Lif2p, a Lif1p-interacting factor essential for NHEJ and downregulated in yeast diploid cells.
Marie Frank-Vaillant, Stéphane Marcand
SBGM, CEA/Saclay, Bat. 142, Gif-sur-Yvette, 91191, France

Two double strand breaks (DSB) repair pathways have been identified in eucaryotes: homologous recombination (HR) and non-homologous end-joining (NHEJ). HR requires the presence of homologous sequence elsewhere in the genome (e. g. a homologous chromosome or a sister chromatide) while NHEJ involves interactions between regions sharing little or no homology. In yeast S. cerevisiae DSB-induced HR requires RAD52 and members of the RAD52 epistasis group while NHEJ is RAD52-independent. NHEJ requires the DNA end-binding heterodimer Yku70p-Yku80p and the ligase Dnl4p associated with its cofactor Lif1p. Recent studies have shown that NHEJ efficiency is influenced by ploidy and mating type status but the mechanism of this regulation is unknown. Here we report the identification of Lif2p, a S. cerevisiae protein that interacts with Lif1p; disruption of LIF2 abolishes the capacity of cells to repair DSB by NHEJ. In MATa/MATa cells, Lif2p is downregulated and its overexpression can suppresse the NHEJ defect suggesting that Lif2p is the principal target of NHEJ regulation by mating type and ploidy.

The roles of S. pombe DNA repair functions in determining sensitivity to topoisomerase targeting agents.
Noor-E-Mobeen Malik, John L. Nitiss
Molecular Pharmacology Dept., St. Jude Children's Res. Hosp., 332 N. Lauderdale St, Memphis, TN 38018, USA

Topoisomerases (topos) have been intensively studied as anti-cancer drug targets. Topo drugs kill cells by stabilizing covalent complexes, which consist of the enzyme covalently bound to DNA by a phosphotyrosine linkage. Although the covalent complex is reversible, DNA metabolic processes such as replication can convert the complexes into irreversible DNA damage. Work with S. cerevisiae has shown that recombination repair genes are required for survival following this type of DNA damage. By contrast, S. cerevisiae cells deficient in excision repair are not drug hypersensitive. We have used the fission yeast S. pombe to further define repair pathways that are important for survival following exposure to anti-topo agents. S. pombe strains with mutations in genes such as rad22, (a homologue of S.c. RAD52) are hypersensitive to both camptothecin and the etoposide analog TOP-53. S. pombe cells lacking checkpoint functions are also drug hypersensitive. Interestingly, S. pombe strains lacking excision repair are also hypersensitive to topo drugs. We suggest that in S. pombe, excision repair may process topo:DNA covalent complexes to remove the protein covalently bound to the DNA. Subsequent steps for repairing the damage would require homologous recombination, since excision of the damage would lead to a double strand break. Our results also indicate the usefulness of developing different genetically tractable systems for studying the action of anti-cancer agents.

The genes in Saccharomyces cerevisiae that exert control over the spontaneous mutation rate.
Robert C. von Borstel, Sandra L. O'Keefe, Micah A. Chrenek
Biological Sciences, University of Alberta, B-202 BioSciences, Edmonton, Alberta, T6G 2E9, Canada

Mutation rate differences are under control of DNA repair, replication, and recombination (the three R's of DNA metabolism); we now know that carbon sources also change the spontaneous mutation rate in Saccharomyces cerevisiae and Kluyveromyces lactis. Our objective is to identify all of the genes in Saccharomyces that exert control over the spontaneous mutation rate. The task is challenging because genetic selection has been relaxed with respect to utilization and preservation of yeast, i.e. most strains have different spontaneous mutation rates. We made a rough estimate of number of genes involved after EMS-induction for mutations, followed by the use of the Lassie two-plate search for mutants with increased or decreased base substitution rates. The control was the measurement of frequency of induction of amino acid and base metabolism mutants. About one-half as many mutants for mutation rate change appeared in comparison with the number of metabolism-deficient mutants. Thus, there are approximately 200-300 genes controlling mutator activity present in the genome, and 20-30 genes controlling antimutator activity. We have devised a plasmid to insert into strain BY4730 for each knockout mutant. The plasmid is designed to detect mutators and antimutators for both frameshift and base substitution mutants. Each knockout will be tested with the Lassie Test. This test is sensitive; differences as small as a fifty percent decrease or increase can be detected with certainty.

Investigating the effects of diazaborine treatment in Saccharomyces cerevisiae .
Helmut Jungwirth (1), Brigitte Pertschy (1), Rene Köffel (1), Nicole Hauser (2), Helmut Bergler (1), Gregor Högenauer (1)
(1) Institut für Molekularbiologie, Karl- Franzens-Universität, Universitätsplatz 2, Graz, A-8010, Austria; (2) DKFZ, Heidelberg

The antibacterial drug diazaborine inhibits fatty acid synthesis of gram negative bacteria by interfering with the bacterial enoyl-ACP-reductase. We have shown previously that this drug also inhibits growth of the yeast S. cerevisiae . The mechanism of action however is different in bacteria and yeast. No inhibition of lipid biosynthesis was observed after treatment of S. cerevisiae with diazaborine. In order to identify the target of diazaborine in yeast, we compared the global expression profile of treated and untreated cells using DNA-microarrays. We also present our data from uridine labeling experiments and northern blot analyses which demonstrated that diazaborine treatment has a strong effect on RNA metabolism and processing in S. cerevisiae.

Regulation of the yeast PDR5 ABC transporter during diauxic shift.
Yasmine Mamnun, Christoph Schüller, Karl Kuchler
Molecular Genetics, Inst. of Medical Biochemistry, Dr.Bohrgasse, Vienna, A-1030, Austria

Several yeast ATP-binding cassette (ABC) transporter genes have been implicated in pleiotropic drug resistance (PDR) to cytotoxic drugs and heavy metal resistance. Recent data indicate that certain ABC genes are targets of stress response pathways, since their expression is regulated by adverse conditions. Hence, it is not surprising that genes belonging to this group are under transcriptional control by dedicated regulators. Expression of the drug efflux pump Pdr5p is under tight control by the Cys₆Zn₂ transcription factors Pdr1p and Pdr3p. In addition, PDR5 transcription is also suppressed during conditions of diauxic shift at the level of transcription in a Pdr1p/Pdr3p- dependent manner. Using GFP fusions, we find that both factors constitutively localize to the nucleus. In vivo footprint analysis showed that the Pdr1p/Pdr3p target sites (pleiotropic drug resistance element - PDRE) in the PDR5 promoter are occupied under logarithmic growth both on rich medium and under glucose starvation. From these data, we suggest that Pdr1p/Pdr3p-mediated transcription activation of PDR5 is repressed during glucose starvation. This might be either achieved by binding of a yet unknown repressor to the PDR5 promoter, a modification of Pdr1p and Pdr3p, or by interference with components of the general transcription machinery. Our results indicate that different metabolic states dictate the expression of PDR5, possibly to eliminate its activity during respiratory growth.

Cis-acting elements responsible for GAT1 expression in Saccharomyces cerevisiae .
Roopa Andhare, Kathleen H. Cox, Rajendra Rai, Terrance Cooper
Molecular Sciences, University of Tennessee, 858 Madison Ave., Memphis, TN 38163, U.S.A.

Expression patterns of the GLN3 and GAT1 genes, encoding two GATA- family transcriptional activators are distinctly different. GLN3 expression is largely unresponsive to changes in the cell's nitrogen supply. It is also unaffected by deletion of GAT1, DAL80, and DEH1. GAT1 expression, on the other hand, is Gln3p- dependent, Dal80p-regulated, autogenously-regulated, and responsive to the cell's nitrogen supply, i.e., it is Nitrogen Catabolite Repression (NCR)-sensitive. In many respects, GAT1 expression possesses the characteristics of a typical NCR-sensitive gene. GAT1 is distinct, however, in that a moderate basal level of expression always remains even when glutamine is provided as nitrogen source. To better understand these patterns of expression, we have dissected the GAT1 promoter and localized sequences required for its expression. A cluster of six GATAs are the major supporters of GAT1 transcription with proline as nitrogen source. With glutamine as nitrogen source, GAT1 expression is Gln3p- independent, but the GATA sequences are still required. They function synergistically with one or more additional elements within the promoter to mediate basal level GAT1 expression. We are refining localization of the NCR-insensitive promoter elements upstream of GAT1 and determining whether Gat1p, itself, is responsible for the GATA-sequence-dependent, Gln3p-independent, NCR-insensitive expression with glutamine as nitrogen source. Supported by NIH grant GM- 35642.

A stochastic molecular model of the fission yeast cell cycle.
Akos Sveiczer (1), John J. Tyson (2), Bela Novak (1)
(1) Department of Agricultural Chemical Technology, Budapest University of Technology and Economics, 1111 Budapest, Szt. Gellert ter 4., Hungary; (2) Department of Biology, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA

We propose a stochastic version of a recently published, deterministic model of the molecular mechanism regulating the mitotic cell cycle of fission yeast, Schizosaccharomyces pombe. Stochasticity is introduced in two ways: (i) by considering the known asymmetry of cell division, which produces daughter cells of slightly different sizes; and (ii) by assuming that the nuclear volumes of the two newborn cells may also differ. In this model, the accumulation of cyclins in the nucleus is proportional to the ratio of cytoplasmic to nuclear volumes. We have simulated the cell-cycle statistics of populations of wild-type cells and of wee1 mutant cells. Our results are consistent with well known experimental observations.

Systematic discovery of new genes in the Saccharomyces cerevisiae genome.
Marco Kessler, Qiandong Zeng, Sarah Hogan, Robin Cook, Debra Willins, Arturo Morales, Guillaume Cottarel
Pathogen Genetics, Genome Therapeutics Corp., 100 Beaver Street, Waltham, MA 02453-8443, USA

We developed a computational biology concept and a genome-wide comparative analysis of predicted protein sequences from small coding sequences to identify new genes within the genome of the budding yeast. These new genes named smORFs (for small open reading frames) were confirmed to encode for a transcript. Further analysis of these smORFs included deletion of the open reading frame to determine essentiality and western blot analysis to confirm protein expression. In some cases, the encoded proteins from these smORFs appear to be unique members of novel classes of proteins and are widely conserved from yeast to human. This investigation brings new concepts to the study of genomes in silico and will increase the expected numbers of coding sequences for a specific genome. This study could have a strong impact on the design of functional genomics and proteomics experimental studies.

Sterol uptake and trafficking in the yeast Saccharomyces cerevisiae.
Matthieu REGNACQ, Thierry FERREIRA, Parissa ALIMARDANI, Sylvaine DANDRIEUX, Julien PUARD, Thierry BERGES
UMR CNRS6161, Université de Poitiers, Av Recteur Pineau, POITIERS CEDEX, 86022, FRANCE

In Saccharomyces cerevisiae, depending on culture conditions, sterol originate from the endogenous biosynthetic pathway (in heme-competent cells) or from an exogenous supply (in anaerobiosis). These two sources are exclusive and sterol import is subjected to tight regulation related with heme availability. Aerobic import of sterol can result from mutation (e.g. hem1, upc2) or from constitutive expression of at least one gene, namely SUT1 a putative transcriptional regulator (1). We showed recently that upregulation and constitutive expression of SUT1 leads to the release of negative regulation of hypoxic genes, as a result of direct interaction between Sut1 and Cyc8(Ssn6) (2). However the target genes of Sut1 precisely involved in sterol uptake are still unknown. Our recent data indicate that this phenomenon implicates components of the secretory pathway. Moreover we have started to investigate the phenotypic effects of SUT1 upregulation on membrane properties in vivo with the use of two fluorescent probes. FM4-64 was used to monitor the kinetics of endocytosis, and NBD-cholesterol, a sterol analogue, to trace exogenously supplied sterol taken up by the yeast cells. (1) Ness et al (2001) Eur J Biochem. 268:1585-95. (2) Regnacq et al (2001) Mol Microbiol. 40:1085-96.

Effects of chs3 gene disruption in the bgl2- mutant of Saccharomyces cerevisiae .
Daniela Laurinavichiute (1), Tatyana Kalebina (1), Philipp Gorlovoy (1), Gleb Fominov (2), Igor Kulaev (1)
(1) Molecular Biology Department, Moscow State University, Vorobiovy Gory, Moscow, 119899, Russia; (2) Institute of Experimental Cardiology, Cardiology Research Center, 3rd Cherepkovskaya str. 15A, Moscow 121552, Russia

The BGL2 gene codes 1,3-beta-glucosyltransferase, a major cell wall protein of Saccharomyces cerevisiae. We have previously shown that the level of chitin (in terms of glucosamine) in the bgl2- cell walls increased and mutant cells were more sensitive to Nikkomycin Z. Deletion of BGL2, however, didn't result in significant phenotypic changes. These facts allow us to suggest that activation of chitin synthase 3 (Chs3) in the bgl2- cells might be involved in the compensatory mechanism. In this work we showed that: (1) chs3-bgl2- double mutant cells are found to be more sensitive to the cell wall synthesis inhibitors Calcofluor White and Congo Red; (2) in the chs3- cell walls the level of chitin is strongly reduced; (3) in the chs3-bgl2- mutant walls the chitin level is about two-fold lower than in bgl2-, but it is still higher than in chs3-. Proteinase treatment of the yeast cell walls and further chitin level determination demonstrated that bgl2- cells accumulate proteinase-released chitin, while in chs3-bgl2- cell walls we don't find chitin associated with the cell wall proteins. The data obtained let us to propose that almost all newly synthesized chitin is covalently bound to the glucan network in bgl2-chs3- cell wall. It is tempting to speculate that in the absence of Bgl2p yeast cells are unable to incorporate Chs3-synthesized chitin properly. We can also suggest that besides Chs3 other chitin synthases might be involved in the compensatory mechanism in the bgl2- cells.

Mitochondrial effects of the pleiotropic proteasomal mutation mpr1- 1/rpn11: uncoupling from cell cycle defects in extragenic revertants.
Teresa Rinaldi (1), Ruggero Ricordy (2), Monique Bolotin-Fukuhara (3), Laura Frontali (1)
(1) Pasteur Institute Cenci Bolognetti Foundation, Department of Cell and Developmental Biology, University of Rome La Sapienza, Rome, Italy; (2) Centro di genetica evoluzionistica, CNR Roma; (3) Laboratoire de Génétique Moléculaire Bat. 400, Université Paris-Sud, 91405 Orsay

We have previously characterised 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. Since the mutant strain is heat-sensitive both on glucose and on glycerol, we searched for revertants in order to shed light on the Rpn11/Mpr1p function. Spontaneous revertants able to grow on glucose but not on glycerol at 36°C were isolated, and, only from them, revertants able to grow at 36°C on glycerol were selected. Revertants of the two classes were found to be extragenic. The detailed characterisation of these extragenic suppressors demonstrates that the phenotypes related to cell cycle defects can be dissociated from those concerned with mitochondrial organisation. In addition a further phenotype of mpr1 mutant has been studied involving abnormalities in mating and in alpha-factor sensitivity.

Localization of yeast cell wall protein Crh2p depends on the mechanisms responsible for polarized growth.
Jose M. Rodriguez-Peña (1), Alberto Alvarez (2), Francisco G. Esquer (1), César Nombela (1), Javier Arroyo (1)
(1) Microbiologia II, Universidad Complutense, Pza. Ramón y Cajal , Madrid, 28040, Spain; (2) Centro de Citometría de Flujo y Microscopía Confocal. UCM.

Cell wall protein Crh2p, although localized in the lateral cell wall, it is mainly targeted to polarized growth sites, being preferentially visualized at the budding site in early stages of cell cycle, afterwards as a ring at the base of the bud neck, and concentrated at the septum between mother and daughter cells during cytokinesis. To study the mechanisms that control temporal and spatial distribution of this protein in the cell wall, we have followed localization of the gfp-Crh2 fusion protein by confocal microscopy in different mutant backgrounds in genes related to cell polarity and morphogenesis: bud1, cdc42, cdc10, cdc15-lyt1 and bni4. Localization of Crh2 to polarized growth sites depends on the participation of all these genes at different levels. In fact, Crh2 protein is absent at the septum region in cdc10 mutants, suggesting the requirement of the septin ring integrity for proper localization of Crh2. However, the results with cdc15-lyt1 strain suggest that the absence of septum itself is responsible for the Crh2 mislocalization. We have also studied the implications of other proteins previously related to the transport of chitin sinthase (Chs3) to the cell surface in the correct localization of Crh2. Transport of this protein is impaired in chs5 and sbe2 sbe22 strains while transport of other covalently bound cell wall proteins like Cwp1 is not affected. These data suggest the existence of specialized transport systems for different cell wall mannoproteins.

G2/M arrest caused by actin disruption is a manifestation of the cell size checkpoint in fission yeast.
Ivan Rupeš (1), Bradley A. Webb (2), Alan Mak (2), Paul G. Young (1)
(1) Department of Biology, Queen's University, Barrie Street, Kingston, ON K7L 3N6, Canada; (2) Department of Biochemistry, Queen's University, Kingston, Ontario K7L 3N6, Canada

In budding yeast, actin disruption prevents nuclear division. This has been explained as activation of a morphogenesis checkpoint monitoring the integrity of the actin cytoskeleton. The checkpoint operates through inhibitory tyrosine phosphorylation of Cdc28, a budding yeast Cdc2 homologue. Wild type S. pombe cells also arrest before mitosis following actin depolymerization. Oversize cells, however, enter mitosis uninhibited. We carried out a careful analysis of the kinetics of mitotic initiation following actin disruption in undersize and oversize cells. We show that an inability to reach the mitotic size threshold explains the arrest in smaller cells. Among the regulators that control the level of the inhibitory Cdc2-Tyr15 phosphorylation, the Cdc25 protein tyrosine phosphatase is required to link cell size monitoring to mitotic control. This represents a novel function of the Cdc25 phosphatase. Furthermore, we demonstrate that this cell size monitoring system fulfils the formal criteria of a cell cycle checkpoint.

Influence low-doses cancerogenic agent 3,4 Benzo(@)pyrene (3,4 BP) on growth population of yeast cells.
Victor Samokhvalov (1), Ignatov Vladimir (2), Museykina Natali (3), Melnikov Gennadiy (3)
(1) Dept.Biochemistry, Saratov State University, Saratov Astrakhanskaya str 83, 410026 Russia; (2) Dept Of Biochemistry, Saratov State University, Saratov Astarkhanskaya str 83, $10026 Russia; (3) Dept of Biochemistry, Saratov State University, Saratov Astrakhanskaya str 83, 410026 Russia

Adaptation responsens (Ar) is playing an important role in adapting of the cells to unfovourableconditions.Under thr term Ar , we mean the increase os stability of cells and organism to high damading doses of toxic and genotoxic agents, forming after preleminary affect of these agents in low-doses. According to this, the investigation of intracellular preventive systems of cell and organism is of great perspective. We studied the influence of low-doses of Cancerogenic compounds 3,4 BP on growth of yeast cells Saccharomyces cerevisiae 14. The 3,4 BP was used in concentrations 1,5 mkg/1 ml of Rider^s medium. We have found, that 24-hours contact of yeast cells with 3,4 BP practically didn^t influence the growth of population of yeast cells. However, we found quick increase of economical coefficient(Y>1,5) and substuntial decrease of glucose consumpshin. Apperently, the addition of 3,4 BP initiaten the pathways of yeast cell to side of anabolical direction. The 3,4 BP used in the concehtration 200 mkg/ 1 ml led to the complete ingibition development of yeast population. Nevertheless, the low doses of 3,4 BP are activating adaptation processes stability of yeast cells to 3,4 BP and some toxic agents . In our experiments yeast cells having grown in low-doses 3,4 BP medium appeared to be nonsensitive to hight - doses of 3,4 BP. In other words, the development of yeast population was the same as with intact cell.

Chemical mutagenesis in fission yeast: effects on survival rates and cell morphology.
Mirela M. Cimpeanu (1), Cristian S. Cimpeanu (2)
(1) Faculty of Biology, Genetics

The aim of our study was the identification of specific effects, certain chemical mutagens induced, on fission yeasts. Schizosaccharomyces pombe (S.pombe) strains used were: h90, h+ and h-. Chemical mutagens were: alkylating agents - methylmethanesulfonate (MMS), ethylethanesulfonate (EMS), nitrosoguanidine (NG), and alcaloids - colchicine (COL), caffeine (CAF) and nicotine (NIC), two concentrations each. Mutagenesis was performed according standard procedures. Survival rates (RS) were per cent evaluated. Acetic fuchsin method and microphotography revealed cell morphology. Alkylating and alcaloid agents seem to induce genotoxic and cytotoxic effects on S.pombe. RS exhibit an inverted correlation with time of exposure and mutagen concentration. Despite their potency, alkylating agents seem to induce a very particular response in S.pombe, i.e. high RS on high mutagen concentration, but also, long time of exposure. This response can be evaluated like an 'adaptative' one. The powerful alcaloid mutagens were NIC and CAF. Exposed cells exhibit similar RS like alkylating agents treated cells. Micrographs of mutagenized cultures allowed us to identify a particular set of morphological alterations. Alkylating compounds treatment consequently, mating partners have produced very thin conjugation tubes, improperly for kariogamy, and also septation and cytokinesis defects. Alcaloids frequently have induced abnormal septation, resulting in multinucleate cells, and lateral conjugation.

Isolation and genetic analysis of Saccharomyces cerevisiae mutants resistant to 4-aminopyridine.
Sukhdeep Gill (1), Sham Sunder (2), Balwant Singh (1)
(1) Dept of Molecular Biology and Biochemistry, Guru Nanak Dev University, Amritsar - 143005, Punjab, INDIA; (2) Dept of Biology, Hindu College, Dhab Khatikan, Amritsar-143006, Punjab, INDIA

4-Aminopyridine(4-AP) is a well known K⁺ channel blocker in animal cells. The growth of Saccharomyces cerevisiae cells on synthetic complete medium agar plates containing 7mM 4-AP was found to be completely inhibited. However, the growth was restored when the above medium plates containing inhibitory conc. of 4-AP were supplemented with 0.2M KCl/0.2M NaCl/0.4M mannitol. Further experiments revealed that 4-AP had no effect on the uptake of K⁺ as the intracellular K⁺ contents of 4-AP treated yeast cells as well as of the control cells were of the same order. It was also found that 4-AP caused cell lysis. S.cerevisiae cells were mutagenized using ethyl methane sulfonate(EMS) and the mutants, which were able to grow on medium plates containing inhibitory conc. of 4-AP, were screened. Total 83 mutants were identified and designated as apr (aminopyridine resistant). The mutants were recessive, monogenic and they fell into two complementation groups. To identify genes which might be interacting with different apr alleles, suppressors of apr phenotype were isolated by mutagenizing apr mutants with EMS. The suppressors were unable to grow on the medium containing inhibitory conc. of 4-AP and were designated as sar (suppressors of aminopyridine resistance). They were recessive and ordered into atleast two complementation groups. The suppressor phenotype also segregated from the mutant phenotype.

Functional studies of the BOP1-3 genes.
Guo-Zhen Hu, Hans Ronne
Department of Plant Biology, Swedish Univ of Agric Sciences, Box 7080, Uppsala, SE-75007, Sweden

We have previously cloned the PAM1 gene as a high copy number suppressor of protein phosphatase 2A (1). A second PAM1-related gene, SVL3 is also present in the yeast genome. While a deletion of either gene has no detectable phenotype, we haver found that a deletion of both genes makes the cell temperature sensitive. The temperature sensitive phenotype was used to clone high copy number suppressor genes that permit growth at the restrictive temperature in the absence of both PAM1 and SVL3. A total of 13 suppressor genes were obtained and further characterized. Some of cloned suppressors are previously known genes with a role in signaling and growth control, such as CLN1 encoding a G1 cyclin, or RTS1 encoding a regulatory subunit of PP2A. Two of them, CLN1 and MSB1 were able to suppress loss of PP2A when overexpressed, just like PAM1 itself. Three of the cloned genes are unique open reading frames, with no previously assigned function in yeast or other eukaryotes. We have named them BOP1-3 for Bypass Of PAM1. The BOP1 and BOP3 genes resemble PAM1 in that high level overexpression of these genes inhibits growth and induces a filamentous-like phenotype. 1. Hu G.-Z. and H. Ronne. (1994) J. Biol. Chem. 269, 3429-3435.

Ddc1 localizes to meiotic chromosomes and promotes Mek1-dependent phosphorylation of Red1 in the pachytene checkpoint.
Eun-Jin Erica Hong, Shirleen Roeder
MCDB, Yale University, 266 Whitney Ave., New Haven, CT 06511, U.S.A.

A meiotic checkpoint monitors the status of recombination and chromosome synapsis, processes that are essential for correct homolog segregation at meiosis I. This checkpoint operates in the zip1 mutant of S. cerevisiae, which arrests at the pachytene stage of meiosis with defects in recombination and synaptonemal complex formation. ddc1 was identified as a mutation that alleviates zip1 arrest. We show that Ddc1 is required for the meiotic checkpoint in zip1. Ddc1 becomes phosphorylated and localizes to meiotic chromosomes, and the degree of phosphorylation and number of foci on chromosomes correlate well with the extent of checkpoint activation. Ddc1 localization and phosphorylation depend on the formation and processing of double-strand breaks (DSBs). Ddc1 patrially colocalizes with DSB repair proteins, suggesting that Ddc1 is at the sites of DSB repair and directly monitors the status of recombination. Ddc1 is recruited to meiotic chromosomes by Rad24, Mec3 and Red1, proteins that are also required for the meiotic checkpoint. Mek1, a meiosis-specific kinase, is required for phosphorylation of Ddc1, but not for its chromosomal localization. In turn, Ddc1 promotes the Mek1-dependent phosphorylation of Red1. Thus, Ddc1 and Mek1 appear to be in a feedback loop, with Ddc1 serving both as a substrate and as a mediator of Mek1 kinase activity.

Genetic and biochemical analysis of calcineurin-mediated signalling pathway using fission yeast as a model system.
Reiko Sugiura (1), Hong Cheng (1), Tomoko Yada (1), Hisato Shuntoh (2), Susie Sio (1), Takayoshi Kuno (1)
(1) Genome Sciences, Kobe Univ. Graduate Sch. Med., Kusunoki-cyo chuo- ku, Kobe, 650-0017, JAPAN; (2) Faculty of Health Science, Kobe University School of Medicine

Calcineurin, a highly conserved Ca2+/calmodulin-regulated phosphatase, is a critical component of many calcium-regulated processes in mammalian cells, including T cell activation, heart valve development and memory. Calcineurin is specifically inhibited by the immunosuppressant drugs cyclosporin A and tacrolimus (FK506), and these drugs have served as valuable reagents in identifying the role of calcineurin in a wide variety of cell types. Recently, we have discovered that calcineurin is essential for the maintenance of Cl- homeostasis, and that calcineurin and the Pmk1 MAP kinase signalling pathway play antagonistic functional roles in the fission yeast Schizosaccharomyces pombe. We carried out an isolation and a screening for several cyclosporin A and FK506-sensitive mutants, in order to identify genes that share an essential function for viability with calcineurin. As a consequence, several attractive candidates for the components of the calcineurin pathway are in hand and actively being pursued. For example, we have identified three genes, related to phosphatidylinositol-4-phosphate 5-kinase, GPI-anchor synthase, and the Rab small G protein family. Functional characterization of these genes in relation to calcineurin will be described.

Bax - initiated apoptosis in Saccharomyces cerevisiae ? Truth and facts about the proapoptic BAX mediated yeast 'killing'.
Veronika Fekete, Eva Mäsiarová, Pavol Sulo
Department of Biochemistry , Comenius University, Mlynská Dolina, Bratislava, 842 15, Slovakia

Signaling from mitochondria to the nucleus is probably mediated by metabolic signals or simple molecules that regulate expression of proteins with mitochondrial destination coded by nuclear genes. The only known protein-mediated signalization from mitochondria is programmed cell death – apoptosis, where the nuclear DNA degradation is triggered by cytochrome c release from mitochondria. Yeast growth is blocked by the expression of proapoptic gene BAX from various mammals, even though a natural apoptosis in S. cerevisiae has not been satisfactory proven. However BAX expression under the inducible promoter carried on episomal plasmid does not destroy cells. Plasmid is rather specifically eliminated that used to be misinterpreted as the cell death, because the cured cells cannot grow on the plates lacking leucine (reporter plasmid marker) as well. The process strongly depends on the cell division and only plating on the complete media shows elimination but not death. Moreover S. cerevisiae compromises the expression of this toxic gene through the number of dominant and recessive mutations. Interestingly dominant mutations are effective almost exclusively in respiring yeast. Dominant suppressor genes were isolated from low copy as well as multicopy libraries and identified. In conclusion our results indicates that Bax overexpression does not trigger processes similar to apoptosis but is rather toxic like overexpression of some other own and heterologous proteins.

Analysis of the Saccharomyces cerevisiae H ₂ O ₂ response by genome-wide mRNA profiling.
Benoit Biteau, Marzia Harnois, Bruno Dumas, Michel B Toledano
LSOC, SBGM/DBCM/DSV, CEA-Saclay, Gif sur Yvette, 91191, France

The changes in gene expression underlying the yeast adaptive response to H₂O₂ were analysed by genome wide mRNA profiling using DNA microarrays. This study analyses mRNA profiles before and after 10, 20, 30, 40, and 50 min after cell exposure to H₂O₂ (300microM). The expression of 1020 genes is increased by a factor ≥ 1.5, at three or more time points after exposure, with a return to baseline levels expression at 50 min for most of these genes. This genomic response includes most of the antioxidants and detoxification genes, most of the genes involved in protein degradation, carbohydrate metabolism, iron metabolism, DNA repair, and a strikingly high number of genes encoding various mitochondrial proteins (respiratory chain, translation factors, ribosomal proteins). Concomitantly, the expression of 500 genes is decreased by the same treatment, mainly genes involved in transcription and cytoplasmic translation. These data indicate that the adaptive response to H₂O₂ involves the massive induction of stress and defence genes, protein degradation pathways and a slowdown of cytoplasmic protein biosynthetic processes. It is striking that the mitochondrial protein expression machinery is concurrently stimulated. The contribution of the transcription regulators Yap1, Skn7, Msn2/4, Rpn4, Gcn4, and Met4 to the H₂O₂ genomic response was analysed using single and combination deletion mutants. Altogether, these regulators control more than 200 genes of the H₂O₂ stimulon.

Mechanistic relationship between replicative lifespan and chronological senescence in Saccharomyces cerevisiae .
Dawn L. Maskell (1), Alan I. Kennedy (2), Jeff A. Hodgson (2), Katherine A. Smart (1)
(1) School of BMS, Oxford Brookes University, Gipsy Lane, Oxford, OX3 0BP, UK; (2) Scottish Courage Brewing Limited, Technical Centre, Sugarhouse Close, 160 Canongate, Edinburgh, EH8 8DD, UK.

Eukaryotic cells possess a finite replicative lifespan where the number of divisions is dependent upon genetic and environmental factors. This maximal divisional capacity of the cell is termed the Hayflick limit. The ageing process which accompanies an increase in replicative age can be defined as a progressive decline in homeostasis that leads to a reduction in the ability of an organism to replicate and withstand stress. By contrast eukaryotic cells may also exhibit post-mitotic senescence in which the metric of lifespan is chronologically and not division related and may sometimes be described as extended stationary phase. This form of senescence in yeast has been extensively studied and represents a simpler model system for research yet no comparison of these two forms of senescence has been made using the same strains has previously been conducted. In this study, Saccharomyces cerevisiae polyploid strains have been utilised as model systems to directly compare the mechanisms of both replicative and chronological lifespan and their relationship to stress tolerance. Here we demonstrate that replicative and chronological senescence are not related phenomenon, though both are influenced by both genetic and environmental factors.

Identification of a homologue of the Saccharomyces cerevisiae PHO85 gene incoded a cyclin-dependent protein kinase in Pichia pastoris.
Julia G. Popova, Elena V. Sambuk
Genetics and Breeding, St.Petersburg University, Universitetskaya nab, Saint-Petersburg, 199034, Russia

Investigation of Pichia pastoris cell metabolism regulation is needed for development of its application in biotechnology. At present, it is a little known about regulation of cellular events involving the cyclin- dependent protein kinase in P. pastoris. Using a construction of plasmid, containing a PHO85 gene of S. cerevisiae disrupted by inserting of HIS4 gene of P. pastoris, we received a transformants. We carried out a description of phenotypes of the P.pastoris deletion of pho85. We discovered that the P.pastoris deletion pho85 had a constitutive activity of acid phosphatase, and had small size of colonies in comparison with the wild type strain GS115. We carried out the PCR with DNA of wild strain GS115 using the primers to S.cerevisiae PHO85 gene. We received a product of PCR. At this time DNA sequence analysis of the PCR product is in progress. Our results indicate that P.pastoris has a homologue of PHO85 gene. At the same time it proves a conservatism of structure and function of the cyclin-dependent protein kinase Pho85p in evolution.

Analysis of the segregation mechanism of the 2µ plasmid in Saccharomyces cerevisiae .
C.M.V.L Wong (1), S. Scott-Drew (2), M.J. Hayes (2), J.A.H Murray (2)
(1) Department of Biotechnology , Universiti Putra Malaysia , 43400 Serdang , Selangor , Malaysia; (2) Institute of Biotechnology, University of Cambridge, Cambridge CB2 1QT, United Kingdom

The 2µ plasmid of Saccharomyces cerevisiae is inherited stably due to an efficient plasmid segregation mechanism. 2µ plasmid-encoded genes, REP1 and REP2 in conjunction with one of its cis-acting loci, STB, are involved in plasmid segregation but the mechanism of segregation is not fully understood. A defect in any one of these three loci disrupts plasmid segregation, resulting in an asymmetrical segregation where the plasmid is retained in mother cells producing daughter cells that do not inherit any plasmid. In this study, the interaction of the Rep proteins with the STB locus was analysed. Plasmids were tagged with green fluorescent protein and the Rep proteins were immunostained. It was found that plasmids carrying 2µ plasmid STB sequences co-localise with the Rep proteins at discrete foci in the nucleus throughout the cell cycle and that they segregate together at cell division. In contrast, plasmids lacking the STB sequences form foci that do not co-localise with the Rep proteins and do not segregate. These observations suggest that there are two types of plasmid foci: (i) Active plasmid foci, where plasmids are present in groups that associate with Rep proteins via the STB. Subsequently, the Rep proteins segregate the plasmid using an active mechanism at mitosis by attaching to the chromosomes. (ii) Inactive replicating plasmid foci, where plasmids are present in groups but fail to associate with the Rep proteins. These plasmids are retained in the mother at mitosis.

Tail-anchored proteins: some bioinformatics and some biochemistry.
Traude Beilharz (1), Billie Egan (1), Kay Hoffman (2), Trevor Lithgow (1)
(1) Biochemistry&Molecular Biology, University of Melbourne, Royal Parade, Melbourne, 3010, Australia; (2) Bioinformatics Group, MEMOREC Stoffel GmbH, Stoeckheimer Weg 1, D-50829 Köln, Germany

A class of proteins anchored to cytoplasmic membranes by a single carboxy-terminal transmembrane 'tail' provide an interesting problem in protein targeting. The amino-terminal domain of these proteins can fold co-translationally, but the carboxy- terminal segment of these proteins contain sequences necessary for intracellular targeting. Thus, the final events of protein synthesis have occurred before the protein can be inserted into a membrane. We have used bioinformatics to identify a near-complete set of tail- anchored proteins from yeast. The list of fifty-five open-reading frames includes at least twenty novel proteins localised to mitochondria and various membranes of the secretory system, including the transitional endoplasmic reticulum and inner nuclear envelope. The carboxy terminal 35 amino acids, including the predicted transmembrane segment, directs targeting of each tail-anchored protein. While no obvious motif is apparent in the primary structure of these short segments, basic and amphipathic characteristics of the segments appear critical for targeting the mitochondria versus the secretory membranes. We are working to define how the translocation machinery a membrane decodes features of the targeting segment of tail-anchored proteins. Wattenberg B. & Lithgow T. (2001) 'Targeting of C-terminal (tail) anchored proteins: understanding how cytoplasmic activities are anchored to intracellular membranes.' Traffic 2, 66-71

Regulation of the Mcs2 C-type cyclin in fission yeast.
Sophie Bamps (1), Damien Hermand (2), Lionel Tafforeau (1), Tomi P. Mäkelä (3), Jean Vandenhaute (1)
(1) URBM-GEMO, FUNDP , rue de Bruxelles, 61, Namur, 5000, BELGIUM; (2) Laboratoire de Génétique Moléculaire (GEMO) University of Namur (FUNDP), 5000 Namur, BELGIUM, Biomedicum Helsinki PL63-B509b, University of Helsinki, SF00014 Helsinki, FINLAND, Imperial Cancer Research Fund (ICRF), Cell Cycle Lab, WC2A 3PX London, UNITED KINGDOM; (3) Biomedicum Helsinki PL63-B509b, University of Helsinki, SF00014 Helsinki, FINLAND

CDK activation requires activating phosphorylation of a conserved residue in the T-loop by the CAK (CDK-activating kinase). In both fission yeast and higher eucaryote, CAK is a trimeric complex composed of Mcs6-Mcs2-Pmh1 or its homologue [1-3]. Two hybrid assays revealed interaction of both Mcs2 and Pmh1 with Shp1, a subunit of the SCF ubiquitin ligase. This prompted us to test the stability of theses CAK regulators. Though the steady state level of Mcs2 does not seem to change during the cell cycle [4], we show that it is strongly correlated to Mcs6 kinase activity and Mcs2 was nearly undetectable when Mcs6 was strongly overexpressed. Interestingly, this effect is reversed by a mutation in shp1. Taken together, theses results suggest a putative regulation of Mcs2 by Mcs6 phosphorylation and SCF ubiquitin ligase complex. Thus hypothesis is under investigation. 1/Hermand, D., Westerling, T., Pihlak, A., Thuret, J. Y., Vallenius, T., Tiainen, M., Vandenhaute, J., Cottarel, G., Mann, C. and Makela, T. P. (2001) Embo J 20, 82-90. 2/Hermand, D., Pihlak, A., Westerling, T., Damagnez, V., Vandenhaute, J., Cottarel, G. and Makela, T. P. (1998) Embo J 17, 7230-8 3/Kaldis, P. (1999) Cell Mol Life Sci 55, 284-96 4/Molz, L. and Beach, D. (1993) Embo J 12, 1723-32 S.B. is a FNRS fellow research. D.H. is a FNRS scientific research worker. L.T. is recipient of a FRIA fellowship.

Characterrization of the C-less Pho84 High-Affinity Phosphate Transporter of Saccharomyces cerevisiae .
Abraham Berhe (1), Jens O. Lagerstedt (1), Jim Pratt (1), Renata Zvyagilskaya (2), Bengt Persson (1)
(1) Dpt. Biochemistry & BioPhysics, Stockholm University, Lillafrescativ. 7, Stockholm, S-106 91, Sweden; (2) A.N. Bach Institute of Biochemistry, Moscow

One of the ways cells respond to fluctuating environmental conditions is through altered gene expression. Signals on the availablity of vital nutrients are modulated across the plasma membrane to the cell interior. As one of the essential nutrients in living organisms phosphate plays a pivotal role in the building components of several cellular processes. In Saccharomyces cerevisiae signals of presence or absense of repressible amount of extracellular inorganic phosphate are regulated by the PHO regulatory pathway [1] and at least two major systems of transport are known to be involved in the acquisition phosphate. The high-affinity respectively the low-affinity transport systems. The Pho84p is part of the high-affinity uptake system and has been expressed, purified and functionally reconstituted [2]. In order elucidate functional and structural aspects of the Pho84p, a C-less version of the wild-type protein has been created and its kinetics characterized in situ. The results obtained suggest that the native cysteins of the Pho84 protein are functionally dispensible and are not required for correct protein folding. References: 1. Kaffman, A., I. Herskowitz, R. Tjian and E.K. O'Shea. (1994) Science 263: 1153- 1156. 2. Berhe, A. Fristedt, U. and Persson B.L. (1995) Eur. J. Biochem 227: 566-572.

Variations in active transport of cathions and membrane resting potential in Schizosaccharomyces pombe and Saccharomyces cerevisiae during cellular cycle.
Cristian S. Cimpeanu

Our work was carried out in order to characterize the active transport of some cathions (Na+ and K+), through plasma membrane and the cell wall of two yeast species (S.pombe and Sacch.cerevisiae), and also the variations of membrane resting potential.Three different strains of S.pombe and two of Sacch.cerevisiae have used. The intensity of cathions transport was determined by measuring Na+, K+P-ATP-ase. We found important variations in the active transport of cathions, according to the cell cycle. The carriers from plasma membrane concentrate the Na+ and K+ in cytoplasm, during stationary phase and in free ascospores in S.pombe. In Sacch.cerevisiae, these modifications are less evident. The values of resting potential, less studied in yeast cells, may offer important dates about the role of passive and active ion transport in generating the electric changes of plasma membrane. The membrane potential was determined by glass microelectrodes. We obtained a common variation pattern for the resting membrane potential, both for S.pombe and Sacch.cerevisiae: the values are smaller in the first stage of cell cycle and lag phase and increase during the terminal stages. Constantly, the values are smaller in S.pombe comparative to Sacch.cerevisiae. The size of membrane resting potential mainly depends of active and passive ions transport modifications, during the cell cycle of these microorganisms.

Pantothenate Uptake and Nuclear Division in Schizosaccharomyces pombe .
Juergen Stolz (1), Thomas Caspari (2), Norbert Sauer (3)
(1) Dept. of Cell Biology and Plant Physiology, University Regensburg, Universitaetsstr. 31, D-93040 Regensburg; (2) MRC Cell Mutation Unit, University of Sussex, Falmer Brighton BN1 9RR, United Kingdom; (3) Dept. of Botany II, University Erlangen-Nuernberg, Staudtstr. 5, D- 91058 Erlangen, Germany

Hydroxyurea inhibits ribonucleotide reductase and causes wild-type cells of in Schizosaccharomyces pombe to arrest at the G2/S checkpoint of the cell division cycle. Mutants in the liz1+ gene proceed into mitosis and display multiple phenotypes such as cell separation in the absence of nuclear separation (cut phenotype), or produce daughter nuclei with unequal size (lsd phenotype). Liz1p is a plasma membrane protein with homology to the S. cerevisiae allantoate transporter family. Here we show that Liz1p is a high-affinity transporter for pantothenate, the precursor of coenzyme A. Cell cycle defects of liz1- mutants are rescued by high extracellular concentrations of pantothenate, indicating that the mutant phenotype is indeed caused by a lack of intracellular pantothenate. Moreover we show that the reductive pathway of uracil degradation is a source of pantothenate in S. pombe. Hydroxyurea is likely to interfere with this pathway, explaining the synthetic phenotypes of inhibition of ribonucleotide reductase and deletion of liz1 and the partial uracil auxotrophy observed for liz1- mutants. The nuclear phenotype of liz1- cells is highly similar to that of mutants in acetyl-CoA carboxylase and fatty acid synthase. This indicates that liz1- mutants are compromised in some aspect of fatty acid biosynthesis. We are currently investigating if the general capacity to newly synthesise fatty acids or a specific class of lipids is required for normal karyokinesis.

MOLECULAR STUDIES ON A KLUYVEROMYCES MARXIANUS MALATE INDUCIBLE PLASMA MEMBRANE PROTEIN.
Odilia Queiros (1), Margarida Casal (2), Pedro Moradas-Ferreira (1), Cecilia Leao (2)
(1) Microorganism Stress Unit, IBMC, Rua do Campo Alegre, Porto, 4150- 180, Portugal; (2) CCA-Biologia, University of Minho, 4710-057, Braga, Portugal

Malate has a number of key roles in the metabolism, including its function as a tricarboxylic acid cycle intermediate, and as a participant in the malate-aspartate shuttle. In addition, malic enzymes catalyze the oxidative decarboxylation of malate to pyruvate. As a sole carbon source, the yeast Saccharomyces cerevisiae uses malic acid very inefficiently, due to the absence of a plasma membrane transporter for the acid. Malic acid can be used very efficiently as sole carbon and energy source by other yeast species. This study aimed at cloning genes of the yeast Kluyveromyces marxianus involved in malic acid transport across the plasma membrane, considered the first step of the metabolism of the acid. A mutant of K. marxianus unable to transport malic acid by a mediated mechanism (Mal7) led to the identification of a 28 kDa plasma membrane polypeptide (Queirós et al, 1998, Yeast 14: 401-407), which probably is involved in the transport system of malate and other dicarboxylates. This protein was purified, digested with tripsin and analysed by mass spectrometry. The peptidic map obtained showed no similarities with the ones available in the databases. One of the peptides was partially sequenced. Based on the data coming from the sequence, a degenerated oligonucleotide with 27 bp was used to search a genomic library of K. marxianus by colony hybridisation and PCR analysis. Several positive clones were identified and the plasmids recovered for further DNA sequence analysis.

LYSIN superproduction mutations are introduced into industrial yaest strains genome.
Vera Stepanova (1), Svetlana Davydenko (2), Vladimir Donich (3), Svetlana Smolina (1), Olga Kurennaja (4), Boris Yarovoy (1)
(1) Konstantinov Institute of Nuclear Physics, RAS, Gatchina, 188350, Russia; (2) Mytosis genetics, Institute of Cytology, RAS, 4 Tikhoretsky av., St.Petersburg, 194064, Russian Federation

Lysine is an important amino acid, which is not produced in mammalian organisms. Possible ways of enrichment of food is the selection and usage yeast strains with increased production of inter and extracellular lysine. We've obtained 2198 mutants, resistant to thyolysine (chemical analog of lysine), one of them secreted the maximum amount of lysine in media -0,45g/l. Intracellular content of lysine was also increased to 30% in this strain. We investigated the genetic nature of lysine superproduction phenotype. Resulting in the growth zone of lysine deficient mutant around colonies producing lysine in media. It was shown that the increased secretion of lysine is determined at least by two different genes: first gene , controlling thyolysine resistance with a pleiotropic effect of lysine superproduction (THL) and second gene involved in regulation of lysine production (PRL). Linkage groups for these genes are found: the first is located on the IV and the second is on the XV chromosome. LYS21 is also located on the IV. The possibility of allelism of THL and LYS21 was not tested. After several backcrossers both genetic properties are introduced in the genotype of industrial yeast strains for baking industry. Genome stabilization of new strains was confirmed by elektrokaryotyping. The secretion of lysine into media could be possibly used in beer production, resulting in appearing of new sorts of lysine enriched beer without any extra expenses.

A physiological comparison between an industrial, recombinant, xylose utilising Saccharomyces sp. and Pichia stipitis .
Ricardo R. Cordero Otero (1), Fredrik C. Wahlbom (2), Willem H. van Zyl (3), Bärbel Hahn-Hägerdal (2)
(1) Institute for Wine Biotechnology, Stellenbosch University, Matieland 7602, South Africa; (2) Applied Microbiology, Lund University, S-221 00, Sweden; (3) Department of Microbiology, Stellenbosch University, Matieland 7602, South Africa

D-xylose is the major constituent of the xylan component, amounting to 25% of the dry biomass. Saccharomyces cerevisiae can utilise D- xylulose, but not D-xylose. The phosphorylated xylulose is metabolised through the pentose-phosphate pathway and converted to ethanol via glycolysis. Yeast xylulokinase has been reported to play an important role in yeast xylulose metabolism, possibly being the rate-limiting enzyme. D-Xylose utilising wine yeasts have already been constructed by heterologous expression of the Pichia stipitis XYL1 and XYL2 genes coding for xylose reductase (XR) and xylitol dehydrogenase (XDH), and the S. cerevisiae xylulokinase (XK) gene, respectively. The resultant strains can produce ethanol from xylose, but not at an economical rate. Saccharomyces sp. TMB 3400 (EMS mutant) and P. stipitis CBS 6054 were grown on xylose in two separate cultivation experiments: During the first cultivation, complete aerobic conditions were maintained. The stirring speed was kept constant in the second cultivation and thereby the culture made itself gradually more oxygen limited as the cell growth proceeded. In the end, anaerobic fermentations were implemented by sparging with nitrogen. The most important parameters during aerobic, oxygen limited and anaerobic fermentations for both yeasts will be discussed.

Expression of biologically active, human soluble CD14 in Yarrowia lipolytica .
Christof Gysler (1), Peter van den Broek (1), Philippe Duboc (1), George Bou-Habib (1), Karine Vidal (1), Jean-Marc Nicaud (2), Eduardo J. Schiffrin (1), Peter Niederberger (1)
(1) Bioscience Department, Centre de Recherche Nestlé, Vers-chez-les- blanc, Lausanne 26, CH-1000, Switzerland; (2) Lab. Génétique des Microorganismes, INRA centre de Grignon BP 01, F- 78850 Thiverval-Grignon , France

CD14 is a 55kDa glycoprotein which mediates cellular immune response towards the lipopolysaccharide (LPS, endotoxin), a cell wall component of Gram-negative bacteria. Soluble sCD14 is present in human serum and milk. However, its low concentration in milk, its hydrophobic nature as well as the presence of numerous other proteins make the purification of milligram quantities, usually required for bioactivity studies, an extremely difficult task. As an alternative source we expressed a sCD14 protein carrying a Histidine tag in the food-grade yeast Yarrowia lipolytica. Yeast transformants harbouring multiple copies of an expression/secretion cassette with the human CD14 cDNA under the control of Yarrowia lipolytica derived transcription- and secretion signal sequences were grown in fed-batch. High levels of protein showing N core-glycosylation were secreted into the medium. The Histidine tag allowed purification of preparative amounts of the protein by immobilized metal affinity chromatography (IMAC). Recombinant CD14, purified to single band on SDS-PAGE, displayed biological activity in an in vitro assay of CD14-dependant LPS-reponsiveness of an astrocytoma cell line.

SECRETION AND INHIBITION OF ASPARTIC PROTEINASES BY CLINICAL IZOLATES OF CANDIDA.
Jiri Dostal (1), Olga Hruskova- Heidingsfeldova (1), Petr Hamal (2), Iva Pichova (1)
(1) Biochemistry, UOCHB AV CR, Flemingovo nam. 2, Prague, 16610, Czech Republic; (2) Institue of Microbiology, Faculty of Medicine, Palacky University, Hnevotinska 3, 77515,Olomouc,CZ

During the past two decades, Candida infections have increased in number and severity.The pathogenic Candida spp. produce extracellular aspartic proteinases (Saps), which are considered as one of virulence factors.The role of Saps is to degrade a number of host substrates as are collagen, keratin, hemoglobin and immunoglobin.Thus, proteinases of Candida are attractive therapeutic target for combating the candidal infection. We use the Saps production for the diagnostic purposes. We describe macromorphological, micromorphological features and ability of clinical Candida isolates to produce extracellular proteinases.The set of 15 Candida species was tested for growth and extracellular proteinase production on a solid medium with hemoglobin as sole nitrogen source.The strains were compared according to ability to utilise hemoglobin for growth and proteinase activity.Furthermore we designed, synthesized and tested in vitro the set of peptidomimetic inhibitors of Saps, based on the structure of pepstatin A. We have also tested HIV proteinase inhibitors clinically used for inhibitory activity of proteinase.We have developed new method for study of inhibitors of Saps on the agar plates with hemoglobin.The results have shown differences between pathogenic Candida species.These results can provide new information for the methodological progress in Candida diagnosis in clinical work.