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We report the development of a homologous transformation system for Cephalosporium acremonium using the niaD gene of the nitrate assimilation (NA) pathway. Mutants in the NA pathway were selected on the basis of chlorate resistance by conventional means. Screening procedures were developed to differentiate between nitrate reductase apoprotein structural gene mutants (niaD) and molybdenum cofactor gene mutants (cnx) as wt. C. acremonium, unlike most filamentous fungi, fails to grow on minimal medium with hypoxanthine as a sole source of nitrogen. Phage clones carrying the niaD gene were isolated from a C. acremonium library constructed in λEMBL3 using the A. nidulans niaD gene as a heterologous probe. An 8.6-kb EcoRI fragment was subcloned into pUC18, and designated pSTA700. pSTA700 was able to transform stable niaD mutants to NA at a frequency of up to 40 transformants per μg DNA. Transformants were easily visible since the background growth was low and no abortives were observed. Gene replacements, single copy homologous integration and complex multiple integrations were observed. The niaD system was used to introduce unselected markers for hygromycin B resistance and benomyl resistance into C. acremonium by cotransformation.  相似文献   

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Abstract. The application of molecular approaches such as mutant analysis and recombinant DNA technology, in conjunction with immunology, are set to revolutionize our understanding of the nitrate assimilation pathway. Mutant analysis has already led to the identification of genetic loci encoding a functional nitrate reduction step and is expected to lead ultimately to the identification of genes encoding nitrate uptake and nitrite reduction. Of particular significance would be identification of genes whose products contribute to regulatory networks controlling nitrogen metabolism. Recombinant DNA techniques are particularly powerful and have already allowed the molecular cloning of the genes encoding the apoprotein of nitrate reductase and nitrite reductase. These successes allow for the first lime the possibility to study directly the role of environmental factors such as type of nitrogen source (NO3 or NH4+) available to the plant, light, temperature water potential and CO2 and O2 tensions on nitrate assimilation gene expression and its regulation at the molecular level. This is an important advance since our current understanding of the regulation of nitrate assimilation is based largely on changes of activity of the component steps. The availability of mutants, cloned genes, and gene transfer systems will permit attempts to manipulate the nitrate assimilation pathway.  相似文献   

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In order to undertake a comparative analysis of carbon catabolite repression in two Aspergillus species, the creA gene has been isolated from A. niger by cross hybridization, using the cloned A. nidulans gene. The A. niger gene has been shown to be functional in A. nidulans by heterologous complementation of the creA204 mutation of A. nidulans. Overall, the genes show 90% sequence similarity (82% identity) at the amino acid (aa) level. There were some striking similarities between the aa sequences encoded by the two fungal creA genes and two genes involved in carbon catabolite repression in Saccharomyces cerevisiae. The zinc-finger regions showed 96% similarity (84% identity) with the zinc-finger region of the MIG1 gene of S. cerevisiae. The CREA protein contains a stretch of 42 aa that is identical in A. niger and A. nidulans, and these show 81% similarity (33% identity) with a region of the S. cerevisiae RGR1 gene.  相似文献   

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