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A J Clutterbuck

Publications and source records attributed to A J Clutterbuck.

At least 19 recordsLinked to original sources
PubMed1997

Autonomous plasmid replication in Aspergillus nidulans: AMA1 and MATE elements.

With few exceptions, in eukaryotic organisms the presence of a chromosomal replicator on a circular vector molecule is not sufficient to confer on it the ability to persist and replicate extrachromosomally. However, it is possible to isolate from genomes of some filamentous fungi DNA fragments which can provide extrachromosomal maintenance of plasmids. In Aspergillus nidulans, two functional classes of such sequences can be distinguished: effective plasmid replicators (e.g., AMA1) and transformation enhancers (e.g., ANS1 or MATEs), which apparently are able to initiate aberrant replication, leading to vector rearrangement and multimerization and eventually resulting in chromosomal integration. We discuss the similarity of these events to DNA amplification in other eukaryotes. A model is suggested which accounts for the formation of effective replicating plasmids as a result of sequence amplification. The model is based on the observation that in some organisms, including A. nidulans and Schizosaccharomyces pombe, duplication of an inefficient replicator enhances its efficiency dramatically. Some structural traits of transformation enhancers in A. nidulans imply a role for topoisomerases in amplification and replication of circular DNA molecules. We discuss practical applications of replicative vectors for gene cloning and expression studies.

Amino Acid Sequence↗
PubMed1997

The validity of the Aspergillus nidulans linkage map.

The Aspergillus nidulans linkage map is reviewed as a background to physical mapping by cosmid cross-hybridization and genome sequencing. DNA-based methods depend on contiguity, so that the resulting maps are only as good as the weakest link, whereas each new marker added to a linkage map can provide independent confirmation of the positions of its neighbors. For all eight chromosomes of A. nidulans a reliable framework has been provided by analysis of mitotic crossing over, in many cases substantiated by the study of translocation disomics. Building on this framework, there is a backbone of loci linked by reliable three-point meiotic mapping and a second set of less precisely mapped loci. The result is a map with a high degree of self-consistency although some areas of uncertainty or conflict are also noted.

Aspergillus nidulans↗
PubMed1996

Gene expression from replicating plasmids in Aspergillus nidulans.

Plasmids bearing the AMA1 replicator from Aspergillus nidulans are capable of extrachromosomal replication in this fungus as well as in other species. Synthetic plasmids bearing the moderately expressed argB gene and the highly expressed, inducible beta-galactosidase gene (bgaS) were introduced into fungal cells. Expression of both genes was monitored by Northern hybridization. It was demonstrated that transcription of bgaS is induced and repressed normally, irrespective of whether the gene is integrated into the chromosome or maintained on an extrachromosomal supercoiled plasmid. Transcription of the strongly expressed bgaS gene stimulates transcription of the argB gene located on the same replicating plasmid irrespective of orientation. This effect also occurs with chromosomally integrated vectors, but to a lesser extent. Episomal vectors are present in 10 copies per nucleus, and the expression level of the argB gene is approximately proportional to copy number. However, the amount of mRNA transcribed from the highly expressed bgaS gene on the multi-copy replicating plasmid does not exceed that from single-copy integrants. High levels of expression of the plasmid-borne gene do not affect plasmid mitotic stability or copy number.

Aspergillus nidulans↗
PubMed1996

The plasmid replicator AMA1 in Aspergillus nidulans is an inverted duplication of a low-copy-number dispersed genomic repeat.

The AMA1 sequence was isolated from a genomic library of Aspergillus nidulans on the basis of its ability to enhance transformation frequency and generate phenotypically unstable transformants in this fungus. These properties were previously shown to be the result of extrachromosomal replication of AMA1-bearing plasmids. Here we demonstrate that AMA1 is an inverted duplication of a sequence which has other isolated genomic copies. These sequences (mobile Aspergillus transformation enhancers, or MATEs) share a high degree of sequence similarity and exhibit some features characteristic of mobile elements, including a potential Met-tRNA priming site, similar to that found in retrotransposons of the Ty-copia group. The nucleotide sequence does not encode any extended polypeptides but contains ARS-consensus matches and a multiply repeated 'Spe' motif, which may be described as a symmetrically duplicated topoisomerase I recognition site. This motif was shown to be a target for illegitimate recombination events. The mobility of members of the MATE family is inferred from the observation that their chromosomal locations are highly variable between wild Aspergillus isolates. The inverted duplication AMA1 is present in laboratory strains derived from the Glasgow isolate but not in other wild isolates tested. This indicates that the inverted duplication AMA1 is of recent evolutionary origin and probably does not exert any conserved function in the chromosome. We discuss possible connections between structural features of AMA1 and its ability to promote extrachromosomal plasmid replication.

Amino Acid Sequence↗
PubMed1995

Integrative and replicative transformation of Penicillium canescens with a heterologous nitrate-reductase gene.

A wild isolate of Penicillium canescens was subjected to mutagenesis, and 150 chlorate-resistant mutants were isolated and classified in respect of their ability to utilize various nitrogen sources. Strains supposedly deficient in nitrate reductase have been transformed with the nitrate-reductase gene from Aspergillus niger. Transformation probably occurred by non-homologous integration of the transforming vector into the chromosome. Co-transformation with the AMA 1 replicating element from A. nidulans enhanced transformation frequency up to 2000-fold, and was shown to result in autonomous maintenance of replicating concatenates, one of which was re-isolated by transformation of E. coli.

DNA, Fungal↗
PubMed1995

Recombinational stability of replicating plasmids in Aspergillus nidulans during transformation, vegetative growth and sexual reproduction.

Plasmids containing the AMA1 replicon are capable of autonomous maintenance in Aspergillus nidulans. It has been reported previously that these plasmids can form concatenates by recombination in a transformed mycelium, and up to 10% of molecules are involved in such events. The present study demonstrates that plasmid recombination, although frequent during transformation, rarely occurs during vegetative growth. As a result, the structure and phenotypic stability of AMA1 plasmids generally remains unaltered for many asexual (conidial) generations. It is also evident that plasmid replication does not require specific recombination events in the AMA1 palindrome. However, during sexual reproduction, autonomous plasmids exhibit increased recombination, which results in both plasmid concatenation and integration into the chromosome.

Aspergillus nidulans↗
PubMed1994

An 'instant gene bank' method for gene cloning by mutant complementation.

We describe a new method of gene cloning by complementation of mutant alleles which obviates the need for construction of a gene library in a plasmid vector in vitro and its amplification in Escherichia coli. The method involves simultaneous transformation of mutant strains of the fungus Aspergillus nidulans with (i) fragmented chromosomal DNA from a donor species and (ii) DNA of a plasmid without a selectable marker gene, but with a fungal origin of DNA replication ('helper plasmid'). Transformant colonies appear as the result of the joining of chromosomal DNA fragments carrying the wild-type copies of the mutant allele with the helper plasmid. Joining may occur either by ligation (if the helper plasmid is in linear form) or recombination (if it is cccDNA). This event occurs with high efficiency in vivo, and generates an autonomously replicating plasmid cointegrate. Transformants containing Penicillium chrysogenum genomic DNA complementing A. nidulans niaD, nirA and argB mutations have been obtained. While some of these cointegrates were evidently rearranged or consisted only of unaltered replicating plasmid, in other cases plasmids could be recovered into E. coli and were subsequently shown to contain the selected gene. The utility of this "instant gene bank" technique is demonstrated here by the molecular cloning of the P. canescens trpC gene.

Aspergillus nidulans↗
PubMed1994

Enhancers of conidiation mutants in Aspergillus nidulans.

Mutants at a number of loci, designated sthenyo, have been isolated as enhancers of the oligoconidial mutations at the medA locus. Two loci have been mapped: sthA on linkage group I, and sthB on linkage group V. Two probable alleles have been identified at each locus but two further mutants were unlinked to either sthA or sthB. Neither sthA nor sthB mutants have conspicuous effects on morphology on their own, nor could the sthA1 sthB2 double mutant be distinguished from wild type. Mutants at both loci also interact with the temperature-sensitive brlA42 mutant at the permissive temperature to give a phenotype described as "Abacoid." sthA1 also induces a slight modification of the phenotype of an abaA mutant. We conclude that sthenyo genes act mainly at the phialide stage of conidiation. We also describe the isolation of new medA mutants arising spontaneously as outgrowths on brlA42 colonies.

Alleles↗
PubMed1994

Mutants of Aspergillus nidulans deficient in nuclear migration during hyphal growth and conidiation.

Anucleate primary sterigmata (aps) mutants of Aspergillus nidulans are partially blocked in conidiation (asexual sporulation) due to failure of the organized migration of nuclei into the conidiophore metulae. The mutants also have a slightly reduced hyphal growth rate and irregular distribution of nuclei in vegetative hyphae; the hyphal phenotype appears somewhat more variable than the conidiation defect. The mutants fall into two complementation groups, apsA and apsB, mapping on chromosomes IV and VI, respectively. apsB mutants are also partially defective in sexual reproduction.

Aspergillus nidulans↗
PubMed1994

Homology at the amino acid level between plant phytochromes and a regulator of asexual sporulation in Emericella (= Aspergillus) nidulans.

Protein sequence comparison between the N-terminal regions of the BRLA (bristle A) protein of the ascomycete fungus Aspergillus nidulans and a number of plant phytochromes has demonstrated a moderate level of sequence similarity. The region of similarity corresponds to the phytochrome domains believed to be responsible for photoreception and which undergo light-induced conformational changes, although a putative chromophore-binding site is not evident. Over 22% of residues are conserved and 24% conservatively substituted between residues 1 and 272 of BRLA and the N-terminal domains of Type 1 phytochromes from dicotyledonous species. A lower level of similarity, but over the same region, is observed in comparison with a wider range of phytochromes. Given the known role of BRLA as a transcriptional activator involved in conidiation, and the red/far-red reversible photoregulation of this developmental process, the similarity with phytochromes may be significant.

Amino Acid Sequence↗
PubMed1993

Co-transformation with autonomously-replicating helper plasmids facilitates gene cloning from an Aspergillus nidulans gene library.

Autonomously-replicating, marker-less "helper" plasmids were added to transformations of Aspergillus nidulans with plasmids which normally transform by chromosomal integration. This resulted in as much as a 200-fold increase in transformation efficiency. Recovery of autonomously-replicating plasmid co-integrates indicated that co-transformation involves recombination between integrating and helper plasmids, which occurs at a high frequency. Increasing DNA sequence-homology between pairs of plasmids used in simultaneous transformations enhanced co-transformation efficiency. Using helper plasmids and an A. nidulans gene library in a normally-integrating vector, the genes adC and adD were cloned as part of such a co-integrate. In effect, the addition of helper plasmid converts an integrating into an autonomously-replicating gene library in vivo.

Aspergillus nidulans↗
PubMed1992

An intragenic map of the brlA locus of Aspergillus nidulans.

We have constructed an intragenic map for the Aspergillus nidulans brlA gene, mutants in which are distinguishable by visual criteria only. Most of the leaky phenotype mutants map near the right (3') end. The gene shows distinct recombinational polarity consistent with recombination initiation at the promoter (centromere-proximal) end of the gene. brlA12 and brlA20 mutants gave abnormal DNA restriction patterns consistent with the III; VIII and VI; VIII translocations, respectively, determined by haploidization.

Alleles↗
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↗