Nitrate reactive structural gene mutants of Aspergillus nidulans

Two strains characterized as niaD structural gene mutants in Aspergillus nidulans produce a nitrate reductase which retains the ability to react with nitrate while lacking the ability to oxidize its naturally occurring substrate NADPH. Fifteen such nitrate reactive niaD strains exhibited strong interallelic complementation when tested against strains bearing point mutations in eleven other loci essential to induction and synthesis of nitrate reductase in Aspergillus. Fourteen representatives of this phenotype formed enzyme with a molecular weight equivalent to that of the wild type (200 kD) and also remained inducible by nitrate and repressible by ammonium. The mutation appears to alter the NADPH binding domain of the nitrate reductase since the affinity for the dinucleotide fold in Affigel blue and the dissociation constant (Ks) for enzyme isolated from the mutants on the basis of reduced methyl viologen-nitrate reductase activity is significantly less than that observed for the native enzyme from the wild type.     

A third unlinked gene controlling the pyruvate dehydrogenase complex in Aspergillus nidulans.

Pyruvate dehydrogenase complex mutants of Aspergillus nidulans were obtained by ultraviolet treatment and enrichment procedures. Among 160 glycolytic mutants, 86 pyruvate dehydrogenase complex mutants (including some temperature-sensitive mutants) were found. In addition to genes pdhA and pdhB, which are described in previous studies, a third gene, pdhC, controlling the function of the enzyme complex, was identified. The three genes were not linked and were mapped in the following linkage groups: pdhA in group I, pdhB in group V, and pdhC in group VIII, where it was the first marker on the left arm.     

The regulation of NADP-linked isocitrate dehydrogenase in Aspergillus nidulans

The regulation of NADP-linked isocitrate dehydrogenase (NADP-IDH) has been studied in wild-type and mutant strains of Aspergillus nidulans. In the wild-type strain studied, the levels of NADP-IDH vary in a similar way to those of acetamidase, acetyl-CoA synthase, isocitrate lyase and malate synthase under all growth conditions used. Similarly, fac mutants, which are altered in the regulation of these enzymes of acetate utilization, are affected in NADP-IDH levels in a parallel fashion, as are cre mutants, which show altered carbon catabolite repression of this group of enzymes. Possible functions of the NADP-IDH enzyme are considered.     

The genetic control of molybdoflavoproteins in Aspergillus nidulans. A xanthine dehydrogenase I half-molecule in cnx- mutant strains of Aspergillus nidulans.

The cnx- group of mutants of Aspergillus nidulans lacks xanthine dehydrogenase (xanthine: NAD+ oxidoreductase, EC 1.2.1.37) and nitrate reductase (EC 1.6.6.3) activities and are thought to be defective in the synthesis of a molybdenum-containing cofactor, 'cnx', common to xanthine dehydrogenase and nitrate reductase [Pateman, J.A., Rever, B.M., Cove, D.J. and Roberts, D.B. (1964) Nature (Lond.) 201, 58-60]. The cnx cofactor has a role in maintaining the aggregated multimeric structure of nitrate reductase [MacDonald, D.W., Cove, D.J. and Coddington, A. (1974) Mol. Gen. Genet. 128, 187-199]. We report here that, in cnx- mutants grown under conditions inducing xanthine dehydrogenase I, a species cross-reacting with antisera to the native enzyme and of half its molecular weight is present, together with cross-reacting molecules of similar molecular weight to the native enzyme. This suggests that the cnx cofactor has a role in maintaining the aggregated structure of xanthine dehydrogenase I. Both cross-reacting species are capable of passing reducing equivalents from NADH to a tetrazolium salt, showing that the cnx cofactor is not necessary for enzymic activity towards NADH.     

Two unlinked genes for the pyruvate dehydrogenase complex in Aspergillus nidulans.

The activity of the overall pyruvate dehydrogenase complex was found to be similar in extracts of Aspergillus nidulans after growth on either sucrose or acetate. Eight mutants lacking the activity of this complex were found among some 200 glycolytic mutants selected for their inability to grow on sucrose. The absence of pyruvate dehydrogenase complex activity was also confirmed for a mutant, g6 (pdhA1), isolated previously. Studies with the mutants supported the existence of two unlinked genes, pdhA and pdhB, controlling the function of the complex. In vivo and in vitro complementation between mutations at the two loci were shown by the ability of forced heterokaryons to grow on sucrose and by the restoration of overall pyruvate dehydrogenase complex activity in mixed cell-free extracts. The mutations were recessive to their wild-type alleles, and the pdhA and pdhB loci were assigned to linkage groups I and V, respectively.     

Identification of a gene for alpha-tubulin in Aspergillus nidulans.

This paper demonstrates that revertants of temperature-sensitive benA (β-tubulin) mutations in Aspergillus nidulans can be used to identify proteins which interact with β-tubulin. Three benomyl-resistant benA (β-tubulin) mutants of Aspergillus nidulans, BEN 9, BEN 15 and BEN 19, were found to be temperature-sensitive (ts^?) for growth. Temperature sensitivity co-segregated with benomyl resistance among the progeny of outcrosses of BEN 9, 15 and 19 to a wild-type strain, FGSC#99, indicating that temperature sensitivity was caused by mutations in the benA gene in these strains. Eighteen revertants to ts⁺ were isolated by selection at the restrictive temperature. Four had back-mutations in the benA gene and fourteen carried extragenic suppressor mutations. Two of the back-mutated strains had β-tubulins which differed from the β-tubulins of their parental strains by one (1^?) or two (2^?) negative charges on two-dimensional gel electrophoresis. Although the β-tubulins of the extragenic suppressor strains were all electrophoretically identical to those of the parental strains, one of the suppressor strains, BEN 9R7, had an electrophoretic abnormality in α1-tubulin (1⁺). A heterozygous diploid between this strain and a strain with wild-type α1-tubulin was found to have both wild-type and mutant (1⁺) α1-tubulins. This experiment rules out post-translational modification as a possible cause of the α1-tubulin abnormality. Thus the suppressor mutation in BEN 9R7 must be in a structural gene for α1-tubulin. We propose that this gene be designated tubA to denote that it is a gene for α1-tubulin in A. nidulans.     

Identification of a gene for beta-tubulin in Aspergillus nidulans.

The tubulins of Aspergillus nidulans have been characterized in wild-type and ben A, B and C benomyl-resistant strains by two-dimensional gel electrophoresis, co-polymerization with porcine brain tubulin and peptide mapping. Four α-tubulins and at least four β-tubulins were resolved by two-dimensional gel electrophoresis of wild-type proteins. Eighteen of 26 benA mutants studied had electrophoretically abnormal β-tubulins. In these strains, one or more of the β-tubulins had either an altered isoelectric point or an altered electrophoretic mobility in the SDS gel dimension, or was diminished in amount. The a-tubulins were normal. Two-dimensional gels of protein extracts of a ben A/wild-type diploid strain demonstrated co-expression of the wild-type β-tubulins with the variant ben A tubulin. This experiment rules out post-translational modification as the source of the β-tubulin abnormalities in the benA mutants. We therefore conclude that benA must be a structural gene for β-tubulin. Due to the variety of abnormalities affecting β-tubulins in ben A mutants, and the absence of abnormalities affecting α-tubulins in any of the benomyl-resistant mutants, we also believe that the benomyl binding site must be located on the β-subunit of the tubulin dimer. The benA mutants of A. nidulans promise to be useful not only for characterizing the biochemical determinants of the benomyl binding site of tubulin but also for understanding the relationship between tubulin structure and function.     

构巢曲霉奎尼酸脱氢酶基因qutB在大肠杆菌中的克隆与表达

从莽草酸途径合成奎尼酸（QA）,奎尼酸脱氢酶（QDHase)是必须的,大肠杆菌基因组不含有该酶的编码基因,在构巢曲霉（Aspergillus nidulans)中存在qutB基因编码此酶。以构巢曲染色体DNA为模板,采用PCR扩增得到qutB基因,在大肠杆菌中进行了克隆表达。结果表明,该基因在λ噬菌体的PRPL串联启动子驱动下实现了QDHase的表达。SDS－PAGE显示,重组菌热诱导后出大小约36000的特异蛋白,表达量占菌体总蛋白的19．81％。经酶活性测定,表达产物具有生物学活性,与对照菌株相比,其酶活性提高了27．8倍,为进一步奎尼酸生物合成研究了奠定了基础。     

A mutant of Aspergillus nidulans defective in NAD-linked glutamate dehydrogenase.

A mutation leading to partial loss of NAD-linked ("catabolic') glutamate dehydrogenase does not affect the regulation of ammonium-repressible activities in Aspergillus nidulans. This mutation has been used to show that NAD-linked glutamate dehydrogenase does not normally participate in ammonium assimilation. A mutation leading to loss of NADP-linked ("anabolic') glutamate dehydrogenase has been used to show that NADP-linked glutamate dehydrogenase is not normally involved in glutamate catabolism. Strains defective in either enzyme are useful for determining which amino acids are metabolised via transamination to yield glutamate rather than via deamination to yield ammonium.     

Developmental gene regulation in Aspergillus nidulans

The Ascomycete fungus Aspergillus nidulans reproduces asexually by differentiating conidiophores and conidia. Gene regulation during asexual reproduction was investigated by comparing poly(A) RNA populations derived from somatic hyphae, conidiating cultures and purified conidia. Single-copy and complementary DNA hybridization experiments showed that vegetative cells contained 5600–6000 diverse, average-sized poly(A) RNA sequences distributed into three prevalence classes. cDNA hybridization experiments indicated that a significant proportion of the poly(A) RNA derived from either conidiating cultures or spores consisted of sequences absent from somatic hyphae. To assess accurately the degree to which the poly(A) RNA populations differed, cDNA preparations were isolated which were complementary to sequences present only in conidia or in conidiating cultures. Hybridization of these cDNAs with poly(A) RNA from conidiating cultures showed that approximately 18.5% of the poly(A) RNA mass comprised 1300 diverse sequences not present in somatic cells. Of these, about 300 were present only in conidia. The remainder were accumulated specifically during sporulation, but were absent from spores. Analogous experiments showed that the great majority of the poly(A) RNA sequences accumulated by vegetative hyphae were also present in conidiating cultures. Thus, cell differentiation during A. nidulans asexual reproduction involves the accumulation of many new poly(A) RNA sequences, but not the loss of preexisting ones.     

Direct and indirect gene replacements in Aspergillus nidulans.

We performed three sets of experiments to determine whether cloned DNA fragments can be substituted for homologous regions of the Aspergillus nidulans genome by DNA-mediated transformation. A linear DNA fragment containing a heteromorphic trpC+ allele was used to transform a trpC- strain to trpC+. Blot analysis of DNA from the transformants showed that the heteromorphic allele had replaced the trpC- allele in a minority of the strains. An A. nidulans trpC+ gene was inserted into the argB+ gene, and a linear DNA fragment containing the resultant null argB allele was used to transform a trpC- argB+ strain to trpC+. Approximately 30% of the transformants were simultaneously argB-. The null argB allele had replaced the wild-type allele in a majority of these strains. The A. nidulans SpoC1 C1-C gene was modified by removal of an internal restriction fragment and introduced into a trpC- strain by transformation with a circular plasmid. A transformant containing a tandem duplication of the C1-C region separated by plasmid DNA was self-fertilized, and trpC- progeny were selected. All of these had lost the introduced plasmid DNA sequences, whereas about half had retained the modified C1-C gene and lost the wild-type copy. Thus, it is possible with A. nidulans to replace chromosomal DNA sequences with DNA fragments that have been cloned and modified in vitro by using either one- or two-step procedures similar to those developed for Saccharomyces cerevisiae.