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A Aleksenko

Publications and source records attributed to A Aleksenko.

11 recordsLinked to original sources
PubMed2001

Genetic and physical mapping of two centromere-proximal regions of chromosome IV in Aspergillus nidulans.

Chromosome IV is the smallest chromosome of Aspergillus nidulans. The centromere-proximal portion of the chromosome was mapped physically using overlapping clones of a cosmid genomic library. Two contiguous segments of a physical map, based on restriction mapping of cosmid clones, were generated, together covering more than 0.4 Mb DNA. A reverse genetic mapping approach was used to establish a correlation between physical and genetic maps; i.e., marker genes were integrated into physically mapped segments and subsequently mapped by mitotic and meiotic recombination. The resulting data, together with additional classical genetic mapping, lead to a substantial revision of the genetic map of the chromosome, including the position of the centromere. Comparison of physical and genetic maps indicates that meiotic recombination is low in subcentromeric DNA, its frequency being reduced from 1 crossover per 0.8 Mb to approximately 1 crossover per 5 Mb per meiosis. The portion of the chromosome containing the functional centromere was not mapped because repeat-rich regions hindered further chromosome walking. The size of the missing segment was estimated to be between 70 and 400 kb.

Aspergillus nidulans↗
PubMed2001

A family of DNA repeats in Aspergillus nidulans has assimilated degenerated retrotransposons.

In the course of a chromosomal walk towards the centromere of chromosome IV of Aspergillus nidulans, several cross-hybridizing genomic cosmid clones were isolated. Restriction mapping of two such clones revealed that their restriction patterns were similar in a region of at least 15 kb, indicating the presence of a large repeat. The nature of the repeat was further investigated by sequencing and Southern analysis. The study revealed a family of long dispersed repeats with a high degree of sequence similarity. The number and location of the repeats vary between wild isolates. Two copies of the repeat contained degenerated long-terminal-repeat (LTR) retrotransposons, which were named Dane1 and Dane2 (for Degenerated Aspergillus nidulans element). The pattern of degeneration suggested that a process similar to the repeat-induced point-mutation (RIP) phenomenon, first described in Neurospora crassa, may have operated in A. nidulans. The data indicate that this family of repeats has assimilated mobile elements that subsequently degenerated but then underwent further duplications as a part of the host repeats.

Amino Acid Sequence↗
PubMed2000

Antisense silencing of the creA gene in Aspergillus nidulans.

Antisense expression of a portion of the gene encoding the major carbon catabolite repressor CREA in Aspergillus nidulans resulted in a substantial increase in the levels of glucose-repressible enzymes, both endogenous and heterologous, in the presence of glucose. The derepression effect was approximately one-half of that achieved in a null creA mutant. Unlike results for that mutant, however, growth parameters and colony morphology in the antisense transformants were not affected.

Aspergillus nidulans↗
PubMed1999

Structural and transcriptional analysis of the pyrABCN, pyrD and pyrF genes in Aspergillus nidulans and the evolutionary origin of fungal dihydroorotases.

The six biochemical steps of the de novo pyrimidine biosynthesis pathway are conserved in all known organisms. However, in animals and fungi, unlike prokaryotes, at least the first two activities are grouped on a multifunctional enzyme. Here, we report cloning, mapping and transcriptional characterization of some pyrimidine biosynthesis genes in the filamentous fungus Aspergillus nidulans. The first two steps of the pathway are performed by a multifunctional enzyme comprising the activities of carbamoyl phosphate synthetase (CPSase) and aspartate transcarbamylase (ATCase). This polypeptide is encoded by a 7 kbp cluster gene, pyrABCN, which has a high degree of nucleotide identity with the Ura2 gene in Saccharomyces cerevisiae. The enzyme of the third step, dihydroorotase (DHOase), is encoded by a separate locus, pyrD. However, the pyrABCN gene apparently contains an evolutionary remnant of a DHOase-encoding sequence, similarly to the Ura2 gene of Saccharomyces cerevisiae. The pyrABCN gene is transcribed as a single 7 kb mRNA species. The level of transcripts of pyrABCN, pyrD and, to a lesser degree, pyrF genes responds to the presence of exogenous pyrimidines and to the conditions of pyrimidine starvation. Derepression of pyrABCN and pyrD under pyrimidine starvation is noticeably enhanced in pyrE mutants that accumulate dihydroorotic acid. The pyrABCN gene maps to the distal portion of the right arm of the chromosome VIII, whereas the pyrD gene, in contrast to early genetic data, is closely linked to the brlA gene and located to the right of it. Our data on mitotic recombination should help to verify the genetic map of the chromosome VIII. Comparison of amino acid sequences of active dihydroorotases with related enzymes and with their non-functional homologues in yeast and Aspergillus indicates that the active dihydroorotases from fungi are more similar to ureases and enzymes of the pyrimidine degradation pathway. The 'silent' dihydroorotase domains of the multifunctional enzymes from fungi and active DHOase domains of the multifunctional enzymes in higher eukaryotes are more closely related to bacterial dehydroorotases.

Aspartate Carbamoyltransferase↗
PubMed1998

In vivo linearization and autonomous replication of plasmids containing human telomeric DNA in Aspergillus nidulans.

Plasmids containing two inverted 0.6-kb stretches of human telomeric repeats transform Aspergillus nidulans at frequencies characteristic of autonomously replicating vectors. Transformation frequency is not affected when the plasmids are linearized in vitro prior to transformation by cutting between the inverted repeats. Southern analysis reveals the presence of a homogeneous pool of linear plasmid molecules in mycelium of transformants. Addition of the AMA1 plasmid replicator to the telomere-containing plasmids has only a minor effect on transformation. The phenotypic stability of the transformants is low. However, unlike conventional replicative transformants containing AMA1-bearing plasmids, these transformants are prone to spontaneous stabilization which occurs predominantly by conversion of the mutant chromosomal allele of the marker gene to the plasmid-borne allele. The data strongly suggest that telomeric DNA can act as a plasmid replicator. An alternative interpretation is that autonomous replication of linear DNA fragments, in contrast to covalently closed supercoiled molecules, does not require any special replicator sequences.

Aspergillus nidulans↗
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↗
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↗
PubMed1996

Multiple copies of MATE elements support autonomous plasmid replication in Aspergillus nidulans.

The AMA1 sequence is an efficient plasmid replicator and transformation enhancer in Aspergillus nidulans. It comprises two long perfect inverted repeats (MATE elements) flanking a short, unique, central spacer. Subclone analysis indicates that the complete inverted duplication, but not the unique central spacer, is necessary for efficient plasmid replication. The smallest fragments able to affect transformation efficiency lie within the AT-rich portions of the inverted repeats. We demonstrate that two or more copies of the repeat in any relative orientation are able to perform the replicator function. A single copy of a MATE element increases transformation frequency to a modest extent but leads to multiple rearrangement, unstable integration or concatenation of vector molecules. Multimeric concatenates generated during this process are more sable mitotically, and when reisolated, transform the fungus at a much higher frequency than the original monomeric vector. Selection for multiple products which resemble amplified DNA in various eukaryotic systems.

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↗