Biomedical subjects

M Bolotin-Fukuhara

Publications and source records attributed to M Bolotin-Fukuhara.

At least 37 records · Page 2Linked to original sources

PubMed1998

A mutation in a novel yeast proteasomal gene, RPN11/MPR1, produces a cell cycle arrest, overreplication of nuclear and mitochondrial DNA, and an altered mitochondrial morphology.

We report here the functional characterization of an essential Saccharomyces cerevisiae gene, MPR1, coding for a regulatory proteasomal subunit for which the name Rpn11p has been proposed. For this study we made use of the mpr1-1 mutation that causes the following pleiotropic defects. At 24 degreesC growth is delayed on glucose and impaired on glycerol, whereas no growth is seen at 36 degreesC on either carbon source. Microscopic observation of cells growing on glucose at 24 degreesC shows that most of them bear a large bud, whereas mitochondrial morphology is profoundly altered. A shift to the nonpermissive temperature produces aberrant elongated cell morphologies, whereas the nucleus fails to divide. Flow cytometry profiles after the shift to the nonpermissive temperature indicate overreplication of both nuclear and mitochondrial DNA. Consistently with the identification of Mpr1p with a proteasomal subunit, the mutation is complemented by the human POH1 proteasomal gene. Moreover, the mpr1-1 mutant grown to stationary phase accumulates ubiquitinated proteins. Localization of the Rpn11p/Mpr1p protein has been studied by green fluorescent protein fusion, and the fusion protein has been found to be mainly associated to cytoplasmic structures. For the first time, a proteasomal mutation has also revealed an associated mitochondrial phenotype. We actually showed, by the use of [rho degrees] cells derived from the mutant, that the increase in DNA content per cell is due in part to an increase in the amount of mitochondrial DNA. Moreover, microscopy of mpr1-1 cells grown on glucose showed that multiple punctate mitochondrial structures were present in place of the tubular network found in the wild-type strain. These data strongly suggest that mpr1-1 is a valuable tool with which to study the possible roles of proteasomal function in mitochondrial biogenesis.

Amino Acid Sequence↗

PubMed1997

The nucleotide sequence of Saccharomyces cerevisiae chromosome XV.

Chromosome XV was one of the last two chromosomes of Saccharomyces cerevisiae to be discovered. It is the third-largest yeast chromosome after chromosomes XII and IV, and is very similar in size to chromosome VII. It alone represents 9% of the yeast genome (8% if ribosomal DNA is included). When systematic sequencing of chromosome XV was started, 93 genes or markers were identified, and most of them were mapped. However, very little else was known about chromosome XV which, in contrast to shorter chromosomes, had not been the object of comprehensive genetic or molecular analysis. It was therefore decided to start sequencing chromosome XV only in the third phase of the European Yeast Genome Sequencing Programme, after experience was gained on chromosomes III, XI and II. The sequence of chromosome XV has been determined from a set of partly overlapping cosmid clones derived from a unique yeast strain, and physically mapped at 3.3-kilobase resolution before sequencing. As well as numerous new open reading frames (ORFs) and genes encoding tRNA or small RNA molecules, the sequence of 1,091,283 base pairs confirms the high proportion of orphan genes and reveals a number of ancestral and successive duplications with other yeast chromosomes.

Base Sequence↗

PubMed1997

The sequence of a 54.7 kb fragment of yeast chromosome XV reveals the presence of two tRNAs and 24 new open reading frames.

A 54,719 bp fragment from the right arm of Saccharomyces cerevisiae chromosome XV has been sequenced from the inserts of two cosmids (pEOA213 and pEOA217). The computer analysis of this sequence has revealed the presence of eight known genes (CKA2, CYC1, ALG8, TCM1, TMP1, UFE1, RTS2 and ASE1) and four open reading frames (ORFs) with strong homologies with known yeast genes (MLP1, SIS2 and HBS1 and the allantoin permease). The characteristics of the other ORFs and of the corresponding proteins do not allow postulation of a precise function. Several have features reminiscent of cytoskeleton or motor elements (keratin-like, myosin-like) and several others have characteristics of proteins which interact with DNA (extremely basic, b-Zip structure and/or acidic domains). Two tRNAs (tRNA(Lys) and tRNA(Pro)) have also been identified on this fragment. Many of these ORFs present similarities with ORFs located on chromosome XI, indicating some information reshuffling between the two chromosomal fragments.

Amino Acid Sequence↗

PubMed1997

Additional copies of the mitochondrial Ef-Tu and aspartyl-tRNA synthetase genes can compensate for a mutation affecting the maturation of the mitochondrial tRNAAsp.

In an attempt to identify new nuclear genes involved in the synthesis and processing of mitochondrial tRNAs, we utilized a multicopy nuclear library to suppress the heat-sensitive phenotype of a Saccharomyces cerevisiae mitochondrial mutant strain. This strain (Ts 932) is defective in the 3'-end processing of the mitochondrial tRNAAsp transcript. The nuclear genes coding for the mitochondrial elongation factor Tuf M and for the mitochondrial aspartyl-tRNA synthetase have been found to restore the temperature-resistant phenotype and to correct the RNA processing defect. Suppression was effective even when the genes were present on a centromeric plasmid.

Aspartate-tRNA Ligase↗

PubMed1997

The MBR1 gene from Saccharomyces cerevisiae is activated by and required for growth under sub-optimal conditions.

The MBR1 gene was isolated as a multicopy suppressor of the phenotype on glycerol medium of a Saccharomyces cerevisiae strain mutant for the Hap2/3/4/5 transactivator complex. In this paper, we show that Mbr1p is a limiting factor for growth on glycerol medium under the following sub-optimal culture conditions: in late growth phase, at low temperature, at high external pH or in the presence of 1,10-phenanthroline. Moreover, deletion of MBR1 protects cells against stress, whilst overexpression of this gene has the opposite effect. MBR1 expression is induced in the late growth phase and is negatively controlled by the cAMP-dependent protein kinase A (PKA). Both activation of PKA or overexpression of SOK1 or SCH9-two genes isolated as multicopy suppressors of a PKA null mutant-suppress the mbr1 growth defect. Our results indicate that Mbr1p is not an essential element of any one of these pathways. Deletion of SAC1, a gene implicated in vesicular transport, in association with MBR1 deletion, causes synthetic lethality. A possible role of Mbr1p in intracellular trafficking is discussed.

Bacterial Proteins↗

PubMed1997

Characterization of an AP-1-like transcription factor that mediates an oxidative stress response in Kluyveromyces lactis.

The KlYAP1 gene, encoding the transcription factor Yap1p from Kluyveromyces lactis, was cloned by functional complementation of the cadmium hypersensitivity phenotype of a Saccharomyces cerevisiae strain lacking functional YAP1 and YAP2 genes. The KlYAP1 gene product is 41% identical to Yap1p, the sequence similarity being centered on the bZip domain and extending into the C-terminal portion of both proteins. When expressed in S. cerevisiae, this gene efficiently complements some of the phenotypes associated with both yap1 and yap2 mutations and also mediates AP-1 response element-dependent transcriptional activation in response to H2O2. Gene disruption experiments in K. lactis indicated that the KlYAP1 gene is involved in both the oxidative and cadmium response pathways. We also demonstrate the existence in K. lactis of inducible protective stress responses to both peroxides and superoxides and investigate the role of the Klyap1p protein in these responses.

Adaptation, Physiological↗

PubMed1996

The nuclear Kluyveromyces lactis MRF1 gene encodes a mitochondrial class I peptide chain release factor that is important for cell viability.

We report the isolation and characterization of the Kluyveromyces lactis MRF1 gene encoding mitochondrial peptide chain release factor mRF-1. Over-expression of the KlMRF1 gene has a strong antisuppressive effect in a Saccharomyces cerevisiae mitochondrial nonsense suppressor strain. Inactivation of KlMRF1 results in a dual phenotype: most cells die after about 10-13 generations, while a small number of cells exceed this limit. We propose that the lethality is related to a loss of mitochondrial genome integrity. Surviving Klmrf1 cells are able to grow slowly on the non-fermentable substrate glycerol, indicating the existence of a second mitochondrial release factor activity. Our previous comparative analysis of class I release factors is refined by the incorporation of KlmRF-1 and ten recently identified prokaryotic release factor sequences.Keywords Kluyveromyces lactis middle dot Mitochondrial release factor middle dot MRF1 middle dot Peptide chain termination

Amino Acid Sequence↗

PubMed1996

Cloning of the ASN1 and ASN2 genes encoding asparagine synthetases in Saccharomyces cerevisiae: differential regulation by the CCAAT-box-binding factor.

Two new yeast genes, named ASN1 and ASN2, were isolated by complementation of the growth defect of an asparagine auxotrophic mutant. Genetical analysis indicates that these two genes are allelic to the asnA and asnB loci described previously. Simultaneous disruption of both genes leads to a total asparagine auxotrophy, while disruption of asn1 or asn2 alone has no effect on growth under tested conditions. Nucleotide sequences of ASN1 and ASN2 revealed striking similarities with genes encoding asparagine synthetase (AS) from other organisms. Regulation of ASN1 and ASN2 expression was studied using lacZ fusions and both genes were found to be several times less expressed in the absence of the transcription activator Gcn4p. The HAP complex, another transcription factor that binds to CCAAT-box sequences, was shown to specifically affect ASN1 expression. Hap2p and Hap3p subunits of the HAP complex are required for optimal expression of ASN1, while the Hap4p regulatory subunit, which is required for regulation by the carbon source, plays a minor role in this process. Consistent with the weak effect of Hap4p, the carbon source does not significantly affect expression of ASN1. Our results show that the role of the HAP complex is not limited to activation of genes required for respiratory metabolism.

Alleles↗

PubMed1996

Glucose uptake in Kluyveromyces lactis: role of the HGT1 gene in glucose transport.

A gene for high-affinity glucose transport, HGT1, has been isolated from the lactose-assimilating yeast Kluyveromyces lactis. Disruption strains showed much-reduced uptake of glucose at low concentrations and growth was particularly affected in low-glucose medium. The HGT1 nucleotide sequence implies that it encodes a typical transmembrane protein with 12 hydrophobic domains and with 26 to 31% amino acid identity with the Hxtp family of glucose transport elements in Saccharomyces cerevisiae. Expression is constitutive (in contrast to RAG1, the major gene for low-affinity glucose uptake in K. lactis) and is controlled by several genes also known to affect expression of RAG1. These include RAG5 (which codes for the single hexokinase of K. lactis), which is required for HGT1 transcription, and RAG4, which has a negative effect. The double mutant deltahgt1deltarag1 showed further reduced glucose uptake but still grew quite well on 2% glucose and was not completely impaired even on 0.1% glucose.

Amino Acid Sequence↗

PubMed1996

The CCAAT box-binding factor stimulates ammonium assimilation in Saccharomyces cerevisiae, defining a new cross-pathway regulation between nitrogen and carbon metabolisms.

In Saccharomyces cerevisiae, carbon and nitrogen metabolisms are connected via the incorporation of ammonia into glutamate; this reaction is catalyzed by the NADP-dependent glutamate dehydrogenase (NADP-GDH) encoded by the GDH1 gene. In this report, we show that the GDH1 gene requires the CCAAT box-binding activator (HAP complex) for optimal expression. This conclusion is based on several lines of evidence: (1) overexpression of GDH1 can correct the growth defect of hap2 and hap3 mutants on ammonium sulfate as a nitrogen source, (ii) Northern (RNA) blot analysis shows that the steady-state level of GDH1 mRNA is strongly lowered in a hap2 mutant, (iii) expression of a GDH1-lacZ fusion is drastically reduced in hap mutants, (iv) NADP-GDH activity is several times lower in the hap mutants compared with that in the isogenic wild-type strain, and finally, (v) site-directed mutagenesis of two consensual HAP binding sites in the GDH1 promoter strongly reduces expression of GDH1 and makes it HAP independent. Expression of GDH1 is also regulated by the carbon source, i.e., expression is higher on lactate than on ethanol, glycerol, or galactose, with the lowest expression being found on glucose. Finally, we show that a hap2 mutation does not affect expression of other genes involved in nitrogen metabolism (GDH2, GLN1, and GLN3 encoding, respectively, the NAD-GDH, glutamine synthetase, and a general activator of several nitrogen catabolic genes). The HAP complex is known to regulate expression of several genes involved in carbon metabolism; its role in the control of GDH1 gene expression, therefore, provides evidence for a cross-pathway regulation between carbon and nitrogen metabolisms.

Ammonium Sulfate↗

PubMed1995

Isolation and characterization of the gene encoding xylose reductase from Kluyveromyces lactis.

The identification of a xylose reductase (XR)-encoding gene (XYL1) from the xylose-assimilating yeast Kluyveromyces lactis (Kl) is described. XYL1 was isolated as a highly expressed fusion clone from a 'lacZ translational fusion library. DNA sequence analysis revealed an open reading frame (ORF) of 987 bp capable of encoding a polypeptide of 329 amino acids (aa). The deduced aa sequence displays a 62% overall identity to that of XR from Pichia stipitis. Gene disruption studies indicate that XYL1 exists as a single copy in the yeast genome and is essential for growth on xylose. Northern blot analysis of the XYL1 transcript and measurement of the XR enzymatic activities show, in contrast to other known XR-encoding genes, a constitutive expression of Kl XYL1.

Aldehyde Reductase↗

PubMed1995

A Saccharomyces cerevisiae gene essential for viability has been conserved in evolution.

To identify the gene coding for the endonuclease which processes the 3' end of mitochondrial (mt) tRNA transcripts in Saccharomyces cerevisiae, nuclear mutations able to complement a mt mutant (Ts932) defective for this process were isolated and analyzed. One of these mutants exhibited a growth defect both on respiratory and fermentable media. Complementation of this phenotype with a S. cerevisiae centrometric wild-type genomic library has allowed us to identify a new essential S. cerevisiae gene strongly conserved in various eukaryotic organisms.

Amino Acid Sequence↗

PubMed1995

The respiratory system of Kluyveromyces lactis escapes from HAP2 control.

A functional homolog of the Saccharomyces cerevisiae HAP2 gene, coding for one element of a transcriptional activator complex, was cloned from the yeast Kluyveromyces lactis and its nucleotide sequence was determined. Inactivation of the gene had no significant effect on respiration-dependent growth, suggesting that the HAP2/3/4 complex has no major control over the formation of the mitochondrial respiratory system in K. lactis.

Amino Acid Sequence↗

PubMed1994

MBR1 and MBR3, two related yeast genes that can suppress the growth defect of hap2, hap3 and hap4 mutants.

Two new yeast genes, named MBR1 and MBR3, were isolated as multicopy suppressors of the growth defect of a strain lacking the HAP2 transcriptional activator. Both genes when overexpressed can also suppress the growth defect of hap3 and hap4 null mutants. However, overexpression of MBR1 cannot substitute for the HAP2/3/4 complex in activation of the CYC1 gene. Nucleotide sequencing of MBR1 and MBR3 revealed that these two genes encode serine-rich, hydrophilic proteins with regions of significant homology. The functional importance of one of these conserved regions was shown by mutagenesis. Disruption of MBR1 leads to a partial growth defect on glycerol medium. Disruption of MBR3 has no major effect but the double disruptant shows a synthetic phenotype suggesting that the MBR1 and MBR3 gene products participate in common function.

Amino Acid Sequence↗

PubMed1994

Structure and regulation of SDH3, the yeast gene encoding the cytochrome b560 subunit of respiratory complex II.

Using an expression library, we have isolated yeast genes activated in the presence of the yeast CCAAT box-binding protein HAP2. One of these genes, SDH3, encodes the cytochrome b560 subunit of respiratory complex II. The SDH3 protein contains three potential transmembrane domains and is more than 30% identical to bovine cytochrome b560 and to a mitochondrially encoded protein from Marchantia polymorpha. Disruption of SDH3 shows that this gene is required for growth on non-fermentable carbon sources. Expression of SDH1, SDH3, and SDH4 is activated in the presence of the HAP2 transcriptional activator.

Amino Acid Sequence↗

PubMed1994

Complete DNA sequence of yeast chromosome XI.

The complete DNA sequence of the yeast Saccharomyces cerevisiae chromosome XI has been determined. In addition to a compact arrangement of potential protein coding sequences, the 666,448-base-pair sequence has revealed general chromosome patterns; in particular, alternating regional variations in average base composition correlate with variations in local gene density along the chromosome. Significant discrepancies with the previously published genetic map demonstrate the need for using independent physical mapping criteria.

Base Sequence↗

PubMed1994

A genetic screen to isolate genes regulated by the yeast CCAAT-box binding protein Hap2p.

We have developed a screening method to isolate yeast genes regulated by a specific transcription activator. The screen is based on the use of expression libraries in which the lacZ reporter gene is placed under control of yeast regulatory elements. Two partially representative libraries, constructed by different methods, were used to isolate genes regulated by the yeast CCAAT-box binding protein Hap2p. Among 26 fusions shown to be regulated by Hap2p only CYT1 was known to be regulated by this activator. Sequence analysis revealed that most of the remaining regulated fusions are in new yeast genes, while some are in previously characterized yeast genes (PTP1, RPM2, SDH1). Optimal expression of these three genes also requires Hap3p and Hap4p and is regulated by carbon source. Hap2p was known to regulate expression of genes involved in Krebs cycle, electron transport and heme biosynthesis. Our results suggest that Hap2p could play a more general role by regulating other mitochondrial processes such as protein import and phosphate transport (PTP1) or maturation of mitochondrial tRNAs (RPM2). Among the remaining regulated fusions, two of them correspond to open reading frames (ORFs) on chromosomes III and XI whose nucleotide sequences have been entirely determined. The use of this approach to functionally analyse ORFs of unknown function is discussed.

Base Sequence↗

PubMed1994

Suppression of a mitochondrial point mutation in a tRNA gene can cast light on the mechanisms of 3' end-processing.

We used a genetic approach to study the nuclear factors involved in the biogenesis of mitochondrial tRNAs. A point mutation in the mitochondrial tRNA(Asp) gene of Saccharomyces cerevisiae had previously been shown to result in a temperature-sensitive respiratory-deficient phenotype as a result of the absence of 3' end-processing of the tRNA(Asp). Analysis of mitochondrial revertants has shown that all revertants sequenced have a G-A compensatory change at position 53, which restores the hydrogen-bond with the mutated nucleotide. We then searched for nuclear suppressors to identify the nuclear gene(s) involved in mitochondrial tRNA 3' end-processing. One such suppressor mutation was further characterized: it restores tRNA(Asp) maturation and growth at 36 degrees C on glycerol medium in heterozygous diploids, but leads to a defective growth phenotype in haploids.

Base Sequence↗