Selection of genes repressed by cAMP that are induced by nutritional limitation in Saccharomyces cerevisiae.

DNA-lacZ fusion libraries of yeast Saccharomyces cerevisiae were used to select genes coordinately regulated by the Ras-cAMP-cAPK signalling pathway. Sixteen new genes (AGP1, APE2, APE3, FPS1, GUT2, MDH2, PLB2, PYK2, RNR3, SUR1, UGA1, YHR033w, YBR006w, YHR143w, YMR086w and YOR173w) were found to be repressed by cAMP. Most of these genes encode for metabolic enzymes and are induced by nutritional limitations. These common properties suggest a role of this pathway in the metabolic adjustment of the cell to nutritional variations. The induction of 10 of these genes is reduced in the msn2,msn4 double mutant, which emphasizes the role of the Msn2/4p transcriptional activators in mediating the Ras-cAMP-cAPK signalling pathway. The Msn2p/Msn4p-independent expression of the six other genes suggests the existence of other regulatory systems under the control of this pathway.

The heat shock response in yeast: differential regulations and contributions of the Msn2p/Msn4p and Hsf1p regulons.

The heat shock transcription factor Hsf1p and the stress-responsive transcription factors Msn2p and Msn4p are activated by heat shock in the yeast Saccharomyces cerevisiae. Their respective contributions to heat shock protein induction have been analysed by comparison of mutants and wild-type strains using [35S]-methionine labelling and two-dimensional gel electrophoresis. Among 52 proteins induced by a shift from 25 degrees C to 38 degrees C, half of them were found to be dependent upon Msn2p and/or Msn4p (including mostly antioxidants and enzymes involved in carbon metabolism), while the other half (including mostly chaperones and associated proteins) were dependent upon Hsf1p. The two sets of proteins overlapped only slightly. Three proteins were induced independently of these transcription factors, suggesting the involvement of other transcription factor(s). The Ras/cAMP/PKA signalling pathway cAMP had a negative effect on the induction of the Msn2p/Msn4p regulon, but did not affect the Hsf1p regulon. Thus, the two types of transcription factor are regulated differently and control two sets of functionally distinct proteins, suggesting two different physiological roles in the heat shock cellular response.

Msn2p and Msn4p control a large number of genes induced at the diauxic transition which are repressed by cyclic AMP in Saccharomyces cerevisiae.

The multicopy suppressors of the snf1 defect, Msn2p and Msn4p transcription factors (Msn2/4p), activate genes through the stress-responsive cis element (CCCCT) in response to various stresses. This cis element is also the target for repression by the cyclic AMP (cAMP)-signaling pathway. We analyzed the two-dimensional gel electrophoresis pattern of protein synthesis of the msn2 msn4 double mutant and compared it with that of the wild-type strain during exponential growth phase and at the diauxic transition. Thirty-nine gene products (including those of ALD3, GDH3, GLK1, GPP2, HSP104, HXK1, PGM2, SOD2, SSA3, SSA4, TKL2, TPS1, and YBR149W) are dependent upon Msn2/4p for their induction at the diauxic transition. The expression of all these genes is repressed by cAMP. Thirty other genes identified during this study are still inducible in the mutant. A subset of these genes were found to be superinduced at the diauxic transition, and others were subject to cAMP repression (including ACH1, ADH2, ALD6, ATP2, GPD1, ICL1, and KGD2). We conclude from this analysis that Msn2/4p control a large number of genes induced at the diauxic transition but that other, as-yet-uncharacterized regulators, also contribute to this response. In addition, we show here that cAMP repression applies to both Msn2/4p-dependent and -independent control of gene expression at the diauxic shift. Furthermore, the fact that all the Msn2/4p gene targets are subject to cAMP repression suggests that these regulators could be targets for the cAMP-signaling pathway.

The Ktr1p, Ktr3p, and Kre2p/Mnt1p mannosyltransferases participate in the elaboration of yeast O- and N-linked carbohydrate chains.

We have determined a role for Ktr1p and Ktr3p as mannosyltransferases in the synthesis of the carbohydrate chains attached to Saccharomyces cerevisiae O- and N-modified proteins. KTR1 and KTR3 encode related proteins that are highly similar to the Kre2p/Mnt1p Golgi alpha1,2-mannosyltransferase (Lussier, M., Camirand, A., Sdicu, A.-M., and Bussey, H. (1993) Yeast 9, 1057-1063; Mallet, L., Bussereau, F., and Jacquet, M. (1994) Yeast 10, 819-831). Examination of the electrophoretic mobility of a specifically O-linked protein from mutants and an analysis of their total O-linked mannosyl chains demonstrates that Ktr1p, Ktr3p, and Kre2p/Mnt1p have overlapping roles and collectively add most of the second and the third alpha1,2-linked mannose residues on O-linked oligosaccharides. Determination of the mobility of the specifically N-linked glycoprotein invertase in different null strains indicates that Ktr1p, Ktr3p, and Kre2p are also jointly involved in N-linked glycosylation, possibly in establishing some of the outer chain alpha1,2-linkages.

The nucleotide sequence of Saccharomyces cerevisiae chromosome XIV and its evolutionary implications.

In 1992 we started assembling an ordered library of cosmid clones from chromosome XIV of the yeast Saccharomyces cerevisiae. At that time, only 49 genes were known to be located on this chromosome and we estimated that 80% to 90% of its genes were yet to be discovered. In 1993, a team of 20 European laboratories began the systematic sequence analysis of chromosome XIV. The completed and intensively checked final sequence of 784,328 base pairs was released in April, 1996. Substantial parts had been published before or had previously been made available on request. The sequence contained 419 known or presumptive protein-coding genes, including two pseudogenes and three retrotransposons, 14 tRNA genes, and three small nuclear RNA genes. For 116 (30%) protein-coding sequences, one or more structural homologues were identified elsewhere in the yeast genome. Half of them belong to duplicated groups of 6-14 loosely linked genes, in most cases with conserved gene order and orientation (relaxed interchromosomal synteny). We have considered the possible evolutionary origins of this unexpected feature of yeast genome organization.

A 43.5 kb segment of yeast chromosome XIV, which contains MFA2, MEP2, CAP/SRV2, NAM9, FKB1/FPR1/RBP1, MOM22 and CPT1, predicts an adenosine deaminase gene and 14 new open reading frames.

A 43,481 bp fragment from the left arm of chromosome XIV of Saccharomyces cerevisiae was sequenced. A gene for tRNA(phe) and 23 non-overlapping open reading frames (ORFs) were identified, seven of which correspond to known yeast genes: MFA2, MEP2, CAP/SRV2, NAM9, FKB1/FPR1/RBP1, MOM22 and CPT1. One ORF may correspond to the yet unidentified yeast adenosine deaminase gene. Among the 15 other ORFs, four exhibit known signatures, which include a protein tyrosine phosphatase, a cytoskeleton-associated protein and two ATP-binding proteins, four have similarities with putative proteins of yeast or proteins from other organisms and seven exibit no significant similarity with amino acid sequences described in data banks. One ORF is identical to yeast expressed sequence tags (EST) and therefore corresponds to an expressed gene. Six ORFs present similarities to human dbESTs, thus identifying motifs conserved during evolution. Nine ORFs are putative transmembrane proteins. In addition, one overlapping and three antisense ORFs, which are not likely to be functional, were detected.

The mitochondrial receptor complex: Mom22 is essential for cell viability and directly interacts with preproteins.

A multisubunit complex in the mitochondrial outer membrane is responsible for targeting and membrane translocation of nuclear-encoded preproteins. This receptor complex contains two import receptors, a general insertion pore and the protein Mom22. It was unknown if Mom22 directly interacts with preproteins, and two views existed about the possible functions of Mom22: a central role in transfer of preproteins from both receptors to the general insertion pore or a more limited function dependent on the presence of the receptor Mom19. For this report, we identified and cloned Saccharomyces cerevisiae MOM22 and investigated whether it plays a direct role in targeting of preproteins. A preprotein accumulated at the mitochondrial outer membrane was cross-linked to Mom22. The cross-linking depended on the import stage of the preprotein. Overexpression of Mom22 suppressed the respiratory defect of yeast cells lacking Mom19 and increased preprotein import into mom19 delta mitochondria, demonstrating that Mom22 can function independently of Mom19. Overexpression of Mom22 even suppressed the lethal phenotype of a double deletion of the two import receptors known so far (mom19 delta mom72 delta). Deletion of the MOM22 gene was lethal for yeast cells, identifying Mom22 as one of the few mitochondrial membrane proteins essential for fermentative growth. These results suggest that Mom22 plays an essential role in the mitochondrial receptor complex. It directly interacts with preproteins in transit and can perform receptor-like activities.

Improved strategy for large-scale DNA sequencing using DNaseI cleavage for generating random subclones.

Large-scale DNA sequencing projects require the use of specific and reliable strategies. Here, we describe an improved strategy using DNaseI cleavage and sequencing strategy using DNaseI cleavage and sequencing from both ends of the plasmid inserts. This strategy yields better results than those obtained using partial digestion with restriction enzymes and cloning in single-stranded vectors.

Complete DNA sequence of yeast chromosome II.

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.

Nucleotide sequence analysis of an 11.7 kb fragment of yeast chromosome II including BEM1, a new gene of the WD-40 repeat family and a new member of the KRE2/MNT1 family.

This paper reports the DNA sequence and analysis of an 11.7 kb segment localized on the right arm of Saccharomyces cerevisiae chromosome II. This fragment contains one incomplete and five long and non-overlapping open reading frames (ORFs) designated from centromere to telomere-proximal side as: YBR1406, 1409, 1410, 1411, 1412 and 1413. YBR1406 corresponds to the 5' end to PG11 encoding phosphoglucoisomerase. YBR1410 encodes a polypeptide of 798 amino acids whose C terminus contains five repeats (WD-40 repeat) similar to those found in the beta-subunits of G proteins and different yeast proteins such as Tup1, Prp4 and Cdc4. The higher similarity score is obtained with dTAFII80, a component of the RNA polymerase II transcriptional complex TFIID. YBR1411 encodes a polypeptide of 464 amino acids which belongs to the family of alpha-mannosyltransferases: KRE2/MNT1, KTR1, KTR2, YUR1 and the product of previously sequenced ORF YBR1445. YBR1412 corresponds to BEM1. The two ORFs, YBR1409 and YBR1413, which do not exhibit significant similarity with any known coding sequences, define new genes.

A 12.8 kb segment, on the right arm of chromosome II from Saccharomyces cerevisiae including part of the DUR1,2 gene, contains five putative new genes.

A 12,820 bp fragment from the right arm of chromosome II of Saccharomyces cerevisiae was sequenced and analysed. This fragment contains six non-overlapping long open reading frames (ORFs) designated from the centromere- to the telomere-proximal ends as: YBR1441, 1443, 1444, 1445, 1446 and 1448. YBR1441 encodes a polypeptide of 845 amino acids which shares a long consensus domain with products of S. cerevisiae MCM2, MCM3, CDC46 and Schizosaccharomyces pombe cdc21+ genes. These genes are involved in DNA replication. YBR1445 encodes a polypeptide of 404 amino acids which has strong similarity with the S. cerevisiae KRE2/MNT1, YUR1, KTR1 gene products. The KRE2/MNT1 protein is an alpha-1,2- mannosyltransferase. The product of YBR1444, which encodes a protein of 375 amino acids, presents a lipase signature sequence and a peroxisomal targeting signal. YBR1448, whose sequence extends further on the telomere-proximal end of the fragment, is identical to the 3' end of the DUR1,2 gene encoding urea amidolyase. The two ORFs, YBR1443 and YBR1446, exhibit no significant similarity with any known gene.

RIM2, MSI1 and PGI1 are located within an 8 kb segment of Saccharomyces cerevisiae chromosome II, which also contains the putative ribosomal gene L21 and a new putative essential gene with a leucine zipper motif.

We report the DNA sequence of an 8 kb segment localized on the right arm of chromosome II from Saccharomyces cerevisiae. The sequence reveals the presence of eight open reading frames (ORFs). Three of them, YBR1402, YBR1405 and YBR1406 are previously sequenced genes, respectively the RIM2 (replication in mitochondria), MSI1 (multicopy suppressor of IRA1 gene) and PGI1 (phosphoglucoisomerase) genes. The predicted product of the ORF YBR1401 could be the putative yeast ribosomal protein L21. A new essential gene, YBR1403, has been identified by disruption; it possesses a leucine zipper motif.

The CCS1 gene from Saccharomyces cerevisiae which is involved in mitochondrial functions is identified as IRA2 an attenuator of RAS1 and RAS2 gene products.

The ccs1-1 mutation of Saccharomyces cerevisiae, which has been previously described, is associated with an increase in cytochrome content, in respiration, and in ATP synthesis. In addition, this mutation leads to the same phenotype as cells de-regulated in the cAMP pathway. From a yeast genomic library, we have isolated a DNA fragment in a recombinant plasmid pCD1 which complements the ccs1-1 mutation. Homologous integration of this DNA in the genome occurs at the CCS1 locus. An 11 kb of the DNA insert is necessary for complementation. Sequencing part of the fragment identifies CCS1 as the IRA2 gene. The IRA2 gene is known to encode an attenuator of RAS gene product activity which stimulates the GTPase activity of the RAS proteins. This result underlines the involvement of cAMP-dependent phosphorylation in mitochondrial function. We present the sequence of 1 kb DNA upstream of the putative ATG of the IRA2/CCS1 gene product which is devoid of an ORF and could contain several regulatory sites.

Monoclonal antibodies to Mokola virus for identification of rabies and rabies-related viruses.

Rabies and rabies-related virus strains were studied by using a panel of monoclonal antibodies directed against either nucleocapsid proteins or cell surface antigens of Mokola virus (Mok-3). Each strain was used in parallel to infect cultured cells and mice. Then, the patterns of reactivity of the different monoclonal antibodies were determined by the immunofluorescent-antibody staining procedure. On cells, the monoclonal antibodies differentiated fixed rabies virus strains (serotype 1) from rabies-related virus strains. The seven fixed strains (CVS, PV4, PM, Flury LEP and HEP, ERA, and SAD) reacted identically. The previous serotype groupings (serotype 2, Lagos-bat virus; serotype 3, Mokola virus; serotype 4, Duvenhage virus) established with anti-rabies monoclonal antibodies were confirmed, except for that of Lagos-bat Kindia, which appeared to be related to the African subtype of the Duvenhage serotype (Duv-2). Within the Mokola (Mok-1, -2, -3, and -5 and Umhlanga) and the Lagos-bat (Lag-1 and -2, Zimbabwe, Pinetown, and Dakar) serotypes, each strain appeared to be distinct. The African subtype of the Duvenhage serotype reacted differently from the European subtype. Within the Duvenhage serotype, subtypes Duv-4, -5, and -6 and Denmark reacted identically, while subtypes Duv-1, -2, and -3 and German Democratic Republic appeared to be distinct. The monoclonal antibodies specific for the cell surface antigens were also used in neutralization tests with all the strains. Two of them neutralized the infectivity of Mokola virus.

Immunological relationships between rabies virus and rabies-related viruses studied with monoclonal antibodies to Mokola virus.

Specific monoclonal antibodies (mAb) were prepared against a rabies-related lyssavirus, Mokola virus. A strain isolated in the Central African Republic, Mok-3, was used as immunogen. After 3 fusions more than 90 hybridoma cultures secreting mAb were identified. According to their different patterns of reactivity against rabies and rabies-related viruses, 61 ascites fluids were obtained. The antibody class was IgM for 2 of them and IgG for 59. They were specific for one of the 4 major viral proteins, as determined by immunofluorescence, neutralization and immunoblotting tests. Their patterns of reactivity were determined against 6 different strains of rabies-related viruses: Lagos-bat virus from Nigeria (Lag-1) and the Central African Republic (Lag-2), Duvenhage virus from the Republic of South Africa (Duv-1) and Federal Republic of Germany (Duv-3), Mokola virus from Nigeria (Mok-1) and Cameroon (Mok-2) and a fixed strain of rabies virus, the challenge virus strain (CVS). According to their reactivities with these strains and the pattern of fluorescence, the mAb were classified into 11 different groups with intracytoplasmic fluorescence and 7 groups with cell surface fluorescence. A differential diagnosis of these lyssaviruses is possible in tissue culture using some of these mAb.

Treatment of rabies in mice and foxes with antiviral compounds.

Thirty four chemical compounds were injected into rabies infected mice by intramuscular (i.m.) route. Twenty four compounds such as well known therapeutic agents: amantadine, lipacids, phenol compounds, didemnin-B, procaine, nucleosides analogues (ribavirin, tiazofurin, pyrazofurin) had no effect. Two compounds had a slight effect not justifying to consider them as possible therapeutic agents: selenazofurin and an analogue of ribavirin (RTA). Eight heteropolyanions (HPA), which have a related chemical structure, were efficient providing 100% protection. Nineteen compounds were injected into rabies infected mice by the intracerebral (i.c.) route. Fourteen compounds such as ribavirin, RTA, selenazofurin, tiazofurin and 9 HPA compounds had no effect. Five other HPA compounds (HPA 23-39-46-51-56) were efficient preventing the development of clinical infection in some mice. Whatever was the treatment route, treated surviving mice developed rabies neutralizing antibodies. No proof of viral multiplication was found in their brains. As some HPA compounds did produce a therapeutic effect in mice, two of them HPA 23 and HPA 39 were administered to rabies-infected foxes. In foxes the compounds prolonged the mean survival time and increased the number of survivors. These data suggest that chemotherapy might be worthwhile when vaccination was impossible.

Isolation and study of temperature-sensitive mutants of rabies virus.

One-hundred and seventeen temperature-sensitive (ts) mutants have been isolated from the challenge virus strain (CVS) of rabies virus (RV). The criterion used for this selection was the absence of plaque-forming units on CER cells under agarose medium at the non-permissive temperature (NPT) of 38.5 degrees C. Of these mutants, 102 were induced by 5-fluorouracil. This compound was much more effective as a mutagen than either nitrous acid or the alkylating agents ethyl methanesulphonate and methyl methanesulphonate. Seventy-six of the ts mutants exhibited residual multiplication at NPT which was less than 2% of that of the wild-type virus. Attempts to demonstrate complementation were unsuccessful, although three phenotypic groups have been established by biochemical and immunological tests. Possible reasons for the failure to obtain complementation are discussed.

Cellular response to rabies virus infection.

The effects of rabies virus on host cells were studied and compared to those obtained with another rhabdovirus, vesicular stomatitis virus [J. Virol. 34, 777-781 (1980)]. We show here: (1) that rabies infection has no effect on cell morphology, while infection with vesicular stomatitis virus caused cell retraction. Thus, only vesicular stomatitis virus induced a depolymerization of the microfilaments; and (2) that microtubules and microfilaments do not play a major role in rabies virus production, as it is suggested by results obtained with several effectors (colcemid, colchicine and cytochalasin-B) which directly or indirectly affect cytoskeleton organization. The same properties were observed with directly or indirectly affect cytoskeleton organization. The same properties were observed with vesicular stomatitis virus. Furthermore, the use of cytochalasin-B shows that an inhibition of glycosylation of the virion spike protein occurs only in rabies infected cells. As vesicular stomatitis viral glycosylation is normal in cytochalasin-B treated cells, results obtained indicate that two types of interactions can occur between a virion and the host-cell depending on the rhabdovirus type.