The cAMP signal transduction pathway controls many processes in fungi. The pkaR gene, encoding the regulatory subunit (PKA-R) of cAMP-dependent protein kinase (PKA), was cloned from the industrially important filamentous fungus Aspergillus niger. To investigate the involvement of PKA in morphology of A. niger, a set of transformants which overexpressed pkaR or pkaC (encoding the catalytic subunit of PKA) either individually or simultaneously was prepared as well as mutants in which pkaR and/or pkaC were disrupted. Strains overexpressing pkaR or both pkaC and pkaR could not be distinguished from the wild-type, suggesting that regulation of PKA activity is normal in these strains. Absence of PKA activity resulted in a two- to threefold reduction in colony diameter on plates. The most severe phenotype was observed in the absence of PKA-R, i.e., very small colonies on plates, absence of sporulation and complete loss of growth polarity during submerged growth. Suppressor mutations easily developed in the DeltapkaR mutant and one of these mutants appeared to lack PKA-C activity. These data suggest that cAMP-dependent protein phosphorylation in A. niger regulates growth polarity and formation of conidiospores.
In this study, a proposal is presented for the allele nomenclature of 17 polymorphic STR loci (AHT4, AHT5, ASB2, ASB17, ASB23, CA425, HMS1, HMS2, HMS3, HMS6, HMS7, HTG4, HTG6, HTG7, HTG10, LEX3 and VHL20) for equine genotyping (Equus caballus). The nomenclature is based on sequence data of the polymorphic region of the STR loci as recommended by the DNA commission of the International Society for Forensic Genetics for human DNA typing. For each STR locus, several alleles were selected and animals homozygous for those alleles were subjected to sequence analysis. The alleles of the 17 STR loci consisted either of simple (10), compound (6) or complex repeat patterns (1). Only a limited number of alleles with the same fragment size showed different repeat structures. The allele designation described here was based on the number of repeats, including all variable regions within the amplified fragment.
The Aspergillus niger hexokinase gene hxkA has been cloned by heterologous hybridisation using the Aspergillus nidulans hexokinase gene as a probe. The DNA sequence of the gene was determined, and the deduced amino acid sequence showed significant similarity to other eukaryotic hexokinase and glucokinase proteins, in particular to those of the budding yeasts. The encoded protein was purified from a multicopy hxkA transformant, and extensively characterised. The hexokinase protein has a molecular mass of 54 090, a pI of 4.9 and is a homodimer. D-Glucose, the glucose analogue 2-deoxy-D-glucose, D-fructose, D-mannose and D-glucosamine are phosphorylated by hexokinase, whereas the hexoses Dgalactose, L-sorbose, methyl A-D-glucoside and the pentoses L-arabinose and D-xylose are not. The enzyme has high affinity for glucose (K m ϭ 0.35 mM at pH 7.5) and for fructose (K m ϭ 2.0 mM at pH 7.5) and is inhibited by ADP. The enzyme is strongly inhibited by physiological concentrations (0.1Ϫ0.2 mM) of trehalose 6-phosphate, which may be of importance for in vivo regulation of the enzyme. Inhibition of A. niger hexokinase by trehalose 6-phosphate is competitive towards the sugar substrate (K i ϭ 0.01 mM). Based on the kinetic constants of hexokinase and glucokinase their relative contribution to in vivo glucose phosphorylation was calculated and found to be strongly dependent on intracellular pH and glucose concentration. At pH 7.5 glucokinase is predominant, whereas at pH 6.5 hexokinase is predominant at glucose concentrations higher than 0.5 mM. Expression of the hexokinase and the glucokinase gene requires active carbon metabolism. Also on carbon sources which are not substrates for hexokinase or glucokinase, clear expression is observed. The hexokinase and glucokinase enzymes are quite stable in vivo. Even in the absence of transcription, active glucokinase and hexokinase remain present in the cells at almost the same level for at least 3Ϫ4 h after depletion of the carbon source.Keywords : Aspergillus niger; hexokinase; glucokinase; purification; expression ; trehalose 6-phosphate.In a recent publication we described the cloning of the Aspergillus niger glucokinase gene and provided evidence for the existence of separate glucokinase and hexokinase enzymes in this industrially important microorganism .Both metabolic modelling (Torres, 1994a, b) and experimental studies (Schreferl-Kunar et al., 1989) indicate that hexose phosphorylation may be of major importance in the regulation of carbon flux in A. niger. Recently, Arisan-Atac et al. (1996) found increased rates of citric acid production in a strain in which the trehalose 6-phosphate synthase gene (ggs1) was disrupted. Saccharomyces cerevisiae hexokinases PI and PII are strongly inhibited by trehalose 6-phosphate, which is also a potent inhibitor of A. niger hexokinase (Arisan-Atac et al., 1996; this report). This inhibition may play an important role in the regulation of glycolytic flux in S. cerevisiae, although the precise mechanism by which trehalose 6-phosphate or ...
The Aspergillus niger glucokinase gene glkA has been cloned using a probe generated by polymerase chain reaction with degenerate oligonucleotides. The DNA sequence of the gene was determined, and the deduced amino acid sequence shows significant similarity to other eukaryotic hexokinase and glucokinase proteins, in particular to the Saccharomyces cerevisiae glucokinase protein. The encoded protein was purified from a multicopy glkA transformant, and extensively characterised. The protein has a molecular mass of 54536 Da and a PI of 5.2. The enzyme has high affinity for glucose (K, 0.063 mM at pH 7.5) and a relatively low affinity for fructose (K, 120 mM at pH 7.5), and in vivo fructose phosphorylation by glucokinase is consequently negligible. The configurations at C1 and C4 of the substrate appear to be essential for substrate specificity. The A. niger glucokinase shows non-competitive inhibition by ADP towards ATP and uncompetitive inhibition by ADP towards glucose. The k,,, (turnover number) decreases rapidly below pH 7.5 (56% at pH 7.0 and 17% at pH 6.5) and this may have important implications for the in vivo regulation of activity. In addition, proof is provided for the presence of a second hexosephosphorylating enzyme in A. niger. This enzyme is probably a hexokinase, since unlike glucokinase, this activity is inhibited by trehalose 6-phosphate.
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