RESUMEN
The ability to form selective cell-cell adhesions is an essential property of metazoan cells. Members of the cadherin superfamily are important regulators of this process in both vertebrates and invertebrates. With the advent of genome sequencing projects, determination of the full repertoire of cadherins available to an organism is possible and here we present the identification and analysis of the cadherin repertoires in the genomes of Caenorhabditis elegans and Drosophila melanogaster. Hidden Markov models of cadherin domains were matched to the protein sequences obtained from the translation of the predicted gene sequences. Matches were made to 21 C. elegans and 18 D. melanogaster sequences. Experimental and theoretical work on C. elegans sequences, and data from ESTs, show that three pairs of genes, and two triplets, should be merged to form five single genes. It also produced sequence changes at one or both of the 5' and 3' termini of half the sequences. In D. melanogaster it is probable that two of the cadherin genes should also be merged together and that three cadherin genes should be merged with other neighbouring genes. Of the 15 cadherin proteins found in C. elegans, 13 have the features of cell surface proteins, signal sequences and transmembrane helices; the other two have only signal sequences. Of the 17 in D. melanogaster, 11 at present have both features and another five have transmembrane helices. The evidence currently available suggests about one-third of the cadherins in the two organisms can be grouped into subfamilies in which all, or parts of, the molecules are conserved. Each organism also has a approximately 980 residue protein (CDH-11 and CG11059) with two cadherin domains and whose sequences match well over their entire length two proteins from human brain. Two proteins in C. elegans, HMR-1A and HMR-1B, and three in D. melanogaster, CadN, Shg and CG7527, have cytoplasmic domains homologous to those of the classical cadherin genes of chordates but their extracellular regions have different domain structures. Other common subclasses include the seven-helix membrane cadherins, Fat-like protocadherins and the Ret-like cadherins. At present, the remaining cadherins have no obvious similarities in their extracellular domain architecture or homologies to their cytoplasmic domains and may, therefore, represent species-specific or phylum-specific molecules.
Asunto(s)
Cadherinas/química , Caenorhabditis elegans/química , Drosophila melanogaster/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Cadherinas/genética , Cadherinas/metabolismo , Calcio/metabolismo , Biología Computacional/métodos , Secuencia Conservada , Factor de Crecimiento Epidérmico/química , Genómica , Laminina/química , Modelos Moleculares , Datos de Secuencia Molecular , Familia de Multigenes , Estructura Terciaria de Proteína , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , VertebradosRESUMEN
An expression vector (CIp10-MAL2p) for use in Candida albicans has been constructed in which a gene of interest can be placed under the control of the CaMAL2 maltase promoter and stably integrated at the CaRP10 locus. Using this vector to express the Candida URA3 gene from the CaMAL2 promoter, we have demonstrated tight regulation of CaURA3 expression by carbon source. Thus under conditions when the CaMAL2 promoter is not induced, expression of Candida URA3 was unable either to complement a C. albicans ura3 mutation or to confer sensitivity to 5-fluoroorotic acid, a compound which is highly toxic to URA3 strains. Since Candida albicans is an obligate diploid organism, analysis of gene function requires manipulation of both copies of any gene of interest. Our expression vector provides a strategy by which the remaining copy of a gene of interest can be placed under CaMAL2 promoter control in a strain where the first copy has been deleted, permitting analysis of gene function by manipulation of carbon source. CIp10-MAL2p should therefore provide a useful means for functional analysis of genes in C. albicans. We have used this strategy with C. albicans DPB2 to demonstrate that the gene is essential and that loss of function leads cells to adopt a hypha-like morphology as they cease proliferation.
Asunto(s)
Antígenos Fúngicos , Candida albicans/genética , Regulación Fúngica de la Expresión Génica , Genes Fúngicos , Regiones Promotoras Genéticas , alfa-Glucosidasas/genética , Candida albicans/crecimiento & desarrollo , Medios de Cultivo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Vectores Genéticos , Glucosa/metabolismo , Maltosa/metabolismo , Proteínas Ribosómicas/genéticaRESUMEN
The CST20 gene of Candida albicans was cloned by functional complementation of a deletion of the STE20 gene in Saccharomyces cerevisiae. CST20 encodes a homolog of the Ste20p/p65PAK family of protein kinases. Colonies of C. albicans cells deleted for CST20 revealed defects in the lateral formation of mycelia on synthetic solid "Spider" media. However, hyphal development was not impaired in some other media. A similar phenotype was caused by deletion of HST7, encoding a functional homolog of the S. cerevisiae Ste7p protein kinase. Overexpression of HST7 partially complemented the deletion of CST20. Cells deleted for CST20 were less virulent in a mouse model for systemic candidiasis. Our results suggest that more than one signaling pathway can trigger hyphal development in C. albicans, one of which has a protein kinase cascade that is analogous to the mating response pathway in S. cerevisiae and might have become adapted to the control of mycelial formation in asexual C. albicans.
Asunto(s)
Candida albicans/fisiología , Candidiasis/fisiopatología , Quinasas de Proteína Quinasa Activadas por Mitógenos , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas de Saccharomyces cerevisiae , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Candida albicans/genética , Candida albicans/patogenicidad , Secuencia de Consenso , Cartilla de ADN , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Eliminación de Gen , Genes Fúngicos , Prueba de Complementación Genética , Biblioteca Genómica , Péptidos y Proteínas de Señalización Intracelular , Quinasas Quinasa Quinasa PAM , Masculino , Ratones , Ratones Endogámicos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Proteínas Quinasas/biosíntesis , Proteínas Quinasas/química , Proteínas Quinasas/fisiología , Proteínas Serina-Treonina Quinasas/biosíntesis , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Homología de Secuencia de Aminoácido , Transducción de Señal , VirulenciaRESUMEN
The Candida albicans clone cDNA10 was isolated on the basis that it encodes a protein which is immunogenic during infections in humans (R. K. Swoboda, G. Bertram, H. Hollander, D. Greenspan, J. S. Greenspan, N. A. R. Gow, G. W. Gooday, and A. J. P. Brown, Infect. Immun. 61:4263-4271, 1993). cDNA10 was used to isolate its cognate gene, and both the cDNA and gene were sequenced, revealing a major open reading frame with the potential to encode a basic protein of 256 amino acids with a predicted molecular weight of 29 kDa. Over its entire length, the open reading frame showed strong homology at both the nucleic acid (75 to 78%) and amino acid (79 to 81%) levels to two Saccharomyces cerevisiae genes encoding the 40S ribosomal protein, Rp10. Therefore, our C. albicans gene was renamed RP10. Northern (RNA) analyses in C. albicans 3153 revealed that RP10 expression is regulated in a manner very similar to that of S. cerevisiae ribosomal genes. The level of the RP10 mRNA decreased upon heat shock (from 25 to 45 degrees C) and was tightly regulated during growth. Maximal levels of the mRNA were reached during mid-exponential phase before they decreased to negligible levels in stationary phase. The level of the RP10 mRNA was induced only transiently during the yeast-to-hyphal morphological transition but did not appear to respond to hyphal development per se.