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1.
Mol Biol Cell ; 18(2): 362-8, 2007 Feb.
Article in English | MEDLINE | ID: mdl-17108321

ABSTRACT

The coherence of mitochondrial biogenesis relies on spatiotemporally coordinated associations of 800-1000 proteins mostly encoded in the nuclear genome. We report the development of new quantitative analyses to assess the role of local protein translation in the construction of molecular complexes. We used real-time PCR to determine the cellular location of 112 mRNAs involved in seven mitochondrial complexes. Five typical cases were examined by an improved FISH protocol. The proteins produced in the vicinity of mitochondria (MLR proteins) were, almost exclusively, of prokaryotic origin and are key elements of the core construction of the molecular complexes; the accessory proteins were translated on free cytoplasmic polysomes. These two classes of proteins correspond, at least as far as intermembrane space (IMS) proteins are concerned, to two different import pathways. Import of MLR proteins involves both TOM and TIM23 complexes whereas non-MLR proteins only interact with the TOM complex. Site-specific translation loci, both outside and inside mitochondria, may coordinate the construction of molecular complexes composed of both nuclearly and mitochondrially encoded subunits.


Subject(s)
Mitochondria/metabolism , Mitochondrial Proteins/metabolism , RNA, Fungal/metabolism , RNA, Messenger/metabolism , Saccharomyces cerevisiae/genetics , Carrier Proteins/genetics , Carrier Proteins/metabolism , In Situ Hybridization, Fluorescence , Membrane Transport Proteins/genetics , Membrane Transport Proteins/metabolism , Mitochondria/chemistry , Mitochondrial Membranes/metabolism , Mitochondrial Precursor Protein Import Complex Proteins , Mitochondrial Proteins/genetics , Protein Transport , RNA, Fungal/analysis , RNA, Messenger/analysis , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism
2.
Mol Cell Biol ; 25(5): 1860-8, 2005 Mar.
Article in English | MEDLINE | ID: mdl-15713640

ABSTRACT

The variety of environmental stresses is probably the major challenge imposed on transcription activators and the transcriptional machinery. To precisely describe the very early genomic response developed by yeast to accommodate a chemical stress, we performed time course analyses of the modifications of the yeast gene expression program which immediately follows the addition of the antimitotic drug benomyl. Similar analyses were conducted with different isogenic yeast strains in which genes coding for relevant transcription factors were deleted and coupled with efficient bioinformatics tools. Yap1 and Pdr1, two well-known key mediators of stress tolerance, appeared to be responsible for the very rapid establishment of a transient transcriptional response encompassing 119 genes. Yap1, which plays a predominant role in this response, binds, in vivo, promoters of genes which are not automatically up-regulated. We proposed that Yap1 nuclear localization and DNA binding are necessary but not sufficient to elicit the specificity of the chemical stress response.


Subject(s)
Benomyl/pharmacology , DNA-Binding Proteins/physiology , Gene Expression Regulation, Fungal/drug effects , Saccharomyces cerevisiae Proteins/physiology , Saccharomyces cerevisiae/genetics , Trans-Activators/physiology , Transcription Factors/physiology , DNA-Binding Proteins/genetics , Down-Regulation/physiology , Gene Expression Profiling , Genome, Fungal , Oligonucleotide Array Sequence Analysis , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae Proteins/genetics , Sequence Deletion/genetics , Stimulation, Chemical , Trans-Activators/genetics , Transcription Factors/genetics , Up-Regulation/physiology
3.
EMBO Rep ; 2(6): 493-8, 2001 Jun.
Article in English | MEDLINE | ID: mdl-11415981

ABSTRACT

We analysed the genome-wide regulatory properties of an artificial transcription activator in which the DNA-binding domain of the yeast transcription factor, Pdr1, was fused to the activation domain of Gal4 (Pdr1*GAD). This Pdr1*GAD chimera was put under the control of the inducible GAL1 promoter. DNA microarray analyses showed that all the target genes upregulated by the well-studied native gain-of-function Pdr1-3 mutant were similarly activated by the chimerical factor Pdr1*GAD upon galactose induction. Additionally, this kinetic approach led us not only to confirm previously published targets, but also to define a hierarchy among members of the Pdr1 regulon. Our observations prove, for the first time at the complete genome level, that the DNA-binding domain of Pdr1 is sufficient to guide its specificity. We propose that this approach could be useful for the study of new transcription factors identified in silico from sequenced organisms. Complete data are available at www.biologie.ens.fr/yeast-publi.html.


Subject(s)
DNA-Binding Proteins/genetics , Genome , Trans-Activators/genetics , Transcription, Genetic , Transcriptional Activation , Cells, Cultured , Galactose/metabolism , Kinetics , Models, Genetic , Oligonucleotide Array Sequence Analysis , Plasmids/metabolism , Protein Structure, Tertiary , Recombinant Fusion Proteins/chemistry , Saccharomyces cerevisiae Proteins , Time Factors , Transcription Factors/chemistry , Up-Regulation
4.
Mol Cell Biol ; 17(9): 5453-60, 1997 Sep.
Article in English | MEDLINE | ID: mdl-9271421

ABSTRACT

In the yeast Saccharomyces cerevisiae, multidrug resistance to unrelated chemicals can result from overexpression of ATP-binding cassette (ABC) transporters such as Pdr5p, Snq2p, and Yor1p. Expression of these genes is under the control of two homologous zinc finger-containing transcription regulators, Pdr1p and Pdr3p. Here, we describe the isolation, by an in vivo screen, of two new Pdr1p-Pdr3p target genes: HXT11 and HXT9. HXT11 and HXT9, encoding nearly identical proteins, have a high degree of identity to monosaccharide transporters of the major facilitator superfamily (MFS). In this study, we show that the HXT11 product, which allows glucose uptake in a glucose permease mutant (rag1) strain of Kluyveromyces lactis, is also involved in the pleiotropic drug resistance process. Loss of HXT11 and/or HXT9 confers cycloheximide, sulfomethuron methyl, and 4-NQO (4-nitroquinoline-N-oxide) resistance. Conversely, HXT11 overexpression increases sensitivity to these drugs in the wild-type strain, an effect which is more pronounced in a strain having both PDR1 and PDR3 deleted. These data show that the two putative hexose transporters Hxt11p and Hxt9p are transcriptionally regulated by the transcription factors Pdr1p and Pdr3p, which are known to regulate the production of ABC transporters required for drug resistance in yeast. We thus demonstrate the existence of genetic interactions between genes coding for two classes of transporters (ABC and MFS) to control the multidrug resistance process.


Subject(s)
DNA-Binding Proteins/genetics , Drug Resistance, Multiple/genetics , Monosaccharide Transport Proteins/genetics , Trans-Activators/genetics , Transcription Factors/genetics , Zinc Fingers/genetics , Gene Expression , Molecular Sequence Data , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins , Transcription, Genetic
5.
Nature ; 387(6632 Suppl): 75-8, 1997 May 29.
Article in English | MEDLINE | ID: mdl-9169867

ABSTRACT

The complete DNA sequence of the yeast Saccharomyces cerevisiae chromosome IV has been determined. Apart from chromosome XII, which contains the 1-2 Mb rDNA cluster, chromosome IV is the longest S. cerevisiae chromosome. It was split into three parts, which were sequenced by a consortium from the European Community, the Sanger Centre, and groups from St Louis and Stanford in the United States. The sequence of 1,531,974 base pairs contains 796 predicted or known genes, 318 (39.9%) of which have been previously identified. Of the 478 new genes, 225 (28.3%) are homologous to previously identified genes and 253 (32%) have unknown functions or correspond to spurious open reading frames (ORFs). On average there is one gene approximately every two kilobases. Superimposed on alternating regional variations in G+C composition, there is a large central domain with a lower G+C content that contains all the yeast transposon (Ty) elements and most of the tRNA genes. Chromosome IV shares with chromosomes II, V, XII, XIII and XV some long clustered duplications which partly explain its origin.


Subject(s)
Chromosomes, Fungal , Saccharomyces cerevisiae/genetics , Base Sequence , DNA, Fungal , Molecular Sequence Data
6.
Yeast ; 12(15): 1587-92, 1996 Dec.
Article in English | MEDLINE | ID: mdl-8972581

ABSTRACT

We have determined the complete nucleotide sequence of a 23 kb segment from the left arm of chromosome IV, which is carried by the cosmid 1L10. This sequence contains the 3' coding region of the STE7 and RET1 (COP1) genes, and 13 complete open reading frames longer than 300 bp, of which ten correspond to putative new genes and three (CLB3, MSH5 and RPC53) have been sequenced previously. The sequence from cosmid IL10 was obtained entirely by a combined subcloning and walking primer strategy.


Subject(s)
Chromosomes/genetics , DNA, Fungal/analysis , Saccharomyces cerevisiae Proteins , Sequence Analysis, DNA , Yeasts/genetics , Amino Acid Sequence , Chromosome Mapping , Cloning, Molecular , Coatomer Protein , Cosmids/genetics , Cyclins/genetics , Electronic Data Processing , Fungal Proteins/genetics , Gene Library , Membrane Proteins/genetics , Molecular Sequence Data , Open Reading Frames , RNA Polymerase III/genetics , Saccharomyces cerevisiae/genetics , Transcription Factors/genetics
7.
Mol Cell Biol ; 15(8): 4043-51, 1995 Aug.
Article in English | MEDLINE | ID: mdl-7623800

ABSTRACT

Simultaneous resistance to an array of drugs with different cytotoxic activities is a property of Saccharomyces cerevisiae, in which the protein Pdr3p has recently been shown to play a role as a transcriptional regulator. We provide evidence that the yeast PDR3 gene, which encodes a zinc finger transcription factor implicated in certain drug resistance phenomena, is under positive autoregulation by Pdr3p. DNase I footprinting analyses using bacterially expressed Pdr3p showed specific recognition by this protein of at least two upstream activating sequences in the PDR3 promoter. The use of lacZ reporter constructs, a mutational analysis of the upstream activating sequences, as well as band shift experiments enabled the identification of two 5'TC CGCGGA3' sequence motifs in the PDR3 gene as consensus elements for the binding of Pdr3p. Several similar sequence motifs can be found in the promoter of PDR5, a gene encoding an ATP-dependent drug pump whose Pdr3p-induced overexpression is responsible for drug resistance phenomena. Recently one of these sequence elements was shown to be the target of Pdr3p to elevate the level of PDR5 transcription. Finally, we provide evidence in the absence of PDR1 for a PDR3-controlled transcriptional induction of the drug pump by cycloheximide and propose a model for the mechanism governing the transcriptional autoregulation of Pdr3p.


Subject(s)
ATP-Binding Cassette Transporters , DNA-Binding Proteins/genetics , Drug Resistance, Microbial/genetics , Gene Expression Regulation, Fungal , Promoter Regions, Genetic/genetics , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/genetics , Trans-Activators , Transcription Factors/genetics , Base Sequence , Binding Sites , Carrier Proteins/biosynthesis , Carrier Proteins/genetics , Consensus Sequence , Cycloheximide/pharmacology , DNA Mutational Analysis , DNA-Binding Proteins/metabolism , Genes, Fungal/genetics , Genes, Reporter , Membrane Proteins/biosynthesis , Membrane Proteins/genetics , Molecular Sequence Data , Protein Binding , RNA, Messenger/biosynthesis , Saccharomyces cerevisiae/drug effects , Transcription Factors/biosynthesis , Transcription Factors/metabolism , Transcription, Genetic
8.
EMBO J ; 13(24): 5795-809, 1994 Dec 15.
Article in English | MEDLINE | ID: mdl-7813418

ABSTRACT

In the framework of the EU genome-sequencing programmes, the complete DNA sequence of the yeast Saccharomyces cerevisiae chromosome II (807 188 bp) has been determined. At present, this is the largest eukaryotic chromosome entirely sequenced. A total of 410 open reading frames (ORFs) were identified, covering 72% of the sequence. Similarity searches revealed that 124 ORFs (30%) correspond to genes of known function, 51 ORFs (12.5%) appear to be homologues of genes whose functions are known, 52 others (12.5%) have homologues the functions of which are not well defined and another 33 of the novel putative genes (8%) exhibit a degree of similarity which is insufficient to confidently assign function. Of the genes on chromosome II, 37-45% are thus of unpredicted function. Among the novel putative genes, we found several that are related to genes that perform differentiated functions in multicellular organisms of are involved in malignancy. In addition to a compact arrangement of potential protein coding sequences, the analysis of this chromosome confirmed general chromosome patterns but also revealed particular novel features of chromosomal organization. Alternating regional variations in average base composition correlate with variations in local gene density along chromosome II, as observed in chromosomes XI and III. We propose that functional ARS elements are preferably located in the AT-rich regions that have a spacing of approximately 110 kb. Similarly, the 13 tRNA genes and the three Ty elements of chromosome II are found in AT-rich regions. In chromosome II, the distribution of coding sequences between the two strands is biased, with a ratio of 1.3:1. An interesting aspect regarding the evolution of the eukaryotic genome is the finding that chromosome II has a high degree of internal genetic redundancy, amounting to 16% of the coding capacity.


Subject(s)
Chromosome Mapping/methods , Chromosomes, Fungal/genetics , DNA, Fungal/genetics , Genes, Fungal/genetics , Saccharomyces cerevisiae/genetics , Base Composition , Base Sequence , Cloning, Molecular , Cosmids/genetics , Molecular Sequence Data , Open Reading Frames , Quality Control , Repetitive Sequences, Nucleic Acid , Reproducibility of Results , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Telomere/genetics
9.
Mol Gen Genet ; 244(5): 501-11, 1994 Sep 01.
Article in English | MEDLINE | ID: mdl-8078477

ABSTRACT

The Saccharomyces cerevisiae PDR3 gene, located near the centromere of chromosome II, has been completely sequenced and characterised. Mutations pdr3-1 and pdr3-2, which confer resistance to several antibiotics can be complemented by a wild-type allele of the PDR3 gene. The sequence of the wild-type PDR3 gene revealed the presence of a long open reading frame capable of encoding a 976-amino acid protein. The protein contains a single Zn(II)2Cys6 binuclear-type zinc finger homologous to the DNA-binding motifs of other transcriptional activators from lower eukaryotes. Evidence that the PDR3 protein is a transcriptional activator was provided by demonstrating that DNA-bound LexA-PDR3 fusion proteins stimulate expression of a nearby promoter containing LexA binding sites. The use of LexA-PDR3 fusions revealed that the protein contains two activation domains, one localised near the N-terminal, cysteine-rich domain and the other localised at the C-terminus. The salient feature of the PDR3 protein is its similarity to the protein coded by PDR1, a gene responsible for pleiotropic drug resistance. The two proteins show 36% amino acid identity over their entire length and their zinc finger DNA-binding domains are highly conserved. The fact that the absence of both PDR1 and PDR3 (simultaneous disruption of the two genes) enhances multidrug sensitivity strongly suggests that the two transcriptional factors have closely related functions.


Subject(s)
Drug Resistance, Microbial/genetics , Gene Expression Regulation, Fungal , Genes, Fungal , Genes, Regulator , Saccharomyces cerevisiae/genetics , Amino Acid Sequence , Base Sequence , Chromosomes, Fungal , DNA, Fungal/genetics , DNA-Binding Proteins/genetics , Microbial Sensitivity Tests , Molecular Sequence Data , Open Reading Frames , Recombinant Fusion Proteins , Restriction Mapping , Saccharomyces cerevisiae Proteins , Sequence Alignment , Sequence Homology, Nucleic Acid , Transcription Factors/genetics , Zinc Fingers
10.
Yeast ; 8(9): 761-8, 1992 Sep.
Article in English | MEDLINE | ID: mdl-1441753

ABSTRACT

A 12,684 bp DNA fragment, between FUS3 and the centromere, from the left arm of chromosome II of Saccharomyces cerevisiae was sequenced as part of the European project to sequence the whole chromosome. This segment contains at least five complete new open reading frames (ORFs) and the beginning (191 first 5' codons) of an ORF whose putative translational product is highly similar to the multidrug resistance PDR1 gene previously characterized by Balzi et al. (1987) on chromosome VII.


Subject(s)
DNA, Fungal/genetics , Genes, Fungal , Saccharomyces cerevisiae/genetics , Amino Acid Sequence , Base Sequence , Chromosome Mapping , Chromosomes, Fungal , Drug Resistance, Microbial/genetics , Fungal Proteins/genetics , Molecular Sequence Data , Open Reading Frames , Protein Biosynthesis , Sequence Homology, Amino Acid
11.
Biochem J ; 277 ( Pt 1): 175-82, 1991 Jul 01.
Article in English | MEDLINE | ID: mdl-1649598

ABSTRACT

Treatment of human beta 2 microglobulin (beta 2m) with defined oxygen-derived species generated by treatment with gamma-radiation was studied. As assessed by SDS/PAGE, the hydroxyl radicals (.OH) caused the disappearance of the protein band at 12 kDa that represents beta 2m, and cross-linked the protein into protein bands stable to both SDS and reducing conditions. However, when .OH was generated under oxygen in equimolar combination with the superoxide anion radical (O2.-), the high-molecular-mass protein products were less represented, and fragmented derivatives were not obviously detectable. Exposure to .OH alone, or to .OH + O2.- in the presence of O2, induced the formation of beta 2m protein derivatives with a more acidic net electrical charge than the parent molecule. In contrast, O2.- alone had virtually no effect on molecular mass or pI. Changes in u.v. fluorescence during .OH attack indicated changes in conformation, as confirmed by c.d. spectrometry. A high concentration of radicals caused the disappearance of the beta-pleated sheet structure and the formation of a random coil structure. Loss of tryptophan and significant production of dityrosine (2,2'-biphenol type) were noted, exhibiting a clear dose-dependence with .OH alone or with .OH + O2.-. The combination of .OH + O2.- induced a pattern of changes similar to that with .OH alone, but more extensive for c.d. and tryptophan oxidation (2 Trp/beta 2m molecule), and more limited for dityrosine formation. Lower levels of these oxidative agents caused the reproducible formation of species at 18 and 25 kDa which were recognized by antibodies against native beta 2m. These findings provide a model for the protein pattern observed in beta 2m amyloidosis described in the literature.


Subject(s)
Hydroxides/pharmacology , Superoxides/pharmacology , beta 2-Microglobulin/chemistry , Amino Acids/analysis , Circular Dichroism , Free Radicals , Gamma Rays , Humans , Hydroxyl Radical , Kinetics , Protein Conformation/drug effects , Spectrometry, Fluorescence , beta 2-Microglobulin/drug effects , beta 2-Microglobulin/radiation effects
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