Integrated statistical analysis of cDNA microarray and NIR spectroscopic data applied to a hemp dataset.

Both cDNA microarray and spectroscopic data provide indirect information about the chemical compounds present in the biological tissue under consideration. In this paper simple univariate and bivariate measures are used to investigate correlations between both types of high dimensional analyses. A large dataset of 42 hemp samples on which 3456 cDNA clones and 351 NIR wavelengths have been measured, was analyzed using graphical representations. For this purpose we propose clustered correlation and clustered discrimination images. Large, tissue-related differences are seen to dominate the cDNA-NIR correlation structure but smaller, more difficult to detect, variety-related differences can be found at specific cDNA clone/NIR wavelength combinations.

Purification and characterization of two different alpha-L-rhamnosidases, RhaA and RhaB, from Aspergillus aculeatus.

Two proteins exhibiting alpha-L-rhamnosidase activity, RhaA and RhaB, were identified upon fractionation and purification of a culture filtrate from Aspergillus aculeatus grown on hesperidin. Both proteins were shown to be N glycosylated and had molecular masses of 92 and 85 kDa, of which approximately 24 and 15%, respectively, were contributed by carbohydrate. RhaA and RhaB, optimally active at pH 4.5 to 5, showed K(m) and V(max) values of 2.8 mM and 24 U/mg (RhaA) and 0.30 mM and 14 U/mg (RhaB) when tested for p-nitrophenyl-alpha-L-rhamnopyranoside. Both enzymes were able to hydrolyze alpha-1,2 and alpha-1,6 linkages to beta-D-glucosides. Using polyclonal antibodies, the corresponding cDNA of both alpha-L-rhamnosidases, rhaA and rhaB, was cloned. On the basis of the amino acid sequences derived from the cDNA clones, both proteins are highly homologous (60% identity).

Differential expression of three alpha-galactosidase genes and a single beta-galactosidase gene from Aspergillus niger.

A gene encoding a third alpha-galactosidase (AglB) from Aspergillus niger has been cloned and sequenced. The gene consists of an open reading frame of 1,750 bp containing six introns. The gene encodes a protein of 443 amino acids which contains a eukaryotic signal sequence of 16 amino acids and seven putative N-glycosylation sites. The mature protein has a calculated molecular mass of 48,835 Da and a predicted pI of 4.6. An alignment of the AglB amino acid sequence with those of other alpha-galactosidases revealed that it belongs to a subfamily of alpha-galactosidases that also includes A. niger AglA. A. niger AglC belongs to a different subfamily that consists mainly of prokaryotic alpha-galactosidases. The expression of aglA, aglB, aglC, and lacA, the latter of which encodes an A. niger beta-galactosidase, has been studied by using a number of monomeric, oligomeric, and polymeric compounds as growth substrates. Expression of aglA is only detected on galactose and galactose-containing oligomers and polymers. The aglB gene is expressed on all of the carbon sources tested, including glucose. Elevated expression was observed on xylan, which could be assigned to regulation via XlnR, the xylanolytic transcriptional activator. Expression of aglC was only observed on glucose, fructose, and combinations of glucose with xylose and galactose. High expression of lacA was detected on arabinose, xylose, xylan, and pectin. Similar to aglB, the expression on xylose and xylan can be assigned to regulation via XlnR. All four genes have distinct expression patterns which seem to mirror the natural substrates of the encoded proteins.

Cloning and biochemical characterisation of Aspergillus niger hexokinase--the enzyme is strongly inhibited by physiological concentrations of trehalose 6-phosphate.

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 54090, 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 D-galactose, L-sorbose, methyl alpha-D-glucoside and the pentoses L-arabinose and D-xylose are not. The enzyme has high affinity for glucose (Km = 0.35 mM at pH 7.5) and for fructose (Km = 2.0 mM at pH 7.5) and is inhibited by ADP. The enzyme is strongly inhibited by physiological concentrations (0.1-0.2 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 (Ki = 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 3-4 h after depletion of the carbon source.

Cloning and biochemical characterisation of an Aspergillus niger glucokinase. Evidence for the presence of separate glucokinase and hexokinase enzymes.

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(m) 0.063 mM at pH 7.5) and a relatively low affinity for fructose (K(m) 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 kcal (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.

Characterisation of the Aspergillus nidulans frA1 mutant: hexose phosphorylation and apparent lack of involvement of hexokinase in glucose repression.

Hexose phosphorylation was studied in Aspergillus nidulans wild-type and in a fructose non-utilising mutant (frA). The data indicate the presence of at least one hexokinase and one glucokinase in wild-type A. nidulans, while the frA1 mutant lacks hexokinase activity. The A. nidulans gene encoding hexokinase was isolated by complementation of the frA1 mutation. The absence of hexokinase activity in the frA1 mutant did not interfere with glucose repression of the enzymes involved in alcohol and L-arabinose catabolism. This suggest that, unlike the situation in yeast where mutation of hexokinase PII abolishes glucose repression, the A. nidulans hexokinase might not be involved in glucose repression.

Regulation of the xylanase-encoding xlnA gene of Aspergillus tubigensis.

A gene encoding an endo-1,4-beta-xylanase from Aspergillus tubigensis was cloned by oligonucleotide screening using oligonucleotides derived from amino acid sequence data obtained from the purified protein. The isolated gene was functional as it could be expressed in the very closely related fungus Aspergillus niger. The xylanase encoded by this gene is synthesized as a protein of 211 amino acids. After cleavage of the presumed prepropeptide this results in a mature protein of 184 amino acids with a molecular weight of 19 kDa and an isoelectric point of 3.6. The regulatory region of the xlnA gene was studied with respect to the response to xylan induction and carbon catabolite repression. By deletion analysis of the 5' upstream region of the gene a 158 bp region involved in the xylan specific induction was identified. To study this regulatory element a reporter system for transcriptional activating sequences was developed that is based on the A. niger glucose oxidase-encoding gene. From the results with this reporter system it is concluded that this 158 bp fragment not only contains the information required for induction of transcription but that it also plays a role in carbon catabolite repression of the xlnA gene. The region directly upstream of this fragment contains four potential CREA target sites; deletion of this region leads to an increase in the level of transcription. These results suggest that carbon catabolite repression of the xlnA gene is controlled at two levels, directly by repression of xlnA gene transcription and indirectly by repression of the expression of a transcriptional activator. This type of mechanism would be similar to the double lock mechanism for the regulation of gene expression of alcA in Aspergillus nidulans. The reporter system was also used to study the regulation of expression via the functions located on this fragment in A. niger and in A. nidulans. Essentially the same pattern of regulation was found in both of these hosts. Therefore, regulation of xylanase gene expression is basically conserved in all three aspergilli.

Induction of glucose oxidase, catalase, and lactonase in Aspergillus niger.

The induction of glucose oxidase, catalase, and lactonase activities was studied both in wild-type and in glucose oxidase regulatory and structural mutants of Aspergillus niger. The structural gene for glucose oxidase was isolated and used for Northern analysis and in transformation experiments using various gox mutations. Wild-type phenotype could be restored in the glucose oxidase-negative mutant (goxC) by transformation with the structural gene. We conclude, therefore, that the goxC marker which is located on chromosome 2 represents the structural gene of glucose oxidase. Glucose and a high oxygen level are necessary for the induction of all three enzyme activities in the wild-type strain and it was shown that both glucose and oxygen effects reflect regulation at the transcriptional level. The goxB mutation results in constitutive expression of all three activities although modulated to some extent by the carbon source. The goxE mutation only has an effect on lactonase and glucose oxidase expression and does not relieve the necessity for a high oxygen level. Catalase and lactonase could not be induced in the glucose oxidase-negative strain (goxC). Addition of H2O2 resulted in the induction of all three enzymes in the wild-type without glucose being present. The H2O2 induction is probably mediated by the goxB product. Besides the H2O2 induction there is still an effect of the carbon source on the induction. A model for induction of glucose oxidase, catalase, and lactonase in A. niger is discussed.(ABSTRACT TRUNCATED AT 250 WORDS)