Olga Groudinsky

LARGE-SCALE PHENOTYPIC ANALYSIS : THE PILOT PROJECT ON YEAST CHROMOSOME III

In 1993, a pilot project for the functional analysis of newly discovered open reading frames, presumably coding for proteins, from yeast chromosome III was launched by the European Community. In the frame of this programme, we have developed a large‐scale screening for the identification of gene/protein functions via systematic phenotypic analysis. To this end, some 80 haploid mutant yeast strains were constructed, each carrying a targeted deletion of a single gene obtained by HIS3 or TRP1 transplacement in the W303 background and a panel of some 100 growth conditions was established, ranging from growth substrates, stress to, predominantly, specific inhibitors and drugs acting on various cellular processes. Furthermore, co‐segregation of the targeted deletion and the observed phenotype(s) in meiotic products has been verified. The experimental procedure, using microtiter plates for phenotypic analysis of yeast mutants, can be applied on a large scale, either on solid or in liquid media. Since the minimal working unit of one 96‐well microtiter plate allows the simultaneous analysis of at least 60 mutant strains, hundreds of strains can be handled in parallel. The high number of monotropic and pleiotropic phenotypes (62%) obtained, together with the acquired practical experience, have shown this approach to be simple, inexpensive and reproducible. It provides a useful tool for the yeast community for the systematic search of biochemical and physiological functions of unknown genes accounting for about a half of the 6000 genes of the complete yeast genome.

Propriétés d'un noyau cytochromique b2 résultant d'une protéolyse de la l-lactate: Cytochrome c oxydoréductase de la levure

Abstract 1. 1. The tryptic hydrolysis of cytochrome b 2 (yeast l -lactate: cytochrome c oxidoreductase, EC 1.1.2.3) liberates a cytochromic polypeptide which can be separated from the other fragments by gel filtration. 2. 2. The properties of this new product, called “noyau cytochromique b 2 ”, have been investigated: the molecular weight is about 11 000 and this molecule is associated with one heme group; its spectral properties are very similar in the visible region to those of cytochrome b 2 . The redox potential is −0.028 V to be compared with the value 0.000 V relative to cytochrome b 2 (pH 7.00; 30°). Severa, different components have been detected by electrophoresis. These data have been used in a discussion on the structural aspects of the active molecule of lactate dehydrogenase.

Novel class of nuclear genes involved in both mRNA splicing and protein synthesis in Saccharomyces cerevisiae mitochondria.

SummaryWe have cloned three distinct nuclear genes, NAM1, NAM7, and NAM8, which alleviate mitochondrial intron mutations of the cytochrome b and COXI (subunit I of cytochrome oxidase) genes when present on multicopy plasmids. These nuclear genes show no sequence homology to each other and are localized on different chromosomes: NAM1 on chromosome IV, NAM7 on chromosome XIII and NAM8 on chromosome VIII. Sequence analysis of the NAM1 gene shows that it encodes a protein of 440 amino acids with a typical presequence that would target the protein to the mitochondrial matrix. Inactivation of the NAM1 gene by gene transplacement leads to a dramatic reduction of the overall synthesis of mitochondrial protein, and a complete absence of the COXI protein which is the result of a specific block in COXI pre-mRNA splicing. The possible mechanisms by which the NAM1 gene product may function are discussed.

Contact-shifted resonances in the 1H NMR spectra of cytochrome b5 Resonance identification and spin density distribution in the heme group

This paper describes the identification of some of the contact-shifted resonances in the 1H NMR spectrum of low spin ferric cytochrome b5. In these experiments comparison with cytochrome b5 which had been reconstituted with deuteroheme IX played an important role. NMR techniques used include double resonance experiments, line width analyses, and studies of the pH-dependence of the 1H NMR chemical shifts. The electronic heme structure derived from these resonance assignments is characterized by a highly anisotropic spin density distribution. This anisotropy is most strikingly manifested in the resonances of the vinyl and propionic acid substituents of the protoheme IX. The experiments described in this paper further revealed the coexistence in aqueous solutions of two different molecular species of cytochrome b5, which can be simultaneously observed in the regions of the 1H NMR spectrum which contain the largely contact-shifted resonances.

The NAM1/MTF2 nuclear gene product is selectively required for the stability and/or processing of mitochondrial transcripts of the atp6 and of the mosaic, cox1 and cytb genes in Saccharomyces cerevisiae

The NAM1/MTF2 gene was firstly isolated as a multicopy suppressor of mitochondrial splicing deficiencies and independently as a gene of which a thermosensitive allele affects mitochondrial transcription in organello. To determine which step in mitochondrial RNA metabolism is controlled in vivo by the NAM1 gene, mitochondrial transcripts of seven transcription units from strains carrying an inactive nam1::URA3 gene disruption in various mitochondrial genetic backgrounds were analysed by Northern blot hybridisations. In a strain carrying an intron-containing mitochondrial genome, the inactivation of the NAM1 gene led to a strong decrease in (or total absence of) the mosaic cytb and cox1 mRNAs and in transcripts of the atp6-rf3/ens2 genes, which are co-transcribed with cox1. Neither the accumulation of unspliced cytb or cox1 pre-mRNAs, nor that of excised circular intron molecules of ai1 or ai2 were observed, but the abundance of the bi1 and ai7 lariats was comparable to that observed in the wild-type strain, thus demonstrating that transcription of the cytb and cox1 genes does occur. In strains carrying the intron-less mitochondrial genome with or without the rf3/ens2 sequence, wild-type amounts of cytb and cox1 mRNAs were detected while the amount of the atp6 mRNA was always strongly decreased. The abundance of transcripts from five other genes was either slightly (21S rRNA) or not at all (cox2, cox3, atp9 and 15S rRNA) affected by the nam1 inactivation. This analysis leads to the conclusion that the NAM1 protein is not a general mitochondrial transcription factor, but rather is predominantly and selectively required for the processing and/or for the stability of cytb and cox1 intron-containing pre-mRNAs and of the atp6 transcripts. Since the original intronic mutations suppressed by the amplification of the NAM1 gene are situated in stem-loop rich structures, we propose that the NAM1 protein is a stem-loop RNA-binding protein that plays a role in determining RNA stability.

Role of Introns in the Yeast Cytochrome- b Gene: Cis - and Trans -acting Signals, Intron Manipulation, Expression, and Intergenic Communications

In a volume on mitochondrial genes it is appropriate to ask questions about the structure and organization (i.e., anatomy), expression and regulation (i.e., physiology), communication and integration (i.e., sociology), and speciation and phylogeny (i.e., history) of a gene. The yeast mitochondrial gene, referred to as cob or box, which specifies the amino acid sequence of cytochrome b (Slonimski and Tzagoloff 1976; Tzagoloff et al. 1976; Claisse et al. 1977; Colson et al. 1979) and controls the synthesis of cytochrome oxidase (Kotylak and Slonimski 1976; Pajot et al. 1976), is an excellent system for investigating such questions. It was shown (Slonimski et al. 1978b), confirmed, and extended (Grivell et al. 1979; Haid et al. 1979; Alexander et al. 1980; Nobrega and Tzagoloff 1980) that the gene displays a mosaic organization composed of exons and introns. Intron mutations arrest RNA splicing (Church et al. 1979; Haid et al. 1980; Halbreich et al. 1980; Van Ommen et al. 1980) and accumulate novel polypeptide chains (Claisse et al. 1978; Kreike et al. 1979; Solioz and Schatz 1979), Introns have different functions from exons because they constitute separate complementation groups (Slonimski et al. 1978a; Kochko et al. 1979; Lamouroux et al. 1980). It was proposed (Church and Gilbert 1980; Jacq et al. 1980a) that introns encode splicing proteins. This was demonstrated (Lazowska et al. 1980) for the second intron, 12; thus, the mRNA-maturase model of splicing homeostasis can be used as a paradigm of the regulation of mosaic-gene expression. The aim of this paper is...

Proton magnetic resonances in cytochrome b2 core structural similarities with cytochrome b5

Abstract The proton NMR spectra at 220 MHz of oxidised and reduced cytochrome b2 core are presented and compared with the previously published NMR data on cytochrome b5. Overall the experiments infer that quite striking structural similarities exist between the two proteins. In particular the heme coordination, details of the heme crevice, and the environment of the only tryptophanyl residue seem to be essentially the same. The NMR experiments provide a means to extend some of the detailed structural information available from the single crystal X-ray structure of cytochrome b5 to cytochrome b2, where at present only parts of the amino acid sequence are known and no atomic coordinates have as yet been reported.

The cytochrome oxidase subunit I split gene in Saccharomyces cerevisiae: genetic and physical studies of the mtDNA segment encompassing the 'cytochrome b-homologous' intron.

SummaryWe have constructed a refined genetic and physical map of 38 oxi3 mutations. With the help of the rho- clones derived from ‘short’ and ‘long’ genes, pairwise crosses between mutants, estimations of their reversion frequencies and analyses of mitochondrially synthesized proteins, we have characterized and localized several mutants in the exon A4 and in the intron aI4. We present genetic and physical evidence that in the ‘long’ gene the exon A5 is split into at least three quite distinct exons, A5-1, A5-2 and A5-3 where numerous mutations are localized. We suggest that a novel 56 Kd polypeptide, which accumulates in some cis-dominant oxi3- mutants results from the translation of the upstream exons and the downstream aI4 intron.

TheNAM8 gene inSaccharomyces cerevisiae encodes a protein with putative RNA binding motifs and acts as a suppressor of mitochondrial splicing deficiencies when overexpressed

SummaryWe have characterized the nuclear geneNAM8 inSaccharomyces cerevisiae. It acts as a suppressor of mitochondrial splicing deficiencies when present on a multicopy plasmid. The suppressed mutations affect RNA folding and are located in both group I and group II introns. The gene is weakly transcribed in wildtype strains, its overexpression is a prerequisite for the suppressor action. Inactivation of theNAM8 gene does not affect cell viability, mitochondrial function or mitochondrial genome stability. TheNAM8 gene encodes a protein of 523 amino acids which includes two conserved (RNP) motifs common to RNA-binding proteins from widely different organisms. This homology with RNA-binding proteins, together with the intronic location of the suppressed mitochondrial mutations, suggests that the NAM8 protein could be a non-essential component of the mitochondrial splicing machinery and, when present in increased amounts, it could convert a deficient intron RNA folding pattern into a productive one.

PETCR46, A GENE WHICH IS ESSENTIAL FOR RESPIRATION AND INTEGRITY OF THE MITOCHONDRIAL GENOME

In the frame of the European Pilot Project for the functional analysis of newly discovered open reading frames (ORFs) from Saccharomyces cerevisiae chromosome III, we have deleted entirely the YCR46C ORF by a one‐step polymerase chain reaction method and replaced it by the HIS3 marker in the strain W303. The deletion has been checked by meiotic segregation and Southern blot analyses. Characterization of the deleted strain indicates that YCR46C is essential for respiration and maintenance of the mitochondrial genome since its deletion leads to the appearance of 100% of cytoplasmic petites. Hybridization with molecular probes from mtDNA of individual clones of such petites showed that about 50% did hybridize (rho− clones) while others did not (possibly rho° clones). The wild‐type gene has been cloned and shown to complement the deletion. The gene, which probably codes for a mitochondrial ribosomal protein, has been called petCR46.