The Aspergillus nidulans uvsB gene encodes an ATM-related kinase required for multiple facets of the DNA damage response

In Aspergillus nidulans, uvsB and uvsD belong to the same epistasis group of DNA repair mutants. Recent observations suggest that these genes are likely to control cell cycle checkpoint responses to DNA damage and incomplete replication. Consistent with this notion, we show here that UVSB is a member of the conserved family of ATM-related kinases. Phenotypic characterization of uvsB mutants shows that they possess defects in additional aspects of the DNA damage response besides checkpoint control, including inhibition of septum formation, regulation of gene expression, and induced mutagenesis. The musN227 mutation partially suppresses the poor growth and DNA damage sensitivity of uvsB mutants. Although musN227 partially suppresses several uvsB defects, it does not restore checkpoint function to uvsB mutants. Notably, the failure of uvsB mutants to restrain septum formation in the presence of DNA damage is suppressed by the musN227 mutation. We propose that UVSB functions as the central regulator of the A. nidulans DNA damage response, whereas MUSN promotes recovery by modulating a subset of the response.     

The Aspergillus nidulans rcoA gene is required for veA-dependent sexual development

The Aspergillus nidulans rcoADelta mutant exhibits growth and developmental defects. We show that the rcoADelta mutant lacks cleistothecia and is self-sterile. In crosses with wild-type strains, rcoADelta nuclei do not contribute to the cleistothecial walls. Furthermore, sexual development resulting from veA overexpression is rcoA dependent, indicating that rcoA lies downstream of veA in the sexual development pathway.     

Aspergillus nidulans stcP encodes an O-methyltransferase that is required for sterigmatocystin biosynthesis.

The Aspergillus nidulans stcP gene was previously identified as a transcribed region associated with a cluster of genes proposed to be involved in sterigmatocystin biosynthesis (D. W. Brown, J.-H. Yu, H. S. Kelkar, M. Fernandes, T. C. Nesbitt, N. P. Keller, T. H. Adams, and T. J. Leonard, Proc. Natl. Acad. Sci. USA 93:1418-1422, 1996). stcP was predicted to encode a methyltransferase responsible for conversion of demethylsterig-matocystin to sterigmatocystin. Here we demonstrate that disruption of stcP in A. nidulans results in strains that accumulate demethylsterigmatocystin.     

The yeast GLC7 gene required for glycogen accumulation encodes a type 1 protein phosphatase.

The glc7 mutant of the yeast Saccharomyces cerevisiae does not accumulate glycogen due to a defect in glycogen synthase activation (Peng, Z., Trumbly, R. J., and Reimann, E.M. (1990) J. Biol. Chem. 265, 13871-13877) whereas wild-type strains accumulate glycogen as the cell cultures approach stationary phase. We isolated the GLC7 gene by complementation of the defect in glycogen accumulation and found that the GLC7 gene is the same as the DIS2S1 gene (Ohkura, H., Kinoshita, N., Miyatani, S., Toda, T., and Yanagida, M. (1989) Cell 57, 997-1007). The protein product predicted by the GLC7 DNA sequence has a sequence that is 81% identical with rabbit protein phosphatase 1 catalytic subunit. Protein phosphatase 1 activity was greatly diminished in extracts from glc7 mutant cells. Two forms of protein phosphatase 1 were identified after chromatography of extracts on DEAE-cellulose. Both forms were diminished in the glc7 mutant and were partly restored by transformation with a plasmid carrying the GLC7 gene. Southern blots indicate the presence of a single copy of GLC7 in S. cerevisiae, and gene disruption experiments showed that the GLC7 gene is essential for cell viability. The GLC7 mRNA was identified as a 1.4-kilobase RNA that increases 4-fold at the end of exponential growth in wild-type cells, suggesting that activation of glycogen synthase is mediated by increased expression of protein phosphatase 1 as cells reach stationary phase.     

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 orIA 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 (26 360 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 of 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 tre-halose-6-phosphatate phosphatase. Disruptants have a nearly-wild-type morphology at 32°C. When germinated at 42°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 orIA 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.     

The Aspergillus nidulans sfaD gene encodes a G protein beta subunit that is required for normal growth and repression of sporulation.

flbA encodes an Aspergillus nidulans RGS (regulator of G protein signaling) domain protein that antagonizes FadA (G(i)alpha-subunit of heterotrimeric G protein)-mediated growth signaling to allow asexual development. We previously defined and characterized five suppressors of flbA (sfa) loss-of-function mutations and showed that one suppressor (sfaB) resulted from a novel dominant-negative allele of fadA. In this report we show that a second suppressor gene (sfaD) is predicted to encode the beta subunit of a heterotrimeric G protein. Deletion of sfaD suppressed all defects resulting from complete loss-of-flbA function mutations, caused a hyperactive sporulation phenotype and severely reduced vegetative growth. However, the sfaD deletion could not suppress the growth activation caused by dominant-activating fadA alleles, indicating that constitutively active FadA can cause proliferative growth in the absence of Gbetagamma signaling. We propose that SfaD and FadA are both positive growth regulators with partially overlapping functions and that FlbA has an important role in controlling the activities of both proteins. Inactivation of signaling events stimulated by both components of the heterotrimeric G protein is essential for both sexual and asexual sporulation.     

PRR1, a homolog of Aspergillus nidulans palF, controls pH-dependent gene expression and filamentation in Candida albicans

The pH of the environment has been implicated in controlling the yeast-hypha transition and pathogenesis of Candida albicans. Several C. albicans genes, including PHR1 and PHR2, are pH dependent in their expression. To investigate the mechanism of pH-dependent expression, we have cloned and characterized PRR1 (for pH response regulator). PRR1 is homologous to palF, a component of the pH response pathway in Aspergillus nidulans. Expression of PRR1 was itself pH dependent, being maximal at acid pH but reduced severalfold at alkaline pH. In a prr1 null mutant the alkaline-induced expression of PHR1 was completely abolished. Conversely, expression of PHR2 was no longer repressed at alkaline pH. A prr1 null mutant exhibited no morphological abnormalities at either pH; however, it lost the ability to form hyphae on medium 199 and on 10% serum plates. The ability to filament on serum was not restored by forced expression of PHR1, indicating that additional PRR1-dependent genes are required for hyphal development. These developmental genes appear to be distinct from those controlled by the developmental regulator EFG1, since the EFG1-dependent gene HWP1 was expressed normally in the prr1 null mutant. We conclude that PRR1 encodes a component of the pH-dependent response pathway in C. albicans and that this pathway regulates the expression of multiple components of hyphal development.     

IAR4, a gene required for auxin conjugate sensitivity in Arabidopsis, encodes a pyruvate dehydrogenase E1alpha homolog

The formation and hydrolysis of indole-3-acetic acid (IAA) conjugates represent a potentially important means for plants to regulate IAA levels and thereby auxin responses. The identification and characterization of mutants defective in these processes is advancing the understanding of auxin regulation and response. Here we report the isolation and characterization of the Arabidopsis iar4 mutant, which has reduced sensitivity to several IAA-amino acid conjugates. iar4 is less sensitive to a synthetic auxin and low concentrations of an ethylene precursor but responds to free IAA and other hormones tested similarly to wild type. The gene defective in iar4 encodes a homolog of the E1alpha-subunit of mitochondrial pyruvate dehydrogenase, which converts pyruvate to acetyl-coenzyme A. We did not detect glycolysis or Krebs-cycle-related defects in the iar4 mutant, and a T-DNA insertion in the IAR4 coding sequence conferred similar phenotypes as the originally identified missense allele. In contrast, we found that disruption of the previously described mitochondrial pyruvate dehydrogenase E1alpha-subunit does not alter IAA-Ala responsiveness or confer any obvious phenotypes. It is possible that IAR4 acts in the conversion of indole-3-pyruvate to indole-3-acetyl-coenzyme A, which is a potential precursor of IAA and IAA conjugates.     

myoA of Aspergillus nidulans encodes an essential myosin I required for secretion and polarized growth

We have identified and cloned a novel essential myosin I in Aspergillus nidulans called myoA. The 1,249-amino acid predicted polypeptide encoded by myoA is most similar to the amoeboid myosins I. Using affinity-purified antibodies against the unique myosin I carboxyl terminus, we have determined that MYOA is enriched at growing hyphal tips. Disruption of myoA by homologous recombination resulted in a diploid strain heterozygous for the myoA gene disruption. We can recover haploids with an intact myoA gene from these strains, but never haploids that are myoA disrupted. These data indicated that myoA encodes an essential myosin I, and this has allowed us to use a unique approach to studying myosin I function. We have developed conditionally null myoA strains in which myoA expression is regulated by the alcA alcohol dehydrogenase promoter. A conditionally lethal strain germinated on inducing medium grows as wild type, displaying polarized growth by apical extension. However, growth of the same myoA mutant strain on repressing medium results in enlarged cells incapable of hyphal extension, and these cells eventually die. Under repressing conditions, this strain also displays reduced levels of secreted acid phosphatase. The mutant phenotype indicates that myoA plays a critical role in polarized growth and secretion.     

The Drosophila embargoed gene is required for larval progression and encodes the functional homolog of schizosaccharomyces Crm1

The CRM1 (Exportin 1) protein is a receptor for leucine-rich nuclear export signal sequences. We have molecularly characterized the Drosophila melanogaster embargoed (emb) gene and find that it encodes a product with 49 and 71% sequence identity to the fission yeast Schizosaccharomyces pombe and human CRM1 proteins, respectively. We show that expression of the emb cDNA is sufficient to suppress the growth phenotype of both conditional-lethal and null S. pombe crm1(-) mutant strains, suggesting that emb encodes the functional homologue of the S. pombe Crm1 protein. Through mutagenesis screens we have recovered a series of recessive lethal emb mutations. There is a substantial maternal contribution of emb mRNA and animals hemizygous for our emb alleles can develop to second instar larvae but persist at this stage and consistently fail to undergo the molt to the third instar stage. We see a nuclear accumulation of endogenous actin in the intestinal epithelial cells of the emb mutant larvae, consistent with a role for the emb gene product in nuclear export of actin protein.     

The Saccharomyces cerevisiae MEC1 gene, which encodes a homolog of the human ATM gene product, is required for G1 arrest following radiation treatment.

The Saccharomyces cerevisiae gene MEC1 represents a structural homolog of the human gene ATM mutated in ataxia telangiectasia patients. Like human ataxia telangiectasia cell lines, mec1 mutants are defective in G2 and S-phase cell cycle checkpoints in response to radiation treatment. Here we show an additional defect in G1 arrest following treatment with UV light or gamma rays and map a defective arrest stage at or upstream of START in the yeast cell cycle.