/citología , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
At the core of chromosome segregation is the centromere, which nucleates the assembly of a macromolecular kinetochore (centromere DNA and associated proteins) complex responsible for mediating spindle attachment. Recent advances in centromere research have led to identification of many kinetochore components, such as the centromeric-specific histone H3 variant, CenH3, and its interacting partner, Scm3. Both are essential for chromosome segregation and are evolutionarily conserved from yeast to humans. CenH3 is proposed to be the epigenetic mark that specifies centromeric identity. Molecular mechanisms that regulate the assembly of kinetochores at specific chromosomal sites to mediate chromosome segregation are not fully understood. In this review, we summarize the current literature and discuss results from our laboratory, which show that restricting the localization of budding yeast CenH3, Cse4, to centromeres and balanced stoichiometry between Scm3 and Cse4, contribute to faithful chromosome transmission. We highlight our findings that, similar to other eukaryotic centromeres, budding yeast centromeric histone H4 is hypoacetylated, and we discuss how altered histone acetylation affects chromosome segregation. This article is part of a Special Issue entitled: Chromatin in time and space.
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Centrómero/genética , Ensamble y Desensamble de Cromatina , Cromatina/genética , Saccharomyces cerevisiae/genética , Animales , Centrómero/metabolismo , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Segregación Cromosómica , Cromosomas Fúngicos/genética , Cromosomas Fúngicos/metabolismo , Inestabilidad Genómica , Histonas/metabolismo , Humanos , Procesamiento Proteico-Postraduccional , Saccharomyces cerevisiae/fisiología , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Fishes are highly perishable, mainly stored and transported under frozen condition; however, they are mainly preferred as fresh or in chilled form for consumption because frozen storage compromises the texture quality and other sensory attributes. Shelf-life enhancement of rohu and seer fish steaks was studied using combination of ajwain extract (various concentrations) and γ-irradiation (various doses) under chilled storage (4 °C). These were analyzed periodically by microbiological, sensory, color and biochemical analyses during storage. Gas chromatography - mass spectrometry (GC-MS) analysis showed thymol as major component. High performance liquid chromatography (HPLC) analysis showed the presence of nine phenolic compounds including thymol in ajwain extract. The best result was achieved when samples were dipped in 10 % ajwain extract with 2 kGy gamma irradiation dose. An extended shelf-life of 5 and 10 days in case of rohu and seer fish steaks were attained respectively using this combination. These results were confirmed by microbial, biochemical and sensory analyses. The present study thus promises potential application of the above protocol in fishery industry for good quality of fish and commercial benefits.
Asunto(s)
Ammi , Apiaceae , Cyprinidae , Perciformes , Animales , Timol , Extractos Vegetales , Esperanza de VidaRESUMEN
Centromere (CEN) identity is specified epigenetically by specialized nucleosomes containing evolutionarily conserved CEN-specific histone H3 variant CENP-A (Cse4 in Saccharomyces cerevisiae, CENP-A in humans), which is essential for faithful chromosome segregation. However, the epigenetic mechanisms that regulate Cse4 function have not been fully defined. In this study, we show that cell cycle-dependent methylation of Cse4-R37 regulates kinetochore function and high-fidelity chromosome segregation. We generated a custom antibody that specifically recognizes methylated Cse4-R37 and showed that methylation of Cse4 is cell cycle regulated with maximum levels of methylated Cse4-R37 and its enrichment at the CEN chromatin occur in the mitotic cells. Methyl-mimic cse4-R37F mutant exhibits synthetic lethality with kinetochore mutants, reduced levels of CEN-associated kinetochore proteins and chromosome instability (CIN), suggesting that mimicking the methylation of Cse4-R37 throughout the cell cycle is detrimental to faithful chromosome segregation. Our results showed that SPOUT methyltransferase Upa1 contributes to methylation of Cse4-R37 and overexpression of UPA1 leads to CIN phenotype. In summary, our studies have defined a role for cell cycle-regulated methylation of Cse4 in high-fidelity chromosome segregation and highlight an important role of epigenetic modifications such as methylation of kinetochore proteins in preventing CIN, an important hallmark of human cancers.
Asunto(s)
Proteínas de Saccharomyces cerevisiae , Saccharomycetales , Humanos , Ciclo Celular , Centrómero/metabolismo , Proteína A Centromérica/metabolismo , Inestabilidad Cromosómica , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Unión al ADN/metabolismo , Metilación , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomycetales/metabolismoRESUMEN
Aim The study aimed to compare the effects of 0.5% bupivacaine injection at pre-incisional port sites and intraperitoneal application on postoperative pain in laparoscopic cholecystectomy cases. Methods After taking ethical clearance, a total of 60 patients of the American Society of Anaesthesia (ASA) grades 1 and 2 scheduled to undergo laparoscopic cholecystectomy were enrolled in the study and were randomized into two groups. Group 1 (n=30) received 20 ml of 0.5% bupivacaine infiltration subcutaneously over the port sites before being given incision. Group 2 (n=30) received 20 ml of 0.5% bupivacaine applied in the intraperitoneal subdiaphragmatic space and in the gall bladder fossa after removal of the gall bladder. The efficacy in terms of abdominal pain, hemodynamics, complications, and total analgesic requirements were assessed at regular intervals throughout the postoperative period for 24 hours. Results No significant difference in terms of demographic variables in the groups. The mean visual analog score (VAS) score for abdominal pain was found to be significantly lower in group 1 from the first postoperative hour till the twenty-fourth hour. Also, no significant difference was seen between the groups regarding hemodynamic parameters. No significant difference between the groups was seen regarding postoperative nausea and vomiting (PONV). The supplemental analgesic requirement was significantly higher in group 2 than in group 1. Conclusion It was observed from this study that pre-incisional infiltration of a local anesthetic agent produces effective postoperative analgesia in the immediate postoperative hours and reduces additional analgesic requirements without causing any adverse reactions.
RESUMEN
R-loops, the byproduct of DNA-RNA hybridization and the displaced single-stranded DNA (ssDNA), have been identified in bacteria, yeasts, and other eukaryotic organisms. The persistent presence of R-loops contributes to defects in DNA replication and repair, gene expression, and genomic integrity. R-loops have not been detected at centromeric (CEN) chromatin in wild-type budding yeast. Here we used an hpr1∆ strain that accumulates R-loops to investigate the consequences of R-loops at CEN chromatin and chromosome segregation. We show that Hpr1 interacts with the CEN-histone H3 variant, Cse4, and prevents the accumulation of R-loops at CEN chromatin for chromosomal stability. DNA-RNA immunoprecipitation (DRIP) analysis showed an accumulation of R-loops at CEN chromatin that was reduced by overexpression of RNH1 in hpr1∆ strains. Increased levels of ssDNA, reduced levels of Cse4 and its assembly factor Scm3, and mislocalization of histone H3 at CEN chromatin were observed in hpr1∆ strains. We determined that accumulation of R-loops at CEN chromatin contributes to defects in kinetochore biorientation and chromosomal instability (CIN) and these phenotypes are suppressed by RNH1 overexpression in hpr1∆ strains. In summary, our studies provide mechanistic insights into how accumulation of R-loops at CEN contributes to defects in kinetochore integrity and CIN.
Asunto(s)
Centrómero/metabolismo , Cromatina/química , Inestabilidad Cromosómica , Cinetocoros/metabolismo , Estructuras R-Loop , Saccharomycetales/metabolismo , Ciclo Celular , ADN de Hongos/metabolismo , Genoma Fúngico , Histonas/metabolismo , Modelos Biológicos , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomycetales/citología , Saccharomycetales/genéticaRESUMEN
Faithful chromosome segregation maintains chromosomal stability as errors in this process contribute to chromosomal instability (CIN), which has been observed in many diseases including cancer. Epigenetic regulation of kinetochore proteins such as Cse4 (CENP-A in humans) plays a critical role in high-fidelity chromosome segregation. Here we show that Cse4 is a substrate of evolutionarily conserved Cdc7 kinase, and that Cdc7-mediated phosphorylation of Cse4 prevents CIN. We determined that Cdc7 phosphorylates Cse4 in vitro and interacts with Cse4 in vivo in a cell cycle-dependent manner. Cdc7 is required for kinetochore integrity as reduced levels of CEN-associated Cse4, a faster exchange of Cse4 at the metaphase kinetochores, and defects in chromosome segregation, are observed in a cdc7-7 strain. Phosphorylation of Cse4 by Cdc7 is important for cell survival as constitutive association of a kinase-dead variant of Cdc7 (cdc7-kd) with Cse4 at the kinetochore leads to growth defects. Moreover, phospho-deficient mutations of Cse4 for consensus Cdc7 target sites contribute to CIN phenotype. In summary, our results have defined a role for Cdc7-mediated phosphorylation of Cse4 in faithful chromosome segregation.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Segregación Cromosómica/fisiología , Proteínas de Unión al ADN/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/fisiología , Centrómero/metabolismo , Proteína A Centromérica/metabolismo , Cromatina/metabolismo , Inestabilidad Cromosómica , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/fisiología , Cromosomas/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , Epigénesis Genética , Histonas/metabolismo , Cinetocoros/metabolismo , Proteínas Nucleares/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/fisiología , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiologíaRESUMEN
Red kidney beans are a staple pulse crop well known for its unique flavor characterized by kidney bean like, smoky, sulfury and earthy aroma notes. However, nature of compounds responsible for the unique beany odor of the cooked pulse has not been established. Steam distillation extracts of red kidney beans were subjected to Gas-chromatography olfactometry (GC-O) techniques namely detection frequency and aroma extract dilution analysis (AEDA). GC-O results suggest that methional with flavor dilution (FD) factor 21 is responsible for imparting the characteristic odor of the cooked red kidney beans. Apart from this p-vinyl guaiacol (FD 13) was identified as most important contributor towards smoky odor note. Sulfury note was mainly contributed by diethyl sulfide (FD 10) while 2-ethyl-3-methyl pyrazine (FD 13) was identified to be responsible for earthy note in cooked red kidney beans. Contribution of these compounds in characteristic aroma of cooked red kidney beans is reported here for the first time.
RESUMEN
Evolutionarily conserved polo-like kinase, Cdc5 (Plk1 in humans), associates with kinetochores during mitosis; however, the role of cell cycle-dependent centromeric ( CEN) association of Cdc5 and its substrates that exclusively localize to the kinetochore have not been characterized. Here we report that evolutionarily conserved CEN histone H3 variant, Cse4 (CENP-A in humans), is a substrate of Cdc5, and that the cell cycle-regulated association of Cse4 with Cdc5 is required for cell growth. Cdc5 contributes to Cse4 phosphorylation in vivo and interacts with Cse4 in mitotic cells. Mass spectrometry analysis of in vitro kinase assays showed that Cdc5 phosphorylates nine serine residues clustered within the N-terminus of Cse4. Strains with cse4-9SA exhibit increased errors in chromosome segregation, reduced levels of CEN-associated Mif2 and Mcd1/Scc1 when combined with a deletion of MCM21. Moreover, the loss of Cdc5 from the CEN chromatin contributes to defects in kinetochore integrity and reduction in CEN-associated Cse4. The cell cycle-regulated association of Cdc5 with Cse4 is essential for cell viability as constitutive association of Cdc5 with Cse4 at the kinetochore leads to growth defects. In summary, our results have defined a role for Cdc5-mediated Cse4 phosphorylation in faithful chromosome segregation.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Segregación Cromosómica/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces/metabolismo , Proteínas de Ciclo Celular/fisiología , Centrómero/metabolismo , Proteína A Centromérica/fisiología , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Unión al ADN/metabolismo , Histonas/metabolismo , Cinetocoros/metabolismo , Mitosis , Proteínas Nucleares/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/fisiología , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiología , Saccharomycetales/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMEN
Shugoshin is an evolutionarily conserved protein, which is involved in tension sensing on mitotic chromosomes, kinetochore biorientation, and protection of centromeric (CEN) cohesin for faithful chromosome segregation. Interaction of the C-terminus of Sgo1 with phosphorylated histone H2A regulates its association with CEN and pericentromeric (peri-CEN) chromatin, whereas mutations in histone H3 selectively compromise the association of Sgo1 with peri-CEN but not CEN chromatin. Given that histone H3 is absent from CEN and is replaced by a histone H3 variant CENP-ACse4, we investigated if CENP-ACse4 interacts with Sgo1 and promotes its association with the CEN chromatin. In this study, we found that Sgo1 interacts with CENP-ACse4 in vivo and in vitro. The N-terminus coiled-coil domain of Sgo1 without the C-terminus (sgo1-NT) is sufficient for its interaction with CENP-ACse4, association with CEN but not the peri-CEN, and this CEN association is cell cycle dependent with maximum enrichment in mitosis. In agreement with the role of CENP-ACse4 in CEN maintenance of Sgo1, depletion of CENP-ACse4 results in the loss of Sgo1 and sgo1-NT from the CEN chromatin. The N-terminus of Sgo1 is required for genome stability as a mutant lacking the N-terminus (sgo1-CT) exhibits increased chromosome missegregation when compared to a sgo1-NT mutant. In summary, our results define a novel role for the N-terminus of Sgo1 in CENP-ACse4 mediated recruitment of Sgo1 to CEN chromatin for faithful chromosome segregation.
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Centrómero/metabolismo , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomycetales/metabolismo , Ciclo Celular , Segregación Cromosómica , Unión Proteica , Dominios Proteicos , Técnicas del Sistema de Dos HíbridosRESUMEN
Centromeric localization of the evolutionarily conserved centromere-specific histone H3 variant CENP-A (Cse4 in yeast) is essential for faithful chromosome segregation. Overexpression and mislocalization of CENP-A lead to chromosome segregation defects in yeast, flies, and human cells. Overexpression of CENP-A has been observed in human cancers; however, the molecular mechanisms preventing CENP-A mislocalization are not fully understood. Here, we used a genome-wide synthetic genetic array (SGA) to identify gene deletions that exhibit synthetic dosage lethality (SDL) when Cse4 is overexpressed. Deletion for genes encoding the replication-independent histone chaperone HIR complex (HIR1, HIR2, HIR3, HPC2) and a Cse4-specific E3 ubiquitin ligase, PSH1, showed highest SDL. We defined a role for Hir2 in proteolysis of Cse4 that prevents mislocalization of Cse4 to noncentromeric regions for genome stability. Hir2 interacts with Cse4 in vivo, and hir2∆ strains exhibit defects in Cse4 proteolysis and stabilization of chromatin-bound Cse4 Mislocalization of Cse4 to noncentromeric regions with a preferential enrichment at promoter regions was observed in hir2∆ strains. We determined that Hir2 facilitates the interaction of Cse4 with Psh1, and that defects in Psh1-mediated proteolysis contribute to increased Cse4 stability and mislocalization of Cse4 in the hir2∆ strain. In summary, our genome-wide screen provides insights into pathways that regulate proteolysis of Cse4 and defines a novel role for the HIR complex in preventing mislocalization of Cse4 by facilitating proteolysis of Cse4, thereby promoting genome stability.
Asunto(s)
Proteína A Centromérica/metabolismo , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Centrómero/metabolismo , Proteína A Centromérica/genética , Cromatina/metabolismo , Segregación Cromosómica , Estudio de Asociación del Genoma Completo , Chaperonas de Histonas/genética , Chaperonas de Histonas/metabolismo , Histonas/metabolismo , Cinetocoros/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Unión Proteica , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Saccharomyces cerevisiae/genética , Saccharomycetales/metabolismo , Ubiquitina-Proteína Ligasas/genética , UbiquitinaciónRESUMEN
Volatile aroma compounds of three varieties of red kidney beans (Phaseolus vulgaris) namely Kashmiri red, Sharmili and Chitra were extracted in raw state using solid-phase microextraction (SPME) and cooked state using simultaneous distillation extraction (SDE). During cooking a significant (p<0.05) reduction in the content of several aldehydes, alcohols and terpene hydrocarbons while an increase in content of various sulfurous compounds, terpene alcohols, ketones and pyrazines was noted. Descriptive sensory analysis showed that the maximum intensity of 'kidney bean', 'earthy' and 'smoky' odour was observed in Kashmiri red while Sharmili variety was characterised by 'sulfurous' odour. Correlation of volatile profile data with descriptive sensory analysis and odour activity values clearly established the role of compounds, such as methanethiol, diethyl sulfide, dimethyl disulfide, methional and dimethyl trisulfide, in contributing to 'cooked kidney bean' aroma, while dimethyl sulfoxide, dimethyl sulfone and ethyl methyl sulfone were responsible for 'sulfurous' aroma.
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Culinaria , Odorantes/análisis , Phaseolus/química , Olfato , Alcoholes/análisis , Aldehídos/análisis , Humanos , Cetonas/análisis , Microextracción en Fase Sólida , Compuestos de Azufre/análisis , Terpenos/análisis , Verduras , Compuestos Orgánicos Volátiles/aislamiento & purificaciónRESUMEN
[This corrects the article on p. 198 in vol. 10, PMID: 27587998.].
RESUMEN
We studied how a neuronal circuit composed of two neuron types connected by chemical and electrical synapses maintains constant its integrative capacities as neurons grow. For this we combined electrophysiological experiments with mathematical modeling in pairs of electrically-coupled Retzius neurons from postnatal to adult leeches. The electrically-coupled dendrites of both Retzius neurons receive a common chemical input, which produces excitatory postsynaptic potentials (EPSPs) with varying amplitudes. Each EPSP spreads to the soma, but also crosses the electrical synapse to arrive at the soma of the coupled neuron. The leak of synaptic current across the electrical synapse reduces the amplitude of the EPSPs in proportion to the coupling ratio. In addition, summation of EPSPs generated in both neurons generates the baseline action potentials of these serotonergic neurons. To study how integration is adjusted as neurons grow, we first studied the characteristics of the chemical and electrical connections onto the coupled dendrites of neuron pairs with soma diameters ranging from 21 to 75 µm. Then by feeding a mathematical model with the neuronal voltage responses to pseudorandom noise currents we obtained the values of the coupling ratio, the membrane resistance of the soma (rm ) and dendrites (r dend), the space constant (λ) and the characteristic dendritic length (L = l/λ). We found that the EPSPs recorded from the somata were similar regardless on the neuron size. However, the amplitude of the EPSPs and the firing frequency of the neurons were inversely proportional to the coupling ratio of the neuron pair, which also was independent from the neuronal size. This data indicated that the integrative constancy relied on the passive membrane properties. We show that the growth of Retzius neurons was compensated by increasing the membrane resistance of the dendrites and therefore the λ value. By solely increasing the dendrite resistance this circuit maintains constant its integrative capacities as its neurons grow.
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The currently available nucleic acid amplification tests (NAATs) for trichomoniasis are accurate, quick and confirmative with superior sensitivity than traditional culture-based microbiology assays. However, these assays are associated with problems of carry over contamination, false positive results, requirement of technical expertise for performance and detection of end product. Hence, a diagnostic assay with easy visualization of the amplified product will be profitable. An in-house, rapid, sensitive, specific molecular-beacon-based PCR assay, using primers against pfoB gene of Trichomonas vaginalis, was developed and evaluated using dry ectocervical swabs (n=392) from symptomatic females with vaginal discharge. Total DNA was isolated and used as template for the PCR assays. The performance and reproducibility of PCR assay was evaluated by composite reference standard (CRS). For easy visualization of the amplified product, molecular-beacon was designed and amplicons were visualized directly using fluorescent handheld dark reader or by Micro-Plate Reader. Molecular-beacons are single-stranded hairpin shaped nucleic acid probes composed of a stem, with fluorophore/quencher pair and a loop region complementary to the desired DNA. The beacon-based PCR assay designed in the present study is highly specific as confirmed by competition experiments and extremely sensitive with detection limit of 20fg of genomic DNA (3-4 pathogens). The minimum infrastructure requirement and ease to perform the assay makes this method highly useful for resource poor countries for better disease management.
Asunto(s)
Colorimetría/instrumentación , Técnicas de Sonda Molecular/instrumentación , Reacción en Cadena de la Polimerasa/instrumentación , Vaginitis por Trichomonas/diagnóstico , Trichomonas vaginalis/genética , Trichomonas vaginalis/aislamiento & purificación , Adolescente , Adulto , Colorimetría/métodos , Sondas de ADN/genética , Diseño de Equipo , Análisis de Falla de Equipo , Femenino , Humanos , Persona de Mediana Edad , Técnicas de Diagnóstico Molecular/instrumentación , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Vaginitis por Trichomonas/parasitología , Adulto JovenRESUMEN
Proteinase inhibitors (PIs) are natural defense proteins of plants found to be active against gut proteases of various insects. A pigeonpea wild relative Cajanus platycarpus was identified as a source of resistance against Helicoverpa armigera, a most devastating pest of several crops including pigeonpea. In the light of earlier studies, trypsin-specific PIs (CpPI 63) were purified from mature dry seeds of C. platycarpus (ICPW-63) and characterized their biochemical properties in contributing to H. armigera resistance. CpPI 63 possessed significant H. armigera gut trypsin-like proteinase inhibitor (HGPI) activity than trypsin inhibitor (TI) activity. Analysis of CpPI 63 using two-dimensional (2-D) electrophoresis and matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry revealed that it contained several isoinhibitors and small oligomers with masses ranging between 6 and 58 kDa. The gelatin activity staining studies suggest that these isoinhibitors and oligomers possessed strong inhibitory activity against H. armigera gut trypsin-like proteases (HGPs). The N-terminal sequence of the isoinhibitors (pI 6.6 and pI 5.6) of CpPI 63 exhibited 80% homology with several Kunitz trypsin inhibitors (KTIs) as well as miraculin-like proteins (MLPs). Further, modification of lysine residue(s) lead to 80% loss in both TI and HGPI activities of CpPI 63. In contrast, the TI and HGPI activities of CpPI 63 were stable over a wide range of temperature and pH conditions. The reported results provide a biochemical basis for pod borer resistance in C. platycarpus.
RESUMEN
Sister chromatid cohesion is essential for tension-sensing mechanisms that monitor bipolar attachment of replicated chromatids in metaphase. Cohesion is mediated by the association of cohesins along the length of sister chromatid arms. In contrast, centromeric cohesin generates intrastrand cohesion and sister centromeres, while highly cohesin enriched, are separated by >800 nm at metaphase in yeast. Removal of cohesin is necessary for sister chromatid separation during anaphase, and this is regulated by evolutionarily conserved polo-like kinase (Cdc5 in yeast, Plk1 in humans). Here we address how high levels of cohesins at centromeric chromatin are removed. Cdc5 associates with centromeric chromatin and cohesin-associated regions. Maximum enrichment of Cdc5 in centromeric chromatin occurs during the metaphase-to-anaphase transition and coincides with the removal of chromosome-associated cohesin. Cdc5 interacts with cohesin in vivo, and cohesin is required for association of Cdc5 at centromeric chromatin. Cohesin removal from centromeric chromatin requires Cdc5 but removal at distal chromosomal arm sites does not. Our results define a novel role for Cdc5 in regulating removal of centromeric cohesins and faithful chromosome segregation.