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M X Caddick

Publications and source records attributed to M X Caddick.

At least 19 recordsLinked to original sources
PubMed1998

An ethanol inducible gene switch for plants used to manipulate carbon metabolism.

Many transgenic plant studies use constitutive promoters to express transgenes. For certain genes, deleterious effects arise from constant expression in all tissues throughout development. We describe a chemically inducible plant gene expression system, with negligible background activity, that obviates this problem. We demonstrate its potential by showing inducible manipulation of carbon metabolism in transgenic plants. Upon rapid induction of yeast cytosolic invertase, a marked phenotype appears in developing leaves that is absent from leaves that developed before induction or after it has ceased.

Alcohol Dehydrogenase↗
PubMed1997

Evolution of transcription-regulating proteins: caveat lector!

The paper of Hawkins et al. [Gene 146 (1994) 145-158] reports incorrect descriptions of mutant phenotypes, omits mention of the absence of a highly relevant glutamine-binding site and contains sequence alignments which might mislead the reader. Extensive sequence analysis reveals as untenable a central hypothesis of the paper concerning a possible evolutionary relationship between anthranilate synthetases and the transcription factors mediating nitrogen metabolite repression in fungi.

Anthranilate Synthase↗
PubMed1996

Molecular characterisation of meaB, a novel gene affecting nitrogen metabolite repression in Aspergillus nidulans.

Mutations within the meaB gene elicit the inappropriate expression of several activities subject to nitrogen metabolite repression in Aspergillus nidulans and also have some unrelated phenotypic effects. We have cloned meaB and isolated a full length cDNA clone. Northern analysis has shown that meaB expression is not subject to nitrogen metabolite repression. meaB encodes a novel protein of 418 amino acids and contains a significantly high number of S/TPXX motifs, a motif common in transcriptional regulatory proteins. We have sequenced three mutations within meaB, two of which have an identical phenotype to that produced by gene disruption.

Amino Acid Sequence↗
PubMed1996

Nitrogen metabolite signalling involves the C-terminus and the GATA domain of the Aspergillus transcription factor AREA and the 3' untranslated region of its mRNA.

AREA is a GATA transcription factor which mediates nitrogen metabolite repression in Aspergillus nidulans in response to intracellular glutamine levels. We have identified and localized three elements important to modulation of AREA function: a region of 13 residues within the DNA-binding GATA domain which forms a putative extended loop structure, the 12 C-terminal residues, and sequences within a 218 nucleotide region of the 3' UTR. The 12 C-terminal residues are also required for transcriptional activation at a subset of loci under areA control. Specific deletions within the 3' UTR and the C-terminus cause similar levels of derepression and the mutations are additive, implicating two principal signal transduction pathways. The contribution of the 3' UTR to AREA modulation is effected at the level of transcript stability such that the areA mRNA is at least five times more stable under nitrogen-derepressing conditions than it is under repressing growth conditions.

Amino Acid Sequence↗
PubMed1996

Mutational analysis of the C-terminal region of AREA, the transcription factor mediating nitrogen metabolite repression in Aspergillus nidulans.

In Aspergillus nidulans the positive-acting, wide domain regulatory gene areA mediates nitrogen metabolite repression. Previous analysis demonstrated that the C-terminal 153 residues of the areA product (AREA) are inessential for at least partial expression of most genes subject to regulation by areA. Paradoxically, areAr2, a -1 frameshift replacing the wild-type 122 C-terminal residues with a mutant peptide of 117 amino acids, leads to general loss of function. To determine the basis for the areAr2 mutant phenotype, and as a means of delineating functional domains within the C-terminal region of AREA, we have selected and characterised areAr2 revertants. Deletion analysis, utilising direct gene replacement, extended this analysis. A mutant areA product truncated immediately after the last residue of the highly conserved GATA (DNA-binding) domain retains partial function. The areAr2 product retains some function with respect to the expression of uaZ (encoding urate oxidase) and the mutant allele is partially dominant with respect to nitrate reductase levels. Consistent with the areAr2 product having a debilitating biological activity, we have demonstrated that a polypeptide containing both the wild-type DNA-binding domain and the mutant C-terminus of AREA2 is able to bind DNA in vitro but no longer shows specificity for GATA sequences.

Amino Acid Sequence↗
PubMed1995

Mutational analysis reveals dispensability of the N-terminal region of the Aspergillus transcription factor mediating nitrogen metabolite repression.

Mutational analysis has enabled identification and localization of an upstream exon of the areA gene of Aspergillus nidulans mediating nitrogen metabolite repression. A mutation in the initiation codon and frameshift mutations, which revert by restoration of the reading frame, established the coding role of the exon and mutations affecting intron splicing in conjunction with DNA sequencing of reverse transcriptase polymerase chain reaction (RT-PCR) products localized the coding region intron. The resulting AREA translation product would have 876 residues. Deletion of the upstream exon such that translation of the remaining areA coding region would yield a protein containing only the 719 C-terminal residues has only a subtle phenotype, very similar to those resulting from single amino acid replacements in upstream exon-encoded regions of strong sequence similarity to the Neurospora crassa and Penicillium chrysogenum homologues. A number of areA mRNAs of different sizes are synthesised and appear to be functionally redundant. Synthesis of at least the smallest mRNA(s) is probably subject to autogenous activation. Suppression of frameshift mutations by compensating mutations preventing intron splicing suggests that insertion of a markedly hydrophobic sequence can impair AREA function. Finally, translational initiation for areA can occur within a region of at least 123 nucleotides.

Amino Acid Sequence↗
PubMed1994

Direct analysis of native and chimeric GATA specific DNA binding proteins from Aspergillus nidulans.

In Aspergillus nidulans the regulatory gene areA is responsible for mediating nitrogen metabolite repression. The areA product (AREA) represents an example of the GATA family of DNA binding proteins, which are characterised by the presence of a GATA domain consisting of a zinc finger within a highly conserved region of 52 amino acids. Among the other transcription factors included in this family is the principal erythroid transcription factor, GATA-1, which contains two GATA domains. In order to demonstrate high specificity binding of native AREA to DNA containing the sequence -GATA-, and investigate the presence in A.nidulans of other proteins with related specificities, we have used gel mobility shift assays. Both AREA-dependent and independent complexes have been identified. Two strains bearing chimeric genes were also characterised. In these, the region encoding the native GATA domain of AREA was replaced by sequences from murine GATA-1 cDNA encoding either the equivalent C-terminal domain or both the N and C-terminal domains. Strains bearing the areA::NC-GATA construct, which includes the sequence encoding both the N and C-terminal domains of GATA-1, leads to a pronounced increase in one of two AREA-dependent complexes and implicates the N-terminal domain of GATA-1 in mediating protein-protein interactions.

Animals↗
PubMed1994

Nitrogen regulation in fungi.

Nitrogen regulation has been extensively studied in fungi revealing a complex array of interacting regulatory genes. The general characterisation of the systems in Aspergillus nidulans and Neurospora crassa shall be briefly described, but much of this paper will concentrate specifically on the recent molecular characterisation of areA, the principle regulatory gene from A. nidulans which mediates nitrogen metabolite repression. Three areas shall be explored in detail, firstly the DNA binding domain, which has been characterised extensively by both molecular and genetic analysis. Secondly we shall report recent analysis which has revealed the presence of related DNA binding activities in A. nidulans. Finally we shall discuss the mechanism by which the nitrogen state of the cell is monitored by the areA product, in particular localisation of the domain within the areA product which mediates the regulatory response within the protein.

Amino Acid Sequence↗
PubMed1993

C-terminal truncation of the transcriptional activator encoded by areA in Aspergillus nidulans results in both loss-of-function and gain-of-function phenotypes.

Mutations truncating as many as 143 C-terminal residues from the transcriptional activator encoded by the areA gene, mediating nitrogen metabolite repression in Aspergillus nidulans, do not significantly reduce the ability of the areA product to activate expression of most genes under areA control. Such mutations can even have a gain-of-function, derepressed phenotype, consistent with a critical role for this region in modulating the activity of the areA protein. However, expression of a few genes under areA control is substantially impaired by such C-terminal truncations, indicating that regions of an activator protein can play differing roles in the control of different structural genes. This underlines the advantages of being able to monitor effects of areA mutations on expression of large numbers of structural genes. Additionally, it is shown that truncation of as many as 153 C-terminal residues, virtually all amino acids C-terminal to the DNA-binding region, is compatible with retention of some areA function.

Amino Acid Sequence↗
PubMed1990

Nitrogen regulation in Aspergillus: are two fingers better than one?

The areA gene, mediating nitrogen metabolite repression in Aspergillus nidulans, encodes a positive-acting regulatory protein with a single putative DNA-binding 'zinc finger' which is remarkably similar to the two 'zinc fingers' of the major regulatory protein of vertebrate erythroid cells (GF-1/Eryf1/NF-E1). The areA-300 mutation alters the specificity of gene activation in that it elevates expression of certain structural genes whilst reducing expression of certain others. It is an 'in-frame' tandem duplication of 417 bp including the entire DNA-binding region. The consequences of areA didactyly are further explored by construction of a double mutant having an altered loop residue in the N-terminal 'finger'.

Alleles↗
PubMed1990

The regulatory gene areA mediating nitrogen metabolite repression in Aspergillus nidulans. Mutations affecting specificity of gene activation alter a loop residue of a putative zinc finger.

The regulatory gene areA mediating nitrogen metabolite repression in Aspergillus nidulans has been sequenced and its transcript mapped and orientated. A single ORF can encode a protein of 719 amino acids. A 52 amino acid region including a putative 'zinc finger' strongly resembles putative DNA binding regions of the major regulatory protein of erythroid cells. The derived protein sequence also contains a highly acidic region possibly involved in gene activation and 22 copies of the motif S(T)PXX, abundant in DNA binding proteins. Analysis of chromosomal rearrangements and transformation with deletion clones identified 342 N-terminal and 124 C-terminal residues as inessential and localized a C-terminal region required for nitrogen metabolite repressibility. A -1 frameshift eliminating the inessential 122 C-terminal amino acids is a surprising loss-of-function mutation. Extraordinary basicity of the replacement C terminus might explain its phenotype. Mutant sequencing also identified a polypeptide chain termination and several missense mutations, but most interesting are sequence changes associated with specificity mutations. A mutation elevating expression of some structural genes under areA control whilst reducing or not affecting expression of others is a leucine to valine change in the zinc finger loop. It reverts to a partly reciprocal phenotype by replacing the mutant valine by methionine.

Amino Acid Sequence↗
PubMed1989

A translocation associated, loss-of-function mutation in the nitrogen metabolite repression regulatory gene of Aspergillus nidulans can revert intracistronically.

The areAr-18 mutation is a loss-of-function mutation in areA, the positive acting regulatory gene mediating nitrogen metabolite repression in Aspergillus nidulans. It results from a reciprocal translocation which splits the coding region into 5' and 3' moieties. Surprisingly, we have selected rare intracistronic revertants of areAr-18. From crosses heterozygous for areAr-18 revertant alleles, duplication-deficiency progeny containing two copies of a substantial portion of chromosome IV but lacking part of chromosome III, including the 5' moiety of areA, have been obtained. For all four revertants analysed genetically, growth properties of these duplication-deficiency strains indicate that the reversion events involve the 3' portion of areA and that the 5' portion of areA is unnecessary for the revertant phenotype. This conclusion was directly confirmed for one revertant using Southern blotting. As all four reversion events involve additional chromosomal rearrangements, they probably fuse functional promoters, ribosome binding sites and 'in frame' initiation codons to the 3' portion of the gene. In the course of characterisation of these mutations, new mapping data for a large region of chromosome IV have been generated, and a new reciprocal translocation activating the cryptic regulatory gene areB, whose product can substitute for that of areA, has been identified.

Aspergillus nidulans↗
PubMed1986

Cloning of the regulatory gene areA mediating nitrogen metabolite repression in Aspergillus nidulans.

The areA gene, which mediates nitrogen metabolite repression in the fungus Aspergillus nidulans, lies sufficiently close to a telomere that no indispensable gene can be distal to it. We were able therefore to exploit the existence of a near terminal pericentric inversion to devise a method for cloning areA plus the region beyond it towards the telomere. In crosses heterozygous for this inversion a class of duplication-deficient progeny lacking areA and the region centromere-distal to it is obtained. We, therefore, sought clones from an A. nidulans gene library in lambda Charon 4 able to hybridize to total genomic DNA from a wild-type strain but not to that from a duplication-deficiency strain. A clone, containing an 11.6-kb insert, which hybridised weakly to duplication-deficiency DNA, overlapped chromosome breakpoints of three different aberration-associated areA alleles and was able to transform an areA mutant to areA+. Southern blotting and genetic analysis established that the transforming sequence had integrated in the region centromere distal to areA. The cloning method yielded other clones from the region centromere-distal to areA which were used to show that the translocation associated with a mutant areA allele is reciprocal rather than non-reciprocal, a fact which could not be established by classical genetics. Finally, analysis of the cloned portion of the dispensable region centromere-distal to areA indicates that this region contains at least 0.5% of the A. nidulans genome.

Aspergillus nidulans↗
PubMed1986

Regulation of gene expression by pH of the growth medium in Aspergillus nidulans.

In the fungus Aspergillus nidulans the levels of a number of enzymes whose location is at least in part extracellular (e.g. acid phosphatase, alkaline phosphatase, phosphodiesterase) and of certain permeases (e.g. that for gamma-amino-n-butyrate) are controlled by the pH of the growth medium. For example, at acidic pH, levels of acid phosphatase are high and those of alkaline phosphatase are low whereas at alkaline pH the reverse is true. Mutations in five genes, palA, B, C, E and F, mimic the effects of growth at acid pH whereas mutations in pacC mimic the effects of growth at alkaline pH. palA, B, C, E and F mutations result in an intracellular pH (pHin) which is more alkaline than that of the wild type whereas pacC mutations result in a pHin more acidic than that of the wild type. This indicates that these mutations exert their primary effects on the regulation of gene expression by pH rather than on the pH homeostatic mechanism but that the expression of at least some component(s) of the pH homeostatic mechanism is subject to the pH regulatory system. It is suggested that pacC might be a wide domain regulatory gene whose product acts positively in some cases (e.g. acid phosphatase) and negatively in others (e.g. alkaline phosphatase). The products of palA, B, C, E and F are proposed to be involved in a metabolic pathway leading to synthesis of an effector molecule able to prevent the (positive and negative) action of the pacC product.(ABSTRACT TRUNCATED AT 250 WORDS)

Acid Phosphatase↗