Donald W. MacDonald

Molybdate metabolism inAspergillus nidulans

SummaryFurther evidence supports the hypothesis that nitrate reductase and xanthine dehydrogenase are molybdo-enzymes inAspergillus nidulans, probably sharing a molybdenum-containing cofactor. This evidence includes (1) five-fold greater toxicity of tungstate on nitrate and hypoxanthine than on other nitrogen sources, (2) locus-specific molybdate reparability of both nitrate reductase and xanthine dehydrogenase at one (cnxE) of five (cnx) loci where mutation can result in pleiotropic loss of both enzyme activities, and (3) an additional class of mutants (molB) which are both molybdate resistant and partially defective in utilization of nitrate and hypoxanthine as nitrogen sources. Moreover, the phenotypes on molybdate-containing media of various mutants altered in the regulation of nitrate reductase synthesis and the ability of nitrate to protect against molybdate toxicity suggest that incorporation of molybdenum into nitrate reductase or into something having the same control properties as nitrate reductase can detoxify molybdate. However, mutations affecting regulation of xanthine dehydrogenase synthesis do not affect growth responses to molybdate. The properties of another class of molybdate resistance mutations (molA) suggest that there is another nitrate-inducible intracellular molybdate detoxification mechanism in addition to the one having identical control properties to nitrate reductase.

A mutant of Aspergillus nidulans lacking NADP-linked glutamate dehydrogenase

SummaryA mutation leading to loss of NADP-linked glutamate dehydrogenase pleiotropically leads to derepression of at least some ammonium-repressible activities in Aspergillus nidulans. It confers hypersensitivity to the toxic ammonium analogue methylammonium and maps independently of the two described loci for mutations to methylamonium resistance.

Regulation of Proline Transport in Aspergillus nidulans

SUMMARY: Aspergillus nidulans has at least two permeases for L-proline. The prnB gene of the prn gene cluster specifies the major proline permease, which is inducible by proline. Synthesis of the prnB permease is subject to repression by ammonium at 37 ° but not at 25 °. A genetically unidentified minor proline permease(s) does not respond to proline induction or ammonium repression but is inhibited by ammonium. areAr mutants are unable to utilize nitrogen sources other than ammonium because they lack a positive-acting regulatory product required for expression of ammonium-repressible activities. However, there are very few cases in which the lack of growth of areAr mutants on a particular nitrogen source can be attributed to a reduction in the level of a particular enzyme activity or permease. Reduced expression of the prnB permease can account for the inability of areAr mutants to utilize proline. This is demonstrated by the ability of cis-acting regulatory mutations designated prnd , which derepress synthesis of the prnB permease, to suppress areAr mutations for proline utilization. The apparent ability of prnd mutations to derepress synthesis of proline oxidase and l-pyrroline-5-carboxylate dehydrogenase is probably an indirect consequence of their ability to derepress synthesis of the prnB permease, preventing inducer exclusion. There is presently no evidence that prnd mutations directly affect expression of the prnA, prnC or prnD genes, but this possibility has not been definitively eliminated.

Gene roles in the prn cluster of Aspergillus nidulans.

SummaryThe roles of the four genes of the prn gene cluster involved in L-proline catabolism in Aspergillus nidulans have been investigated. prnD and prnC encode, respectively, proline exidase and ΔI-pyrroline-5-carboxylate (P5C) dehydrogenase. prnB is almost certainly the structural gene for the proline-inducible major proline permease. The prnA product has no structural role in these activities but is a positive acting regulatory molecule necessary for the expression of prnD, prnC and, to a lesser extent, prnB. Evidence favouring de novo synthesis of P5C dehydrogenase upon induction in the presence of a functional prnA allele is also presented.

Reduced expression of a distal gene of the prn gene cluster in deletion mutants of Aspergillus nidulans: genetic evidence for a dicistronic messenger in an eukaryote.

SummaryThe prn gene cluster involved in L-proline catabolism in Aspergillus nidulans, has the gene order prnA-prnD-regulatory region-prnB-prnC. prnB, prnD, and prnC specify proline permease, proline oxidase, and Δ1-pyrr0line-5-carboxylate (P5C) dehydrogenase, respectively. prnA is probably a positive regulatory gene whose product is necessary for expression of the prn activities. Proline induces proline permease and P5C dehydrogenase in prnD- mutants which lack proline oxidase, showing that proline does not have to be converted to P5C to act as inducer. Deletion mutations extending from within prnD to within prnB result in considerably reduced expression of prnC, whereas a prnD−prnB− double mutant shows normal prnC expression. This strongly suggests that the deletion mutations eliminate a promoter/initiator site for transcription of a dicistronic messenger for prnB and prnC. The fact that the deletions do not eliminate prnC expression altogether indicates that at least one other species of prnC transcript (monocistronic, tricistronic, or tetracistronic) can be made.

A single mutation leads to loss of glutamine synthetase and relief of ammonium repression in Aspergillus

SummaryGlutamine synthetase activity in the ascomycete fungus Aspergillus nidulans is regulated by nitrogen source. The lowest activities are obtained when the fungus is grown on L-glutamine, and the highest activities when grown on L-glutamate + arabinose. Glutamine auxotrophs of the fungus have been isolated, and one of these mutant strains, glnA-1, has been shown to lack the enzyme glutamine synthetase. The mutation is recessive, and is located on the right arm of chromosome II. In addition to abolishing glutamine synthetase activity, the mutation results in the relief of repression for several enzyme activities normally subject to repression by ammonium. These include nitrate reductase, asparaginase, proline uptake and urea uptake.

Behaviour of recombinant plasmids in Aspergillus nidulans: structure and stability

SummaryA pyrG−Aspergillus strain was transformed with plasmid pDJB-1, derived from pBR325 by insertion of the Neurospora crassa pyr4 gene (orotidine 5′-phosphate carboxylase), giving mitotically unstable transformants. Aspergillus DNA which acted as an “autonomously replicating sequence” (ARS) in yeast was inserted into pDJB-1 and the resulting construct, pDJB12.1, gave mitotically stable transformants when introduced into Aspergillus. Transformants obtained with pDJB-1 and pDJB12.1 gave few pyr− progeny in crosses to a pyrG+ strain. Southern hybridisation analysis of pyr+ transformants obtained with pDJB-1 revealed restriction fragments expected for integrated plasmid but transformants obtained with pDJB12-1 showed only bands derived from free plasmid. pDJB-1 and derivatives of pDJB12.1 could be recovered from transformants. These derivatives could not be explained by straightforward excision of integrated pDJB12.1 sequences but could result from recombination between plasmid molecules. Hybridisation of undigested transformant DNAs showed that the transforming DNA was present in a high molecular weight form. These results suggest: (1) pDJB12.1 derivatives and possibly pDJB-1 can replicate autonomously in Aspergillus; (2) A. nidulans DNA acting as an ARS in yeast enhances replication and/or segregation of transforming plasmids in Aspergillus; and (3) recombinant plasmids may undergo rearrangements when introduced into Aspergillus.

glnA mutations define the structural gene for glutamine synthetase in Aspergillus

SummaryFive allelic glutamine auxotrophs of Aspergillus have been assayed for glutamine synthetase activity. The amounts of glutamine synthetase activity present in these strains were proportional to their ability to grow in the absence of L-glutamine, whereas the amount of γ-glutamyl transferase did not correlate with the amount of synthetase or with the growth properties. Two out of this sample of mutants have been shown to have a glutamine synthetase with a Km for L-glutamate which differs from the wild type.

The threonine dehydratase structural gene in Aspergillus nidulans

Abstract Mutations at the ile A locus of Aspergillus nidulans can lead to loss of threonine dehydratase ( l -threonine hydro-lyase (deaminating), EC 4.2.1.16), the first enzyme for isoleucine biosynthesis. A cold-sensitive allele, ile A-13, leads to production of an enzyme having an increased apparent K m for l -threonine and showing inhibition by l -valine at concentrations which slightly stimulate activity of the wild type enzyme. Hence, ile A codes for a structural component of threonine dehydratase. The ability of very high concentrations of l -threonine to supplement ile A-13 strains at non-permissive temperatures would suggest the auxotrophy conferred by ile A-13 results primarily from the increased apparent K m of the mutant enzyme for l -threonine.