Positive-acting Regulatory Gene Research Articles

Expression of a bacterial aspartase gene in Aspergillus nidulans: an efficient system for selecting multicopy transformants.

The Escherichia coli aspartase gene aspA has been expressed in the fungus Aspergillus nidulans using the powerful constitutive gpdA promoter and trpC terminator, both from A. nidulans. Multiple, but not single, copies of aspA overcome nutritional deficiencies resulting from the loss of catabolic NAD-linked glutamate dehydrogenase. They also circumvent certain nutritional deficiencies resulting from loss of the positive-acting regulatory gene product mediating nitrogen metabolite repression. Both of these cases of physiological suppression involve the aspartase-catalyzed catabolism of aspartate to ammonium plus fumarate. No physiological evidence for the opposite reaction leading to aspartate synthesis was obtained as multiple copies of aspA did not affect the phenotype resulting from the loss of anabolic NADP-linked glutamate dehydrogenase. The use of vectors containing aspA and recipients lacking NAD-linked glutamate dehydrogenase is an efficient means of selecting multicopy transformants in A. nidulans and also offers the possibility to select strains having increased aspartase levels from original transformants.

Regulation of branched-chain amino acid biosynthesis in Neurospora crassa: Cloning and characterization of the leu-1 and ilv-3 genes

The genes coding for the branched-chain amino acid biosynthetic enzymes comprise an integrated regulatory system. The expression of the several structural genes coding for enzymes of the isoleucine-valine and leucine pathways is controlled in parallel by the positive-acting regulatory gene, leu-3. The leu-1 and ilv-3 genes, coding for beta-isopropyl-malate dehydrogenase and aceto-hydroxyacid synthase, respectively, were cloned from a cosmid library. Restriction fragment length polymorphism analysis revealed that the two cloned fragments indeed mapped to the genomic locations of the leu-1 and ilv-3 genes, respectively. Northern blot analysis demonstrated that the leu-1 gene is transcribed to give an mRNA of approximately 1.5 kb, whereas the ilv-3 transcript size is 2.6 kb. The expression of both genes appears to be regulated at the transcriptional level. One leu-3 regulatory mutant was greatly deficient in both leu-1 and ilv-3 mRNAs, whereas another leu-3 allele showed an unusual antiparallel pattern of regulation.

Nucleotide sequence and DNA recognition elements of alc, the structural gene which encodes allantoicase, a purine catabolic enzyme of Neurospora crassa

The nitrogen regulatory circuit of Neurospora crassa contains structural genes that encode nitrogen catabolic enzymes which are subject to complex genetic and metabolic regulation. This set of genes is controlled by nitrogen limitation, by specific induction, and by the action of nit-2, a major positive-acting regulatory gene, and nmr, a negative-acting control gene. The complete nucleotide sequence of alc, the gene that encodes allantoicase, a purine catabolic enzyme, is presented. The alc gene contains a single intron, is transcribed from two initiation sites situated approximately 50 nb upstream of the translation start site, and encodes a protein comprised of 354 amino acids. Mobility shift and DNA footprint experiments identified a single binding site for the NIT2 regulatory protein in the alc promoter region. The binding site contains a 10 nucleotide base pair symmetrical sequence which is flanked by two possible core binding sequences, TATCT and TATCG. Mutant NIT2/beta-gal fusion proteins with amino acid substitutions in a putative zinc-finger motif were shown to be completely deficient in the ability to bind to the alc promoter DNA fragment.

Phosphatase regulation inAspergillus nidulans: responses to nutritional starvation

SUMMARYThe regulation of the syntheses of a number of phosphatases in the fungusAspergillus nidulanshas been examined. Levels of the intracellular alkaline phosphatase P11 are increased by starvation for carbon, nitrogen, phosphorus or sulphur. There is, however, no evidence that any of the wide domain regulatory genes which mediate sufficiency-triggered repression for each of these elements involved. A possible interpretation is that all four forms of starvation result in accumulation of an inducing metabolite. ThepalcA gene has been identified as a wide domain, probably positive-acting regulatory gene mediating phosphate repression. ThepalcA product controls the syntheses of alkaline phosphatase PI, acid phosphatases PIII and PV, a phosphodiesterase lacking phosphomonoesterase activity and probably also a phosphate permease. Mutations resulting in derepression of phosphate-repressible activities at acid but not alkaline growth pH define a gene designatedpacJ.pacJ mutations also confer arsenate resistance at low but not high pH. It is likely that phosphate derepression and arsenate resistance result from reduced uptake of H2PO4−. Finally, phosphatase regulation might be less complex than previously thought. Mutations designatedrand mapping at several loci apparently have no effect on phosphatase. They enhance phosphatase colony staining but this occurs even if the phosphatase substrates are omitted from the staining mixtures.rmutations appear to promote reactions converting the diazonium salts used for phosphatase staining to coloured precipitates.

Isolation of the positive-acting regulatory gene PHO4 from Saccharomyces cerevisiae

We have isolated a 10.2-kb fragment of yeast DNA from a genomic library of recombinant centromeric YCp50 plasmids, which complements a mutation in the PH04 gene of Saccharomyces cerevisiae. The identity of the PH04 gene on this plasmid was established by integration of a subfragment into the PH04 region of the yeast chromosome. Analysis of a series of plasmid subclones covering different regions of the original yeast DNA insert localized the PH04 gene within a 2.25-kb sequence. Southern hybridization of total genomic DNA prepared from wild-type strains and from integrative transformants show that the PH04 gene consists of unique yeast DNA sequences and is present at a single copy in the S. cerevisiae genome. RNA blot hybridization mapping of transcripts within this genomic region identify the PH04 transcript as a 1.7-kb, low-abundancy, constitutively expressed and polyadenylated RNA.

Nitrate uptake in Aspergillus nidulans and involvement of the third gene of the nitrate assimilation gene cluster.

In Aspergillus nidulans, chlorate strongly inhibited net nitrate uptake, a process separate and distinct from, but dependent upon, the nitrate reductase reaction. Uptake was inhibited by uncouplers, indicating that a proton gradient across the plasma membrane is required. Cyanide, azide, and N-ethylmaleimide were also potent inhibitors of uptake, but these compounds also inhibited nitrate reductase. The net uptake kinetics were problematic, presumably due to the presence of more than one uptake system and the dependence on nitrate reduction, but an apparent Km of 200 microM was estimated. In uptake assays, the crnA1 mutation reduced nitrate uptake severalfold in conidiospores and young mycelia but had no effect in older mycelia. Several growth tests also indicate that crnA1 reduces nitrate uptake. crnA expression was subject to control by the positive-acting regulatory gene areA, mediating nitrogen metabolite repression, but was not under the control of the positive-acting regulatory gene nirA, mediating nitrate induction.