Biomedical subjects

F Foury

Publications and source records attributed to F Foury.

At least 19 recordsLinked to original sources

PubMed2004

Mitochondrial DNA mutators.

In this article we review our current knowledge of the mechanisms by which point mutations arise in the mitochondrial DNA (mtDNA) of Saccharomyces cerevisiae and discuss to what extent these mechanisms operate in human mtDNA mutagenesis. The 3'-5' exonuclease proofreading activity of Pol gamma ensures accuracy of mtDNA replication in both yeast and humans, while the role of base excision repair in mtDNA error avoidance remains debated. The mitochondrial mismatch repair Msh1 protein, which removes transitions in yeast, is absent in humans, a particularity that might cause accumulation of transitions, while the most frequent substitution in yeast mtDNA is A:T to T:A transversion. Proofreading-deficient mutator human cell lines and knockin mice have been created. They will be useful for studying the mechanisms by which mtDNA mutations accumulate in diseases, ageing, malignancy and drug therapy.

Animals↗

PubMed2003

Search for protein partners of mitochondrial single-stranded DNA-binding protein Rim1p using a yeast two-hybrid system.

RIM1 is a nuclear gene of the yeast Saccharomyces cerevisiae coding for a protein with single-stranded DNA-binding activity that is essential for mitochondrial genome maintenance. No protein partners of Rim1p have been described so far in yeast. To better understand the role of this protein in mitochondrial DNA replication and recombination, a search for protein interactors by the yeast two-hybrid system was performed. This approach led to the identification of several candidates, including a putative transcription factor, Azf1p, and Mph1p, a protein with an RNA helicase domain which is known to influence the mutation rate of nuclear and mitochondrial genomes.

DNA, Mitochondrial↗

PubMed2000

Mitochondrial control of iron homeostasis. A genome wide analysis of gene expression in a yeast frataxin-deficient strain.

Deletion of YFH1, the yeast frataxin homologue gene, elicits mitochondrial iron accumulation and alters cellular iron homeostasis. Here, we report a genome wide analysis of gene expression in a yfh1(DeltaYFH1) deleted strain. Frataxin deficiency results in enhanced expression of some 70 genes including a set of genes, called the iron regulon, that are under the control of the iron-sensing transcription factor AFT1. Five new AFT1-dependent genes, YOR382w, YOR383c, YDR534c, YLR136c, and YLR205c were found. The first three genes presumably encode cell-wall glycosylphosphatidylinositol anchor proteins and exhibit a 30-100-fold increased expression. The triple deletion of these genes decreases efficiency in utilization of the iron of ferrioxamine B by the yeast cell. YLR136c bears homology to tristetraproline proteins, which are post-transcriptional regulators in mammalian cells. Deletion of YLR136c increases the mRNA levels of iron regulon members. YLR205c bears homology to heme oxygenases. Our data show that frataxin deficiency elicits iron mobilization from all iron sources in an AFT1-dependent manner. Wild-type and DeltaYFH1 glycerol-grown cells exhibit similar high respiration rates, no mitochondrial iron accumulation, and high expression of the iron regulon, suggesting that under these conditions little iron is extruded from mitochondria. These data suggest that the activity of Yfh1p is not essential in cells grown on glycerol. This study has also revealed unexpected links between mitochondria and remote metabolic pathways since frataxin deficiency also enhances the expression of genes such as HSP30, that escape to AFT1 control. Finally, no oxidative stress gene is induced.

Ceruloplasmin↗

PubMed2000

In vivo functional analysis of the human mitochondrial DNA polymerase POLG expressed in cultured human cells.

The human gene POLG encodes the catalytic subunit of mitochondrial DNA polymerase, but its precise roles in mtDNA metabolism in vivo have not hitherto been documented. By expressing POLG fusion proteins in cultured human cells, we show that the enzyme is targeted to mitochondria, where the Myc epitope-tagged POLG is catalytically active as a DNA polymerase. Long-term culture of cells expressing wild-type POLG-myc revealed no alterations in mitochondrial function. Expression of POLG-myc mutants created dominant phenotypes demonstrating important roles for the protein in mtDNA maintenance and integrity. The D198A amino acid replacement abolished detectable 3'-5' (proofreading) exonuclease activity and led to the accumulation of a significant load (1:1700) of mtDNA point mutations during 3 months of continuous culture. Further culture resulted in the selection of cells with an inactivated mutator polymerase, and a reduced mutation load in mtDNA. Transient expression of POLG-myc variants D890N or D1135A inhibited endogenous mitochondrial DNA polymerase activity and caused mtDNA depletion. Deletion of the POLG CAG repeat did not affect enzymatic properties, but modestly up-regulated expression. These findings demonstrate that POLG exonuclease and polymerase functions are essential for faithful mtDNA maintenance in vivo, and indicate the importance of key residues for these activities.

Amino Acid Substitution↗

PubMed2000

New features of mitochondrial DNA replication system in yeast and man.

In this review, we sum up the research carried out over two decades on mitochondrial DNA (mtDNA) replication, primarily by comparing this system in Saccharomyces cerevisiae and Homo sapiens. Brief incursions into systems of other organisms have also been achieved when they provide new information.S. cerevisiae and H. sapiens mitochondrial DNA (mtDNA) have been thought for a long time to share closely related architecture and replication mechanisms. However, recent studies suggest that mitochondrial genome of S. cerevisiae may be formed, at least partially, from linear multimeric molecules, while human mtDNA is circular. Although several proteins involved in the replication of these two genomes are very similar, divergences are also now increasingly evident. As an example, the recently cloned human mitochondrial DNA polymerase beta-subunit has no counterpart in yeast. Yet, yeast Abf2p and human mtTFA are probably not as closely functionally related as thought previously. Some mtDNA metabolism factors, like DNA ligases, were until recently largely uncharacterized, and have been found to be derived from alternative nuclear products. Many factors involved in the metabolism of mitochondrial DNA are linked through genetic or biochemical interconnections. These links are presented on a map. Finally, we discuss recent studies suggesting that the yeast mtDNA replication system diverges from that observed in man, and may involve recombination, possibly coupled to alternative replication mechanisms like rolling circle replication.

DNA Replication↗

PubMed2000

Systematic disruption of 456 ORFs in the yeast Saccharomyces cerevisiae.

In the framework of the European Network for Functional Analysis (EUROFAN), five packages of 96 ORFs from chromosomes III, IV, VII, XIII, XIV and XV were subjected to systematic deletions in an isogenic derivative of strain S288c. Deletions were constructed in diploid and haploid strains. Two questionable ORFs overlapping with larger ORFs and seven TY ORFs were discarded. A total of 456 heterozygous and 385 homozygous deletant diploids were obtained. Sixty-nine deletions, 25 of which had never been published before, were lethal in haploid strains and 30 caused slow cellular growth.

Gene Deletion↗

PubMed1999

Two antigens recognized by autologous cytolytic T lymphocytes on a melanoma result from a single point mutation in an essential housekeeping gene.

We have pursued our analysis of antigens recognized by autologous cytolytic T lymphocytes (CTLs) on the melanoma cells of patient LB33. This patient enjoys an unusually favorable evolution, which is associated with a strong and sustained antitumor CTL response. We reported previously the analysis of two melanoma cell lines, MEL.A and MEL.B, which were derived from metastases removed from the patient at 5 years' distance. Autologous CTL clones derived from blood lymphocytes recognized several antigens presented by different HLA class I molecules on MEL.A. The MEL.B cells resisted lysis by these CTLs because they have lost expression of most HLA molecules, suggesting that they were selected in vivo by the anti-MEL.A CTL response. One of the MEL.A antigens was shown to result from a point mutation in the tumor. Here we report the cloning of a gene that encodes two other MEL.A antigens. This new gene, MUM-2, is expressed ubiquitously. In the melanoma cells of patient LB33, it contains a point mutation that changes one amino acid in the translated protein. Two different antigenic peptides, one presented to CTL by HLA-B44 molecules and another by HLA-C6 molecules, overlap and contain the mutated residue. Gene MUM-2 is homologous to an essential yeast gene, bet5, that was recently shown to be implicated in the vesicular transport of proteins from the endoplasmic reticulum to the Golgi. In a mutant yeast with a disrupted bet5 gene, both the wild-type and the mutated MUM-2 genes could complement for bet5 function. These results indicate that the antigenic mutation does not destroy the function of the protein, a function that is conserved in eukaryotic cells. The identification of these antigens suggests that point mutations could be the major cause of the strong immunogenicity of MEL.A cells.

Amino Acid Sequence↗

PubMed1999

Low iron concentration and aconitase deficiency in a yeast frataxin homologue deficient strain.

Deletion of the yeast frataxin homologue, YFH1, elicits accumulation of iron in mitochondria and mitochondrial defects. We report here that in the presence of an iron chelator in the culture medium, the concentration of iron in mitochondria is the same in wild-type and YFH1 deletant strains. Under these conditions, the activity of the respiratory complexes is restored. However, the activity of the mitochondrial aconitase, a 4Fe-4S cluster-containing protein, remains low. The frataxin family bears homology to a bacterial protein family which confers resistance to tellurium, a metal closely related to sulfur. Yfh1p might control the synthesis of iron-sulfur clusters in mitochondria.

Aconitate Hydratase↗

PubMed1999

Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis.

The functions of many open reading frames (ORFs) identified in genome-sequencing projects are unknown. New, whole-genome approaches are required to systematically determine their function. A total of 6925 Saccharomyces cerevisiae strains were constructed, by a high-throughput strategy, each with a precise deletion of one of 2026 ORFs (more than one-third of the ORFs in the genome). Of the deleted ORFs, 17 percent were essential for viability in rich medium. The phenotypes of more than 500 deletion strains were assayed in parallel. Of the deletion strains, 40 percent showed quantitative growth defects in either rich or minimal medium.

Culture Media↗

PubMed1999

Mass-murdering: deletion of twenty-three ORFs from Saccharomyces cerevisiae chromosome XI reveals five genes essential for growth and three genes conferring detectable mutant phenotype.

In the frame of the European Network for Functional Analysis (EUROFAN), two regions from chromosome XI covering 54kb have been subjected to 'mass-murder'. Ten deletions covering 23 novel open reading frames (ORFs) were constructed in haploid and diploid strains. Six deletions were lethal in haploid strains. One deletion caused slow germination of spores and slow cellular growth, and another one was associated with both cellular growth thermosensitivity and poor growth on glycerol. These two defects were assigned to two different genes. All mutant phenotypes were complemented by a single gene, enabling us to identify five genes essential for vegetative growth, three genes with detectable phenotype and 15 dispensable genes under standard physiological conditions.

Cell Division↗

PubMed1999

Disruption and basic phenotypic analysis of 18 novel genes from the yeast Saccharomyces cerevisiae.

In the frame of the European Network for Functional Analysis (EUROFAN) we have deleted 18 yeast open reading frames (ORFs) from chromosomes II, X and XIV using the short flanking homology-PCR strategy. Two diploid strains were used: FY1679 and CEN.PK2. The deletion kanMX6 cassettes with long flanking homology and the cognate gene clones have also been constructed. Heterozygous diploid deletant strains have been sporulated. Tetrad analysis revealed that all the ORFs studied were non-essential. However, four deletant strains exhibited phenotypes. The YBL025w delta strain showed extremely slow cellular growth under all conditions tested. The YJL204c delta strain grew slower than wild-type at 30 degrees C and 37 degrees C, was cold-sensitive, and the homozygous diploids did not sporulate. The YNL213c delta strain did not grow on glycerol and had lost mitochondrial DNA. The deletion of YNL215w caused slower growth on all media but the defect was more pronounced on glucose-minimal and glycerol-rich media than on glucose-rich medium. All deletion mutants were complemented by the corresponding plasmid borne cognate gene. The YJL204w, YNL213c and YNL215w ORFs do not bear significant homology to proteins of known function. YBL025w has recently been identified as RRN10, a gene that encodes an RNA polymerase I-specific transcription initiator factor. The deletion of the remaining fourteen ORFs did not reveal any mutant phenotype in our basic growth tests.

Cold Temperature↗

PubMed1998

The complete sequence of the mitochondrial genome of Saccharomyces cerevisiae.

The currently available yeast mitochondrial DNA (mtDNA) sequence is incomplete, contains many errors and is derived from several polymorphic strains. Here, we report that the mtDNA sequence of the strain used for nuclear genome sequencing assembles into a circular map of 85,779 bp which includes 10 kb of new sequence. We give a list of seven small hypothetical open reading frames (ORFs). Hot spots of point mutations are found in exons near the insertion sites of optional mobile group I intron-related sequences. Our data suggest that shuffling of mobile elements plays an important role in the remodelling of the yeast mitochondrial genome.

Base Composition↗

PubMed1998

The role of 3'-5' exonucleolytic proofreading and mismatch repair in yeast mitochondrial DNA error avoidance.

In the D171G/D230A mutant generated at conserved aspartate residues in the Exo1 and Exo2 sites of the 3'-5' exonuclease domain of the yeast mitochondrial DNA (mtDNA) polymerase (pol-gamma), the mitochondrial genome is unstable and the frequency of mtDNA point mutations is 1500 times higher than in the wild-type strain and 10 times higher than in single substitution mutants. The 10(4)-fold decrease in the 3'-5' exonuclease activity of the purified mtDNA polymerase is associated with mismatch extension and high rates of base misincorporation. Processivity of the purified polymerase on primed single-stranded DNA is decreased and the Km for dNTP is increased. The sequencing of mtDNA point mutations in the wild-type strain and in proofreading and mismatch-repair deficient mutants shows that mismatch repair contributes to elimination of the transitions while exonucleolytic proofreading preferentially repairs transversions, and more specifically A to T (or T to A) transversions. However, even in the wild-type strain, A to T (or T to A) transversions are the most frequent substitutions, suggesting that they are imperfectly repaired. The combination of both mismatch repair and proofreading deficiencies elicits a mitochondrial error catastrophe. These data show that the faithful replication of yeast mtDNA requires both exonucleolytic proofreading and mismatch repair.

Base Sequence↗

PubMed1998

Two-hybrid systematic screening of the yeast proteome.

The yeast two-hybrid system is a genetic method that detects protein-protein interactions. One application is the detection by library screening of new interactors of a protein of known function. In the August issue of Nature Genetics, Fromont-Racine et al. showed for the first time that the construction of the protein interaction map of a complex pathway, such as that of the mRNA splicing machinery, is now possible, because of the combination of recent technical improvements elaborated in several laboratories. With a yeast cell mating procedure that increases screen efficiency, they used their complex yeast genomic library of 5 x 10(6) clones to test 700 x 10(6) interactions against 15 proteins. They identified and classified 170 potential interactors, including approximately 70 proteins of previously unknown function. More than 25% of the interactors are probably biologically relevant. The achievements of Fromont-Racine et al. have opened the way to the systematic analysis of the protein interaction networks of the 6,000 open reading frames-yeast proteome. This task requires, however, automation of the library screens and creation of a two-hybrid library database.

Fungal Proteins↗

PubMed1997

Human genetic diseases: a cross-talk between man and yeast.

A sequence similarity search has been carried out against the complete Saccharomyces cerevisiae genome to identify the yeast homologues of human disease-associated genes. Using the BLAST algorithm (Basic Local Alignment Search Tool), it was found that 52 out of the 170 disease genes identified without reference to chromosomal map position and 22 of the 80 (27.5%) positionally cloned genes match yeast genes with a P-value of <e(-40). The percentage of the disease genes identified by positional cloning which bear homology to yeast is similar to that of a random collection of human cDNAs. The biochemical and physiological functions of the large majority of these human genes remain poorly understood and, even though a strict conservation of function cannot safely be assessed from structural homology analysis without the support of experimental and three-dimensional data, functional analogies can often be established between the human and yeast genes.

Chromosome Mapping↗

PubMed1997

Deletion of the yeast homologue of the human gene associated with Friedreich's ataxia elicits iron accumulation in mitochondria.

Deletion of YDL120, the yeast homologue of the human gene responsible for Friedreich's ataxia, elicits decreased cellular respiration associated with decreased cytochrome c oxidase activity and, in certain nuclear backgrounds, mitochondrial DNA is lost. In the null mutants, the cellular growth is highly sensitive to oxidants, such as H2O2, iron and copper. However, only ferrous sulfate elicits loss of mitochondrial DNA. Mitochondria of the null mutants contain 10 times more iron than wild-type. The neurodegeneration observed in Friedreich's ataxia can be well explained on the basis of a mitochondrial iron overload responsible for an increased production of highly toxic free radicals.

Adenosine Triphosphatases↗

PubMed1997

Mitochondrial DNA polymerases from yeast to man: a new family of polymerases.

We report the sequence of a 4.5-kb cDNA clone isolated from a human melanoma library which bears high amino acid sequence identity to the yeast mitochondrial (mt) DNA polymerase (Mip1p). This cDNA contains a 3720-bp open reading frame encoding a predicted 140-kDa polypeptide that is 43% identical to Mip1p. The N-terminal part of the sequence contains a 13 glutamine stretch encoded by a CAG trinucleotide repeat which is not found in the other DNA polymerases gamma (Pol gamma). Multiple amino acid sequence alignments with Pol gamma from Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris, Drosophila melanogaster, Xenopus laevis and Mus musculus show that these DNA polymerases form a family strongly conserved from yeast to man and are only loosely related to the Family A DNA polymerases.

Amino Acid Sequence↗

PubMed1997

Studies of human, mouse and yeast homologues indicate a mitochondrial function for frataxin.

Friedreich's ataxia is due to loss of function mutations in the gene encoding frataxin (FRDA). Frataxin is a protein of unknown function. In situ hybridization analyses revealed that mouse frataxin expression correlates well with the main site of neurodegeneration, but the expression pattern is broader than expected from the pathology of the disease. Frataxin mRNA is predominantly expressed in tissues with a high metabolic rate, including liver, kidney, brown fat and heart. We found that mouse and yeast frataxin homologues contain a potential mitochondrial targeting sequence in their N-terminal domains and that disruption of the yeast gene results in mitochondrial dysfunction. Finally, tagging experiments demonstrate that human frataxin co-localizes with a mitochondrial protein. Friedreich's ataxia is therefore a mitochondrial disease caused by a mutation in the nuclear genome.

Amino Acid Sequence↗