RESUMEN
Using a yeast two-hybrid screen we isolated a gene from Schizosaccharomyces pombe which corresponds to the previously uncharacterized ORF SPCC1906.01. We have designated this gene as mpg1, based on the putative function of its product as a mannose-1-phosphatase guanyltransferase. Mpg1 shows strong similarity to other GDP-mannose-1-phosphate guanyltransferases involved in the maintenance of cell wall integrity and/or glycosylation. This homology, together with the protein's localization pattern demonstrated in this work, strongly suggests that Mpg1 is involved in cell wall and septum synthesis. Moreover, cells lacking Mpg1 present a defect in glycosylation, are more sensitive to Lyticase, and show an aberrant septum structure from the start of its deposition, indicating that the Mpg1 function is necessary for the correct assembly of the septum. Interestingly, lack of Mpg1 clearly affects cell cycle progression: mpg1 null mutants arrest as septated and bi-nucleated 4C cells, without an actomyosin ring. Wee1 is required for the G2/M arrest induced in the absence of Mpg1, since the blockade is circumvented when Wee1 is inactivated. Wee1 is part of a cell-size checkpoint that prevents entry into mitosis before cells reach a critical size. The results presented in this work demonstrate that the G2/M arrest induced in the absence of Mpg1 is mediated by this cell size checkpoint, since oversized mutant cells enter mitosis. The mpg1 loss-of-function mutant, therefore, provides a good model in which to study how cells coordinate cell growth and cell division.
Asunto(s)
Nucleotidiltransferasas/genética , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/citología , Schizosaccharomyces/genética , Secuencia de Aminoácidos , Secuencia de Bases , Ciclo Celular , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Pared Celular/enzimología , Citoplasma/enzimología , ADN de Hongos/genética , Perfilación de la Expresión Génica , Genes Fúngicos , Datos de Secuencia Molecular , Mutación , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Nucleotidiltransferasas/metabolismo , Proteínas Tirosina Fosfatasas/genética , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Schizosaccharomyces/enzimología , Proteínas de Schizosaccharomyces pombe/metabolismo , Homología de Secuencia de Aminoácido , Técnicas del Sistema de Dos HíbridosRESUMEN
In the fission yeast Schizosaccharomyces pombe, the wos2 gene encodes p23, a highly conserved protein which functions as a co-chaperone for the heat shock protein Hsp90. This p23 protein binds to Hsp90, but its activities and regulatory mechanisms are still unclear. Northern analysis has shown that the wos2 gene produces three transcripts of about 1.1, 0.9 and 0.8 kb, which are expressed differentially depending on the growth temperature. The largest and the smallest transcripts were most abundant at 25 degrees C, whereas the 0.9-kb transcript predominated at 37 degrees C. A time-course analysis indicated that this 0.9-kb species rapidly increased in abundance after a shift from 25 degrees C to 37 degrees C, reaching a maximum after 15 min. A shift back to 25 degrees C resulted in a decline in the amount of this transcript, albeit at a slower rate. Expression analysis of wos2:ura4 and nmt1:wos2 constructs showed that the 3' untranslated region of wos2 alone directs the formation of these multiple, discrete wos2 mRNAs. Sequence analysis of cDNAs derived from these mRNAs showed that the use of different polyadenylation sites results in the production of the three differently sized wos2 transcripts. In the case of the 0.9- and 0.8-kb mRNA species, these sites lie in a predicted hairpin loop in the mRNA, suggesting that polyadenylation signals in wos2 transcripts may be mediated by RNA secondary structure. The possibility that differential thermal stability of these hairpin structures could influence polyadenylation site choice during formation of the 3'-ends of the mRNAs is discussed.
Asunto(s)
Genes Fúngicos , Poli A/metabolismo , ARN de Hongos/metabolismo , ARN Mensajero/metabolismo , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces/genética , Regiones no Traducidas 3' , Proteínas Fúngicas/genética , Chaperonas Moleculares/genética , Fosfoproteínas/genética , Regiones Promotoras Genéticas , Análisis de Secuencia de ARN , TemperaturaRESUMEN
The Wee1 kinase inhibits entry into mitosis by phosphorylation of the Cdc2 kinase. Searching for multicopy suppressors that abolish this inhibition in the fission yeast, we have identified a novel gene, here named wos2, encoding a protein with significant homology to human p23, an Hsp90-associated cochaperone. The deletion mutant has a modest phenotype, being heat-shock sensitive. Using antibodies raised against bacterially produced protein, we determined that Wos2 is very abundant, ubiquitously distributed in the yeast cell, and its expression dropped drastically as cells entered into early stationary phase, indicating that its function is associated with cell proliferation. In proliferating cells, the amount of Wos2 protein was not subjected to cell cycle regulation. However, in vitro assays demonstrated that this Hsp90 cochaperone is potentially regulated by phosphorylation. In addition to suppressing Wee1 activity, overproduction of Wos2 displayed synthetic lethality with Cdc2 mutant proteins, indicating that this Hsp90 cochaperone functionally interacts with Cdc2. The level of Cdc2 protein and its associated H1 kinase activity under synthetic lethal conditions suggested a regulatory role for this Wos2-Cdc2 interaction. Hsp90 complexes are required for CDK regulation; the synergy found between the excess of Wos2 and a deficiency in Hsp90 activity suggests that Wos2 could specifically interfere with the Hsp90-dependent regulation of Cdc2. In vitro analysis indicated that the above genetic interactions could take place by physical association of Wos2 with the single CDK complex of the fission yeast. Expression of the budding yeast p23 protein (encoded by the SBA1 gene) in the fission yeast indicated that Wos2 and Sba1 are functionally exchangeable and therefore that properties described here for Wos2 could be of wide significance in understanding the biological function of cochaperone p23 in eukaryotic cells.
Asunto(s)
Proteínas Fúngicas/genética , Mitosis , Schizosaccharomyces/citología , Secuencia de Aminoácidos , Cartilla de ADN , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Humanos , Datos de Secuencia Molecular , Schizosaccharomyces/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
The eukaryotic translation initiation factor 4A (eIF4A) is an RNA helicase required for translation initiation of eukaryotic mRNAs. By engineering fission yeast mutants with diminished eIF4A activity, we have found that translation of cdc25 mRNAs (a dosage-dependent activator of mitosis in all eukaryotic cells) is particularly sensitive to limitations of protein synthesis mediated by limited eIF4A activity. Genetic and biochemical analysis indicated that a rate-limited translation initiation of cdc25 mRNAs, exerted throughout its unusual 5' untranslated leader, acts as a molecular sensor to ensure that a minimum cell mass (protein synthesis) is attained before mitosis occurs. The Cdc13 cyclin B is also among the limited pool of proteins whose translation is sensitive to reduced translation initiation activity. Interestingly, the 5' leader sequences of cdc25 and cdc13 mRNAs have conserved features which are unusual in other yeast mRNAs, suggesting that common mechanisms operate in the expression of these two key mitotic activators at the translational level.
Asunto(s)
Biosíntesis de Proteínas , Schizosaccharomyces/citología , Schizosaccharomyces/genética , Fosfatasas cdc25/genética , Regiones no Traducidas 5' , Secuencia de Bases , División Celular/efectos de los fármacos , División Celular/fisiología , Ciclina B/genética , Cartilla de ADN/genética , Factor 4A Eucariótico de Iniciación , Genes Fúngicos , Mitosis/genética , Mitosis/fisiología , Conformación de Ácido Nucleico , Iniciación de la Cadena Peptídica Traduccional , Factores de Iniciación de Péptidos/metabolismo , ARN de Hongos/química , ARN de Hongos/genética , ARN de Hongos/metabolismo , ARN Mensajero/química , ARN Mensajero/genética , ARN Mensajero/metabolismo , Schizosaccharomyces/metabolismoRESUMEN
The chromosome complement of Danio rerio was investigated by Giemsa staining and C-banding, Ag-NORs and replication banding. The diploid number of this species is 2n = 50 and the arm number (NF) = 100. Constitutive heterochromatin was located at the centromeric position of all chromosome pairs. Nucleolus organizer regions appeared in the terminal position of the long arms of chromosomes 1, 2 and 8. Replication banding pattern allowed the identification of each chromosome pair.