RESUMEN
A subtraction library was prepared from cultures of Aspergillus niger that had or had not been exposed to dithiothreitol (DTT), in order to identify genes involved in the unfolded protein response (UPR) or in the response to reductive stress. A large fraction of the clones in the library (40%) encoded two putative methyltransferases (MTs) whose function has yet to be determined. Other stress-responsive genes included a homologue of the Mn2+-containing superoxide dismutase gene (sodB) and a number of genes predicted to code for products that function in protein turnover and in intra- and extracellular transport of molecules. Transcriptional microarray analysis was carried out with a group of 15 genes, comprising 11 from the cDNA library, two genes linked to the putative MT genes but not represented in the library, and two UPR control genes (bipA and pdiA). Eleven of the 15 genes were inducible with DTT. This was either reflected by the presence of transcripts in cells subjected to DTT stress compared to absence under control conditions, or by an induction ratio of between 1.4 and 8.0 in cases where transcripts were already detectable under control conditions. The MT genes were among the four most highly induced. None of the genes, apart from bipA and pdiA, showed significant induction in response to other stresses that are known to induce the UPR in fungi. We conclude that DTT alone does not provide for specific induction of UPR genes and that other stress conditions must also be examined.
Asunto(s)
Aspergillus niger/genética , Aspergillus niger/metabolismo , Ditiotreitol/química , Regulación Fúngica de la Expresión Génica , Secuencia de Aminoácidos , ADN Complementario/metabolismo , Proteínas Fúngicas/química , Biblioteca de Genes , Datos de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Estrés Oxidativo , Plásmidos/metabolismo , Pliegue de Proteína , Saccharomyces cerevisiae/metabolismo , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Transcripción GenéticaRESUMEN
Mortierella alpina was transformed successfully to hygromycin B resistance by using a homologous histone H4 promoter to drive gene expression and a homologous ribosomal DNA region to promote chromosomal integration. This is the first description of transformation in this commercially important oleaginous organism. Two pairs of histone H3 and H4 genes were isolated from this fungus. Each pair consisted of one histone H3 gene and one histone H4 gene, transcribed divergently from an intergenic promoter region. The pairs of encoded histone H3 or H4 proteins were identical in amino acid sequence. At the DNA level, each histone H3 or H4 open reading frame showed 97 to 99% identity to its counterpart but the noncoding regions had little sequence identity. Unlike the histone genes from other filamentous fungi, all four M. alpina genes lacked introns. During normal vegetative growth, transcripts from the two histone H4 genes were produced at approximately the same level, indicating that either histone H4 promoter could be used in transformation vectors. The generation of stable, hygromycin B-resistant transformants required the incorporation of a homologous ribosomal DNA region into the transformation vector to promote chromosomal integration.
Asunto(s)
ADN Ribosómico/genética , Vectores Genéticos/genética , Histonas/genética , Mortierella/genética , Regiones Promotoras Genéticas/genética , Transformación Genética , ADN de Hongos/genética , Ácidos Grasos Insaturados/metabolismo , Datos de Secuencia Molecular , Mortierella/metabolismo , Aceites/química , Aceites/metabolismoRESUMEN
Genes encoding two distinct fatty acid delta9-desaturases were isolated from strains of the oleaginous fungus Mortierella alpina. Two genomic sequences, delta9-1 and delta9-2, each containing a single intron, were cloned from strain CBS 528.72 while one cDNA clone, LM9, was isolated from strain CBS 210.32. The delta9-1 gene encoded a protein of 445 aa which shared 99% identity with the LM9 gene product. These proteins also showed 40-60% identity to the delta9-desaturases (Ole1p) of other fungi and contained the three conserved histidine boxes, C-terminal cytochrome b5 fusion and transmembrane domains characteristic of endoplasmic reticulum membrane-bound delta9-desaturases. LM9 and delta9-1 are therefore considered to represent the same gene (ole1). The ole1 gene was transcriptionally active in all M. alpina strains tested and its function was confirmed by complementation of the Saccharomyces cerevisiae ole1 mutation. Fatty acid analysis of yeast transformants expressing the CBS 210.32 ole1 gene showed an elevated level of oleic acid (18:1) compared to palmitoleic acid (16:1), the major fatty acid component of wild-type S. cerevisiae. This indicated that the M. alpina delta9-desaturase had a substrate preference for stearic acid (18:0) rather than palmitic acid (16:0). Genomic clone delta9-2 (ole2) also encoded a protein of 445 aa which had 86% identity to the delta9-1 and LM9 proteins and whose ORF also complemented the yeast ole1 mutation. The transcript from this gene could only be detected in one of the six M. alpina strains tested, suggesting that its expression may be strain-specific or induced under certain physiological conditions.