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A J Clutterbuck

Publications and source records attributed to A J Clutterbuck.

34 records · Page 2Linked to original sources
PubMed1991

An autonomously replicating plasmid transforms Aspergillus nidulans at high frequency.

From an unstable Aspergillus nidulans colony, resulting from transformation with an A. nidulans gene bank, a plasmid was reisolated which transformed A. nidulans at a frequency of 20,000 transformants per 10(6) protoplasts at near saturation levels of transforming DNA. This represents a 250-fold enhancement of transformation efficiency over that found for typical integrative vectors such as pILJ16, the plasmid used in gene bank constructions. The plasmid, designated ARp1, is 11.5 kb in size, and consists of sequences derived from the 5.4-kb gene bank vector pILJ16, which carries the A. nidulans gene argB, and a 6.1-kb insert, designated AMA1. Southern analysis of transformant DNA showed ARp1 to be maintained in free form and not integrated into the chromosome. It has a mean copy number of 10-30 per haploid genome, and is mitotically unstable, being lost from 65% of asexual progeny of transformants. It shows similar transformational properties in A. niger and A. oryzae.

Aspergillus nidulans↗
PubMed1991

Isolation and developmentally regulated expression of an Aspergillus nidulans phenol oxidase-encoding gene, ivoB.

Ivory (ivo) mutants of Aspergillus nidulans lack conidiophore pigmentation. We have cloned ivoB which codes for a conidiophore-specific phenol oxidase (AHTase) via the adjacent selectable ureD gene. Gene-library transformants of a ureD4 strain proved defective for the vector, but we recovered both ureD and ivoB from a lambda library of transformant DNA. The ivoB transcription unit was localized to a SalI-XbaI 3-kb fragment and its 5' end was located by hybridization with an oligodeoxyribonucleotide corresponding to the N-terminal polypeptide sequence of AHTase. Expression of the ivoB 1.4-kb mRNA corresponded temporally with AHTase in conidiating cultures, and the levels of both mRNA and AHTase in leaky brlA mutants implied transcriptional control by brlA. A second developmentally regulated locus of unknown function adjacent to ivoB was also transcriptionally dependent on brlA, but was expressed 4 h later.

Aspergillus nidulans↗
PubMed1990

Isolation and characterisation of the crnA-niiA-niaD gene cluster for nitrate assimilation in Aspergillus nidulans.

Genomic clones containing the entire crnA-niiA-niaD gene cluster of Aspergillus nidulans have been isolated, and the structures of the niiA and niaD genes have been determined by nucleotide sequence analysis. This gene cluster is required for the assimilation of nitrate in A. nidulans, and the three genes encode a product required for nitrate uptake and the enzymes, nitrite reductase and nitrate reductase, respectively. The putative coding sequences, as deduced by comparison to cDNA clones of both niiA and niaD, are interrupted by multiple small introns, and the two genes are divergently transcribed. Identification and characterization of specific mRNAs involved in nitrate assimilation indicates that only monocistronic transcripts are involved, and that the approximate sizes of these transcripts are 1.6 kb, 3.4 kb and 2.8 kb for crnA, niiA and niaD, respectively. The results also indicate that control of niiA and niaD gene expression is mediated by the levels of mRNA accumulation, in response to the source of nitrogen in the growth medium. Two types of transcripts for niiA were observed.

Amino Acid Sequence↗
PubMed1990

N-acetyl-6-hydroxytryptophan oxidase, a developmentally controlled phenol oxidase from Aspergillus nidulans.

We have purified a specific phenol oxidase which is produced during conidiophore development in the fungus Aspergillus nidulans. Two active forms (A and B) have molecular masses of 50 and 48 kDa respectively; they have identical N-termini (24 residues). We have analysed the metal ion content of the B form; it is unusual in consisting of one zinc and two copper atoms per molecule. A temperature-sensitive mutant (ivoB192) produces a thermolabile enzyme, implying that ivoB is the structural locus. The natural substrate of the enzyme is N-acetyl-6-hydroxytryptophan, but it can be assayed colorimetrically or polarographically using hydroquinone monomethyl ether (HME) as substrate. It will also oxidize p-cresol, but not tyrosine, 3,4-dihydroxyphenylalanine or o-methoxyphenol. Colour development with HME substrate is strongly enhanced by high ammonium ion concentrations. Activity against HME is inhibited by 2,3-dihydroxynaphthalene, phenylhydrazine, diethyl dithiocarbamate and 8-hydroxyquinolene.

Amino Acid Sequence↗
PubMed1990

The genetics of conidiophore pigmentation in Aspergillus nidulans.

The grey-brown pigmentation of Aspergillus nidulans conidiophores depends on the functions of two 'ivory' loci. ivoB codes for a developmental specific phenol oxidase, and mutants accumulate its substrate N-acetyl-6-hydroxytryptophan. ivoA mutants are unable to make this substrate. ygA mutants are also poorly pigmented, and extracts require copper salts to activate both the phenol oxidase and conidial laccase. ivoA and ivoB mutants partially suppress the spore colour phenotype of ygA mutants. Comparisons of morphology, phenol oxidase and substrate accumulation in morphological mutants at the brlA locus suggest that the brlA protein regulates ivoA, ivoB and morphogenetic loci independently. The medA locus, which also affects morphology and pigmentation, may code for a modifier of brlA function. abaA mutants which are blocked at a later stage of development than brlA or medA mutants have low phenol oxidase levels, implying that by this stage of development the activity of the ivoB locus is declining.

5-Hydroxytryptophan↗
PubMed1985

Cloning an Aspergillus nidulans developmental gene by transformation.

We have developed a transformation system for Aspergillus nidulans giving a frequency of transformation high enough to screen a gene bank from which we were able to isolate and clone the A. nidulans developmental gene brlA by visual selection. The vector contains the selective marker argB+, and with it a frequency of transformation of 500 stable transformants/micrograms plasmid DNA can regularly be achieved. The evidence suggests that transformation is by integration but spontaneous excision of integrated plasmids is apparently frequent enough to allow the recovery of transforming plasmids in Escherichia coli.

Aspergillus nidulans↗
PubMed1983

A polyamine-sensitive mutant of Aspergillus nidulans.

A mutation designated spsA1 has been induced in the putrescine (puA2) auxotroph of Aspergillus nidulans which enables this mutant to grow on low concentrations of spermidine in place of putrescine. In addition, the spsA1 mutant, irrespective of putrescine requirement, is abnormally sensitive to high concentrations of spermidine, spermine or the polyamine analogue methylglyoxal bis(guanylhydrazone). When spsA1 strains are grown on medium containing spermidine, uptake of the polyamine continues at a high level for a longer period than in the wild-type and leads to a doubled intracellular spermidine pool. A similar increase in the intracellular spermine pool results from growth on spermine.

Aspergillus nidulans↗
PubMed1982

Polyamine transport in Aspergillus nidulans.

The uptake of putrescine, spermidine and spermine was studied in Aspergillus nidulans using 14C-labelled polyamines. Active transport systems, inhibited by azide and regulated by nitrogen availability, exist at least for putrescine and spermidine. Putrescine is taken up two to three times more rapidly than spermidine, reflecting a lower Km for the former substrate. The two uptake systems appear to be independent, spermidine uptake being inhibited by both putrescine and spermine, while putrescine uptake is not inhibited by the other two polyamines. The relationships of these transport systems to the tenfold or greater difference between spermidine and putrescine concentrations required to support growth of the putrescine auxotroph are discussed.

Aspergillus nidulans↗