Regulation of gene expression by pH of the growth medium in Aspergillus nidulans

Summary In the fungus Aspergillus nidulans the levels of a number of enzymes whose location is at least in part extracellular (e.g. acid phosphatase, alkaline phosphatase, phosphodiesterase) and of certain permeases (e.g. that for -amino-n-butyrate) are controlled by the pH of the growth medium. For example, at acidic pH, levels of acid phosphatase are high and those of alkaline phosphatase are low whereas at alkaline pH the reverse is true. Mutations in five genes, palA, B, C, E and F, mimic the effects of growth at acid pH whereas mutations in pacC mimic the effects of growth at alkaline pH. palA, B, C, E and F mutations result in an intracellular pH (pH_in) which is more alkaline than that of the wild type whereas pacC mutations result in a pH_in more acidic than that of the wild type. This indicates that these mutations exert their primary effects on the regulation of gene expression by pH rather than on the pH homeostatic mechanism but that the expression of at least some component(s) of the pH homeostatic mechanism is subject to the pH regulatory system. It is suggested that pacC might be a wide domain regulatory gene whose product acts positively in some cases (e.g. acid phosphatase) and negatively in others (e.g. alkaline phosphatase). The products of palA, B, C, E and F are proposed to be involved in a metabolic pathway leading to synthesis of an effector molecule able to prevent the (positive and negative) action of the pacC product.These genes are, to our knowledge, the first examples of genes involved in the regulation of extracellular enzyme and permease synthesis by the pH of the growth medium to be described in any organism.     

A mutation affecting amdS expression in Aspergillus nidulans contains a triplication of a cis-acting regulatory sequence

Summary In Aspergillus nidulans expression of the acetamidase structural gene, amdS, is under the control of at least four regulatory genes including the trans-acting amdA regulatory gene. A cis-acting mutation (amdI66) consisting of an 18 by duplication in the 5 region of the amdS gene results in very high levels of acetamidase activity but only in strains carrying semi-dominant mutations in the amdA gene. In selecting for increased amdS expression in an amdI66 amdA ^– strain, an A. nidulans strain with a mutation in the 5 region of the amdS gene was isolated. The nucleotide sequence was determined of the region containing the mutation, designated amdI666. The mutant strain carries three tandem copies of the 18 by sequence that is duplicated in the amdI66 mutation. Thus, from a strain carrying a duplication of an apparent regulatory protein binding site with little effect on gene expression, a strain has been derived that carries a triplication of the site with consequent major effects on regulation. The multiple copies of regulatory sites present in many genes may have been generated by a similar mechanism.     

An intragenic map of the brlA locus of Aspergillus nidulans.

Summary We have constructed an intragenic map for the Aspergillus nidulans brlA gene, mutants in which are distinguishable by visual criteria only. Most of the leaky phenotype mutants map near the right (3) end. The gene shows distinct recombinational polarity consistent with recombination initiation at the promoter (centromereproximal) end of the gene. brlA12 and brlA20 mutants gave abnormal DNA restriction patterns consistent with the III; VIII and VI; VIII translocations, respectively, determined by haploidization.     

Genetical and biochemical aspects of quinate breakdown in the filamentous fungus Aspergillus nidulans

In the ascomycetous fungus Aspergillus nidulans, the expression of two inducible, contiguous or closely linked genes (qutB and qutC) which encode enzymes for quinate breakdown to protocatechuate, appears to be controlled by the product of a tightly linked third gene (qutA). The qut gene cluster locates on chromosome VIII. The catalytic steps required for this conversion are dehydrogenase, dehydroquinase, and dehydratase, and these activities are induced by the presence of quinate in a similar manner. The dehydroquinase enzyme has been purified and shown to be multimeric, consisting of 20–22 identical subunits of approximately 10,000 MW. The enzyme has a pI value of 5.84, a K _m of 5×10^–4 m, and an amino acid composition that lacks tryptophan and cysteine. The enzyme also cross-reacts with rabbit antibodies raised against Neurospora crassa catabolic dehydroquinase.This work was supported in part by European Molecular Biology Organisation grants to J.R.K. and A.R.H. and by National Institutes of Health Grant GM23051 to N.H.G.     

Sequence, organization and expression of the core histone genes of Aspergillus nidulans

Summary The core histone gene family ofAspergillus nidulans was characterized. The H2A, H2B and H3 genes are unique in theA. nidulans genome. In contrast there are two H4 genes, H4.1 and H4.2. As previously reported for the H2A gene (May and Morris 1987) introns also interrupt the other core histone genes. The H2B gene, like the H2A gene, is interrupted by three introns, the H3 and H4.1 gene are each interrupted by two introns and the H4.2 gene contains one intron. The position of the single intron in H4.2 is the same as that the first intron of the H4.1 gene. The H2A and H2B genes are arranged as a gene pair separated by approximately 600 by and are divergently transcribed. The H3 and H4.1 genes are similarly arranged and are separated by approximately 800 bp. The H4.2 gene is not closely linked to either the H2A-H2B or H3-H4.1 gene pairs. Using pulse field gel electrophoresis an electrophoretic karyotype was established forA. nidulans. This karyotype was used to assign the H3–H4.1 gene pair and the H4.2 gene to linkage group VIII and the H2A–H2B gene pair to either linkage group III or VI. The abundance of each of the histone messenger RNAs was determined to be cell cycle regulated but the abundance of the H4.2 mRNA appears to be regulated differently from the others.     

Localisation of several chromosome I genes of Aspergillus nidulans: implications for mitotic recombination

Summary Another laboratory previously reported that the vast majority of mitotic recombinants in chromosome I disomics of Aspergillus nidulans arise from double exchange events involving the centromeric region and a far distal, possibly telomeric, region. This conclusion was based on the assumption that the camC gene is located in a position far distal to the centromere on the left arm of chromosome. I. As a left arm location for camC distal to the centromere was possibly in conflict with mapping data obtained in the context of an unrelated project, camC was partially mapped along with three other previously unlocated chromosome I genes, davA, ornD and uapA. The data presented here indicate that camC is located in a position far distal to the centromere but on the right arm of chromosome I, a conclusion also supported by the previous data. The positioning of uapA and camC in far distal locations on the right arm of chromosome I indicates the existence of a vast, otherwise nearly unmapped region on this chromosome arm.     

Isolation of the facA (acetyl-coenzyme A synthetase) and acuE (malate synthase) genes of Aspergillus nidulans

Summary Acetate inducible genes of Aspergillus nidulans were cloned via differential hybridization to cDNA probes. Using transformation of mutant strains the genes were identified as facA (acetyl-Coenzyme A synthetase) and acuE (malate synthase). The levels of RNA encoded by these genes were shown to be acetate inducible and subject to carbon catabolite repression. Induction is abolished in a facB mutant and carbon catabolite repression is relieved in a creA mutant.     

Transformation of Aspergillus nidulans with a cloned, oligomycin-resistant ATP synthase subunit 9 gene

Summary An allele (oliC31) of the A. nidulans oliC gene has been cloned using homology with the equivalent gene from N. crassa. OliC31 codes for an oligomycin-resistant, triethyltin-hypersensitive form of subunit 9 of the mitochondrial ATP synthase complex. Direct selection for oligomycin-resistance was possible following transformation of A. nidulans with the oliC31 gene. The phenotypes of transformants cultured in the presence of oligomycin were indicative of the position of integration of the transforming plasmid within the genome. Subsequent recombination events involving the integrated oliC31 gene were also apparent from altered levels of resistance to oligomycin or triethyltin. This gene should prove useful as a marker for transformation of strains lacking auxotrophic lesions and in gene replacement or disruption experiments.     

Characterization of phiA, a gene essential for phialide development in Aspergillus nidulans

We have previously identified genes and proteins involved in the fungal response to the Streptomyces-produced antibiotics, bafilomycin B1 and concanamycin A, known inhibitors of V-ATPases. Using mRNA differential display we identified an Aspergillus nidulans gene with 30-fold up-regulated expression in the presence of bafilomycin. This gene, here denoted phiA, and its gene product, were further characterized by targeted gene disruption and immunohistochemistry. Phenotypically, the phiA mutation resulted in reduced growth and severely reduced sporulation. The abnormality could be traced to the phialides, which divided several times instead of forming a single flask-shaped cell. The importance of phiA for phialide and conidium development was supported by immunohistochemistry experiments that showed the protein to be mainly present in these two cell types. Attempts to relate phiA to inhibition of V-ATPases did not result in unambiguous conclusions, but suggest the possibility that changed expression of phiA is correlated with growth arrest caused by inhibited V-ATPases.     

Gene pacA+ codes for the multiple active forms of Pi-repressible acid phosphatase in the mould Aspergillus nidulans

The acid phosphatase secreted by the biA1 strain of the mould Aspergillus nidulans was separated into at least nine isoforms by isoelectric focusing (IEF). The components visualized by activity were predominantly acidic proteins with isoelectric points ranging from pH 4.0 to 6.5. Almost the same isoforms were secreted by strains pabaA1 and palD8 biA1. Furthermore, the isoforms secreted by strain pacA1 biA1 were not visualized by staining after IEF, indicating that these isoforms are encoded by gene pacA. Treatment of the secreted enzyme with endoglycosidase H also reduced the number of isoforms visualized by staining after IEF and enhanced the R_f (electrophoretic mobility) value of this enzyme visualized after PAGE.     

Cotransformation of Aspergillus nidulans: a tool for replacing fungal genes

Summary When a non-selected DNA sequence was added during the transformation of amdS320 deletion strains of Aspergillus nidulans with a vector containing the wild-type amdS gene the AmdS⁺ transformants were cotransformed at a high frequency. Cotransformation of an amdS320, trpC801 double mutant strain showed that both the molar ratio of the two vectors and the concentration of the cotransforming vector affected the cotransformation frequency. The maximum frequency obtained was defined by the gene chosen as selection marker for transformation. Cotransformation was used to induce a gene replacement in A. nidulans. An amdS320 strain was transformed to AmdS⁺ and cotransformed with a DNA fragment containing a fusion between a non-functional A. nidulans trpC gene and the Escherichia coli lacZ gene. Ten AmdS⁺, LacZ⁺ transformants with a Trp^– mutant phenotype were selected. All of these strains could be transformed with a functional copy of the A. nidulans trpC gene, but only two strains yielded TrpC⁺ transformants which, with a low frequency, had a LacZ^– phenotype. These latter transformants had also lost the AmdS⁺ phenotype. Southern blotting analysis of DNA from these transformants confirmed the inactivation of the wild-type trpC gene, but revealed that amdS vector sequences were also involved in the gene replacement events.     

Demonstration of an altered phenylalanyl-tRNA synthetase in an analogue-resistant mutant of Aspergillus nidulans

Summary We have isolated and characterized a new class of p-fluorophenylalanine (FPA)-resistant mutant in Aspergillus nidulans using a phenA strain as the wild type, by optimizing the conditions of growth. All four spontaneous mutants selected on a medium containing FPA were found to be recessive to their wild-type alleles in heterozygous diploids. Complementation analyses and linkage data showed that they were allelic and mapped at a single locus (fpaU) in the facA-riboD interval on the right arm of linkage group V. Partial purification and characterization of Phe-tRNA synthetase from wild-type and mutant strains revealed that the mutant enzyme had a greatly reduced ability to activate the analogue. It is suggested that mutation in the fpaU gene brings about a structural alteration in Phe-tRNA synthetase.Abbreviations FPA DL-p-fluorophenylalanine - phenA auxotroph of phenylalanine - Phe-tRNA synthetase phenylalanyl-transfer ribonucleic acid synthetase Current address: Department of Biological Sciences (M/C 066) The University of Illinois at Chicago, Box 4348, Chicago, IL 60680, USA