Cloning and characterization of the cpyA gene encoding intracellular carboxypeptidase from Aspergillus nidulans.

Carboxypeptidase Y (CPY) has been used as a maker enzyme for investigations on intracellular transport of vacuolar proteins and on vacuolar biogenesis in Saccharomyces cerevisiae. We describe the cloning and characterization of the CPY homologue encoding gene (cpyA) from the filamentous fungus Aspergillus nidulans. The cpyA gene has one intron and encodes a protein with 552 amino acids containing a putative signal sequence and pro-sequence. The predicted CpyA protein is highly similar in sequence with carboxypeptidases from several yeast species and contains a catalytic triad (Asp-His-Ser) like that of serine carboxypeptidase. The cpyA disruptant cells showed reduced levels of intracellular carboxypeptidase. These results suggest that the cpyA gene encodes a vacuolar carboxypeptidase in A. nidulans.     

Identification and molecular cloning of a gene encoding Phospholipase A2 (plaA) from Aspergillus nidulans

The plaA gene encoding a protein that contains the cytosolic Phospholipase A(2) (cPLA(2)) motif is cloned for the first time from the filamentous fungus, Aspergillus nidulans. The translated 837 amino acid protein product of plaA comprises conserved lipase regions that are present in most mammalian cPLA(2) homologs. High expression of plaA was observed in glucose-lactose medium by Northern blot analyses. Deletion mutants of plaA grew and formed conidia similar to the wild-type strain, but showed decreased PLA(2) activity. Expression of the N-terminal truncated form of plaA in yeast cells resulted in increased Ca(2+)-dependent PLA(2) activity with (14)C-labeled phosphatidylcholine (PC) and phosphatidylethanolamine (PE) as substrates, compared with vector-transformed cells. In conclusion, we have identified and cloned a phospholipid-hydrolyzing novel cPLA(2) protein from A. nidulans for the first time.     

Cloning and characterization of the gene encoding Aspergillus nidulans DNA topoisomerase II.

We have determined the complete nucleotide sequence of a 5544bp genomic DNA fragment from Aspergillus nidulans that encodes DNA topoisomerase II (topo II). It contains a single open reading frame of 4740bp that codes for 1579 amino acid residues with a molecular weight of 178kDa; when expressed in Escherichia coli and Saccharomyces cerevisiae the molecular weight was 180kDa. The gene (TOP2) is divided into three exons. Two introns, 54bp and 60bp in length, are located at nucleotide positions 187 and 3214 respectively. Comparison of the deduced amino acid sequence with other eukaryotic topo II sequences showed a higher degree of identity with other fungal enzymes than the human topo IIalpha. One of monoclonal antibodies raised against human topo II, 6H8, can cross-react with Aspergillus topo II.     

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.     

Molecular cloning and characterization of a gene encoding glutaminase from Aspergillus oryzae

 A glutaminase from Aspergillus oryzae was purified and its molecular weight was determined to be 82,091 by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Purified glutaminase catalysed the hydrolysis not only of l-glutamine but also of d-glutamine. Both the molecular weight and the substrate specificity of this glutaminase were different from those reported previously [Yano et al. (1998) J Ferment Technol 66: 137–143]. On the basis of its internal amino acid sequences, we have isolated and characterized the glutaminase gene (gtaA) from A. oryzae. The gtaA gene had an open reading frame coding for 690 amino acid residues, including a signal peptide of 20 amino acid residues and a mature protein of 670 amino acid residues. In the 5′-flanking region of the gene, there were three putative CreAp binding sequences and one putative AreAp binding sequence. The gtaA structural gene was introduced into A. oryzae NS4 and a marked increase in activity was detected in comparison with the control strain. The gtaA gene was also isolated from Aspergillus nidulans on the basis of the determined nucleotide sequence of the gtaA gene from A. oryzae. Received: 23 August 1999 / Received last revision: 7 January 2000 / Accepted: 14 January 2000     

Purification and characterization of a neutral endoxylanase from Aspergillus nidulans

Abstract A neutral endoxylanase from a culture filtrate of Aspergillus nidulans grown on oat spelt xylan was purified to apparent homogeneity. The purified enzyme showed a single band on SDS-PAGE with a molecular mass of 22,000 and had an isoelectric point of 6.4. The enzyme was a non-debranching endoxylanase highly specific for xylans and completely free from cellulolytic activity. The xylanase showed an optimum activity at pH 5.5 and 62°C and had a K m of 4.2 mg oat spelt xylan per ml and a V max of 710 μmol min⁻¹ (mg protein)⁻¹.     

Isolation and functional analysis of a gene,tcsB, encoding a transmembrane hybrid-type histidine kinase from Aspergillus nidulans

We cloned and characterized a novel Aspergillus nidulans histidine kinase gene, tcsB, encoding a membrane-type two-component signaling protein homologous to the yeast osmosensor synthetic lethal N-end rule protein 1 (SLN1), which transmits signals through the high-osmolarity glycerol response 1 (HOG1) mitogen-activated protein kinase (MAPK) cascade in yeast cells in response to environmental osmotic stimuli. From an A. nidulans cDNA library, we isolated a positive clone containing a 3,210-bp open reading frame that encoded a putative protein consisting of 1,070 amino acids. The predicted tcsB protein (TcsB) has two probable transmembrane regions in its N-terminal half and has a high degree of structural similarity to yeast Sln1p, a transmembrane hybrid-type histidine kinase. Overexpression of the tcsB cDNA suppressed the lethality of a temperature-sensitive osmosensing-defective sln1-ts yeast mutant. However, tcsB cDNAs in which the conserved phosphorylation site His(552) residue or the phosphorelay site Asp(989) residue had been replaced failed to complement the sln1-ts mutant. In addition, introduction of the tcsB cDNA into an sln1delta sho1delta yeast double mutant, which lacked two osmosensors, suppressed lethality in high-salinity media and activated the HOG1 MAPK. These results imply that TcsB functions as an osmosensor histidine kinase. We constructed an A. nidulans strain lacking the tcsB gene (tcsBdelta) and examined its phenotype. However, unexpectedly, the tcsBdelta strain did not exhibit a detectable phenotype for either hyphal development or morphology on standard or stress media. Our results suggest that A. nidulans has more complex and robust osmoregulatory systems than the yeast SLN1-HOG1 MAPK cascade.     

A murine model for type III tyrosinemia: lack of immunologically detectable 4-hydroxyphenylpyruvic acid dioxygenase enzyme protein in a novel mouse strain with hypertyrosinemia.

We have characterized a new mutant strain of mouse that has hypertyrosinemia. The blood tyrosine level was persistently high, and increased amounts of 4-hydroxyphenylpyruvic acid and its derivatives were excreted into the urine. Succinylacetone was not detected in urine samples from these mice. All the animals were apparently healthy, and there was no evidence of hepatorenal dysfunction. The hypertyrosinemia was transmitted through an autosomal recessive inheritance. Analyses of hepatic enzymes related to tyrosine metabolism revealed that 4-hydroxyphenylpyruvic acid dioxygenase activity was virtually absent, while fumarylacetoacetase and tyrosine aminotransferases (cytosolic and mitochondrial forms) were normal in these mutant mice. Immunoblot analysis of 4-hydroxyphenylpyruvic acid dioxygenase protein in the liver indicated that the subunit protein of the enzyme was absent. It would appear that hypertyrosinemia in this mutant strain was caused by a genetic defect in 4-hydroxyphenylpyruvic acid dioxygenase. These features are similar to type III tyrosinemia in humans. Analysis of this mutant strain of mouse is expected to provide valuable information on the pathogenesis of human type III tyrosinemia and can also serve as a useful system for studies on tyrosine metabolism.     

Suppression of the bimC4 mitotic spindle defect by deletion of klpA, a gene encoding a KAR3-related kinesin-like protein in Aspergillus nidulans

To investigate the relationship between structure and function of kinesin-like proteins, we have identified by polymerase chain reaction (PCR) a new kinesin-like protein in the filamentous fungus Aspergillus nidulans, which we have designated KLPA. DNA sequence analysis showed that the predicted KLPA protein contains a COOH terminal kinesin-like motor domain. Despite the structural similarity of KLPA to the KAR3 and NCD kinesin-like proteins of Saccharomyces cerevisiae and Drosophila melanogaster, which also posses COOH-terminal kinesin-like motor domains, there are no significant sequence similarities between the nonmotor or tail portions of these proteins. Nevertheless, expression studies in S. cerevisiae showed that klpA can complement a null mutation in KAR3, indicating that primary amino acid sequence conservation between the tail domains of kinesin-like proteins is not necessarily required for conserved function. Chromosomal deletion of the klpA gene exerted no observable mutant phenotype, suggesting that in A. nidulans there are likely to be other proteins functionally redundant with KLPA. Interestingly, the temperature sensitive phenotype of a mutation in another gene, bimC, which encodes a kinesin-like protein involved in mitotic spindle function in A. nidulans, was suppressed by deletion of klpA. We hypothesize that the loss of KLPA function redresses unbalanced forces within the spindle caused by mutation in bimC, and that the KLPA and BIMC kinesin-like proteins may play opposing roles in spindle function.     

Isolation and characterization of the positively acting regulatory gene QUTA from Aspergillus nidulans.

The positively acting regulator gene QUTA from Aspergillus nidulans has been identified and located within a cluster of quinic acid utilisation (QUT) genes isolated within a recombinant phage lambda (lambda Q1). The DNA sequence of the QUTA gene reveals a single uninterrupted reading frame coding for a protein of mw 90.416 Kd. The QUTA protein sequence has a protein motif in the form of a putative "DNA finger" that shows strong homology to other such motifs in the GAL4, PPR1, ARGRII, LAC9 and QA1F regulatory gene products of S. cerevisiae, K. lactis and N. crassa. The data presented confirm the view deduced by genetical analysis that the QUTA gene of A. nidulans encodes a protein capable of interacting with QUT specific DNA sequences.