Can J Microbiol, 2000 Mar, 46(3), 195 - 210 Ribosomal DNA, species structure, and biogeography of the cactophilic yeast Clavispora opuntiae; Lachance MA et al.; The ribosomal DNA of the cactophilic yeast species Clavispora opuntiae was studied in order to clarify the global distribution of the yeast . Over 500 strains, including isolates from several new localities worldwide, were characterized by rDNA restriction mapping . An unusual restriction pattern previously encountered only in one strain, from Conception Island in the Bahamas, was found in several Brazilian isolates . Sequences of the D1/D2 and D7/D8 divergent domains of the large subunit (LSU) and of the intergenic spacers (IGS) confirmed that these strains represent a genetically distinct variety of Clavispora opuntiae . This divergence had previously been hypothesized on the basis of reduced genetic recombination in inter-varietal crosses and the presence of a polymorphic ApaI restriction site located in the LSU . The exact position of the ApaI site in the D8 divergent domain and the nature of the variation that it reveals were determined . The complete sequences of 12 intergenic spacers clarified the significance of the species-wide variation uncovered by restriction mapping . Most of the polymorphic sites occur in the IGS1 and IGS2 regions, on either side of the 5S gene, and the variation is largely due to differences in the numbers and the sequences of internal repeats . Two other polymorphic sites are located in the external transcribed spacer (ETS) region . The reliability of various sites as indicators of overall spacer sequence divergence differed from one case to another . Variety-specific probes were devised and used to screen 120 strains for the presence of recombinant rDNA spacers . Three strains gave ambiguous results, but these did not constitute evidence that inter-varietal recombination has taken place in nature . The hypothesis that the global movement of Clavispora opuntiae has been influenced by the worldwide biological control of prickly pear with Cactoblastis cactorum, a moth of Argentinian origin, has received additional support from the demonstration that Argentinian strains have rDNAs similar to those found where the moth has been introduced . A dramatic founder effect was identified in a yeast population collected in cacti (Maui, Hawaii) in a site where the moth had been recently introduced. J Biol Chem, 2000 May 26, 275(21), 15895 - 904 Functional interactions among yeast Rad51 recombinase, Rad52 mediator, and replication protein A in DNA strand exchange; Song B et al.; Rad51-catalyzed DNA strand exchange is greatly enhanced by the single-stranded (ss) DNA binding factor RPA if the latter is introduced after Rad51 has already nucleated onto the initiating ssDNA substrate . Paradoxically, co-addition of RPA with Rad51 to the ssDNA to mimic the in vivo situation diminishes the level of strand exchange, revealing competition between RPA and Rad51 for binding sites on ssDNA . Rad52 promotes strand exchange but only when there is a need for Rad51 to compete with RPA for loading onto ssDNA . Rad52 is multimeric, binds ssDNA, and targets Rad51 to ssDNA . Maximal restoration of pairing and strand exchange requires amounts of Rad52 substoichiometric to Rad51 and involves a stable, equimolar complex between Rad51 and Rad52 . The Rad51-Rad52 complex efficiently utilizes a ssDNA template saturated with RPA for homologous pairing but does not appear to be more active than Rad51 when an RPA-free ssDNA template is used . Rad52 does not substitute for RPA in the pairing and strand exchange reaction nor does it lower the dependence of the reaction on Rad51 or RPA. J Biol Chem, 2000 Jun 9, 275(23), 17249 - 55 The transcriptional response of yeast to saline stress; Posas F et al.; Adaptation to changes in extracellular salinity is a critical event for cell survival . Genome-wide DNA chip analysis has been used to analyze the transcriptional response of yeast cells to saline stress . About 7% of the genes encoded in the yeast genome are induced more than 5-fold after a mild and brief saline shock (0.4 m NaCl, 10 min) . Interestingly, most responsive genes showed a very transient expression pattern, as mRNA levels dramatically declined after 20 min in the presence of stress . A quite similar set of genes increased expression in cells subjected to higher saline concentrations (0.8 m NaCl), although in this case the response was delayed . Therefore, our data show that cells respond to saline stress by inducing the expression of a very large number of genes and suggest that stress adaptation requires regulation of many cellular aspects . The transcriptional induction of most genes that are strongly responsive to salt stress was highly or fully dependent on the presence of the stress-activated mitogen-activated protein kinase Hog1, indicating that the Hog1-mediated signaling pathway plays a key role in global gene regulation under saline stress conditions. J Biol Chem, 2000 Jun 9, 275(23), 17718 - 27 Distinct protein domains of the yeast Golgi GDP-mannose transporter mediate oligomer assembly and export from the endoplasmic reticulum; Gao XD et al.; The substrates for glycan synthesis in the lumen of the Golgi are nucleotide sugars that must be transported from the cytosol by specific membrane-bound transporters . The principal nucleotide sugar used for glycosylation in the Golgi of the yeast Saccharomyces cerevisiae is GDP-mannose, whose lumenal transport is mediated by the VRG4 gene product . As the sole provider of lumenal mannose, the Vrg4 protein functions as a key regulator of glycosylation in the yeast Golgi . We have undertaken a functional analysis of Vrg4p as a model for understanding nucleotide sugar transport in the Golgi . Here, we analyzed epitope-tagged alleles of VRG4 . Gel filtration chromatography and co-immunoprecipitation experiments demonstrate that the Vrg4 protein forms homodimers with specificity and high affinity . Deletion analyses identified two regions essential for Vrg4p function . Mutant Vrg4 proteins lacking the predicted C-terminal membrane-spanning domain fail to assemble into oligomers (Abe, M., Hashimoto, H., and Yoda, K . (1999) FEBS Lett . 458, 309-312) and are unstable, while proteins lacking the N-terminal cytosolic tail are stable and multimerize efficiently, but are mislocalized to the endoplasmic reticulum (ER) . Fusion of the N terminus of Vrg4p to related ER membrane proteins promote their transport to the Golgi, suggesting that sequences in the N terminus supply information for ER export . The dominant negative phenotype resulting from overexpression of truncated Vrg4-DeltaN proteins provides strong genetic evidence for homodimer formation in vivo . These studies are consistent with a model in which Vrg4p oligomerizes in the ER and is subsequently transported to the Golgi via a mechanism that involves positive sorting rather than passive default. J Biol Chem, 2000 Jun 9, 275(23), 17762 - 70 A plant plasma membrane H+-ATPase expressed in yeast is activated by phosphorylation at its penultimate residue and binding of 14-3-3 regulatory proteins in the absence of fusicoccin; Maudoux O et al.; The Nicotiana plumbaginifolia plasma membrane H(+)-ATPase isoform PMA2, equipped with a His(6) tag, was expressed in Saccharomyces cerevisiae and purified . Unexpectedly, a fraction of the purified tagged PMA2 associated with the two yeast 14-3-3 regulatory proteins, BMH1 and BMH2 . This complex was formed in vivo without treatment with fusicoccin, a fungal toxin known to stabilize the equivalent complex in plants . When gel filtration chromatography was used to separate the free ATPase from the 14-3-3.H(+)-ATPase complex, the complexed ATPase was twice as active as the free form . Trypsin treatment of the complex released a smaller complex, composed of a 14-3-3 dimer and a fragment from the PMA2 C-terminal region . The latter was identified by Edman degradation and mass spectrometry as the PMA2 C-terminal 57 residues, whose penultimate residue (Thr-955) was phosphorylated . In vitro dephosphorylation of this C-terminal fragment prevented binding of 14-3-3 proteins, even in the presence of fusicoccin . Mutation of Thr-955 to alanine, aspartate, or a stop codon prevented PMA2 from complementing the yeast H(+)-ATPase . These mutations were also introduced in an activated PMA2 mutant (Gln-14 --> Asp) characterized by a higher H(+) pumping activity . Each mutation directly modifying Thr-955 prevented 14-3-3 binding, decreased ATPase specific activity, and reduced yeast growth . We conclude that the phosphorylation of Thr-955 is required for 14-3-3 binding and that formation of the complex activates the enzyme. J Biol Chem, 2000 Jun 2, 275(22), 16963 - 8 Limited proteolysis of yeast elongation factor 3 . Sequence and location of the subdomains; Kambampati R et al.; Elongation factor 3 (EF-3) is an ATPase essential for polypeptide chain synthesis in a variety of yeasts and fungi . We used limited proteolysis to study the organization of the subdomains of EF-3 . Trypsinolysis of EF-3 at 30 degrees C resulted in the formation of three fragments with estimated molecular masses of 90, 70, and 50 kDa . Yeast ribosomes protected EF-3 and the large fragments from further degradation . ATP exposed a new tryptic cleavage site and stabilized the 70- and 50-kDa fragments . The conformation of EF-3 as measured by fluorescence spectroscopy did not change upon ATP binding . Poly(G) stimulated proteolysis and quenched the intrinsic fluorescence of EF-3 . Using gel mobility shift, we demonstrated a direct interaction between EF-3 and tRNA . Neither tRNA nor rRNA altered the tryptic cleavage pattern . The proteolytic products were sequenced by mass spectrometric analysis . EF-3 is blocked NH(2)-terminally by an acetylated serine . The 90-, 70-, and 50-kDa fragments are also blocked NH(2)-terminally, confirming their origin . The 50-kDa fragment (Ser(2)-Lys(443)) is the most stable domain in EF-3 with no known function . The 70-kDa fragment (Ser(2)-Lys(668)) containing the first nucleotide-binding sequence motif forms the core ATP binding subdomain within the 90-kDa domain . The primary ribosome binding site is located near the loosely structured carboxyl-terminal end. J Biol Chem, 2000 Jun 9, 275(23), 17754 - 61 Analysis of the role of the hypervariable region of yeast Ras2p and its farnesylation in the interaction with exchange factors and adenylyl cyclase; Crechet JB et al.; Ras proteins from Saccharomyces cerevisiae differ from mammalian Ha-Ras in their extended C-terminal hypervariable region . We have analyzed the function of this region and the effect of its farnesylation with respect to the action of the GDP/GTP exchange factors (GEFs) Cdc25p and Sdc25p and the target adenylyl cyclase . Whereas Ras2p farnesylation had no effect on the interaction with purified GEFs from the Cdc25 family, this modification became a strict requirement for stimulation of the nucleotide exchange on Ras using reconstituted cell-free systems with GEFs bound to the cell membrane . Determination of GEF effects showed that in cell membrane the Cdc25p dependent activity on Ras2p was predominant over that of Sdc25p . In contrast to full-length GEFs, a membrane-bound C-terminal region containing the catalytic domain of Cdc25p was still able to react productively with unfarnesylated Ras2p . These results indicate that in membrane-bound full-length GEF the N-terminal moiety regulates the interaction between catalytic domain and farnesylated Ras2p.GDP . Differently from GEF, full activation of adenylyl cyclase did not require farnesylation of Ras2p.GTP, even if this step of maturation was found to facilitate the interaction . The use of Ha-Ras/Ras2p chimaeras of different length emphasized the key role of the hypervariable region of Ras2p in inducing maximum activation of adenylyl cyclase and for a productive interaction with membrane-bound GEF. J Biol Chem, 2000 May 26, 275(21), 15709 - 16 Functional role of charged residues in the transmembrane segments of the yeast plasma membrane H+-ATPase; Petrov VV et al.; As defined by hydropathy analysis, the membrane-spanning segments of the yeast plasma membrane H(+)-ATPase contain seven negatively charged amino acids (Asp and Glu) and four positively charged amino acids (Arg and His) . To explore the functional role of these residues, site-directed mutants at all 11 positions and at Glu-288, located near the cytoplasmic end of M3, have been constructed and expressed in yeast secretory vesicles . Substitutions at four of the positions (Glu-129, Glu-288, Asp-833, and Arg-857) had no significant effect on ATP hydrolysis or ATP-dependent proton pumping, substitutions at five additional positions (Arg-695, His-701, Asp-730, Asp-739, and Arg-811) led to misfolding of the ATPase and blockage at an early stage of biogenesis, and substitutions of Asp-143 allowed measurable biogenesis but nearly abolished ATP hydrolysis and proton transport . Of greatest interest were mutations of Glu-703 in M5 and Glu-803 in M8, which altered the apparent coupling between hydrolysis and transport . Three Glu-703 mutants (E703Q, E703L, E703D) showed significantly reduced pumping over a wide range of hydrolysis values and thus appeared to be partially uncoupled . At Glu-803, by contrast, one mutant (E803N) was almost completely uncoupled, while another (E803Q) pumped protons at an enhanced rate relative to the rate of ATP hydrolysis . Both Glu-703 and Glu-803 occupy positions at which amino acid substitutions have been shown to affect transport by mammalian P-ATPases . Taken together, the results provide growing evidence that residues in membrane segments 5 and 8 of the P-ATPases contribute to the cation transport pathway and that the fundamental mechanism of transport has been conserved throughout the group. J Biol Chem, 2000 May 5, 275(18), 13171 - 4 Atomic force microscopy reveals two conformations of the 20 S proteasome from fission yeast; Osmulski PA et al.; The proteasome is a major cytosolic proteolytic complex, indispensable in eukaryotic cells . The barrel-shaped core of this enzyme, the 20 S proteasome, is built from 28 subunits forming four stacked rings . The two inner beta-rings harbor active centers, whereas the two outer alpha-rings play a structural role . Crystal structure of the yeast 20 S particle showed that the entrance to the central channel was sealed . Because of this result, the path of substrates into the catalytic chamber has remained enigmatic . We have used tapping mode atomic force microscopy (AFM) in liquid to address the dynamic aspects of the 20 S proteasomes from fission yeast . We present here evidence that, when observed with AFM, the proteasome particles in top view position have either open or closed entrance to the central channel . The preferred conformation depends on the ligands present . Apparently, the addition of a substrate to the uninhibited proteasome shifts the equilibrium toward the open conformation . These results shed new light on the possible path of the substrate into the proteolytic chamber. J Biol Chem, 2000 May 26, 275(21), 15609 - 12 A lecithin cholesterol acyltransferase-like gene mediates diacylglycerol esterification in yeast; Oelkers P et al.; The terminal step in triglyceride biosynthesis is the esterification of diacylglycerol . To study this reaction in the model eukaryote, Saccharomyces cerevisiae, we investigated five candidate genes with sequence conservation to mammalian acyltransferases . Four of these genes are similar to the recently identified acyl-CoA diacylglycerol acyltransferase and, when deleted, resulted in little or no decrease in triglyceride synthesis as measured by incorporation of radiolabeled oleate or glycerol . By contrast, deletion of LRO1, a homolog of human lecithin cholesterol acyltransferase, resulted in a dramatic reduction in triglyceride synthesis, whereas overexpression of LRO1 yielded a significant increase in triglyceride production . In vitro microsomal assays determined that Lro1 mediated the esterification of diacylglycerol using phosphatidylcholine as the acyl donor . The residual triglyceride biosynthesis that persists in the LRO1 deletion strain is mainly acyl-CoA-dependent and mediated by a gene that is structurally distinct from the previously identified mammalian diacylglycerol acyltransferase . These mechanisms may also exist in mammalian cells. Biochemistry, 2000 Apr 11, 39(14), 4199 - 205 Environmental study of subunit i, a F(o) component of the yeast ATP synthase; Paumard P et al.; The topology of subunit i, a component of the yeast F(o)F(1)-ATP synthase, was determined by the use of cysteine-substituted mutants . The N(in)-C(out) orientation of this intrinsic subunit was confirmed by chemical modification of unique cysteine residues with 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid . Near-neighbor relationships between subunit i and subunits 6, f, g, and d were demonstrated by cross-link formation following sulfhydryl oxidation or reaction with homobifunctional and heterobifunctional reagents . Our data suggest interactions between the unique membrane-spanning segment of subunit i and the first transmembranous alpha-helix of subunit 6 and a stoichiometry of 1 subunit i per complex . Cross-linked products between mutant subunits i and proteins loosely bound to the F(o)F(1)-ATP synthase suggest that subunit i is located at the periphery of the enzyme and interacts with proteins of the inner mitochondrial membrane that are not involved in the structure of the yeast ATP synthase. Biochemistry, 2000 Apr 11, 39(14), 4082 - 9 Yeast resolving enzyme CCE1 makes sequential cleavages in DNA junctions within the lifetime of the complex; Fogg JM et al.; CCE1 is a DNA junction-resolving enzyme of Saccharomyces cerevisiae . Such enzymes are required to make two symmetrically paired cleavages in order to resolve the four-way junction productively . Using a cruciform assay, we show here that CCE1 introduces two unilateral cleavages in a sequential manner . This requires that the protein remains bound to the junction, preventing branch migration of the point of strand exchange . From a detailed kinetic analysis, we find that the CCE1 cleavage at a given site is accelerated by a factor of 5-10 when it occurs subsequently to the initial cleavage . These properties ensure a productive resolution of the four-way junction and may be general for junction-resolving enzymes. Biochemistry, 2000 Apr 11, 39(14), 3943 - 54 Characterization of the DNA-binding domains from the yeast cell-cycle transcription factors Mbp1 and Swi4; Taylor IA et al.; The minimal DNA-binding domains of the Saccharomyces cerevisiae transcription factors Mbp1 and Swi4 have been identified and their DNA binding properties have been investigated by a combination of methods . An approximately 100 residue region of sequence homology at the N-termini of Mbp1 and Swi4 is necessary but not sufficient for full DNA binding activity . Unexpectedly, nonconserved residues C-terminal to the core domain are essential for DNA binding . Proteolysis of Mbp1 and Swi4 DNA-protein complexes has revealed the extent of these sequences, and C-terminally extended molecules with substantially enhanced DNA binding activity compared to the core domains alone have been produced . The extended Mbp1 and Swi4 proteins bind to their cognate sites with similar affinity {K(A) approximately (1-4) x 10(6) M(-)(1)} and with a 1:1 stoichiometry . However, alanine substitution of two lysine residues (116 and 122) within the C-terminal extension (tail) of Mbp1 considerably reduces the apparent affinity for an MCB (MluI cell-cycle box) containing oligonucleotide . Both Mbp1 and Swi4 are specific for their cognate sites with respect to nonspecific DNA but exhibit similar affinities for the SCB (Swi4/Swi6 cell-cycle box) and MCB consensus elements . Circular dichroism and (1)H NMR spectroscopy reveal that complex formation results in substantial perturbations of base stacking interactions upon DNA binding . These are localized to a central 5'-d(C-A/G-CG)-3' region common to both MCB and SCB sequences consistent with the observed pattern of specificity . Changes in the backbone amide proton and nitrogen chemical shifts upon DNA binding have enabled us to experimentally define a DNA-binding surface on the core N-terminal domain of Mbp1 that is associated with a putative winged helix-turn-helix motif . Furthermore, significant chemical shift differences occur within the C-terminal tail of Mbp1, supporting the notion of two structurally distinct DNA-binding regions within these proteins. Genetics, 2000 Apr, 154(4), 1561 - 76 A yeast taf17 mutant requires the Swi6 transcriptional activator for viability and shows defects in cell cycle-regulated transcription; Macpherson N et al.; In Saccharomyces cerevisiae, the Swi6 protein is a component of two transcription factors, SBF and MBF, that promote expression of a large group of genes in the late G1 phase of the cell cycle . Although SBF is required for cell viability, SWI6 is not an essential gene . We performed a synthetic lethal screen to identify genes required for viability in the absence of SWI6 and identified 10 complementation groups of swi6-dependent lethal mutants, designated SLM1 through SLM10 . We were most interested in mutants showing a cell cycle arrest phenotype; both slm7-1 swi6Delta and slm8-1 swi6Delta double mutants accumulated as large, unbudded cells with increased 1N DNA content and showed a temperature-sensitive growth arrest in the presence of Swi6 . Analysis of the transcript levels of cell cycle-regulated genes in slm7-1 SWI6 mutant strains at the permissive temperature revealed defects in regulation of a subset of cyclin-encoding genes . Complementation and allelism tests showed that SLM7 is allelic with the TAF17 gene, which encodes a histone-like component of the general transcription factor TFIID and the SAGA histone acetyltransferase complex . Sequencing showed that the slm7-1 allele of TAF17 is predicted to encode a version of Taf17 that is truncated within a highly conserved region . The cell cycle and transcriptional defects caused by taf17(slm7-1) are consistent with the role of TAF(II)s as modulators of transcriptional activation and may reflect a role for TAF17 in regulating activation by SBF and MBF. Genetics, 2000 Apr, 154(4), 1485 - 95 Regulation of the yeast INO1 gene . The products of the INO2, INO4 and OPI1 regulatory genes are not required for repression in response to inositol; Graves JA et al.; The ino2Delta, ino4Delta, opi1Delta, and sin3Delta mutations all affect expression of INO1, a structural gene for inositol-1-phosphate synthase . These same mutations affect other genes of phospholipid biosynthesis that, like INO1, contain the repeated element UAS(INO) (consensus 5' CATGTGAAAT 3') . In this study, we evaluated the effects of these four mutations, singly and in all possible combinations, on growth and expression of INO1 . All strains carrying an ino2Delta or ino4Delta mutation, or both, failed to grow in medium lacking inositol . However, when grown in liquid culture in medium containing limiting amounts of inositol, the opi1Delta ino4Delta strain exhibited a level of INO1 expression comparable to, or higher than, the wild-type strain growing under the same conditions . Furthermore, INO1 expression in the opi1Delta ino4Delta strain was repressed in cells grown in medium fully supplemented with both inositol and choline . Similar results were obtained using the opi1Delta ino2Delta ino4Delta strain . Regulation of INO1 was also observed in the absence of the SIN3 gene product . Therefore, while Opi1p, Sin3p, and the Ino2p/Ino4p complex all affect the overall level of INO1 expression in an antagonistic manner, they do not appear to be responsible for transmitting the signal that leads to repression of INO1 in response to inositol . Various models for Opi1p function were tested and no evidence for binding of Opi1p to UAS(INO), or to Ino2p or Ino4p, was obtained. Genetics, 2000 Apr, 154(4), 1463 - 71 The fission yeast git5 gene encodes a Gbeta subunit required for glucose-triggered adenylate cyclase activation; Landry S et al.; Fission yeast adenylate cyclase is activated by the gpa2 Galpha subunit of a heterotrimeric guanine-nucleotide binding protein (G protein) . We show that the git5 gene, also required for this activation, encodes a Gbeta subunit . In contrast to another study, we show that git5 is not a negative regulator of the gpa1 Galpha involved in the pheromone response pathway . While 43% identical to mammalian Gbeta's, the git5 protein lacks the amino-terminal coiled-coil found in other Gbeta subunits, yet the gene possesses some of the coding capacity for this structure 5' to its ORF . Although both gpa2 (Galpha) and git5 (Gbeta) are required for adenylate cyclase activation, only gpa2 is needed to maintain basal cAMP levels . Strains bearing a git5 disruption are derepressed for fbp1 transcription and sexual development even while growing in a glucose-rich environment, although fbp1 derepression is half that observed in gpa2 deletion strains . Multicopy gpa2 partially suppresses the loss of git5, while the converse is not true . These data suggest that Gbeta is required for activation of adenylate cyclase either by promoting the activation of Galpha or by independently activating adenylate cyclase subsequent to Galpha stimulation as seen in type II mammalian adenylate cyclase activation. EMBO J, 2000 Apr 3, 19(7), 1650 - 60 U snRNP assembly in yeast involves the La protein; Xue D et al.; In all eukaryotic nuclei, the La autoantigen binds nascent RNA polymerase III transcripts, stabilizing these RNAs against exonucleases . Here we report that the La protein also functions in the assembly of certain RNA polymerase II-transcribed RNAs into RNPs . A mutation in a core protein of the spliceosomal snRNPs, Smd1p, causes yeast cells to require the La protein Lhp1p for growth at low temperatures . Precursors to U1, U2, U4 and U5 RNAs are bound by Lhp1p in both wild-type and mutant cells . At the permissive temperature, smd1-1 cells contain higher levels of stable U1 and U5 snRNPs when Lhp1p is present . At low temperatures, Lhp1p becomes essential for the accumulation of U4/U6 snRNPs and for cell viability . When U4 RNA is added to extracts, the pre-U4 RNA, but not the mature RNA, is bound by Smd1p . These results suggest that, by stabilizing a 3'-extended form of U4 RNA, Lhp1p facilitates efficient Sm protein binding, thus assisting formation of the U4/U6 snRNP. Bioinformatics, 1999 Dec, 15(12), 980 - 6 Segmentation of yeast DNA using hidden Markov models; Peshkin L et al.; MOTIVATION: Compositionally homogeneous segments of genomic DNA often correspond to meaningful biological units . Simple sliding window analysis is usually insufficient for compositional segmentation of natural sequences . Hidden Markov models (HMM) with a small number of states are a natural language for description of compositional properties of chromosome-size DNA sequences . RESULTS: The algorithms were applied to yeast Saccharomyces cerevisiae chromosomes (YC) I, III, IV, VI and IX . The optimal number of HMM states is found to be four . The optimal four-state HMMs for all chromosomes are very similar, as well as the reconstructed segmentations . In most cases the models with k + 1 states are obtained by 'splitting' one of the states in the model with k states, and the corresponding increase of the level of detail in segmentation . The high AT states usually correspond to intergenic regions . We also explore the model's likelihood landscape and analyze the dynamics of the optimization process, thus addressing the problem of reliability of the obtained optima and efficiency of the algorithms. Curr Biol, 2000 Mar 23, 10(6), 329 - 32 Phosphorylation and spindle pole body localization of the Cdc15p mitotic regulatory protein kinase in budding yeast; Xu S et al.; Cdc15p is an essential protein kinase and functions with a group of late mitotic proteins that includes Lte1p, Tem1p, Cdc14p and Dbf2p/Dbf20p to inactivate Cdc28p-Clb2p at the end of mitosis in budding yeast {1} {2} . Cdc14p is activated and released from the nucleolus at late anaphase/telophase to dephosphorylate important regulators of Cdc28p-Clb2p such as Hct1p/Cdh1p, Sic1p and Swi5p in a CDC15-dependent manner {3} {4} {5} {6} {7} . How Cdc15p itself is regulated is not known . Here, we report that both the phosphorylation and localization of Cdc15p are cell cycle regulated . The extent of phosphorylation of Cdc15p gradually increases during cell-cycle progression until some point during late anaphase/telophase when it is rapidly dephosphorylated . We provide evidence suggesting that Cdc14p is the phosphatase responsible for the dephosphorylation of Cdc15p . Using a Cdc15p fusion protein coupled at its carboxyl terminus to green fluorescent protein (GFP), we found that Cdc15p, like its homologue Cdc7p {8} in fission yeast, localizes to the spindle pole bodies (SPBs) during mitosis . At the end of telophase, a portion of Cdc15p is located at the mother-bud neck, suggesting a possible role for Cdc15p in cytokinesis. J Biol Chem, 2000 Apr 7, 275(14), 10655 - 60 Impaired proteasome function rescues thermosensitivity of yeast cells lacking the coatomer subunit epsilon-COP; Kimata Y et al.; Formation of COPI-coated transport vesicles requires a cytosolic protein complex consisting of seven subunits: alpha-, beta-, beta'-, gamma-, delta-, epsilon- and zeta-COP, collectively designated coatomer . The yeast Saccharomyces cerevisiae gene encoding the epsilon-COP subunit is known as SEC28/ANU2 . anu2 null mutant cells (anu2Delta) are temperature-sensitive, and alpha-COP is rapidly degraded in these cells when they are shifted to the restrictive temperature . We isolated extragenic suppressors that rescue the temperature-sensitive growth defect of anu2Delta cells . Genetic analysis revealed that one of the suppressors is allelic to PRE8 (PRS4), which encodes a 20 S proteasome subunit . In the presence of a proteasome inhibitor, MG132, anu2Delta cells did not cease growth even at the restrictive temperature . Furthermore, MG132 inhibited the rapid decrease of alpha-COP levels in anu2Delta cells shifted to the restrictive temperature . However, secretion of certain proteins by these cells was impaired even in the presence of MG132 . In conclusion, impairment of proteasome-dependent proteolysis rescued some, but not all, temperature-sensitive defects of anu2Delta cells . These results are discussed in terms of evidence that epsilon-COP plays a critical role in maintaining the structural integrity of alpha-COP. RNA, 2000 Mar, 6(3), 449 - 57 The yeast antiviral proteins Ski2p, Ski3p, and Ski8p exist as a complex in vivo; Brown JT et al.; The yeast superkiller (SKI) genes were originally identified from mutations allowing increased production of killer toxin encoded by M "killer" virus, a satellite of the dsRNA virus L-A . XRN1 (SKI1) encodes a cytoplasmic 5'-exoribonuclease responsible for the majority of cytoplasmic RNA turnover, whereas SKI2, SKI3, and SKI8 are required for normal 3'-degradation of mRNA and for repression of translation of poly(A) minus RNA . Ski2p is a putative RNA helicase, Ski3p is a tetratricopeptide repeat (TPR) protein, and Ski8p contains five WD-40 (beta-transducin) repeats . An xrn1 mutation in combination with a ski2, ski3, or ski8 mutation is lethal, suggesting redundancy of function . Using functional epitope-tagged Ski2, Ski3, and Ski8 proteins, we show that Ski2p, Ski3p, and Ski8p can be coimmunoprecipitated as an apparent heterotrimeric complex . With epitope-tagged Ski2p, there was a 1:1:1 stoichiometry of the proteins in the complex . Ski2p did not associate with Ski3p in the absence of Ski8p, nor did Ski2p associate with Ski8p in the absence of Ski3p . However, the Ski3p/Ski8p interaction did not require Ski2p . In addition, ski6-2 or ski4-1 mutations or deletion of SKI7 did not affect complex formation . The identification of a complex composed of Ski2p, Ski3p, and Ski8p explains previous results showing phenotypic similarity between mutations in SKI2, SKI3, and SKI8 . Indirect immunofluorescence of Ski3p and subcellular fractionation of Ski2p and Ski3p suggest that Ski2p and Ski3p are cytoplasmic . These data support the idea that Ski2p, Ski3p, and Ski8p function in the cytoplasm in a 3'-mRNA degradation pathway. RNA, 2000 Mar, 6(3), 352 - 68 Splicing enhancement in the yeast rp51b intron; Libri D et al.; Splicing enhancement in higher eukaryotes has been linked to SR proteins, to U1 snRNP, and to communication between splice sites across introns or exons mediated by protein-protein interactions . It has been previously shown that, in yeast, communication mediated by RNA-RNA interactions between the two ends of introns is a basis for splicing enhancement . We designed experiments of randomization-selection to isolate splicing enhancers that would work independently from RNA secondary structures . Surprisingly, one of the two families of sequences selected was essentially composed of 5' splice site variants . We show that this sequence enhances splicing independently of secondary structure, is exportable to heterologous contexts, and works in multiple copies with additive effects . The data argue in favor of an early role for splicing enhancement, possibly coincident with commitment complex formation . Genetic compensation experiments with U1 snRNA mutants suggest that U1 snRNP binding to noncanonical locations is required for splicing enhancement. Prog Cell Cycle Res, 2000, 4, 97 - 106 Functions of Pho85 cyclin-dependent kinases in budding yeast; Moffat J et al.; Pho85 is a multifunctional cyclin-dependent kinase (Cdk) in Saccharomyces cerevisiae that has emerged as an important model for the role of Cdks in both cell cycle control and other processes . Pho85 was originally discovered as a regulator of phosphate metabolism but roles for Pho85 in glycogen biosynthesis, actin regulation and cell cycle progression have since been discovered . Ten genes encoding known or putative Pho85 cyclins (Pcls) have been identified and the Pcls appear to target Pho85 to specific cellular functions and substrates . In this chapter, we review the functions of the various Pcl-Pho85 complexes in budding yeast . We focus on the known biological roles of Pho85 with an emphasis on Pho85 substrates and cyclin-Cdk specificity. Prog Cell Cycle Res, 2000, 4, 61 - 9 A Cdc7p-Dbf4p protein kinase activity is conserved from yeast to humans; Johnston LH et al.; DBF4 and CDC7 were identified as budding yeast cell cycle mutants that arrest immediately before S phase . The Dbf4p and Cdc7p proteins interact to form a protein kinase, Cdc7p being the catalytic subunit and Dbf4p is a cyclin-like molecule that activates the kinase in late G1 . Dbf4p also targets Cdc7p to origins of replication where likely substrates include the Mcm proteins . Dbf4p and Cdc7p related proteins occur in the fission yeast and in metazoans . These also phosphorylate Mcm proteins and preliminary evidence indicates a similar function to Dbf4p/Cdc7p in budding yeast . The Dbf4p/Cdc7p activity will therefore very likely be conserved in all eukaryotes. Acta Crystallogr D Biol Crystallogr, 2000 Apr, 56 ( Pt 4), 492 - 4 Crystallization and preliminary X-ray crystallographic analysis of yeast arginyl-tRNA synthetase-yeast tRNAArg complexes; Delagoutte B et al.; Three different crystal forms of complexes between arginyl-tRNA synthetase from the yeast Saccharomyces cerevisae (yArgRS) and the yeast second major tRNA(Arg) (tRNA(Arg)(ICG)) isoacceptor have been crystallized by the hanging-drop vapour-diffusion method in the presence of ammonium sulfate . Crystal form II, which diffracts beyond 2.2 A resolution at the European Synchrotron Radiation Facility ID14-4 beamline, belongs to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 129.64, b = 107.47, c = 71 . 38 A . This crystal form presents the highest resolution obtained for an active form of an aminoacyl-tRNA synthetase-tRNA complex . The estimated V(m) of 2.6 A(3) Da(-1) indicates one molecule of complex in the asymmetric unit . The three crystal forms were solved by the molecular-replacement method using the coordinates of the free yArgRS. Acta Crystallogr D Biol Crystallogr, 2000 Apr, 56 ( Pt 4), 421 - 30 MAD analyses of yeast 5-aminolaevulinate dehydratase: their use in structure determination and in defining the metal-binding sites; Erskine PT et al.; MAD experiments attempting to solve the structure of 5--aminolaevulinic acid dehydratase using Zn and Pb edges are described . The data obtained proved insufficient for a complete structure solution but were invaluable in subsequent identification of metal-binding sites using anomalous difference Fourier analyses once the structure of the enzyme had been solved . These sites include the highly inhibitory substitution of an enzymic cofactor Zn(2+) ion by Pb(2+) ions, which represents a major contribution towards understanding the molecular basis of lead poisoning . The MAD data collected at the Pb edge were also used with isomorphous replacement data from the same Pb co-crystal and a Hg co-crystal to provide the first delineation of the enzyme's quaternary structure . In this MADIR analysis, the Hg co-crystal data were treated as native data . Anomalous difference Fouriers were again used, revealing that Hg(2+) had substituted for the same Zn(2+) cofactor ion as had Pb(2+), a finding of fundamental importance for the understanding of mercury poisoning . In addition, Pt(2+) ions were found to bind at the same place in the structure . The refined structures of the Pb- and the Hg-complexed enzymes are presented at 2.5 and 3.0 A resolution, respectively. Proteins, 2000 May 15, 39(3), 204 - 11 Conversion of yeast phosphoglycerate kinase into amyloid-like structure; Damaschun G et al.; Yeast phosphoglycerate kinase is a structurally well-characterized enzyme consisting of 415 amino acids without disulfide bonds . Anion-induced refolding from its acid-unfolded state gives rise to the formation of worm-like amyloid fibrils with a persistence length of 73 nm . Electron microscopy and small-angle X-ray scattering data indicate that the fibrils have an elliptical cross-section with dimensions of 10.2 nm x 5.1 nm . About half of all amino acids are organized in form of cross-beta structure which gives rise to typical infrared spectra, X-ray diffraction and yellow-green birefringence after Congo red staining . The kinetics of amyloid formation, monitored by infrared spectroscopy, dynamic light scattering and X-ray scattering, was found to be strongly dependent on protein concentration . The infrared data indicate that the formation of cross-beta structure practically comes to an end already after some hours, whereas the length-growth of the amyloid fibrils, monitored by small-angle X-ray scattering, was not yet completed after 1,300 hours. J Biochem Biophys Methods, 2000 Mar 16, 42(3), 133 - 5 A simple approach for the simultaneous isolation and immobilization of invertase using crude extracts of yeast and Jack bean meal; Melo JS et al.; Crude cell-free extract of yeast cells was mixed with sufficient amount of Jack bean meal extract so as to precipitate all the invertase . The precipitate was then cross-linked using 2% glutaraldehyde retaining over 60% of the activity . The immobilized invertase could be reused for over ten batches without loss in activity. Biochemistry, 2000 Apr 4, 39(13), 3699 - 707 Kinetic analysis of oxygen utilization during cofactor biogenesis in a copper-containing amine oxidase from yeast; Schwartz B et al.; A detailed kinetic analysis of oxygen consumption during TPQ biogenesis has been carried out on a yeast copper amine oxidase . O(2) is consumed in a single, exponential phase, the rate of which responds linearly to dissolved oxygen concentration . This behavior is observed up to conditions of maximally obtainable oxygen concentrations . In contrast, no viscosity effect is observed on rate, implicating a high K(m) for O(2) . Binding of oxygen appears to occur faster than its consumption and to result in displacement of the precursor tyrosine onto copper to form a charge-transfer species, described in the the preceding paper of this issue {Dove, J . E., Schwartz, B., Williams, N . K., and Klinman, J . P . (2000) Biochemistry 39, 3690-3698) . Reaction between this intermediate and O(2) is proposed to occur in a rate-limiting step, and to proceed more rapidly when the tyrosine is deprotonated . This rate-limiting step in cofactor biogenesis does not display a solvent isotope effect and is, thus, uncoupled from proton transfer . Comparisons are drawn between the proposed biogenesis mechanism and that for the oxidation of reduced cofactor during catalytic turnover in the mature enzyme. Biochemistry, 2000 Apr 4, 39(13), 3611 - 23 X-ray crystallographic and analytical ultracentrifugation analyses of truncated and full-length yeast copper chaperones for SOD (LYS7): a dimer-dimer model of LYS7-SOD association and copper delivery; Hall LT et al.; Copper-zinc superoxide dismutase (CuZnSOD) acquires its catalytic copper ion through interaction with another polypeptide termed the copper chaperone for SOD . Here, we combine X-ray crystallographic and analytical ultracentrifugation methods to characterize rigorously both truncated and full-length forms of apo-LYS7, the yeast copper chaperone for SOD . The 1.55 A crystal structure of LYS7 domain 2 alone (L7D2) was determined by multiple-isomorphous replacement (MIR) methods . The monomeric structure reveals an eight-stranded Greek key beta-barrel similar to that found in yeast CuZnSOD, but it is substantially elongated at one end where the loop regions of the beta-barrel come together to bind a calcium ion . In agreement with the crystal structure, sedimentation velocity experiments indicate that L7D2 is monomeric in solution under all conditions and concentrations that were tested . In contrast, sedimentation velocity and sedimentation equilibrium experiments show that full-length apo-LYS7 exists in a monomer-dimer equilibrium under nonreducing conditions . This equilibrium is shifted toward the dimer by approximately 1 order of magnitude in the presence of phosphate anion . Although the basis for the specificity of the LYS7-SOD interaction as well as the exact mechanism of copper insertion into SOD is unknown, it has been suggested that a monomer of LYS7 and a monomer of SOD may associate to form a heterodimer via L7D2 . The data presented here, however, taken together with previously published crystallographic and analytical gel filtration data on full-length LYS7, suggest an alternative model wherein a dimer of LYS7 interacts with a dimer of yeast CuZnSOD . The advantages of the dimer-dimer model over the heterodimer model are enumerated. Nucleic Acids Res, 2000 Apr 15, 28(8), 1700 - 6 Test of intron predictions reveals novel splice sites, alternatively spliced mRNAs and new introns in meiotically regulated genes of yeast; Davis CA et al.; Correct identification of all introns is necessary to discern the protein-coding potential of a eukaryotic genome . The existence of most of the spliceosomal introns predicted in the genome of Saccharomyces cerevisiae remains unsupported by molecular evidence . We tested the intron predictions for 87 introns predicted to be present in non-ribosomal protein genes, more than a third of all known or suspected introns in the yeast genome . Evidence supporting 61 of these predictions was obtained, 20 predicted intron sequences were not spliced and six predictions identified an intron-containing region but failed to specify the correct splice sites, yielding a successful prediction rate of <80% . Alternative splicing has not been previously described for this organism, and we identified two genes (YKL186C/ MTR2 and YML034W) which encode alternatively spliced mRNAs; YKL186C/ MTR2 produces at least five different spliced mRNAs . One gene (YGR225W/ SPO70 ) has an intron whose removal is activated during meiosis under control of the MER1 gene . We found eight new introns, suggesting that numerous introns still remain to be discovered . The results show that correct prediction of introns remains a significant barrier to understanding the structure, function and coding capacity of eukaryotic genomes, even in a supposedly simple system like yeast. Mol Cell Biol, 2000 Apr, 20(8), 2827 - 38 The role of nuclear cap binding protein Cbc1p of yeast in mRNA termination and degradation; Das B et al.; The cyc1-512 mutation in Saccharomyces cerevisiae causes a 90% reduction in the level of iso-1-cytochrome c because of the lack of a proper 3'-end-forming signal, resulting in low levels of eight aberrantly long cyc1-512 mRNAs which differ in length at their 3' termini . cyc1-512 can be suppressed by deletion of either of the nonessential genes CBC1 and CBC2, which encode the CBP80 and CBP20 subunits of the nuclear cap binding complex, respectively, or by deletion of the nonessential gene UPF1, which encodes a major component of the mRNA surveillance complex . The upf1-Delta deletion suppressed the cyc1-512 defect by diminishing degradation of the longer subset of cyc1-512 mRNAs, suggesting that downstream elements or structures occurred in the extended 3' region, similar to the downstream elements exposed by transcripts bearing premature nonsense mutations . On the other hand, suppression of cyc1-512 defects by cbc1-Delta occurred by two different mechanisms . The levels of the shorter cyc1-512 transcripts were enhanced in the cbc1-Delta mutants by promoting 3'-end formation at otherwise-weak sites, whereas the levels of the longer cyc1-512 transcripts, as well as of all mRNAs, were slightly enhanced by diminishing degradation . Furthermore, cbc1-Delta greatly suppressed the degradation of mRNAs and other phenotypes of a rat7-1 strain which is defective in mRNA export . We suggest that Cbc1p defines a novel degradation pathway that acts on mRNAs partially retained in nuclei. Bioorg Med Chem, 2000 Mar, 8(3), 571 - 80 Efficient syntheses, human and yeast farnesyl-protein transferase inhibitory activities of chaetomellic acids and analogues; Singh SB et al.; Chaetomellic acids are a class of alkyl dicarboxylic acids that were isolated from Chaetomella acutiseta . They are potent and highly specific farnesyl-pyrophosphate (FPP) mimic inhibitors of Ras farnesyl-protein transferase . We have previously described the first biogenetic type aldol condensation-based total synthesis of chaetomellic acid A . Modification of the later steps of that synthesis resulted in the efficient syntheses of chaetomellic acids A and B in three steps with 75-80% overall yield . In this report, details of the original total syntheses of chaetomellic acids A, B and C, the new syntheses of acids A and B and structure-activity relationship of these compounds against various prenyl transferases including human and yeast FPTase and bovine and yeast GGPTase I are described . Chaetomellic acids are differentially active against human and yeast FPTase . Chaetomellic acid A inhibited human and yeast FPTase activity with IC50 values of 55 nM and 225 microM, respectively . In contrast, chaetomellic acid C showed only a 10-fold differential in inhibitory activities against human versus yeast enzymes . In keeping with molecular modeling-based predictions, the compounds with shorter alkyl side chains (C-8) were completely inactive against FPTase. Mol Gen Genet, 2000 Feb, 263(1), 90 - 5 Acquisition of a potential marker for insect transformation: isolation of a novel alcohol dehydrogenase gene from Bactrocera oleae by functional complementation in yeast; Benos P et al.; The alcohol dehydrogenase genes make up one of the best studied gene families in Drosophila, both in terms of expression and evolution . Moreover, alcohol dehydrogenase genes constitute potential versatile markers in insect transformation experiments . However, due to their rapid evolution, these genes cannot be cloned from other insect genera by DNA hybridization or PCR-based strategies . We have therefore explored an alternative strategy: cloning by functional complementation of appropriate yeast mutants . Here we report that two alcohol dehydrogenase genes from the medfly Ceratitis capitata can functionally replace the yeast enzymes, even though the medfly and yeast genes have evolved independently, acquiring their enzymatic function convergently . Using this method, we have cloned an alcohol dehydrogenase gene from the olive pest Bactrocera oleae . We conclude that functional complementation in yeast can be used to clone alcohol dehydrogenase genes that are unrelated in sequence to those of yeast, thus providing a powerful tool for isolation of dominant insect transformation marker genes. Mol Gen Genet, 2000 Feb, 263(1), 81 - 9 Involvement of the inverted repeat of the yeast 2-micron plasmid in Flp site-specific and RAD52-dependent homologous recombination; Storici F et al.; Site-specific recombination within the Saccharomyces cerevisiae 2-micron DNA plasmid is catalyzed by the Flp recombinase at specific Flp Recognition Target (FRT) sites, which lie near the center of two precise 599-bp Inverted Repeats (IRs) . However, the role of IR DNA sequences other than the FRT itself for the function of the Flp reaction in vivo is not known . In the present work we report that recombination efficiency differs depending on whether the FRT or the entire IR serves as the substrate for Flp . We also provide evidence for the involvement of the IR in RAD52-dependent homologous recombination . In contrast, the catalysis of site-specific recombination between two FRTs does not require the function of RAD52 . The efficiency of Flp site-specific recombination between two IRs cloned in the same orientation is about one hundred times higher than that obtained when only the two FRTs are present . Moreover, we demonstrate that a single IR can activate RAD52-dependent homologous recombination between two flanking DNA regions, providing new insights into the role of the IR as a substrate for recombination and a new experimental tool with which to study the molecular mechanism of homologous recombination. Mol Gen Genet, 2000 Feb, 263(1), 48 - 59 Activation of basal transcription by a mutation in SIN4, a yeast global repressor, occurs through a mechanism different from activator-mediated transcriptional enhancement; Mizuno T et al.; The Saccharomyces cerevisiae protein Sin4 has been suggested to affect the transcription of various genes by locally altering chromatin structure . Previous studies have defined two classes of promoters: those which are activated by loss of SIN4 function (termed sin4-responsive promoters) and those which are not activated by sin4 mutations (termed sin4 non-responsive promoters) . We analyzed the mechanism of this differential response of the two classes of promoters to a sin4 mutation . The sin4 non-responsive promoters were activated when upstream elements in the promoter region were eliminated . The upstream elements of sin4 non-responsive promoters were, in turn, found to repress the activity of the sin4-responsive promoters in an orientation-independent manner . The sin4-mediated activation was repressed by the Rme1- but not by the Tup1-Ssn6-mediated repression system . Activation of sin4-responsive promoters by Pho4 and the sin4 mutation was additive, and enhancement of transcription driven by sin4-responsive promoters was found to be due to an increase in the basal rate of transcription . The upstream regions in the sin4 non-responsive promoters contained elements that were able to inhibit activation of basal transcription . Based on these observations, we suggest that activation of basal transcription by a mutation in a gene for a global repressor, SIN4, occurs through a mechanism that differs from that responsible for activator-mediated transcriptional enhancement, and we therefore propose that basal transcription and activator-mediated transcription are repressed by different mechanisms. Science, 2000 Mar 24, 287(5461), 2257 - 9 Molecular linkage underlying microtubule orientation toward cortical sites in yeast; Korinek WS et al.; Selective microtubule orientation toward spatially defined cortical sites is critical to polarized cellular processes as diverse as axon outgrowth and T cell cytotoxicity . In yeast, oriented cytoplasmic microtubules align the mitotic spindle between mother and bud . The cortical marker protein Kar9 localizes to the bud tip and is required for the orientation of microtubules toward this region . Here, we show that Kar9 directs microtubule orientation by acting through Bim1, a conserved microtubule-binding protein . Bim1 homolog EB1 was originally identified through its interaction with adenomatous polyposis coli (APC) tumor suppressor, raising the possibility that an APC-EB1 linkage orients microtubules in higher cells. Genomics, 2000 Mar 1, 64(2), 211 - 5 CFTR intron 1 increases luciferase expression driven by CFTR 5'-flanking DNA in a yeast artificial chromosome; Mogayzel PJ Jr et al.; The DNA elements that account for the highly regulated expression of the cystic fibrosis transmembrane conductance regulator gene (CFTR) are poorly understood . The goal of this study was to assess the feasibility of using a yeast artificial chromosome (YAC)-based reporter gene construct to define these elements further . An approximately 350-kb YAC (y5'luc) was constructed by replacing CFTR with a luciferase reporter gene (luc) . A second YAC (y5'lucI) was similarly constructed but included a putative positive regulatory element from CFTR intron 1 . Stable Chinese hamster ovary (CHO-K1) cell clones were derived using each YAC to assess the role that luc copy number and the presence of intron 1 played in luc expression . The CHO-K1 clonal cell lines demonstrated a wide range of luciferase activity . On average, this activity was significantly higher in clones derived from y5'lucI . After correcting for luc copy number, the presence of intron 1 was still associated with an increase in luciferase activity (P < 0.05), despite the fact that luciferase activity did not correlate with luc copy number in y5'luc-derived clones (r = -0.12) . In contrast, the luciferase activity correlated well with luc copy number in the clones derived from y5'luc (r = 0 . 75) . These data are consistent with a positive role for intron 1 in regulating CFTR expression, but suggest that copy number is not the only factor that determines expression levels, particularly when this element is present . This YAC-based reporter system will provide a unique strategy for further assessment of the cis-acting elements that control CFTR expression . Biochem J, 2000 Mar 1, 346 Pt 2, 329 - 36 Zinc-regulated ubiquitin conjugation signals endocytosis of the yeast ZRT1 zinc transporter; Gitan RS et al.; The yeast ZRT1 zinc transporter is regulated by zinc at both transcriptional and post-translational levels . At the post-translational level, zinc inactivates ZRT1 by inducing the removal of the protein from the plasma membrane by endocytosis . The zinc transporter is subsequently degraded in the vacuole . This regulatory system allows for the rapid shut off of zinc uptake activity in cells exposed to high zinc concentrations, thereby preventing overaccumulation of this potentially toxic metal . In this report, we examine the role of ubiquitin conjugation in this process . First, we show that ZRT1 is ubiquitinated shortly after zinc treatment and before endocytosis . Secondly, mutations in various components of the ubiquitin conjugation pathway, specifically the RSP5 ubiquitin-protein ligase and the UBC4 and UBC5 ubiquitin conjugating enzymes, inhibit both ubiquitination and endocytosis . Finally, mutation of a specific lysine residue in ZRT1 blocks both ubiquitination and endocytosis . This critical lysine, Lys-195, is located in a cytoplasmic loop region of the protein and may be the residue to which ubiquitin is attached . These results demonstrate that ubiquitin conjugation is a critical step in the signal transduction pathway that controls the rate of ZRT1 endocytosis in response to zinc. Biochimie, 2000 Feb, 82(2), 139 - 45 Dimerization of the Epstein-Barr virus ZEBRA protein in the yeast two-hybrid system . Comparison Of a ZEBRA variant with the B95-8 form; Martel-Renoir D et al.; Epstein-Barr virus (EBV) is a herpes virus associated with several human tumors . The EBV protein, ZEBRA, is a transactivator of the basic leucine zipper family (bZip) . It binds to specific sequences on DNA and is able to interact with cellular proteins such as p53 . The interaction of the ZEBRA protein with its cognate DNA sequences is stable as long as the dimerization domain is functional . Recent work from this laboratory identified a ZEBRA variant (Z206) with a single amino acid change at residue 206 . An alanine is substituted for a serine, and this replacement is present in 72% of nasopharyngeal carcinoma from Europe and North Africa . As amino acid 206 lies within the dimerization domain it could be instrumental in interactions with other proteins . The yeast two-hybrid system was used to study ZEBRA-protein interactions . As ZEBRA by itself is a transactivator in yeast, it cannot be used directly in this assay . This paper describes modifications in ZEBRA amino acid sequences, rendering it usable in the yeast two-hybrid assay . We compared the dimerization capacity of the Z206 variant to that of ZEBRA from B95-8 (Z95) and observed that reporter gene activity with Z206 was consistently lower than that of Z95 (P < 0.05) . Furthermore, no interaction was found to occur between either form of ZEBRA (Z206 or Z95) and the tumor suppressor, p53 in the yeast two-hybrid system. J Dent Res, 2000 Feb, 79(2), 732 - 9 Molecular mapping of statherin- and histatin-binding domains in human salivary mucin MG1 (MUC5B) by the yeast two-hybrid system; Iontcheva I et al.; MGI is a high-molecular-weight mucin secreted by mucous acinar cells in human submandibular and sublingual glands . We have recently shown that the tracheobronchial mucin MUC5B is a major component of MG1 . MUC5B is organized into cysteine-rich N- and C-terminal regions that flank a central tandem-repeat region containing cysteine-rich subdomains and imperfect 29-residue tandem repeats . In earlier work, we have shown that this mucin selectively forms heterotypic complexes with amylase, proline-rich proteins, statherin, and histatins in salivary secretions, and the aim of this study was to identify specific binding domains within MUC5B using the yeast two-hybrid system . Interactions of cysteine-rich domains in the tandem-repeat region (Cys1-Cys4) and C-terminal region (Cys8a, Cys8b, Cys8c) of MUC5B with statherin and histatins were investigated . These studies indicated that histatin 1 selectively bound to Cysl and Cys2, whereas statherin and histatin 1, 3, and 5 selectively bound to Cys8a . Analysis of the primary sequences of the identified binding domains suggests that these domains most probably can fold into globular-like structures in the native mucin . A ProDom blast search revealed that sequences in Cys1, Cys2, and Cys8a exhibit similarity to domains in evolutionarily diverse extracellular proteins known to participate in a wide variety of protein-protein interactions. Biochemistry, 2000 Mar 28, 39(12), 3185 - 91 Identification of 12-lipoxygenase interaction with cellular proteins by yeast two-hybrid screening; Tang K et al.; The platelet isoform of 12-lipoxygenase (12-LOX) is expressed in a variety of human tumors . 12-LOX metabolizes arachidonic acid to 12(S)-hydroxyeicosateraenoic acid (12(S)-HETE), which induces a number of cellular responses associated with tumor progression and metastasis . Little is known about 12-LOX regulation and no direct regulators of 12-LOX activity have been identified . To identify potential regulators of 12-LOX, we isolated cDNAs encoding 12-LOX interacting proteins using the yeast two-hybrid system . We screened a yeast two-hybrid interaction library from human epidermoid carcinoma A431 cells and identified four cellular proteins that interact specifically with 12-LOX . We identified type II keratin 5, lamin A, the cytoplasmic domain of integrin beta4 subunit and a phosphoprotein C8FW as 12-LOX interacting proteins . Here, we demonstrated that keratin 5, a 58 kD protein required for formation of 8 nm intermediate filaments, binds to 12-LOX in human tumor cells and may contribute to the regulated trafficking of 12-LOX . We also showed that lamin A binds 12-LOX in human tumor cells . These proteins provide the first candidate regulators of 12-LOX. Ukr Biokhim Zh, 1999 Sep-Oct, 71(5), 104 - 9 {Characteristics of biologically active mannan substances, isolated from fodder yeast}; Kovalenko AG et al.; The component composition and antiviral properties of the mannan-containing preparations were studied . These preparations were extracted from some laboratory and commercial specimens of the fodder yeast cultivated on different substrates . It was shown, that the main component of pure preparation was the mannan which had molecular mass near 13 kDa . The monosaccaride composition of mannan component was varied depending on the source, degree of purification, methods of purification of the preparations . However, the crude and pure mannan preparations activity relatively to VTV was approximately equal. Proc Natl Acad Sci U S A, 2000 Mar 28, 97(7), 3219 - 23 Signal transduction by a nondissociable heterotrimeric yeast G protein; Klein S et al.; Many signal transduction pathways involve heterotrimeric G proteins . The accepted model for activation of heterotrimeric G proteins states that the protein dissociates to the free G(alpha) (GTP)-bound subunit and free G(betagamma) dimer . On GTP hydrolysis, G(alpha) (GDP) then reassociates with G(betagamma) {Gilman, A . G . (1987) Annu . Rev . Biochem . 56, 615-649} . We reexamined this hypothesis, by using the mating G protein of the yeast Saccharomyces cerevisiae encoded by the genes GPA1, STE4, and STE18 . In the absence of mating pheromone, the G(alpha) (Gpa1) subunit represses the mating pathway . On activation by binding of pheromone to a serpentine receptor, the G(betagamma) (Ste4, Ste18) dimer transmits the signal to a mitogen-activated protein kinase cascade, leading to gene activation, arrest in the G(1) stage of the cell cycle, production of shmoos (mating projections), and cell fusion . We found that a Ste4-Gpa1 fusion protein transmitted the pheromone signal and activated the mating pathway as effectively as when Ste4 (G(beta)) and Gpa1 (G(alpha)) were coexpressed as separate proteins . Hence, dissociation of this G protein is not required for its activation . Rather, a conformational change in the heterotrimeric complex is likely to be involved in signal transduction. Proc Natl Acad Sci U S A, 2000 Mar 28, 97(7), 3696 - 701 Activation of the human estrogen receptor by the antiestrogens ICI 182,780 and tamoxifen in yeast genetic systems: implications for their mechanism of action; Dudley MW et al.; The antiestrogens tamoxifen and ICI 182,780 have been portrayed as competitive antagonists of the estrogen binding site of the alpha-form of the human estrogen receptor (ER) . However, in functional studies, neither compound has consistently been able to block estradiol-induced transcription . In this report, three yeast genetic systems were used to investigate the effects of tamoxifen and ICI 182,780 on ER dimerization, transcriptional activation, and the interaction of the receptor with a coactivator, RIP140 . Tamoxifen and ICI 182,780 were able to induce ER dimerization and ER-dependent transcription, albeit at up to 15,000-fold higher concentrations than that of estradiol . In the presence of RIP140, the transcription response maximum was increased up to 30-fold for estradiol and both antiestrogens . Whole yeast cell {(3)H}estradiol binding studies demonstrated that tamoxifen could displace the estradiol from the ER, whereas ICI 182,780 treatment resulted in a 4-fold increase in {(3)H}estradiol binding to the receptor . No antagonism of estradiol was observed with tamoxifen or ICI 182,780 in any of the yeast models employed . We have concluded that the antiestrogen activity of compounds like tamoxifen and ICI 182,780 is not caused by their ability to competitively antagonize estradiol binding to the hormone binding site, but possibly by their ability to induce ER-dependent transcription, which in mammalian systems would result in receptor down-regulation . Compounds such as tamoxifen act through the hormone binding site, whereas ICI 182,780 may cause receptor activation through an allosteric binding site. J Cell Biol, 2000 Mar 20, 148(6), 1231 - 8 The docking stage of yeast vacuole fusion requires the transfer of proteins from a cis-SNARE complex to a Rab/Ypt protein; Price A et al.; The homotypic fusion of yeast vacuoles requires Sec18p (NSF)-driven priming to allow vacuole docking, but the mechanism that links priming and docking is unknown . We find that a large multisubunit protein called the Vam2/6p complex is bound to cis-paired SNAP receptors (SNAREs) on isolated vacuoles . This association of the Vam2/6p complex with the cis-SNARE complex is disrupted during priming . The Vam2/6p complex then binds to Ypt7p, a guanosine triphosphate binding protein of the Rab family, to initiate productive contact between vacuoles . Thus, cis-SNARE complexes can contain Rab/Ypt effectors, and these effectors can be mobilized by NSF/Sec18p-driven priming, allowing their direct association with a Rab/Ypt protein to activate docking. J Cell Sci, 2000 Apr, 113 ( Pt 8), 1447 - 58 A fission yeast general translation factor reveals links between protein synthesis and cell cycle controls; Grallert B et al.; In two independent screens we isolated fission yeast mutations with phenotypes suggesting defects in B-cyclin function or expression . These mutations define a single gene which we call ded1 . We show that ded1 encodes a general translation factor that is related in sequence and function to RNA helicases required for translation in other species . Levels of the B-cyclins Cig2 and Cdc13 are dramatically reduced upon inactivation of Ded1, and this reduction is independent of degradation by the anaphase promoting complex . When a ded1 mutant is grown under semi-restrictive conditions, the translation of Cig2 (and to a lesser extent Cdc13), is impaired relative to other proteins . We show that B-cyclin translation is specifically inhibited upon nitrogen starvation of wild-type cells, when B-cyclin/Cdc2 inactivation is a prerequisite for G(1) arrest and subsequent mating . Our data suggest that translational inhibition of B-cyclin expression represents a third mechanism, in addition to cyclin degradation and Rum1 inhibition, that contributes to Cdc2 inactivation as cells exit from the mitotic cell cycle and prepare for meiosis. Res Microbiol, 2000 Jan-Feb, 151(1), 43 - 52 At acidic pH, the diminished hypoxic expression of the SRP1/TIR1 yeast gene depends on the GPA2-cAMP and HOG pathways; Bourdineaud JP; The hypoxic SRP1/TIR1 gene encodes a stress-response cell wall mannoprotein, which is shown to be necessary for yeast growth at acidic pH in the presence of sodium dodecyl sulfate . However, the hypoxic expression of SRP1 is shown to be downregulated at acidic pH . The stress-responsive HOG pathway appeared necessary to maintain hypoxic SRP1 expression, but only at acidic pH . However, unlike known HOG pathway-dependent genes, SRP1 was under positive cAMP control and was positively modulated by protein kinase A at neutral and acidic pH . In addition, the HOG-independent hypoxic HEM13 gene was also positively regulated by cAMP levels . Therefore, the positive cAMP control of the hypoxic SRP1 and HEM13 genes was uncoupled from the HOG pathway . Surprisingly, this positive cAMP control was found to be mediated by GPA2 but not by RAS2, so the Gpa2p requirement appears critical at acidic pH . Although RAS2 is not involved in the regulation of SRP1 expression, the guanine nucleotide exchange factor Cdc25, which is known to control the GTP/GDP ratio on the Ras proteins, was nevertheless required for hypoxic SRP1 expression . Furthermore, the Ras proteins did not compensate for Gpa2p requirement in a delta gpa2 mutated strain . These results suggest that the Cdc25 factor might also control Gpa2p. J Biol Chem, 2000 Mar 24, 275(12), 9035 - 42 Comparison of nucleosome remodeling by the yeast transcription factor Pho4 and the glucocorticoid receptor; Then Bergh F et al.; Chromatin reorganization of the PHO5 and murine mammary tumor virus (MMTV) promoters is triggered by binding of either Pho4 or the glucocorticoid receptor (GR), respectively . In order to compare the ability of Pho4 and GR to remodel chromatin and activate transcription, hybrid promoter constructs were created by insertion of the MMTV B nucleosome sequence into the PHO5 promoter and then transformed into a yeast strain expressing GR . Activation of either Pho4 (by phosphate depletion) or GR (by hormone addition) resulted in only slight induction of hybrid promoter activity . However, simultaneous activation of both Pho4 and GR resulted in synergistic activation to levels exceeding that of the wild type PHO5 promoter . Under these conditions, Pho4 completely disrupted the nucleosome containing its binding site . In contrast, GR had little effect on the stability of the MMTV B nucleosome . A minimal transactivation domain of the GR fused to the Pho4 DNA-binding domain is capable of efficiently disrupting the nucleosome with a Pho4-binding site, whereas the complementary hybrid protein (Pho4 activation domain, GR DNA-binding domain) does not labilize the B nucleosome . Therefore, we conclude that significant activation by Pho4 requires nucleosome disruption, whereas equivalent transcriptional activation by GR is not accompanied by overt perturbation of nucleosome structure . Our results show that the DNA-binding domains of the two factors play critical roles in determining how chromatin structure is modified during promoter activation. J Biol Chem, 2000 Mar 24, 275(12), 8382 - 8 Identification of a lumenal sequence specifying the assembly of Emp24p into p24 complexes in the yeast secretory pathway; Ciufo LF et al.; The p24 proteins are transmembrane proteins of the endomembrane system that play a poorly defined role in vesicle traffic between the endoplasmic reticulum and the Golgi apparatus . Various lines of evidence indicate that p24 proteins fall into four subfamilies (alpha, beta, gamma, and delta) and that tetramers are assembled containing one representative from each subfamily; however, the nature of the protein-protein interactions within these hetero-oligomers is unknown . We have identified a lumenal segment of yeast p24beta (Emp24p) that is necessary for its assembly into p24 complexes . Replacement of 52 C-terminal residues of Emp24p with the corresponding sequence from Erv25p (p24delta) generates a chimeric protein able to replace Emp24p in p24 complexes that retain partial function in vivo, ruling out a role for the transmembrane and cytosolic domains in specifying p24 interactions . Substitution of a further 50 residues, encompassing a heptad repeat region, abolishes the ability of the chimera to replace Emp24p but instead creates a protein that resembles its Erv25p parent in its requirement for stabilization by Emp24p . These data point to a role for coiled-coil interactions in directing subfamily-specific assembly of p24 oligomers that project into the lumen of transport vesicles, where they may act to exclude secretory cargo from coat protein complex type I-coated retrograde transport vesicles. Toxicol Lett, 2000 Mar 15, 112-113, 239 - 44 Mutator phenotype due to loss of heterozygosity in diploid yeast strains with mutations in MSH2 and MLH1; Drotschmann K et al.; Mutations in mismatch repair (MMR) genes predispose humans to cancer . Particularly prevalent are frameshift and point mutations in MSH2 and MLH1, two genes whose products are required for the early steps in MMR . In normal tissues of persons predisposed to hereditary non-polyposis colon cancer (HNPCC), these mutations are usually present in only one allele . In tumor cells of these patients, the second, wild type allele is typically found to be deleted or inactivated by point mutation . This suggests that loss of heterozygosity (LOH) results in a strong mutator phenotype that could eventually lead to the onset of disease . Here we demonstrate that diploid yeast strains that are heterozygous for MSH2 and MLH1 alleles have an elevated mutation rate . We further show that this effect results not from saturation of the MMR capacity of all cells in the population, but rather from loss of the wild type allele in a subpopulation of heterozygous cells . These results have implications for understanding the mechanisms of carcinogenesis in humans. EMBO J, 2000 Mar 15, 19(6), 1357 - 65 Three conserved members of the RNase D family have unique and overlapping functions in the processing of 5S, 5.8S, U4, U5, RNase MRP and RNase P RNAs in yeast; van Hoof A et al.; The biogenesis of a number of RNA species in eukaryotic cells requires 3' processing . To determine the enzymes responsible for these trimming events, we created yeast strains lacking specific 3' to 5' exonucleases . In this work, we describe the analysis of three members of the RNase D family of exonucleases (Rex1p, Rex2p and Rex3p) . This work led to three important conclusions . First, each of these exonucleases is required for the processing of distinct RNAs . Specifically, Rex1p, Rex2p and Rex3p are required for 5S rRNA, U4 snRNA and MRP RNA trimming, respectively . Secondly, some 3' exonucleases are redundant with other exonucleases . Specifically, Rex1p and Rex2p function redundantly in 5.8S rRNA maturation, Rex1p, Rex2p and Rex3p are redundant for the processing of U5 snRNA and RNase P RNA, and Rex1p and the exonuclease Rrp6p have an unknown redundant essential function . Thirdly, the demonstration that the Rex proteins can affect reactions that have been attributed previously to the exosome complex indicates that an apparently simple processing step can be surprisingly complex with multiple exonucleases working sequentially in the same pathway. Proc Natl Acad Sci U S A, 2000 Mar 28, 97(7), 3254 - 9 Lipid rafts function in biosynthetic delivery of proteins to the cell surface in yeast; Bagnat M et al.; Lipid rafts, formed by lateral association of sphingolipids and cholesterol, have been implicated in membrane traffic and cell signaling in mammalian cells . Sphingolipids also have been shown to play a role in protein sorting in yeast . Therefore, we wanted to investigate whether lipid rafts exist in yeast and whether these membrane microdomains have an analogous function to their mammalian counterparts . We first developed a protocol for isolating detergent-insoluble glycolipid-enriched complexes (DIGs) from yeast cells . Sequencing of the major protein components of the isolated DIGs by mass spectrometry allowed us to identify, among others, Gas1p, Pma1p, and Nce2p . Using lipid biosynthetic mutants we could demonstrate that conditions that impair the synthesis of sphingolipids and ergosterol also disrupt raft association of Gas1p and Pma1p but not the secretion of acid phosphatase . That endoplasmic reticulum (ER)-to-Golgi transport of Gas1p is blocked in the sphingolipid mutant lcb1-100 raised the question of whether proteins associate with lipid rafts in the ER or later as shown in mammalian cells . Using the sec18-1 mutant we found that DIGs are present already in the ER . Taken together, our results suggest that lipid rafts are involved in the biosynthetic delivery of proteins to the yeast plasma membrane. Gene, 2000 Mar 7, 245(1), 109 - 17 Rad25p, a DNA helicase subunit of yeast transcription factor TFIIH, is required for promoter escape in vivo; Ostapenko D et al.; The general transcription factor TFIIH is required for initial DNA unwinding and promoter escape by RNA polymerase II in vitro . We examined whether Rad25p, a DNA helicase subunit of TFIIH, mediates promoter opening and promoter escape in the yeast Saccharomyces cerevisiae . DNA unwinding was probed with an in vivo permanganate reactivity assay, in a temperature-sensitive mutant of RAD25 . The consequences of Rad25p inactivation were promoter-specific . Whereas in the TDH2 promoter permanganate reactivity was entirely abolished, the reactivity at the GAL1 and GAL10 promoter regions was only moderately affected . In the GAL genes permanganate reactivity uniformly decreased downstream of the transcription start site, indicating that progression of RNA polymerase II to this region was impaired . Our results suggest that in yeast cells, promoter opening is not sufficient for productive initiation and that Rad25p-mediated promoter escape may be a limiting step in the transcription of some promoters. Neurosci Lett, 2000 Mar 17, 282(1-2), 65 - 8 Proteolytic processing and degradation of human presenilin-1 expressed in yeast; Song S et al.; Numerous mutations causing early-onset familial Alzheimer's disease have been identified in the presenilin-1 gene . Presenilin-1 protein is produced as a 47 kDa holoprotein and proteolytically processed to an N-terminal 28 kDa and a C-terminal 19 kDa fragments by unidentified presenilinase in mammalian cells . We have demonstrated that this proteolytic processing also occurs in yeast . We also show that degradation of C-terminal fragment of presenilin-1 is dependent of proteasomal function . This yeast system will be a good tool to identify presenilinase and to study the role of presenilin-1 in amyloid precursor protein processing. J Biol Chem, 2000 Mar 17, 275(11), 8233 - 9 Specificity of DNA lesion bypass by the yeast DNA polymerase eta; Yuan F et al.; DNA polymerase eta (Pol(eta), xeroderma pigmentosum variant, or Rad30) plays an important role in an error-free response to unrepaired UV damage during replication . It faithfully synthesizes DNA opposite a thymine-thymine cis-syn-cyclobutane dimer . We have purified the yeast Pol(eta) and studied its lesion bypass activity in vitro with various types of DNA damage . The yeast Pol(eta) lacked a nuclease or a proofreading activity . It efficiently bypassed 8-oxoguanine, incorporating C, A, and G opposite the lesion with a relative efficiency of approximately 100:56:14, respectively . The yeast Pol(eta) efficiently incorporated a C opposite an acetylaminofluorene-modified G, and efficiently inserted a G or less frequently an A opposite an apurinic/apyrimidinic (AP) site but was unable to extend the DNA synthesis further in both cases . However, some continued DNA synthesis was observed in the presence of the yeast Pol(zeta) following the Pol(eta) action opposite an AP site, achieving true lesion bypass . In contrast, the yeast Pol(alpha) was able to bypass efficiently a template AP site, predominantly incorporating an A residue opposite the lesion . These results suggest that other than UV damage, Pol(eta) may also play a role in bypassing additional DNA lesions, some of which can be error-prone. J Biol Chem, 2000 Mar 17, 275(11), 8143 - 53 Ubiquitination of the PEST-like endocytosis signal of the yeast a-factor receptor; Roth AF et al.; A 58-residue-long, PEST-like sequence within the yeast a-factor receptor (Ste3p) specifies the ubiquitination, endocytosis, and consequent vacuolar degradation of the receptor protein (Roth, A . F., Sullivan, D . M., and Davis, N . G . (1998) J . Cell Biol . 142, 949-961) . The present work investigates three lysyl residues that map within this sequence as the potential ubiquitin acceptor sites . Lys --> Arg substitution mutants were tested for effects on both ubiquitination and endocytosis . Results indicate that the three lysines function redundantly; a severe blockade to both ubiquitination and endocytosis is seen only for receptors having all three lysines replaced . Of the three, Lys(432) plays the predominant role; ubiquitination and turnover are significantly impaired for receptors having just the K432R mutation . CNBr fragmentation of the receptor protein, used for the physical mapping of the ubiquitin attachment sites, showed PEST-like sequence lysines to be modified both with single ubiquitin moieties as well with short multi-ubiquitin chains, two or three ubiquitins long . Thus, in addition to being the signal for ubiquitination, the Ste3p PEST-like sequence also provides the site for ubiquitin attachment . To test if this endocytosis signal functions solely for ubiquitination, we have asked if the requirement for the PEST-like sequence in endocytosis might be bypassed through pre-attachment of ubiquitin to the receptor protein . Indeed, Ste3-ubiquitin translational fusions that have a ubiquitin moiety fused to the receptor in place of the PEST-like signal do undergo rapid endocytosis and vacuolar turnover . We conclude that ubiquitin alone, with no required contribution from receptor sequences, provides the sufficient signal for initiating uptake . In addition, our results confirm conclusions originally drawn from studies with the alpha-factor receptor (Terrell, J., Shih, S., Dunn, R., and Hicke, L . (1998) Mol . Cell 1, 193-202), namely that mono-ubiquitin, and not multi-ubiquitin chains provide the primary recognition determinant for uptake . Although mono-ubiquitination suffices, our results indicate that multi-ubiquitination serves to augment the rate of uptake. J Biol Chem, 2000 Mar 17, 275(11), 7980 - 7 A mutation in yeast topoisomerase II that confers hypersensitivity to multiple classes of topoisomerase II poisons; Dong J et al.; A mutation was constructed in the CAP homology domain of yeast topoisomerase II that resulted in hypersensitivity to the intercalating agent N-{4-(9-acridinylamino)-3-methoxy-phenyl}methanesulfonamide and the fluoroquinolone 6, 8-difluoro-7-(4'-hydroxyphenyl)-1-cyclopropyl-4-quinolone-3-carboxyli c acid, but not to etoposide . This mutation, which changes threonine at position 744 to proline, also confers hypersensitivity to anti-bacterial fluoroquinolones . The purified T744P mutant protein had wild type enzymatic activity in the absence of drugs, and no alteration in drug-independent DNA cleavage . Enhanced DNA cleavage in the presence of N-{4-(9-acridinylamino)-3-methoxy-phenyl}methanesulfonamide and fluoroquinolones was observed, in agreement with the results observed in vivo . DNA cleavage was also seen in the presence of norfloxacin and oxolinic acid, two quinolones that are inactive against eukaryotic topoisomerase II . The hypersensitivity was not associated with heat-stable covalent complexes, as was seen in another drug-hypersensitive mutant . Molecular modeling suggests that the mutation in the CAP homology domain may displace amino acids that play important roles in catalysis by topoisomerase II and may explain the drug-hypersensitive phenotype. J Biol Chem, 2000 Mar 17, 275(11), 7626 - 32 CCC1 suppresses mitochondrial damage in the yeast model of Friedreich's ataxia by limiting mitochondrial iron accumulation; Chen OS et al.; Deletion of YFH1 in Saccharomyces cerevisiae leads to a loss of respiratory competence due to excessive mitochondrial iron accumulation . A suppressor screen identified a gene, CCC1, that maintained respiratory function in a Deltayfh1 yeast strain regardless of extracellular iron concentration . CCC1 expression prevented excessive mitochondrial iron accumulation by limiting mitochondrial iron uptake rather than by increasing mitochondrial iron egress . Expression of CCC1 did not result in sequestration of iron in membranous compartments or cellular iron export . CCC1 expression in wild type cells resulted in increased expression of the high affinity iron transport system composed of FET3 and FTR1, suggesting that intracellular iron is not sensed by the iron-dependent transcription factor Aft1p . Introduction of AFT1(up), a constitutive allele of the iron transcription factor, AFT1, that also leads to increased high affinity iron transport did not prevent Deltayfh1 cells from becoming respiratory-incompetent . Although the mechanism by which CCC1 expression affects cytosolic iron is not known, the data suggest that excessive mitochondrial iron accumulation only occurs when cytosolic free iron levels are high. Curr Biol, 2000 Mar 9, 10(5), 231 - 40 The cyclin-dependent kinase Cdc28p regulates distinct modes of Cdc6p proteolysis during the budding yeast cell cycle; Drury LS et al.; BACKGROUND: Cdc28p, the major cyclin-dependent kinase in budding yeast, prevents re-replication within each cell cycle by preventing the reassembly of Cdc6p-dependent pre-replicative complexes (pre-RCs) once origins have fired . Cdc6p is a rapidly degraded protein that must be synthesised in each cell cycle and is present only during the G1 phase . RESULTS: We found that, at different times in the cell cycle, there are distinct modes of Cdc6p proteolysis . Before Start, Cdc6p proteolysis did not require either the anaphase-promoting complex (APC/C) or the SCF complex, which mediate the major cell cycle regulated ubiquitination pathways, nor did it require Cdc28p activity or any of the potential Cdc28p phosphorylation sites in Cdc6p . In fact, the activation of B cyclin (Clb)-Cdc28p kinase inactivated this pathway of Cdc6p degradation later in the cell cycle . Activation of the G1 cyclins (Clns) caused Cdc6p degradation to become extremely rapid . This degradation required the SCF(CDC4) and Cdc28p consensus sites in Cdc6p, but did not require Clb5 and Clb6 . Later in the cell cycle, SCF(CDC4)-dependent Cdc6p proteolysis remained active but became less rapid . CONCLUSIONS: Levels of Cdc6p are regulated in several ways by the Cdc28p cyclin-dependent kinase . The Cln-dependent elimination of Cdc6p, which does not require the S-phase-promoting cyclins Clb5 and Clb6, suggests that the ability to assemble pre-RCs is lost before, not concomitant with, origin firing. Mol Cell Biol, 2000 Apr, 20(7), 2455 - 65 Genetic analysis of the YDR1-BUR6 repressor complex reveals an intricate balance among transcriptional regulatory proteins in yeast; Kim S et al.; A transcriptional repressor complex encoded by two essential genes, YDR1 and BUR6, was isolated from Saccharomyces cerevisiae and shown to be the functional counterpart of the human repressor complex Dr1-DRAP1 . To elucidate the mechanism of repression by this complex, altered forms of Ydr1 and Bur6 were studied in vitro and in vivo . Deletion of the C-terminal 41 amino acids of Ydr1 resulted in loss of repressor activity and a growth defect, suggesting that the C-terminal domain of Ydr1 functions as a potent transcriptional repressor . A screen for extragenic suppressors of a cold-sensitive ydr1 (ydr1(cs)) mutant led to the identification of recessive mutations in the SIN4 gene, which encodes a component of the SRB-MED complex . The sin4 alleles suppressed not only ydr1(cs) mutations but also bur6(cs) mutations . In contrast, deletion of the gal11 gene, whose product is also a member of the SRB-MED complex, failed to suppress ydr1(cs) and bur6(cs) mutations, indicating that suppression is not due to general defects in the SRB-MED complex . Moreover, one of the sin4 alleles, but not the sin4 deletion, was found to specifically suppress the inviability of a ydr1 deletion, demonstrating that the essential function of Ydr1 becomes dispensable in a sin4 mutant background . Biochemical analysis of the SRB-MED complex from the sin4 suppressor strain revealed a structurally distinct form of the SRB-MED complex that lacks a subset of mediator subunits . These results define a delicate balance between positive and negative regulators of transcription operating through the Ydr1-Bur6 repressor complex. Mol Cell Biol, 2000 Apr, 20(7), 2385 - 99 Impaired core promoter recognition caused by novel yeast TAF145 mutations can be restored by creating a canonical TATA element within the promoter region of the TUB2 gene; Tsukihashi Y et al.; The general transcription factor TFIID, which is composed of TATA-binding protein (TBP) and an array of TBP-associated factors (TAFs), has been shown to play a crucial role in recognition of the core promoters of eukaryotic genes . We isolated Saccharomyces cerevisiae yeast TAF145 (yTAF145) temperature-sensitive mutants in which transcription of a specific subset of genes was impaired at restrictive temperatures . The set of genes affected in these mutants overlapped with but was not identical to the set of genes affected by a previously reported yTAF145 mutant (W.-C . Shen and M . R . Green, Cell 90:615-624, 1997) . To identify sequences which rendered transcription yTAF145 dependent, we conducted deletion analysis of the TUB2 promoter using a novel mini-CLN2 hybrid gene reporter system . The results showed that the yTAF145 mutations we isolated impaired core promoter recognition but did not affect activation by any of the transcriptional activators we tested . These observations are consistent with the reported yTAF145 dependence of the CLN2 core promoter in the mutant isolated by Shen and Green, although the CLN2 core promoter functioned normally in the mutants we report here . These results suggest that different promoters require different yTAF145 functions for efficient transcription . Interestingly, insertion of a canonical TATA element into the TATA-less TUB2 promoter rescued impaired transcription in the yTAF145 mutants we studied . It therefore appears that strong binding of TBP to the core promoter can alleviate the requirement for at least one yTAF145 function. Mol Cell Biol, 2000 Apr, 20(7), 2367 - 77 The yeast ULP2 (SMT4) gene encodes a novel protease specific for the ubiquitin-like Smt3 protein; Li SJ et al.; Yeast Smt3 and its vertebrate homolog SUMO-1 are ubiquitin-like proteins (Ubls) that are reversibly ligated to other proteins . Like SMT3, SMT4 was first isolated as a high-copy-number suppressor of a defective centromere-binding protein . We show here that SMT4 encodes an Smt3-deconjugating enzyme, Ulp2 . In cells lacking Ulp2, specific Smt3-protein conjugates accumulate, and the conjugate pattern is distinct from that observed in a ulp1(ts) strain, which is defective for a distantly related Smt3-specific protease, Ulp1 . The ulp2Delta mutant exhibits a pleiotropic phenotype that includes temperature-sensitive growth, abnormal cell morphology, decreased plasmid and chromosome stability, and a severe sporulation defect . The mutant is also hypersensitive to DNA-damaging agents, hydroxyurea, and benomyl . Although cell cycle checkpoint arrest in response to DNA damage, replication inhibition, or spindle defects occurs with normal kinetics, recovery from arrest is impaired . Surprisingly, either introduction of a ulp1(ts) mutation or overproduction of catalytically inactive Ulp1 can substantially overcome the ulp2Delta defects . Inactivation of Ulp2 also suppresses several ulp1(ts) defects, and the double mutant accumulates far fewer Smt3-protein conjugates than either single mutant . Our data suggest the existence of a feedback mechanism that limits Smt3-protein ligation when Smt3 deconjugation by both Ulp1 and Ulp2 is compromised, allowing a partial recovery of cell function. Mutat Res, 2000 Feb 16, 459(1), 55 - 64 Mitotic viability and metabolic competence in UV-irradiated yeast cells; Conconi A et al.; Colony formation is the classic method for measuring survival of yeast cells . This method measures mitotic viability and can underestimate the fraction of cells capable of carrying out other DNA processing events . Here, we report an alternative method, based on cell metabolism, to determine the fraction of surviving cells after ultraviolet (UV) irradiation . The reduction of 2,3,5-triphenyl tetrazolium chloride (or TTC) to formazan in mitochondria was compared with cell colony formation and DNA repair capacity in wt cells and two repair-deficient strains (rad1Delta and rad7Delta) . Both TTC reduction and cell colony formation gave a linear response with different ratios of mitotically viable cells and heat-inactivated cells . However, monitoring the formation of formazan in non-dividing yeast cells that are partially (rad7Delta) or totally (wt) proficient at DNA repair is a more accurate measure of cell survival after UV irradiation . Before repair of UV photoproducts (cis-syn cyclobutane pyrimidine dimers or CPDs) is complete, these two assays give very different results, implying that many damaged cells are metabolically competent but cannot replicate . For example, only 25% of the rad7Delta cells are mitotically viable after a UV dose of 12 J/m(2)75% of these cells are metabolically competent and remove over 55% of the CPDs from their genomic DNA . Moreover, repair of CPDs in wt cells dramatically decreases after the first few hours of liquid holding (L.H.; incubation in water) and correlates with a substantial decrease in cell metabolism over the same time period . In contrast, cell colony formation may be the more accurate indicator of cell survival after UV irradiation of rad1Delta cells (i.e., cells with little DNA repair activity) . These results indicate that the metabolic competence of UV-irradiated, non-dividing yeast cells is a much better indicator of cell survival than mitotic viability in partially (or totally) repair proficient yeast cultures. Mol Cell, 2000 Jan, 5(1), 163 - 72 Rnq1: an epigenetic modifier of protein function in yeast; Sondheimer N et al.; Two protein-based genetic elements (prions) have been identified in yeast . It is not clear whether other prions exist, nor is it understood how one might find them . We established criteria for searching protein databases for prion candidates and found several . The first examined, Rnq1, exists in distinct, heritable physical states, soluble and insoluble . The insoluble state is dominant and transmitted between cells through the cytoplasm . When the prion-like region of Rnq1 was substituted for the prion domain of Sup35, the protein determinant of the prion {PSI+}, the phenotypic and epigenetic behavior of {PSI+} was fully recapitulated . These findings identity Rnq1 as a prion, demonstrate that prion domains are modular and transferable, and establish a paradigm for identifying and characterizing novel prions. Appl Microbiol Biotechnol, 2000 Feb, 53(2), 152 - 8 Synthesis of alpha-ketoglutaric acid by Yarrowia lipolytica yeast grown on ethanol; Chernyavskaya OG et al.; The ability of yeast to synthesize alpha-ketoglutaric acid (KGA) from ethanol has been studied . Thiamine-auxotrophic yeasts of different genera and species may be able to produce KGA; the main condition of synthesis is growth limitation by thiamine . Using a model culture, mutant Yarrowia lipolytica N 1, the principal conditions affecting KGA oversynthesis were identified . These were: thiamine concentration in medium and in cells, nitrogen and oxygen concentration in medium, and pH level . A KGA concentration of 49 g/l and a yield from ethanol consumed of 42% were achieved . Based on the results of the analysis of the activities of the key enzymes participating in ethanol metabolism and KGA synthesis, a concept of the mechanism of KGA biosynthesis by Y . lipolytica yeast is suggested and discussed. Biochem Biophys Res Commun, 2000 Mar 16, 269(2), 474 - 80 Identification of a family of noncanonical ubiquitin-conjugating enzymes structurally related to yeast UBC6; Lester D et al.; Ubiquitin-conjugating enzymes (UBCs) selectively target proteins for proteasomal degradation by the covalent attachment of ubiquitin moieties . Yeast UBC6 is unusual in having an active site distinct from all other UBCs and in possessing a transmembrane domain that anchors it to the cytoplasmic surface of the endoplasmic reticulum . During a differential display analysis on chick growth plate chondrocytes we isolated a cDNA encoding a noncanonical ubiquitin-conjugating enzyme (NCUBE1) structurally similar to yeast UBC6 . Chick NCUBE1 transcripts were detected in all tissues examined and decreased threefold during chondrocyte terminal differentiation . Database searches identified other related proteins; the human and mouse orthologues of NCUBE1, a second human homologue of yeast UBC6 (NCUBE2), and related proteins from S . pombe, C . elegans, and P . mariana . Together with yeast UBC6 these proteins constitute a distinct family of UBCs sharing a conserved noncanonical active site sequence and a C-terminal transmembrane domain . By analogy with yeast UBC6 they are likely to be localised to the endoplasmic reticulum where they may be involved in targeting retrotranslocated, ER-associated proteins for proteasomal degradation . Nucleic Acids Res, 2000 Apr 1, 28(7), 1656 - 64 In vivo, high-resolution analysis of yeast and mammalian RNA-protein interactions, RNA structure, RNA splicing and ribozyme cleavage by use of terminal transferase-dependent PCR; Chen HH et al.; We have investigated the analysis of RNA by use of terminal transferase-dependent PCR (TDPCR), a procedure previously used for the analysis of DNA and chromatin {J.Komura and A.D.Riggs, Nucleic Acids Res.,26, 1807-1811 (1998)} . When preceded by reverse transcription (RT), TDPCR provides an extremely sensitive, versatile, quantitative and nucleotide-level assay for detecting RNA lesions or structures that block primer extension during the RT step . The procedure is: (i) RT using a gene-specific oligonucleotide; (ii) ribo-tailing of the single-stranded cDNA product by use of terminal deoxy-nucleotidyl transferase; (iii) ligation of a DNA linker to the tailed cDNA by use of T4 DNA ligase; and (iv) PCR using a nested, gene-specific primer and a linker-specific primer . This procedure combines the versatility of a primer extension assay with nucleotide-level resolution, the specificity of nested primers and the sensitivity of PCR . Band patterns obtained are reproducible and quantifiable . We successfully used the technique for the study of yeast RNA structure, splicing intermediates and ribozyme cleavage . Also, in vivo footprint experiments, using mammalian cells and RNase T1, revealed the binding of iron-responsive element binding protein to iron responsive elements in the mRNAs of transferrin receptor and ferritin H-chain. Nucleic Acids Res, 2000 Apr 1, 28(7), 1594 - 603 Positive and negative mutant selection in the human histone hairpin-binding protein using the yeast three-hybrid system; Martin F et al.; We have used the yeast three-hybrid system in a positive selection for mutants of the human histone hairpin-binding protein (HBP) capable of interacting with non-canonical hairpins and in a negative selection for loss-of-binding mutants . Interestingly, all mutations from the positive selection are located in the N- and C-terminal regions flanking a minimal RNA-binding domain (RBD) previously defined between amino acids 126 and 198 . Further, in vitro binding studies demonstrate that the RBD, which shows no obvious similarity to other RNA-binding motifs, has a relaxed sequence specificity compared to full-length HBP, allowing it to bind to mutant hairpin RNAs not normally found in histone genes . These findings indicate that the sequences flanking the RBD are important for restricting binding to the highly conserved histone hairpin structure . Among the loss-of-binding mutations, about half are nonsense mutations distributed throughout the N-terminal part and the RBD whereas the other half are missense mutations restricted to the RBD . Whereas the nonsense mutations permit a more precise definition of the C-terminal border of the RBD, the missense mutations identify critical residues for RNA binding within the RBD. Nucleic Acids Res, 2000 Apr 1, 28(7), 1576 - 84 Scp160p, a multiple KH-domain protein, is a component of mRNP complexes in yeast; Lang BD et al.; Scp160p is a 160 kDa protein in the yeast Saccharomyces cerevisiae that contains 14 repeats of the hnRNP K-homology (KH) domain, and demonstrates significant sequence homology to a family of proteins collectively known as vigilins . As a first step towards defining the function of Scp160p, we have characterized the subcellular distribution and in vivo interactions of this protein . Using sucrose gradient fractionation studies we have demonstrated that Scp160p in cytoplasmic lysates is predominantly associated with polyribosomes . Furthermore, we have found that Scp160p is released from polyribosomes by EDTA in the form of a large complex of> or =1300 kDa that is sensitive both to RNase and NaCl . Using affinity-chromatography to isolate these complexes, we have identified two protein components other than Scp160p: poly(A) binding protein, Pab1p, and Bfr1p . The presence of Pab1p confirms these complexes to be mRNPs . The presence of Bfr1p is intriguing because the null phenotype for this gene is essentially the same as that reported for scp160 -null cells: increased cell size and aberrant DNA content . These results demonstrate that Scp160p associates with polyribosome-bound mRNP complexes in vivo, implicating a role for this protein in one or more levels of mRNA metabolism in yeast. Microbiology, 2000 Feb, 146 ( Pt 2), 385 - 91 The yeast Chs4 protein stimulates the trypsin-sensitive activity of chitin synthase 3 through an apparent protein-protein interaction; Ono N et al.; Inducible overexpression of the CHS4 gene under the control of the GAL1 promoter increased Chs3p (chitin synthase 3) activity in Saccharomyces cerevisiae several fold . Approximately half of the Chs3p activity in the membranes of cells overexpressing Chs4p was extracted using CHAPS and cholesteryl hemisuccinate . The detergent-extractable Chs3p activity appeared to be non-zymogenic because incubation with trypsin decreased enzyme activity in both the presence and absence of the substrate, UDP-N-acetylglucosamine . Western blotting confirmed that Chs3p was extracted from membranes by CHAPS and cholesteryl hemisuccinate and revealed that Chs4p was also solubilized using these detergents . Yeast two-hybrid analysis with truncated Chs4p demonstrated that the region of Chs4p between amino acids 269 and 563 is indispensable not only for eliciting the non-zymogenic activity of Chs3p but also for binding of Chs4p to Chs3p . Neither the EF-hand motif nor a possible prenylation site in Chs4p was required for these activities . Thus, it was demonstrated that stimulation of non-zymogenic Chs3p activity by Chs4p requires the amino acid region from 269 to 563 of Chs4p, and it seems that Chs4p activates Chs3p through protein-protein interaction. Biosci Biotechnol Biochem, 2000 Jan, 64(1), 89 - 95 Requirement for lysine-19 of the yeast mitochondrial ATPase inhibitor for the stability of the inactivated inhibitor-F1Fo complex at higher pH; Ichikawa N et al.; The ATPase inhibitor is a regulatory subunit of mitochondrial ATP synthase . In this study, the role of Lys19 of the yeast ATPase inhibitor was examined by site-directed mutagenesis . Two amino acids (Gln and Glu) were substituted for the Lys19 . The purified mutant inhibitor (Lys19-->Gln) had similar ATPase inhibitory activity to that of the wild-type inhibitor at pH 6.5, but was less active at pH 7.4 . ATP synthesis in mutant mitochondria was normally activated by the addition of ADP and succinate, but the inactivated ATPase complex in the mutant mitochondria was activated more readily than that in control cells by raising pH . These results show that Lys19 of the yeast ATPase inhibitor is not essential for ATPase inhibitory activity, but increases the stability of the inhibitor-F1Fo complex at higher pH. Diagn Microbiol Infect Dis, 2000 Feb, 36(2), 137 - 8 Rapid identification of clinical yeast isolates using the colorimetric AUXACOLOR system; Romney MG et al.; The AUXACOLOR colorimetric system (Sanofi Diagnostics Pasteur, Marnes-la-Coquette, France) for the identification of clinical yeast isolates, was compared in its identification of 100 yeast strains to conventional identification methods . Of the 94 correctly identified isolates, 47% (n = 44) were identified by 24 h, and 100% (n = 94) were identified by 48 h . AUXACOLOR is a simple, rapid and accurate method for the identification of yeast pathogens. J Steroid Biochem Mol Biol, 1999 Dec 31, 71(5-6), 239 - 46 Pregnenolone metabolized to 17alpha-hydroxyprogesterone in yeast: biochemical analysis of a metabolic pathway; Degryse E et al.; The cDNA coding for the human 3beta-hydroxy-5-ene steroid dehydrogenase/5-ene-4-ene steroid isomerase (3beta-HSD) has been expressed in yeast . When expressed from identical vectors except for the coding sequence, the specific activity of the type I is lower than that of the type II enzyme . A mutant of the human 3beta-HSD type II lacking the putative membrane spanning domain 1 was generated by site directed mutagenesis: its apparent K(m) for pregnenolone (PREG) is significantly increased and its V reduced to the level of the type I enzyme . The influence of the kinetic properties of 3beta-HSD in the accumulation of 17alpha-hydroxyprogesterone was probed by co-expression of the bovine 17alpha-hydroxylase cytochrome P450 (P45017alpha) cDNA . The metabolism of PREG was followed with time using the membrane fraction . Kinetic properties of the 3beta-HSD were modulated such that its activity was in excess, limiting or balanced with respect to the activity of the P45017alpha and the accumulation of intermediates and products recorded . Conditions for the generation of the by-products resulting from the 17,20-Lyase activity of the P45017alpha were found . The potential applications of the system are discussed. Biotechnol Bioeng, 2000 Apr 5, 68(1), 44 - 51 Efficient bioconversion of ethanol to acetaldehyde using a novel mutant strain of the methylotrophic yeast Hansenula polymorpha; Moroz OM et al.; We report the isolation of mutant strains of the methylotrophic yeast Hansenula polymorpha that are able to efficiently oxidize ethanol to acetaldehyde in an intact cell system . The oxidation reaction is catalyzed by alcohol oxidase (AOX), a key enzyme in the methanol metabolic pathway that is typically present only in H . polymorpha cells growing on methanol . At least three mutations were introduced in the strains . Two of the mutations resulted in high levels of AOX in glucose-grown cells of the yeast . The third mutation introduced a defect in the cell's normal ability to degrade AOX in response to ethanol, and thus stabilizing the enzyme in the presence of this substrate . Using these strains, conditions for bioconversion of ethanol to acetaldehyde were examined . In addition to pH and buffer concentration, we found that the yield of acetaldehyde was improved by the addition of the proteinase inhibitor phenylmethylsulfonyl fluoride (PMSF) and by permeabilization of the cells with digitonin . Under optimal shake-flask conditions using one of the H . polymorpha mutant strains, conversion of ethanol to acetaldehyde was nearly quantitative . J Pharm Biomed Anal, 1999 Feb, 19(1-2), 115 - 25 Using entropies of reaction to predict changes in protein stability: tyrosine-67-phenylalanine variants of rat cytochrome c and yeast Iso-1 cytochromes c; Feinberg BA et al.; Using the voltammetric method of square-wave voltammetry, a direct electrochemical examination was made of the wild type and Tyr67Phe mutant of both rat cytochrome c and yeast iso-1-cytochrome c . In addition to determining the equilibrium reduction potential (E0') for each cytochrome, the entropy of reaction, deltaS0'(Rxn)(deltaS0'(Rxn) = S0'(Red) - S0'(Ox)), for the reduction process was determined via the non-isothermal method . Having determined deltaS0'(Rxn) and E0', deltaH0' was calculated . For rat cytochrome c, it was found that deltaS0'(Rxn) = -43 J mol(-1) K(-1) for the wild type and -53 J mol(-1) K(-1) for the Tyr67Phe variant, with the deltaH0' for both the wild type and variant nearly identical, indicating that the changes in reduction potential and probably stability are due to changes in deltaS0'(Rxn) . In contrast the measured deltaS0'(Rxn) for yeast iso-1-cytochrome c demonstrated significant changes in both entropic and enthalpic contributions in going from wild type to mutant cytochrome c . The entropy of reaction provides information regarding the relative degree of solvation, and very likely the degree of compactness, of the oxidized state versus the reduced state of the redox protein . A thermodynamic scheme and stability derivation are presented that show how the entropies of reaction of wild type versus variant cytochromes contribute to and predict changes in stability in going from oxidized to reduced protein . For yeast iso-1-cytochrome c, the thermodynamically predicted change in stability was very close to the experimentally observed value, based on previous differential scanning calorimetric stability measurements . While such data is not available for rat cytochrome c, consideration of the enormously increased local stability of the rat oxidized cytochrome c variant predicts that the reduced rat variant will be even more stable than the already stabilized oxidized variant. Antonie Van Leeuwenhoek, 2000 Jan, 77(1), 43 - 7 The isolation and classification of Candida mokoenaii sp . nov . A new yeast isolate fro