Biomedical subjects

M Bolotin-Fukuhara

Publications and source records attributed to M Bolotin-Fukuhara.

At least 19 recordsLinked to original sources

PubMed2006

Mutations in yeast mt tRNAs: specific and general suppression by nuclear encoded tRNA interactors.

Mutations in mitochondrial tRNA genes can produce alterations in tRNA structure resulting in defective mitochondrial protein synthesis and hence respiratory defects. Such defects are often at the origin of neurodegenerative diseases in humans and can be easily studied in yeast since respiratory deficient mutants are viable. Several nuclear encoded tRNA interactors have been shown to rescue the mitochondrial defects due to mutations in mitochondrial tRNAs. Among these, we have identified the gene for the mitochondrial protein synthesis elongation factor EF-Tu and the specific mt aminoacyl-tRNA synthetases. We also observed that the respiratory defects and the effect of the TUF1 over-expression were strongly strain dependent. The importance of the nuclear background in which the mitochondrial mutation is expressed was investigated by changing the nuclear context. Finally, we demonstrated, using the RT-PCR method, the existence of significantly variable levels of the TUF1 transcript among strains with functional and dysfunctional mitochondria.

Amino Acyl-tRNA Synthetases↗

PubMed2004

A new Hansenula polymorpha HAP4 homologue which contains only the N-terminal conserved domain of the protein is fully functional in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, the HAP transcriptional complex is involved in the fermentation-respiration shift. This complex is composed of four subunits. Three subunits are necessary for DNA-binding, whereas the Hap4p subunit, glucose-repressed, contains the transcriptional activation domain. Hap4p is the key regulator of the complex activity in response to carbon sources in S. cerevisiae. To date, no HAP4 homologue has been identified, except in Kluyveromyces lactis. Examination of these two HAP4 sequences led to the identification of two very short conserved peptides also identified in other yeasts. In the yeast Hansenula polymorpha, two possible HAP4 homologues have been found. Their deduced amino acid sequences are similar to the ScHap4p and KlHap4p proteins only in the N-terminal 16-amino-acid basic motif. Since molecular genetic tools exist and complete genome sequence is known for this yeast, we expressed one of these putative HpHap4 proteins in S. cerevisiae and showed that this protein is able to restore the growth defect of the S. cerevisiae hap4-deleted strain. A set of experiments was performed to confirm the functional homology of this new gene with ScHAP4. The discovery of a Hap4-regulatory protein in H. polymorpha with only the N-terminal conserved domain of the S. cerevisiae protein indicates that this domain may play a crucial role during evolution.

CCAAT-Binding Factor↗

PubMed2003

The yeast counterparts of human 'MELAS' mutations cause mitochondrial dysfunction that can be rescued by overexpression of the mitochondrial translation factor EF-Tu.

We have taken advantage of the similarity between human and yeast (Saccharomyces cerevisiae) mitochondrial tRNA(Leu)(UUR), and of the possibility of transforming yeast mitochondria, to construct yeast mitochondrial mutations in the gene encoding tRNA(Leu)(UUR) equivalent to the human A3243G, C3256T and T3291C mutations that have been found in patients with the neurodegenerative disease MELAS (for mitochondrial 'myopathy, encephalopathy, lactic acidosis and stroke-like episodes'). The resulting yeast cells (bearing the equivalent mutations A14G, C26T and T69C) were defective for growth on respiratory substrates, exhibited an abnormal mitochondrial morphology, and accumulated mitochondrial DNA deletions at a very high rate, a trait characteristic of severe mitochondrial defects in protein synthesis. This effect was specific at least in the pathogenic mutation T69C, because when we introduced A or G instead of C, the respiratory defect was absent or very mild. All defective phenotypes returned to normal when the mutant cells were transformed by multicopy plasmids carrying the gene encoding the mitochondrial elongation factor EF-Tu. The ability to create and analyse such mutated strains and to select correcting genes should make yeast a good model for the study of tRNAs and their interacting partners and a practical tool for the study of pathological mutations and of tRNA sequence polymorphisms.

Amino Acid Substitution↗

PubMed2002

Mitochondrial effects of the pleiotropic proteasomal mutation mpr1/rpn11: uncoupling from cell cycle defects in extragenic revertants.

We have previously characterized a Saccharomyces cerevisiae mutant which contains a mutation in the essential rpn11/mpr1 gene coding for the proteasomal regulatory subunit Rpn11. The mpr1-1 mutation shows the phenotypic characteristics generally associated with proteasomal mutations, such as cell cycle defects and accumulation of polyubiquitinated proteins. However, for the first time, mitochondrial defects have also been found to be a consequence of a mutation in a proteasomal gene (Mol. Biol. Cell 9 (1998) 2917-2931). Since the mutant strain is thermosensitive both on glucose and on glycerol, we searched for revertants in order to shed light on the Rpn11/Mpr1 functions. Spontaneous revertants able to grow on glucose but not on glycerol at 36 degrees C were isolated, and, only from them, revertants able to grow at 36 degrees C on glycerol were selected. Revertants of the two classes were found to be extragenic. The detailed characterization of these extragenic suppressors demonstrates that the phenotypes related to cell cycle defects can be dissociated from those concerned with mitochondrial organization.

Amino Acid Sequence↗

PubMed2001

Reintroduction of a characterized Mit tRNA glycine mutation into yeast mitochondria provides a new tool for the study of human neurodegenerative diseases.

We report the identification and characterization of a new mutation (ts9) in the Saccharomyces cerevisiae mitochondrial genome, which was first genetically mapped in the tRNAgly region and further identified by means of sequencing as consisting of a G to A transition at position 30 in the tRNA. The mutation causes an almost complete disappearance of mature tRNAgly, while a second mitochondrial mutation with a compensatory C to T change restores it in normal quantities; this points to the importance of the strong bond between bases 30 and 40 of the anticodon stem in the stabilization of the tRNA. In addition to resulting in a clear-cut heat-sensitive phenotype, the ts9 mutation creates a new EcoRV restriction site. Both properties were used as markers to monitor the successful (re) introduction of the mutated allele into a wild-type mitochondrial genome through biolistic transformation. The mutant frequency in the progeny as well as the correct integration of the mutated allele at its proper site demonstrate the feasibility of this method for creating and investigating specific mitochondrial tRNA mutations. The method will provide important applications for the use of yeast as a model system of human mitochondrial pathologies.

Bacterial Proteins↗

PubMed2001

A new family of yeast vectors and S288C-derived strains for the systematic analysis of gene function.

The yeast genome has been shown to contain a significant number of gene families with more than three members. In order to study these families it is often necessary to generate strains carrying deletions of all members of the family, which can require a wide range of auxotrophic markers. To facilitate such studies, we have generated yeast strains containing deletions of a selection of nutritional marker genes (ade2, ade4, ade8, met3 and met14). We have also cloned the corresponding cognate genes, allowing their use in PCR-based gene disruptions. Two new pRS family Saccharomyces cerevisiae-Escherichia coli shuttle vectors containing ADE8 (one low-copy, pRS4110, and one high-copy, pRS4210) have been produced for use in conjunction with the new strains. A system for easier synthetic lethal screening using one of these new markers is also presented. The ADE8 and HIS3 genes have been cloned together on a high-copy vector (pRS4213), providing a plasmid for red-white colour screening in the ade2 Delta 0 ade8 Delta 0 strains we have generated. In contrast to some conventional systems, this plasmid allows for screening using gene libraries constructed in URA3 plasmids.

Base Sequence↗

PubMed2000

Genomic exploration of the hemiascomycetous yeasts: 1. A set of yeast species for molecular evolution studies.

The identification of molecular evolutionary mechanisms in eukaryotes is approached by a comparative genomics study of a homogeneous group of species classified as Hemiascomycetes. This group includes Saccharomyces cerevisiae, the first eukaryotic genome entirely sequenced, back in 1996. A random sequencing analysis has been performed on 13 different species sharing a small genome size and a low frequency of introns. Detailed information is provided in the 20 following papers. Additional tables available on websites describe the ca. 20000 newly identified genes. This wealth of data, so far unique among eukaryotes, allowed us to examine the conservation of chromosome maps, to identify the 'yeast-specific' genes, and to review the distribution of gene families into functional classes. This project conducted by a network of seven French laboratories has been designated 'Génolevures'.

Ascomycota↗

PubMed2000

Genomic exploration of the hemiascomycetous yeasts: 3. Methods and strategies used for sequence analysis and annotation.

The primary analysis of the sequences for our Hemiascomycete random sequence tag (RST) project was performed using a combination of classical methods for sequence comparison and contig assembly, and of specifically written scripts and computer visualization routines. Comparisons were performed first against DNA and protein sequences from Saccharomyces cerevisiae, then against protein sequences from other completely sequenced organisms and, finally, against protein sequences from all other organisms. Blast alignments were individually inspected to help recognize genes within our random genomic sequences despite the fact that only parts of them were available. For each yeast species, validated alignments were used to infer the proper genetic code, to determine codon usage preferences and to calculate their degree of sequence divergence with S. cerevisiae. The quality of each genomic library was monitored from contig analysis of the DNA sequences. Annotated sequences were submitted to the EMBL database, and the general annotation tables produced served as a basis for our comparative description of the evolution, redundancy and function of the Hemiascomycete genomes described in other articles of this issue.

Amino Acid Sequence↗

PubMed2000

Genomic exploration of the hemiascomycetous yeasts: 4. The genome of Saccharomyces cerevisiae revisited.

Since its completion more than 4 years ago, the sequence of Saccharomyces cerevisiae has been extensively used and studied. The original sequence has received a few corrections, and the identification of genes has been completed, thanks in particular to transcriptome analyses and to specialized studies on introns, tRNA genes, transposons or multigene families. In order to undertake the extensive comparative sequence analysis of this program, we have entirely revisited the S. cerevisiae sequence using the same criteria for all 16 chromosomes and taking into account publicly available annotations for genes and elements that cannot be predicted. Comparison with the other yeast species of this program indicates the existence of 50 novel genes in segments previously considered as 'intergenic' and suggests extensions for 26 of the previously annotated genes.

Ascomycota↗

PubMed2000

Genomic exploration of the hemiascomycetous yeasts: 11. Kluyveromyces lactis.

Random sequencing of the Kluyveromyces lactis genome allowed the identification of 2235-2601 open reading frames (ORFs) homologous to S. cerevisiae ORFs, 51 ORFs which were homologous to genes from other species, 64 tRNAs, the complete rDNA repeat, and a few Ty1- and Ty2-like sequences. In addition, the complete sequence of plasmid pKD1 and a large coverage of the mitochondrial genome were obtained. The global distribution into general functional categories found in Saccharomyces cerevisiae and as defined by MIPS is well conserved in K. lactis. However, detailed examination of certain subcategories revealed a small excess of genes involved in amino acid metabolism in K. lactis. The sequences are deposited at EMBL under the accession numbers AL424881-AL430960.

Ascomycota↗

PubMed2000

Genomic exploration of the hemiascomycetous yeasts: 18. Comparative analysis of chromosome maps and synteny with Saccharomyces cerevisiae.

We have analyzed the evolution of chromosome maps of Hemiascomycetes by comparing gene order and orientation of the 13 yeast species partially sequenced in this program with the genome map of Saccharomyces cerevisiae. From the analysis of nearly 8000 situations in which two distinct genes having homologs in S. cerevisiae could be identified on the sequenced inserts of another yeast species, we have quantified the loss of synteny, the frequency of single gene deletion and the occurrence of gene inversion. Traces of ancestral duplications in the genome of S. cerevisiae could be identified from the comparison with the other species that do not entirely coincide with those identified from the comparison of S. cerevisiae with itself. From such duplications and from the correlation observed between gene inversion and loss of synteny, a model is proposed for the molecular evolution of Hemiascomycetes. This model, which can possibly be extended to other eukaryotes, is based on the reiteration of events of duplication of chromosome segments, creating transient merodiploids that are subsequently resolved by single gene deletion events.

Ascomycota↗

PubMed2000

Genomic exploration of the hemiascomycetous yeasts: 19. Ascomycetes-specific genes.

Comparisons of the 6213 predicted Saccharomyces cerevisiae open reading frame (ORF) products with sequences from organisms of other biological phyla differentiate genes commonly conserved in evolution from 'maverick' genes which have no homologue in phyla other than the Ascomycetes. We show that a majority of the 'maverick' genes have homologues among other yeast species and thus define a set of 1892 genes that, from sequence comparisons, appear 'Ascomycetes-specific'. We estimate, retrospectively, that the S. cerevisiae genome contains 5651 actual protein-coding genes, 50 of which were identified for the first time in this work, and that the present public databases contain 612 predicted ORFs that are not real genes. Interestingly, the sequences of the 'Ascomycetes-specific' genes tend to diverge more rapidly in evolution than that of other genes. Half of the 'Ascomycetes-specific' genes are functionally characterized in S. cerevisiae, and a few functional categories are over-represented in them.

Ascomycota↗

PubMed2000

Genomic exploration of the hemiascomycetous yeasts: 20. Evolution of gene redundancy compared to Saccharomyces cerevisiae.

We have evaluated the degree of gene redundancy in the nuclear genomes of 13 hemiascomycetous yeast species. Saccharomyces cerevisiae singletons and gene families appear generally conserved in these species as singletons and families of similar size, respectively. Variations of the number of homologues with respect to that expected affect from 7 to less than 24% of each genome. Since S. cerevisiae homologues represent the majority of the genes identified in the genomes studied, the overall degree of gene redundancy seems conserved across all species. This is best explained by a dynamic equilibrium resulting from numerous events of gene duplication and deletion rather than by a massive duplication event occurring in some lineages and not in others.

Ascomycota↗

PubMed2000

Genomic exploration of the hemiascomycetous yeasts: 21. Comparative functional classification of genes.

We explored the biological diversity of hemiascomycetous yeasts using a set of 22000 newly identified genes in 13 species through BLASTX searches. Genes without clear homologue in Saccharomyces cerevisiae appeared to be conserved in several species, suggesting that they were recently lost by S. cerevisiae. They often identified well-known species-specific traits. Cases of gene acquisition through horizontal transfer appeared to occur very rarely if at all. All identified genes were ascribed to functional classes. Functional classes were differently represented among species. Species classification by functional clustering roughly paralleled rDNA phylogeny. Unequal distribution of rapidly evolving, ascomycete-specific, genes among species and functions was shown to contribute strongly to this clustering. A few cases of gene family amplification were documented, but no general correlation could be observed between functional differentiation of yeast species and variations of gene family sizes. Yeast biological diversity seems thus to result from limited species-specific gene losses or duplications, and for a large part from rapid evolution of genes and regulatory factors dedicated to specific functions.

Ascomycota↗

PubMed2000

Regulation of primary carbon metabolism in Kluyveromyces lactis.

In the recent past, through advances in development of genetic tools, the budding yeast Kluyveromyces lactis has become a model system for studies on molecular physiology of so-called "Nonconventional Yeasts." The regulation of primary carbon metabolism in K. lactis differs markedly from Saccharomyces cerevisiae and reflects the dominance of respiration over fermentation typical for the majority of yeasts. The absence of aerobic ethanol formation in this class of yeasts represents a major advantage for the "cell factory" concept and large-scale production of heterologous proteins in K. lactis cells is being applied successfully. First insight into the molecular basis for the different regulatory strategies is beginning to emerge from comparative studies on S. cerevisiae and K. lactis. The absence of glucose repression of respiration, a high capacity of respiratory enzymes and a tight regulation of glucose uptake in K. lactis are key factors determining physiological differences to S. cerevisiae. A striking discrepancy exists between the conservation of regulatory factors and the lack of evidence for their functional significance in K. lactis. On the other hand, structurally conserved factors were identified in K. lactis in a new regulatory context. It seems that different physiological responses result from modified interactions of similar molecular modules.

Journal Article↗

PubMed2000

A large-scale study of Yap1p-dependent genes in normal aerobic and H2O2-stress conditions: the role of Yap1p in cell proliferation control in yeast.

Yeast genes regulated by the transcriptional activator Yap1p were screened by two independent methods: (i) use of a LacZ-fused gene library and (ii) high-density membrane hybridization. Changes in transcriptome profile were examined in the presence and in the absence of Yap1p, as well as under normal and H2O2-mediated stress conditions. Both approaches gave coherent results, leading to the identification of many genes that appear to be directly or indirectly regulated by Yap1p. Promoter sequence analysis of target genes revealed that this regulatory effect is not always dependent upon the presence of a Yap1p binding site. The results show that the regulatory role of Yap1p is not restricted to the activation of stress response but that this factor can act as a positive or a negative regulator, both under normal and oxidative stress conditions. Among the targets, a few genes participating in growth control cascades were detected. In particular, the RPI1 gene, a repressor of the ras-cAMP pathway, was found to be downregulated by Yap1p during the early phase of growth, but upregulated in the stationary phase or after oxidative stress.

Aerobiosis↗

PubMed1999

HAP4, the glucose-repressed regulated subunit of the HAP transcriptional complex involved in the fermentation-respiration shift, has a functional homologue in the respiratory yeast Kluyveromyces lactis.

In Saccharomyces cerevisiae, the heteromeric HAP transcription factor is necessary for optimal growth on respiratory carbon sources. One of its components, the Hap4p protein, is necessary for transcriptional activation. The same protein is also the regulatory part of the complex in response to carbon sources, as HAP4 is strongly induced during the shift from fermentative to respiratory metabolism in S. cerevisiae. We report here the characterization of a new gene from the respiratory yeast Kluyveromyces lactis, obtained by heterologous complementation of a delta hap4 S. cerevisiae mutant strain. The deduced sequence of the protein (643 amino acids) exhibits two small domains (11 and 16 amino acids respectively) highly homologous to corresponding domains of ScHap4p, while the overall similarity is rather weak. Additional experiments were performed to confirm the functional homology of this new gene with ScHAP4, which we named KIHAP4. The importance of the small highly conserved N-terminal sequence was confirmed by in vitro mutagenesis. All the mutations that interfere with the Hap4p-Hap2/3/5 interaction were localized in it. The discovery of the same regulatory protein in two metabolically distinct yeast species raises the question of its functional significance during evolution.

Amino Acid Sequence↗

PubMed1998

Ts mutations in mitochondrial tRNA genes: characterization and effects of two point mutations in the mitochondrial gene for tRNAphe in Saccharomyces cerevisiae.

Two new mitochondrial mutations conferring heat sensitivity on glycerol medium to the cells that carry them and affecting mitochondrial protein synthesis were investigated. Both map in the mitochondrial tRNAphe gene and have C-to-U transitions, one at position 2 (ts22b16) and the other at 62 (ts1345). The latter mutation clearly affects the 3' end-maturation of tRNAphe, while the former presents normal patterns of both tRNA processing and amino-acylation. The defective phenotype resulting from the ts22b16 mutation can be corrected by over-expressing either the mitochondrial elongation factor EF-Tu or the mutated form of the tRNA. These results suggest that this mutation's primary effect might involve modified interactions during the ternary complex formation.

Base Sequence↗