The essential Aspergillus nidulans gene pmaA encodes an homologue of fungal plasma membrane H(+)-ATPases

The genetic pathways underlying the induction and anterior-posterior patterning of the heart are poorly understood. The recent emergence of the zebrafish model system now allows a classical genetic approach to such challenging problems in vertebrate development. Two large-scale screens for mutations affecting zebrafish embryonic development have recently been completed; among the hundreds of mutations identified were several that affect specific aspects of cardiac morphogenesis, differentiation, and function. However, very few mutations affecting induction and/or anterior-posterior patterning of the heart were identified. We hypothesize that a directed approach utilizing molecular markers to examine these particular steps of heart development will uncover additional such mutations. To test this hypothesis, we are conducting two parallel screens for mutations that affect either the induction or the anterior-posterior patterning of the zebrafish heart. As an indicator of cardiac induction, we examine expression of nkx2.5, the earliest known marker of precardiac mesoderm; to assess anterior-posterior patterning, we distinguish ventricle from atrium with antibodies that recognize different myosin heavy chain isoforms. In order to expedite the examination of a large number of mutations, we are screening the haploid progeny of mosaic F1 females. In these ongoing screens, we have identified four mutations that affect nkx2.5 expression as well as 21 that disrupt either ventricular or atrial development and thus far have recovered several of these mutations, demonstrating the value of our approach. Future analysis of these and other cardiac mutations will provide further insight into the processes of induction and anterior-posterior patterning of the heart.     

The orlA gene from Aspergillus nidulans encodes a trehalose-6-phosphate phosphatase necessary for normal growth and chitin synthesis at elevated temperatures

A cosmid carrying the orlA gene from Aspergillus nidulans was identified by complementation of an orlA1 mutant strain with DNA from the pKBY2 cosmid library. An orlA1 complementing fragment from the cosmid was sequenced. orlA encodes a predicted polypeptide of 227 amino acids (26360 Da) that is homologous to a 211-amino-acid domain from the polypeptide encoded by the Saccharomyces cerevisiae TPS2 gene and to almost the entire Escherichia coli otsB-encoded polypeptide. TPS2 and otsB each specify a trehalose-6-phosphate phosphatase, an enzyme that is necessary for trehalose synthesis. orlA disruptants accumulate trehalose-6-phosphate and have reduced trehalose-6-phosphatate phosphatase levels, indicating that the gene encodes a trehalose-6-phosphatate phosphatase. Disruptants have a nearly-wild-type morphology at 32 degrees C. When germinated at 42 degrees C, the conidia and hyphae from disruptants are chitin deficient, swell excessively, and lyse. The lysis is almost completely remedied by osmotic stabilizers and is partially remedied by N-acetylglucosamine (GlcNAc). The activity of glutamine:fructose-6-phosphate amido-transferase (GFAT), the first enzyme unique to aminosugar synthesis, is reduced and is labile in orlA disruption strains. The findings are consistent with the hypothesis that trehalose-6-phosphate reduces the temperature stability of GFAT and other enzymes of chitin metabolism at elevated temperatures. The results extend to filamentous organisms the observation that mutations in fungal trehalose synthesis are highly pleiotropic and affect aspects of carbohydrate metabolism that are not directly related to trehalose synthesis.     

Molecular characterization of the Aspergillus nidulans treA gene encoding an acid trehalase required for growth on trehalose

Aspergillus nidulans conidiospores contain high levels of the non-reducing disaccharide trehalose. We show that upon induction of conidiospore germination, the trehalose pool is rapidly degraded and a glycerol pool is transiently accumulated. A trehalase with an acidic pH optimum was purified from conidiospores. Characterization of the treA gene encoding this trehalase shows that it is homologous to Saccharomyces cerevisiae vacuolar acid trehalase, the product of the ATH1 gene, and to two related proteins of unknown function identified in Mycobacterium tuberculosis and Mycobacterium leprae. A. nidulans mutants that lack acid trehalase activity were constructed by gene replacement at the treA locus. Analysis of these mutants suggests that the treA gene product is localized in the conidiospore wall, is required for growth on trehalose as a carbon source, and is not involved in the mobilization of the intracellular pool of trehalose. Therefore, it is proposed that a cytoplasmic regulatory trehalase is controlling this latter process.     

Characterization of the Aspergillus nidulans nmrA gene involved in nitrogen metabolite repression

The gene nmrA of Aspergillus nidulans has been isolated and found to be a homolog of the Neurospora crassa gene nmr-1, involved in nitrogen metabolite repression. Deletion of nmrA results in partial derepression of activities subject to nitrogen repression similar to phenotypes observed for certain mutations in the positively acting areA gene.     

The effects of temperature on genetic instability in Aspergillus nidulans

Previous work has shown that strains of Aspergillus nidulans with a chromosome segment in duplicate (one in normal position, one translocated to another chromosome) are unstable. Deletions occur from either duplicate segment. The present work has shown that most deletions occur from the translocated duplicate segment. Furthermore, it has been found that the overall frequency of deletions from a duplication is dependent upon the temperature of growth. The overall frequency of deletions from a chromosome III duplication is greatly enhanced by low temperatures, while the overall frequency of deletions from a chromosome I duplication is markedly enhanced by high temperatures. A temperature of 39.5 degrees C appears to enhance to overall frequency of deletions from the I duplication to the greatest extent. With regard to the non-translocated duplicate I segment, an increase in temperature progressively enhances the frequency of those deletions to which it is subject to far more deletions during a particular period of growth than during any other period, and at 42 degrees C, a section of the III duplication is subject to far more deletions during a given period of growth than during any other period. Comparisons with other cases of genetic instability are made and common underlying connections are proposed.     

Cloning and characterization of a eukaryotic pantothenate kinase gene (panK) from Aspergillus nidulans

Pantothenate kinase (PanK) is the key regulatory enzyme in the CoA biosynthetic pathway. The PanK gene from Escherichia coli (coaA) has been previously cloned and the enzyme biochemically characterized; highly related genes exist in other prokaryotes. We isolated a PanK cDNA clone from the eukaryotic fungus Aspergillus nidulans by functional complementation of a temperature-sensitive E. coli PanK mutant. The cDNA clone allowed the isolation of the genomic clone and the characterization of the A. nidulans gene designated panK. The panK gene is located on chromosome 3 (linkage group III), is interrupted by three small introns, and is expressed constitutively. The amino acid sequence of A. nidulans PanK (aPanK) predicted a subunit size of 46.9 kDa and bore little resemblance to its bacterial counterpart, whereas a highly related protein was detected in the genome of Saccharomyces cerevisiae. In contrast to E. coli PanK (bPanK), which is regulated by CoA and to a lesser extent by its thioesters, aPanK activity was selectively and potently inhibited by acetyl-CoA. Acetyl-CoA inhibition of aPanK was competitive with respect to ATP. Thus, the eukaryotic PanK has a distinct primary structure and unique regulatory properties that clearly distinguish it from its prokaryotic counterpart.     

Characterization of Aspergillus nidulans peroxisomes by immunoelectron microscopy

We investigated the possible role of enterohepatic recirculation in prolongation of the half-life of elimination for Adriamycin, a commonly prescribed anticancer agent. We sought to determine whether enterohepatic recirculation of Adriamycin and its metabolites occurs using a linked-rat model. Two rats, a donor and a receiver, were linked via a catheter from the bile duct of the donor rat to the duodenum of the receiver. Control experiments were conducted with intact rats (without a bile duct cannula, control A) in order to estimate the half-life of elimination and with bile duct-cannulated rats (control B) to determine the amounts of Adriamycin and its metabolites in the bile. [14C-14]-Adriamycin was injected intravenously via the femoral vein to control A, control B and donor rats. The biological half-life of Adriamycin in the intact rats (control A, 10 h) was significantly higher than in the bile-duct-cannulated rats (control B, 4 h). The cumulative amount of Adriamycin and its metabolites excreted in the urine of the control A rats was also greater than from control B rats, indicating higher levels of the drug in their systemic circulation. Biological samples (bile, urine, plasma, blood cells and the major organs heart, liver and kidney) of the receivers contained significant amounts of Adriamycin and its metabolites. The total radioactivity recovered in the bile of the receivers accounted for 0.1% to 8% of the Adriamycin dose that was administered to the donors. Adriamycin and its metabolites appeared there only after a lag time that was consistent among all the receivers. Doxorubicinol aglycone was the major metabolite found in the bile and urine of the receivers. Low but constant levels of radioactivity were also detected in the plasma and blood cells of the receivers. The presence of unchanged Adriamycin in the bile and urine of the receivers suggested absorption of the parent drug from the intestine of the receivers. Overall, we estimated that about 22% of the dose injected to the donors was absorbed from the intestine of the receivers. Taken together, these findings clearly demonstrate a significant role for enterohepatic recirculation of Adriamycin and its metabolites, which may contribute to the ability of these compounds to induce cumulative cardiac damage and/or to increase the efficacy of Adriamycin.     

The Aspergillus nidulans gltA gene encoding glutamate synthase is required for ammonium assimilation in the absence of NADP-glutamate dehydrogenase

Mutants of Aspergillus nidulans lacking NADP-glutamate dehydrogenase activity grow more poorly than wild-type strains on ammonium as a sole nitrogen source. The leaky growth of these mutants is indicative of an alternative pathway of ammonium assimilation and glutamate biosynthesis. We have PCR-amplified a portion of the A. nidulans gene encoding glutamate synthase and used this sequence to inactivate the genomic copy. This gene, designated gltA, was found to be dispensable for growth on ammonium in the presence of NADP-glutamate dehydrogenase activity. However, a strain carrying the gltA inactivation together with an NADP-glutamate dehydrogenase structural gene mutation (gdhA) was unable to grow on ammonium or on nitrogen sources metabolized via ammonium. The gltA gene was located to linkage group V of the A. nidulans genetic map.     

RNA-mediated transformation in Aspergillus nidulans recovers gene functions lost by deletion or by point mutations

This work presents the results on two different approaches of RNA-mediated transformation in A. nidulans: a) the receptor strain was an argB2 (III) mutant deficient in arginine (OTCase deficient), and b) the receptor was an A. nidulans mutant defective in nitrate reductase synthesis due to a deletion in the niaD gene (VIII). The analyses of the arg+ and the nia+ retrotransformants allowed an insight on the fate and inheritance of the newly acquired characteristics. The occurrence and the study of Gene Inactivation Mechanism (RIP-like) inactivating the expression of extra copies of genes ectopically scattered over the receptor genome, was a byproduct of this research. Retrotransformants were also used as RNA-donor for a second turn of retrotransformation of the argB and niaD receptor strains. Genetic analyses of the new retrotransformants proved that the retrotransformation ability is kept by the re-extracted RNA when used in a second round of transformation process. This is the best genetic evidence that the newly acquired genetic characteristics were cDNA inserted, precisely transcribed and expressed. These are the first in vivo evidences of genetic information transference mediated by homologous RNA in lower eukaryotes.     

Constitutive activation of endocytosis by mutation of myoA, the myosin I gene of Aspergillus nidulans

Class I myosins function in cell motility, intracellular vesicle trafficking and endocytosis. Recently, it was shown that class I myosins are phosphorylated by a member of the p21-activated kinase (PAK) family. PAK phosphorylates a conserved serine or threonine residue in the myosin heavy chain. Phosphorylation at this site is required for maximal activation of the actin-activated Mg2+-ATPase activity in vitro. This serine or threonine residue is conserved in all known class I myosins of microbial origin and in the human and mouse class VI myosins. We have investigated the in vivo significance of this phosphorylation by mutating serine 371 of the class I myosin heavy chain gene myoA of Aspergillus nidulans. Mutation to glutamic acid, which mimics phosphorylation and therefore activation of the myosin, results in an accumulation of membranes in growing hyphae. This accumulation of membranes results from an activation of endocytosis. In contrast, mutation of serine 371 to alanine had no discernible effect on endocytosis. These studies are the first to demonstrate the in vivo significance of a regulatory phosphorylation on a class I myosin. Furthermore, our results suggest that MYOA has two functions, one dependent and one independent of phosphorylation.     

The murI gene of Escherichia coli is an essential gene that encodes a glutamate racemase activity

The murI gene of Escherichia coli was recently identified on the basis of its ability to complement the only mutant requiring D-glutamic acid for growth that had been described to date: strain WM335 of E. coli B/r (P. Doublet, J. van Heijenoort, and D. Mengin-Lecreulx, J. Bacteriol. 174:5772-5779, 1992). We report experiments of insertional mutagenesis of the murI gene which demonstrate that this gene is essential for the biosynthesis of D-glutamic acid, one of the specific components of cell wall peptidoglycan. A special strategy was used for the construction of strains with a disrupted copy of murI, because of a limited capability of E. coli strains grown in rich medium to internalize D-glutamic acid. The murI gene product was overproduced and identified as a glutamate racemase activity. UDP-N-acetylmuramoyl-L-alanine (UDP-MurNAc-L-Ala), which is the nucleotide substrate of the D-glutamic-acid-adding enzyme (the murD gene product) catalyzing the subsequent step in the pathway for peptidoglycan synthesis, appears to be an effector of the racemase activity.     

Amino acid alterations in the benA (beta-tubulin) gene of Aspergillus nidulans that confer benomyl resistance

We report the cloning and sequencing of 18 mutant alleles of the benA, beta-tubulin gene of Aspergillus nidulans that confer resistance to the benzimidazole antifungal, antimicrotubule compounds benomyl, carbendazim, nocodazole, and thiabendazole. In 12 cases, amino acid 6 was changed from histidine to tyrosine or leucine. In four cases, amino acid 198 was changed from glutamic acid to aspartic acid, glutamine, or lysine. In two cases, amino acid 200 was altered from phenylalanine to tyrosine. These data, along with previous data indicating that amino acid 165 is involved in the binding of the R2 group of these compounds [Jung and Oakley, 1990: Cell Motil. Cytoskeleton 17:87-94], suggest that regions of beta-tubulin containing amino acids 6, 165, and 198-200 interact to form the binding site of benzimidazole antimicrotubule agents. These results also suggest that the presence of phenylalanine at amino acid 200 contributes to the great sensitivity of many fungi to benzimidazole antimicrotubule agents.