Biomedical subjects

M R Mautino

Publications and source records attributed to M R Mautino.

7 recordsLinked to original sources

PubMed1998

Analysis of models involving enzymatic activities for the occurrence of C-->T transition mutations during repeat-induced point mutation (RIP) in Neurospora crassa.

The phenomenon of repeat-induced point mutation (RIP), acting during the sexual phase of the model eukaryote Neurospora crassa, is considered to study the putative in vivo relationships existing between cellular levels of S-adenosylmethionine (SAM), cytosine methylation and the occurrence of C-->T transition mutations. We analyse the kinetic behaviour of the different enzymatic models proposed to explain the underlying mutagenic mechanisms of RIP. The dependence of the mutation rate on the cellular levels of the methyl group donor SAM was evaluated for the models of mutation catalysed by a DNA-cytosine deaminase, a DNA-(5-methylcytosine) deaminase, a DNA-(5-cytosine) methyltransferase, and for a model combining the activities of the last two enzymes. We propose that these models can be distinguished by studying the dependence of RIP on intracellular SAM levels.

Models, Biological↗

PubMed1997

Mapping chromosome landmarks in the centromere I region of Neurospora crassa.

Chromosome translocation breakpoints, RFLP heterozygosity in partial chromosome duplications, and RFLP-marked crossover events have been used as chromosomal landmarks to find the position and orientation of cloned regions flanking centromere I of Neurospora crassa. Determination of physical:genetic ratios in genomic regions flanking the loci mei-3, un-2, and his-2 supports previous evidence indicating that recombinational activity is lower in regions flanking centromere I than in the general N. crassa genome. The homogeneous distribution of crossover events found in these regions suggests that there is not a gradient of crossover inhibition in the vicinity of centromere I. Thus, a largely extended centromeric effect and/or a general crossover inhibitory effect operating on linkage group I (LGI) could constitute the basis of these abnormal physical:genetic ratios. A DNA element containing about 76% A+T was isolated from the centromeric end of a cloned region on LGIR. The fragment includes a previously undescribed DNA sequence, highly repeated in the Neurospora genome, which may correspond to centromeric DNA.

Base Composition↗

PubMed1996

Cloning and sequence of the Ascobolus immersus S-adenosyl-L-methionine synthetase-encoding gene.

The structural gene encoding S-adenosyl-L-methionine synthetase (SAM-S) in the fungus Ascobolus immersus has been cloned and sequenced. It contains a 1179-bp ORF, interrupted by three introns, encoding a 393-amino-acid protein (42 978 Da) that is 90% homologous to the SAM-S of the filamentous fungus Neurospora crassa, indicating that these fungi are closely related species.

Amino Acid Sequence↗

PubMed1996

eth-1, the Neurospora crassa locus encoding S-adenosylmethionine synthetase: molecular cloning, sequence analysis and in vivo overexpression.

Intense biochemical and genetic research on the eth-1r mutant of Neurospora crassa suggested that this locus might encode S-adenosylmethionine synthetase (S-Adomet synthetase). We have used protoplast transformation and phenotypic rescue of a thermosensitive phenotype associated with the eth-1r mutation to clone the locus. Nucleotide sequence analysis demonstrated that it encodes S-Adomet synthetase. Homology analyses of prokaryotic, fungal and higher eukaryotic S-Adomet synthetase polypeptide sequences show a remarkable evolutionary conservation of the enzyme. N. crassa strains carrying S-Adomet synthetase coding sequences fused to a strong heterologous promoter were constructed to assess the phenotypic consequences of in vivo S-Adomet synthetase overexpression. Studies of growth rates and microscopic examination of vegetative development revealed that normal growth and morphogenesis take place in N. crassa even at abnormally high levels of cellular S-Adomet. The degree of cytosine methylation of a naturally methylated genomic region was dependent on the cellular levels of S-Adomet. We conclude that variation in S-Adomet levels in N. crassa cells, which in addition to the status of genomic DNA methylation could modify the flux of other S-Adomet-dependent metabolic pathways, does not affect growth rate or morphogenesis.

Alleles↗

PubMed1996

A dominant negative effect of eth-1r, a mutant allele of the Neurospora crassa S-adenosylmethionine synthetase-encoding gene conferring resistance to the methionine toxic analogue ethionine.

eth-1r, a thermosensitive allele of the Neurospora crassa S-adenosylmethionine (AdoMet) synthetase gene that confers ethionine resistance, has been cloned and sequenced. Replacement of an aspartic amino acid residue (D48-->N48), perfectly conserved in prokaryotic, fungal and higher eukaryotic AdoMet synthetases, was found responsible for both thermosensitivity and ethionine resistance conferred by eth-1r. Gene fusion constructs, designed to overexpress eth-1r in vivo, render transformant cells resistant to ethionine. Dominance of ethionine resistance was further demonstrated in eth-1+/eth-1r partial diploids carrying identical gene doses of both alleles. Heterozygous eth-1+/eth-1r cells have, at the same time, both the thermotolerance conferred by eth-1+ and the ethionine-resistant phenotype conferred by eth-1r. AdoMet levels and AdoMet synthetase activities were dramatically decreased in heterozygous eth-1+/ eth-1r cells. We propose that this negative effect exerted by eth-1r results from the in vivo formation of heteromeric eth-1+/eth-1r AdoMet synthetase molecules.

Alleles↗

PubMed1994

A computer program for construction of circular restriction maps.

The computer program COSMAP for construction of circular restriction maps has been developed. The generator is based on a permutational algorithm that puts forward all the possible maps that can be made with two enzymes, and the consistency checker compares the calculated and experimental double-digest fragment sizes within a predefined error bound. The output consists of the complete set of solutions ordered by a score number that is based on the matching degree between experimental and calculated double restriction fragment sizes. The program allows the application of heuristic rules which reduces the number of permutations to be checked and the number of sound solutions.

DNA, Circular↗

PubMed1993

Physical mapping of meiotic crossover events in a 200-kb region of Neurospora crassa linkage group I.

We propose a general restriction fragment length polymorphism-based strategy to analyze the distribution of meiotic crossover events throughout specific genetic intervals. We have isolated 64 recombinant chromosomes carrying independent meiotic crossover events in the genetic interval eth-1-un-2 on linkage group I of Neurospora crassa. Thirty-eight crossover events were physically mapped with reference to a 200-kb region cloned by chromosome walking, using N. crassa lambda and cosmid libraries. Crossovers were homogeneously distributed at intervals of 5.0 +/- 2.3 kb along the entire cloned interval. The ratio of physical to genetic distance appears to be higher in the region than in the overall N. crassa genome, suggesting that recombinational activity is less in large chromosomes than in small ones. The present work provides a method for defining the centromeric-telomeric orientation of single cloned DNA fragments. Their physical distance can also be estimated with respect to linked loci, provided that crossover events are distributed homogeneously in the interval. This strategy overcomes typical difficulties in defining the position and direction of chromosome walking steps on conventional linkage maps.

Chromosome Mapping↗