RESUMEN
The Aspergillus niger and Trichoderma reesei genes encoding the functional homologues of the small GTP-binding protein SAR1p, which is involved in the secretion pathway in Saccharomyces cerevisiae, have been cloned and characterised. The A. niger gene (sarA) contains five introns, whereas the T. reesei gene (sar1) has only four. In both cases the first intron is at the same position as the single S. cerevisiae SAR1 intron. The encoded proteins show 70-80% identity to the SAR1 protein. Complementation of S. cerevisiae sar1 and sec12 mutants by expression vectors carrying the A. niger sarA and T. reesei sar1 cDNA clones confirmed that the cloned genes are functional homologues of the S. cerevisiae SAR1 gene. Three mutant alleles of the A. niger sarA gene (D29G, E109K, D29G/E109K), generated by site-directed mutagenesis, revealed a thermosensitive dominant-negative phenotype in the presence of the wild-type sarA allele. This result contrasts with the situation in S. cerevisiae, where similar mutations have a thermosensitive phenotype. Taken together, our results indicate that the sarA gene is involved in an essential function in A. niger.
Asunto(s)
Aspergillus niger/genética , Proteínas Fúngicas/genética , Proteínas de Unión al GTP/genética , Genes Fúngicos , Proteínas de Unión al GTP Monoméricas , Proteínas de Saccharomyces cerevisiae , Trichoderma/genética , Alelos , Secuencia de Aminoácidos , Secuencia de Bases , ADN de Hongos , Proteínas Fúngicas/química , Proteínas de Unión al GTP/química , Prueba de Complementación Genética , Datos de Secuencia Molecular , Mutación , Saccharomyces cerevisiae , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Proteínas de Transporte VesicularRESUMEN
The sequence of the poliovirus genome encoding 3CD (a protease) was transferred to the yeast Saccharomyces cerevisiae on expression vectors with either a constitutive or an inducible promoter. Transformants could only be obtained with vectors carrying the inducible transcription unit. Extracts of induced cells were able to cleave cell-free synthesized P1, the precursor of the poliovirus capsid proteins, into VP0, VP3 and VP1. In yeast cells constitutively expressing P1, induction of 3CD expression resulted in only trace amounts of processed products. Processing could be improved considerably by simultaneous induction of both P1 and 3CD expression. Analysis of extracts of such induced cells revealed the presence of particles that resembled authentic subviral particles.
Asunto(s)
Cápside/biosíntesis , Cisteína Endopeptidasas/genética , Poliovirus/metabolismo , Saccharomyces cerevisiae/genética , Proteínas Virales , Proteasas Virales 3C , Secuencia de Bases , Cápside/genética , Cápside/aislamiento & purificación , Cápside/metabolismo , Cisteína Endopeptidasas/biosíntesis , Cisteína Endopeptidasas/metabolismo , Inducción Enzimática , Expresión Génica , Datos de Secuencia Molecular , Poliovirus/genética , Vacuna Antipolio de Virus Inactivados/genética , Precursores de Proteínas/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes de Fusión/biosíntesisRESUMEN
Although the application of filamentous fungi, such as Aspergillus niger for the production of extracellular proteins is well established for several decades, hardly any information is available about the molecular mechanisms of the process of protein secretion in these organisms. Two lines of research initiated towards a systematic analysis of the mechanism of protein targeting and secretion are presented in this paper. 1--To study routing and targeting of proteins in filamentous fungi the availability of a versatile reporter/carrier protein will be of considerable importance. Experiments towards the identification of such a protein are presented. 2--In analogy to the situation in Saccharomyces cerevisiae, the availability of defined (conditional) mutations in the secretion pathway will provide very important information about the organisation of the pathway. Therefore, based on results obtained in S. cerevisiae, the cloning of several fungal 'secretion' genes was started. The results of the cloning and characterisation of one of these genes is presented.
Asunto(s)
Proteínas Fúngicas/metabolismo , Hongos/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , ADN de Hongos/genética , Escherichia coli/enzimología , Escherichia coli/genética , Proteínas Fúngicas/genética , Hongos/genética , Hongos/ultraestructura , Genes Fúngicos , Genes Reporteros , Glucuronidasa/genética , Microscopía Electrónica , Datos de Secuencia MolecularRESUMEN
Rabbit reticulocyte lysates were programmed with either RNA extracted from purified poliovirus or a mixture of mRNAs encoding the capsid precursor, P1, and proteinase 3CD. In both cases, 14S subunits were formed at 30 degrees C and empty capsids at 37 degrees C. Both the 14S subunits and empty capsids had the expected polypeptide composition and neutralization epitopes. It is concluded that the proteinase 3CD gene is the only viral genetic information needed for the correct processing of P1 and the formation of 14S subunits, and their assembly into antigenically correct empty capsids.
Asunto(s)
Poliovirus/genética , Biosíntesis de Proteínas , Proteínas Virales , Proteasas Virales 3C , Animales , Antígenos Virales/genética , Antígenos Virales/inmunología , Cápside/genética , Cápside/inmunología , Sistema Libre de Células , Cisteína Endopeptidasas/genética , Cisteína Endopeptidasas/inmunología , Poliovirus/inmunología , Precursores de Proteínas/genética , Precursores de Proteínas/inmunología , ARN Viral/metabolismo , ConejosRESUMEN
The effect of several transcriptional regulatory elements on gene expression in mammalian cells was investigated. As a reporter gene we have used the bacterial gene merA coding for the enzyme mercuric reductase. Several plasmids were constructed with different promoter/enhancer sequences (pSV/E, pSV/L, pMT, pRSV or pAd) at the 5' end and different splicing (small intron of the T antigen of SV40 or the second intron of the rabbit beta-globin gene) and/or polyadenylation signals (AEn, ALn or AR beta Gn) at the 3' end of the merA gene. Expression was measured in five different mammalian cell lines. In COS cells the highest level of expression is obtained with pSV/L and the lowest level with pSV/E. In HeLa, CV-1, Ltk-, and CHO cells merA expression is relatively high, under control of pRSV and pMT and relatively low under control of pSV/L and pAd. The introns studied have a negative effect on the expression of merA. The presence of a polyadenylation signal downstream from the gene is essential for its expression. The three different polyadenylation signals studied give a similar stimulatory effect on the level of expression of the merA gene.