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Induced segregation in interspecific hybrids of Aspergillus nidulans and Aspergillus rugulosus obtained by protoplast fusion.

Interspecific hybrids produced by polyethylene glycol induced fusion of protoplasts from auxotrophic mutants of Aspergillus nidulans and Aspergillus rugulosus were grown in the presence of the recombinogens benomyl and chloral hydrate to stimulate segregation. The A. nidulans parental strains used had a known genetic marker in each linkage group. Hybrids grown on complete medium containing benomyl yielded more segregants. Analysis of the segregants showed that the distribution of A. nidulans linkage groups was random. No specific linkage group appeared in all the segregants. The two parents are closely related taxonomically and the findings from these experiments suggest that a high degree of chromosomal homology may exist between them.

Aspergillus

Hybridization and selection for increased penicillin titre in wild-type isolates of Aspergillus nidulans.

Repeated hybridization and selection among wild-type isolates produced strains of Aspergillus nidulans with increased penicillin titre. Four independent selection lines were established, each originating from a sexual cross between two different heterokaryon-incompatible wild-type isolates. In each generation, two selected high-titre sister strains were crossed to produce the next generation. An initial increase in titre was obtained in each line, but after four or five generations of selection the genetic variation was considerably reduced and the rate of response to selection had decreased. From a base population of wild-type isolates with a mean titre of 8-6 units/ml the progeny mean titre was raised to between 16 and 20 units/ml in each line. The gradual nature of the response suggests that a number of genes determine penicillin titre in the wild-type isolates used. The gene action throughout the selection programme was predominantly additive.

Aspergillus nidulans

Cis-dominant regulatory mutations affecting the expression of GABA permease in Aspergillus nidulans.

In Aspergillus nidulans expression of the gabA gene, the probable structural gene for the gamma-amino-n-butyrate (GABA) permease, is controlled by induction, via the intA gene, ammonium repression, mediated by the areA gene, and probably carbon catabolite repression. Regulatory mutations, tightly linked to gabA, were selected by reverting an areAr-2 strain on GABA as nitrogen source. These mutations, gabI-1, gabI-2, and gabI-3 result in increased gabA expression and are cis-dominant in their effects on the gabA gene. Mapping data show that the regulatory mutations map on one side of all gabA- alleles tested.

Aspergillus nidulans

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.

Aspergillus nidulans

[Presence of sexual forms (cleistothecia and Hülle cells) in a case of human maxillary sinus aspergillosis caused by Aspergillus nidulans in association with Aspergillus fumigatus].

The authors observed, in a biopsic sample from a human maxillary sinus, the coexistence, in parasitic stages, of two different Aspergillus species: A. fumigatus and A. nidulans. After having described the morphologies of each species, in sections on slides, and particularly those of cleistothecia and Hülle-cells for the second, they draw attention to the three interests of such an observation: -- rarety of observing Aspergillus nidulans in a parasitic stage, in spite of its particular thermotolerant character. -- association of two different Aspergillus species in a same sample, in parasitic stages, observed in man for the first time, at least according their informations. -- presence of reproductive sexual forms, observed in vivo for the first time, in an Aspergillus species.

Adult

Reversion in variants from a duplication strain of Aspergillus nidulans.

Strains of Aspergillus nidulans with a chromosome segment in duplicate, one in normal position and one translocated to another chromosome, are unstable at mitosis. In addition to variants which result from deletions in either of the duplicate segments, which usually have improved morphology, they produce variants with deteriorated morphology. Three deteriorated variants reverted frequently to parental type morphology, both spontaneously and after ultra-violet treatment. Of six reversions analysed genetically, five were due to suppressors and one was probably due to back mutation. The suppressors segregated as single genes and were not linked to the mutation which they suppress. The instability of these so-called "deteriorate"variants is discussed in relation to mitotic instability phenomena in A. nidulans.

Aspergillus nidulans

Assimilation of ammonia and growth of biotin deficient Aspergillus nidulans.

Biotin deficiency in Aspergillus nidulans has been found to increase the uptake of ammonium ions, associated with a marked increase in the activity of NADP-linked glutamate dehydrogenase, which is found to be the major route of ammonia assimilation in this culture. The results obtained are discussed with respect to the growth of Aspergillus nidulans during biotin deficiency.

Alanine

Factors affecting protein synthesis during biotin deficiency in Aspergillus nidulans.

Biotin deficiency in Aspergillus nidulans resulted in a 70% increase of the protein content and increased levels of free and bound aspartate, glutamate, serine, leucine and methionine. Likewise, the activities of NADP+ glutamate dehydrogenase, NAD+ gluatmate dehydrogenase, aspartate aminotransferase and alanine aminotransferase were significantly increased. The total RNA content increased while the DNA content was unaffected. The rRNA/tRNA ratio remained higher in biotin-deficient cells. Supplementation of glutamate, aspartate, serine, leucine and methionine to the culture medium raised the rRNA/tRNA ratio, and the difference observed in the qualitative and the quantitative patterns of protein and dry cell mass between normal and biotin-deficient cultures was abolished.

Alanine Transaminase

Multiple independent control mechanisms affecting the acetamidase of Aspergillus nidulans.

The acetamidase of Aspergillus nidulans is induced by sources of acetyl CoA, benzoate and benzamide and by beta-alanine and other omega-amino acids. The effects of these groups of inducers are appromimately additive. The cis-acting control site mutant, amdI9, affects induction by sources of acetyl-CoA specifically. Lesions in the amdR and gatA genes affect induction by omega-amino acids specifically. Mutations in the amdA gene can lead to elevated acetamidase levels which still respond to the various inducers. The induction controls act independently of repression control by nitrogen metabolites and are not altered by the areA102 mutation. The properties of double mutants with lesions affecting the different control mechanisms also indicate their independence of each other. It is suggested that the acetamidase is subject to complex control by multiple regulatory circuits and that functionally independent control sites adjacent to the structural gene occur.

Acetamides

Genetic damage induced by ethyl alcohol in Aspergillus nidulans.

Heterozygous diploid conidia of Aspergillus nidulans were treated during germination with ethyl alcohol in concentrations ranging from 0.25% to 20% (v/v). The diploid strain carried three recessive conidial color mutations, in addition to genetic markers on all eight pairs of linkage groups. It was thereby possible to detect events of crossing over, non-disjunction, and mutation. An increase in the dose of ethanol was associated with a decrease in conidial viability and an increase in the relative and absolute frequencies of formation of (a) normal colonies which produced colored sectors and (b) phenotypically abnormal colonies, the majority of which (83.1%) produced normal sectors. At a concentration of 5% (v/v) ethanol, the survivors included 17.59% of the former and 44.7% of the latter colonies. Genetic analysis of the various segregants suggested that the frequencies of both mitotic crossing over and non-disjunction or the misdistribution of chromosomes were increased by ethanol. Among 133 abnormal colonies which segregated normal clones, 79 (59.4%) were associated with one of these genetic events. A total of 297 haploids and 130 diploids arose as normal segregants from the abnormal colonies. There were 31 recognizable events of non-disjunction and 14 crossing over in linkage groups I and II, where these events could be distinguished. These data suggested that the predominant effect of ethanol was a disruption of chromosome distribution. A cytological examination of ethanol-treated, germinating conidia revealed an interference with the mitotic spindle apparatus. The frequency of detectable spindles decreased more than 3-fold after 8 h exposure to 5% (v/v) ethanol. This finding supported the conclusion that ethanol disrupted chromosome distribution, and suggested the mechanism by which it does so. Human clinical data on alcohol consumption were examined in light of these findings.

Aspergillus nidulans

Nucleosome structure in Aspergillus nidulans.

The structure of chromatin from Aspergillus nidulans was studied using micrococcal nuclease and DNAase I. Limited digestion with micrococcal nuclease revealed a nucleosomal repeat of 154 base pairs for Aspergillus and 198 base pairs for rat liver. With more extensive digestion, both types of chromatin gave a similar quasi-limit product with a prominent fragment at 140 base pairs. The similarity of the two limit digests suggests that the structure of the 140 base pair nucleosome core is conserved. This implies that the difference in nucleosome repeat lengths between Aspergillus and rat liver is caused by a difference in the length of the DNA between two nucleosome cores. Digestion of Aspergillus chromatin with DNAase I produced a pattern of single-stranded fragments at intervals of 10 bases which was similar to that produced from rat liver chromatin.

Animals

Transmission and recombination of extranuclear genes during sexual crosses in Aspergillus nidulans.

Three extranuclear mitochondrial mutations in Aspergillus nidulans, (oliA1), (camA1) and (cs67), were used as markers in sexual crosses to provide information on the frequencies of transmission and recombination of the mitochondrial genome. Any individual perithecium contained ascospores of only one extranuclear genotype. Using mono-, bi- and trifactorial crosses it was found that all three markers could be recovered from the progeny, although the transmission frequencies were different for each marker. This bias was present irrespective of the nuclear background or the presence of selective agents in the medium on which the cross was established. These findings enable a series of "transmission strength" to be established, as shown below:-- (camA1) greater than (cs67, camA1) greater than (+) = (cs67) greater than (oliA1, cs67) greater than (oliA1) greater than (oliA1, camA1). However, the numbers of recombinants isolated were so variable as to make this form of analysis unsuitable for mapping the mitochondrial genome.

Aspergillus nidulans

Basic and neutral amino acid transport in Aspergillus nidulans.

Arginine and methionine transport by Aspergillus nidulans mycelium was investigated. A single uptake system is responsible for the transport of arginine, lysine and ornithine. Transport is energy-dependent and specific for these basic amino acids. The Km value for arginine is 1 X 10(-5) M, and Vmax is 2-8 nmol/mg dry wt/min; Km for lysine is 8 X 10(-6) M; Kt for lysine as inhibitor of arginine uptake is 12 muM, and Ki for ornithine is mM. On minimal medium, methionine is transported with a Km of 0-I mM and Vmax about I nmol/mg dry wt/min; transport is inhibited by azide. Neutral amnio acids such as serine, phenylalanine and leucine are probably transported by the same system, as indicated by their inhibition of methionine uptake and the existence of a mutant specifically impaired in their transport. The recessive mutant nap3, unable to transport neutral amino acids, was isolated as resistant to selenomethionine and p-fluorophenylanine. This mutant has unchanged transport of methionine by general and specific sulphur-regulated permeases.

Amino Acids

A mutation defective in the xanthine alternative pathway of Aspergillus nidulans: its use to investigate the specificity of uaY mediated induction.

In Aspergillus nidulans uric acid can be produced from xanthine via purine hydroxylase I (xanthine dehydrogenase) or via the xanthine alternative pathway (Darlington and Scazzocchio, Biochem. Biophys. Acta, 166, 569--571; 1968). A mutation defective in the xanthine alternative pathway of Aspergillus nidulans is described. By combining this mutation with hxB-20 which results in complete loss of purine hydroxylase I and II activities, but which conserves cross-reacting material, it is possible to block completely uric acid production and thus investigate which are the effective in vivo inducers of three enzymes under the control of the positive regulatory gene uaY: adenine deaminase, purine hydroxylase I (measured as cross-reacting material) and urate oxidase. It is concluded that uric acid is the only effective physiological inducer, while its 2 and 8 thio-analogues serve as gratuitous inducers.

Aspergillus nidulans

Restriction endonuclease cleavage map of mitochondrial DNA from Aspergillus nidulans.

Mitochondrial DNA of the ascomycete fungus Aspergillus nidulans, a circular molecule of 31 500 base pairs, is cleaved by restriction endonucleases Eco R I, Hind II, Hind III and Bgl II into 3, 7, 9 and 5 fragments, respectively. The relative positions of the cleavage sites could be mapped by analysis of fragments obtained by double enzyme digestions of whole DNA and by complete and partial redigestion of isolated restriction fragments.

Aspergillus nidulans

Effects of ethidium bromide in diploid and duplication strains of Aspergillus nidulans.

Unstable duplication and diploid strains of Aspergillus nidulans were treated with ethidium bromide, and it was shown that this drug reduces the number of sectors produced by such strains. The mechanisms which could be responsible for the partial stabilization of the strains are discussed and it is suggested that a similar mechanism is responsible for the production of sectors in both strains. It is also suggested that ethidium bromide could be useful for the reduction of instability of industrial strains.

Aspergillus nidulans

In-Silico and Functional Characterization of EcdLp, an ABC Transporter of Aspergillus nidulans NRRL11440.

Echinocandin B (ECB) biosynthesis in Aspergillus nidulans is primarily governed by multiple genes located within the biosynthetic echinocandin (ecd) gene cluster. The contributory functions of many genes, including transcription factors and tailoring enzymes of the ecd gene cluster, have been previously studied. The present study focused on determining the role of transporter proteins, EcdLp, EcdCp, and EcdDp, in ECB efflux using in silico and biochemical approaches. The molecular docking analysis revealed that ECB relatively showed higher binding affinity for EcdLp than the other co-clustered MFS transporters EcdCp and EcdDp, suggesting a preferred substrate of EcdLp. These results were further confirmed by heterologous integration of the ecdL gene in the ABC transporters-deficient Saccharomyces cerevisiae AD1-8u⁻, confirming active efflux. However, the binding of ECB in EcdLp is distinct from the R6G binding, overlapping the promiscuous site of farnesol, resulting in inhibition of R6G efflux in a dose-dependent manner. In conclusion, these results decipher the ECB binding and efflux mechanism and unveil the evolutionarily specialized architecture of EcdLp that permits targeted metabolite export in addition to environmental responsiveness, and lay the groundwork for optimizing ECB production via transporter engineering.

Aspergillus nidulans