16TH INT. CONF. ON YEAST GENETICS AND MOLECULAR BIOLOGY
REGULATION OF PHOSPHOLIPID BIOSYNTHESIS IN YEAST J. Lopes, T. Gill, :M. Nikoloff, J. Ambroziak, M. Swede, K. Hudak, M. Kanipes, Q. Ouyang, N. Bachhawat
Department of Biological Sciences, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, PA 15213 USA Our laboratory is involved in a comprehensive analysis of the coordinate regulation of phospholipid biosynthesis in yeast. Previously, we had shown that a number of phospholipid biosynthetic genes, including the IN02 gene, are regulated in a coordinated fashion at a transcriptional level (Review, M. Nikoloff and S. Henry, 1991, Annu. Rev. Genet. 25: 559433). This regulation occurs in response to the availability of
soluble precursors, including inositol, and in response to the products of the INO2, I N 0 4 and OPIZ regulatory genes. The transcriptional regulation in response to inositol
occurs only if phosphatidylcholine synthesis is ongoing.
We will report recent progress in defining both the cis- and trans- acting elements that are involved in control of the I N 0 2 promoter. In particular, we will report on progress in defining the relationship between the formation of specific protein complexes with DNA sequences found within the I N 0 2 promoter and the in vivo regulation of this gene. We will also report on an extensive analysis of new classes of regulatory mutants that affect I N 0 2 expression, including mutants involved in the response to ongoing phosphatidylcholine synthesis.
0749-503X/92/Spec. ISS. OOOI-01 $05.50 0 1992 by John Wiley & Sons Ltd.
16TH INT. CONF. ON YEAST GENETICS AND MOLECULAR BIOLOGY SI-2
UNIPOLAR CELL DIVISION FILAMENTOUS GROWTH
IN THE YEAST S. CEREVISIAE LEAD TO
B.R. Fink, C.J. Gimeno, P.O. Ljungdahl and C.A. Styles Whitehead Institute for Biomedical Research, Nine Cambridge Center, Cambridge, MA 02142 Diploid S. cerevisiae strains undergo a dimorphic transition that involves changes in cell shape and the pattern of cell division and results in invasive filamentous growth in response to starvation for nitrogen. Cells become long and thin and form pseudohyphae that grow away from the colony and invade the agar medium. Pseudohyphal growth allows yeast cells to forage for nutrients. This mode of growth requires the polar budding pattern of ala diploid cells; haploid axially budding cells of identical genotype cannot undergo this dimorphic transition. Constitutive activation of RAS2 or mutation of SHRS,a gene required for amino acid uptake, enhance the pseudohyphal phenotype; a dominant mutation in RSRl /BUD1 that causes random budding suppresses pseudohyphal growth. Some laboratory strains have lost the ability to make this dimorphic switch and are locked in the unicellular form.
0749-503X192/Spec.ISS. 0002-01 $05.50 0 1992 by John Wiley & Sons Ltd.
16TH INT. CONF. ON YEAST GENETICS AND MOLECULAR BIOLOGY
MOLECULAR ANATOMY OF SACCHAROMYCES CEREVISIAE D. Botstein',
Mulhol and' and D. Preuss2
'Department of Genetics Stanford University School of Medicine, Stanford, CA
2Department of Biochemistry, Stanford University School of Medicine, Stanford, CA
Determination of the intracellular location of a protein is a useful tool in studying its func1:ion.
This has proved to be particularly useful in
yeast, even though localization has generally been accomplished by immunofluorescence microscopy, with a spatial resolution that is quite limited when compared to the small total dimensions of yeast cells. By optimizing conditions, we have been able to apply immunoelectron microscopy to Saccharomyces, with irhe result that we can localize within the location of proteins at the level of ultrastructure. Using immunoelectron microscopy, we have been able to study the morphology of many subcellular structures in yeast, including especially the actin cytoskeleton. the endoplasmic reticulum, the Golgi apparatus and the vacuole.
In each case, we were able
to associate structures visible in the electron microscope with their cognate functions on the basis of the proteins they contain. In each case, the appearance of the organelles was easily reconciled with the patterns of staining seen with immunofluorescence microscopy. Results to be described include ultrastructure of the cortical actin patches in young buds; the distribution through the cell of endoplasmic reticulum and the Gclgi, especially the entry of both kinds of secretory organelles into buds, very early in the cell cycle: and the ultrastructure of the vacuole, which suggests an endocytic role for this organelle.
0749-503X/92/Spec. ISS. 0003-01 $05.50 0 1992 by John Wiley & Sons Ltd.
16TH INT. CONF. ON YEAST GENETICS AND MOLECULAR BIOLOGY s2-1
HISTONES H3 AND H4 REGULATE TRANSCRIPTIONIN YEAST Michael Gnmstein, Jeff Thompson, Lianna Johnson, Randal/ K. Mann, Grace Fisher-Adam, Bo Thomen, Andrew Carmen and Jackson Wan.
Molecular Biology Institute and thc Departmen1of Biology, University of California, Los Angclcs, CA 90024 Iiivtonc H4 contains two domains at its amino tcrminus involved in genetic rcgulation, Residues 4-23 contain a domain required for activation of GAL1 and PH05. Residues 16-29 contain clements necessnry for thc repression of the silcnt mating loci. Mutations in this latter region havc much greater effcctq on the dercprcssion of the silent mating locus HMLa lhan on HMRa. We havc determined that this is due neither to lhe genetic information nt thcsc loci, nor is the main reason the presence of thc addcd ABFl rcgulatory site at HMR. By transposing the teIomerc associated HML and HMR ioci to various chromosomal positions we havc found that therc is a unique interaction k l w c n HMR and the telomcrc which causes silencing of HMR and which is capable of overiding silencing defects in histone H4 mutations. In contrast to the effecu of histone H4 on genelic rcgulation, histono H3 which forms a telrnmcr with H4 in the nucleosome, is hardly involved in silencing of Ihc mating loci. Nor is its amino terminus rcquired for the activalion of GALL Instead, H3 N-tcrminal dclctions hyperactivate GALl. Thcso results suggest that the amino tcrminal tails of H3 and H4 arc involved in unique intcractions with protcin and/orDNA components of thc chromosome thercby regulating promoter function diffcxntially. At present wc BTC attempting to obtain evidence for some of these inlerctions and their effects on promoter chromatin structure.
0749-503X/92/Spec.Iss. ooo4-01 $05.50 0 1992 by John Wiley P Sons Ltd.
16TH INT. CONF. ON YEAST GENETICS AND MOLECULAR BIOLOGY
SUPPRESSION OF TELOMERIC POSITION EFFECT BY INCREASED DOSAGE OF H M L , H M R , OR TELOMERES: IS THERE A HIERARCHY OF SILENCING IN S. CEREVISIAE?
D. Gottschling and M. Mahowald Molecular Genetics & Cell Biology, University of Chicago, 920 E. 58th. Chicago, IL 60637, USA. Cytological observations in many organisms indicate that chromosomal regions are condensed to different extents. These regions may be divided into two maih types: euchromatin and heterochromatin. Euchromatin is where most gene transcription occurs In heterocbromatin, little gene and exists in a condensed state only during mitosis. expression is observed and the chromatin is condensed throughout the cell cycle. Heterochromatin can he further subdivided into a and fi types, which correspond to different degrees of chromatin compaction. Heterochromatin has two additional characteristics: it replicates late in S phase, and exerts a position effect on euchromatic genes that are placed near or within the heterochromatic region. The position effect is manifested as transcriptional repression of the gene. In S. ccrrvisiae three loci possess heterochromatic traits: telomeres and the silent mating type cassettes, H M L and H M R . A number of components are shared between telomeres and the H M loci. Each silent locus shares a cis element, the RAP1 binding site. In trans, each locus requires SIR2, SIR3, SIR4. NATI. A R D I . and HHF2 (histone H4) for complete silencing. Thus a common mechanism of silencing is likely at work at all three loci. However, there are several differences between the loci. For instance, S I R 1 plays no apparent role in silencxng at telomeres, yet it is required for complete silencing at the H M loci. These and other results led us to suggest that telomeres exhibit a basal level of transcriptional repression, and that silencing at H M L and H M R is based on the same mechanism(s), but is strengthened and regulated by additional elements [Cell 66: 12791. In order to further explore the relationship between telomeres and the H M loci, an assay was designed to test whether: 1. there were limiting amounts of silencing factor(s) present in the cell. and 2. what the relative affinity of each of the position effect loci was for these silencing factor(s). The rqlative degree of silencing upon a telomere-linked gene was examined in cells in which extra copies of H M L . HMR, or telomeres were introduced. A plasmid vector (FAT303) was made which could be maintained at either -20 copieskell (high copy) or at -200 copies/cell (very high copy). Three derivatives of FAT303 were made: a linear version to which telomeric DNA sequences were ligated on the ends to generate L-FAT303, and two circular versions that contained either H M L (FATHML) or H M R (FAT-HMR). Each plasmid was transformed into strain UCC1001. This strain has U R A 3 adjacent to the VII-L telomere, and thus U R A 3 serves as a marker for the telomeric position effect. Approximately 0.30 of the cells in a population of UCClOOl are 5FOAR. When FAT303 was present in UCC1001, there was no change in the level of 5-FOAR at either high or very high copy levels of the plasmid. A modest suppression of telomeric silencing was observed when extra telomeres were introduced into the cells; at very high cells were 5-FOAR. Greater suppression was seen for extra copy levels of L-FAT303, cells were 5-FOAR with high copy levels and were 5-FOAR with copies of H M L : very high copy levels. Greatest suppression resulted from increased dosage of H M R : -10-3 were 5-FOAR with high copy FAT-HMR and when at the plasmid was at very high COPY. These results suggest silencing factor(s) are in limiting amounts within the cell, and that they may be competed away from the telomeric domain. In addition, the H M R locus has the greatest affinity for these factor(& telomeres the least, and H M L an intermediate affinity. These experiments provide a groundwork for dissecting the shared and distinct mechanisms of transcriptional silencing within the yeast genome, which may define different classes of heterochromatin.
0749-503X192/Spr. ISS. 0005--01 $05.50 0 1992 by John Wiley & Sons Id.
16TH INT. CONF. ON YEAST GENETICS AND MOLECULAR BIOLOGY S2-3
In Vivo Function of a Golgi-Specific Protein Transport Factor in Yeast
V. A. Bankaitis
Department of Cell Biology, University of Alabama at Birmingham, Birmingham, Alabama, USA The SEC14p is the yeast phosphatidylinositol (PI)/phosphatidylcholine(PC) transfer protein and this polypeptide plays an essential role in stimulating yeast Golgi secretory function. Genetic analysis of SEC14p function have demonshated that the SEC14p functions to alleviate the inherently toxic effects in Golgi membrane function caused by PC synthesis via the CDPcholine pathway. Biochemical analysis of the phospholopid content of highly enriched yeast Golgi membranes indicated that wild-Golgi membranes exhibited rather high PYPC ratios relative to bulk membranes and that, under conditions of SEC14p dysfunction the PYPC ratio of dysfunctional Golgi membranes markedly dearas& The cumulative data are interpreted to indicate that the SEC14p functions, perhaps in a sensory capacity, to maintain an appropriate PYPC content in Golgi membranes that is a critical determinant of the secretory competence of these membranes.
0749--503X/92/Spec. Iss. ooo6-01 $05.50 6 3 1 9 9 2 by John Wiley & Sons Ltd.
16TH INT. CONF. ON YEAST GENETICS AND MOLECULAR BIOLOGY
PROTEIN SORTING TO MITOCHONDRIA COMPONENTS AND PATHWAYS Walter Neupert, Thomas Sollner, Hans Koll, Roland Lill, Nikolaus Pfanner Institut fur Physiologische Chemie, Universitat Miinchen, Germany We characterize the mitochondrial machinery for the import of cytosolically synthesized precursor proteins. The precursor proteins carry signal sequences that are often present in aminoterminal presequences. Two receptor proteins of 19 kd and 72 kd, termed MOM19 and MOM72 (MOM = mitochondrial outer membrane), were identified in the mitochondrial outer membrane. After interaction with the receptors, the precursor proteins are inserted into and translocated across the outer membrane at the so-called general insertion protein/pore (GIP). GIP seems to be formed by at least four proteins, termed MOM7, MOM8, MOM30 and MOM38, and is present in a high molecular weight complex together with the two receptors (mitochondrial receptor complex) (1). Preproteins are transported from the outer membrane to the inner membrane at sites where both membranes are in close proximity (translocation contact sites). By accumulation of translocation intermediates in vital yeast cells, we demonstrated that protein import occurs via translocation contact sites in vivo and moreover thai preproteins have to be in a not fully folded ("unfolded") conformation to be competent for membrane translocation in vivo (2). Transport from the outer membrane to the inner membrane does not occur through a sealed continuous channel. The preproteins are exposed to the intermembrane space. We propose a dynamic model in that the import machineries of the outer and inner membranes are transiently connected during transfer of preproteins (translocation contact sites) (3). The machineries of both membranes can function independently of each other. A typical example is the import pathway of cytochrome c heme lyase. This preprotein uses the receptor MOM19 and GIP to reach its functional location in the intermembrane space, but does not require the inner membrane import machinery (4). Many proteins that are destined for the mitochondrial inner membrane or intermembrane space are first translocated into the matrix with the help of the heat shock protein hsp70 (matrix-hsp70). The proteins then interact with hsp60 that possesses antifolding activity to prevent premature folding of the proteins in the matrix (5). Thus hsp60 has a dual function: prevention of folding of proteins that are retranslocated to the inner membrane (mediated by a prokaryotic-type export signal), and folding of proteins that functionally end up in the matrix (and lack the hydrophobic export signal). (1) Sollner, T., Rassow, I., Wiedmann, M., SchloBmann, J., Keil, P., Neupert, W., Pfanner, N. (1992). Nature 355, 84-87. (2) Wienhues, U., Becker, K., Schleyer, M., Guiard, B., Tropschug, M., Horwich, A.L., Pfanner, N., Neupert, W. (1991). J. Cell Bid. 115, 1601-1609. (3) Pfanner, N., Rassow, J., van der Klei, I.J., Neupert, W. (1992). Cell 68, in press. (4) Lill, R., Stuart, R.A., Drygas, M.E., Nargang, F.E., Neupert, W. (1992).EMBO 1. 11,449-456. (5)Koll, H., Guiard, B., IRassow, J., Ostermann, J., Horwich, A.L., Neupert, W., Hartl, F.-U. (1992). Cell 68, in press.
0749-503X/92/Spec.ISS. 0007-01 $05.50 0 1992 by John Wiley & Sons Ltcl.
16TH INT.CONF.ON YEAST GENETICS AND MOLECULAR BIOLOGY S3-2
EXPLORING GENES THAT ARE REQUIRED FOR VACUOLAR MORPHOGENESIS AND VACUOLAR ACIDIFICATION IN S-ACCHAROMYCESCEREVISIAE* Yasuhiro Anraku Department of Biology, University of Tokyo, Hongo, Tokyo 113, Japan The fungal vacuole is an acidic compartment and plays indispensable roles in metabolic storage and in cytosolic ion and pH homeostasis,in addition to functioning in endolytic macromolecular degradation like the phagocytotic animal lysosome. Dufing the last ten years, it has become known that a new, distinct class of H -translocating ATPase exists ubiquitously in vacuolysosomal organelles and archaebacteria. The vacuolar H -ATPase of the yeast 5. cerevisiae is a DCCD- and bafilomycin Al-sensitive electrogenic proton pump that generates a proton motive force of 180 mV, inside positive and acidic, in the vacuolar membrane vesicles. Current studies from our laboratory have-shown that the vacuolar membrane of yeast is equipped with two distinct C1 transport systems, which each contributes to the formation of a chemical gradient of protons across the vacuolar membrane by shunting the membrane potential generated by the H -ATPa$e. Vacuolar acidifi$at$on is a prerequisite fvr operation of amino acid/H -antiporters, a Ca /H antiporter, and a K -channel. In the case where this ability of acidification is lost, vacuolar protein transport and non-specific fluid phase endocytosis are considerably affected. We have suggested that the large volume of the vacuole probably endows it with a high capacity for these chemiosmotic work that direst ionic homeostasis in the cytoplasm. Thus, the mechanisms underlying establishment of the vacuolar morphology are another important prerequisite for expression of the vacuolar function. To identify the genetic components involved in acquisition and maintenance of large vacuoles, we developed genetic methods to detect mutations that affect normal vacuolar morphogenesis: Mutants with a defect in vacuolar morphogenesis were enriched by treatment with an acido trophic reagent, chloroquine, or screened for pigmentation of endogenous ade fluorochrome. The vam mutants (for vacuolar morphology) were isolated by cytological screeningusing light microscopy. By genetic analyses 18 alleles were identified, which were further classified into 9 complementation groups and two classes. The class I mutations ( v a m 1 , ~ , 8 , ~show ) pleiotropic phenotypes: The mutants contain a few small vesicles, have defects in maturation of vacuolar marker proteins, and are sensitive to 100 mM CaC12 or a temperature of 37OC. The class I1 mutants (vam2,3,f1,5,1) contain numerous small vesicles and mature forms of the vacuolar marker proteins, and do not show any apparent growth defect in the presence of 100 mM CaC12 or at 37OC. The VAM5 and VAM7 genes encode a polypeptide of 691 and 316 amino acid residucrespe-ely. Disruption of either or was not lethal and a mutant with vam5 vam7 null mutations has neither prominent vacuolar structures nor mature forms of vacuolar marker proteins. The yeast vacuolar Hi -ATPase contains at least 9 subunits. Nine&V genes (for vacuolar Fembrane ATPase) have proven to be essential for expression encode periof the enzyme activity: VMAl, VMAZ, VMAl3, VMA5, VMAC and pheral subunits with a calculated molecular mass of 67.7, 57.7, 54.4, 42.3, 36 and 26.6 kDa, respectively; and VMAll encode proteolipids (16.4 and 17.0 kDa, respectively) and VMA12 encodes an integral 25.3 kDa polypeptide. All the chromosomal VMA-disrupted mutants show a Pet cis phenotype (they cannot grow on YP-glucose plate containing 100 mM CaC12 and YP-plates containing nonfermentable carbon sources such as glycerol and succinatq ) and have defects of vacuolar acidification in vivo and ATP-dependent Ca 'uptake into isolated vacuoles. Cell biological issues regarding biogenesis of the ATPase will be discussed. (*supported in part by a Grant-in-Aid for Scientific Research on Priority Areas from the Monbusho, Japan).
0749-503X/92/Sps Iss. ooO8-01 $05.50 0 1 5 9 2 by John Wiley & Sons Ltd.
16TH INT. CONF. ON YEAST GENETICS AND MOLECULAR BIOLOGY
s3 -3 The Role of the BET and BOS Gene Products in ER to Golgi Transport
S. Ferro-Novick, G. RossI., A. Newman, Y. Jiang, J. P. Lian, M. Groesch, P. Mancini and K. Kolstatl
Department of Cell Biology, Yale University School of Medicine, New Haven. C T
06510, USA We have identified several new genes, BETandBOS, whose-producrsare required for transport from the ER to the Go@ complex in the v a t Sacchammyces cerevisiae. BETI genetically interacts with BOSI and SEC22, another gene wnose product mediates transport at this stage of the pathway. DNA sequence analysis has revealed that BETI, BOSI,and SEC22 encode small hydrophilic proteins that conrain a hydrophobic stretch of amino acids that are potentially membrane spanning. BETI and SEC22 are structurally similar to synautobrevin,a constiruent of synapcic vesicies, and compensate for the loss of Yptl function when overexpressed. We are utilizin,~an in vim assay that reconstitutes ER to Golgi transport to determine the role of the Bet and Bos proteins. These in vim, studies have shown that Bosl is required at a late step in ER to Golgi transport,subsequent to the formation of cyrier vesicles. Betl and Bosl appear to reside on the vesicles as well as the ER membrane. but they are not associated with the Golgi compartment. Thus, when the transpm vesicles fuse with the Golgi complex, these proteins are either recycled back to the ER or rapidly degraded. Betl, Bosl, and possibly Sec22, may be components of a membrane-bound complex that interacts with cytosolic factors required for nanspon. Recently, we have identified several new genes (BOS2, BOS3, and BOS4) whose products genetically interact with both BET1 and SEC22. Transport vesicles may 'bedirected to their appropriate target membrane by GTP-binding proteins such as Yptl. Recently, we have shown that the BET2 gene encodes a geranylgeranyltransferasethat modifies the cytoplasmicform of Ypt 1, enabling it to attach to membranes. Currently, we are characterizing this prenyltransferaseand determining the role of isoprenylation in membrane naffic.
0749-503X/92/Spec.ISS. ooo9-01 $05.50 0 1992 by John Wiley & Sons Ltd.
16TH INT. CONF. ON YEAST GENETICS AND MOLECULAR BIOLOGY s4- I
REGULATION OF SEXUAL DIFFERENTIATION IN SCHIZOSACCHAROMYCES POMBE
M. Yamamoto Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113, Japan Nutritional starvation, particularly that for nitrogen, induces sexual development in the fission yeast Schizosaccharomyces pombe. Many genes are regulated in an organized manner during this differentiation process. I will discuss how nitrogen starvation induces concerted gene expression that commits & pombe cells to sexual development, focusing on the following points. 1) Nitrogen starvation results in a decrease in the intracellular cAMP level, which in turn functions as a signal for induction of the genes that are essential for mating or meiosis. 2) Expression of stell, which depends on nitrogen starvation and a concomitant decrease in the CAMP level, is necessary and sufficient for transcriptional activation of several other nitrogen starvation-responsive genes and results in induction of sexuBl development. The stell gene encodes an HMG-box protein that binds to TTCTTTGTTY, a cis element commonly seen in the upstream regions of nitrogen starvation-responsive genes. The stell gene product hence appears to be a key transcription factor that controls gene expression required for initiation of mating and meiosis. 3) The ste6 gene, which is a homologue of S, cerevisiae CDC25 and encodes an activator of Ras protein, is also regulated by stell. Thus, nitrogen starvation leads to activation of Ras via which is essential for the pheromone recognition induction of pathway to transmit the signal properly. 4) Two genes encoding alpha-subunits of G protein have been cloned. One, named g p a l , is essential for mating, and its gene product apparently couples to both the P-factor receptor (Mam2) and the M-factor receptor (Map3). In contrast, loss of function of the other, gpa2, promotes mating and sporulation in rich medium, like loss of function of cyrl coding for adenylyl cyclase. Evidence indicates that the gpa2 gene product is indeed involved in determination of the cAMP level in fission yeast cells, probably by regulating the activity of adenylyl cyclase. This is a curious observation because mammalian adenylyl cyclase, which is regulated by G proteins, is very different from the S . pombe enzyme whereas S. cerevisiae adenylyl cyclase, which shares homology with the S, E m b e one, is regulated by Ras protein rather than G protein.
0749-503X/92/Spec.Iss. 0010 -01 $05.50 0 1992 by John Wiley & Sons Ltd.
16TH INT. CONF. ON YEAST GENETICS AND MOLECULAR BIOLOGY
Regulation of cell cycle progression in fission yeast S. L. Forsburg, S. Moreno, T. Enoch and P. Nurse ICRF Cell Cycle Group, Biochemistry Department, Oxford University. The cell cycle consists a period of DNA replication (S phase) and Mitosis (M phase), separated by gaps in which growth occurs (GI and G2). Regulation of these events must be precise, in order to maintain the integrity of the cell and its genetic material. The decision to undergo a complete cycle of division is influenced by numerous signals, including cell size, available nutrients, and presence of signalling molecules such as growth factors or pheromones. Once in the cycle, various mechanisms must operate to ensure the dependency of M on S, and S on M, is maintained. Progression through the cell cycle in fission yeast may be regulated at two transition points: one at the GI/S boundary, called START, and the second at the G2/M boundary. Both these transitions require the function of the p34Ck2 protein kinase, functional homologues of which have been identified in many eukaryotes. Fission yeast regulates its cycle chiefly at the G2/M transition under laboratory growth conditions, and the regulation of the p34cdc2 protein khase at this point in the cycle has been extensively studied. We are particularly interested in elements that control entry into the cycle and maintain its temporal order in fission yeast. We have identified several new gene functions involved in this regulation of cell cycle progression. The first of the genes we have identified is pucl+ @ombe unusual cyclin), a homologue of the budding yeast S. cerevisiue G1 cyclins. pucl+ was isolated by complementation in budding yeast. Evidence for its role as a cyclin in fission yeast will be presented. The second gene, ruml + (replication uncoupled from mitosis), w.as identified in a screen for over-replication. Overexpression of ruml + causes multiple rounds of DNA replication. Finally, a screen for mutants deficient in the checkpoint control that ensures S phase is complete before M phase begins identified several hus (hydroxyurea sensitive) genes. Characterisation of these genes and discussion of their role in the fission yeast cell cycle will be presented.
0749-503X/92/Spec. IS. 001 1-01 $05.50 0 1992 by John Wiley & Sons Ltd.
16TH INT. CONF. ON YEAST GENETICS AND MOLECULAR BIOLOGY
CELL-CELL INTERACTIONS INVOLVED IN MATING IN SACCHAROMYCES C€R€V/S/AE
bL!wal Department of Microbiology and Molecular Genetics, Given Building, University of Vermont, Burlington, VT 05405 U.S.A. Two types of cell-cell interaction are involved in the mating of a and a cells to produce the a/a diploid in the yeast Saccharomyces cerevisiae. One interaction mediates adhesion between a and a cells to produce cellular a gregates. A direct interaction between cell wall glycoproteins expressed by a and a cells, called a- an61a-agglutinin, respectively, results in this adhesion. The second interaction involves the secretion of the pheromones a-factor and a-factor by a and a cells, respectively. Each of the pheromones binds to a receptor present on the opposite mating type and elicits responses that are essential for mating. The genes that encode the agglutinins were obtained by first isolating agglutination-defectivemutants and cloning the genes by complementation. These genes are being used to study two aspects of agglutinin function, cell-surface attachment and binding to the opposite agglutinin. a-agglutininin is composed of two 0-glycosylated polypeptides, a binding subunit and a core or cell-surface attachment subunit. AGA7 encodes the core subunit, and AGA2 (identified by W. Tanner) encodes the binding subunit. Analysis of these genes should allow the examination of the interaction between the agglutinins that mediates adhesion. a-agglutinin is composed of a single N-glycosylated polypeptide, which is encoded by the A& 7 gene. Preliminary evidence suggests that a phosphatidyl inositol glycan (GPI) anchor is involved in localization and attachment of a-agglutinin to the cell wall. A& 7 contains a hydrophobic C-terminus, which is a prominent feature of the precursors of eukaryotic proteins that are linked to the plasma membrane by GPI anchors. A& 7 truncations that remove this hydrophobic C-terminus or replace it with a hydrophilic sequence result in secretion of a-agglutinin, indicating that this region is involved in cell-surface anchorage. The secretion of truncated proteins that retain a-agglutinin binding activity provides a convenient assay to characterize the binding domain of a-agglutinin, i.e. the domain that binds a-agglutinin. Analysis of truncations indicate that the C-terminal boundary for the binding domain is between amino acids 280 and 350 of the 650 amino acid open reading frame. The region between amino acids 210 to 310 shows proposed structural features and matches to the consensus sequence for V-type immunoglobulinfold domains, which are present in many mammalian adhesion proteins, suggesting that this structure feature is a highly conserved feature of adhesion proteins. Reponse to pheromone is mediated by a G protein homologous to the G proteins involved in wellcharacterized mammalian signal transduction pathways.The SCGl (also called GPA 7). STE4, and STE78 genes encode homologs to the a, D, and y subunits of mammalian G proteins. Scgl plays a negative role and Ste4 and Stel8 play positive roles in the pheromone response pathway. The current model for the action of these components is that fly acts downstream of a to activate a currently unidentified effector in the presence of pheromone. In the absence of pheromone, a binds to fly to keep the pathway inactive, consistent with its negative role. Identification of the effector in the pathway is an important area of inquiry. Mutations in SCGl can result in either of two opposite phenotypes, constitutive activation of the pheromone response pathway (similar to the null phenotype), resulting in defects in growth and cellular morphology, or defects in pheromone response and mating. Mutations in the putative guanine nucleotide binding domain should help elucidate functional aspects of this protein. Several C-terminal mutations result in defects in pheromone response and mating, consistent with the proposed role of the C-terminus in receptor interactions suggested by experiments with mammalian G proteins. One particular mutation results in pheromone response and mating defects in a cells but only a mild effect in a cells. The G protein is proposed directly with the two non-homologouspheromone receptors, Ste2 and Ste3; the differential effect of this mutation in the two mating types suggests that the specificity for the interactions with the two receptors may be different. These and other mutants should be useful in investigating interactions between components of the system.
0749 -503X/92/Spec. Iss. 0010 -0 1 $05.50 0 1992 by John Wiley & Sons Ltd.
16TH INT. CONF. ON YEAST GENETICS AND MOLECULAR BIOLOGY
PROTEIN SERINE-THREQNINE PHOSPHATASES AND CELL CYCLE CONTROL IN SACCHAROMYCES CEREVISIAE Stark, M. J. R. Department of Biochemistry, The University, Dundee. DDl 4HN. U.K. Evidence from a variety of eukaryotic systems has revealed that protein serindthreonine phosphatases play critical roles in the regulation of events in the cell cycle. We have therefore commenced studies of such activities in Succhuromyces cerevisiue to determine their roles in budding yeast cell cycle execution and regulation. S. cerevisiae contains a single essential gene (DIS2 ) encoding protein phosphatase 1 (PPl). Overexpression of this gene from the GAL promoter is highly detrimental to the cell and causes a range of aberrant morphological changes, culminating in the accumulation of very large unbudded cells after prolonged induction. In fission yeast, an R+Q mutation at position 245 results in a cold-sensitive defect in chromosome disjunction [l]. We have made the corresponding mutation in the budding yeast gene. While the mutant allele fails to complement a dis2::LEUZ knockout in single copy, when present on a multicopy plasmid in a wild-type strain it leads to a severe growth defect. Expression of either of the PP2A genes  of budding yeast (PPHZI, PPH22 ) from the GAL promoter is also detrimental to growth and l a d s to a phenotype with most cells showing elongated buds. The cbrresponding R+Q mutation in either gene also fails to provide PP2A function. In contrast to fission yeast , multiple copies of this mutant allele failed to produce a cold-sensitive phenotype in wild-type strains. However, a PPH2I pph22A strain carrying multiple copies of the PPH21 R+Q mutant arrested proliferation at lower growth temperatures. Cells relying on a copy of PPH2l lacking the first 16 codons showed no obvious defects, indicating that the amino-terminal region of the product is dispensable. The two PP2A genes have been tagged with an epitope from SV40 large T antigen, enabling detection of their products in the cell. A chimeric gene comprising part of the IgG-binding region of S. uureus protein A and CMDl (the S . cerevisiue calmodulin gene) has been constructed and the product (ProtA-CaM) expressed in bacteria. ProtACaM has been used to isolate genes encoding Ca2+/calmodulin-binding proteins by screening ligtll yeast expression libraries . Initially three calmodulin target protein genes, namely CMK2 (encoding a calmodulinregulated protein kinase) and CMPI , CMP2 (encoding protein phosphatase 2B (PP2B) catalytic subunits) and were isolated. The products of these genes interact with ProtA-CaM in a Ca2+-dependent manner but fail to interact with the corresponding ProtA-CaM expressed from a fusion gene constructed with cmdl-3 , whose product does not bind Ca2+. However, since cmplA cmp2A strains are viable, PP2B activity appears to be dispensable under normal growth conditions. The isolation of these genes demonstrates the utility of the approach for identifying genes iencoding additional calmodulin-regulated activities. Protein A-PP1 fusions have been used to identify polypeptides which interact with the DIS2 product using a similar approach. Budding yeast also carry additional genes encoding protein phosphatases with sequences related to PPl and PP2A. The products of several such genes have large amino-terminal domains which are distinct from the protein phosphatase catalytic region and which may target or regulate the activity of the latter domain. [l] Ohkura, H., Kinoshita, N., Miyatani, S., Toda, T. and Yanagida, M. (1989). Cell 5 7 , 997-1007.  Sneddon, A., Cohen, P. T. W. and Stark, M. (1990). EMBO J. 9,4339-4346.  Kinoshita, N., Ohkura, H. and Yanagida, M. (1990). Cell 63,405-415.  Stirling, D, Petrie, A., Pulford, D, Paterson, D. and Stark, M (1992). MolMicrobiol 6, 703-713.  Geiser, J., van Tuinen, D., Brockerhoff, S, Neff, M. and Davis, T. (1991). Cell 65,949-959. 0749-503X/92/Spe~. ISS. 0013-01 $05.50 0 1992 by John Wiley & Sons Ltd.
16TH INT. CONF. ON YEAST GENETICS AND MOLECULAR BIOLOGY S5-1
DEVELOPMENT OF CELL POLARITY IN SACWMJ?OHYCES CgREVIsIAB
J. R. Pringle
Department of Biology, University of North Carolina, Chapel Hill, North Carolina, 27599, USA In Saccharomyces cerevisiae, both the mitotic cell cycle and mating involve the development of cell polarity. Recent studies in several laboratories have elucidated some aspects of this process. Polarity establishment during budding appears to involve at least three stages, namely the selection of the budding site, the organization of that site and establishment of an associated axis of cytoskeletal polarity, and the eventual localized growth of the cell surface to produce the bud. Numerous components involved in each stage have now been identified, primarily by application of a variety of genetic strategies. These components appear to define a morphogenetic hierarchy. At each level in the hierarchy, low molecular weight GTPbinding proteins and the factors that modify them or modulate their activity appear to play central roles; it appears that the switching of these proteins from the GDP-bound to the GTP-bound form facilitates the next stage in the assembly of the appropriate complexes. This presentation will focus on five issues: (1) Is the concept of a morphogenetic hierarchy valid? (2) If so, what is the nature of the communication between the different levels in the hierarchy? (3) What role(s) do the GTP-binding proteins play in this communication? (4) How are the cytoskeletal components ultimately recruited into the approprite organization? (5) What is the ultimate source of the positional information governing the choice of the next bud site?
0749-503X/92/Spef.Iss. 0014-01 $05.50 0 1992 by John Wiley & Sons Ltd.
16TH INT. CONF. ON YEAST GENETICS AND MOLECULAR BIOLOGY
PROTEIN GLYCOSYLATION, AGGLUTININS AND THE CELL CYCLE W. Tanner, Lehrstuhl fur Zellbiologie und Pflanzenphysiologie, Universiat Regensburg, Universiatsstde 31, 8400 Regensburg, FRG Glycosylation of proteins is the most extensive and complex protein modification known. Why proteins are glycosylated, however, remains essentially a great enigma. When protein N-glycosylation is prevented in yeast cells, they arrest in a pseudo G1-state: nonbudding cells with a G2 nucleus (1,2). Thus, protein N-glycosylation Seems to be required in some way for bud initiation and for the transition from G2 to mitosis. Protein 0-glycosylation in fungi proceeds by a special pathway not found in higher eucaryotes. The key enzyme is an ER membrane protein that transfers mannosyl residues cotranslationally from Dol-P-Man to seryl/threonyl residues of nascent proteins. To see whether this protein modification is t:ssential, the mannosyl transferase has been purified (3), and via peptide and oligonucleotide sequences the corresponding gene has been cloned; gene disruptions are in progress. Two cell surface glycoproteins, the synthesis of which is induced by mating pheromones, are responsible for mating type specific agglutinations. The two proteins have been purified to homogeneity and using peptide information the corresponding genes have been cloned (4-6). The a agglutinin is a small, solely 0-glycosylated protein (4) which is attached to a cell wall core protein (7) by S-S linkage. The a agglutinin is a large highly N-glycosylated protein. For the interaction of the two isolated proteins an in vitro test has been worked out; the proteins interact with a 1: 1 stoichiometry and His273 of a agglutinin is essential. And the carbohydrates?
1. F. Klebl, T. Huffaker, 'W. Tanner. Exp. Cell Research 309-313, 1984 2. M. Vaj, L. Popolo, L. .4lberghina. Exp. Cell Research 171,448-459, 1987 185-190, 1991 3. S. Strahl-Bolsinger, W. Tanner. Eur. J. Biochem. 4. M.Watzele, F. Klis, W. Tanner. EMBO J. 5, 1483-1488, 1988 5. K. Hauser, W. Tanner. FEBS Letters m, 290-294, 1989 6. C. Cappellaro, K. Hauser, V. Mrsa, M. Watzele, G. Watzele, C. Gruber, W. Tanner. The EMBO Journal 4081-4088, 1991 A. Roy, C.F. Lu, D.L. Marykwas, P. Lipke, J. Kurjan. Mol. Cell. Biol. 11,41967. 4206, 1991
0749-503X/92/Spe~. ISS. 0015-01 $05.50 0 1992 by John Wiley & Sons Ltd.
16TH INT. CONF. ON YEAST GENETICS AND MOLECULAR BIOLOGY S6- 1
YEAST SPINDLE POLE BODY COMPONENTS. J.V.Kilmartin and A.Donaldson. MRC Laboratory of Molecular Biology, Hills Rd., Cambridge, CB2 2QH, U.K. Four components of the yeast (S. cerevisiue) spindle pole body (SPB) have been identified by preparing monoclonal antibodies against enriched SPBs, these have apparent molecular weights of 42,85,90, and 110kD. All localise to different and discrete parts of the SPB by immunoelectron microscopy. Genes for the 110 and 42kD components have been cloned and sequenced and are called SPBllO and SPB42 respectively. Both are essential genes, SPB42 shows no significant homology to other sequences, SPBllO shows homology to proteins with coiled-coil structure for about 6OkD of it's central part. Both genes have MluI sites in their promoter regions, suggesting that transcription of these SPB genes is regulated in the same way as some genes involved in DNA synthesis. These all have one or more MluI sites in their promoters which increase transcription at the GI/S boundary, this is the point in the cell cycle where the SPB is assembled suggesting that SPB genes could also make use of this control system. Preliminary Northern blots of SPBllO transcripts do show cell cycle periodicity with a peak at G1/S. The role of this periodic transcription in the regulation of SPB assembly is currently being investigated. We are also investigating the function of the individual SPB components in the SPB structure. SPBIIO localises to the nuclear side of the SPB between the central plaque which is in the plane of the nuclear envelope and the more ill-defined inner plaque from which the nuclear microtubules emerge. In SPBs extracted with DEAE-dextran there appear to be filaments connecting the two plaques, since SPBI 10 may have a coiled-coil domain long enough to bridge this gap and localises to this position, it might be forming these filamentous structures. We decided to test this by replacing the normal SPBllO gene with truncated versions containing normal N- and C-termini but with varying amounts of the central possible coiled-coil domain removed. We found that deletions of up to 60kD of the central domain could substitute for the normal SPBllO protein and electron microscopy of SPBs from this strain showed that microtubules, instead of initiating at a distance from the central plaque as in normal cells, now appeared to initiate directly from the central plaque. This suggests that SPBllO acts as part of a spacer element in the SPB.
0749-503X/92/Spec. Iss. 0016-01 $05.50 0 1992 by John Wiley & Sons Ltd.
16TH INT.CONF. O N YEAST GENETICS AND MOLECULAR BIOLOGY
ANCHORS AND WINCHES: THE SPINDLE POLE BODY AND KINESIN-RELATED PROTEINS IN YEAST MITOSIS AND MATING M.D. Rose, E.A. Vallen, P.B. Meluh, and D. Roof Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544 U.S.A. The spindle pole body (SPB) is the sole microtubule organizing center in yeast. In order to dissect the functions of the SPB and microtubules, we have been examining the func:tions of several yeast genes required during nuclear fusion. These and related genes are also required during various steps in mitosis.
KARl is required for both nuclear fusion and SPB duplication and these two functions are mediated by separate domains of the KARl protein. An internal region required only for SPB duplication will target hybrid proteins to the SPB. This same region interacts genetically with another protein required for SPB duplication, CDC31. CDC31 is a calmodulin-relatedprotein whose closest relative is caltractin, a component of the Chlamydomonas basal body. The region of KARl required for nuclear fusion may interact with KAR3, a kinesin-relatedproteir.. Both nuclear fusion and SPB duplication require the hydrophobic carboxyl terminus of KARl, which appears to act as a membrane spanning domain for the nuclear envelope. In total, we have identified four kinesin-relatedgenes in S. cerevisiae. KAR3 is essential for nuclear fusion and meiosis, but only partially required for mitosis. Using the homology between KAR3 and kinesin, we have isolated two more genes, KIPl and KIP2. Although both proteins are expressed in yeast, single mutations in KIPl or KIP2 have no phenotype. Using a plasmid loss strategy, we have isolated mutations that are lethal only in combination with mutations in KIPl or KIP2. One gene identified in this manner, KSLP, is identical to CINB, another kinesin-like protein involved in chromosome segregation (A. Hoyt). Using temperature sensitive mutations in KIPl and a null mutation in CINB, we (and Andy Hoyt's laboratory) have shown that these two genes are required for separation of the SPB's early in mitosis. Epitope tagging experiments localized KIPl protein to the nuclear microtubules during SPB separation, prior to spindle elongation. During mating, KAR3 is required for the function of the cytoplasmic microtubules, but during mitosis, KAR3 is important for nuclear spindle elongation. The difference between these two activities correlates with a shift in the subcellular localization of KAR3 protein, from the nucleus in vegetative cells, to the cytoplasm of mating cells. The shift in localization occurs as part of the r,esponseto mating pheromone and is not solely the result of cell cycle arrest. The molecular nature of the shift will be discussed.
0749-503X/92/Spe~. ISS. 0017-01 $35.50 0 1992 by John Wiley & Sons Ltd.
16TH INT. CONF. ON YEAST GENETICS AND MOLECULAR BIOLOGY S6-3
THE MEMBRANE CYTOSKELETON OF BUDDING YEAST AND ITS ROLE IN MORPHOGENESIS
D.Drubin, D. Holtzman, H. Jones, T. Lila, A. Moon, D. Vinh, M. Welch, K. Wertman Department of Molecular and Cell Biology, 455 Life Sciences Addition, University of California, Berkeley, California 94720 In budding yeast, asymmetric localization (or activation) of cell surface proteins, including enzymes involved in cell wall remodeling and (in mating cells) pheromone receptors and adhesion molecules, is required for polarized growth and mating. By analogy to more complex eukaryotes, these proteins might be localized to specific regions of the cell cortex by transmembrane linkages to a membrane cytoskeleton. In yeast, cortical actin structures are concentrated at actively growing cortical regions which in mating cells contain the highest concentration of pheromone receptors and adhesion molecules. Actin and actin-binding protein mutants are defective in development of surface polarity. We have used actin filament affinity chromatography and biochemical assays to identify yeast actin-binding proteins. Genetics of yeast actin and actin-binding proteins performed in a number of laboratories has demonstrated that these proteins are responsible for delivery secretory vesicles to a particular plasma membrane surface so that yeast cells can grow in a polarized manner to form buds or mating projections. There are two kinds of actin structures in yeast, cytoplasmic cables and a membrane cytoskeleton organized as punctate actincontaining foci. Yeast lacking actin cables can grow in a polarized manner, but at a reduced rate. However, yeast defective in polarized assembly of the cortical actin structures are defective in polarized growth. One function of the yeast cortical actin cytoskeleton, therefore, seems to be to spatially organize growth of the cell surface. Yeast actin and the actin-binding proteins cofilin, fimbrin, and Abpl p have all been shown to be concentrated at the growing region of the cell cortex. The ABPl gene encodes a 65 kD protein located at the cell cortex. When overproduced, Abpl p causes actin to assemble inappropriate1 on surfaces of the mother cell rather than exclusively in the bud as is normal or wild type cells. This causes cell surface growth in the mother cell which normally does not grow. Deletion of the ABPl gene was found to have no effect on the polarized growth of the yeast cell. This suggested that yeast might contain a second protein with a function redundant with Abpl p function. A genetic screen to identify mutations that enhance the severity of a deficiency in Abpl p function, identified two SLA genes. sla mutants are defective in assembly of the cortical cytoskeleton. Rather than assembling a dispersed punctate membrane cytoskeleton. a few large actin aggregates appear on the cell cortex even at the permissive temperature. Moreover, at the non-permissivetemperature sla mutants grow isotropically rather than in a polarized manner.
0749-503X/92/Spec.ISS. 0018-01 $05.50
0 1992 by John Wiley
& Sons Ltd.
16TH INT. CONF. ON YEAST GENETICS AND MOLECULAR BIOLOGY
DIVERSE STRATEGIES REGULATING THE PROGRESSION OF MEIOSIS R. Easton Espositol, R. Strich2, R. Suroskyl, S. Honigbergl, R. McCarrolll, L. Buckinghaml, and C. Atchesonl 1 Department of Molecular Genetics and Cell Biology, University of Chicago, 920 E. 58th St., Chicago, IL 60637, U.S.A..
2 Institute for Cancer Resemh, Fox Chase Cancer Center, 7701 Burholme Ave. Philadelphia, PN 191 11, 1J.S.A. The dramatic changes in chromosome behavior that occur during meiosis offer a unique opportunity to investigate the regulatory mechanisms that control both cell division and cell differentiation. In particulx, our laboratory has been addressing the following questions: 1) How is the progression of meiotic events coordinated? 2) By what mechanisms are meiotic functions suppressed during mitotic growth? and 3) How are cell division controls regulated during the meiotic process, and specifically, what functions are involved in "commitment to meiosis'' ?. Our studies thus far have revealed that several regulatory strategies operate during the meiotic process to confer exquisite control over meiotic gene expression. These include transcriptional repression and induction controls that are specific for the various meiotic gene expression classes, a specific regulatory system that causes extremely rapid mRNA turnover for genes expressed early in the process, and probably post-transcriptional modification of particular negative regulators that repress meiotic transcription during mitotic cell division. Finally, we believe that a novel control mechanism,which we have termed "kinetic choice", may drive the commitment process. The evidence for these various regulatory strategies has come principally from studies of the structure, function and regulation of several genes previously identified in our laboratory, that are required either for genetic recombination (SPOI I ) , the meiosis I division ( S P 0 1 3 ) , commitment to meiosis (Sf0 1 4 ) ,or negative regulation of particular meiotic-specific genes during mitotic cell division (UME'I to UME6). The analysis of these genes has demonstrated that both transcriptional induction and repression are mediated by URS 1, a ubiquitous cis-acting element, previously found in a variety of other genes normally expressed during vegetative growth. This element resides upstream 1of most of the early expression class of meiosis-specific genes (e.g. SP013, IME2, HOPI, RELII) except for S P O l l where it is located within the coding sequence. Mutation of the element results in constitutive derepression (-10-fold) which is substantially lower than meiotic induction levels (75-100-fold). Gel retardation studies, and genetic epistatic analysis combining m e lesions with the URSl mutations have shown that the protein products of at least two of the UME genes (UME4 and UME6) interact (directly or indirectly) with this element. Two of the other LIME genes (UME2 and UMES) are required for the rapid degradation of eary meiotic mRNAs. All four of these U M E loci have been cloned, and their sequences have been or are being determined. The functions of the SPOI3 and SP014 genes are of particular interest with regard to the coordination of meiotic events once initiation has occurred. Evidence will be presented that SP014 is involved in commitment to meiosis based upon the finding that mutants in this gene can retum to growth directly from late stages of meiotic development, even after meiosis I and/or meiosis I1 have occurred. Finally, rectmt studies will be presented that demonstrate that the wild type SP013 gene when overexpressed during vegetative growth acts as a cell-cycle-specific negative regulator of mitosis, and a model discussed in which this gene functions in a similar way during meiosis, as a transient inhibitor of the meiosis I1 division.
0749-503X192/Spe~, ISS. 0019-01 $05.50 0 1992 by John Wiley & Sons Ltd.
16TH INT. CONF. ON YEAST GENETICS AND MOLECULAR BIOLOGY S7-2
THE ARG4 HOT SPOT OF MEIOTIC GENE CONVERSION M. Vedel and A. N i c o b B. de Massy, E.RO~CO, Institut de GMtique et Microbiologie,B i t 400, Universit6 Paris-Sud 91405 Orsay Cedex, France. Several genetic and molecular studies strongly suggest that the 5’ region of the ARG4 gene contains an initiation site for meiotic gene conversion (1-3).The lines of evidences are: (a) the studies of the pattern of gene conversion events in this region. It revealed decreasing gradients of frequencies extending on both sides of the ARG4 promoter as the result of a single meiotic event (L3.4). The peak of highest frequency (17% of total meiosis) is located around position -119with respect to the ARG4 translation SM
site (+l); (b) An extensivedeletion analysis which maps a neceSSary cis-acting region to
the promozr of ARG4 between positions -319/-37 (3.5); (c) The identification of a meiotic specific DNA double-strand break (DSB) event occuting in this region during the time of recombination (6-8). in agreement with the DSB rqak model for homologousrecombination (9). Our recent studies concern the functional transplacement of the ARG4 initiator in the yeast
genome an& further c h a r a c h t i o n of meiotic DSBs. To test whether the -319/-37 sequence is sufficient to promote gene conversion in a novel chromosomal context, we inverted on the chromosome different
DNA fragments including this region and the ARG4 coding region. Surprisingly, we found that some inversions result in the loss of the normal recombination properties and associated DSB, due to the presence of flanking transcription overlapping the ARG4 5’ region. The recombinationalactivity and DSB were restored in the presence of a functional transcription terminator upslream of the site. We conclude that the ARG4 initiafor is active only when localized in a non transcribed region and, that the necessary and sufficient cis-acting region is located between positions -4654-37.The functional sigruficance of the location of this initiator of recombination within the entire ARG4DED81 intergenic region and otha chromosomal context effects found during transplacement studies will be discussed. To study the correlation between ARG4 site specific initiation of gene conversion and DSB lesion, we have analysed various deletions of the ARG4 region in addition to the above insertions. We found a good correlation between the high level of gene conversion and the pmence of a detectable meiotic DSB strongly suggesting the role for DSBs in the process of gene conversion at ARM. The identification of the cis acting element(s) that control the occurence of the ARG4 DSB. its frequency and position are under investigation, in parallel to the identification of otha sites of meiotic DSB in the genome. References: 1) Fogel et al.. 1981 The molecular Biology of Yeast (CSH); 2) Petes et al.. 1991
The Molecular and Cellular Biology of the yeast Saccharomyces(CSH); 3) Nicolas et al.. 1989 Name
338.35; 4) Schultes and SzoscaL, 1990 Genetics 126.813; 5)Schultes and Szostalr, 1991 Mol. Cell. Biol. 11.322.6) Sun et al., 1989 Name, 338.87; 7)Sun et al.. 1991 Cell 64,1155;8) Cao et al..
1990 Cell 61. 1089;9) Szostak et al., 1983 Cell 33.25.
0749-503X/92/Spec. ISS. 0020-01 $05.50 0 1992 by John Wiley & Sons Ltd.
16TH INT. CONF. ON YEAST GENETICS AND MOLECULAR BIOLOGY
Chromosome pairing during meiosis in S. cerevisiue. Nancy Kleckner. Department of Biochemistry and Molecular Biology Harvard University, Cambridge, MA. USA. The pairing of and recombination between homologous chromosomes are important and essential features of meiosis. We have developed physical assays for changes at the DNA level that are relevant to these processes and have examined the temporal and functional relationships between these changes and cytologically observable events of meiosis. These observations are compatible with a model, based on a proposal originally made by Oliver Smithies, in which chromosomes initially pair by DNA/DNA interactions at a time when they are relatively decondensed; chromosome condensation then serves to bring homologues into approximate alignment and synaptonemal complex formation occurs thereafter. We have recently identified a new meiosis-specific gene, DMCI, which is a structural and evolutionary homologue of bacterial RecA protein @.K.Bishop et al., in press). Mutations in this gene cause a block in both recombination and SC formation at what would normally be the early zygotene stage and also cause meiosis to arrest. dmcl mutants accumulate site-specific double strand breaks with long 3' single stranded tails, form long axial cores but only a small amount of tripartite SC structure, and arrest prior to separation of SPBs in preparation for meiosis I. DMCl is highly homologous to RADSI, and thus presumably represents a case in which a meiosisspecific gene has evolved from a mitotic DNA repair gene. Observations will be presented regarding the functional relationships among RecA, DMCl, and RADSl, new meiosis-specific mutations that cause an intermediate block in prophase, detection of physical connections between homologues at early stages of meiosis, and the role of cdc28 in control of the meiotic cell cycle.
0749-503X192/Spec,ISS. 002 1-01 $05.50 0 1992 by John Wiley & Son!; Ltd.
16TH INT. CONF. ON YEAST GENETICS AND MOLECULAR BIOLOGY S8-1
SACCHAROMYCES CEREVISIAE CENTROMERE GENETICS P. Hieter, K. Floy, J. Puziss, D. Sears, F. Spencer Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, 725 N. Wolfe St., Baltimm, MD 21205, USA
Proper centromere function requires structural components (CEN DNA and associated proteins) as well as coordination of centromere activity within the meiotic and mitotic cell cycles. We have previously analyzed a large set of CEN DNA mutations for their effects on mitotic segregation using marker chromosome fragments and a visual assay. These CEN mutants have provided useful reagents for developing several genetic strategies to study mitotic and meiotic centromere function. A genetic screen was employed to recover extragenic dosage suppressors of a CEN DNA mutation. Increased dosage of a gene, M C K l , specifically suppressed CEN DNA mutations in CDEIII, but not comparably defective CEN DNA mutations in CDEI or CDEII. MCKI was identified and cloned independently for its involvement in the induction of meiosis (Neigebom and Mitchell) and is identical to a gene that encodes a phosphotyrosyl protein with protein kinase activity (Dailey, et al). Several unique genes that suppress the cold sensitive phenotype resulting from the deletion of MCKI have been isolated. One of these genes, SMCIO, appears to encode a novel protein kinase. Another approach to identifying genes important for centromere function involves transcriptional readthrough by RNA polymerase II. The ability of transcription to proceed through a centromere sequence inserted into the intron of an actin-LACZ fusion gene is used to assess kinetochore structure. A 185 bp wild-type CDEI+II+III sequence effectively blocks production of B-galactosidase; introduction of a point mutation at position 15 of CDEIII allows LACZ expression. CDEIII alone is not sufficient for the transcriptional block. This assay provides a screen for potential kinetochore mutants. A CEN DNA mutation carried on a single chromosome can induce a cell cycle delay observed as retarded mitosis. A 31 base pair deletion within centmmere DNA element II (CDEII ~ 3 1that ) causes chromosome missegregation in only 1% of cell divisions elicited a dramatic mitotic delay phenotype. Single division pedigree analysis of strains containing the CDEII 831 CEN mutation indicated that most (and possibly all) cells experienced delay in each cell cycle and that the delay was not due to increased chromosome copy number. Furthermore, a synchronous population of cells containing the CDEII a31 mutation underwent DNA synthesis on schedule with wild-type kinetics, but subsequently exhibited late chromosomal separation and concomitant late cell separation. We speculate that this delay in cell cycle progression, before the onset of anaphase, provides a mechanism for the stabilization of chromosomes with defective kinetochore structure.
To analyze meiotic centromere function, we have developed a system that utilizes human DNAderived yeast artificial chromosomes (YACs) as marker chromosomes. Since aneuploidy for the YACs does not affect spore viability all classes of meiotic missegregation can be scored in fourviable-spore tetrads including precocious sister separation, meiosis I nondisjunction, meiotic chromosome loss and meiosis 11nondisjunction. Segregation of the homologous pair of marker YACs, each 360kb in length, was shown to occur with high fidelity in the first meiotic division and was associated with a high frequency of recombination within the human DNA segment. Thirteen CEN mutants have been tested for effects on meiotic segregation. CDEII A 3 1 causes a dramatic increase in missegregation of the YAC during meiosis I. In addition, it was observed that gene conversion events occurring several kb from a wildtype CEN segment were associated with high levels of meiosis I nondisjunction and precocious sister separation.
0749-503X/92/Spec. ISS. 0022-01 $05.50 0 1992 by John Wiley & Sons Ltd.
16TH INT. CONF. ON YEAST GENETICS AND MOLECULAR BIOLOGY
CONTROL OF REPLICATION ORIGINS IN YEAST B. J. Brewer, B. M. Ferguson*, M. K. Raghuraman, J. D. Diller and W. L. Fangman Department of Genetics, University of Washington, Seattle, WA 98 195, USA. Present address: Institute of Molecular Biology, University of Oregon, Eugene, OR 97403- 1229, USA. The chromosomes of Saccharomyceserevisiae have a high density of potential replication origins; however, not all origins are equivalent. Some origins are infrequently used and others show differences in their time of activation. We are studying the efficiency of origin activation using two-dimensional(2-D) agarose gels and the time of their activation by Meselson-Stahlexperimentswith synchronouscell cultures. We have identified an origin of replication (ARSSO1) near the right telomere of chromosomeV that is activated late in the S phase. Investigationof the temporal control of ARSSO 1 has led to the conclusion that the late activation is determined by chromosomalcontext (Cell 68 [ 19921333). Three lines of evidence suggest that late activation is a consequence of proximity to the chromosome end. First, ARSSOI is activated in late S when it is on a linear plasmid containingC1-fi telomeric sequencesbut early when it is on a circular plasmid containing the same telomeric sequences. Second, ARSl is an early activated origin when assayed in its normal chromosomal location near the centromere on chromosome N ;however, it is late activated when transplantedto the late region at the right end of chromosomeV or placed on a linear plasmid. Third, the repeated telomeric sequence, Y', contains an ARS elements that is also activated late in S phase. Late activation of origins near a chromosome end could be caused by the functional telomere or by some topological property associated with a DNA end. To distinguish between these possibilities, a circular A R S 1 plasmid was linearized at a specificsite during the G 1 phase and the time or replicationwas determined during the following S phase. The plasmid contains, in addition to ARSl and CEN4, the gene for the HO endonuclease and the recognition and cut site for the HO endonuclease. The results indicate that a cut end is not sufficient to generate a late-activation signal. Experimentsare in progress to determine whether the late-activationsignal that spreads from the telomere can be abolished by cutting the telomere away from a neighboring origin prior to the onset of the S phase. We have identified a second late activated origin that resides 200 kb from the left telomere of chromosome XIV. We are interested in the possibility that this origin is surrounded by early replicating DNA and that late activation of thii origin is caused by a cis-acting element other than the telomere.
ARS 1 is a very efficientlyactivated chromosomal origin. However, when two copies of ARSl are placed 180° apart on a 7 kb plasmid, 2-D gel analysis shows that activation of both origins on the same molecule is rare. In such close proximity, activation of one origin may directly interfere with the activation of the other. Alternatively, initiation of the two (identical) origins may be sufficientlystaggered in time that replication forks from one simply reach the other before it can be activated. Experiments to distinguishbetween these possibilities are in progress. When a single origin is activated in this two-origin plasmid, there is a strong preference (>80%) for one of the copies of ARS 1. The cis-acting element responsible for this preferential activation resides in a segment of the URA3 gene that serves as one of the selectable markers on this plasmid. Deletion of this segment results in equivalent activation of the two origins, although double activations are still rare. We are investigating the possibility that one of the copies of ARSl is preferentially activated because the URA3 segment alters the initiation times of the two ARSs.
0749-503X/92/Spe~. ISS. 0023-01 $05.50 0 1992 by John Wiley & Sons Ltd.
16TH INT. CONF. ON YEAST GENETICS AND MOLECULAR BIOLOGY S8-3
YEAST DNA REPLICATION: FUNCTIONS DNA POLYMERASE a-PRIMASE COMPLEX
CONTROLS IN THE
G.. -.Lucchinif-eand P. Plevani'
'Dipartimento di Genetica e Biologia dei Microrganismi, Universita di Milano. Via Celoria 26, 20133 Milano, Italy eIstituto di Genetica, Universita di Sassari. Via Mancini 5, 07100 Sassari, Italy Nuclear DNA replication in Saccharornyces cerevisiae is a very complex process, which requires a number of still partially undefined protein-protein and protein-DNA interactions. In this context, both "in vitro" and "in vivo" data strongly indicate that the DNA polymerase a-primase complex is essential for initiation of DNA replication at an origin and formation of the Okazaki fragments on the lagging strand. Production and characterization of conditionnl mutants in the single essential yeast genes encoding DNA polymerase a ( P O L I ) and the two primase subunits p48 ( P R I 1 ) and p58 ( P R I Z ) has allowed us to assess the critical role of regions, where amino acid residues are conserved in the same enzymes from other eukaryotes. Despite of the high amino acid sequence conservation, neither mouse able to substitute "in vivo" f o r the primase subunit is corresponding yeast polypeptide, as expected for proteins involved in species-specific interactions. Biochemical characterization of the yeast primase mutants suggests different roles for the two subunits inside the complex. Furthermore, among revertants of a p r i 2 temperature-sensitive mutant, we have found at least one class o f external suppressors, possibly identifying a new protein interacting with the p58 polypeptide. Transcription of all the so far analyzed DNA replication genes fluctuates during cell cycle, and a critical role in this transcriptional control might be played by the common cis-acting element 5lACGCGT3'. We have identified and partially characterized trans-acting mutations affecting the expression of a CYC1-lacZ fusion only when controlled by a UAS containing the above sequence. Our attempts to correlate fluctuation of P O L 1 transcription and level of DNA polymerase a during cell-cycle will also be discussed.
0749 -503X/92/Spe~.ISS. 0024 -01 $05 S O 0 1992 by John Wiley & Sons Ltd.
16TH INT. CONF. ON YEAST GENETICS AND MOLECULAR BIOLOGY
YEAST MITOSIS; IN VlTRO AND IN VlVO J. Kingsbury, A. Yamamoto,
Carnegie Institution of Washington, Department of Embryology, 115 West University Parkway, Baltimore, Maryland 21210 USA Previously we have shown that minichromosomes isolated from Saccharomyces cerevisiae can bind to bovine microtubules. This binding is centromere dependent. The fraction of minichromosomes that bind to microtubules is 7 fold higher in nocodazole arrested cells than in alpha factor arrested cells, suggesting that centromere activity may be cell cycle regulated. We have used this assay to begin to examine the physical properties of centromere-microtubule interaction as well as its apparent cell cycle regulation. The ability of the centromere to bind to microtubules is very similar for circular and linear minichromosomes suggesting that the assembly and function of the centromere complex is unaffected by DNA topology or neighboring telomere complexes. The a b i l i of the centromere to bind microtubules is unaffected by excess competing centromere DNA suggesting that the interaction between centromere DNA and its proteins are very stable. We are currently analyzing centromere activity in several different mutants including those defective in cell cycle regulation. Before the onset of anaphase, the yeast spindle apparatus is always positioned with one spindle pole at, or through, the neck between the mother cell and the growing bud. This spindle orientation enables proper chromosome segregation to occur during anaphase; allowing one replicated genome to be segregated into the bud and the other to remain in the mother cell. We synchronized a population of cells prior to the onset of anaphase such that greater than 90% of the cells in the population had spindles with the correct orientation, and then disrupted specific cytoskeletal elements using temperature-sensitive mutations. Disruption of either the astral microtubules or actin function resulted in improper spindle orientation in approximately 40-50% of the cells. When cells with disrupted astral microtubules or actin function entered into anaphase, there was a 100 to 200 fold increase in the frequency of binucleatedcell bodies. Thus, the maintenance of proper spindle orientation by these cytoskeletal elements was essential for proper chromosome segregation. These data are consistent with the model that proper spindle orientation is maintained by tethering the astral microtubules to the actin cytoskeleton. We have identified temperature-sensitive mutations in new gene that appears to affect spindle orientation. At the nonpermissive temperature, these mutants contains both astral microtubules and normal actin distribution suggesting that the product of the new gene identifies an additional component required for spindle orientation. After nuclear migration but prior to anaphase, bulk chromosome movement occurs within the nucleus apparently because the chromosomes are attached to a mobile spindle. The frequency and magnitude of bulk chromosome movement is greatly diminished by disruption of the astral microtubules but not by disruption of the non kinetochore spindle microtubules. These results suggest that astral microtubules are not only important for spindle orientation prior to anaphase, but they also mediate force on the spindle, generating spindle displacement and in turn chromosome movement. Potential roles for this force in spindle assembly and orientation are discussed. 0749-503X/92/Spec.ISS. 0025-01 $05.50 0 1992 by John Wiley & Sons Ltd
16TH INT. CONF. ON YEAST GENETICS AND MOLECULAR BIOLOGY S9-1
The Tyl transposon J. D. Boekel, C. L. Bakerl, L. T. Braitermanl, A. S. Bystromz, E. M. Caputol, K. B. Chapmanl, A. Gabriell, H. L. Jil, J. B. Keeney, V. Lauermannl, K. Naml, G. Natsoulisl, and D. F. Voytasl 1 Department of Molecular Biology & Genetics, The Johns Hopkins University School of Medicine, 725 N. Wolfe St., Baltimore, MD 21205, USA 2 Department
of Microbiology, Umea University, S-90187 UmeA, Sweden
Retrotransposon Tyl is a multicopy transposable element scattered around the yeast genome. The mechanism of its transposition closely resembles aspects of the retroviral multiplication process. Specifically, Ty1 RNA is encapsidated within a special structure, the viruslike particle (VLP), where it is converted into double-stranded DNA by an element-encoded reverse transcriptase, making use of at least one host-encoded primer, the initiator methionine tRNA. The linear cDNA is then integrated into the target DNA via the Tyl integrase protein; the resultant product, which is presumably flanked by 5 bp gaps, is then repaired to generate a progeny transposon copy. We are studying Tyl retrotransposition through a combination of in vitro and genetic approaches. In vitro systems exist to examine reverse transcription in general, as well as priming of the minus strand. New systems for the assay of integration have been developed that indicate that 5% or more of Tyl DNA substrates can be converted into recombinant products in vitro, a reaction mediated by VLPs. A variety of genetic approaches have been used to study the Tylencoded components themselves as well as the less known contributions of host components. Host functions are known to be involved in the following steps in retrotransposition: Tyl transcription (SPT genes), TYAlTYB frameshifting (leucyl and arginyl tRNAs), and reverse transcription (initiator tRNA). An additional host function, lariat debranching enzyme, has an as yet undiscovered role in retrotransposition. Host functions are suspected to be involved in regulation of transposition, VLP assembly, transport of transposition intermediates to the nucleus, and the repair of gaps adjacent to new transposition events.
In addition to serving as a direct intermediate in the transposition process, VLPs can be used as an expression system to detect enzymatic activities encoded in a piece of DNA. We have used this system to demonstrate that heterologous retrotransposons that lack LTRs (LINEfamily) from a uypanosomatid and frpm human encode functional reverse transcriptases. In addition, we have shown that nucleases can be targeted to VLPs. The latter approach forms the basis for a new type of antiviral strategy, capsid-targeted viral inactivation, in which virus particles are inactivated by virtue of the fact that during assembly, they incorporate viral/enzyme fusion proteins which are toxic to the viral particle.
0749-503X/92/Spe~, ISS. 0026-01 $05.50 0 1992 b y John Wiley & Sons Ltd.
16TH INT. CONF. ON YEAST GENETICS AND MOLECULAR BIOLOGY
TY3,A POSITION-SPECIFICRETROVIRUSLIKEELEMENT IN YEAST D. Chalker, J. Kirchner, P. Kinsey, and S. Sandmever Department of Microbiology and Molecular Genetics, University of California, Irvine; Irvlne, California 92717,IJ.S.A. Ty3 is a retroviruslike element which occurs in the yeast S. cerevkiue. It is 5.4 kbp in len th and is composed of an internal domain flanked by long terminal re eats of 340 bp. f y 3 contains two long open reading frames referred to as GAG3 and 8OL.3. These reading frames encode nucleoeapsid and capsid structural proteins and protease, reverse transcriptase, and integrase catalytic proteins, which are required for replication and integration, respectively. High levels of expression of Ty3 results in transposition of the element which can be monitored genetically. Unlike most retrotransposons and retroviruses, Ty3 has position specificity. Naturally occurring Ty3 elements are observed in the vicinity of tRNA gene transcripbon initiation. T 3 insertions onto an artificial plasmid target have been observed near the initiation sites o!tRNA, U6, and 5s genes. PolII-transcribed genes on this plasmid are not used as targets at an observable frequency. Several approaches are being used to investigate the basis of insertion specificity of the Ty3 element. These include mutagenesis of the target genes and the Ty3 integrase. The efficiency of use of a target is not absolutely linked to its level of transcription. Mutagenesis of the target sequence has indicated that while the box A and box B promoter elements of tRNA genes are required, but that the box B sequence of U6 may not be absolutely essential for transposition. Mutations in a domain of the integrase which is not conserved between Ty3 and retroviral integrases or among retroviral integrases dramatically decrease the efficiency of transposition and the effect of these on specificity is under investigation.
0749-503X/92/Spe~. ISS. 0027-01 $05.50 0 1992 by John Wiley & Sons Ltd.
16TH INT. CONF. ON YEAST GENETICS AND MOLECULAR BIOLOGY s10- 1
INTERACTIONS OF PRP2 AND PRP8 PROTEINS IN PRE-mRNA SPLICING COMPLEXES
J. D. Beges, J. D. Brown, M. Plumpton and D. J. Jamieson Institute of Cell and Molecular Biology, Division of Biology, University of Edinburgh, King's Buildings, Mayfield Road, Edinburgh EH9 3JR, U.K. The process of nuclear pre-mRNA splicing involves many trans-acting factors that assemble in an ATPdependent manner to form the spliceosome complex in which the two transesterification reactions of splicing take place. In vifro, the assembly of this complex is highly organised with various components interacting in a defined temporal order. The best characterised of the splicing factors are the four small nuclear ribonucleoproteinparticles (snRNPs) containing the U1, U2, U4/U6 and U5 RNAs. U1 snRNP interacts first with the pre-mRNA, binding at the 5' splice site, then U2 snRNP binds to the branchpoint region of the intron to form a pre-spliceosome. The U5 and U4/U6 snRNPs then appear to associate simultaneously with the pre-spliceosome in the form of a single U4/U6.U5 triple-snRNP particle. In the spliceosome a conformational change occurs that destabilises the U4:U6 base-pairing interaction. This has been proposed to result in the exposure of residues of U6 important - possibly catalytic - in the splicing reaction. Indeed, there are a number of base-pairing interactions that need to be disrupted during the splicing reaction and the action of RNA helicases could at least in part account for the ATP requirement of splicing and could ensure a uni-directionality in the process. PRP8 protein is a 280kDa yeast splicing factor that is specifically associated with the U5 snRNP and with U4/U6.U5 triple-snRNPs. Several observations indicate that PRP8 protein is a pivotal component of the spliceosome: 1) over a diverse range of eukaryotic organisms PRP8 is highly conserved both immunologically and in its very large size as well as in its association with U5 snRNPs; 2) it is present in the spliceosome throughout the splicing reaction and in a post-splicing complex containing excised intron; 3) PRP8 and its human homologue can be cross-linked to pre-mRNA, indicating a close contact with the substrate RNA; 4) genetic interactions have been found between PRP8 and several putative R N A helicases including one, PRP28, which may be responsible for unwinding U4 and U6; 5) using in vivu depletion, heat-inactivation and antibody inhibition to eliminate the function of the protein, we have shown that PRP8 protein is required for the stable formation of U4/U6.U5 triple-snRNPs, without which spliceosomes fail to form, and that in the absence of functional PRP8 protein in vivu the levels of U4, U5 and U6 snRNAs decline dramatically. PRP2 protein is a member of the DEAH box family of RNA-stimulated ATPases, that are closely related to the DEAD box RNA helicase family. It is a non-snRNP splicing factor that is not required for spliceosome assembly, but is essential for the first cleavage-ligationreaction, and associates transiently with spliceosomes at this stage. The highly transient association of PRP2 with spliceosomes has hindered biochemical studies of the interactions of this protein, and therefore we isolated dominant negative alleles of PRP2 which when over-expressed from an inducible promoter cause a defect in pre-mRNA splicing. One of the dominant negative alleles encodes a single amino acid change in a highly conserved motif characteristic of DEAH proteins. The inhibitory effect of this protein is also dominant in virru, and assembled spliceosomescontaining the mutant PRP2 protein stall in an inactive form. W e are using biochemical and genetic methods to investigate possible effects of this mutation on the predicted RNA-stimulated ATPase activity and on the molecular interactions of PRP2 with other splicing factors in this system. Reviews: Green, M.R. (1991) Ann. Rev. Cell. Biol. 2, 559-599. Ruby, S.W. and Abelson, J. (1991) TIG 2, 79-85. Beggs, J.D. and Plumpton, M. (1992) Nucl. Acids Mol. Biol. 4 Springer-Verlag in press. 0749-503X/92/Spec.Iss. 0028-01 505.50 0 1992 hy John Wiley .4 Sons Lid.
16TH INT. CONF. ON YEAST GENETICS AND MOLECULAR BIOLOGY
THE N-END RULE PATHWAY OF PROTEIN DEGRADATION
Alexander Varshavsky Division of Biology, California Institute of Technology, Pasadena, California 91125, USA The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. Distinct versions of the N-end rule have been shown to operate in all organisms examined, from mammals to yeast and bacteria. The N-end rule is the manifestation of a degradation signal called the N-degron. In eukaryotes, the N-degron comprises two distinct determinants: a destabilizing N-terminal residue and a specific internal lysine residue (or residues). The latter is a site of attachment of a multiubiquitin chain, whose formation is required for the degradation of at least some N-end rule substrates. I will discuss the hierarchical structure of the N-end rule, the modular organization of the N-degron, the function of ubiquitin, cis-fmns recognition and subunitspecific degradation of N-end rule substrates, mechanistic dissection of the yeast N-end rule pathway, and the current understanding of its functions.
References 1. Varshavsky, A. (1992:).The N-end rule. Cell (in press). 2. Ota, I. and Varshavsky, A. (1992). A gene encoding a putative tyrosine
phosphatase suppresses lethality of an N-end rule-dependent mutant. Proc. Natl. Acad. Sci. USA 89,2355-2359. 3. Tobias, J.W., Shrader, T.E., h a p , G. and Varshavsky, A. (1991). The
N-end rule in bacteria. Science 254,1374-1377. 4. hhmen, R.J.,Madura, K.,Bartel, B. and Varshavsky, A. (1991). The N-end rule is mediated by the Ubc2(Rad6) ubiquitin-conjugatingenzyme. Proc. Natl. Acad. Sci. 88,7351-7355.
5. Baker, R.T. and Varshavsky, A. (1991). Inhibition of the N-end rule pathway in living cells. Proc. Natl. Acad. Sci. USA 88,1090-1094.
0749-503X/92/Spec.ISS. 0029-01 W5.50 0 1992 by John Wiley & Sons Ltd.
16TH INT.CONF.ON YEAST GENETICS AND MOLECULAR BIOLOGY S10-3
Recombinational repair and cell cycle control Leland Hartwell, Lisa Kadyk, and Todd Seeley Dept of Genetics, University of Washington, Seattle Washington 98195, USA The cell cycle is controlled in response to DNA damage in both G1 and G2. We have identified genes responsible for G2 arrest in response to DNA breaks and incomplete DNA replication and will report on mutants that may be defective in cell cycle delay in G1 in response to unexcised W dimers. These cell cycle checkpoints exist, presumably, to allow cells time to repair lesions or errors before chromosomes are segregated in 62 or replicated in G1. We have investigated the recombinational repair pathways that act at the G1, S and 62 stages in concert with these cell cycle controls. X-ray induced lesions are repaired in G1 by recombination between homologous chromosomes and in G2 almost exclusively by recombination between sisterchromatids. We have evidence for another repair pathway that acts during S phase between sister chromatids that responds to unexcised W dimers and may be analogous to sister chromatid exchange visualized in mammalian cells by BUdr incorporation.
0749-503X192/Spe~. ISS. 0030-01 $05.50 0 1992 by John Wiley & Sons Ltd.
16TH INT. CONF. ON YEAST GENETICS AND MOLECULAR BIOLOGY
TRANSCRIPTIONAL MODEL IN YEAST
G. Thireos, D Tavernarakis.
Maniataki and N.
Institute of Molecular Biology and Biotechnology, PO Box 1527, Heraklion 711 10, Crete, Greece. One important feature in biological systems is their ability to reprogram gene expression in response to environmental, hormonal or intercellular signals. Yeast cells adapt to amino acid limitation by increasing the levels of transcription of genes encoding amino acid biosynthetic enzymes. This increase is mediated by the binding of the GCN4 transcriptional activator at a specific DNA site located within the promoter region of these genes. Underlying this transcriptional regulatory mechanism is a translational control system necessary to mediate the response. When amino acids are limited, translation of the GCN4 mRNA is increased while the rates of the overall protein synthesis are reduced. The regulated step in this response is the formation of preinitiation complexes between the 40s ribosomal subunit and the initiator met tRNA (43s complexes). We have established that the amino acid starvation signal is transduced to the protein synthetic machinery through the mediation of the GCN2 protein kinase. This protein kinase when activated (quantitatively and qualitatively) phosphorylates the translation initiation factor eIF2. This phosphorylation then results in the reduction of the formation of 435 preinitiation complexes. The translational activation of the GCN4 mRNA under these conditions is explained by the unusual structure of this message. Its 5’ untranslated region contains four small open reading frames which engage the 40s ribosomal subunits to repeated rounds of translational reinitiation events. This accounts for the low levels of translational initiation at the GCNl coding AUG when the growth conditions are normal. When the recharging of the 40s subunits with met tRNA is limited (i.e.under amino acid limitation conditions) these reinitiation events are reduced and thus more 43s complexes can establish translation initiation events at the GCN4 coding AUG. Translational activation of the GCN4 mRNA is necessary but not sufficient f o r the transcriptional enhancement of the amino acid biosynthetic genes. We have recently established that the GCN4 mediated transcriptional activation requires the function of the GCNS protein. This protein is a transcriptional coactivator which shares a region of similarity with other proteins such as SNF2/SWI2, SPTl, STHl in yeast, Brm1,Fshl in Drosophila and FSH1,CCGI in mammals. The requirement for the GCN5 protein correlates with the access of the GCN4 protein to its target and its absence also affects the function of other transcriptional activators such as HAP2/HAP3. The possible interactions of the GCN5 protein with these activators and the core transcriptional machinery is under investigation.
0749-503X/92/Spec,ISS. 0031 -01 $05.50 @ 1992 by John Wiley & Sons Lrd.
16TH INT. CONF. ON YEAST GENETICS AND MOLECULAR BIOLOGY S10-5
TRANSCRIPTIONAL CONTROL OF GLUCOSE REPRESSED GENES M. Carlson, B.C. Laurent, E.J.A. Hubbard, X. Yang and J. Tu
Dept. of Genetics and Development, Columbia University, College of Physicians & Surgeons, 701 W. 168th St., New York, NY 10032 USA Genetic analysis has identified a host of genes required for the transcriptional regulation of genes controlled by glucose repression. Some of these regulatory genes play very general roles in the cell, affecting transcription of diversely regulated genes; for example, SNF2/SWI2, SNFS and SNF6 are involved in global transcriptional activation, and SSN6 and Tupl are broadly acting negative regulators. Other genes appear to be more directly involved in signalling the availability of glucose. The SNFl protein kinase and its associated activator, SNF4, are required for release from glucose repression. To identify other components of the SNFl pathway, we have used the twehybrid system to identify proteins that interact physically with SNFl, and we have isolated genes that in multicopy suppress defects caused by reduced SNFl kinase activity. The SIP1 gene, which was obtained by both approaches, encodes a protein that coimmunoprecipitateswith SNFl and is phosphorylated in vitro. Several other genes that are functionally related to SNFl have also been characterized.
0749-503X/92/Spec.ISS. 0032-01 $05.50 0 1992 by John Wiley Sr Sons Ltd.