Yeasts

Gene, 1997 Jun 3, 191(2), 161 - 6
Cloning and characterization of mouse Dhm2 cDNA, a functional homolog of budding yeast SEP1; Shobuike T et al.; We have isolated mouse Dhm2 cDNAs encoding a homolog of budding yeast SEP1, whose product is involved in many cellular processes including meiosis, cellular senescence, and telomere maintenance . The putative Dhm2 protein (Dhm2p), which consists of 1687 amino acids and whose molecular weight is 191,400, matches the size of Sep1p and shares extensive homology with Sep1p especially in their N-terminal regions . A multicopy plasmid containing of the Dhm2 cDNA complements the slow growth phenotype, sporulation defect, and DNA recombination defect caused by the sep1 mutation in yeast, indicating that Dhm2 is a functional homolog of SEP1 . Since Dhm1, another SEP1 homolog we reported previously, only partially compensates for the sep1 mutation, we conclude that Dhm2 is a true homolog of SEP1 . Northern analysis revealed that 5.8 kb mRNA corresponding to Dhm2 open reading frame is produced highly in testis . These results strongly suggest that Dhm2p participates in gametogenesis in mouse.

Biochemistry, 1997 Jun 3, 36(22), 6803 - 13
Metal-ion-mediated allosteric triggering of yeast pyruvate kinase . 2 . A multidimensional thermodynamic linked-function analysis; Mesecar AD et al.; A role has been proposed for the free divalent metal in triggering the allosteric responses of yeast pyruvate kinase based upon a kinetic linked-function analysis {Mesecar, A . D., & Nowak, T . (1997a) (preceding paper in this series)} . The major conclusion from the analysis is that the allosteric activator, fructose 1,6-diphosphate (FDP), does not directly communicate with the substrate, phosphoenolpyruvate (PEP), at the active site of the enzyme: it is Mn2+ that mediates the allosteric communication between the PEP and FDP sites in an allosteric relay mechanism . Assumptions were necessary to treat kinetic parameters as thermodynamic parameters, and the presence of the substrate ADP was necessary for the kinetic analysis . In this study, the influence of FDP on the interactions of PEP and Mn2+ and the influence of PEP and Mn2+ on the interaction of FDP with YPK were measured, where possible, by direct binding methods in the absence of ADP . Direct binding data were then subjected to a thermodynamic linked-function analysis for a heterotropic, three ligand coupled system in order to ascertain the two and three ligand coupling free energies . The two ligand coupling free energies deltaG(Mn-PEP), deltaG(Mn-FDP), and deltaG(PEP-FDP) are -3.88, -1.09, and -0.22 kcal/mol, respectively . These values indicate that positive, heterotropic interactions exist between each of these ligand pairs . The three ligand coupling free energy term, deltaG(Mn-PEP-FDP), indicates that simultaneous binding of Mn2+, PEP, and FDP is considerably favored over the sum of their independent binding free energies by -6.6 kcal/mol . These results demonstrate the key role of the metal in the modulation of ligand binding and are consistent with the values and the relationships of the kinetic parameters obtained from the kinetic linked-function analysis.

Biochemistry, 1997 Jun 3, 36(22), 6792 - 802
Metal-ion-mediated allosteric triggering of yeast pyruvate kinase . 1 . A multidimensional kinetic linked-function analysis; Mesecar AD et al.; Regulation of the glycolytic pathway is considered to be primarily achieved by the carbon metabolites resulting from glucose metabolism {e.g., fructose 1,6-diphosphate (FDP), phosphoenolpyruvate (PEP), and citrate} and by the ATP charge of the cell . The divalent cations (e.g., Mg2+ and Mn2+) have not been considered as having regulatory roles in glycolysis, although they are involved in almost every enzyme-catalyzed reaction in the pathway . Using a kinetic linked-function analysis of steady-state kinetic data for the interactions of PEP, FDP, and Mn2+ with yeast pyruvate kinase (YPK), we have found that the divalent metal is the principal trigger of the allosteric responses observed with this enzyme . The interaction of Mn2+ to YPK enhances the interaction of FDP by -1.6 kcal/mol and the interaction of PEP by -2.8 kcal/mol . The simultaneous interaction of all three of these ligands to YPK is favored by -4.3 kcal/mol over the sum of their independent binding free energies . Surprisingly, the binding of the allosteric activator FDP does not directly influence the binding of the substrate PEP since a coupling free energy near zero was calculated for these two ligands . Thus, communication between the PEP and FDP sites occurs structurally through the metal by an allosteric relay mechanism . These conclusions are supported by results of a thermodynamic linked-function analysis of direct binding data for the interactions of PEP, FDP, and Mn2+ with YPK {Mesecar, A . D., & Nowak, T . (1997) Biochemistry (following paper in this series)} . Our findings raise important questions as to the possible roles of divalent metals in modulating multiligand interactions with YPK and in the regulation of the glycolytic pathway.

Appl Biochem Biotechnol, 1997 Jun, 66(3), 263 - 8
Photosensitization of the yeast Phaffia rhodozyma at a low temperature for screening carotenoid hyperproducing mutants; An GH; Phaffia rhodozyma strain Ant-1 produces more carotenoids, known as antioxidants, but it was more sensitive to light plus toluidine blue O (TBO), a superoxide producer, than wild strain 67-385 at 20 degrees C . Carotenoid hyperproducing mutants (CHMs), Ant-1 and 2A2N, exhibited decreased activity of superoxide dismutase (SOD) compared to 67-385, and this is in part responsible for hypersensitivity of the mutants to photosensitization . Light plus TBO at 2 degrees C allowed carotenoid hyperproducing mutants to produce higher colony-forming units than the wild-type . Photosensitization with limited cell metabolism by a low temperature, provides an idea of selective conditions for carotenoid hyperproducers of P . rhodozyma.

Cell Struct Funct, 1997 Jun, 22(3), 365 - 77
Immunocytochemical analysis of peptide hormone processing: importance of the positively charged N-terminal domain of signal peptide in correct ER targeting in yeast cells; Cheong KH et al.; We used a morphological approach to determine the topogenic role of the signal peptide in mediating the ER translocation of yeast prepro-alpha-factor . In prepro-alpha-factor-somatostatin hybrids, changes in the N-terminal amino acid sequence from wild-type NH2-Met-Arg-Phe (MRF) to NH2-Met-Phe-Lys (MFK) caused a subtle difference in protein trafficking in yeast cells . Immunofluorescence microscopy on semithin cryosections and immunoelectron microscopy on ultrathin sections showed that the transposition of the charged amino acid at N-terminus caused the precursors to be associated with either nucleus or mitochondria . This suggests that the secretory proteins are mistargeted to the irrelevant organelles as the result of inefficient ER translocation . Structural aspects of nuclear or mitochondrial targeting proteins and common principles in membrane translocation systems account for the mistargeting of overexpressed mutant hybrid precursors that are not rapidly translocated into the ER . Based on our immunocytochemical study on individual cells, we propose here that the positively charged N-terminal domain of signal peptide is important not merely in the efficiency of ER translocation, but also in appropriate targeting of peptide hormone precursors in yeast cells where post-translational ER translocation is known to occur frequently.

J Enzyme Inhib, 1997 Jun, 12(2), 143 - 54
Modification of an essential amino group of glutathione reductase from yeast by pyridoxal 5'-phosphate; Pandey A et al.; Yeast glutathione reductase is inactivated by pyridoxal 5'-phosphate (PLP) . The reactivation of the enzyme by dilution as well as a characteristic absorption peak at 325 nm exhibited by NaBH4-reduced-PLP modified enzyme show that the inactivation is due to the specific modification of the epsilon-amino group of lysine residue . The maximum of 70% inactivation was observed at 7mM PLP and the equilibrium was reached within 3 min . Kinetic and equilibrium analysis of inactivation data derived at different PLP concentrations showed that a noncovalent intermediate is formed prior to inactivation . From the studies on the effect of pH on the inactivation rate, the pKa of epsilon-amino group of the reactive lysine residue was calculated to be 7.3 . Among various protecting agents tried, only NADP was found to be effective . The apparent stoichiometry of the reaction was one to one as the incorporation of 0.65 mole PLP/mole of enzyme led to 70% inactivation at saturating PLP concentration.

J Enzyme Inhib, 1997 Jun, 12(2), 101 - 21
Yeast hexokinase inhibitors designed from the 3-D enzyme structure rebuilding; Willson M et al.; This work describes a search for hexokinase inhibitors based on the interactions analysis at the active site of the X-ray resolved o-tolulyl-glucosamine-hexokinase (OTG-HK) complex structure . As the actual enzyme sequence was unknown when the X-ray structure was made (only 30% homology), the structure of the complex was rebuilt by modelling on the X-ray structure frame which allowed residues in close vicinity to the inhibitor to be defined, particularly Glu249 and Gln278 . Compounds with inhibitor-bearing groups able to interact with these residues were synthesized and assayed . Some of them revealed strong affinities, in the Km range for glucose . Kinetic analysis of their behaviour towards glucose and ATP together with spectroscopic studies using NMR, allowed the determination of the corresponding inhibition patterns and provided complementary information on HK.

Mol Gen Genet, 1997 Jun, 255(2), 157 - 65
A soluble 12-kDa protein of the mitochondrial intermembrane space, Mrs11p, is essential for mitochondrial biogenesis and viability of yeast cells; Jarosch E et al.; We have isolated an essential yeast gene termed MRS11, which codes for a soluble protein of the mitochondrial intermembrane space . Interestingly, this new gene shares many similarities with the previously characterized MRS5 gene: when expressed from a multicopy plasmid, MRS11 like MRS5 restores respiration competence to yeast strains defective in the splicing of mitochondrial group II introns . Both genes are essential for viability of yeast cells, as the disruption of either of them is lethal . The proteins encoded by MRS5 and MRS11, which display 35%, sequence identity are both located in the mitochondrial intermembrane space . Depletion of Mrs11p results in a phenotype similar to that observed in Mrs5p-depleted cells: accumulation of the precursor form of mitochondrial hsp60, inability to form spectrophotometrically detectable amounts of cytochromes and changes in the mitochondrial morphology . Although similar in sequence and function, Mrs5p and Mrs11p are not functionally equivalent and neither can substitute for the other, even when overexpressed . Taken together, our data suggest a cooperative mode of action of Mrs11p and Mrs5p in mitochondrial protein import or other related essential mitochondrial processes.

Curr Opin Genet Dev, 1997 Jun, 7(3), 405 - 9
From gene to screen with yeast; Oliver SG; With the complete sequence now available, the yeast genome project enters a post-sequencing phase that will concentrate on a comprehensive determination of gene function . Novel techniques have been developed to undertake genome-wide functional analysis at the levels of phenotype, transcript and protein . These include techniques for the efficient deletion of individual genes while tagging the deletants with specific oligonucleotide signatures, as well as strategies to quantify the physiological effects of such deletions by comparing growth rates and metabolite profiles under a range of conditions . Comprehensive approaches to the study of gene expression include hybridization array technology to identify and quantify transcripts, and the exploitation of mass spectometry to identify proteins resolved by two-dimensional gel electrophoresis . Yeast presents opportunities for the discovery of new human medicines both via the recognition of functional homologies between human and yeast genes and by the use of yeast to express human coding sequences specifying potential drug targets.

Plant J, 1997 Jun, 11(6), 1237 - 51
Isolation of a novel class of bZIP transcription factors that interact with ABA-responsive and embryo-specification elements in the Dc3 promoter using a modified yeast one-hybrid system; Kim SY et al.; Dc3 is a carrot lea class gene that is abundantly expressed during somatic and zygotic embryogenesis . Its expression is normally embryo-specific and also can be induced by abscisic acid . The regulatory elements mediating the embryo-specific expression of Dc3 reside within the proximal promoter region (-117 to +26), which is also essential for ABA-induced expression . In this study, an optimized version of the yeast one-hybrid system has been used to clone factors that bind to the promoter region of the Dc3 gene . Twenty-five million yeast transformants were screened in a single experiment, and nine independent cDNA clones were isolated from a sunflower library that encode proteins that specifically bind to functional cis-regulatory elements in the Dc3 promoter . Analysis of these clones showed that they are derived from three different mRNA species that encode two basic leucine zipper proteins . The basic regions of these proteins, named DPBF-1 and 2 (Dc3 Promoter-Binding Factor-1 and 2), respectively, are nearly identical to each other and are similar to the plant G-box binding factor GBF-4 . Outside the basic region, however, DPBF-1 and 2 diverge significantly from each other and from other known factors . Both factors have transcriptional activity in yeast, and bind to DNA as dimers . Unlike other plant bZIP factors, DPBF-1 and 2 recognize sequences containing the ACACNNG core . Cloning of these factors demonstrates the power of the one-hybrid approach when optimally applied.

Bioorg Med Chem, 1997 Jun, 5(6), 1001 - 9
Strategy for RNA recognition by yeast histidyl-tRNA synthetase; Rudinger J et al.; Histidine aminoacylation systems are of interest because of the structural diversity of the RNA substrates recognized by histidyl-tRNA synthetases . Among tRNAs participating in protein synthesis, those specific for histidine all share an additional residue at their 5'-extremities . On the other hand, tRNA-like domains at the 3'--termini of some plant viruses can also be charged by histidyl-tRNA synthetases, although they are not actors in protein synthesis . This is the case for the RNAs from tobacco mosaic virus and its satellite virus but also those of turnip yellow and brome mosaic viruses . All these RNAs have intricate foldings at their 3'-termini differing from that of canonical tRNAs and share a pseudoknotted domain which is the prerequisite for their folding into structures mimicking the overall L-shape of tRNAs . This paper gives an overview on tRNA identity and rationalizes the apparently contradictory structural and aminoacylation features of histidine-specific tRNAs and tRNA-like structures . The discussion mainly relies on histidylation data obtained with the yeast synthetase, but the conclusions are of a more universal nature . In canonical tRNA(His), the major histidine identity element is the 'minus' 1 residue, since its removal impairs histidylation and conversely its addition to a non-cognate tRNA(Asp) confers histidine identity to the transplanted molecule . Optimal expression of histidine identity depends on the chemical nature of the -1 residue and is further increased and/or modulated by the discriminator base N73 and by residues in the anticodon . In the viral tRNA-like domains, the major identity determinant -1 is mimicked by a residue from the single-stranded L1 regions of the different pseudoknots . The consequences of this mimicry for the function of minimalist RNAs derived from tRNA-like domains are discussed . The characteristics of the histidine systems illustrate well the view that the core of the amino acid accepting RNAs is a scaffold that allows proper presentation of identity nucleotides to their amino acid identity counterparts in the synthetase and that different types of scaffoldings are possible.

Plant Cell, 1997 Jun, 9(6), 909 - 23
Modification of seed oil content and acyl composition in the brassicaceae by expression of a yeast sn-2 acyltransferase gene; Zou J et al.; A putative yeast sn-2 acyltransferase gene (SLC1-1), reportedly a variant acyltransferase that suppresses a genetic defect in sphingolipid long-chain base biosynthesis, has been expressed in a yeast SLC deletion strain . The SLC1-1 gene product was shown in vitro to encode an sn-2 acyltransferase capable of acylating sn-1 oleoyl-lysophosphatidic acid, using a range of acyl-CoA thioesters, including 18:1-, 22:1-, and 24:0-CoAs . The SLC1-1 gene was introduced into Arabidopsis and a high erucic acid-containing Brassica napus cv Hero under the control of a constitutive (tandem cauliflower mosaic virus 35S) promoter . The resulting transgenic plants showed substantial increases of 8 to 48% in seed oil content (expressed on the basis of seed dry weight) and increases in both overall proportions and amounts of very-long-chain fatty acids in seed triacylglycerols (TAGs) . Furthermore, the proportion of very-long-chain fatty acids found at the sn-2 position of TAGs was increased, and homogenates prepared from developing seeds of transformed plants exhibited elevated lysophosphatidic acid acyltransferase (EC 2.3.1.51) activity . Thus, the yeast sn-2 acyltransferase has been shown to encode a protein that can exhibit lysophosphatidic acid acyltransferase activity and that can be used to change total fatty acid content and composition as well as to alter the stereospecific acyl distribution of fatty acids in seed TAGs.

Curr Genet, 1997 Jun, 31(6), 469 - 80
The VPS4 gene is involved in protein transport out of a yeast pre-vacuolar endosome-like compartment; Finken-Eigen M et al.; Four yeast mutants were isolated in a screen for dominant-negative vacuolar protein-sorting mutants, secreting a carboxypeptidase Y-invertase hybrid protein . In addition to defects in the sorting/transport of soluble vacuolar hydrolases, the mutants accumulated a pre-vacuolar endosome-like compartment . The mutant alleles causing the defects were identified as the members of the VPS4 gene locus, each harbouring single-point mutations leading to amino-acid exchanges at positions 233 (E233Q), 211 (E211 K), and 178 (G178D) . These mutations all reside within a 200 amino-acid-long ATPase module, common to members of the AAA-protein family . The VPS4 gene product shows homology to the yeast Sec18p (50% similarity and 25% identity), which is involved in several vesicle-mediated protein transport steps and homotypic membrane fusion events . Disruption of the VPS4 gene leads to a recessive vacuolar protein-sorting phenotype . About 40% of newly synthesized CPY is secreted as the Golgi-modified p2CPY precursor form . Transport of secretory proteins to the plasma membrane is normal as demonstrated by the secretion of invertase and alpha-factor . The alpha-factor, however, is secreted as a partially processed precursor, caused by defects in late Golgi function . The vps4 mutants also exhibit defects in fluid-phase endocytosis, as demonstrated by the accumulation of Lucifer Yellow in a pre-vacuolar endosome-like compartment . Based on the pleiotropic phenotype of the vps4 mutants and on the sequence homology to NSF/Sec18p, we propose that the VPS4 gene product is required for efficient transport out of the pre-vacuolar endosome-like compartment.

Eur J Biochem, 1997 Jun 1, 246(2), 283 - 90
Evidence for channeling of intermediates in the oxidative pentose phosphate pathway by soybean and pea nodule extracts, yeast extracts, and purified yeast enzymes; Debnam PM et al.; Evidence is presented that intermediates of the oxidative pentose phosphate pathway (OPPP) are channeled from one pathway enzyme to the next . CO2 produced from {1-14C}glucose in the presence of unlabelled pathway intermediates contained much more radioactivity than predicted by a model in which pathway-produced intermediates are in equilibrium with identical molecules in the bulk phase . This was the case whether glucose 6-phosphate (Glc6P), 6-phosphogluconolactone, or 6-phosphogluconate was added . Assumptions involved in calculating the amount of 14CO2 predicted for free mixing of 14C-labelled and unlabelled intermediates are discussed, together with the following results . (a) 14CO2 production by pea nodules in the presence of 3 mM 6-phosphogluconate was higher than in its absence . (b) Apparent channeling of intermediates was much higher for purified yeast enzymes than for yeast extract . (c) 6-Phosphogluconate and 6-phosphogluconolactone were channeled between yeast Glc6P dehydrogenase and 6-phosphogluconate dehydrogenase despite the absence of 6-phosphogluconolactonase in the purified yeast enzyme mixture . (d) When purified yeast hexokinase was physically separated from Glc6P dehydrogenase and 6-phosphogluconate dehydrogenase by a dialysis membrane, there was no apparent channeling . (e) Poly(ethylene glycol), high salt and detergents had little effect on apparent channeling of OPPP intermediates, which is consistent with a stable complex of enzymes . On the other hand, density gradient centrifugation experiments suggested a more transient interaction between the enzymes . Taken together, the results support channeling of OPPP pathway intermediates.

Mol Biol Cell, 1997 Jun, 8(6), 1089 - 104
A novel Sec18p/NSF-dependent complex required for Golgi-to-endosome transport in yeast; Burd CG et al.; The vacuolar protein-sorting (VPS) pathway of Saccharomyces cerevisiae mediates localization of proteins from the trans-Golgi to the vacuole via a prevacuolar endosome compartment . Mutations in class D vacuolar protein-sorting (vps) genes affect vesicle-mediated Golgi-to-endosome transport and result in secretion of vacuolar proteins . Temperature-sensitive-for-function (tsf) and dominant negative mutations in PEP12, encoding a putative SNARE vesicle receptor on the endosome, and tsf mutations in VAC1, a gene implicated in vacuole inheritance and vacuolar protein sorting, were constructed and used to demonstrate that Pep12p and Vac1p are components of the VPS pathway . The sequence of Vac1p contains two putative zinc-binding RING motifs, a zinc finger motif, and a coiled-coil motif . Site-directed mutations in the carboxyl-terminal RING motif strongly affected vacuolar protein sorting . Vac1p was found to be tightly associated with membranes as a monomer and in a large SDS-resistant complex . By using Pep12p affinity chromatography, we found that Vac1p, Vps45p (SEC1 family member), and Sec18p (yeast N-ethyl maleimide-sensitive factor, NSF) bind Pep12p . Consistent with a functional role for this complex in vacuolar protein sorting, double pep12tsfvac1tsf and pep12tsf vps45tsf mutants exhibited synthetic Vps- phenotypes, the tsf phenotype of the vac1tsf mutant was rescued by overexpression of VPS45 or PEP12, overexpression of a dominant pep12 allele in a sec18-1 strain resulted in a severe synthetic growth defect that was rescued by deletion of PEP12 or VAC1, and subcellular fractionation of vac1 delta cells revealed a striking change in the fractionation of Pep12p and Vps21p, a rab family GTPase required for vacuolar protein sorting . The functions of Pep12p, Vps45p, and Vps21p indicate that key aspects of Golgi-to-endosome trafficking are similar to other vesicle-mediated transport steps, although the role of Vac1p suggests that there are also novel components of the VPS pathway.

Mol Biol Cell, 1997 Jun, 8(6), 957 - 72
Centromere position in budding yeast: evidence for anaphase A; Guacci V et al.; Although general features of chromosome movement during the cell cycle are conserved among all eukaryotic cells, particular aspects vary between organisms . Understanding the basis for these variations should provide significant insight into the mechanism of chromosome movement . In this context, establishing the types of chromosome movement in the budding yeast Saccharomyces cerevisiae is important since the complexes that mediate chromosome movement (microtubule organizing centers, spindles, and kinetochores) appear much simpler in this organism than in many other eukaryotic cells . We have used fluorescence in situ hybridization to begin an analysis of chromosome movement in budding yeast . Our results demonstrate that the position of yeast centromeres changes as a function of the cell cycle in a manner similar to other eukaryotes . Centromeres are skewed to the side of the nucleus containing the spindle pole in G1; away from the poles in mid-M and clustered near the poles in anaphase and telophase . The change in position of the centromeres relative to the spindle poles supports the existence of anaphase A in budding yeast . In addition, an anaphase A-like activity independent of anaphase B was demonstrated by following the change in centromere position in telophase-arrested cells upon depolymerization and subsequent repolymerization of microtubules . The roles of anaphase A activity and G1 centromere positioning in the segregation of budding yeast chromosomes are discussed . The fluorescence in situ hybridization methodology and experimental strategies described in this study provide powerful new tools to analyze mutants defective in specific kinesin-like molecules, spindle components, and centromere factors, thereby elucidating the mechanism of chromosome movement.

Genome Res, 1997 Jun, 7(6), 657 - 60
Centromeric and noncentromeric ADE2-selectable fragmentation vectors for yeast artificial chromosomes in AB1380; Heus JJ et al.; We have constructed a set of fragmentation vectors for the truncation of either the centromeric or the noncentromeric end of YACs containing a human DNA insert . These vectors carry ADE2 or HIS5 as the selectable marker, enabling direct use in AB1380, the host strain of most publicly available YAC libraries . Centromeric fragmentation vectors for AB1380 have not been reported previously; the noncentromeric vectors show high frequencies of fragmentation.

Curr Biol, 1997 Jun 1, 7(6), R366 - 8
Genetic redundancy: screening for selection in yeast; Brookfield JF; An unexpectedly large proportion of eukaryotic genes yield no obvious mutant phenotype when inactivated . An ingenious new approach using yeast allows all genes to be screened simultaneously for the presence of weak selection against lack-of-function mutations.

Nat Struct Biol, 1997 Jun, 4(6), 477 - 82
A molecular clamp in the crystal structure of the N-terminal domain of the yeast Hsp90 chaperone; Prodromou C et al.; Hsp90 is a highly specific chaperone for many signal transduction proteins, including steroid hormone receptors and a broad range of protein kinases . The crystal structure of the N-terminal domain of the yeast Hsp90 reveals a dimeric structure based on a highly twisted sixteen stranded beta-sheet, whose topology suggests a possible 30-domain-swapped structure for the intact Hsp90 dimer . The opposing faces of the beta-sheets in the dimer define a potential peptide-binding cleft, suggesting that the N-domain may serve as a molecular 'clamp' in the binding of ligand proteins to Hsp90.

J Infect Dis, 1997 Jun, 175(6), 1494 - 7
Immunogenicity of two doses of yeast recombinant hepatitis B vaccine in healthy older adults; Gellin BG et al.; To determine the immunogenicity of two doses of yeast recombinant hepatitis B virus (HBV) vaccine containing surface (S) protein, an open-label, multicenter trial was conducted in 199 healthy HBV-seronegative adults > or = 40 years old . Volunteers were randomly assigned to 1 of 5 groups to receive a total of three 10-microg doses, at 0, 1, and 6 months, or a total of two doses of 20 microg and 10 microg, 20 microg and 20 microg, 40 microg and 10 microg, or 40 microg and 20 microg at 0 and 6 months . The 40-microg/20-microg regimen elicited the highest rate of seroprotection (96.2%), with a geometric mean titer of antibody against the S protein of 369 mIU/mL, not significantly different from the 536 mIU/mL achieved with three doses . These results suggest that a two-dose regimen can achieve seroprotection similar to that of the three-dose regimen . Whether a shorter interval can be used or a booster dose will be needed later to confer durable immunity are unknown.

Protein Expr Purif, 1997 Jun, 10(1), 132 - 40
Yeast chromatin reconstitution system using purified yeast core histones and yeast nucleosome assembly protein-1; Pilon J et al.; Transcription regulation in the cell occurs in the context of chromatin . It follows that a thorough investigation of the mechanism of transcription regulation must take into account the role of chromatin structure . Through classical and molecular genetic experiments in yeast, great strides have been made in understanding the role of chromatin in eukaryotic gene regulation . To achieve a more detailed understanding of the biochemical mechanism of transcription regulation, a yeast chromatin reconstitution system is needed . This need drove us to develop a yeast core histone purification procedure for the reconstitution of these histones into chromatin templates using components wholly derived from yeast . We have purified native yeast core histones in milligram quantities and we have shown these histones to be competent for reconstitution of chromatin templates using yeast nucleosome assembly protein-1 . This accomplishment sets the stage for studies using the full power of yeast as an experimental organism to investigate the role of chromatin in transcription regulation.

Blood, 1997 Jun 1, 89(11), 4196 - 203
Recombinant sickle hemoglobin containing a lysine substitution at Asp-85(alpha): expression in yeast, functional properties, and participation in gel formation; Himanen JP et al.; Clinical modalities based on inhibition of gelation of HbS are hindered by the lack of quantitative information on the extent of participation of different amino acid residues in the aggregation process . One such site is Asp-85(alpha), which is involved in a parallel interdouble strand ionic interaction with Lys-144(beta) according to the crystal structure of HbS, but electron microscopy does not specifically show Asp-85(alpha) as a contact site for fiber formation . Using a yeast recombinant system, we have substituted this site by Lys to abolish ion pairing and to make a quantitative determination of its participation in aggregation . The purified double mutant was shown to have the expected pI, the calculated molecular weight, correct amino acid composition, and peptide map . The recombinant double mutant has an oxygen affinity of 10 mm Hg, which is identical to that for HbA and HbS under the same conditions; it also has high cooperativity with an average n value of 2.7 . The change in P50 in response to chloride ions was about 25% less than that for HbA or HbS and is ascribed to the introduction of a new positive charge near one of the major oxygen-linked chloride binding sites of hemoglobin . The gelation concentration of the double mutant was measured by a new procedure (Bookchin et al, 1994); the maximal amount of soluble hemoglobin (Csat) in the presence of dextran indicated a decreased tendency for gelation with a Csat of 53 mg/mL compared with 34 mg/mL for HbS . This inhibitory effect is smaller than that of the E6V(beta)/L88A(beta) (Csat, 67 mg/mL) and the E6V(beta)/K95I(beta) (Csat, 90 mg/mL) recombinant hemoglobins . Thus, we would classify Asp-85(alpha) as a moderate contributor to the strength of the HbS aggregate . This wide range of gelation values demonstrates that some sites are more important than others in promoting HbS aggregation.

Curr Opin Cell Biol, 1997 Jun, 9(3), 329 - 36
A comparison of mammalian and yeast pre-mRNA 3'-end processing; Keller W et al.; Many components of the mammalian and yeast pre-mRNA 3'-end-processing machinery have recently been purified and cDNAs or genes coding for these factors have been cloned . Most of the factors consist of multiple subunits, some of which serve to bind the RNA substrate, others of which are involved in forming a complex network of protein-protein interactions . Most of the mammalian 3'-end-processing factors are similar in their amino acid sequence to the yeast factors, indicating that they have a common evolutionary history.

Curr Opin Cell Biol, 1997 Jun, 9(3), 383 - 7
Molecular model for telomeric heterochromatin in yeast; Grunstein M; A molecular model for the formation of yeast core telomeric heterochromatin has been proposed recently . The RAP1 protein provides the specificity for the localization of heterochromatin through its recognition of telomeric DNA sequences . Its complexing with silencing information regulators (SIR2, SIR3 and SIR4) and histones H3 and H4 generates a folded-back DNA structure . This not only represses adjacent genes through SIR-protein-histone interactions, but also enables condensation and protection of the telomeric end . The SIR2 and SIR4 levels at the core differ from those in the extended telomeric heterochromatin produced when the limiting protein, SIR3, is overexpressed.

Mol Cell Biol, 1997 Jun, 17(6), 3388 - 97
A ubiquitin-conjugating enzyme in fission yeast that is essential for the onset of anaphase in mitosis; Osaka F et al.; A cDNA encoding a ubiquitin-conjugating enzyme designated UbcP4 in fission yeast was isolated . Disruption of its genomic gene revealed that it was essential for cell viability . In vivo depletion of the UbcP4 protein demonstrated that it was necessary for cell cycle progression at two phases, G2/M and metaphase/anaphase transitions . The G2 arrest of UbcP4-depleted cells was dependent upon chk1, which mediates checkpoint pathway . UbcP4-depleted cells arrested at metaphase had condensed chromosomes but were defective in separation . However, septum formation and cytokinesis were not restrained during the metaphase arrest . Overexpression of UbcP4 specifically rescued the growth defect of cut9ts cells at a restrictive temperature . cut9 encodes a component of the anaphase-promoting complex (APC) which is required for chromosome segregation at anaphase and moreover is defined as cyclin-specific ubiquitin ligase . Cdc13, a mitotic cyclin in fission yeast, was accumulated in the UbcP4-depleted cells . These results strongly suggested that UbcP4 is a ubiquitin-conjugating enzyme working in conjunction with APC and mediates the ubiquitin pathway for degradation of "sister chromatid holding protein(s)" at the onset of anaphase and possibly of mitotic cyclin at the exit of mitosis.

Mol Cell Biol, 1997 Jun, 17(6), 3081 - 93
Structure-function analysis of TAF130: identification and characterization of a high-affinity TATA-binding protein interaction domain in the N terminus of yeast TAF(II)130; Bai Y et al.; We report structure-function analyses of TAF130, the single-copy essential yeast gene encoding the 130,000-Mr yeast TATA-binding protein (TBP)-associated factor TAF(II)130 (yTAF(II)130) . A systematic family of TAF130 mutants was generated, and these mutant TAF130 alleles were introduced into yeast in both single and multiple copies to test for their ability to complement a taf130delta null allele and support cell growth . All mutant proteins were stably expressed in vivo . The complementation tests indicated that a large portion (amino acids 208 to 303 as well as amino acids 367 to 1037) of yTAF(II)130 is required to support cell growth . Direct protein blotting and coimmunoprecipitation analyses showed that two N-terminal deletions which remove portions of yTAF(II)130 amino acids 2 to 115 dramatically decrease the ability of these mutant yTAF(II)130 proteins to bind TBP . Cells bearing either of these two TAF130 mutant alleles also exhibit a slow-growth phenotype . Consistent with these observations, overexpression of TBP can correct this growth deficiency as well as increase the amount of TBP interacting with yTAF(II)130 in vivo . Our results provide the first combined genetic and biochemical evidence that yTAF(II)130 binds to yeast TBP in vivo through yTAF(II)130 N-terminal sequences and that this binding is physiologically significant . By using fluorescence anisotropy spectroscopic binding measurements, the affinity of the interaction of TBP for the N-terminal TBP-binding domain of yTAF(II)130 was measured, and the Kd was found to be about 1 nM . Moreover, we found that the N-terminal domain of yTAF(II)130 actively dissociated TBP from TATA box-containing DNA.

Nucleic Acids Res, 1997 Jun 1, 25(11), 2146 - 52
Functional and physical interaction between the yeast splicing factors Slu7 and Prp18; Zhang X et al.; We show that the requirement for Prp18 during the second step of actin pre-mRNA splicing in vitro is dictated by the distance between the branch point and the 3'splice site . Prp18 is dispensable for splicing of precursor RNAs in which the interval between the branch point and 3'splice site is <12 nt . This resembles the requirement for another second step factor, Slu7 . Excess Slu7 protein can bypass the need for Prp18 in vitro , suggesting that Slu7 and Prp18 function in a concerted manner . Physical interaction between Slu7 and Prp18 was demonstrated by using the two-hybrid assay . Deletion mutants of SLU7 were tested for their ability to support growth of a slu7 null strain . Removal of 199 amino acids from the N-terminus of the 382 amino acid Slu7 protein did not affect cell viability at 25 degrees C . A more extensive N-terminal deletion of 221 amino acids was lethal, as was a C-terminal deletion of 47 amino acids . Deleted versions of Slu7 were also tested for interaction with Prp18 in the two-hybrid system . We define a segment of Slu7 from residue 200 to 224 that is necessary for interaction with Prp18.

J Mol Biol, 1997 May 30, 269(1), 67 - 81
Differential effects of aromatic and charged residue substitutions in the RNA binding domains of the yeast poly(A)-binding protein; Deardorff JA et al.; The yeast poly(A)-binding protein (Pab1p) contains four RNA recognition motifs (RRMs) . Site-directed mutations were introduced into each of these RRMs in order to investigate their relative contributions to specific and non-specific RNA binding, and to determine the consequences of these mutations on the ability of Pab1p to support viability . Specifically, a charged and an aromatic residue that were predicted to be involved in RNA binding were mutated in each RRM . These mutations revealed that the second RRM is primarily responsible for poly(A) binding, while the fourth RRM is primarily responsible for non-specific polypyrimidine RNA binding . The mutated aromatic residues in each RRM contributed to both modes of binding whereas the mutated charged residues contributed primarily to non-specific RNA binding . RNA binding in vivo correlated with the in vitro binding measurements . Furthermore, RNA binding, but not high-affinity poly(A) binding, correlated with the ability of Pab1p to sustain yeast cell viability . These data suggest that a single aromatic substitution in Pab1p can significantly reduce its RNA binding ability, that the capacity of Pab1p to bind poly(A) as well as other RNAs is mediated by distinct residues within different RRMs, and that Pab1p does not require high affinity poly(A) tail binding to perform its essential function.

J Biol Chem, 1997 May 30, 272(22), 14356 - 64
SHY1, the yeast homolog of the mammalian SURF-1 gene, encodes a mitochondrial protein required for respiration; Mashkevich G et al.; C173 and W125 are pet mutants of Saccharomyces cerevisiae, partially deficient in cytochrome oxidase but with elevated concentrations of cytochrome c . Assays of electron transport chain enzymes indicate that the mutations exert different effects on the terminal respiratory pathway, including an inefficient transfer of electrons between the bc1 and the cytochrome oxidase complexes . A cloned gene capable of restoring respiration in C173/U1 and W125 is identical to reading frame YGR112w of yeast chromosome VII (GenBank Z72897Z72897) . The encoded protein is homologous to the product of the mammalian SURF-1 gene . In view of the homology, the yeast gene has been designated SHY1 (Surf Homolog of Yeast) . An antibody against the carboxyl-terminal half of Shy1p has been used to localize the protein in the inner mitochondrial membrane . Deletion of part of SHY1 produces a phenotype similar to that of G91 mutants . Disruption of SHY1 at a BamHI site, located approximately 2/3 of the way into the gene, has no obvious phenotypic consequence . This evidence, together with the ability of a carboxyl-terminal coding sequence starting from the BamHI site to complement a shy1 mutant, suggests that the Shy1p contains two domains that can be separately expressed to form a functional protein.

J Biol Chem, 1997 May 30, 272(22), 14208 - 13
Purification and characterization of Fet3 protein, a yeast homologue of ceruloplasmin; de Silva D et al.; The FET3 gene product of Saccharomyces cerevisiae is an essential component of the high affinity iron transport system . Based on FET3 sequence homology to the multicopper oxidase family and iron oxidation studies in spheroplasts (De Silva, D . M., Askwith, C . C., Eide, D., and Kaplan, J . (1995) J . Biol . Chem . 270, 1098-1101), it was hypothesized that the Fet3 protein (Fet3p) was a cell surface ferroxidase . To further characterize the protein, we have isolated Fet3p from yeast membranes and purified the protein to apparent homogeneity . Consistent with its localization at the plasma membrane, Fet3p is a glycosylated protein . SDS-polyacrylamide gel electrophoresis analysis showed that the protein was present in two differentially glycosylated forms of approximately 120 and 100 kDa . Purified Fet3p is a copper-containing protein that is able to catalyze the oxidation of a variety of organic compounds in addition to ferrous iron . Azide and metal chelators strongly inhibited enzyme activity . Iron appeared to be the best substrate for the enzyme, and the apparent Km for ferrous oxidation was 2 microM . Interestingly, Fet3p was able to effectively catalyze the incorporation of iron onto apotransferrin . We conclude that Fet3p is a ferro-O2-oxidoreductase in yeast, homologous to the human plasma protein ceruloplasmin.

Nature, 1997 May 29, 387(6632 Suppl), 7 - 65
Overview of the yeast genome; Mewes HW et al.; The collaboration of more than 600 scientists from over 100 laboratories to sequence the Saccharomyces cerevisiae genome was the largest decentralised experiment in modern molecular biology and resulted in a unique data resource representing the first complete set of genes from a eukaryotic organism . 12 million bases were sequenced in a truly international effort involving European, US, Canadian and Japanese laboratories . While the yeast genome represents only a small fraction of the information in today's public sequence databases, the complete, ordered and non-redundant sequence provides an invaluable resource for the detailed analysis of cellular gene function and genome architecture . In terms of throughput, completeness and information content, yeast has always been the lead eukaryotic organism in genomics; it is still the largest genome to be completely sequenced.

Biochemistry, 1997 May 27, 36(21), 6367 - 76
Yeast protein farnesyltransferase: a pre-steady-state kinetic analysis; Mathis JR et al.; Protein farnesyltransferase catalyzes alkylation of the cysteine in a carboxy-terminal CaaX motif where a is typically an aliphatic amino acid and X is alanine, methionine, serine, glutamine, or cysteine by a farnesyl residue . The modification enhances the lipophilicity of farnesylated proteins and promotes their association with membranes as part of their normal cellular function . Among the proteins modified by farnesyl residues is Ras, an important component in the signal transduction network for cell division that has been implicated in several forms of human cancer . In this paper, we describe isotope trapping, rapid quench, and single turnover experiments with the yeast enzyme using farnesyl diphosphate and the short peptide RTRCVIA as substrates . The kinetic constants for substrate binding, chemistry, and product release were determined from a fit of the differential equations describing the minimal catalytic mechanism to the kinetic data by numerical integration . Rate constants for chemistry and product release were 10.5 and 3.5 s(-1), respectively . The dissociation rate constant (33 s(-1)) for release of peptide from the ternary enzyme-substrate complex was three times larger than the rate constant for chemistry . The enthalpy of reaction, delta Hrxn = -17 +/- 1 kcal/mol for farnesylation of cysteine, was measured by microcalorimetry . Isotope trapping experiments revealed that the enzyme-farnesyl diphosphate complex was efficiently trapped by peptide but that the enzyme-peptide complex was not trapped by farnesyl diphosphate . These results are consistant with an ordered mechanism for formation of a catalytically competent ternary enzyme-farnesyl diphosphate-peptide complex.

Proc Natl Acad Sci U S A, 1997 May 27, 94(11), 5820 - 5
Human BRCA1 inhibits growth in yeast: potential use in diagnostic testing; Humphrey JS et al.; Germline-inactivating mutations of BRCA1 result in a hereditary predisposition to breast and ovarian cancer . Truncating mutations of BRCA1 predispose to cancer and can be ascertained by protein truncation testing or sequencing . However, cancer-predisposing missense mutations of BRCA1 are difficult to distinguish from polymorphisms by genetic testing methods currently used . Here we show that expression of BRCA1 or BRCA1 fused to a GAL4 activation domain in Saccharomyces cerevesiae inhibits growth, resulting in small colonies easily distinguishable from vector-transformed controls . The growth inhibitory effect can be localized to sequences encoding the recently described BRCA1 C-terminal domains . Growth suppression by a BRCA1 fusion protein is not influenced by introduction of neutral polymorphisms but is diminished or abolished by frameshift, nonsense, or disease-associated missense mutations located in the C-terminal 305 amino acids of BRCA1 . These observations may permit the functional significance of many BRCA1 sequence changes to be assessed in yeast . Additionally, the correlation of growth suppression with wild-type forms of BRCA1 suggests that the assay may be capable of detecting functionally conserved interactions between the evolutionarily conserved BRCA1 C-terminal domains and cellular elements found in both human and yeast cells.

Proc Natl Acad Sci U S A, 1997 May 27, 94(11), 5679 - 84
The I/LWEQ module: a conserved sequence that signifies F-actin binding in functionally diverse proteins from yeast to mammals; McCann RO et al.; Talin is an actin-binding protein involved in integrin-mediated cell adhesion and spreading . The C-terminal 197 amino acids of vertebrate talin are 45% similar to the C-terminal residues of Sla2, a yeast protein implicated in polarized assembly of the yeast actin cytoskeleton . Talin is also homologous in this region to nematode talin, cellular slime mold filopodin, and an Sla2 homolog from nematode . Analysis of the conserved C-terminal sequences of these five proteins with BLOCK MAKER reveals a series of four blocks, which we name the I/LWEQ module after the conserved initial residues in each block . Experiments presented here show that the conserved protein domain represented by the I/LWEQ module competes quantitatively with native talin for binding to F-actin in vitro . Furthermore, the corresponding domain of Sla2 binds to both yeast and vertebrate F-actin in vitro . Mutation of one of the conserved residues in the fourth conserved block abolishes the interaction of the Sla2 I/LWEQ module with F-actin . These results establish the location of an F-actin binding domain in native talin, demonstrate that direct interaction of Sla2 with actin is a possible basis for its effect on the actin cytoskeleton in vivo, and define the I/LWEQ consensus as a new actin-binding motif.

Proc Natl Acad Sci U S A, 1997 May 27, 94(11), 5662 - 6
Characterization of VPS41, a gene required for vacuolar trafficking and high-affinity iron transport in yeast; Radisky DC et al.; Mutations in the yeast gene VPS41 give rise to poor growth on low iron medium, severe alterations in vacuolar morphology, and cause the missorting of membranous and soluble vacuolar proteins . Our studies predict that VPS41 encodes a hydrophilic protein of 992 amino acids that contains no obvious signal sequence or hydrophobic domains . The deduced Vps41p sequence contains a domain rich in glutamic and aspartic residues, as well as a domain with resemblance to a region of clathrin heavy chain . We have also identified and sequenced putative VPS41 homologues from Caenorhabditis elegans, plants, and humans . The VPS41 homologues (but not the yeast VPS41 itself) contain a conserved cysteine-rich RING-H2 zinc finger at their COOH termini . Biochemical experiments suggest that VPS41 functions in post-Golgi protein processing: the deletion mutant exhibits defective high affinity transport due to impaired Fet3p activity and also exhibits defects in the processing and sorting of multiple vacuolar hydrolases.

Proc Natl Acad Sci U S A, 1997 May 27, 94(11), 5611 - 6
Cdc6p-dependent loading of Mcm proteins onto pre-replicative chromatin in budding yeast; Donovan S et al.; The Cdc6 protein is essential for the assembly of pre-replicative complexes (pre-RCs) at origins of DNA replication in the budding yeast Saccharomyces cerevisiae . This reaction is blocked in vivo by the cyclin-dependent kinase Cdc28p, together with its regulatory subunits, the B type cyclins that are present throughout S, G2, and M phases . Because the destruction of B type cyclins and the consequent inactivation of the kinase are essential for exit from mitosis, pre-RC formation can only occur after passage through mitosis . Therefore, pre-RC formation has been proposed to be essential for coupling S phase and mitosis and for limiting DNA replication to once per cell cycle . The Mcm2-7 family of proteins has been implicated in limiting replication to once per cell cycle from experiments with Xenopus egg extracts . Here we show that the Mcm proteins of budding yeast are abundant and are quantitatively found in a chromatin-enriched fraction specifically during the G1 phase of the cell cycle . This chromatin binding depends on the de novo synthesis of Cdc6p, providing evidence that a conserved biochemical pathway plays a critical role in coordinating DNA replication with mitosis in both yeast and higher eukaryotes . Cdc6p and the origin recognition complex can be selectively removed from this chromatin-enriched fraction without removing the Mcm proteins . From these results, we propose that Cdc6p (and the origin recognition complex) nucleates the binding of Mcm proteins to chromatin, but once bound, the Mcm proteins appear to interact tightly with some other component of chromatin.

Proc Natl Acad Sci U S A, 1997 May 27, 94(11), 5550 - 5
Copper-mediated repression of the activation domain in the yeast Mac1p transcription factor; Graden JA et al.; The expression of a number of genes encoding products involved in copper ion uptake in yeast is specifically inhibited by copper ions . We show here that copper metalloregulation occurs through Cu-dependent repression of the transactivation activity of Mac1p . A segment of the yeast transcription factor Mac1p was identified that activated transcription in vivo in a heterologous system using fusion polypeptides with the yeast Gal4 DNA-binding domain . The Gal4/Mac1p hybrid exhibits transactivation activity that is repressed in cells cultured in the presence of copper salts and derepressed in cells with reduced copper uptake . The repressive effect is specific for copper ions . The concentration dependency of the Cu-inactivation of Gal4/Mac1p is similar to that of Cu-inhibition of CTR1 expression, a known Cu-regulated gene in vivo . Copper inhibition of gene expression is not observed with a Gal4/Mac1p chimera containing the MAC1(up1) substitution within the transactivation domain . Cells harboring the MAC1(up1) allele fail to attenuate FRE1 and CTR1 expression in a Cu-dependent manner . Additional MAC1(up) alleles exist within the first of two cysteine-rich sequence motifs adjacent to the His --> Gln MAC1(up1) encoded substitution . Thus, Cu-regulation of Mac1p function arises from a novel Cu-specific repression of the transactivation domain function . Models for the mechanism of Cu-repression of Mac1p function will be discussed.

J Mol Biol, 1997 May 23, 268(5), 816 - 21
A histidine variant of yeast iso-1-cytochrome c that strongly affects the energetics of the denatured state; Godbole S et al.; Iso-1-cytochrome c has been engineered to remove all histidine residues not involved in heme ligation in the native state to produce a variant designated TM . Single histidine residues were then introduced at positions 26, TM + His26, and 54, TM + His54 . Since histidine residues not involved in native state heme ligation are known to replace the methionine 80 heme ligand in denatured cytochrome c, these variants were expected to affect the structure of the denatured state . Guanidine hydrochloride denaturations were performed to assess the stability of these proteins relative to the wild-type protein . The free energy difference for heme ligation in the denatured state was assessed by pH titration . The experimentally observed mutation-induced change (delta deltaG(D-state)) in the free energy of heme ligation for unfolded TM + His54 versus TM + His26 is -0.4 kcal/mol . The expected mutation-induced change in delta deltaG(D-state) calculated for a random coil unfolded state is +2 kcal/mol . Thus, unfolded TM + His54 has residual structure stabilizing its denatured state by -2.4 kcal/mol relative to TM + His26 . The results imply that the denatured state can contribute significantly to mutation-induced changes in the free energy of unfolding of a protein.

Science, 1997 May 23, 276(5316), 1252 - 5
Ndj1p, a meiotic telomere protein required for normal chromosome synapsis and segregation in yeast; Conrad MN et al.; The Saccharomyces cerevisiae gene NDJ1 (nondisjunction) encodes a protein that accumulates at telomeres during meiotic prophase . Deletion of NDJ1 (ndj1Delta) caused nondisjunction, impaired distributive segregation of linear chromosomes, and disordered the distribution of telomeric Rap1p, but it did not affect distributive segregation of circular plasmids . Induction of meiotic recombination and the extent of crossing-over were largely normal in ndj1Delta cells, but formation of axial elements and synapsis were delayed . Thus, Ndj1p may stabilize homologous DNA interactions at telomeres, and possibly at other sites, and it is required for a telomere activity in distributive segregation.

J Biol Chem, 1997 May 23, 272(21), 13786 - 92
The yeast Fre1p/Fre2p cupric reductases facilitate copper uptake and are regulated by the copper-modulated Mac1p activator; Georgatsou E et al.; Fre1p and Fre2p are ferric reductases which account for the total plasma membrane associated activity, a prerequisite for iron uptake, in Saccharomyces cerevisiae . The two genes are transcriptionally induced by iron depletion . In this communication, we provide evidence that Fre2p has also cupric reductase activity, as has been previously shown for Fre1p (Hassett, R., and Kosman, D.J . (1995) J . Biol . Chem . 270, 128-134) . Both Fre1p and Fre2p enzymes are functionally significant for copper uptake, as monitored by the accumulation of the copper-regulated CUP1 and CTR1 mRNAs in fre1Delta, fre2Delta, and fre1Deltafre2Delta mutant strains . However, only Fre1p activity is induced by copper depletion, even in the presence of iron . This differential copper-dependent regulation of Fre1p and Fre2p is exerted at the transcriptional level of the two genes . We have shown that Mac1p, known to affect the basal levels of FRE1 gene expression (Jungmann, J., Reins, H.-A., Lee, J., Romeo, A., Hassett, R., Kosman, D., and Jentsch, S . (1993) EMBO J . 12, 5051-5056), accounts for both the copper-dependent induction of FRE1 and down-regulation of FRE2 gene . Finally, Mac1p transcriptional activation function is itself modulated by the availability of copper.

J Biol Chem, 1997 May 23, 272(21), 13766 - 71
Cell cycle-dependent expression and spindle pole localization of a novel human protein kinase, Aik, related to Aurora of Drosophila and yeast Ipl1; Kimura M et al.; Mutations in Aurora of Drosophila and related Saccharomyces cerevisiae Ipl1 kinase are known to cause abnormal chromosome segregation . We have isolated a cDNA encoding a novel human protein kinase of 402 amino acids with a predicted molecular mass of 45.9 kDa, which shares high amino acid identities with the Aurora/Ipl1 protein kinase family; hence the cDNA is designated as aik (aurora/IPL1-related kinase) . Amino acid sequence of C-terminal kinase domain of Aik shares 86, 86, 72, 59, and 49% identity with those of Xenopus XLP46APK and XLP46BPK, mouse STK-1, Aurora of Drosophila, and yeast Ipl1, respectively, whereas N-terminal domain of Aik shares high homology only with those of XLP46APK and XLP46BPK . Northern and Western blotting analyses revealed that Aik is expressed highly in testis and various proliferating cells including HeLa cells . In HeLa cells, the endogenous levels of aik mRNA and protein contents are tightly regulated during cell cycle progression . Both of these levels are low in G1/S, accumulate during G2/M, and reduce rapidly after mitosis . Its protein kinase activity is also enhanced at mitosis as inferred by exogenous casein phosphorylation . Immunofluorescence studies using a specific antibody have shown that Aik is localized to the spindle pole during mitosis, especially from prophase through anaphase . These results strongly suggest that Aik is a novel member of a protein kinase family possibly involved in a centrosome function(s) such as chromosome segregation or spindle formation.

Mol Gen Genet, 1997 May 20, 254(5), 555 - 61
Enhancement of Ty transposition at the ADH4 and ADH2 loci in meiotic yeast cells; Ribeiro-dos-Santos G et al.; Genome polymorphism in the yeast Saccharomyces cerevisiae is frequently the result of transposition and recombination events involving Ty elements . The activity of these retrotransposons is closely integrated with the life cycle of the host . Ty transcription is repressed in diploid, but not haploid, cells and is induced by certain stress conditions . We have found that Ty transposition at the ADH4 and ADH2 loci is not only active, but 50-fold more frequent in meiotic yeast than in mitotic cells . These data provide a further example of the success of Ty elements in maximising their own chances of spread and survival while minimising the risks to the host yeast population.

Biochemistry, 1997 May 20, 36(20), 6090 - 9
The angle between the anticodon and aminoacyl acceptor stems of yeast tRNA(Phe) is strongly modulated by magnesium ions; Friederich MW et al.; Many tRNAs undergo tertiary folding transitions at temperatures well below the main thermally induced (hyperchromic) transition . Such transitions are essentially isochromic and isoenthalpic and display an absolute requirement for divalent cations; however, the nature of the structural transition is not known for any tRNA . Using a combination of transient electric birefringence (TEB) and gel electrophoretic measurements, we have characterized the influence of magnesium ions on the apparent angle between the anticodon and acceptor stems of a yeast tRNA(Phe) construct . TEB is a particularly sensitive method for quantifying the bends introduced in RNA by various nonhelix elements . In the current instance, the tRNA construct comprises an unmodified tRNA(Phe) molecule in which the anticodon and acceptor stems have been extended by approximately 70 bp to more effectively "report" the interstem angles . Upon the addition of sub-millimolar concentrations of magnesium ions, the tRNA core undergoes a substantial rearrangement in tertiary structure, passing from an open form with an apparent interstem angle of approximately 150 degrees to a conformation with an interstem angle of approximately 70 degrees (200 microM Mg2+) . Further addition of magnesium ions results in a minor adjustment of the apparent interstem angle to approximately 80-90 degrees, in line with earlier results . Finally, the magnesium-induced structural transition is essentially isochromic, in agreement with previous observations with native tRNAs . The current results suggest that changes in local divalent ion concentration in the ribosome could profoundly affect the global conformations of tRNAs during the translation cycle.

J Biol Chem, 1997 May 16, 272(20), 13372 - 9
QSR1, an essential yeast gene with a genetic relationship to a subunit of the mitochondrial cytochrome bc1 complex, codes for a 60 S ribosomal subunit protein; Dick FA et al.; QSR1 (quinol-cytochrome c reductase subunit-requiring) is a highly conserved, essential gene in Saccharomyces cerevisiae that was identified through a synthetic lethal screen by its genetic relationship to QCR6, the gene for subunit 6 (Qcr6p) of the mitochondrial cytochrome bc1 complex . The function of the QSR1-encoded protein (Qsr1p) and its relationship to the QCR6-encoded protein are unknown . When yeast cell lysates are fractionated by density gradient centrifugation, Qsr1p separates from organelles and sediments with a uniformly sized population of particles that are similar to eukaryotic ribosomes upon velocity gradient centrifugation . When 40 S and 60 S ribosomal subunits are separated on velocity gradients, Qsr1p is found exclusively with the 60 S subunits, where it is a stoichiometric component . Extracts prepared from qsr1-1 cells are defective in in vitro translation assays relative to the wild type . In yeast cell lysates in which QCR6 rescues an otherwise lethal qsr1-1 mutation, Qcr6p is found only in mitochondria, both in respiratory-competent cells and in rho0 cells in which the bc1 complex is no longer present . These results suggest that suppression of the qsr1-1 mutation by QCR6 occurs by a trans-relationship across the outer mitochondrial membrane.

J Biol Chem, 1997 May 16, 272(20), 13165 - 70
Distinct regulation of osmoprotective genes in yeast and mammals . Aldose reductase osmotic response element is induced independent of p38 and stress-activated protein kinase/Jun N-terminal kinase in rabbit kidney cells; Kultz D et al.; In yeast glycerol-3-phosphate dehydrogenase 1 is essential for synthesis of the osmoprotectant glycerol and is osmotically regulated via the high osmolarity glycerol (HOG1) kinase pathway . Homologous protein kinases, p38, and stress-activated protein kinase/Jun N-terminal kinase (SAPK/JNK) are hyperosmotically activated in some mammalian cell lines and complement HOG1 in yeast . In the present study we asked whether p38 or SAPK/JNK signal synthesis of the osmoprotectant sorbitol in rabbit renal medullary cells (PAP-HT25), analogous to the glycerol system in yeast . Sorbitol synthesis is catalyzed by aldose reductase (AR) . Hyperosmolality increases AR transcription through an osmotic response element (ORE) in the 5'-flanking region of the AR gene, resulting in elevated sorbitol . We tested if AR-ORE is targeted by p38 or SAPK/JNK pathways in PAP-HT25 cells . Hyperosmolality (adding 150 mM NaCl) strongly induces phosphorylation of p38 and of c-Jun, a specific target of SAPK/JNK . Transient lipofection of a dominant negative mutant of SAPK kinase, SEK1-AL, into PAP-HT25 cells specifically inhibits hyperosmotically induced c-Jun phosphorylation . Transient lipofection of a dominant negative p38 kinase mutant, MKK3-AL, into PAP-HT25 cells specifically suppresses hyperosmotic induction of p38 phosphorylation . We cotransfected either one of these mutants or their empty vector with an AR-ORE luciferase reporter construct and compared the hyperosmotically induced increase in luciferase activity with that in cells lipofected with only the AR-ORE luciferase construct . Hyperosmolality increased luciferase activity equally (5-7-fold) under all conditions . We conclude that hyperosmolality induces p38 and SAPK/JNK cascades in mammalian renal cells, analogous to inducing the HOG1 cascade in yeast . However, activation of p38 or SAPK/JNK pathways is not necessary for transcriptional regulation of AR through the ORE . This finding stands in contrast to the requirement for the HOG1 pathway for hyperosmotically induced activation of yeast GPD1.

J Biol Chem, 1997 May 16, 272(20), 13159 - 64
The yeast SPC22/23 homolog Spc3p is essential for signal peptidase activity; Meyer HA et al.; In eucaryotic cells signal sequences of secretory and membrane proteins are cleaved by the signal peptidase complex during their transport into the lumen of the endoplasmic reticulum . The signal peptidase complex in yeast consists of four subunits . To date, three of these subunits have been functionally characterized . One of them, the Sec11p, is essential for viability of yeast cells . It shows significant homology to the mammalian SPC18 and SPC21 as well as to bacterial leader peptidases . Two other subunits, Spc1p and Spc2p, have been shown to be homologous to mammalian SPC12 and SPC25, respectively, and are not essential for protein translocation or signal peptide cleavage . We have purified and analyzed the fourth subunit of yeast signal peptidase, Spc3p . The protein is essential for viability of yeast cells . Depletion of SPC3 leads to accumulation of precursors of secretory proteins in vivo and to the loss of the signal peptidase activity in vitro . Therefore, in contrast to the bacterial leader peptidases, yeast signal peptidase requires a second subunit for its function.

J Biol Chem, 1997 May 16, 272(20), 13152 - 8
In addition to SEC11, a newly identified gene, SPC3, is essential for signal peptidase activity in the yeast endoplasmic reticulum; Fang H et al.; Among the three characterized subunits comprising the signal peptidase complex of the yeast Saccharomyces cerevisiae (Sec11p, Spc1p, and Spc2p), only Sec11p is essential for cell growth, signal peptide cleavage, and signal peptidase-dependent protein degradation . Here we report the cloning of the SPC3 gene encoding the homolog to mammalian signal peptidase subunit SPC22/23 . We find that Spc3p is also required for cell growth and signal peptidase activity within the yeast endoplasmic reticulum.

J Biol Chem, 1997 May 16, 272(20), 12961 - 7
Casein kinase II catalyzes tyrosine phosphorylation of the yeast nucleolar immunophilin Fpr3; Wilson LK et al.; In the yeast Saccharomyces cerevisiae, the nucleolar immunophilin, Fpr3, is phosphorylated at tyrosine and dephosphorylated by the phosphotyrosine-specific phosphoprotein phosphatase, Ptp1 . In Ptp1-deficient cells, Fpr3 contains phospho-Tyr at a single site (Tyr184), but also contains phospho-Ser and phospho-Thr . Ser186 (adjacent to Tyr184) is situated within a canonical site for phosphorylation by casein kinase II (CKII) . Yeast cell lysates contain an activity that binds to Fpr3 in vitro and phosphorylates Fpr3 at Ser, Thr, and Tyr; this activity was found to be dependent on expression of functional yeast CKII . Moreover, purified Fpr3 was phosphorylated on Tyr184 in vitro by either purified yeast CKII or purified, bacterially-expressed human CKII . Likewise, phosphorylation of Fpr3 at tyrosine in vivo was markedly enhanced in yeast cells overexpressing a heterologous (Drosophila) CKII, but was undetectable in yeast cells carrying only a temperature-sensitive allele of the endogenous CKII, even when the cells were grown at a permissive temperature . Phosphorylation of Fpr3 at Tyr184 by CKII in vitro lagged behind phosphorylation of Fpr3 at Ser, and was accelerated by pre-phosphorylation of Fpr3 at Ser using CKII . Furthermore, synthetic peptides corresponding to the sequence surrounding Tyr184 that contained P-Ser (or Glu) at position 186 were much more efficient substrates for CKII phosphorylation of Tyr184 than a synthetic peptide containing Ala at position 186 . These findings indicate that CKII phosphorylates Fpr3 at tyrosine and serine both in vivo and in vitro and thus possesses dual specificity . These results also indicate that Tyr184 is phosphorylated by CKII via a two-step process, in which phosphorylation at the +2 position provides a negatively-charged specificity determinant that allows subsequent phosphorylation of Tyr184.

EMBO J, 1997 May 15, 16(10), 2769 - 82
Novel Golgi to vacuole delivery pathway in yeast: identification of a sorting determinant and required transport component; Cowles CR et al.; More than 40 vacuolar protein sorting (vps) mutants have been identified which secrete proenzyme forms of soluble vacuolar hydrolases to the cell surface . A subset of these mutants has been found to show selective defects in the sorting of two vacuolar membrane proteins . Under non-permissive conditions, vps45tsf (SEC1 homolog) and pep12/vps6tsf (endosomal t-SNARE) mutants efficiently sort alkaline phosphatase (ALP) to the vacuole while multiple soluble vacuolar proteins and the membrane protein carboxypeptidase yscS (CPS) are no longer delivered to the vacuole . Vacuolar localization of ALP in these mutants does not require transport to the plasma membrane followed by endocytic uptake, as double mutants of pep12tsf and vps45tsf with sec1 and end3 sort and mature ALP at the non-permissive temperature . Given the demonstrated role of t-SNAREs such as Pep12p in transport vesicle recognition, our results indicate that ALP and CPS are packaged into distinct transport intermediates . Consistent with ALP following an alternative route to the vacuole, isolation of a vps41tsf mutant revealed that at non-permissive temperature ALP is mislocalized while vacuolar delivery of CPS and CPY is maintained . A series of domain-swapping experiments was used to define the sorting signal that directs selective packaging and transport of ALP . Our data demonstrate that the amino-terminal 16 amino acid portion of the ALP cytoplasmic tail domain contains a vacuolar sorting signal which is responsible for the active recognition, packaging and transport of ALP from the Golgi to the vacuole via a novel delivery pathway.

EMBO J, 1997 May 15, 16(10), 2693 - 702
Regulation of B-type cyclin proteolysis by Cdc28-associated kinases in budding yeast; Amon A; In budding yeast, stability of the mitotic B-type cyclin Clb2 is tightly cell cycle-regulated . B-type cyclin proteolysis is initiated during anaphase and persists throughout the G1 phase . Cln-Cdc28 kinase activity at START is required to repress B-type cyclin-specific proteolysis . Here, we show that Clb-dependent kinases, when expressed during G1, are also capable of repressing the B-type cyclin proteolysis machinery . Furthermore, we find that inactivation of Cln- and Clb-Cdc28 kinases is sufficient to trigger Clb2 proteolysis and sister-chromatid separation in G2/M phase-arrested cells, where the B-type cyclin-specific proteolysis machinery is normally inactive . Our results suggest that Cln- and Clb-dependent kinases are both capable of repressing B-type cyclin-specific proteolysis and that they are required to maintain the proteolysis machinery in an inactive state in S and G2/M phase-arrested cells . We propose that in yeast, as cells pass through START, Cln-Cdc28-dependent kinases inactivate B-type cyclin proteolysis . As Cln-Cdc28-dependent kinases decline during G2, Clb-Cdc28-dependent kinases take over this role, ensuring that B-type cyclin proteolysis is not activated during S phase and early mitosis.

Cancer Res, 1997 May 15, 57(10), 1970 - 80
A persistent double-strand break destabilizes human DNA in yeast and can lead to G2 arrest and lethality; Bennett CB et al.; Double-strand breaks (DSBs) are an important source of genomic change in many organisms . We have examined the consequences of a persistent versus a rapidly repaired DSB on cell progression, viability, and stability of human DNA contained in dispensable yeast artificial chromosomes (YACs) within the yeast Saccharomyces cerevisiae . An Alu-URA3-YZ integrating plasmid was used to target the YZ sequence to repetitive Alu sequences within the human YAC . The YZ site can be cut by an inducible HO-endonuclease resulting in a DSB . Two classes of DSBs had been identified previously: those that could be rapidly repaired (RR-DSB), through recombination between flanking Alus; and persistent DSBs (C . B . Bennett et al., Mol . Cell . Biol., 16: 4414-4425, 1996) . These persistent DSBs (type 1) resulted in G2 delay and lethality . A third class of DSB is now identified corresponding to a persistent DSB that does not lead to G2 arrest or lethality (type 2) . Unlike YACs in which the DSB was rapidly repaired, the two types of persistent DSBs destabilized the human YAC DNA, resulting in a high likelihood of YAC loss (approximately 85% of surviving colonies) . Furthermore, both types of persistent DSBs could be misrepaired, resulting in mostly large internal or terminal deletions in the retained YACs . Therefore, recovery of these altered YACs can occur regardless of the effect of the DSBs on G2 arrest and cell lethality . If similar events occur in mammalian cells, persistent DSBs could be the initiating events that lead to a loss of heterozygosity and the expression of recessive oncogenes seen in malignant cells.

J Immunol, 1997 May 15, 158(10), 4908 - 15
Neutrophils as a source of putative restriction proteases: degradation of mammalian and yeast proteins monitored by two-dimensional gel electrophoresis; Lefkovits I et al.; We have compared the ability of intact neutrophils to degrade a complex substrate of proteins from mammalian and yeast origin . The substrate was obtained by biosynthetic labeling, and subsequent lysis of K562 cells (leukemic cell line) and of yeast culture . The mammalian substrate consisted of 619 and the yeast substrate of 185 different polypeptides, as visualized and represented on two-dimensional gel patterns . Upon incubation of the mammalian substrate with neutrophils, the bulk of spots disappeared so rapidly that after 240 min of incubation only 21 spots were detectable . Just one spot remained unaltered in its intensity throughout the whole period of incubation . About 440 spots reveal a t1/2 shorter than 8 min . Yeast substrate is represented by a smaller number of the starting polypeptides (185) from which 55 spots "survive" the neutrophil treatment . About 30 spots have a t1/2 shorter than 8 min . We conclude that neutrophils are equipped with a potent proteolytic apparatus, and this is capable of eliminating various proteins in a highly efficient manner . The system is much less effective in eliminating proteins from distant species, like yeast . Although the cells governing and regulating the immune system are clearly of lymphoid origin, it might well be that the preimmune task of eliminating self antigens in a manner as predicted in the restriction protease hypothesis is performed by neutrophils.

Nucleic Acids Res, 1997 May 15, 25(10), 2039 - 40
Yeast colony size reflects YAC copy number; Popov AV et al.; A novel strategy for separation of co-cloned YACs was developed . For this, yeast cells were grown under non-selective conditions to allow the mitotic loss of multiple YACs . Yeast colonies of different size appear on 'drop-out' selection plates with small clones consistently containing a single-copy YAC . Different auxotrophic marker genes can be used to separate co-cloned YACs or reduce their copy number, which is essential for most YAC-modification procedures.

Proc Natl Acad Sci U S A, 1997 May 13, 94(10), 5213 - 8
Clustering of meiotic double-strand breaks on yeast chromosome III; Baudat F et al.; In the yeast Saccharomyces cerevisiae, meiotic recombination is initiated by transient DNA double-strand breaks (DSBs) that are repaired by interaction of the broken chromosome with its homologue . To identify a large number of DSB sites and gain insight into the control of DSB formation at both the local and the whole chromosomal levels, we have determined at high resolution the distribution of meiotic DSBs along the 340 kb of chromosome III . We have found 76 DSB regions, mostly located in intergenic promoter-containing intervals . The frequency of DSBs varies at least 50-fold from one region to another . The global distribution of DSB regions along chromosome III is nonrandom, defining large (39-105 kb) chromosomal domains, both hot and cold . The distribution of these localized DSBs indicates that they are likely to initiate most crossovers along chromosome III, but some discrepancies remain to be explained.

J Biol Chem, 1997 May 9, 272(19), 12801 - 8
Alterations in the catalytic activity of yeast DNA topoisomerase I result in cell cycle arrest and cell death; Megonigal MD et al.; Eukaryotic DNA topoisomerase I catalyzes the relaxation of supercoiled DNA through a concerted mechanism of DNA strand breakage and religation . The cytotoxic activity of camptothecin results from the reversible stabilization of a covalent enzyme-DNA intermediate . Mutations in two conserved regions of yeast DNA topoisomerase I induced a similar mechanism of cell killing, albeit through different effects on enzyme catalysis . In Top1T722Ap, substituting Ala for Thr722 reduced enzyme specific activity by 3-fold, yet enhanced the stability of the covalent enzyme-DNA complex . In contrast, Top1R517Gp was 1,000-fold less active and camptothecin resistant . Nevertheless, salt-stable DNA-enzyme intermediates were detected . Mutation of the active-site tyrosine abrogated mutant enzyme activity and cytotoxicity, while sublethal levels of top1T722A expression increased rDNA recombination . In checkpoint proficient cells, pGAL1-induced top1 expression coincided with the accumulation of a terminal G2-arrested phenotype . Although the acquisition of this phenotype did not require Rad9p, Top1R517Gp- and Top1T722Ap-induced lethality was enhanced in rad9Delta strains . Thus, despite mechanistic differences between Top1R517Gp and Top1T722Ap, the DNA lesions resulting from the enhanced stability of the covalent enzyme-DNA intermediates were sufficient to cause cell cycle arrest and cell death.

J Biol Chem, 1997 May 9, 272(19), 12544 - 50
Cloning and characterization of a cDNA encoding a protein synthesis initiation factor-2alpha (eIF-2alpha) kinase from Drosophila melanogaster . Homology To yeast GCN2 protein kinase; Santoyo J et al.; Phosphorylation of the alpha subunit of the eukaryotic initiation factor 2 (eIF-2alpha) is one of the best-characterized mechanisms for downregulating protein synthesis in mammalian cells in response to various stress conditions . In Drosophila, such a regulatory mechanism has not been elucidated . We report the molecular cloning and characterization of DGCN2, a Drosophila eIF-2alpha kinase related to yeast GCN2 protein kinase . DGCN2 contains all of the 12 catalytic subdomains characteristic of eukaryotic Ser/Thr protein kinases and the conserved sequence of eIF-2alpha kinases in subdomain V . A large insert of 94 amino acids, which is characteristic of eIF-2alpha kinases, is also present between subdomains IV and V . It is particularly notable that DGCN2 possesses an amino acid sequence related to class II aminoacyl-tRNA synthetases, a unique feature of yeast GCN2 protein kinase . DGCN2 expression is developmentally regulated . During embryogenesis, DGCN2 mRNA is dynamically expressed in several tissues . Interestingly, at later stages this expression becomes restricted to a few cells of the central nervous system . Affinity-purified antibodies, raised against a synthetic peptide based on the predicted DGCN2 sequence, specifically immunoprecipitated an eIF-2alpha kinase activity and recognized an approximately 175 kDa phosphoprotein in Western blots of Drosophila embryo extracts.

J Biol Chem, 1997 May 9, 272(19), 12366 - 72
Subunit interactions in the Na,K-ATPase explored with the yeast two-hybrid system; Colonna TE et al.; Subunit interactions of the alpha1- and beta1-subunits of the chicken Na,K-ATPase were explored with the yeast two-hybrid system . Gal4-fusion proteins containing domains of the alpha1- and beta1-subunits were designed for examining both intersubunit and intrasubunit protein-protein interactions . Regions of the alpha- and beta-subunits known to be involved in alpha-beta-subunit assembly were positive in two-hybrid assay, supporting the validity of the assays . A library of beta-subunit ectodomains with C-terminal truncations was screened to find the maximal truncation retaining an interaction with the alpha-subunit extracellular H7H8 loop (where H7 refers to the seventh membrane span, and so on) . The maximal truncation removed all the cysteines involved in disulfide bridges, leaving only 63 amino acids of the beta-subunit ectodomain . Scanning alanine mutagenesis led to identification of an evolutionarily conserved sequence of four amino acids (SYGQ) in the extracellular H7H8 loop of the alpha-subunit that is crucial to alpha-beta-intersubunit interactions . Oligomerization studies with single domains failed to detect self-association of either of the two large cytosolic loops (H2H3 and H4H5) within the alpha-subunit . However, evidence was found for an interaction between these two cytoplasmic loops.

Biochem Biophys Res Commun, 1997 May 8, 234(1), 162 - 6
Irreversible metabolic transitions: the glucose 6-phosphate metabolism in yeast cell-free extracts; Coevoet MA et al.; The steady-state and dynamic behavior of a partial glycolytic reaction sequence are investigated in cell-free extracts of yeast . Pyruvate kinase, adenylate kinase and glucose 6-phosphate isomerase cooperate to a multienzyme system centered around the 6-phosphofructokinase (6-PFK) and fructose 1,6-bisphosphatase (FBPase) cycle . The reaction system operates under thermodynamically open conditions maintained by a continuous supply of substrates, i.e., glucose 6-phosphate (Glc6P), ATP and phosphoenolpyruvate (PPrv) in a flow-through reaction chamber . Appropriate conditions lead to the occurrence of (two) coexisting and markedly different time-independent states in the metabolite concentrations and fluxes . For particular experimental conditions, changes in the influx adenylic energy charge, {AEC}IN, may cause transitions between these alternative steady states which are either reversible as it occurs in classical hysteresis phenomena, or, more importantly, irreversible (irreversible transitions, IT) where the system is not able to switch back to its previous state even when the perturbation is reverted . The emergence of these irreversible transitions do not result from artificial or non-realistic experimental constraints, but are a potential intrinsic property of any non-linear dynamic system exhibiting bi- or multistability . These one-way transitions may well have important biological implications with respect to switching, adaptation and memory phenomena.

Biochemistry, 1997 May 6, 36(18), 5538 - 45
Cryoenzymic studies on yeast 3-phosphoglycerate kinase . Attempt to obtain the kinetics of the hinge-bending motion; Geerlof A et al.; This is a continuation of a study on the 3-phosphoglycerate kinase (PGK) reaction in the direction of 1,3-bisphosphoglycerate (bPG) formation: ATP + 3-phosphoglycerate (PG) <==> ADP + bPG {Schmidt, P . P., Travers, F., & Barman, T . (1995) Biochemistry 34, 824-832} . We showed that species containing bPG accumulate in the steady state, but their low concentrations and rapid kinetics of formation precluded a full study, even under cryoenzymic conditions in 40% ethylene glycol . Here we studied the PGK reaction in 30% methanol . The transient kinetics of bPG formation were obtained by chemical sampling: PGK was mixed with PG and {gamma-32P}ATP in a rapid flow quench apparatus, the mixture aged 4 ms up and quenched in acid, and the {1-(32)P}bPG was determined . The time course consisted of a rapid rise of bPG (kinetics k(obs)) and a steady state phase . In methanol, the amplitude of the rise was large (>50% of the PGK in the steady state), and k(obs) was measurable . Fluorescence stopped flow was used to study the formation of the binary E x PG and E x ATP . The affinities of PGK for ATP and PG were high in methanol (Kd = 102 and 1.5 microM, respectively), but the kinetics of the formation of E x PG and E x ATP were too rapid to be measured . From these and the chemical sampling experiments, we propose a reaction scheme for PGK: a rapid formation of the collision complex E x PG x ATP (K1), a slow isomerisation to E* x PG x ATP (k2,k(-2)), a rapid phosphorylation transfer step to E x bPG x ADP (K3), and a slow release of the products (k4) . In our scheme, k(obs) is the reflection mainly of k2 and k(-2) and the steady state of k4 . Using a computer simulation procedure, k2/K1 = 0.37 microM(-1) s(-1), k(-2) = 33 s(-1), K3 = 4, and k4 = 7.1 s(-1) . We propose that k(obs) measures the kinetics of the putative hinge-bending motion of PGK, i.e., the conformational change that is necessary for the substrates to line up for phosphoryl transfer.

J Cell Biol, 1997 May 5, 137(3), 563 - 80
Two new Ypt GTPases are required for exit from the yeast trans-Golgi compartment; Jedd G et al.; Small GTPases of the Ypt/rab family are involved in the regulation of vesicular transport . These GTPases apparently function during the targeting of vesicles to the acceptor compartment . Two members of the Ypt/rab family, Ypt1p and Sec4p, have been shown to regulate early and late steps of the yeast exocytic pathway, respectively . Here we tested the role of two newly identified GTPases, Ypt31p and Ypt32p . These two proteins share 81% identity and 90% similarity, and belong to the same protein subfamily as Ypt1p and Sec4p . Yeast cells can tolerate deletion of either the YPT31 or the YPT32 gene, but not both . These observations suggest that Ypt31p and Ypt32p perform identical or overlapping functions . Cells deleted for the YPT31 gene and carrying a conditional ypt32 mutation exhibit protein transport defects in the late exocytic pathway, but not in vacuolar protein sorting . The ypt31/ 32 mutant secretory defect is clearly downstream from that displayed by a ypt1 mutant and is similar to that of sec4 mutant cells . However, electron microscopy revealed that while sec4 mutant cells accumulate secretory vesicles, ypt31/32 mutant cells accumulate aberrant Golgi structures . The ypt31/32 phenotype is epistatic to that of a sec1 mutant, which accumulates secretory vesicles . Together, these results indicate that the Ypt31/32p GTPases are required for a step that occurs in the trans-Golgi compartment, between the reactions regulated by Ypt1p and Sec4p . This step might involve budding of vesicles from the trans-Golgi . Alternatively, Ypt31/32p might promote secretion indirectly, by allowing fusion of recycling vesicles with the trans-Golgi compartment.

J Mol Biol, 1997 May 2, 268(2), 303 - 21
Complete transcriptional map of yeast chromosome XI in different life conditions; Richard GF et al.; Systematic sequencing of the genome of Saccharomyces cerevisiae has demonstrated the existence of many novel genes, whose functions need to be studied . Entire chromosome sequences also offer the possibility to examine functional properties of the genome at a higher hierarchical level than the genes themselves . We used ordered DNA fragments of chromosome XI to systematically probe yeast DNA and total RNA extracted from MAT a, MAT alpha and diploid cells grown under three different conditions . Taking into account transcript sizes and uniqueness of probes, we attributed 94 transcripts to sequence-predicted open reading frames (ORFs) or tRNA genes; another 83 being tentatively assigned . The remaining 187 ORFs on chromosome XI do not correspond to transcripts detected under our conditions . More than 80% of transcripts are constitutively expressed, others are regulated by medium composition or cell type, the most frequent regulations being determined by carbon source (glycerol/glucose) or rich versus synthetic medium . Moreover, we show that transcript levels and regulation patterns are not statistically different between ORFs of unknown function, which constitute ca . 40% of the total, and previously identified genes (ca . 30%) or their structural homologues.

J Mol Biol, 1997 May 2, 268(2), 229 - 34
RNA polymerase I transcription termination: similar mechanisms are employed by yeast and mammals; Mason SW et al.; Termination of RNA polymerase I (Pol I) transcription requires the interaction of a specific DNA binding factor with terminator elements downstream of the pre-rRNA coding region . Both the terminator elements and the respective termination factors are distinct in yeast and mammals, and differences in the mechanism of transcription termination have been postulated . We have compared in vitro transcription termination of yeast and mouse Pol I using both the murine factor TTF-I, and the yeast homolog Reb1p . We show that, similar to TTF-I, Reb1p was sufficient for pausing of Pol I from either species, but was unable to cause release of the nascent transcripts from the paused ternary complex . The deficiency of Reb1p to mediate transcript release from Pol I of either species was complemented by the recently characterized murine release factor . Thus, both yeast and mouse Pol I termination requires a trans-acting factor that, in conjunction with the T-rich flanking sequence, releases the transcripts and Pol I from the template . The observation that the murine factor causes dissociation of ternary transcription complexes arrested by Reb1p suggests that the mechanism of Pol I termination is highly conserved from yeast to mammals.

J Biol Chem, 1997 May 2, 272(18), 12132 - 7
Study of yeast DNA topoisomerase II and its truncation derivatives by transmission electron microscopy; Benedetti P et al.; The 1429-amino acid residue long yeast DNA topoisomerase II and three of its deletion derivatives, a C-terminal truncation containing residues 1-1202, a 92-kDa fragment spanning residues 410-1202, and an A'-fragment spanning residues 660-1202, were examined by transmission electron microscopy . Analysis of rotary-shadowed images of these molecules shows that the full-length enzyme assumes a tripartite structure, in which a large globular core comprising the carboxyl parts of the dimeric enzyme is connected to a pair of smaller spherical masses comprising the ATPase domains of the enzyme . The linkers bridging the large globular structure and each of the smaller spheres are not visible in most of the images but appear to be sufficiently stiff to keep the relative positions of the connected parts . The angle extended by the pair of spherical masses is variable and falls in a range of 50-100 degrees for the majority of the images . On binding of a nonhydrolyzable ATP analog to the enzyme, this angle is significantly reduced as the two spherical masses swing into contact . These observations, together with results from previous biochemical and x-ray crystallographic studies of the enzyme, provide a sketch of the molecular architecture and conformational states of a catalytically active type II DNA topoisomerase.

J Biol Chem, 1997 May 2, 272(18), 12091 - 9
Camptothecin sensitivity is mediated by the pleiotropic drug resistance network in yeast; Reid RJ et al.; The antineoplastic alkaloid camptothecin interferes with the catalytic cycle of DNA topoisomerase I rendering it a cellular poison . Camptothecin stabilizes a covalent enzyme-DNA intermediate that is converted into lethal double strand DNA lesions during S phase of the cell cycle . Yeast SCT1 mutants were isolated in a screen for mutations in genes other than TOP1 that result in camptothecin resistance . Here we report SCT1 is allelic to PDR1 and that a Thr-879 to Met substitution in the PDR1-101 transcription factor confers multiple drug resistance . PDR1 regulates the expression of several gene products including the ATP-binding cassette transmembrane transport proteins PDR5, YOR1, and SNQ2 . The PDR1 T879M mutant increased PDR5 transcription compared with wild-type PDR1 strains . Deletion of PDR1 or the downstream effector SNQ2 increased cell sensitivity to camptothecin, whereas deletion of YOR1 or PDR5 had little effect on camptothecin sensitivity . However, the camptothecin resistance accompanying GAL1-promoted overexpression of PDR5 suggests some substrate promiscuity among the ATP-binding cassette transporters . These data underscore the role of the pleiotropic drug resistance network in regulating camptothecin toxicity and are consistent with a model of decreased intracellular concentrations of camptothecin resulting from the increased expression of the SNQ2 transporter.

J Biol Chem, 1997 May 2, 272(18), 11770 - 7
Characterization of the FET4 protein of yeast . Evidence for a direct role in the transport of iron; Dix D et al.; The low affinity Fe2+ uptake system of Saccharomyces cerevisiae requires the FET4 gene . In this report, we present evidence that FET4 encodes the Fe2+ transporter protein of this system . Antibodies prepared against FET4 detected two distinct proteins with molecular masses of 63 and 68 kDa . In vitro synthesis of FET4 suggested that the 68-kDa form is the primary translation product, and the 63-kDa form may be generated by proteolytic cleavage of the full-length protein . Consistent with its role as an Fe2+ transporter, FET4 is an integral membrane protein present in the plasma membrane . The level of FET4 closely correlated with uptake activity over a broad range of expression levels and is itself regulated by iron . Furthermore, mutations in FET4 can alter the kinetic properties of the low affinity uptake system, suggesting a direct interaction between FET4 and its Fe2+ substrate . Mutations affecting potential Fe2+ ligands located in the predicted transmembrane domains of FET4 significantly altered the apparent Km and/or Vmax of the low affinity system . These mutations may identify residues involved in Fe2+ binding during transport.

J Biol Chem, 1997 May 2, 272(18), 11750 - 6
Site-directed mutagenesis of the yeast V-ATPase A subunit; Liu Q et al.; To investigate the function of residues at the catalytic nucleotide binding site of the V-ATPase, we have carried out site-directed mutagenesis of the VMA1 gene encoding the A subunit of the V-ATPase in yeast . Of the three cysteine residues that are conserved in all A subunits sequenced thus far, two (Cys284 and Cys539) appear essential for correct folding or stability of the A subunit . Mutation of the third cysteine (Cys261), located in the glycine-rich loop, to valine, generated an enzyme that was fully active but resistant to inhibition by N-ethylmalemide, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole, and oxidation . To test the role of disulfide bond formation in regulation of vacuolar acidification in vivo, we have also determined the effect of the C261V mutant on targeting and processing of the soluble vacuolar protein carboxypeptidase Y . No difference in carboxypeptidase Y targeting or processing is observed between the wild type and C261V mutant, suggesting that disulfide bond formation in the V-ATPase A subunit is not essential for controlling vacuolar acidification in the Golgi . In addition, fluid phase endocytosis of Lucifer Yellow, quinacrine staining of acidic intracellular compartments and cell growth are indistinguishable in the C261V and wild type cells . Mutation of G250D in the glycine-rich loop also resulted in destabilization of the A subunit, whereas mutation of the lysine residue in this region (K263Q) gave a V-ATPase complex which showed normal levels of A subunit on the vacuolar membrane but was unstable to detergent solubilization and isolation and was totally lacking in V-ATPase activity . By contrast, mutation of the acidic residue, which has been postulated to play a direct catalytic role in the homologous F-ATPases (E286Q), had no effect on stability or assembly of the V-ATPase complex, but also led to complete loss of V-ATPase activity . The E286Q mutant showed labeling by 2-azido-{32P}ATP that was approximately 60% of that observed for wild type, suggesting that mutation of this glutamic acid residue affected primarily ATP hydrolysis rather than nucleotide binding.

Biol Signals, 1997 May-Jun, 6(3), 157 - 65
Novel features of the functional site and expression of the yeast deoxyhypusine synthase; Abid R et al.; A unique amino acid, hypusine, is formed posttranslationally in the precursor of eukaryotic translation initiation factor 5A (eIF-5A) . Deoxyhypusine synthase catalyzes the first of two steps in the biosynthesis of hypusine . We reported earlier that the DYS1 gene encoding deoxyhypusine synthase is essential for cell viability and proliferation in yeast . Here, we show by deletion studies that both N- and C-terminal regions, which are not so well conserved, are necessary for the activity of the yeast enzyme . Of the seven cysteine residues present in the yeast enzyme, only one cysteine (position 252; C252) appeared to be essential for its activity . Moderate overexpression of DYS1 showed very little effects on cell growth and no obvious effects on the intracellular level of eIF-5A . However, repression of the expression of DYS1 resulted in near-complete depletion of eIF-5A 24 h after the initiation of repression and was followed by cell growth arrest after another 24 h . This novel finding suggests that the major role of deoxyhypusine synthase in cell proliferation is mediated not only through its modification of the eIF-5A precursor, but also through its regulation of intracellular eIF-5A levels.

Genes Cells, 1997 May, 2(5), 329 - 43
Xenopus cyclin A1 can associate with Cdc28 in budding yeast, causing cell-cycle arrest with an abnormal distribution of nuclear DNA; Funakoshi M et al.; BACKGROUND: Cyclins play a regulatory role in cell cycle progression, associated with cyclin-dependent kinases . We have investigated the structure-function relationships of cyclin A, mainly using Xenopus egg extracts in vitro . To further analyse the function and structure of cyclin A in vivo, we expressed Xenopus cyclin A1 in the budding yeast Saccharomyces cerevisiae . RESULTS: We herein show that vertebrate cyclin A1 can associate with endogenous Cdc28 to form histone H1 kinase . The growth of the yeast was inhibited by the expression of indestructible cyclin A1, but not by a non-Cdk binding cyclin A1 mutant . The induction of cyclin A1 expression in yeast caused cell cycle arrest with an abnormal distribution of nuclear DNA to the daughter bud . Suppressors of the cyclin A1-mediated growth arrest were identified as new alleles of the cdc28 mutation that reduced the binding of cyclin A1 and possessed different affinities for the cyclin-Cdc28 complexes . The temperature-sensitivity of the cdc28 mutation was thus preferentially suppressed by the endogenous cyclins CLN2 and CLB2 . CONCLUSIONS: These results suggest that the Cdc28 protein kinase activity mediated by vertebrate cyclin A1 may be involved in the process of nuclear movement in the yeast, and thereby affect the dependence of the M phase on the completion of the S phase through a preferential binding affinity of the cyclin-Cdc28 complex.

Genetika, 1997 May, 33(5), 610 - 5
{Fusion of glutathione S-transferase with the N-terminus of yeast Sup35p protein inhibits its prion-like properties}; Dagkesamanskaia AR et al.; The yeast Saccharomyces cerevisiae SUP35 gene that encodes the Sup35p protein homologous to the translation termination eRF3 factor of higher eukaryotes is essential to replication of the nonchromosomally inherited {psi+} determinant . The nonsense suppressor phenotype of this determinant was assumed to be dependent on a specific conformational state of the Sup35p protein; the transition to this state leads to partial inactivation of this protein . In terms of this hypothesis, the Sup35p protein can, like mammalian prions, induce its own specific conformation via protein-protein interactions in the newly synthesized Sup35p molecules; in this way, inheritance of the {psi+} phenotype is ensured in a series of cell generations . In recent years, this hypothesis has been experimentally verified . Allele substitution of the wild-type SUP35 gene by its chimeric GST-SUP35 version, which encodes the glutathione S-transferase sequence fused with the N end of Sup35p, was shown to cause elimination of the {psi+} determinant . The ability to eliminate {psi+} is a recessive trait, because fusions heterozygous for the GST-SUP35 allele did not lose this trait . Elimination of {psi+} seems to be caused by inability of the chimeric protein to bring about oligomerization . The obtained data indicate that the chimeric protein manifests attenuated terminating activity but can interact with the eRF1 translation termination factor encoded by the SUP45 gene.

Appl Microbiol Biotechnol, 1997 May, 47(5), 476 - 81
Continuous sucrose hydrolysis by yeast cells immobilized to wool; Krastanov A; A novel immobilized biocatalyst with invertase activity was prepared by adhesion of yeast cells to wool using-glutaraldehyde . Yeast cells could be immobilized onto wool by treating either the yeast cells or wool or both with glutaraldehyde . Immobilized cells were not desorbed by washing with 1 M KCl or 0.1 M buffers . pH 3.5-7.5 . The biocatalyst shows a maximum enzyme activity when immobilized at pH 4.2-4.6 and 7.5-8.0 . The immobilized biocatalyst was tested in a tubular fixed-bed reactor to investigate its possible application for continuous full-scale sucrose hydrolysis . The influence of temperature, sugar concentration and flow rate on the productivity of the reactor and on the specific productivity of the biocatalyst was studied . The system demonstrates a very good productivity at a temperature of 70 degrees C and a sugar concentration of 2.0 M . The increase of the volume of the biocatalyst layer exponentially increases the productivity . The productivity of the immobilized biocatalyst decreases no more than 50% during 60 days of continuous work at 70 degrees C and 2.0 M sucrose, but during the first 30 days it remains constant . The cumulative biocatalyst productivity for 60 days was 4.8 x 10(3) kg inverted sucrose/kg biocatalyst . The biocatalyst was proved to be fully capable of continuous sucrose hydrolysis in fixed-bed reactors.

Bioorg Med Chem, 1997 May, 5(5), 821 - 32
Stereochemistry of yeast delta 24-sterol methyl transferase; Acuna-Johnson AP et al.; S-Adenosyl-l-methionine: delta 24-sterol methyl transferase (24-SMT) mediates introduction of the C-28 carbon of yeast sterols . It has been shown that sulfonium analogues of the presumptive cationic intermediates of the methylenation reaction are potent in vivo and in vitro inhibitors of this process . In the presence of these inhibitors, cultures of yeast produced increased proportions of zymosterol, the natural substrate of the enzyme, while proportions of ergosterol and ergostatetraenol were decreased . New C27-sterol metabolites were also found . The in vivo inhibitory power of the analogues {I50 (microM)} was determined from the proportion of C-24 methylated sterols to C-24 nonmethylated sterols in treated cultures to be in the following order: 25-thiacholesterol iodide (0.07) > 24(S)-methyl-25-thiacholesteryl iodide (0.14) > 24(R)-methyl-25-thiacholesteryl iodide (0.25) . Kinetic inhibition as revealed by radiolabeled S-adenosyl-l-methionine (SAM), crude enzyme and 25-thiacholesteryl iodide revealed this inhibitor to be uncompetitive with respect to zymosterol and competitive with respect to SAM . The greater inhibitory power of 24(S)-methyl-25-thiacholesteryl iodide compared to 24(R)-methyl-25-thiacholesteryl iodide suggests that methyl donation to delta 24 occurs from the si face . When considered in conjunction with Arigoni's previous work, the present results infer the methylenation mediated by yeast 24-SMT proceeds by alkylation from the si face of delta 24 followed by migration of a hydrogen from C-24 to C-25 across the re face and final loss of a hydrogen from C-28 on the re face.

Plant Mol Biol, 1997 May, 34(2), 325 - 30
Cloning and characterisation of a carrot cDNA coding for a WD repeat protein homologous to Drosophila fizzy, human p55CDC and yeast CDC20 proteins; Luo M et al.; The present study describes the isolation of a cDNA coding for a carrot protein of 450 amino acids that contains WD repeats (DcWD1) and is homologous to Drosophila melanogaster fizzy protein, mammalian p55CDC and yeast Cdc20p . As for the known related proteins, sequence conservation concerned the majority of the polypeptide except the far N-terminus . Results of Southern blot analysis with genomic DNA under high stringency conditions showed the occurrence of a single gene . Northern blot analyses revealed the accumulation of DcWD1 mRNA in all tested tissues (leaves, petioles and hypocotyls, apical meristems, roots and suspension cultured cells), though at a different extent . Lack of induction of relevant transcripts in proliferating auxin-stimulated hypocotyls suggests a mode of expression not strictly related to the cell proliferation.

Antonie Van Leeuwenhoek, 1997 May, 71(4), 375 - 8
Candida novakii, sp . nov . a new anamorphic yeast species of ascomycetous affinity; Peter G et al.; Two strains of an undescribed species of the genus Candida were isolated from decaying wood of Quercus sp . A description of the new species Candida novakii is given.

Yeast, 1997 May, 13(6), 583 - 9
Sequence analysis of a 33.2 kb segment from the left arm of yeast chromosome XV reveals eight known genes and ten new open reading frames including homologues of ABC transporters, inositol phosphatases and human expressed sequence tags; Tzermia M et al.; The complete nucleotide sequence of a 33221 bp segment, contained in cosmid pEOA1044, derived from the left arm of chromosome XV of Saccharomyces cerevisiae, appears in public databases between coordinates 177013 and 210234 . Computer analysis of that sequence revealed the presence of the previously known genes IRA2, DEC1, NUF2, HST1, RTG1, RIB2 and HAL2, one previously partially sequenced open reading frame (ORF) of unknown function (SCORFAC) and ten newly identified ORFs . One of the new ORFs is similar to the Drosophila melanogaster white gene and other transmembrane ABC transporters, another one has similarities to inositol phosphatases and others are similar to ORFs of unknown function from various organisms, including human Expressed Sequence Tags (ESTs) . Potential transmembrane regions, ATP/GTP-binding and WD motifs have also been identified . The existence of yeast ESTs for two of the newly identified ORFs indicates that they are transcribed.

Yeast, 1997 May, 13(6), 541 - 9
Role of the cytoskeleton in endocytosis of the yeast maltose transporter; Penalver E et al.; Certain components of the cytoskeleton play a role in yeast fluid-phase endocytosis as well as in endocytosis of the alpha-factor when this pheromone is bound to its 7-transmembrane segment receptor . The yeast maltose transporter is a 12-transmembrane segment protein that, under certain physiological conditions, is degraded in the vacuole after internalization by endocytosis . In this work, the possible role of the cytoskeleton in endocytosis of this transporter has been investigated . Using mutants defective in beta-tubulin, actin and the actin-binding proteins Sac6 and Abp85 . as well as nocodazole, which inhibits formation of microtubules, we have shown that actin microfilaments are involved in endocytosis of the maltose transporter whereas microtubules are not.

J Cell Sci, 1997 May, 110 ( Pt 9), 1063 - 72
The yeast VPS5/GRD2 gene encodes a sorting nexin-1-like protein required for localizing membrane proteins to the late Golgi; Nothwehr SF et al.; Genetic analysis of late Golgi membrane protein localization in Saccharomyces cerevisiae has uncovered a large number of genes (called GRD) that are required for retention of A-ALP, a model late Golgi membrane protein . Here we describe one of the GRD genes, VPSS/GRD2, that encodes a hydrophilic protein similar to human sorting nexin-1, a protein involved in trafficking of the epidermal growth factor receptor . In yeast cells containing a vps5 null mutation the late Golgi membrane proteins A-ALP and Kex2p were rapidly mislocalized to the vacuolar membrane . A-ALP was delivered to the vacuole in vps5 mutants in a manner independent of a block in the early endocytic pathway . vps5 null mutants also exhibited defects in both vacuolar morphology and in sorting of a soluble vacuolar protein, carboxypeptidase Y . The latter defect is apparently due to an inability to localize the carboxypeptidase Y sorting receptor, Vps10p, to the Golgi since it is rapidly degraded in the vacuole in vps5 mutants . Fractionation studies indicate that Vps5p is distributed between a free cytosolic pool and a particulate fraction containing Golgi, transport vesicles, and possibly endosomes, but lacking vacuolar membranes . Immunofluorescence microscopy experiments show that the membrane-associated pool of Vps5p localizes to an endosome-like organelle that accumulates in the class E vps27 mutant . These results support a model in which Vps5p is required for retrieval of membrane proteins from a prevacuolar/late endosomal compartment back to the late Golgi apparatus.

EMBO J, 1997 May 1, 16(9), 2441 - 51
Assembly of a bZIP-bHLH transcription activation complex: formation of the yeast Cbf1-Met4-Met28 complex is regulated through Met28 stimulation of Cbf1 DNA binding; Kuras L et al.; Transcriptional activation of sulfur amino acid metabolism in yeast is dependent on a multi-functional factor, the centromere-binding factor 1 (Cbf1) and on two specific transcription factors, Met4 and Met28 . Cbf1 belongs to the basic helix-loop-helix DNA-binding protein family while Met4 and Met28 are two basic leucine zipper (bZIP) factors . We have shown previously that in cell extracts, the three factors are found in a high molecular weight complex . By using mobility shift assays, we report here that the in vitro reconstitution of the Cbf1-Met4-Met28 complex on MET16UAS can be obtained with purified recombinant proteins . DNase I protection experiments confirm that the Cbf1-Met4-Met28 complex is formed over the TCACGTG sequence . The experiments also show that both Met4 and Met28 bind to DNA only in the presence of Cbf1 . Moreover, Met28 is shown to enhance the DNA-binding activity of Cbf1 . Analysis of MET28 gene regulation reveals that its expression requires Met4 . Thus the biochemical activity of Met28 allows the establishment of a positive regulatory loop . The results thus provide evidence of a new functional relationship between bHLH and bZIP proteins and demonstrate that the association of such factors may serve to discriminate between the different TCACGTG sequences found in the chromosomes.

Microbiology, 1997 May, 143 ( Pt 5), 1673 - 80
beta-Glucosylated proteins in the cell wall of the black yeast Exophiala (Wangiella) dermatitidis; Montijn RC et al.; Wild-type cells of the pathogenic black yeast Exophiala (Wangiella) dermatitidis grown in a low-pH ascorbate medium became less melanized and less resistant to Zymolyase . This was accompanied by increased staining with fluorescently labelled concanavalin A . The sugar composition of wild-type and mutant cell walls was, except for the presence of galactose, similar to that of Saccharomyces cerevisiae . Digestion of mutant cell walls with laminarinase released galactomannoproteins . In addition, the released cell wall proteins contained glucose and reacted with affinity-purified 1,6-beta-glucan antiserum, indicating that they are linked to 1,6-beta-glucan . It is proposed that 1,6-beta-glucosylated cell wall proteins generally occur among ascomycetes.

Mol Biol Cell, 1997 May, 8(5), 871 - 95
Pep7p provides a novel protein that functions in vesicle-mediated transport between the yeast Golgi and endosome; Webb GC et al.; Saccharomyces cerevisiae pep7 mutants are defective in transport of soluble vacuolar hydrolases to the lysosome-like vacuole . PEP7 is a nonessential gene that encodes a hydrophilic protein of 515 amino acids . A cysteine-rich tripartite motif in the N-terminal half of the polypeptide shows striking similarity to sequences found in many other eukaryotic proteins . Several of these proteins are thought to function in the vacuolar/lysosomal pathway . Mutations that change highly conserved cysteine residues in this motif lead to a loss of Pep7p function . Kinetic studies demonstrate that Pep7p function is required for the transport of the Golgi-precursors of the soluble hydrolases carboxypeptidase Y, proteinase A, and proteinase B to the endosome . Integral membrane hydrolase alkaline phosphatase is transported to the vacuole by a parallel intracellular pathway that does not require Pep7p function . pep7 mutants accumulate a 40-60-nm vesicle population, suggesting that Pep7p functions in a vesicle consumption step in vesicle-mediated transport of soluble hydrolases to the endosome . Whereas pep7 mutants demonstrate no defects in endocytic uptake at the plasma membrane, the mutants demonstrate defects in transport of receptor-mediated macromolecules through the endocytic pathway . Localization studies indicate that Pep7p is found both as a soluble cytoplasmic protein and associated with particulate fractions . We conclude that Pep7p functions as a novel regulator of vesicle docking and/or fusion at the endosome.

FEMS Microbiol Lett, 1997 May 1, 150(1), 121 - 6
Immobilization and ethanol stress induce the same molecular response at the level of the cell wall in growing yeast; Parascandola P et al.; The effect of immobilization on the cell wall of Saccharomyces cerevisiae cells was investigated in comparison with freely suspended batch grown cells . The pattern of mannoproteins released from the cell wall after Zymolyase digestion showed the presence of new mannoprotein species when cell growth takes place in a state of immobilization . The same result was obtained by exposure of freely suspended cells to a stressful concentration of ethanol, showing that two different adverse culture conditions induce a similar molecular response at the cell wall level.

Oncogene, 1997 May 1, 14(17), 2019 - 24
Yeast two-hybrid in vivo association of the Src kinase Lyn with the proto-oncogene product Cbl but not with the p85 subunit of PI 3-kinase; Dombrosky-Ferlan PM et al.; Ligand binding of multi-chain antigen receptors and hematopoietin/cytokine receptors results in rapid activation of protein tyrosine kinase (PTK)-dependent signalling molecules such as phosphatidylinositol 3-kinase (PI 3-kinase) . Co-precipitation studies have shown that Src-related PTK, such as Lyn, associates with the p85 regulatory subunit of PI 3-kinase via SH2 and SH3 domain binding with their cognate ligands . More recent studies have shown that the proto-oncogene product Cbl co-precipitates with p85 following engagement of cytokine and antigen receptors . As opposed to in vitro co-precipitation studies, the yeast two-hybrid screen reveals in vivo protein-protein interactions . Using the yeast two-hybrid screen, we demonstrate an in vivo association of Lyn's SH3 and SH2 domains with the proline-rich domain of Cbl . Lyn's SH3 and SH2 domains do not interact with p85 in the yeast two-hybrid screen, as would be predicted from glutathione-S-transferase (GST) fusion protein pull-down or co-immunoprecipitation studies from whole cell lysates . However, the SH3 domain of p85 interacts with the proline-rich domain of Cbl . When yeast were transformed with catalytic Lyn, an interaction between p85's SH2 domain and Cbl occurred . From the data, we propose the following three step process of PI 3-kinase activation: (1) complexes of Lyn-Cbl and Cbl-p85 exist without ligand stimulation, (2) upon ligand binding, Lyn becomes active and phosphorylates Cbl, and (3) Cbl's tyrosine phosphorylated residue serves as a docking site for the SH2 domains of p85 - thereby stabilizing the complex and activating PI 3-kinase . The yeast two-hybrid system can be used to dissect the precise mechanisms of in vivo protein-protein interactions, including those between phosphotyrosine and SH2-containing proteins.

Nat Struct Biol, 1997 May, 4(5), 366 - 9
Structure determination of yeast cofilin; Fedorov AA et al.; Cofilin, a ubiquitous 15,000 M(r) protein, plays a central role in regulating cytoskeletal dynamics . Cofilin binds to actin monomers and filaments, and has a pH-dependent actin severing activity . The structure will allow for a detailed analysis of cofilin function.

Arch Biochem Biophys, 1997 May 1, 341(1), 70 - 4
Structure of a cell wall mannan from the pathogenic yeast, Candida catenulata: assignment of 1H nuclear magnetic resonance chemical shifts of the inner alpha-1,6-linked mannose residues substituted by a side chain; Kobayashi H et al.; We performed an enzyme-linked immunosorbent assay of the cell wall mannan purified from the pathogenic yeast, Candida catenulata, using antisera to factors of the genus Candida . The results suggest that mannan possesses a linear backbone consisting of alpha-1, 6-linked mannose residues and side chains possessing nonreducing terminal alpha-1,2- and alpha-1,3-linked mannose residues . The chemical structure of the mannan was analyzed by two-dimensional homonuclear Hartmann-Hahn and two-dimensional nuclear Overhauser enhancement and exchange spectroscopy . The sequential assignments of the cross-peaks caused by J-coupling and the nuclear Overhauser effect from these terminal mannose residues demonstrate that the H1 signal of an inner alpha-1,6-linked mannose residue substituted by an alpha-oligomannosyl side chain or a single mannose through the C-2 position in an alpha-anomer configuration undergoes a significant downfield shift (delta delta = 0.16 or 0.19 ppm, respectively) compared with that of unsubstituted residues . We therefore propose the exact overall structure of the antigenic mannan obtained from C . catenulata . The assignment data in the present study are useful for the determination of the exact overall structure of various yeast mannans using the two-dimensional nuclear magnetic resonance analysis without the need for harsh procedures.

Genetics, 1997 May, 146(1), 263 - 73
Dominant mutations of Drosophila MAP kinase kinase and their activities in Drosophila and yeast MAP kinase cascades; Lim YM et al.; Eight alleles of Dsor1 encoding a Drosophila homologue of mitogen-activated protein (MAP) kinase kinase were obtained as dominant suppressors of the MAP kinase kinase kinase D raf . These Dsor1 alleles themselves showed no obvious phenotypic consequences nor any effect on the viability of the flies, although they were highly sensitive to upstream signals and strongly interacted with gain-of-function mutations of upstream factors . They suppressed mutations for receptor tyrosine kinases (RTKs); torso (tor), sevenless (sev) and to a lesser extent Drosophila EGF receptor (DER) . Furthermore, the Dsor1 alleles showed no significant interaction with gain-of-function mutations of DER . The observed difference in activity of the Dsor1 alleles among the RTK pathways suggests Dsor1 is one of the components of the pathway that regulates signal specificity . Expression of Dsor1 in budding yeast demonstrated that Dsor1 can activate yeast MAP kinase homologues if a proper activator of Dsor1 is coexpressed . Nucleotide sequencing of the Dsor1 mutant genes revealed that most of the mutations are associated with amino acid changes at highly conserved residues in the kinase domain . The results suggest that they function as suppressors due to increased reactivity to upstream factors.

Genetics, 1997 May, 146(1), 245 - 52
Isolation of mutations in the Drosophila homologues of the human Neurofibromatosis 2 and yeast CDC42 genes using a simple and efficient reverse-genetic method; Fehon RG et al.; Reverse genetic analysis in Drosophila has been greatly aided by a growing collection of lethal P transposable element insertions that provide molecular tags for the identification of essential genetic loci . However, because the screens performed to date primarily have generated autosomal P-element insertions, this collection has not been as useful for performing reverse genetic analysis of X-linked genes . We have designed a reverse genetic screen that takes advantage of the hemizygosity of the X chromosome in males together with a cosmid-based transgene that serves as an autosomally linked duplication of a small region of the X chromosome . The efficacy and efficiency of this method is demonstrated by the isolation of mutations in Drosophila homologues of two well-studied genes, the human Neurofibromatosis 2 tumor suppressor and the yeast CDC42 gene . The method we describe should be of general utility for the isolation of mutations in other X-linked genes, and should also provide an efficient method for the isolation of new allcles of existing X-linked or autosomal mutations in Drosophila.

Genetics, 1997 May, 146(1), 79 - 88
Karyotype variability in yeast caused by nonallelic recombination in haploid meiosis; Loidl J et al.; Chromosomes of altered size were found in the meiotic products of a haploid Saccharomyces cerevisiae strain by pulsed field gel electrophoretic separation of whole chromosomes . About 7% of haploid meioses produced chromosomes that differed by > or = 10 kb from their wild-type counterparts . Chromosomes most often became enlarged or shortened due to recombination events between sister chromatids at nonallelic sequences . By this mechanism chromosome III acquired tandem arrays of up to eight extra copies of the approximately kb MAT-HMR segment during repeated rounds of haploid meioses . Enlarged chromosomes III were unstable and changed their size during meiosis more often than remaining unchanged . Altered chromosomes appeared also as the products of intrachromatid recombination and of reciprocal translocations caused by ectopic recombination between nonhomologous chromosomes . In diploid meiosis, chromosomes of altered size occurred at least 10 times less frequently, whereas in mitotic cultures cells with altered karyotypes were virtually absent . The results show that various forms of ectopic recombination are promoted by the absence of allelic homologies.

Genetics, 1997 May, 146(1), 69 - 78
Meiotic crossing over between nonhomologous chromosomes affects chromosome segregation in yeast; Jinks-Robertson S et al.; Meiotic recombination between artificial repeats positioned on nonhomologous chromosomes occurs efficiently in the yeast Saccharomyces cerevisiae . Both gene conversion and crossover events have been observed, with crossovers yielding reciprocal translocations . In the current study, 5.5-kb ura3 repeats positioned on chromosomes V and XV were used to examine the effect of ectopic recombination on meiotic chromosome segregation . Ura3 random spores were selected and gene conversion vs . crossover events were distinguished by Southern blot analysis . Approximately 15% of the crossover events between chromosomes V and XV were associated with missegregation of one of these chromosomes . The missegregation was manifest as hyperploid spores containing either both translocations plus a normal chromosome, or both normal chromosomes plus one of the translocations . In those cases where it could be analyzed, missegregation occurred at the first meiotic division . These data are discussed in terms of a model in which ectopic crossovers compete efficiently with normal allelic crossovers in directing meiotic chromosome segregation.

Nat Biotechnol, 1997 May, 15(5), 453 - 7
High-level expression of a sweet protein, monellin, in the food yeast Candida utilis; Kondo K et al.; We describe the heterologous expression of monellin in the yeast Candida utilis . A single-chain monellin gene was expressed under the control of the glyceraldehyde-3-phosphate dehydrogenase {correction of decarboxylase} gene promoter from C . utilis . A promoter-deficient marker gene allowed high-copy-number integration of vectors into either the rDNA locus or the URA3 gene locus . Monellin was produced at a high level, accounting for > 50% of the soluble protein . No significant decrease in the production level of monellin was detected in transformants after 50 generations of nonselective growth.

Mol Cell Biol, 1997 May, 17(5), 2897 - 907
Analysis of the receptor binding domain of Gpa1p, the G(alpha) subunit involved in the yeast pheromone response pathway; Kallal L et al.; The Saccharomyces cerevisiae G protein alpha subunit Gpa1p is involved in the response of both MATa and MAT alpha cells to pheromone . We mutagenized the GPA1 C terminus to characterize the receptor-interacting domain and to investigate the specificity of the interactions with the a- and alpha-factor receptors . The results are discussed with respect to a structural model of the Gpa1p C terminus that was based on the crystal structure of bovine transducin . Some mutants showed phenotypes different than the pheromone response and mating defects expected for mutations that affect receptor interactions, and therefore the mutations may affect other aspects of Gpa1p function . Most of the mutations that resulted in pheromone response and mating defects had similar effects in MATa and MAT alpha cells, suggesting that they affect the interactions with both receptors . Overexpression of the pheromone receptors increased the mating of some of the mutants tested but not the wild-type strain, consistent with defects in mutant Gpa1p-receptor interactions . The regions identified by the mating-defective mutants correlated well with the regions of mammalian G(alpha) subunits implicated in receptor interactions . The strongest mating type-specific effects were seen for mutations to proline and a mutation of a glycine residue predicted to form a C-terminal beta turn . The analogous beta turn in mammalian G(alpha) subunits undergoes a conformational change upon receptor interaction . We propose that the conformation of this region of Gpa1p differs during the interactions with the a- and alpha-factor receptors and that these mating type-specific mutations preclude the orientation necessary for interaction with one of the two receptors.

Mol Cell Biol, 1997 May, 17(5), 2888 - 96
A yeast TATA-binding protein mutant that selectively enhances gene expression from weak RNA polymerase II promoters; Blair WS et al.; We describe a unique gain-of-function mutant of the TATA-binding protein (TBP) subunit of Saccharomyces cerevisiae TFIID that, at least in part, renders transcriptional transactivators dispensable for efficient mRNA expression . The yTBPN69S mutant enhances transcription from weaker yeast promoter elements by up to 50-fold yet does not significantly increase gene expression directed by highly active promoters . Therefore, this TBP mutant and transcriptional transactivators appear to affect a common rate-limiting step in transcription initiation . Consistent with the hypothesis that this step is TFIID recruitment, tethering of TBP to a target promoter via a heterologous DNA binding domain, which is known to bypass the need for transcriptional transactivators, also nullifies the enhancing effect exerted by the N69S mutation . These data provide genetic support for the hypothesis that TFIID recruitment represents a rate-limiting step in the initiation of mRNA transcription that is specifically enhanced by transcriptional transactivators.

Mol Cell Biol, 1997 May, 17(5), 2859 - 65
Hypermutability of homonucleotide runs in mismatch repair and DNA polymerase proofreading yeast mutants; Tran HT et al.; Homonucleotide runs in coding sequences are hot spots for frameshift mutations and potential sources of genetic changes leading to cancer in humans having a mismatch repair defect . We examined frameshift mutations in homonucleotide runs of deoxyadenosines ranging from 4 to 14 bases at the same position in the LYS2 gene of the yeast Saccharomyces cerevisiae . In the msh2 mismatch repair mutant, runs of 9 to 14 deoxyadenosines are 1,700-fold to 51,000-fold, respectively, more mutable for single-nucleotide deletions than are runs of 4 deoxyadenosines . These frameshift mutations can account for up to 99% of all forward mutations inactivating the 4-kb LYS2 gene . Based on results with single and double mutations of the POL2 and MSH2 genes, both DNA polymerase epsilon proofreading and mismatch repair are efficient for short runs while only the mismatch repair system prevents frameshift mutations in runs of > or = 8 nucleotides . Therefore, coding sequences containing long homonucleotide runs are likely to be at risk for mutational inactivation in cells lacking mismatch repair capability.

Mol Cell Biol, 1997 May, 17(5), 2851 - 8
Microsatellite instability in yeast: dependence on repeat unit size and DNA mismatch repair genes; Sia EA et al.; We examined the stability of microsatellites of different repeat unit lengths in Saccharomyces cerevisiae strains deficient in DNA mismatch repair . The msh2 and msh3 mutations destabilized microsatellites with repeat units of 1, 2, 4, 5, and 8 bp; a poly(G) tract of 18 bp was destabilized several thousand-fold by the msh2 mutation and about 100-fold by msh3 . The msh6 mutations destabilized microsatellites with repeat units of 1 and 2 bp but had no effect on microsatellites with larger repeats . These results argue that coding sequences containing repetitive DNA tracts will be preferred target sites for mutations in human tumors with mismatch repair defects . We find that the DNA mismatch repair genes destabilize microsatellites with repeat units from 1 to 13 bp but have no effect on the stability of minisatellites with repeat units of 16 or 20 bp . Our data also suggest that displaced loops on the nascent strand, resulting from DNA polymerase slippage, are repaired differently than loops on the template strand.

Mol Cell Biol, 1997 May, 17(5), 2825 - 34
Template topology and transcription: chromatin templates relaxed by localized linearization are transcriptionally active in yeast; Liang CP et al.; To address the role of transient torsional stress in transcription, we have utilized the regulated expression of HO endonuclease in yeast to create double-strand breaks in DNA templates in vivo at preselected sites . Linearization of circular minichromosomes, either 2 kb upstream or immediately downstream of a lacZ reporter gene controlled by the yeast metallothionein gene (CUP1) promoter, did not alter the copper induction profile of lacZ RNA transcripts compared to that of nonlinearized controls . Constructs site-specifically integrated into yeast chromosome II gave similar results . In vivo cross-linking with psoralen as a probe for negative DNA supercoiling demonstrated that template linearization efficiently dissipated DNA supercoiling induced by transcription . Therefore, the efficient transcription of linearized, relaxed templates found here demonstrates that transient torsional tension is not required for transcription of chromatin templates in yeast.

Mol Cell Biol, 1997 May, 17(5), 2798 - 805
Interaction between yeast Sup45p (eRF1) and Sup35p (eRF3) polypeptide chain release factors: implications for prion-dependent regulation; Paushkin SV et al.; The SUP45 and SUP35 genes of Saccharomyces cerevisiae encode polypeptide chain release factors eRF1 and eRF3, respectively . It has been suggested that the Sup35 protein (Sup35p) is subject to a heritable conformational switch, similar to mammalian prions, thus giving rise to the non-Mendelian {PSI+} nonsense suppressor determinant . In a {PSI+} state, Sup35p forms high-molecular-weight aggregates which may inhibit Sup35p activity, leading to the {PSI+} phenotype . Sup35p is composed of the N-terminal domain (N) required for {PSI+} maintenance, the presumably nonfunctional middle region (M), and the C-terminal domain (C) essential for translation termination . In this study, we observed that the N domain, alone or as a part of larger fragments, can form aggregates in {PSI+} cells . Two sites for Sup45p binding were found within Sup35p: one is formed by the N and M domains, and the other is located within the C domain . Similarly to Sup35p, in {PSI+} cells Sup45p was found in aggregates . The aggregation of Sup45p is caused by its binding to Sup35p and was not observed when the aggregated Sup35p fragments did not contain sites for Sup45p binding . The incorporation of Sup45p into the aggregates should inhibit its activity . The N domain of Sup35p, responsible for its aggregation in {PSI+} cells, may thus act as a repressor of another polypeptide chain release factor, Sup45p . This phenomenon represents a novel mechanism of regulation of gene expression at the posttranslational level.

Mol Cell Biol, 1997 May, 17(5), 2511 - 20
The peroxin Pex17p of the yeast Yarrowia lipolytica is associated peripherally with the peroxisomal membrane and is required for the import of a subset of matrix proteins; Smith JJ et al.; PEX genes encode peroxins, which are required for the biogenesis of peroxisomes . The Yarrowia lipolytica PEX17 gene encodes the peroxin Pex17p, which is 671 amino acids in length and has a predicted molecular mass of 75,588 Da . Pex17p is peripherally associated with the peroxisomal membrane . The carboxyl-terminal tripeptide, Gly-Thr-Leu, of Pex17p is not necessary for its targeting to peroxisomes . Synthesis of Pex17p is low in cells grown in glucose-containing medium and increases after the cells are shifted to oleic acid-containing medium . Cells of the pex17-1 mutant, the original mutant strain, and the pex17-KA mutant, a strain in which most of the PEX17 gene is deleted, fail to form normal peroxisomes but instead contain numerous large, multimembraned structures . The import of peroxisomal matrix proteins in these mutants is selectively impaired . This selective import is not a function of the nature of the peroxisomal targeting signal . We suggest a regulatory role for Pex17p in the import of a subset of matrix proteins into peroxisomes.

Nucleic Acids Res, 1997 May 1, 25(9), 1872 - 3
Rapid isolation of yeast plasmids as native chromatin; Martinez-Campa C et al.; Many regions of chromatin are subject to dynamic changes . We have developed a rapid method for isolation of small chromatin templates from yeast which will facilitate biochemical analysis of chromatin composition . Using the PHO5 promoter we show that templates prepared from cells grown in inducing or repressing conditions show native chromatin structures . This method may be widely applicable as the chromatin structures at a centromere, at ARS1 and at part of the lacZ region on two other plasmids are preserved after chromatin isolation.

Nucleic Acids Res, 1997 May 1, 25(9), 1859 - 61
Matrix-assisted laser desorption/ionisation mass spectrometry of transfer ribonucleic acids isolated from yeast; Gruic-Sovulj I et al.; tRNATyr and tRNASer purified from bulk brewer's yeast tRNA were subjected to analysis by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry . Choosing a mixture of 2,4,6- and 2,3,4-trihydroxy-acetophenone and diammonium citrate as matrix a mass resolution of up to 220 (FWHM) was achieved in the linear mode of operation . Cation adduct suppression by addition of cation exchange beads and a chelating agent (CDTA) is shown to substantially improve mass resolution for this class of molecules.

Gene, 1997 Apr 29, 190(1), 77 - 85
The presence of two tightly bound Zn2+ ions is essential for the structural and functional integrity of yeast RNA polymerase II; Mayalagu S et al.; DNA-dependent RNA polymerases (RNApol) are Zn2+ metalloproteins where the Zn2+ ion plays both catalytic and structural roles . Although the ubiquitous presence of Zn2+ with the RNApol from eukaryotes had already been established, the exact stoichiometry of Zn2+ ion(s) per mole enzyme is not well documented, and its role in enzymatic function remains elusive . We show here that RNApolII from Saccharomyces cerevisiae has two Zn2+ ions tightly associated with it which are necessary for its transcriptional activity . Upon prolonged dialysis against 10 mM EDTA for 4-5 h, the enzyme loses one Zn2+, as well as partial activity . However, Zn2+ can be added back to the enzyme, but without recovering its total activity . 5 mM orthophenanthroline (OP) removes one Zn2+ within 2 h; the enzyme, however, cannot be reconstituted back with Zn2+ . Circular dichroism (CD) studies showed that the conformation of the native enzyme is unique and cannot be reproduced with Zn2+-reconstituted RNApolII . Similarly, the rate of abortive synthesis of a dinucleotide product over a non-specific template is faster when catalyzed by two Zn2+-native enzymes . Zn2+-reconstituted RNApolII or one Zn2+-RNApolII showed a slower abortive synthesis rate . 65Zn2+-blotting experiments indicated that the removal of one Zn2+ from the enzyme destroys the Zn2+-binding ability of the larger subunits of yeast RNApolII . In order to check whether the presence of Zn2+ ions has any effect on substrate recognition, we followed the binding of (gamma-AmNS)UTP, a fluorescent substrate analog to RNApolII . It was observed that OP-treated enzyme showed non-specific substrate recognition, whereas two Zn2+-native RNApol binds substrate at a single site.

Gene, 1997 Apr 29, 190(1), 69 - 76
Characterisation of 3' end formation of the yeast HIS3 mRNA; Mahadevan S et al.; The nucleotide (nt) sequence of the 3' end of the yeast HIS3 mRNA was determined by PCR amplification of the 3' end . Analysis of 28 individual clones revealed that at least 13 distinct polyadenylation sites are present . The sites of polyadenylation are extremely heterogeneous and do not show any obvious similarity other than that they occur after pyrimidine residues in most cases . Most mutants carrying internal deletions of the 3' untranslated region (3' UTR) did not abolish 3' end formation and showed polyadenylation at normal sites . Deletion of a 90-nt region that contains an A+T-rich sequence close to the 3' end of the HIS3 coding sequence and a subset of processing sites resulted in a drastic reduction in the levels of full-length HIS3 mRNA and concomitant transcription past the normal HIS3 3' end . The 90-nt region appears to be sufficient to direct the formation of at least a subset of the HIS3 3' ends since mutants that carry deletions of flanking regions of this sequence show detectable levels of HIS3 mRNA . Spacing between the upstream A-T sequence and the site of processing is variable . In the light of the extreme heterogeneity of the sites, a possible mechanism for 3' processing is discussed.

Biochemistry, 1997 Apr 29, 36(17), 5251 - 9
Proton NMR assignments and magnetic axes orientations for wild-type yeast iso-1-ferricytochrome c free in solution and bound to cytochrome c peroxidase; Sukits SF et al.; Extensive proton hyperfine-shifted resonance assignments have been made for wild-type yeast iso-1-ferricytochrome c when it is free in solution and when it is noncovalently complexed to resting state cytochrome c peroxidase . Complete heme proton resonance assignments were made for free iso-1-ferricytochrome c, while for CcP-complexed iso-1-ferricytochrome c, 70% of heme proton assignments were made . Additional proton resonance assignments were made for hyperfine-shifted protons of amino acids near the heme . These assignments allowed identification of the most extensive set of complex-induced proton shifts yet reported for CcP/cytochrome c complexes . Several purely dipolar-shifted resonances from heme vicinity amino acid protons were also assigned in both free and complexed iso-1-ferricyt c . Both sets of resonance assignments allowed assessment of the origin of proton complex-induced shifts . Using the assigned dipolar-shifted proton resonances as a basis, the orientations of the principal axis systems of the paramagnetic susceptibility tensors for free and cytochrome c peroxidase-bound iso-1-ferricytochrome c were elucidated . The results indicated that the iso-1-ferricytochrome c magnetic axis system orientation shifts significantly upon complex formation . The direction of the complex-induced shifts for heme proton resonances is largely accounted for by the magnetic anisotropy changes . However, analysis of heme complex-induced shifts also reveals local changes in magnetic environment for two heme substituents, presumably through a specific structure change.

Proc Natl Acad Sci U S A, 1997 Apr 29, 94(9), 4463 - 8
Two active states of the Ras-related Bud1/Rsr1 protein bind to different effectors to determine yeast cell polarity; Park HO et al.; Cells of budding yeast organize their cytoskeleton in a highly polarized manner during vegetative growth . Selection of a site for polarization requires a group of proteins including a Ras-like GTPase, Bud1, and its regulators . Another group of proteins, which includes a Rho-like GTPase (Cdc42), its guanine nucleotide exchange factor (Cdc24), and Bem1, is necessary for organization of the actin cytoskeleton and for cell polarization . We have proposed previously that the Bud1 protein, through its GTPase cycle, determines the localization of one or more of the cell polarity proteins to the bud site . Herein we demonstrate that Bud1 directly interacts with Cdc24 and Bem1: Bud1 in its GTP-bound form associates preferentially with Cdc24, whereas the GDP-bound form of Bud1 associates with Bem1 . We also present subcellular fractionation data for Bud1 that is consistent with the idea that Bud1 can travel between the site for budding on the plasma membrane and the cytosol . We propose that Bud1 can exist in two active states for association with different partners and that the switch from Bud1-GTP to Bud1-GDP provides a regulatory device for ordered assembly of a macromolecular complex at the bud site.

DNA Res, 1997 Apr 28, 4(2), 127 - 31
Yeast artificial chromosome clones of rice chromosome 2 ordered using DNA markers; Umehara Y et al.; Yeast artificial chromosome (YAC) clones were ordered for the physical mapping of rice chromosome 2, the last of the 12 rice chromosomes to be assigned YACs by the Rice Genome Research Program . A total of 128 restriction fragment length polymorphism markers and 4 sequence-tagged site (STS) markers located on our high-density genetic map were used for YAC clone landing . By colony/Southern hybridization and polymerase chain reaction screening, a total of 239 individual YACs were selected from our YAC library of 6934 clones covering six genome equivalents . The YACs located on the corresponding marker positions in the linkage map formed 43 contigs and islands and were estimated to encompass about 50% of the length of rice chromosome 2.

Biochem Pharmacol, 1997 Apr 25, 53(8), 1161 - 72
Transcriptional activation of the human estrogen receptor by DDT isomers and metabolites in yeast and MCF-7 cells; Chen CW et al.; In this study, we determined whether the DDT isomers p,p'-DDT {1,1,1,-trichloro-2,2-bis(p-chlorophenyl)ethane}, o,p'-DDT {1,1,1-trichloro-2(p-chlorophenyl)-2-(o-chlorophenyl)ethane}, and their metabolites p,p'-DDD {1,1-dichloro-2,2-bis(p-chlorophenyl)ethane}, o,p'-DDD {1,1-dichloro-2-(p-chlorophenyl)-2-(o-chlorophenyl)ethane}, p,p'-DDE {1,1,-dichloro-2,2-bis(p-chlorophenyl)ethylene}, o,p'-DDE {1,1-dichloro-2-(p-chlorophenyl)-2-(o-chlorophenyl)ethylene}, and p,p'-DDA {2,2-bis(p-chlorophenyl)acetic acid}, could bind to and transcriptionally activate the human estrogen receptor (hER) . Novel results from competitive binding assays showed that o,p'-DDD, o,p'-DDE, and p,p'-DDT, as well as the established environmental estrogen o,p'-DDT, were able to bind specifically to the hER with approximately 1000-fold weaker affinities for the hER than that of estradiol . In contrast, only o,p'-DDT, but not p,p'-DDT, bound to the rat estrogen receptor . Moreover, two yeast expression-reporter systems, constructed to test if the DDT isomers and metabolites could transcriptionally activate the hER, demonstrated that an o,p'-DDT metabolite could transactivate the hER or LexA-hER fusion protein with just a 140- to 300-fold weaker potency than that of estradiol . The DDT isomers and metabolites that bound the hER in vitro triggered estrogen receptor-mediated transcription of the lacZ reporter gene in the yeast systems . Furthermore, the DDT isomers and metabolites that transactivated the hER elicited an additive response when given together or with estradiol . The DDT isomers and metabolites that triggered transcription of the yeast expression-reporter systems also stimulated two estrogenic endpoints in estrogen-responsive MCF-7 cells: the induction of the progesterone receptor and the down-regulation of the hER . Thus, in MCF-7 cells and in yeast expression-reporter systems, certain DDT isomers and metabolites act directly as agonists and transactivate the hER at concentrations found in human tissues.

Mol Cell Endocrinol, 1997 Apr 25, 129(1), 63 - 71
o,p'-DDT and its metabolites inhibit progesterone-dependent responses in yeast and human cells; Klotz DM et al.; Using a combination of in vitro assays we have evaluated whether DDT metabolites can interact with the progesterone receptor pathway in yeast expressing human progesterone receptor (hPR) and in T47D human breast cancer cells which express endogenous hPR . In transactivation assays using both yeast and T47D cells, o,p'-DDT and the metabolites p,p'-DDT, o,p'-DDD, p,p'-DDD, o,p'-DDE, p,p'-DDE, p,p'-DDA, and DDOH inhibited progesterone-induced reporter gene activity in a dose-dependent manner . None of the DDT metabolites functioned as hPR agonists . Whole cell competition binding assays using T47D cells indicated that the inhibitory effects of DDT metabolites on progesterone-dependent activites may occur through both hPR-dependent and hPR-independent pathways . Our results and previous reports of DDT metabolites interacting with estrogen and androgen receptors suggests that this class of environmental chemicals may interact with numerous hormone receptor signaling pathways.

J Cell Biol, 1997 Apr 21, 137(2), 399 - 416
High rates of actin filament turnover in budding yeast and roles for actin in establishment and maintenance of cell polarity revealed using the actin inhibitor latrunculin-A; Ayscough KR et al.; We report that the actin assembly inhibitor latrunculin-A (LAT-A) causes complete disruption of the yeast actin cytoskeleton within 2-5 min, suggesting that although yeast are nonmotile, their actin filaments undergo rapid cycles of assembly and disassembly in vivo . Differences in the LAT-A sensitivities of strains carrying mutations in components of the actin cytoskeleton suggest that tropomyosin, fimbrin, capping protein, Sla2p, and Srv2p act to increase actin cytoskeleton stability, while End3p and Sla1p act to decrease stability . Identification of three LAT-A resistant actin mutants demonstrated that in vivo effects of LAT-A are due specifically to impairment of actin function and implicated a region on the three-dimensional actin structure as the LAT-A binding site . LAT-A was used to determine which of 19 different proteins implicated in cell polarity development require actin to achieve polarized localization . Results show that at least two molecular pathways, one actin-dependent and the other actin-independent, underlie polarity development . The actin-dependent pathway localizes secretory vesicles and a putative vesicle docking complex to sites of cell surface growth, providing an explanation for the dependence of polarized cell surface growth on actin function . Unexpectedly, several proteins that function with actin during cell polarity development, including an unconventional myosin (Myo2p), calmodulin, and an actin-interacting protein (Bud6/Aip3p), achieved polarized localization by an actin-independent pathway, revealing interdependence among cell polarity pathways . Finally, transient actin depolymerization caused many cells to abandon one bud site or mating projection and to initiate growth at a second site . Thus, actin filaments are also required for maintenance of an axis of cell polarity.

J Mol Biol, 1997 Apr 18, 267(5), 1124 - 38
Folding of the presequence of yeast pAPI into an amphipathic helix determines transport of the protein from the cytosol to the vacuole; Martinez E et al.; To investigate the role of the 17 residues long presequence (p17) in the transport of the precursor of yeast API (pAPI) from the cytosol to the vacuole we have studied the effects of point mutations upon its conformation and on the process of transport . 1H NMR analysis of p17 indicates that in aqueous solution 26% of the molecules have the 4-12 segment folded into an helix . The hydrophobic environment provided by SDS micelles promotes the folding of 54% of the p17 molecules into a 5-16 amphipathic alpha-helix . Both Schiffer-Edmunson helical wheel analysis of segment 4-12 and residue hydrophobic moments calculated considering all possible side-chain orientations between 80 and 120 degrees, indicate the amphipathic character of the helixes assembled in water and detergent . Charge interactions between the dipole pairs N-Glu2Glu3 and C-Lys12Lys13 are essential for helix stability and condition pAPI transport . Substitution of either Pro2Pro3 or Lys2Lys3 for Glu2Glu3, results in moderate destabilization of the helix, decreases protein targeting to the vacuolar membrane and partly inhibits translocation of the protein to the vacuolar lumen . Replacement of either Pro12Pro13 or Glu12Glu13 for Lys12Lys13, causes a major disruption of the helix, decreases protein targeting and blocks completely the translocation of the protein to the vacuolar lumen . Replacement of Gly7 for Ile7, a substitution which is known to destabilize alpha-helixes in peptides and proteins as a result of the peptide bond to the solvent at Gly residues, produces similar effects as the substitutions for the K12K13 pair . The effects of Gly7 on helix stability and protein transport are partly reversed by introduction of Asp residues at positions 2 and 3 and Ala at position 4 . Replacements such as Arg2 for Glu2, or Arg6 for Glu6, which change the net and local charges of the presequence without altering its conformation, have no effect on the protein transport . These results provide direct evidence of the involvement of the presequence in the transport of pAPI from the cytosol to the vacuole . They show that folding of the pAPI presequence is conditioned by the physical/chemical properties of the environment and is critical for targeting the protein to the vacuolar membrane and for its translocation to the vacuolar lumen.

Mol Gen Genet, 1997 Apr 16, 254(3), 284 - 90
Transcription elongation factor S-II is not required for transcription-coupled repair in yeast; Verhage RA et al.; Two different subpathways play a role in removal of UV-induced cyclobutane pyrimidine dimers (CPDs) by nucleotide excision repair (NER) . The relatively slow global genome repair subpathway operates on all CPDs irrespective of their position in the DNA, whereas the transcription-coupled repair subpathway is responsible for the rapid removal of CPDs from transcribed strands . In Saccharomyces cerevisiae, the RAD26 gene is implicated in transcription-coupled repair . However, transcription-coupled repair is not completely absent in rad26 mutants, and therefore other gene products are possibly involved in this subpathway . Based on in vitro experiments with purified components, the transcription elongation factor S-II appeared to be a candidate for a function in transcription-coupled repair . To investigate a possible role of S-II in transcription-coupled repair in vivo in yeast, S-II null mutations were introduced into various genetic backgrounds differing in NER capacity . UV sensitivity was not altered by disruption of the S-II gene in a RAD+ (NER proficient) strain, or in rad26 (impaired in efficient transcription-coupled repair), rad7 (lacking global genome repair), or rad7 rad26 (lacking global genome repair, but having residual transcription-coupled repair capacity) mutants . Moreover, S-II did not influence the repair rate on the transcribed strand of the RPB2 gene, either in repair-proficient or in rad7 rad26 backgrounds . Hence, transcription-coupled repair is fully functional in yeast cells lacking the gene encoding S-II . Furthermore, S-II is not required for the Rad26-independent residual transcription-coupled repair in vivo.

Mol Gen Genet, 1997 Apr 16, 254(3), 267 - 74
Screening and identification of yeast sequences that cause growth inhibition when overexpressed; Akada R et al.; To isolate genes that negatively regulate cell growth, we constructed a galactose-inducible expression library with partially digested Saccharomyces cerevisiae genomic DNA fragments inserted downstream of the GAL10 promoter . In all, 240,000 yeast transformants were screened for lethality on galactose medium . From 17 such transformants identified, 16 nonoverlapping DNA sequences were obtained . Restriction mapping and determination of DNA sequences adjacent to the GAL10 promoter indicated that the inserts encoded part or all of the URA2, RBP1, TPK3, SAC7, BOI1, and BNI1 genes, and also open reading frames (ORFs) from chromosomes IV, V, IX, XI, and XIII . Some of the identified sequences lacked the amino-terminal sequences of the ORFs, suggesting that truncated forms of the proteins might be necessary for growth inhibition . The sequence of the pGA108 insert was highly homologous to the telomeric X-element and contained an ARS consensus sequence, suggesting a possible growth inhibitory effect of an RNA molecule . Overexpression of the BNI1 deltaN and BOI1 deltaN genes, which lacked amino-terminal sequences, was associated with phenotypes similar to those of mutants defective in bud formation . Overexpression of the GIN4 and GIN12 sequences induced elongated buds and a G2/M arrest-like phenotype, respectively . The phenotypes induced by the overexpression of our cloned sequences could result from either a dominant-positive or a dominant-negative effect and, unexpectedly, in one case from an effect of an RNA.

Biochem J, 1997 Apr 15, 323 ( Pt 2), 409 - 13
Bivalent cations stabilize yeast alcohol dehydrogenase I; De Bolle X et al.; The thermostability of yeast alcohol dehydrogenase (ADH) I is strongly dependent on the presence of NaCl, a salt that is almost neutral on the Hofmeister scale, which suggests that solvent-accessible electrostatic repulsion might play a role in the inactivation of the enzyme . Moreover, CaCl2 and MgCl2 are able to stabilize the enzyme at millimolar concentrations . Ca2+ stabilizes yeast ADH I by preventing the dissociation of the reduced form of the enzyme and by preventing the unfolding of the oxidized form of the enzyme . An analysis of several chimaeric ADHs suggests that Ca2+ is fixed by the Asp-236 and Glu-101 side chains in yeast ADH I, but that Ca2+ can be displaced by replacing Met-168 by an Arg residue, as suggested by a three-dimensional model of the enzyme structure . These results indicate that electrostatic repulsion can cause protein unfolding and/or dissociation . It is proposed that yeast ADH I binds Mg2+ in vivo.

EMBO J, 1997 Apr 15, 16(8), 2119 - 29
Site-specific deoxynucleotide substitutions in yeast U6 snRNA block splicing of pre-mRNA in vitro; Kim CH et al.; We have identified 2'-hydroxyl groups of the U6 phosphate-ribose backbone which are required for reconstitution of splicing activity in U6-depleted yeast extract . To screen the 2'-hydroxyls of yeast U6 at nucleotides 39-88, spanning the conserved central domain, synthetic U6 RNAs were constructed with deoxyribonucleotides incorporated site specifically . Only four individual deoxynucleotide substitutions blocked splicing activity: dA51 (in the ACAGAG sequence), dA62 (next to the AGC triad), and dU70 and dC72 (both in the loop of the 3' intramolecular stem-loop) . Native gel analysis revealed that these deoxy-substituted U6 RNAs were competent for assembly of spliceosomes . Interestingly, a 2'-O-methyl substituent at A51, A62, U70 or C72 did not inhibit splicing activity, indicating that the essential 2'-OH groups at these positions in U6 act as hydrogen bond acceptors or neutral coordinated ligands . The requisite 2'-hydroxyls at A62, U70 and C72 show both similarities and differences relative to the positions of essential 2'-hydroxyls of catalytic domain V of group II ribozymes . The identification of the essential 2'-hydroxyls at positions 62, 70 and 72 corroborates that the 3' intramolecular stem-loop in U6 plays an important role in pre-mRNA splicing.

EMBO J, 1997 Apr 15, 16(8), 2054 - 60
mof, a putative acetyl transferase gene related to the Tip60 and MOZ human genes and to the SAS genes of yeast, is required for dosage compensation in Drosophila; Hilfiker A et al.; Dosage compensation is a regulatory process that insures that males and females have equal amounts of X-chromosome gene products . In Drosophila, this is achieved by a 2-fold enhancement of X-linked gene transcription in males, relative to females . The enhancement of transcription is mediated by the activity of a group of regulatory genes characterized by the male-specific lethality of their loss-of-function alleles . The products of these genes form a complex that is preferentially associated with numerous sites on the X chromosome in somatic cells of males but not of females . Binding of the dosage compensation complex is correlated with a significant increase in the presence of a specific histone isoform, histone 4 acetylated at Lys16, on this chromosome . Experimental results and sequence analysis suggest that an additional gene, males-absent on the first (mof), encodes a putative acetyl transferase that plays a direct role in the specific histone acetylation associated with dosage compensation . The predicted amino acid sequence of MOF exhibits a significant level of similarity to several other proteins, including the human HIV-1 Tat interactive protein Tip60, the human monocytic leukemia zinc finger protein MOZ and the yeast silencing proteins SAS3 and SAS2.

EMBO J, 1997 Apr 15, 16(8), 1832 - 41
Transmembrane domain-dependent sorting of proteins to the ER and plasma membrane in yeast; Rayner JC et al.; Sorting of membrane proteins between compartments of the secretory pathway is mediated in part by their transmembrane domains (TMDs) . In animal cells, TMD length is a major factor in Golgi retention . In yeast, the role of TMD signals is less clear; it has been proposed that membrane proteins travel by default to the vacuole, and are prevented from doing so by cytoplasmic signals . We have investigated the targeting of the yeast endoplasmic reticulum (ER) t-SNARE Ufe1p . We show that the amino acid sequence of the Ufe1p TMD is important for both function and ER targeting, and that the requirements for each are distinct . Targeting is independent of Rer1p, the only candidate sorting receptor for TMD sequences currently known . Lengthening the Ufe1p TMD allows transport along the secretory pathway to the vacuole or plasma membrane . The choice between these destinations is determined by the length and composition of the TMD, but not by its precise sequence . A longer TMD is required to reach the plasma membrane in yeast than in animal cells, and shorter TMDs direct proteins to the vacuole . TMD-based sorting is therefore a general feature of the yeast secretory pathway, but occurs by different mechanisms at different points.

EMBO J, 1997 Apr 15, 16(8), 1820 - 31
Endosomal transport function in yeast requires a novel AAA-type ATPase, Vps4p; Babst M et al.; In a late-Golgi compartment of the yeast Saccharomyces cerevisiae, vacuolar proteins such as carboxypeptidase Y (CPY) are actively sorted away from the secretory pathway and transported to the vacuole via a pre-vacuolar, endosome-like intermediate . The vacuolar protein sorting (vps) mutant vps4 accumulates vacuolar, endocytic and late-Golgi markers in an aberrant multilamellar pre-vacuolar compartment . The VPS4 gene has been cloned and found to encode a 48 kDa protein which belongs to the protein family of AAA-type ATPases . The Vps4 protein was purified and shown to exhibit an N-ethylmaleimide-sensitive ATPase activity . A single amino acid change within the AAA motif of Vps4p yielded a protein that lacked ATPase activity and did not complement the protein sorting or morphological defects of the vps4 delta1 mutant . Indeed, when expressed at normal levels in wild-type cells, the mutant vps4 gene acted as a dominant-negative allele . The phenotypic characterization of a temperature-sensitive vps4 allele showed that the immediate consequence of loss of Vps4p function is a defect in vacuolar protein delivery . In this mutant, precursor CPY was not secreted but instead accumulated in an intracellular compartment, presumably the pre-vacuolar endosome . Electron microscopy revealed that upon temperature shift, exaggerated stacks of curved cisternal membranes (aberrant endosome) also accumulated in the vps4ts mutant . Based on these and other observations, we propose that Vps4p function is required for efficient transport out of the pre-vacuolar endosome.

Eur J Biochem, 1997 Apr 15, 245(2), 266 - 72
Intracellular location, complex formation, and function of the transporter associated with antigen processing in yeast; Urlinger S et al.; Peptide transport across the membrane of the endoplasmic reticulum (ER) gains increasing importance in view of its potential function in selective protein degradation and antigen processing . An example for peptide transport in the ER is the transporter associated with antigen processing (TAP), which supplies peptides for the formation of major-histocompatibility-complex class-I complexes . Here, we have expressed human TAP1 and TAP2 in the yeast Saccharomyces cerevisiae . Expression of both genes resulted in the formation of a stable TAP heterodimer that was localized mainly in the ER . Although a minor fraction of TAP is found in the plasma membrane, TAP is unable to restore a-factor secretion in a mutant cell line that lacks the yeast mating-factor transporter Ste6 . Nevertheless, in vitro studies with microsomal vesicles demonstrated that the TAP complex is fully functional in the ER membrane in terms of selective peptide binding, ATP-dependent transport, and specific inhibition by the viral protein of herpes simplex virus ICP47 . This offers opportunities for topological, structural and mechanistic studies as well as genetic screenings for TAP functionality.

Arch Biochem Biophys, 1997 Apr 15, 340(2), 250 - 6
Unusual usage of noncomplementary dinucleotide primers by the yeast mitochondrial RNA polymerase; Biswas TK; The mitochondrial RNase P RNA gene in yeast Saccharomyces cerevisiae is transcribed from a variant mitochondrial promoter (SP) . The sequence of this SP promoter {TATAAGAAG (+2)} differs from the conserved mitochondrial promoter sequence {TATAAGTAA (+2)} by-1T-->A and +2A-->G nucleotide substitutions . To determine the effect of these nucleotide alterations in mitochondrial promoter function, an in vitro transcription analysis was carried out . In the presence of high concentrations of rNTPs (i.e., 125 microM), transcription initiation on the wild-type or variant promoter occurred at the conventional 3' adenine nucleotide . However, at low rNTP concentrations (i.e., 5 microM) and in the presence of a complementary dinucleotide primer corresponding to positions -1 + 1, the mitochondrial RNA polymerase started transcription one nucleotide upstream of the conventional start site . Surprisingly, in the presence of some noncomplementary dinucleotides (i.e., GpA or CpA), which do not have perfect Watson-Crick base pairing with the initiator sequence, transcriptional initiation also occurred with the SP promoter but not with the conserved promoter sequence . This finding is the first example of utilization of noncomplementary dinucleotide primer by an RNA polymerase . Further analysis of mitochondrial promoter function by site-directed mutagenesis determined that the guanine nucleotide at position +2 is mainly responsible for this unusual function of the SP promoter.

Genes Dev, 1997 Apr 15, 11(8), 1008 - 22
The Mcs4 response regulator coordinately controls the stress-activated Wak1-Wis1-Sty1 MAP kinase pathway and fission yeast cell cycle; Shieh JC et al.; The fission yeast Sty1 MAP kinase is required for cell cycle control, initiation of sexual differentiation, and protection against cellular stress . Like the mammalian JNK/SAPK and p38/CSBP1 MAP kinases, Sty1 is activated by a range of environmental insults including osmotic stress, hydrogen peroxide, menadione, heat shock, and the protein synthesis inhibitor anisomycin . We have identified an upstream regulator that mediates activation of the Sty1 MAP kinase by multiple environmental stresses as the product of the mitotic catastrophe suppressor, mcs4 . Mcs4 is structurally and functionally homologous to the budding yeast SSK1 response regulator, suggesting that the eukaryotic stress-activated MAP kinase pathway is controlled by a conserved two-component system . Mcs4 acts upstream of Wak1, a homolog of the SSK2 and SSK22 MEK kinases, which transmits the stress signal to the Wis1 MEK . We show that the Wis1 MEK is controlled by an additional pathway that is independent of both Mcs4 and the Wak1 MEK kinase . Furthermore, we demonstrate that Mcs4 is required for the correct timing of mitotic initiation by mechanisms both dependent and independent on Sty1, indicating that Mcs4 coordinately controls cell cycle progression with the cellular response to environmental stress.

Biochemistry, 1997 Apr 15, 36(15), 4552 - 7
Yeast protein geranylgeranyltransferase type-I: steady-state kinetics and substrate binding; Stirtan WG et al.; Protein geranylgeranyltransferase type-I (PGGTase-I) catalyzes alkylation of the cysteine residue in proteins containing a consensus C-terminal CaaX sequence ending in Leu or Phe by the C20 hydrocarbon moiety in geranylgeranyl diphosphate (GGPP) . A kinetic study of the alkylation reaction was conducted with a continuous assay based on the fluorescence enhancement that accompanies geranylgeranylation of dansyl-GCIIL . The kinetic constants k(cat) = 0.34 +/- 0.01 s(-1), K(M)(G) = 0.86 +/- 0.05 microM for GGPP, and K(M)(D) = 1.6 +/- 0.1 microM for dansyl-GCIIL were calculated from initial rates measured at varying concentrations of the substrates . Inhibitor studies were conducted with dead-end inhibitors for GGPP and the peptide substrate . Double reciprocal plots for the peptide mimic Cys-AMBA-Leu gave a competitive pattern when plotted against varying concentrations of dansyl-GCIIL and an uncompetitive pattern against GGPP . Similar plots for 1-phosphono-(E,E,E)-geranylgeraniol, a dead-end inhibitor for GGPP, gave a competitive double reciprocal plot for varied concentrations of GGPP and induced potent substrate inhibition by dansyl-GCIIL when dansyl-GCIIL was the varied substrate . The dissociation constant (K(D)) for the PGGTase-I x GGPP complex was 120 +/- 20 nM . These results are consistent with an ordered binding mechanism for PGGTase-I where GGPP adds before peptide.

Proc Natl Acad Sci U S A, 1997 Apr 15, 94(8), 3888 - 92
The k43 gene, required for chorion gene amplification and diploid cell chromosome replication, encodes the Drosophila homolog of yeast origin recognition complex subunit 2; Landis G et al.; Lethal alleles of the Drosophila k43 gene result in small or missing imaginal discs, greatly reduced mitotic index, and fragmented and abnormally condensed chromosomes . A female-sterile allele of k43 specifically reduces chorion gene amplification in ovarian follicle cells . k43 was cloned by chromosomal walking, and the identification of the k43 gene was confirmed by phenotypic rescue and sequence analysis of mutant alleles . The sequence analyses reveal that the k43 gene encodes the Drosophila homolog of the yeast origin recognition complex subunit 2 (Orc2p), a protein required for replication origin function and transcriptional silencing in yeast . These results suggest an evolutionarily conserved role for Orc2p in eukaryotic chromosomal DNA replication.

Proc Natl Acad Sci U S A, 1997 Apr 15, 94(8), 3732 - 5
Resinless section electron microscopy reveals the yeast cytoskeleton; Penman J et al.; The cytoskeleton of Saccharomyces cerevisiae is essentially invisible using conventional microscopy techniques . A similar problem was solved for the mammalian cell cytoskeleton using resinless section electron microscopy, a technique applied here to yeast . In the resinless image, soluble proteins are no longer cloaked by embedding medium and must be removed by selective detergent extraction . In yeast, this requires breaching the cell wall by digesting with Zymolyase sufficiently to allow detergent extraction of the plasma membrane lipids . Gel electropherograms show that the extracted or "soluble" proteins are distinct from the retained or "structural" proteins that presumably comprise the cytoskeleton . These putative cytoskeleton proteins include the major portions of a 43-kDa protein, which is presumably actin, and of proteins in a band appearing at 55 kDa, as well as numerous less abundant, nonactin proteins . Resinless section electron micrographs show a dense, three-dimensional web of anastomosing, polymorphic filaments bounded by the remnant cell wall . Although the filament network is very heterogenous, there appear to be two principal classes of filament diameters-5 nm and 15-20 nm-which may correspond to actin and intermediate filaments, respectively . A large oval region of lower filament density probably corresponds to the vacuole, and an electron dense spheroidal body, 300-500 nm in diameter, is likely the nucleus . The techniques detailed in this report afford new approaches to the study of yeast cytoarchitecture.

Proc Natl Acad Sci U S A, 1997 Apr 15, 94(8), 3697 - 702
Yeast hormone response element assays detect and characterize GRIP1 coactivator-dependent activation of transcription by thyroid and retinoid nuclear receptors; Walfish PG et al.; The mouse glucocorticoid receptor-interacting protein (GRIP1) is a member of the ERAP160 family of nuclear receptor (NR) coactivators (including SRC-1 and TIF2) which function as bridging proteins between ligand-activated NRs bound to cognate hormone-response elements (HREs) and the transcription initiation apparatus (TIA) . Although these coactivators bind to several NRs, studies overexpressing these coactivators with these NRs in mammalian cells have not uniformly observed a corresponding enhancement of ligand-dependent transactivation . Here, we show that GRIP1 interacts in vitro in a ligand-dependent manner with thyroid receptor, retinoic acid receptor, and retinoid X receptor . Additionally, in yeast (Saccharomyces cerevisiae) GRIP1 coactivator protein markedly increased the ability of these full-length class II NRs to transactivate beta-galactosidase reporter genes containing cognate HREs . The magnitude of GRIP1 enhancement of liganded NR homodimer was dependent upon NR subtype and HRE configuration . For most HRE configurations, thyroid receptor and retinoic acid receptor homodimers were essentially unresponsive or very weakly active in the absence of GRIP1, but GRIP1 dramatically restored the ligand-dependent function of these NRs . Although GRIP1 exerted no significant effect on NR homodimers in the absence of their cognate ligands, it increased the transactivation of unliganded NR heterodimers . Whether GRIP1 increased ligand-dependent transactivation of a heterodimer to levels greater than that of the cognate homodimer was determined by HRE configuration and copy number . Compared with the limitations of yeast two-hybrid and mammalian coexpression systems, the yeast HRE-assay systems described in this report facilitated both the detection of putative mammalian NR coactivator function and the elucidation of their mechanisms of transactivational enhancement.

Nucleic Acids Res, 1997 Apr 15, 25(8), 1597 - 604
The Cbp2 protein stimulates the splicing of the omega intron of yeast mitochondria; Shaw LC et al.; The Cbp2 protein is encoded in the nucleus and is required for the splicing of the terminal intron of the mitochondrial COB gene in Saccharomyces cerevisiae . Using a yeast strain that lacks this intron but contains a related group I intron in the precursor of the large ribosomal RNA, we have determined that Cbp2 protein is also required for the normal accumulation of 21S ribosomal RNA in vivo . Such strains bearing a deletion of the CBP2 gene adapt slowly to growth in glycerol/ethanol media implying a defect in derepression . At physiologic concentrations of magnesium, Cbp2 stimulates the splicing of the ribosomal RNA intron in vitro . Nevertheless, Cbp2 is not essential for splicing of this intron in mitochondria nor is it required in vitro at magnesium concentrations >5 mM . A similar intron exists in the large ribosomal RNA (LSU) gene of Saccharomyces douglasii . This intron does need Cbp2 for catalytic activity in physiologic magnesium . Similarities between the LSU introns and COB intron 5 suggest that Cbp2 may recognize conserved elements of the these two introns, and protein-induced UV crosslinks occur in similar sites in the substrate and catalytic domains of the RNA precursors.

J Biol Chem, 1997 Apr 11, 272(15), 9895 - 901
PMR1, a Ca2+-ATPase in yeast Golgi, has properties distinct from sarco/endoplasmic reticulum and plasma membrane calcium pumps; Sorin A et al.; PMR1, a P-type ATPase cloned from the yeast Saccharomyces cerevisiae, was previously localized to the Golgi, and shown to be required for normal secretory processes (Antebi, A., and Fink, G.R . (1992) Mol . Biol . Cell 3, 633-654) . We provide biochemical evidence that PMR1 is a Ca2+-transporting ATPase in the Golgi, a hitherto unusual location for a Ca2+ pump . As a starting point for structure-function analysis using a mutagenic approach, we used the strong and inducible heat shock promoter to direct high level expression of PMR1 from a multicopy plasmid . Yeast lysates were separated on sucrose density gradients, and fractions assayed for organellar markers . PMR1 is found in fractions containing the Golgi marker guanosine diphosphatase, and is associated with an ATP-dependent, protonophore-insensitive 45Ca2+ uptake activity . This activity is virtually abolished in the absence of the expression plasmid . Furthermore, replacement of the active site aspartate within the phosphorylation domain had the expected effect of abolishing Ca2+ transport activity entirely . Interestingly, the mutant enzymes (Asp-371 --> Glu and Asp-371 --> Asn) demonstrated proper targeting to the Golgi, unlike analogous mutations in the related yeast H+-ATPase . Detailed characterization of calcium transport by PMR1 showed that sensitivity to inhibitors (vanadate, thapsigargin, and cyclopiazonic acid) and affinity for substrates (MgATP and Ca2+) were different from the previously characterized sarco/endoplasmic reticulum and plasma membrane Ca2+-ATPases . PMR1 therefore represents a new and distinct P-type Ca2+-ATPase . Because close homologs of PMR1 have been cloned from rat and other organisms, we suggest that Ca2+-ATPases in the Golgi will form a discrete subgroup that are important for functioning of the secretory pathway.

Biochemistry, 1997 Apr 8, 36(14), 4067 - 73
Effect of yeast and human DnaJ homologs on clathrin uncoating by 70 kilodalton heat shock protein; King C et al.; We recently found that the DnaJ homolog auxilin is required for Hsc70 to uncoat clathrin baskets . In the present study, we investigated the effect of two other DnaJ homologs, YDJ1 from yeast and HDJ1 from humans, on the uncoating activity of Hsc70 . Neither YDJ1 nor HDJ1 substituted for auxilin in supporting uncoating . Rather, in the presence of auxilin, both YDJ1 and HDJ1 strongly inhibited uncoating at pH 7 and also prevented the binding of Hsc70 to clathrin baskets at pH 6 . Both YDJ1, as shown previously, and HDJ1 catalytically induce polymerization of Hsc70 into large polymers in ATP, and the YDJ1 concentration required to inhibit uncoating was similar to the concentration required for polymerization . However, uncoating was almost completely inhibited even at low concentrations of Hsc70 where only partial polymerization occurs, suggesting that YDJ1 inhibits uncoating not only by polymerizing the Hsc70 but also by some other mechanism as well . The effects of YDJ1 and HDJ1 were completely reversible; when they were removed, the Hsc70 regained full activity . Since both YDJ1 and HDJ1 inhibited the uncoating of clathrin baskets by brain cytosol as well as by purified Hsc70, this could be a physiological phenomenon which could affect other activities of Hsc70 in addition to uncoating.

Biochem Biophys Res Commun, 1997 Apr 7, 233(1), 139 - 46
Several synthetic chemicals inhibit progesterone receptor-mediated transactivation in yeast; Jin L et al.; The human progesterone receptor (hPR) B-form and a progesterone-sensitive reporter were expressed in yeast and used to screen a library of synthetic chemicals for their ability to function as agonists or antagonists of hPR . The transcriptional activity of hPR was not increased in the presence of over 40 individual chemicals . Seven chemicals decreased progesterone-dependent activity in yeast . The most effective chemicals were 6-hydroxychrysene, 1-hydroxypyrene, 4-hydroxy, 2',4',6'-trichloro biphenyl, and 4-hydroxy, 2',3',4',5'-tetrachloro biphenyl . The decrease of progesterone-mediated transactivation strongly correlated with their displacement of {3H}progesterone from hPR . The absence of the hydroxyl group on the above chemicals completely abolished their inhibitory activity . The other chemicals which decreased progesterone activity were endosulfan II, endosulfan sulfate, and lindane . These chemicals did not inhibit {3H}progesterone binding, suggesting that they inhibit progesterone action by interacting with a region of hPR distinct from binding {3H}progesterone or by a mechanism independent of hPR . These results highlight the utility of yeast for screening hormonally-active chemicals . In addition, hydroxylation appears to be essential for the interaction of some chemicals with hPR.

J Cell Biol, 1997 Apr 7, 137(1), 141 - 53
The yeast gene, MDM20, is necessary for mitochondrial inheritance and organization of the actin cytoskeleton; Hermann GJ et al.; In Saccharomyces cerevisiae, the growing bud inherits a portion of the mitochondrial network from the mother cell soon after it emerges . Although this polarized transport of mitochondria is thought to require functions of the cytoskeleton, there are conflicting reports concerning the nature of the cytoskeletal element involved . Here we report the isolation of a yeast mutant, mdm20, in which both mitochondrial inheritance and actin cables (bundles of actin filaments) are disrupted . The MDM20 gene encodes a 93-kD polypeptide with no homology to other characterized proteins . Extra copies of TPM1, a gene encoding the actin filament-binding protein tropomyosin, suppress mitochondrial inheritance defects and partially restore actin cables in mdm20 delta cells . Synthetic lethality is also observed between mdm20 and tpm1 mutant strains . Overexpression of a second yeast tropomyosin, Tpm2p, rescues mutant phenotypes in the mdm20 strain to a lesser extent . Together, these results provide compelling evidence that mitochondrial inheritance in yeast is an actin-mediated process . MDM20 and TPM1 also exhibit the same pattern of genetic interactions; mutations in MDM20 are synthetically lethal with mutations in BEM2 and MYO2 but not SAC6 . Although MDM20 and TPM1 are both required for the formation and/or stabilization of actin cables, mutations in these genes disrupt mitochondrial inheritance and nuclear segregation to different extents . Thus, Mdm20p and Tpm1p may act in vivo to establish molecular and functional heterogeneity of the actin cytoskeleton.

Cell, 1997 Apr 4, 89(1), 55 - 62
RNA polymerase II holoenzyme recruitment is sufficient to remodel chromatin at the yeast PHO5 promoter; Gaudreau L et al.; We examine transcriptional activation and chromatin remodeling at the PHO5 promoter in yeast by fusion proteins that are thought to act by recruiting the RNA polymerase II holoenzyme to DNA in the absence of a classic activating region . These hybrid proteins (e.g., Gal11+Pho4 or Gal4(58-97)+Pho4 in the presence of a GAL11P allele) efficiently activated transcription and remodeled chromatin . Similar chromatin remodeling was observed at a PHO5 promoter deleted for TATA and thus unable to support transcription . We conclude that recruitment of the holoenzyme or associated proteins suffices for chromatin remodeling . We also show that the SWI/SNF complex is required neither for efficient transcription of the wild-type PHO5 nor the GAL1 promoters, and we observe nearly complete chromatin remodeling at PHO5 in the absence of Snf2.

Science, 1997 Apr 4, 276(5309), 118 - 22
Bni1p, a yeast formin linking cdc42p and the actin cytoskeleton during polarized morphogenesis; Evangelista M et al.; The Saccharomyces cerevisiae BNI1 gene product (Bni1p) is a member of the formin family of proteins, which participate in cell polarization, cytokinesis, and vertebrate limb formation . During mating pheromone response, bni1 mutants showed defects both in polarized morphogenesis and in reorganization of the underlying actin cytoskeleton . In two-hybrid experiments, Bni1p formed complexes with the activated form of the Rho-related guanosine triphosphatase Cdc42p, with actin, and with two actin-associated proteins, profilin and Bud6p (Aip3p) . Both Bni1p and Bud6p (like Cdc42p and actin) localized to the tips of mating projections . Bni1p may function as a Cdc42p target that links the pheromone response pathway to the actin cytoskeleton.

Biochim Biophys Acta, 1997 Apr 3, 1325(1), 8 - 12
Molecular cloning of a mammalian homologue of the yeast vesicular transport protein vps45; El-Husseini AE et al.; We have identified the rat homologue (rvps45) of the yeast vps45 protein, a member of the Sec1 family of proteins involved in intracellular vesicle trafficking . Sequence analysis of the full-length rvps45 cDNA obtained from a rat brain library predicts a protein of 570 amino acids which shares 36% identity with the yeast vps45 protein . The sequence shows less homology with other mammalian Sec1 family proteins . Northern blotting identified a 2.3 kb mRNA highly expressed in brain and testis . RT-PCR analysis showed that the rvps45 gene product is expressed throughout the brain . The homology of this protein with the yeast vps45 together with its high expression in brain suggests a role for rvps45 in transport from the Golgi complex to synaptic vesicles.

Nature, 1997 Apr 3, 386(6624), 463 - 71
Structure of 20S proteasome from yeast at 2.4 A resolution; Groll M et al.; The crystal structure of the 20S proteasome from the yeast Saccharomyces cerevisiae shows that its 28 protein subunits are arranged as an (alpha1...alpha7, beta1...beta7)2 complex in four stacked rings and occupy unique locations . The interior of the particle, which harbours the active sites, is only accessible by some very narrow side entrances . The beta-type subunits are synthesized as proproteins before being proteolytically processed for assembly into the particle . The proforms of three of the seven different beta-type subunits, beta1/PRE3, beta2/PUP1 and beta5/PRE2, are cleaved between the threonine at position 1 and the last glycine of the pro-sequence, with release of the active-site residue Thr 1 . These three beta-type subunits have inhibitor-binding sites, indicating that PRE2 has a chymotrypsin-like and a trypsin-like activity and that PRE3 has peptidylglutamyl peptide hydrolytic specificity . Other beta-type subunits are processed to an intermediate form, indicating that an additional nonspecific endopeptidase activity may exist which is important for peptide hydrolysis and for the generation of ligands for class I molecules of the major histocompatibility complex.

Biochemistry (Mosc), 1997 Apr, 62(4), 377 - 85
Comparative kinetic characterization of catalases from Candida boidinii yeast and bovine liver; Metelitza DI et al.; Catalase with molecular weight 230 +/- kD was isolated and purified from methylotrophic yeasts Candida boidinii by ion-exchange chromatography . The kinetic characteristics of yeast and bovine liver catalases were compared in the reaction of H2O2 decomposition using a wide range of H2O2 concentrations (up to 0.12 M) and PH (2-10) . First order rates constants (k, sec-1) were determined for both enzymes from semi-logarithmic anamorphoses of kinetic curves of H2O2 utilization . Anamorphoses of complete kinetic curves as a function of 1/ln({H2O2}0/{H2O2}t) versus 1/t were used for calculation of the effective rate constants of catalase inactivation during the reaction (k(in), sec-1) and the rate constants of interaction of catalase complex I with the second molecule of H2O2 (k2, M-1.sec-1) . The effects of initial catalase concentrations, H2O2, and pH on k, k2, and k(in) were similar for both enzymes . Catalytic constant, k2, and the efficacy expressed as a ratio kcat/Km were 1.87-, 1.45-, and 1.3-fold, respectively, higher for bovine catalase than that of yeast catalase . Operational stability of yeast catalase is 3.5-fold higher than the stability of bovine catalase and much higher during cyclic decomposition of 50 mM H2O2 . Enhanced operational stability and inexpensive source of its preparation open prospects for practical applications of yeast catalase for co-immobilization with superoxide dismutase on non-toxic carriers.

Mol Biol Cell, 1997 Apr, 8(4), 729 - 53
Aip3p/Bud6p, a yeast actin-interacting protein that is involved in morphogenesis and the selection of bipolar budding sites; Amberg DC et al.; A search for Saccharomyces cerevisiae proteins that interact with actin in the two-hybrid system and a screen for mutants that affect the bipolar budding pattern identified the same gene, AIP3/BUD6 . This gene is not essential for mitotic growth but is necessary for normal morphogenesis . MATa/alpha daughter cells lacking Aip3p place their first buds normally at their distal poles but choose random sites for budding in subsequent cell cycles . This suggests that actin and associated proteins are involved in placing the bipolar positional marker at the division site but not at the distal tip of the daughter cell . In addition, although aip3 mutant cells are not obviously defective in the initial polarization of the cytoskeleton at the time of bud emergence, they appear to lose cytoskeletal polarity as the bud enlarges, resulting in the formation of cells that are larger and rounder than normal . aip3 mutant cells also show inefficient nuclear migration and nuclear division, defects in the organization of the secretory system, and abnormal septation, all defects that presumably reflect the involvement of Aip3p in the organization and/or function of the actin cytoskeleton . The sequence of Aip3p is novel but contains a predicted coiled-coil domain near its C terminus that may mediate the observed homo-oligomerization of the protein . Aip3p shows a distinctive localization pattern that correlates well with its likely sites of action: it appears at the presumptive bud site prior to bud emergence, remains near the tips of small bund, and forms a ring (or pair of rings) in the mother-bud neck that is detectable early in the cell cycle but becomes more prominent prior to cytokinesis . Surprisingly, the localization of Aip3p does not appear to require either polarized actin or the septin proteins of the neck filaments.

Biosci Rep, 1997 Apr, 17(2), 189 - 207
The GS-X pump in plant, yeast, and animal cells: structure, function, and gene expression; Ishikawa T et al.; This review addresses the recent molecular identification of several members of the glutathione S-conjugate (GS-X) pump family, a new class of ATP-binding cassette (ABC) transporters responsible for the elimination and/or sequestration of pharmacologically and agronomically important compounds in mammalian, yeast and plant cells . The molecular structure and function of GS-X pumps encoded by MRP, cMOAT, YCF1, and AtMRP genes, have been conserved throughout molecular evolution . The physiologic function of GS-X pumps is closely related with cellular detoxification, oxidative stress, inflammation, and cancer drug resistance . Coordinated expression of GS-X pump genes, e.g., MRP1 and YCF1, and gamma-glutamylcystaine synthetase, a rate-limiting enzyme of cellular glutathione (GSH) biosynthesis, has been frequently observed.

Leukemia, 1997 Apr, 11 Suppl 3, 3 - 6
Transcriptional activation and self-association in yeast: protein-protein dimerization as a pleiotropic mechanism of HTLV-I Tax function; Jin DY et al.; The yeast one-hybrid and two-hybrid systems for the detection of protein-DNA and protein-protein interactions were used as an in vivo approach to investigate the functional characteristics of HTLV-1 Tax expressed in yeast . Tax, when targeted to the upstream activating sequence (UAS) via the DNA-binding domain of Gal4 (Gal4BD), was found to activate a minimal promoter in yeast, indicating the presence of a functionally intact activation domain . Using the two-hybrid assay in which Tax was fused to either Gal4BD or Gal4 activation domain (Gal4AD), we demonstrate that Tax self-associates in the nucleus of yeast cells . Mutational analysis was performed to delineate the functional domain(s) necessary for Tax-mediated trans-activation and self-association . Based on our results, we propose a pleiotropic mechanism in which Tax facilitates protein-protein dimerization of various cellular partners.

J Photochem Photobiol B, 1997 Apr, 38(2-3), 184 - 8
Laser flash photolysis studies of electron transfer in complex III from yeast mitochondria; de la Rosa FF; The kinetics of reduction of cytochrome b and cytochrome c1 of yeast Complex III by 5-deazariboflavin semiquinone, generated by laser flash photolysis under anaerobic conditions, have been investigated . The reduction of cytochrome b occurs in two phases with first-order rate constants of 1300 and 670 s-1, whereas the reduction of cytochrome c1 appears as a unique exponential phase with an intermediate value of 800 s-1 . Under these experimental conditions, about 50% of cytochrome b is reduced in comparison with cytochrome c1 . After photoreduction, the re-oxidation of the cytochromes by internal re-equilibrium occurs in both cases, following pseudo-first-order kinetics at a rate constant of 43 s-1 for cytochrome b and 39 s-1 for cytochrome c1 . These results, which agree with the data from the rapid mixing technique (A.-L . Tsai, J.S . Olson, G . Palmer, J . Biol . Chem . 262 (1987) 8677-8684), have implications for the mechanistic understanding of inner Complex III electron transfer . One of the goals of the investigation reported here is to provide direct evidence for the hypothesis of a proton-motive Q cycle for the mechanism of electron transfer in Complex III . Moreover, these results demonstrate the usefulness of laser flash photolysis in studying the redox kinetic properties of mitochondrial Complex III.

Regul Toxicol Pharmacol, 1997 Apr, 25(2), 176 - 88
The rodent uterotrophic assay: critical protocol features, studies with nonyl phenols, and comparison with a yeast estrogenicity assay; Odum J et al.; The major protocol features of the immature rat uterotrophic assay have been evaluated using a range of reference chemicals . The protocol variables considered include the selection of the test species and route of chemical administration, the age of the test animals, the maintenance diet used, and the specificity of the assay for estrogens . It is concluded that three daily oral administrations of test chemicals to 21- to 22-day-old rats, followed by determination of absolute uterus weights on the fourth day, provide a sensitive and toxicologically relevant in vivo estrogenicity assay . Rats are favored over mice for reasons of toxicological practice, but the choice of test species is probably not a critical protocol variable, as evidenced by the similar sensitivity of rats and mice to the uterotrophic activity of methoxychlor . Vaginal opening is shown to be a useful, but nondefinitive, adjunct to the uterotrophic assay . The ability of test chemicals to reduce or abolish the uterotrophic response of estradiol is suggested to provide a useful extension of the uterotrophic assay for the purpose of detecting antiestrogens . The results of a series of studies on the environmental estrogen nonyl phenol (NP), and its linear isomer n-nonyl phenol, confirm that branching of the aliphatic side chain is important for activity . 17beta-Desoxyestradiol is shown to be of similar activity to estradiol in the uterotrophic assay and is suggested to represent the "parent" estrogen of NP . Benzoylation of NP and 17-desoxyestradiol did not affect their uterotrophic activity, in contrast to the enhancing effect of benzoylation on estradiol . Selected chemicals shown to be active in the immature rat uterotrophic assay were also evaluated in an in vitro yeast human estrogen receptor transactivation assay . Most of the chemicals gave similar qualitative responses to those seen in the uterotrophic assay, and the detection of the estrogen methoxychlor by the yeast assay evidenced a degree of intrinsic metabolic competence . However, the assay had a reduced ability (compared to rodents) to hydrolyze the benzoate ester of estradiol, and the estrogenic benzoate derivative of NP was not active in the yeast assay . These last results indicate that current metabolic deficiencies of in vitro estrogenicity assays will limit the value of negative data for the immediate future . The results described illustrate the intrinsic complexity of evaluating chemicals for estrogenic activities and confirm the need for rigorous attention to experimental design and criteria for assessing estrogenic activity.

Vaccine, 1997 Apr-May, 15(6-7), 637 - 47
Comparison between hepatitis B surface antigen (HBsAg) particles derived from mammalian cells (CHO) and yeast cells (Hansenula polymorpha): composition, structure and immunogenicity; Diminsky D et al.; The composition, structure and immunogenicity of hepatitis B surface antigen (HBsAg) particles derived from Chinese hamster ovary (CHO) cells and from cells of the yeast Hansenula polymorpha were compared . The particles were similar in size distribution (mean 20-33 nm), in shape (spherical), in gross composition (protein to lipid weight ratio of 60:40), and in types of lipids (phospholipids > > sterols = sterol esters = triacylglycerols) . Differences related to genetic engineering and type of host cells were found in peptide and lipid compositions . CHO-HBsAg has three peptides: S, M and L, each in two forms of glycosylation, while the Hansenula-HBsAg has only the nonglycosylated S peptide . The electrical surface potential at the lipid/water interface of HBsAg derived from Hansenula is more negative than that of HBsAg derived from CHO, which was close to neutrality . Although the numbers of cysteine residues (all in the S peptides) are identical (14), 11 of them are free thiols in the CHO-HBsAg, compared with three to four in the Hansenula-HBsAg . The fact that 85% of the phospholipids are hydrolyzed by phospholipase C and that all the aminophospholipids react with trinitrobenzenesulfate suggests that the particles derived from both cell types are either leaky vesicles or have a lipoprotein-like structure . Subcutaneous injection into mice of fluorescein-isothiocyanate-labeled HBsAg particles from both sources resulted in their accumulation in the marginal sinus of lymph nodes . The humoral responses to subcutaneous injection into mice of CHO- and Hansenula-HBsAg were similar: however, the cytotoxic T lymphocyte response to CHO-HBsAg was lower.

Appl Biochem Biotechnol, 1997 Spring, 63-65, 483 - 93
Performance of coimmobilized yeast and amyloglucosidase in a fluidized bed reactor for fuel ethanol production; Sun MY et al.; The performance of coimmobilized Saccharomyces cerevisiae and amyloglucosidase (AG) was evaluated in a fluidized-bed reactor . Soluble starch and yeast extracts were used as feed stocks . Conversion of soluble starch streams to ethanol has potential practical applications in corn dry and wet milling and in developmental lignocellulosic processes . The biocatalyst performed well, and demonstrated no significant loss of activity or physical integrity during 10 wk of continuous operation . The reactor was easily operated and required no pH control . No operational problems were encountered from bacterial contaminants even though the reactor was operated under nonsterile conditions over the entire course of experiments . Productivities ranged between 25 and 44 g ethanol/L/h/ . The experiments demonstrated that ethanol inhibition and bed loading had significant effects on reactor performance.

Yeast, 1997 Apr, 13(5), 483 - 7
Sequencing analysis of a 36.8 kb fragment of yeast chromosome XV reveals 26 open reading frames including SEC63, CDC31, SUG2, GCD1, RBL2, PNT1, PAC1 and VPH1; Poirey R et al.; The complete sequence of a 36775 bp DNA segment located on the right arm of chromosome XV of Saccharomyces cerevisiae has been determined and analysed . The sequence encodes 26 open reading frames of at least 100 amino acids . Eight of these correspond to known genes, whereas 18 correspond to new genes.

Yeast, 1997 Apr, 13(5), 479 - 82
Sequence and analysis of a 36.2 kb fragment from the right arm of yeast chromosome XV reveals 19 open reading frames including SNF2 (5' end), CPA1, SLY41, a putative transport ATPase, a putative ribosomal protein and an SNF2 homologue; Poirey R et al.; The complete sequence of a 36 196 bp DNA segment located on the right arm of chromosome XV of Saccharomyces cerevisiae has been determined and analysed . The sequence includes the 5' coding region of the SNF2 gene, the CPA1 leader peptide sequence and 17 open reading frames (ORFs) of at least 100 amino acids . Two of these correspond to previously known genes (CPA1, SLY41), whereas 15 correspond to new genes . The putative translation products of three ORFs show significant similarity with known proteins: one is a putative transport ATPase, another appears to be a ribosomal protein, and the third is an Snf2p homologue.

Yeast, 1997 Apr, 13(5), 449 - 62
A conditional sterol esterification defect in yeast having either a sec1 or sec5 mutation in the secretory pathway; Tomeo ME et al.; Two temperature-conditional secretory mutations, sec1 and sec5, cause the accumulation of post-Golgi vesicles when strains containing these mutations are grown at 37 degrees C . In addition to accumulating vesicles, the mutants do not esterify free sterol on rich media at the restrictive temperature . It is the high level of inositol in the media that causes this condition in the yeast Saccharomyces cerevisiae, not a defective steryl ester synthase or lack of substrates . When strains containing the sec1 or sec5 mutation were transformed separately with a plasmid carrying SEC1 and SEC5, the esterification and secretory defects were alleviated . Double mutants containing sec6, sec14 or sec18 with either a sec1 or sec5 mutation have normal esterification levels . Strains with suppressor mutations were isolated that grew at 37 degrees C, esterified sterols and had diminished accumulation of vesicles, when grown at the restrictive temperature on defined media with additional inositol . Electron microscopy was used to examine vesicle accumulation, the number of lipid droplets, and to further characterize the esterification defect . When grown at 37 degrees C on defined medium, the strains with sec5 or sec1 accumulated the usual secretory vesicles, but when grown under similar conditions with elevated levels of inositol, accumulated an additional vesicular-like body.

Biochem Mol Biol Int, 1997 Apr, 41(5), 933 - 40
Dicarbanonaborates in yeast respiration and membrane transport; Kotyk A et al.; Two derivatives of carborates, sodium 5,6-dichloro-7,8-dicarbanonaborate (CB-Cl) and sodium 5-mercapto-7,8-dicarbanonaborate (CB-SH) were found to inhibit endogenous as well as glucose-induced respiration of the yeast Saccharomyces cerevisiae . Both substances slightly increased endogenous acid production, were neutral toward H(+)-ATPase-associated acidification but pronouncedly inhibited the K(+)-stimulated acidification . The same effects were observed also with an ATPase-deficient mutant of the yeast . The ATP-hydrolyzing activity of yeast plasma membranes in vitro was severely reduced . The membrane potential was substantially increased toward more negative values . The H(+)-symporting uptake of glutamic acid was considerably decreased, that of adenine was diminished much less . The effects of the dicarbanonaborates are obviously pleiotropic but their inhibition of ATP hydrolysis and of uptake of H(+)-symported substances, on the one hand, and absolute lack of effect on ATPase-catalyzed acidification, on the other, pose an unresolved problem.

J Cell Sci, 1997 Apr, 110 ( Pt 7), 899 - 910
Novel functions of clathrin light chains: clathrin heavy chain trimerization is defective in light chain-deficient yeast; Huang KM et al.; Clathrin is a major coat protein involved in sorting and retention of proteins at the late Golgi and in endocytosis from the cell surface . The clathrin triskelion contains three heavy chains, which provide the structural backbone of the clathrin lattice and three light chains, which are thought to regulate the formation or disassembly of clathrin coats . To better understand the function of the clathrin light chain, we characterized yeast strains carrying a disruption of the clathrin light chain gene (CLC1) . Light chain-deficient cells showed phenotypes similar to those displayed by yeast that have a disruption in the clathrin heavy chain gene (CHC1) . In clc1-delta cells, the steady state level of the clathrin heavy chain was reduced to 20%-25% of wild-type levels and most of the heavy chain was not trimerized . If CHC1 was overexpressed in clc1-delta cells, heavy chain trimers were detected and several clc1-delta phenotypes were partially rescued . These results indicate that the light chain is important for heavy chain trimerization and the heavy chain still has some function in the absence of the light chain . In yeast, deletion of CHC1 is lethal in strains carrying the scd1-i allele, while strains carrying the scd1-v allele can survive without the heavy chain . In previous studies we isolated several multicopy suppressors of inviability of chc1-delta scd1-i cells . Surprisingly, one of these suppressors, SCD4, is identical to CLC1 . Overexpression of CLC1 in viable chc1-delta scd1-v strains rescued some but not all of the phenotypes displayed by these cells . In the absence of the heavy chain, the light chain was not found in a high molecular mass complex, but still associated with membranes . These results suggest that the light chain can function independently of the clathrin heavy chain in yeast.

Gene, 1997 Apr 1, 188(2), 169 - 74
Construction of a pilot human YAC library in a recombination-defective yeast strain; Palmieri G et al.; Using high-molecular-weight DNA fragments from a human lymphoblastoid cell line, a pilot collection of 2500 YACs was constructed in YKK115, a recombination-deficient strain of Saccharomyces cerevisiae carrying mutations in both the rad51 and rad52 genes . Analysis of 520 clones from the current library by pulsed-field gel electrophoresis revealed more than 97% single YACs with an insert size averaging 340 kb . Fluorescent in situ hybridization (FISH) performed with 37 clones on metaphase chromosomes suggested a high proportion mapping at centromeric (7) or telomeric (4) locations . The results are consistent with the stabilization of YACs in strains disarmed in recombination functions {Kohno, K., Oshiro, T., Kishine, H., Wada, M., Takeda, H., Ihara, N., Imamoto, F., Kano, Y . and Schlessinger, D . (1997) Human YACs unstable in a rad52 single mutant strain become stable in rad51rad52 double mutant . Gene, 000, 000-000 (GENE 10429)}, and further suggest that the YACs may include regions that have been difficult to clone in other strains.

EMBO J, 1997 Apr 1, 16(7), 1759 - 71
In vivo commitment to splicing in yeast involves the nucleotide upstream from the branch site conserved sequence and the Mud2 protein; Rain JC et al.; Pre-mRNA splicing is a stepwise nuclear process involving intron recognition and the assembly of the spliceosome followed by intron excision . We previously developed a pre-mRNA export assay that allows the discrimination between early steps of spliceosome formation and splicing per se . Here we present evidence that these two assays detect different biochemical defects for point mutations . Mutations at the 5' splice site lead to pre-mRNA export, whereas 3' splice site mutations do not . A genetic screen applied to mutants in the branch site region shows that all positions in the conserved TACTAAC sequence are important for intron recognition . An exhaustive analysis of pre-mRNA export and splicing defects of these mutants shows that the in vivo recognition of the branch site region does not involve the base pairing of U2 snRNA with the pre-mRNA . In addition, the nucleotide preceding the conserved TACTAAC sequence contributes to the recognition process . We show that a T residue at this position allows for optimal intron recognition and that in natural introns, this nucleotide is also used preferentially . Moreover, the Mud2 protein is involved in the recognition of this nucleotide, thus establishing a role for this factor in the in vivo splicing pathway.

Mol Cell Biol, 1997 Apr, 17(4), 2279 - 90
Regulation of the myeloid-cell-expressed human gp91-phox gene as studied by transfer of yeast artificial chromosome clones into embryonic stem cells: suppression of a variegated cellular pattern of expression requires a full complement of distant cis elements; Lien LL et al.; Identifying the full repertoire of cis elements required for gene expression in mammalian cells (or animals) is challenging, given the moderate sizes of many loci . To study how the human gp91-phox gene is expressed specifically in myeloid hematopoietic cells, we introduced yeast artificial chromosome (YAC) clones and derivatives generated in yeast into mouse embryonic stem cells competent to differentiate to myeloid cells in vitro or into mouse chimeras . Fully appropriate regulation was recapitulated with a 130-kb YAC containing 60 and 30 kb of 5' and 3' flanking sequences, respectively . Immunodetection of human gp91-phox protein revealed uniform expression in individual myeloid cells . The removal of upstream sequences led to decreased overall expression which reflected largely a variegated pattern of expression, such that cells were either "on" or "off," rather than pancellular loss of expression . The proportion of clones displaying marked variegation increased with progressive deletion . DNase I mapping of chromatin identified two hypersensitive clusters, consistent with the presence of multiple regulatory elements . Our findings point to cooperative interactions of complex regulatory elements and suggest that the presence of an incomplete set of elements reduces the probability that an open chromatin domain (or active transcriptional complex) may form or be maintained in the face of repressive influences of neighboring chromatin.

Mol Cell Biol, 1997 Apr, 17(4), 2235 - 46
Yrb2p, a Nup2p-related yeast protein, has a functional overlap with Rna1p, a yeast Ran-GTPase-activating protein; Noguchi E et al.; The Ran-GTPase cycle is important for nucleus-cytosol exchange of macromolecules and other nuclear processes . We employed the two-hybrid method to identify proteins interacting with Ran and the Ran GTP/GDP exchange factor . Using PRP20, encoding the Ran GTP/GDP exchange factor, we identified YRB1, previously identified as a protein able to interact with human Ran GTP/GDP exchange factor RCC1 in the two-hybrid system . Using GSP1, encoding the yeast Ran, as bait, we isolated YRB2 . YRB2 encodes a protein containing a Ran-binding motif similar to that found in Yrb1p and Nup2p . Yrb1p is located in the cytosol whereas Nup2p is nuclear . Similar to Yrb1p, Yrb2p bound to GTP-Gsp1p but not to GDP-Gsp1p and enhanced the GTPase-activating activity of Rna1p . However, unlike Yrb1p, Yrb2p did not inhibit the nucleotide-releasing activity of Prp20p . While overproduction of Yrb1p inhibited the growth of a mutant possessing a PRP20 mutation (srm1-1) and suppressed the rna1-1 mutation, overproduction of Yrb2p showed no effect on the growth of these mutants . Disruption of YRB2 made yeast cold sensitive and was synthetically lethal with rna1-1 but not with nup2delta . Nuclear protein import and the mRNA export were normal in strains possessing mutations of YRB2 . We propose that Yrb2p is involved in the nuclear processes of the Ran-GTPase cycle which are not related to nucleus-cytosol exchange of macromolecules.

Mol Cell Biol, 1997 Apr, 17(4), 2136 - 42
A yeast replicative helicase, Dna2 helicase, interacts with yeast FEN-1 nuclease in carrying out its essential function; Budd ME et al.; We have recently described a new helicase, the Dna2 helicase, that is essential for yeast DNA replication . We now show that the yeast FEN-1 (yFEN-1) nuclease interacts genetically and biochemically with Dna2 helicase . FEN-1 is implicated in DNA replication and repair in yeast, and the mammalian homolog of yFEN-1 (DNase IV, FEN-1, or MF1) participates in Okazaki fragment maturation . Overproduction of yFEN-1, encoded by RAD27/RTH1, suppresses the temperature-sensitive growth of dna2-1 mutants . Overproduction of Dna2 suppresses the rad27/rth1 delta temperature-sensitive growth defect . dna2-1 rad27/rth1 delta double mutants are inviable, indicating that the mutations are synthetically lethal . The genetic interactions are likely due to direct physical interaction between the two proteins, since both epitope-tagged yFEN-1 and endogenous yFEN-1 coimmunopurify with tagged Dna2 . The simplest interpretation of these data is that one of the roles of Dna2 helicase is associated with processing of Okazaki fragments.

Mol Cell Biol, 1997 Apr, 17(4), 2090 - 8
Stability of a CTG/CAG trinucleotide repeat in yeast is dependent on its orientation in the genome; Freudenreich CH et al.; Trinucleotide repeat expansion is the causative mutation for a growing number of diseases including myotonic dystrophy, Huntington's disease, and fragile X syndrome . A (CTG/CAG)130 tract cloned from a myotonic dystrophy patient was inserted in both orientations into the genome of Saccharomyces cerevisiae . This insertion was made either very close to the 5' end or very close to the 3' end of a URA3 transcription unit . Regardless of its orientation, no evidence was found for triplet-mediated transcriptional repression of the nearby gene . However, the stability of the tract correlated with its orientation on the chromosome . In one orientation, the (CTG/CAG)130 tract was very unstable and prone to deletions . In the other orientation, the tract was stable, with fewer deletions and two possible cases of expansion detected . Analysis of the direction of replication through the region showed that in the unstable orientation the CTG tract was on the lagging-strand template and that in the stable orientation the CAG tract was on the lagging-strand template . The orientation dependence of CTG/CAG tract instability seen in this yeast system supports models involving hairpin-mediated polymerase slippage previously proposed for trinucleotide repeat expansion.

Mol Cell Biol, 1997 Apr, 17(4), 1995 - 2004
An origin of replication and a centromere are both needed to establish a replicative plasmid in the yeast Yarrowia lipolytica; Vernis L et al.; Two DNA fragments displaying ARS activity on plasmids in the yeast Yarrowia lipolytica have previously been cloned and shown to harbor centromeric sequences (P . Fournier, A . Abbas, M . Chasles, B . Kudla, D . M . Ogrydziak, D . Yaver, J.-W . Xuan, A . Peito, A.-M . Ribet, C . Feynerol, F . He, and C . Gaillardin, Proc . Natl . Acad . Sci . USA 90:4912-4916, 1993; and P . Fournier, L . Guyaneux, M . Chasles, and C . Gaillardin, Yeast 7:25-36, 1991) . We have used the integration properties of centromeric sequences to show that all Y . lipolytica ARS elements so far isolated are composed of both a replication origin and a centromere . The sequence and the distance between the origin and centromere do not seem to play a critical role, and many origins can function in association with one given centromere . A centromeric plasmid can therefore be used to clone putative chromosomal origins coming from several genomic locations, which confer the replicative property on the plasmid . The DNA sequences responsible for initiation in plasmids are short (several hundred base pairs) stretches which map close to or at replication initiation sites in the chromosome . Their chromosomal deletion abolishes initiation, but changing their chromosomal environment does not.

Mol Cell Biol, 1997 Apr, 17(4), 1986 - 94
Plant farnesyltransferase can restore yeast Ras signaling and mating; Yalovsky S et al.; Farnesyltransferase (FTase) is a heterodimeric enzyme that modifies a group of proteins, including Ras, in mammals and yeasts . Plant FTase alpha and beta subunits were cloned from tomato and expressed in the yeast Saccharomyces cerevisiae to assess their functional conservation in farnesylating Ras and a-factor proteins, which are important for cell growth and mating . The tomato FTase beta subunit (LeFTB) alone was unable to complement the growth defect of ram1 delta mutant yeast strains in which the chromosomal FTase beta subunit gene was deleted, but coexpression of LeFTB with the plant alpha subunit gene (LeFTA) restored normal growth, Ras membrane association, and mating . LeFTB contains a novel 66-amino-acid sequence domain whose deletion reduces the efficiency of tomato FTase to restore normal growth to yeast ram1 delta strains . Coexpression of LeFTA and LeFTB in either yeast or insect cells yielded a functional enzyme that correctly farnesylated CaaX-motif-containing peptides . Despite their low degree of sequence homology, yeast and plant FTases shared similar in vivo and in vitro substrate specificities, demonstrating that this enzymatic modification of proteins with intermediates from the isoprenoid biosynthesis pathway is conserved in evolutionarily divergent eukaryotes.

Mol Cell Biol, 1997 Apr, 17(4), 1787 - 95
A34.5, a nonessential component of yeast RNA polymerase I, cooperates with subunit A14 and DNA topoisomerase I to produce a functional rRNA synthesis machine; Gadal O et al.; A34.5, a phosphoprotein copurifying with RNA polymerase I (Pol I), lacks homology to any component of the Pol II or Pol III transcription complexes . Cells devoid of A34.5 hardly affect growth and rRNA synthesis and generate a catalytically active but structurally modified enzyme also lacking subunit A49 upon in vitro purification . Other Pol I-specific subunits (A49, A14, and A12.2) are nonessential for growth at 30 degrees C but are essential (A49 and A12.2) or helpful (A14) at 25 or 37 degrees C . Triple mutants without A34.5, A49, and A12.2 are viable, but inactivating any of these subunits together with A14 is lethal . Lethality is rescued by expressing pre-rRNA from a Pol II-specific promoter, demonstrating that these subunits are collectively essential but individually dispensable for rRNA synthesis . A14 and A34.5 single deletions affect the subunit composition of the purified enzyme in pleiotropic but nonoverlapping ways which, if accumulated in the double mutants, provide a structural explanation for their strict synthetic lethality . A34.5 (but not A14) becomes quasi-essential in strains lacking DNA topoisomerase I, suggesting a specific role of this subunit in helping Pol I to overcome the topological constraints imposed on ribosomal DNA by transcription.

Curr Opin Genet Dev, 1997 Apr, 7(2), 176 - 81
Mechanisms of transcriptional activation: differences and similarities between yeast, Drosophila, and man; Sauer F et al.; Activation of transcription requires an interplay between enhancer-binding factors and components of the general transcription machinery . New developments within the past few years suggest that novel cofactors are required for relaying specific activation signals to the RNA polymerase II transcription complex in order to achieve enhanced levels of mRNA synthesis . The role of these different cofactors in mediating activation and potential differences in their utilization by divergent organisms, however, raise new questions about the mechanisms of transcriptional regulation.

FEBS Lett, 1997 Apr 1, 405(3), 337 - 44
Extracellular K+ and Ba2+ mediate voltage-dependent inactivation of the outward-rectifying K+ channel encoded by the yeast gene TOK1; Vergani P et al.; Gating of the yeast K+ channel encoded by the Saccharomyces cerevisiae gene TOK1, unlike other outward-rectifying K+ channels that have been cloned, is promoted by membrane voltage (inside positive-going) and repressed by extracellular K+ . When expressed in Xenopus laevis oocytes, the TOK1p current rectified strongly outward, its activation shifting in parallel with the K+ equilibrium potential when the external K+ concentration ({K+}o) was increased above 3 mM . Analysis of the TOK1p current indicated that two kinetic components contributed to the conductance and the voltage sensitivity of the conductance . By contrast, the {K+}o sensitivity of the current was accommodated entirely within the slow-relaxing component; it was diminished near 1 mM {K+}o, and at submillimolar concentrations the voltage dependence of the TOK1p conductance was insensitive to {K+}o . External Rb+, the K+ channel blockers Cs+ and Ba2+--but not Na+, Ca2+ or Mg2+--substituted for K+ in control of TOK1p activation, indicating a specificity in cation interaction with the TOK1p gate . These and additional results indicate that external K+ acts as a ligand to inactivate the TOK1p channel, and they implicate a gating process mediated by a single cation binding site within the membrane electric field, but distinct from the permeation pathway.

FEMS Microbiol Lett, 1997 Apr 1, 149(1), 17 - 24
Fatty acid accumulation in the yeast Sporidiobolus salmonicolor during batch production of gamma-decalactone; Feron G et al.; This paper provides new information about the metabolism of various fatty acids and gamma-decalactone production by yeast . An analysis of the fatty acid composition of the yeast Sporidiobolus salmonicolor during batch production of lactone with ricinoleic acid methyl ester as a precursor showed an accumulation of the gamma-decalactone precursor inside the cells . Electron microscopy of the yeasts showed the presence of large internal inclusions leading to membrane and organelle lysis and, consequently, death of the yeast . S . salmonicolor cultivated with methyl oleate did not produce gamma-decalactone and is viable during the whole culture . Analysis of the long chain fatty acid fraction showed incorporation of methyl oleate.

Int J Syst Bacteriol, 1997 Apr, 47(2), 307 - 12
Dipodascus starmeri sp . nov., a new species of yeast occurring in cactus necroses; Phaff HJ et al.; In a previous publication describing the geographic distribution of yeasts associated with cactus necroses (W . T . Starmer, M.-A . Lachance, H . J . Phaff, and W . B . Heed, Evol . Biol . 24:253-296, 1990), 127 isolates were identified as strains of Candida ingens van der Walt et van Kerken on the basis of morphology and certain phenotypic characteristics . Here we show by using DNA hybridization and additional phenotypic properties that these strains were misidentified and that they represent a minimum of three separate species that can be differentiated from C . ingens and from each other by utilization of 2-propanol or acetone, sensitivity to digitonin, utilization of L-lysine as a sole nitrogen source, vitamin dependence, NaCl tolerance, lipolytic activity, and habitat . One of the new species is haploid and heterothallic, and its teleomorph represents the genus Dipodascus . We describe Dipodascus starmeri sp . nov . The phylogenetic relationship of D . starmeri with other members of the genus Dipodascus and its anamorph, the genus Geotrichum, was estimated from ribosomal DNA nucleotide sequence divergence . The type strain, a heterothallic haploid isolate, is UCD-FST 72-316 (= CBS 780.96 = ATCC 200546 = NRRL Y-17816) . The complementary mating type is UCD-FST 81-513.3 (= CBS 781.96 = ATCC 200547 = NRRL Y-17817).

Protein Sci, 1997 Apr, 6(4), 882 - 91
An engineered amino-terminal domain of yeast phosphoglycerate kinase with native-like structure; Sherman MA et al.; Previous studies have suggested that the carboxy-terminal peptide (residues 401-415) and interdomain helix (residues 185-199) of yeast phosphoglycerate kinase, a two-domain enzyme, play a role in the folding and stability of the amino-terminal domain (residues 1-184) . A deletion mutant has been created in which the carboxy-terminal peptide is attached to the amino-terminal domain (residues 1-184) plus interdomain helix (residues 185-199) through a flexible peptide linker, thus eliminating the carboxy-terminal domain entirely . CD, fluorescence, gel filtration, and NMR experiments indicated that, unlike versions described previously, this isolated N-domain is soluble, monomeric, compactly folded, native-like in structure, and capable of binding the substrate 3-phosphoglycerate with high affinity in a saturable manner . The midpoint of the guanidine-induced unfolding transition was the same as that of the native two-domain protein (Cm approximately 0.8 M) . The free energy change associated with guanidine-induced unfolding was one-third that of the native enzyme, in agreement with previous studies that evaluated the intrinsic stability of the N-domain and the contribution of domain-domain interactions to the stability of PGK . These observations suggest that the C-terminal peptide and interdomain helix are sufficient for maintaining a native-like fold of the N-domain in the absence of the C-domain.

Steroids, 1997 Apr, 62(4), 365 - 72
The estrogenic and antiestrogenic activities of phytochemicals with the human estrogen receptor expressed in yeast; Collins BM et al.; We have used the expression of the human estrogen receptor (hER) and two estrogen response elements linked to the lacZ gene in yeast (YES) to study the estrogenic and antiestrogenic activities of various phytochemicals . Coumestrol, alpha-zearalenol, or genistein could produce beta-galactosidase activity comparable to estradiol, but these required concentrations 100 to 1000-fold greater than estradiol . These compounds did not possess antiestrogenic activity . Narigenin, kaempferide, phloretin, biochanin A, flavone, or chrysin only partially induced beta-galactosidase activity in the YES at any concentration tested . When narigenin, kaempferide, or phloretin was given concurrently with estradiol, the estradiol-dependent beta-galactosidase activity was not inhibited by more than 50% . However, biochanin A, flavone, or chrysin could inhibit the activity of estradiol in a dose-response manner with IC50 values of 500 nM, 2 microM, and 10 microM, respectively . Combinations of biochanin A, chrysin, and flavone decreased estradiol-dependent beta-galactosidase activity in an additive fashion . Similar to the antiestrogens tamoxifen or ICI 182, 780, the antiestrogenic activity of these compounds with the exception of chrystin involved the disruption of hER dimerization, as demonstrated in the yeast two-hybrid system . Biochanin A, chrysin, or flavone were less effective in inhibiting the activity of an estrogenic polychlorinated biphenyl than they were inhibiting the activity of estradiol . Interestingly, this latter group of antiestrogenic phytocompounds did not inhibit the estrogenic activity of such phytochemicals as coumestrol or genistein . These results suggest that the antiestrogenic activity of biochanin A and flavone occurs by a mechanism similar to tamoxifen or ICI 182,780 . Moreover, it seems that phytochemicals functioning as antiestrogens do not inhibit the activity of all estrogenic chemicals to the same extent . This suggests that conformational changes induced by different estrogens bound to the hER may regulate the antiestrogenic activity of a compound.

Nat Genet, 1997 Apr, 15(4), 381 - 4
Rhizomelic chondrodysplasia punctata is caused by deficiency of human PEX7, a homologue of the yeast PTS2 receptor; Purdue PE et al.; The rhizomelic form of chondrodysplasia punctata (RCDP) is an autosomal recessive disease of peroxisome biogenesis characterized by deficiencies in several peroxisomal proteins, including the peroxisomal enzymes of plasmalogen biosynthesis and peroxisomal 3-ketoacyl thiolase . In cultured fibroblasts from patients with this disorder, both the peroxisomal targeting and proteolytic removal of the amino-terminal type 2 peroxisomal targeting sequence (PTS2) of thiolase are defective, whereas the biogenesis of proteins targeted by carboxyterminal type 1 peroxisomal targeting sequences (PTS1) is unimpaired . We have previously isolated a Saccharomyces cerevisiae peroxisomal biogenesis mutant, pex7 (formerly peb1/pas7), which demonstrates a striking similarity to the cellular phenotype of RCDP fibroblasts in that PTS1 targeting is functional, but the peroxisomal packaging of PTS2 targeted thiolase is lacking . Complementation of this mutant has led to the identification of the protein ScPex7p, a PTS2 receptor . In this paper we report cloning of the human orthologue of ScPEX7, and demonstrate that this is the defective gene in RCDP . We show that expression of human PEX7 in RCDP cells rescues PTS2 targeting and restores some activity of dihydroxyacetone phosphate acyltransferase (DHAP-AT), a peroxisomal enzyme of plasmalogen biosynthesis, and we identify the mutations responsible for loss of function of PEX7 in a compound heterozygote RCDP patient . These results imply that several peroxisomal proteins are targeted by PTS2 signals and that the various biochemical and clinical defects in RCDP result from a defect in the receptor for this class of PTS.

Mol Cell Biochem, 1997 Apr, 169(1-2), 95 - 106
The dual role of mRNA half-lives in the expression of the yeast ALG7 gene; Lennon K et al.; The yeast ALG7 gene functions by initiating the synthesis of the dolichol-linked oligosaccharide precursor and plays an important role in the control of protein N-glycosylation . The levels of ALG7 multiple transcripts are modulated by the physiological status of the cell and environmental cues, and deregulation of their abundance is deleterious to several cellular functions . Since ALG7 mRNAs are unstable, we investigated the role of these transcripts' half-lives in determining their steady-state levels . Using a temperature-sensitive RNA polymerase II mutant, we demonstrate that increased stability was the primary determinant of higher ALG7 mRNA abundance in response to glucose limitation or treatment with tunicamycin . In contrast, at the G1/G0 transition point, changes in the decay rates were inversely related to ALG7 transcript accumulation: the decreased abundance of ALG7 mRNAs following exit from the mitotic cycle was associated with lengthening of the decay rates, while their increased accumulation after growth stimulation correlated with decreased stability . This suggests that, depending on the circumstance, mRNA half-lives can either directly determine the level of ALG7 transcript accumulation or oppose regulatory changes at other control levels.

Endocrinology, 1997 Apr, 138(4), 1520 - 7
Estrogenic activity of a dieldrin/toxaphene mixture in the mouse uterus, MCF-7 human breast cancer cells, and yeast-based estrogen receptor assays: no apparent synergism; Ramamoorthy K et al.; The estrogenic activity of dieldrin, toxaphene, and an equimolar mixture of both compounds (dieldrin/toxaphene) was investigated in the 21-day-old B6C3F1 mouse uterus, MCF-7 human breast cancer cells, and in yeast-based reporter gene assays . Treatment of the animals with 17beta-estradiol (E2) (0.0053 kg/day x3) resulted in a 3.1-, 4.8-, and 7.8-fold increase in uterine wet weight, peroxidase activity, and progesterone receptor binding, respectively . In contrast, treatment with 2.5, 15 and 60 micromol/kg (x3) doses of toxaphene, dieldrin, or dieldrin/toxaphene (equimolar) did not significantly induce a dose-dependent increase in any of the E2-induced responses . The organochlorine pesticides alone and the binary mixture did not bind to the mouse uterine estrogen receptor (ER) in a competitive binding assay using {3H}E2 as the radioligand . In parallel studies, estrogenic activities were determined in MCF-7 cells by using a cell proliferation assay and by determining induction of chloramphenicol acetyl transferase (CAT) activity in MCF-7 cells transiently transfected with plasmids containing estrogen-responsive 5'-promoter regions from the rat creatine kinase B and human cathepsin D genes . E2 caused a 24-fold increase in CAT activity in MCF-7 cells transiently transfected with creatine kinase B and a 3.8-fold increase in cells transiently transfected with the human cathepsin D construct . Treatment of MCF-7 cells with dieldrin, toxaphene, or an equimolar mixture of dieldrin plus toxaphene (10(-8)-10(-5) M) did not significantly induce cell proliferation or CAT activity in the transient transfection experiment with both plasmids . The relative competitive binding of the organochlorine pesticides was determined by incubating MCF-7 cells with 10(-9) M {3H}E2 in the presence or absence of 2 x 10(-7) M unlabeled E2 (to determine nonspecific binding), toxaphene (10(-5) M), dieldrin (10(-5) M), and equimolar concentrations of the dieldrin plus toxaphene mixture (10(-5) M) . The binding observed for {3H}E2 in the whole cell extracts was displaced by unlabeled E2, whereas the organochlorine pesticides and binary mixture exhibited minimal to nondetectable competitive binding activity . E2 caused a 5000-fold induction of beta-galactosidase (beta-gal) activity in yeast transformed with the human ER and a double estrogen responsive element upstream of the beta-gal reporter gene . Treatment with 10(-6)-10(-4) M chlordane, dieldrin, toxaphene, or an equimolar mixture of dieldrin/toxaphene did not induce activity, whereas 10(-4) M endosulfan caused a 2000-fold increase in beta-gal activity . Diethylstilbestrol caused a 20-fold increase in activity in yeast transformed with the mouse ER and a single estrogen responsive element upstream of the beta-gal reporter gene . Dieldrin, chlordane, toxaphene, and endosulfan induced a 1.5- to 4-fold increase in activity at a concentration of 2.5 x 10(-5) M . Synergistic transactivation was not observed for any equimolar binary mixture of the pesticides at concentrations of either 2.5 x 10(-5) M or 2.5 x 10(-4) M . The results of this study demonstrate that for several estrogen-responsive assays in the mouse uterus, MCF-7 human breast cancer cells, and yeast-based reporter gene assays, the activities of both dieldrin and toxaphene were minimal, and no synergistic interactions were observed with a binary mixture of the two compounds.

Yeast, 1997 Mar 30, 13(4), 379 - 90
The sequence of a 54.7 kb fragment of yeast chromosome XV reveals the presence of two tRNAs and 24 new open reading frames; Valens M et al.; A 54,719 bp fragment from the right arm of Saccharomyces cerevisiae chromosome XV has been sequenced from the inserts of two cosmids (pEOA213 and pEOA217) . The computer analysis of this sequence has revealed the presence of eight known genes (CKA2, CYC1, ALG8, TCM1, TMP1, UFE1, RTS2 and ASE1) and four open reading frames (ORFs) with strong homologies with known yeast genes (MLP1, SIS2 and HBS1 and the allantoin permease) . The characteristics of the other ORFs and of the corresponding proteins do not allow postulation of a precise function . Several have features reminiscent of cytoskeleton or motor elements (keratin-like, myosin-like) and several others have characteristics of proteins which interact with DNA (extremely basic, b-Zip structure and/or acidic domains) . Two tRNAs (tRNA(Lys) and tRNA(Pro)) have also been identified on this fragment . Many of these ORFs present similarities with ORFs located on chromosome XI, indicating some information reshuffling between the two chromosomal fragments.

Yeast, 1997 Mar 30, 13(4), 365 - 8
The sequence of a 8 kb segment on the right arm of yeast chromosome VII identifies four new open reading frames and the genes for yTAFII145; Ruzzi M et al.; We report the sequence of a 8,061 bp fragment of Saccharomyces cerevisiae chromosome VII . Five open reading frames (ORFs) of at least 100 amino acids were identified . Three show similarities to the amino-acid sequence of known gene products . ORF G9374 corresponds to the gene coding for the yTAFII145 protein: a TBP-associated factor whose amino-acid sequence was previously reported (Reese et al., 1994) . The remaining ORF does not display similarities to known sequences.

Yeast, 1997 Mar 30, 13(4), 353 - 6
Construction of a yeast strain deleted for the TRP1 promoter and coding region that enhances the efficiency of the polymerase chain reaction-disruption method; Baudin-Baillieu A et al.; The sequence of the genome of Saccharomyces cerevisiae was recently determined . As well as all the informations concerning the structure of the chromosomes the scientific community had to deal with the discovery of dozens of new open reading frames (ORFs) of unknown function . The study of these ORFs requires the development of simple procedures that can be used on a large scale . In the framework of a European Pilot Project we have described a new approach for deleting ORFs . This method is based on transformation with a polymerase chain reaction product but is limited by the use of a strain deleted for the auxotropic marker . We present here the construction of a new recipient strain that lacks the TRP1 region and that allows a high efficiency of gene deletion.

J Biol Chem, 1997 Mar 28, 272(13), 8679 - 85
Characterization of a novel mammalian RGS protein that binds to Galpha proteins and inhibits pheromone signaling in yeast; Chen C et al.; Genetic studies of molecules that negatively regulate G-coupled receptor functions have led to the identification of a large gene family with an evolutionarily conserved domain, termed the RGS domain . It is now understood that RGS proteins serve as GTPase-activating proteins for subfamilies of the heterotrimeric G-proteins . We have isolated from mouse pituitary a full-length cDNA clone encoding a novel member of the RGS protein family, termed RGS16, as well as the full-length cDNA of mRGS5 and mRGS2 . Tissue distribution analysis shows that the novel RGS16 is predominantly expressed in liver and pituitary, and that RGS5 is preferentially expressed in heart and skeletal muscle . In contrast, RGS2 is widely expressed . Genetic analysis using the pheromone response halo assay and FUS1 gene induction assay show that overexpression of the RGS16 gene dramatically inhibits yeast response to alpha-factor, whereas neither RGS2 nor RGS5 has any discernible effect on pheromone sensitivity, pointing to a possible functional diversity among RGS proteins . In vitro binding assays reveal that RGS5 and RGS16 bind to Galphai and Galphao subunits of heterotrimeric G-proteins, but not to Galphas . Based on mutational analysis of the conserved residues in the RGS domain, we suggest that the G-protein binding and GTPase-activating protein activity may involve distinct functional structures of the RGS proteins, indicating that RGS proteins may exert a dual function in the attenuation of signaling via G-coupled receptors.

J Biol Chem, 1997 Mar 28, 272(13), 8402 - 9
The yeast homeodomain protein MATalpha2 shows extended DNA binding specificity in complex with Mcm1; Zhong H et al.; The MATalpha2 (alpha2) repressor interacts with the Mcm1 protein to turn off a-cell type-specific genes in the yeast Saccharomyces cerevisiae . We compared five natural alpha2-Mcm1 sites with an alpha2-Mcm1 symmetric consensus site (AMSC) for their relative strength of repression and found that the AMSC functions slightly better than any of the natural sites . To further investigate the DNA binding specificity of alpha2 in complex with Mcm1, symmetric substitutions at each position in the alpha2 half-sites of AMSC were constructed and assayed for their effect on repression in vivo and DNA binding affinity in vitro . As expected, substitutions at positions in which there are base-specific contacts decrease the level of repression . Interestingly, substitutions at other positions, in which there are no apparent base-specific contacts made by the protein in the alpha2-DNA co-crystal structure, also significantly decrease repression . As an alternative method to examining the DNA binding specificity of alpha2, we performed in vitro alpha2 binding site selection experiments in the presence and absence of Mcm1 . In the presence of Mcm1, the consensus sequences obtained were extended and more closely related to the natural alpha2 sites than the consensus sequence obtained in the absence of Mcm1 . These results demonstrate that in the presence of Mcm1 the sequence specificity of alpha2 is extended to these positions.

J Cell Biol, 1997 Mar 24, 136(6), 1185 - 99
In vivo dynamics of nuclear pore complexes in yeast; Bucci M et al.; While much is known about the role of nuclear pore complexes (NPCs) in nucleocytoplasmic transport, the mechanism of NPC assembly into pores formed through the double lipid bilayer of the nuclear envelope is not well defined . To investigate the dynamics of NPCs, we developed a live-cell assay in the yeast Saccharomyces cerevisiae . The nucleoporin Nup49p was fused to the green fluorescent protein (GFP) of Aequorea victoria and expressed in nup49 null haploid yeast cells . When the GFP-Nup49p donor cell was mated with a recipient cell harboring only unlabeled Nup49p, the nuclei fused as a consequence of the normal mating process . By monitoring the distribution of the GFP-Nup49p, we could assess whether NPCs were able to move from the donor section of the nuclear envelope to that of the recipient nucleus . We observed that fluorescent NPCs moved and encircled the entire nucleus within 25 min after fusion . When assays were done in mutant kar1-1 strains, where nuclear fusion does not occur, GFP-Nup49p appearance in the recipient nucleus occurred at a very slow rate, presumably due to new NPC biogenesis or to exchange of GFP-Nup49p into existing recipient NPCs . Interestingly, in a number of existing mutant strains, NPCs are clustered together at permissive growth temperatures . This has been explained with two different hypotheses: by movement of NPCs through the double nuclear membranes with subsequent clustering at a central location; or, alternatively, by assembly of all NPCs at a central location (such as the spindle pole body) with NPCs in mutant cells unable to move away from this point . Using the GFP-Nup49p system with a mutant in the NPC-associated factor Gle2p that exhibits formation of NPC clusters only at 37 degrees C, it was possible to distinguish between these two models for NPC dynamics . GFP-Nup49p-labeled NPCs, assembled at 23 degrees C, moved into clusters when the cells were shifted to growth at 37 degrees C . These results indicate that NPCs can move through the double nuclear membranes and, moreover, can do so to form NPC clusters in mutant strains . Such clusters may result by releasing NPCs from a nuclear tether, or by disappearance of a protein that normally prevents pore aggregation . This system represents a novel approach for identifying regulators of NPC assembly and movement in the future.

Cell, 1997 Mar 21, 88(6), 865 - 74
Mobility of yeast mitochondrial group II introns: engineering a new site specificity and retrohoming via full reverse splicing; Eskes R et al.; The mobile group II introns aI1 and aI2 of yeast mtDNA encode endonuclease activities that cleave intronless DNA target sites to initiate mobility by target DNA-primed reverse transcription . For aI2, sense-strand cleavage occurs mainly by a partial reverse splicing reaction, whereas for aI1, complete reverse splicing occurs, leading to insertion of the linear intron RNA into double-stranded DNA . Here, we show that aI1 homing and reverse splicing depend on the EBS1 (RNA)/IBS1(DNA) pairing and that target specificity can be changed by compensatory changes in the target site and the donor intron . Using well-marked strains to follow coconversion of flanking DNA, we show that homing occurs by both RT-dependent and -independent pathways . Remarkably, in most RT-dependent events, the reverse spliced intron is the initial template for first-strand cDNA synthesis.

Science, 1997 Mar 21, 275(5307), 1781 - 4
SBF cell cycle regulator as a target of the yeast PKC-MAP kinase pathway; Madden K et al.; Protein kinase C (PKC) signaling is highly conserved among eukaryotes and has been implicated in the regulation of cellular processes such as cell proliferation and growth . In the budding yeast, PKC1 functions to activate the SLT2(MPK1) mitogen-activated protein (MAP) kinase cascade, which is required for the maintenance of cell integrity during asymmetric cell growth . Genetic studies, coimmunoprecipitation experiments, and analysis of protein phosphorylation in vivo and in vitro indicate that the SBF transcription factor (composed of Swi4p and Swi6p), an important regulator of gene expression at the G1 to S phase cell cycle transition, is a target of the Slt2p(Mpk1p) MAP kinase . These studies provide evidence for a direct role of the PKC1 pathway in the regulation of the yeast cell cycle and cell growth and indicate that conserved signaling pathways can act to control key regulators of cell division.

Oncogene, 1997 Mar 20, 14(11), 1307 - 13
Determining mutational fingerprints at the human p53 locus with a yeast functional assay: a new tool for molecular epidemiology; Inga A et al.; In order to isolate experimentally induced p53 mutations, a yeast expression vector harbouring a human wild-type p53 cDNA was treated in vitro with the antineoplastic drug chloroethyl-cyclohexyl-nitroso-urea (CCNU) and transfected into a yeast strain containing the ADE2 gene regulated by a p53-responsive promoter . p53 mutations were identified in 32 out of 39 plasmids rescued from independent ade- transformants . Ninety-two percent of CCNU induced mutations were GC-targeted single base pair substitutions, and GC > AT transitions represented 73% of all single base pair substitutions . In 70% of the cases the mutated G was preceded 5' by a purine . The distribution of the mutations along the p53 cDNA was not random: positions 734 and 785 appeared as CCNU mutational hotspots (n=3, P<0.0003) and CCNU induced only GC > AT transitions at those positions . The features of these CCNU-induced mutations are consistent with the hypothesis that O6-alkylguanine is the major causative lesion . One third of the CCNU-induced mutants were absent from a huge collection of 4496 p53 mutations in human tumours and cell lines, thus demonstrating that CCNU has a mutational spectrum which is uniquely different from that of naturally selected mutations . This strategy allows direct comparison of observed natural mutation spectra with experimentally induced mutation spectra and opens the way to a more rigorous approach in the field of molecular epidemiology.

Proc Natl Acad Sci U S A, 1997 Mar 18, 94(6), 2449 - 53
Detection of heterozygous truncating mutations in the BRCA1 and APC genes by using a rapid screening assay in yeast; Ishioka C et al.; The detection of inactivating mutations in tumor suppressor genes is critical to their characterization, as well as to the development of diagnostic testing . Most approaches for mutational screening of germ-line specimens are complicated by the fact that mutations are heterozygous and that missense mutations are difficult to interpret in the absence of information about protein function . We describe a novel method using Saccharomyces cerevisiae for detecting protein-truncating mutations in any gene of interest . The PCR-amplified coding sequence is inserted by homologous recombination into a yeast URA3 fusion protein, and transformants are assayed for growth in the absence of uracil . The high efficiency of homologous recombination in yeast ensures that both alleles are represented among transformants and achieves separation of alleles, which facilitates subsequent nucleotide sequencing of the mutated transcript . The specificity of translational initiation of the URA3 gene leads to minimal enzymatic activity in transformants harboring an inserted stop codon, and hence to reliable distinction between specimens with wild-type alleles and those with a heterozygous truncating mutation . This yeast-based stop codon assay accurately detects heterozygous truncating mutations in the BRCA1 gene in patients with early onset of breast cancer and in the APC gene in patients with familial adenomatous polyposis . This approach offers a rapid and reliable method for genetic diagnosis in individuals at high risk for germ-line mutations in cancer susceptibility genes.

Proc Natl Acad Sci U S A, 1997 Mar 18, 94(6), 2427 - 32
Mutational studies of yeast transcription factor IIB in vivo reveal a functional surface important for gene activation; Shaw SP et al.; Recent experiments in yeast (Saccharomyces cerevisiae) cells have identified a species-specific region of yeast transcription factor IIB (TFIIB) located at residues 144-157 . According to the human TFIIB structure, this region is part of a solvent-exposed helix in the first repeat of the carboxyl-terminal core domain . In this report, we systematically analyze four positions in this region (Lys-147, Cys-149, Lys-151, and Glu-152) that together have been shown previously to be important for yeast TFIIB's function in vivo . Our experiments suggest that all of these four positions, and in particular positions 151, 149, and 152, are critical for yeast TFIIB's ability to support cell growth . In addition, we describe an intragenic suppressor screening experiment to identify mutations that reverse, or partially reverse, the temperature-sensitive phenotype of a yeast TFIIB derivative bearing amino acid changes at these four positions to human residues . The suppressor mutations reveal changes at positions 115, 117, and 182 that are located outside the species-specific region of yeast TFIIB, suggesting an extended surface available to interact with other proteins . Finally, we demonstrate that the suppressor mutations restore gene activation in vivo, further supporting the idea that one important function of yeast TFIIB in living cells is to mediate gene activation.

Biochem Biophys Res Commun, 1997 Mar 17, 232(2), 503 - 7
Association of elongation factor 1 alpha and ribosomal protein L3 with the proline-rich region of yeast adenylyl cyclase-associated protein CAP; Yanagihara C et al.; CAP is a multifunctional protein; the N-terminal region binds adenylyl cyclase and controls its response to Ras while the C-terminal region is involved in cytoskeletal regulation . In between the two regions, CAP possesses two proline-rich segments, P1 and P2, resembling a consensus sequence for binding SH3 domains . We have identified two yeast proteins with molecular sizes of 48 and 46 kDa associated specifically with P2 . Determination of partial protein sequences demonstrated that the 48-kDa and 46-kDa proteins correspond to EF1 alpha and rL3, respectively, neither of which contains any SH3-domain-like sequence . Deletion of P2 from CAP resulted in loss of the activity to bind the two proteins either in vivo or in vitro . Yeast cells whose chromosomal CAP was replaced by the P2-deletion mutant displayed an abnormal phenotype represented by dissociated localizations of CAP and F-actin, which were colocalized in wild-type cells . These results suggest that these associations may have functional significance.

Eur J Biochem, 1997 Mar 15, 244(3), 953 - 8
Characterization of recombinant yeast dolichyl mannosyl phosphate synthase and site-directed mutagenesis of its cysteine residues; Forsee WT et al.; Dolichyl mannosyl phosphate synthase is associated with membranes of the rough endoplasmic reticulum and catalyzes mannosyl transfer from GDP-mannose to the hydrophobic long-chain acceptor dolichyl-phosphate . The gene for the yeast enzyme encodes a protein with a molecular mass of 30.36 kDa containing three cysteine residues, at positions 93, 172 and 259 {Orlean, P., Albright, C . & Robbins, P . W . (1988) J . Biol . Chem . 263, 17499-17507} . Inhibition of the synthase by thiol-specific reagents, including N-ethylmaleimide, p-hydroxymercuribenzoate, 5,5'-dithiobis(2-nitrobenzoic acid) (Nbs2), and lucifer yellow iodoacetamide (LYI), suggests that sulfhydryl groups might play a role in the catalytic mechanism of the enzyme . Titration of the synthase with Nbs2 or LYI indicated that 1 mol sulfhydryl/mol protein was accessible to these reagents, and that saturation of this site completely inhibited enzyme activity . To ascertain the reactive group and its possible function in enzyme catalysis, each of the cysteine residues was replaced individually by site-directed mutagenesis . The mutant enzymes had specific activities comparable to that of the wild-type enzyme, demonstrating that none of the cysteine residues were essential for catalytic activity . All of the mutant proteins except those containing a substitution at Cys93 were inhibited by thiol-blocking reagents, indicating that Cys93 might be physically located near the catalytic site of the enzyme . GDP-mannose, dolichyl phosphate and substrate analogs were found to protect against Nbs2 inactivation, further suggesting that Cys93 was physically near, or within, the substrate-binding site of the enzyme.

Nucleic Acids Res, 1997 Mar 15, 25(6), 1248 - 53
Stabilization of yeast artificial chromosome clones in a rad54-3 recombination-deficient host strain; Le Y et al.; The cloning and propagation of large fragments of DNA on yeast artificial chromosomes (YACs) has become a routine and valuable technique in genome analysis . Unfortunately, many YAC clones have been found to undergo rearrangements or deletions during the cloning process . The frequency of transformation-associated alterations and mitotic instability can be reduced in a homologous recombination-deficient yeast host strain such as a rad52 mutant . RAD52 is one member of an epistatic group of genes required for the recombinational repair of double-strand breaks in DNA . rad52 mutants grow more slowly and transform less efficiently than RAD + strains and are therefore not ideal hosts for YAC library construction . We have investigated the ability of both null and temperature-sensitive alleles of RAD54 , another member of the RAD52 epistasis group, to prevent rearrangements of human YAC clones containing tandemly repeated DNA sequences . Our results show that the temperature-sensitive rad54-3 allele blocks mitotic recombination between tandemly repeated DYZ3 satellite sequences and significantly stabilizes a human DYZ5 satellite-containing YAC clone . Yeast carrying the rad54-3 mutation can undergo meiosis, have growth and transformation rates comparable with RAD + strains, and therefore represent improved YAC cloning hosts.

Yeast, 1997 Mar 15, 13(3), 275 - 80
Sequencing of a 9.9 kb segment on the right arm of yeast chromosome VII reveals four open reading frames, including PFK1, the gene coding for succinyl-CoA synthetase (beta-chain) and two ORFs sharing homology with ORFs of the yeast chromosome VIII; Guerreiro P et al.; A 9.9 kb DNA fragment from the right arm of chromosome VII of Saccharomyces cerevisiae has been sequenced and analysed . The sequence contains four open reading frames (ORFs) longer than 100 amino acids . One gene, PFK1, has already been cloned and sequenced and the other one is the probable yeast gene coding for the beta-subunit of the succinyl-CoA synthetase . The two remaining ORFs share homology with the deduced amino acid sequence (and their physical arrangement is similar to that) of the YHR161c and YHR162w ORFs from chromosome VIII.

J Biol Chem, 1997 Mar 14, 272(11), 7540 - 5
Critical role of the second stirrup region of the TATA-binding protein for transcriptional activation both in yeast and human; Kim TK et al.; We previously identified three TATA-binding protein (TBP) point mutations (L114K, L189K, and K211L) that have severe effects on transcriptional activation by acidic activators, but no effect on basal transcription, in a yeast-derived TBP-dependent in vitro transcription system (Kim, T . K., Hashimoto, S., Kelleher, R . J., III, Flanagan, P . M., Kornberg, R . D., Horikoshi, M., and Roeder, R . G . (1994) Nature 369, 252-255) . These activation defects were also demonstrated in vivo in yeast cells (Lee, M., and Struhl, K . (1995) Mol . Cell . Biol . 15, 5461-5469) . Here, the transcriptional activities of these and other TBP mutations were examined in human by both in vitro and in vivo assays . Mutations L189K and E188K, which lie in the second stirrup region of TBP, show defective activation by acidic activators both in yeast and human . Somewhat surprisingly, mutations L114K and K211L have almost no demonstrable effect on activation by acidic activators in human, in contrast to their severe effects on defective activator responses in yeast . The implications of these results for TBP structure and function are discussed.

J Biol Chem, 1997 Mar 14, 272(11), 6876 - 81
Expression, secretion, and processing of rice alpha-amylase in the yeast Yarrowia lipolytica; Park CS et al.; The gene encoding rice alpha-amylase in Oryza sativa was expressed in the yeast Yarrowia lipolytica, which is a potential host system for heterologous protein expression . For efficient secretion, the strong and inducible XPR2 promoter was used in the construction of four kinds of expression vectors with the following configurations between the XPR2 promoter and terminator: 1) XPR2 prepro-region-rice alpha-amylase coding sequence, 2) rice alpha-amylase signal peptide-rice alpha-amylase coding sequence, 3) XPR2 signal peptide-rice alpha-amylase coding sequence, and 4) XPR2 signal peptide-dipeptide stretch-rice alpha-amylase coding sequence . Secretion of active recombinant rice alpha-amylase into the culture medium was achieved only in the first two cases, demonstrating that the XPR2 signal peptide is not sufficient to direct the secretion of heterologous protein . Furthermore, our study shows that the XPR2 prepro-region causes imprecise processing (after Pro150-Ala151 or Val135-Leu136 instead of Lys156-Arg157) and leads to N-terminal amino acid sequences that differ from that of native rice alpha-amylase . Secondary structure analysis proposed that the structural form in the vicinity of the KEX2-like endopeptidase processing site in the XPR2 pro-region might play a critical role in the processing of heterologous proteins . These results suggest that the XPR2 pro-region is dispensable for obtaining the precise N-terminal amino acid in heterologous protein secretion . In contrast, utilizing the rice alpha-amylase signal peptide was sufficient in directing secretion of recombinant protein with the expected N-terminal sequence, indicating that the signal peptide of rice alpha-amylase was effectively recognized and processed by the Y . lipolytica secretory pathway.

Mutat Res, 1997 Mar 12, 383(2), 155 - 65
Expression of yeast but not human apurinic/apyrimidinic endonuclease renders Chinese hamster cells more resistant to DNA damaging agents; Tomicic M et al.; Abasic sites represent ubiquitous DNA lesions that arise spontaneously or are induced by DNA-damaging agents . They block DNA replication and are considered to be cytotoxic and mutagenic . The key enzymes involved in the repair of abasic sites are apurinic/apyrimidinic (AP) endonucleases which process these lesions in an error-free mechanism . To analyze the role of AP endonuclease in the protection of mammalian cells against DNA damaging agents, we have transfected both the human (APE) and the yeast (APN1) AP endonuclease in Chinese hamster cells and compared the effects of expression of these genes in stable transfectants as to survival of cells and formation of chromosomal aberrations . Although APE was markedly expressed on RNA and protein level, nuclear extracts of human APE transfectants did not show a higher AP endonuclease activity than the parental line and became not more resistant to the cell killing and clastogenic effect of methyl methanesulfonate (MMS) and hydrogen peroxide (H2O2) . In contrast, cells transfected with the yeast APN1 gene expressed higher AP endonuclease activity and became clearly more resistant to the cytotoxic and chromosome breakage inducing activity of the agents . The results indicate that the excision repair capacity and correspondingly the mutagen resistance can be elevated by introducing, in mammalian cells, a yeast DNA repair gene and verify that AP sites are both cytotoxic and clastogenic lesions.

Biochemistry, 1997 Mar 11, 36(10), 2884 - 97
Kinetics and motional dynamics of spin-labeled yeast iso-1-cytochrome c: 1 . Stopped-flow electron paramagnetic resonance as a probe for protein folding/unfolding of the C-terminal helix spin-labeled at cysteine 102; Qu K et al.; The kinetics of chemically induced folding and unfolding processes in spin-labeled yeast iso-1-cytochrome c were measured by stopped-flow electron paramagnetic resonance (EPR) . Stopped-flow EPR, based on a new dielectric resonator structure {Sienkiewicz, A., Qu, K., & Scholes, C . P . (1994) Rev . Sci . Instrum . 65, 68-74}, gives a new temporal component to probing nanosecond molecular tumbling motions that are modulated by macromolecular processes requiring time resolution of milliseconds to seconds . The stopped-flow EPR technique presented in this work is a kinetic technique that has not been previously used with such a time resolution on spin-labeled systems, and it has the potential for application to numerous spin-labeled sites in this and other proteins . The cysteine-specific spin-label, methanethiosulfonate spin-label (MTSSL), was attached to yeast iso-1-cytochrome c at the single naturally occurring cysteine102, and the emphasis for this work was on this disulfide-attached spin-labeled prototype . This probe has the advantage of reflecting the protein tertiary fold, as shown by recent, systematic site-directed spin labeling of T4 lysozyme {Mchaourab, H . S . Lietzow, M . A., Hideg, K., & Hubbell, W . L . (1996) Biochemistry 35, 7692-7704}, and protein backbone dynamics, as also shown by model peptide studies {Todd, A . P., & Millhauser, G . L . (1991) Biochemistry 30, 5515-5523} . The C-terminal cytochrome c helix where the label is attached is thought to be critical in the initial steps of protein folding and unfolding . Stopped-flow EPR resolved the monoexponential, guanidinium-induced unfolding process at pH 6.5 with an approximately 20 ms time constant; this experiment required less than 150 microL of 80 microM spin-labeled protein . We observed an approximately 50-fold decrease of this unfolding time from the 1 s range to the 20 ms time range as the guanidinium denaturant concentration was increased from 0.6 to 2.0 M . The more complex refolding kinetics of our labeled cytochrome were studied by stopped-flow EPR at pH 5.0 and 6.5 . The spin probe showed a fast kinetic process compatible with the time range over which hydrogen/deuterium amide protection indicates helix formation; this process was monoexponential at pH 5.0 . At pH 6.5, there was evidence of an additional slower kinetic phase resolved by stopped-flow EPR and by heme-ligation-sensitive UV-Vis that indicated a slower folding where heme misligation may be involved . Since the disulfide-attached probe has reported folding and backbone dynamics in other systems, the implication is that our kinetic experiments were directly sensing events of the C-terminal helix formation and possibly the N- and C-terminal helical interaction . The cysteine-labeled protein was also studied under equilibrium conditions to characterize probe mobility and the effect of the probe on protein thermodynamics . The difference in spin probe mobility between folded and denatured protein was marked, and in the folded protein, the motion of the probe was anisotropically restricted . The motion of the attached nitroxide in the folded protein appears to be restricted about the carbon and sulfur bonds which tether it to the cysteine . The original point of cysteine sulfur attachment is approximately 11 A from the heme iron within the C-terminal helix near its interface with the N-terminal helix, but the low-temperature EPR spin probe line width showed that the probe lies more distant (> 15 A) from the heme iron . By all physical evidence, the protein labeled at cysteine102 folded, but the spin probe in this prototype system perturbed packing which lowered the thermal melting temperature, the free energy of folding, the guanidinium concentration at the midpoint of the unfolding transition, the m parameter of the denaturant, and the helical CD signature . This study prepares the way for study of protein folding/unfolding kinetics using EPR spectroscopy of spin-labels placed at specific cysteine-mutated sites within

J Cell Biol, 1997 Mar 10, 136(5), 969 - 82
The yeast CDC37 gene interacts with MPS1 and is required for proper execution of spindle pole body duplication; Schutz AR et al.; The MPS1 gene from Saccharomyces cerevisiae encodes an essential protein kinase required for spindle pole body (SPB) duplication and for the mitotic spindle assembly checkpoint . Cells with the mps1-1 mutation fail early in SPB duplication and proceed through monopolar mitosis with lethal consequences . We identified CDC37 as a multicopy suppressor of mps1-1 temperature-sensitive growth . Suppression is allele specific, and synthetic lethal interactions occur between mps1 and cdc37 alleles . We examined the cdc37-1 phenotype for defects related to the SPB cycle . The cdc37-1 temperature-sensitive allele causes unbudded, G1 arrest at Start (Reed, S.I . 1980 . Genetics . 95: 561-577) . Reciprocal shifts demonstrate that cdc37-1 arrest is interdependent with alpha-factor arrest but is not a normal Start arrest . Although the cells are responsive to alpha-factor at the arrest, SPB duplication is uncoupled from other aspects of G1 progression and proceeds past the satellite-bearing SPB stage normally seen at Start . Electron microscopy reveals side-by-side SPBs at cdc37-1 arrest . The outer plaque of one SPB is missing or reduced, while the other is normal . Using the mps2-1 mutation to distinguish between the SPBs, we find that the outer plaque defect is specific to the new SPB . This phenotype may arise in part from reduced Mps1p function: although Mps1p protein levels are unaffected by the cdc37-1 mutation, kinase activity is markedly reduced . These data demonstrate a requirement for CDC37 in SPB duplication and suggest a role for this gene in G1 control . CDC37 may provide a chaperone function that promotes the activity of protein kinases.

J Biol Chem, 1997 Mar 7, 272(10), 6377 - 81
Functional subdomains of yeast elongation factor 3 . Localization of ribosome-binding domain; Kambampati R et al.; Elongation factor 3 (EF-3) is an essential requirement of the fungi for translational elongation . EF-3 is an ATPase, and the hydrolytic activity is stimulated 2 orders of magnitude by yeast ribosomes . Limited trypsinolysis of EF-3 results in the cleavage of a single peptide bond between residues 774 (Arg) and 775 (Gln), generating polypeptides of approximate molecular mass 90 and 30 kDa . The 90-kDa fragment is relatively resistant to proteolysis and retains ribosome-independent ATPase activity . The 30-kDa fragment is further proteolyzed into smaller fragments and retains the specificity for binding to yeast ribosomes . Both the intact EF-3 and the 30-kDa fragment are protected from proteolysis by yeast ribosomes . EF-3 is NH2 terminally blocked, and so is the 90-kDa fragment . The COOH terminally derived 30-kDa fragment contains glutamine (residue 775) at the NH2-terminal end . A construct was designed representing the COOH-terminal domain of EF-3 (30-kDa fragment), subcloned, and expressed as a glutathione S-transferase fusion in yeast . The glutathione S-transferase-30-kDa peptide remains stringently associated with ribosomes . Isolated fusion peptide rebinds to yeast ribosomes with high affinity . Based on these results, we propose that at least one of the ribosome-binding sites of EF-3 resides at the COOH-terminal end of the protein.

J Biol Chem, 1997 Mar 7, 272(10), 6252 - 60
Functional differences among wheat voltage-dependent anion channel (VDAC) isoforms expressed in yeast . Indication for the presence of a novel VDAC-modulating protein?
Elkeles A, Breiman A, Zizi M.
VDAC is a voltage-gated anion channel located in the mitochondrial outer membrane, presumably participating in controlling aerobic metabolism . Three distinct wheat vdac cDNAs were expressed in a vdac-minus yeast strain and successfully complemented its defective phenotype . The growth curves of these transformants were different . The wheat channel isoforms were functionally characterized following purification from yeast mitochondria and reconstitution into soybean phospholipid planar membranes . All three isoforms yielded voltage-dependent anion channels with electrophysiological parameters comparable to known VDACs . Isoform-related functional features (specific conductance levels, kinetics, and gating behaviors) are reported for the first time in VDACs . The presence (or absence) of protease inhibitors during the purification procedure, and the use of Pronase on reconstituted channels, strongly suggest that some of the unique wheat VDAC properties are due to co-purification of a yeast channel-modulating protein . Its effects, different from the reported functional interactions of the channel with hexo- or creatine kinases, could not be mimicked by the protein termed VDAC modulator, indicating the presence of a novel VDAC modulator . In addition to strengthening VDAC presumed role in metabolism, the functional diversity of the channels (as shown here in two different systems) implies a highly dynamic outer membrane permeability . Our results are consistent with VDAC functioning as a heteromer including one pore protein and other modulating subunits.

Biochem Biophys Res Commun, 1997 Mar 6, 232(1), 204 - 8
Role of the fission yeast nim 1 protein kinase in the cell cycle response to nutritional signals; Belenguer P et al.; The fission yeast cdr1/nim1 protein kinase phosphorylates and inactivates the weel cdc2-inhibitory kinase . We have investigated the role played by cdr1/nim1 in the connection between nutritional signals and the cell cycle machinery . We show that loss of nim1 activity impairs the appropriate cellular adaptation to nutritional changes . However, the reduction in cell size at division in response to nitrogen starvation is independent of nim1 . Moreover, we report that nim1 is an unstable protein that is rapidly degraded upon starvation, through a mechanism that is dependent upon protein synthesis . We propose that nim1, as a constitutive indirect activator of cdc2 at mitosis, favors the cellular response to starvation but does not actively participate in it . On the contrary, upon nitrogen starvation nim1 must be actively destroyed to protect the cells from a commitment into the cell cycle under unfavourable growth conditions.

Biochemistry, 1997 Mar 4, 36(9), 2642 - 8
Sequence requirement for trimethylation of yeast cytochrome c; Takakura H et al.; Lysine 72 (using the vertebrate numbering system) is trimethylated in cytochromes c from fungi and plants but not from higher animals . We have investigated the characteristics of an amino acid sequence required for trimethylation of lysine 72 by examining 21 altered iso-1-cytochromes c from Saccharomyces cerevisiae having single replacements in the region encompassing residues 67 through 77 . These results indicated that tyrosine 74 is critical for trimethylation of lysine 72, whereas replacements at other positions did not produce significant diminutions . Various replacements of tyrosine 74 resulted in different levels of inhibition, with the Y74F replacement causing no significant reduction, and the Y74E and Y74K replacements completely or almost completely preventing trimethylation of lysine 72 . However, other similarly spaced lysine and tyrosine residues at other sites in the protein did not result in trimethylation of the lysine residue . Thus, a properly situated aromatic residue, determined by the overall conformation of apocytochrome c in the vicinity of lysine 72, appears to be essential for trimethylation.

EMBO J, 1997 Mar 3, 16(5), 1114 - 21
A novel inhibitor of cap-dependent translation initiation in yeast: p20 competes with eIF4G for binding to eIF4E; Altmann M et al.; In the yeast Saccharomyces cerevisiae a small protein named p20 is found associated with translation initiation factor eIF4E, the mRNA cap-binding protein . We demonstrate here that p20 is a repressor of cap-dependent translation initiation . p20 shows amino acid sequence homology to a region of eIF4G, the large subunit of the cap-binding protein complex eIF4F, which carries the binding site for eIF4E . Both, eIF4G and p20 bind to eIF4E and compete with each other for binding to eIF4E . The eIF4E-p20 complex can bind to the cap structure and inhibit cap-dependent but not cap-independent translation initiation: the translation of a mRNA with the 67 nucleotide omega sequence of tobacco mosaic virus in its 5' untranslated region (which was previously shown to render translation cap-independent) is not inhibited by p20 . Whereas the translation of the same mRNA lacking the omega sequence is strongly inhibited by p20 . Disruption of CAF20, the gene encoding p20, stimulates the growth of yeast cells, overexpression of p20 causes slower growth of yeast cells . These results show that p20 is a regulator of eIF4E activity which represses cap-dependent initiation of translation by interfering with the interaction of eIF4E with eIF4G, e.g . the formation of the eIF4F-complex.

FEBS Lett, 1997 Mar 3, 404(1), 51 - 5
Functional mapping reveals the importance of yeast cytochrome b C-terminal region in assembly and function of the bc1 complex; Edderkaoui B et al.; Genetic and molecular analyses have been undertaken for four respiratory deficient mutants (mit-) . The four mutations affect the C-terminal region of apocytochrome b . The frameshift (L263STOP) and non-sense (Q338STOP) mutations give rise to a truncated apocytochrome b . The mutant G337R conserves only 32% of its NADH oxidase activity which suggests that the presence of a positively charged amino acid in the transmembranous helix 7 of cytochrome b alters, either directly or indirectly, the bc1 function, without affecting its assembly . The mutation G352V has a 65% loss of cytochrome b spectral content and prevents all of the mitochondrial respiratory activity . This leads us to believe that the glycine, conserved in position 352, may play a crucial role in bc1 complex function.

Mol Biol Rep, 1997 Mar, 24(1-2), 17 - 26
ATPase and ubiquitin-binding proteins of the yeast proteasome; Rubin DM et al.; The 26S proteasome is a 2-Megadalton proteolytic complex with over 30 distinct subunits . The 19S particle, a subcomplex of the 26S proteasome, is thought to confer ATP-dependence and ubiquitin-dependence on the proteolytic core particle of the proteasome . Given the complexity of the 19S particle, genetic approaches are likely to play an important role in its analysis . We have initiated biochemical and genetic studies of the 19S particle in Saccharomyces cerevisiae . Here we describe the localization to the proteasome of several ATPases that were previously proposed to be involved in transcription . Independent studies indicate that the mammalian 26S proteasome contains closely related ATPases . We have also found that the multiubiquitin chain binding protein Mcb1, a homolog of the mammalian S5a protein, is a subunit of the yeast proteasome . However, contrary to expectation, MCB1 is not an essential gene in yeast . The mcb1 mutant grows at a nearly wild-type rate, and the breakdown of most ubiquitin-protein conjugates is unaffected in this strain . One substrate, Ub-Proline-beta gal, was found to require MCB1 for its breakdown, but it remains unclear whether Mcb1 serves as a ubiquitin receptor in this process . Our data suggest that the recognition of ubiquitin conjugates by the proteasome is a complex process which must involve proteins other than Mcb1.

Electrophoresis, 1997 Mar-Apr, 18(3-4), 418 - 23
Identification of two-dimensional gel electrophoresis resolved yeast proteins by matrix-assisted laser desorption ionization mass spectrometry; Larsson T et al.; Protein extract from yeast cells growing exponentially in saline medium was separated by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), with the separation in the first dimension on a wide range immobilized pH (3-10) gradient . From one preparative 2-D gel a number of previously identified proteins were used as test material for our initial matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) efforts on large scale rapid protein spot identification . Sample preparation via in-gel trypsin digestion was slightly modified to be compatible to MS analysis, and via this modified procedure MS generated peptide mass profiles could, in most cases with good precision, identify the protein in question . Preferential ionization was tested on a yeast aldehyde dehydrogenase (ALD7), and it was shown that the ionization of some peptides was clearly suppressed by the presence of others . Roughly 50% of the observed peptide masses was found by the search routines in the database, and the mass measurement accuracy of the peptides was within 0.5 Da . Silver-stained gels could be used with good results for the generation of peptides to be analyzed by MALDI-MS . For one of the 2-D resolved proteins, glycerol 3-phosphatase (GPP1), the post-source decay (PSD) spectrum proved crucial in identification.

Plant Cell Physiol, 1997 Mar, 38(3), 366 - 70
Heterologous expression and subcellular localization of pumpkin seed tonoplast intrinsic proteins (TIP) in yeast cells; Inoue K et al.; Two tonoplast intrinsic proteins (TIP) of pumpkin seeds, pMP23 and MP28, were expressed in yeast cells under control of the GAL1 promoter, and the subcellular localization of the proteins was analyzed . The pMP23 and MP28 stably accumulated in the yeast vacuolar membrane when the proteins were expressed in the proteinase A-deficient strain (pep4), which lacks the activities of vacuolar proteases . However, pMP23 and MP28 did not accumulate in the wild-type strain; the expressed pMP23 and MP28 were degraded in a proteinase A-dependent manner . These results indicate that pMP23 and MP28 are transported to the vacuolar membrane when expressed in yeast.

Hum Mol Genet, 1997 Mar, 6(3), 487 - 95
HIP-I: a huntingtin interacting protein isolated by the yeast two-hybrid system; Wanker EE et al.; We report the discovery of the huntingtin interacting protein I (HIP-I) which binds specifically to the N-terminus of human huntingtin, both in the two-hybrid screen and in in vitro binding experiments . For the interaction in vivo, a protein region downstream of the polyglutamine stretch in huntingtin is essential . The HIP1 cDNA isolated by the two-hybrid screen encodes a 55 kDa fragment of a novel protein . Using an affinity-purified polyclonal antibody raised against recombinant HIP-I, a protein of 116 kDa was detected in brain extracts by Western blot analysis . The predicted amino acid sequence of the HIP-I fragment exhibits significant similarity to cytoskeleton proteins, suggesting that HIP-I and huntingtin play a functional role in the cell filament networks . The HIP1 gene is ubiquitously expressed in different brain regions at low level . HIP-I is enriched in human brain but can also be detected in other human tissues as well as in mouse brain . HIP-I and huntingtin behave almost identically during subcellular fractionation and both proteins are enriched in the membrane containing fractions.

Hum Mol Genet, 1997 Mar, 6(3), 349 - 55
Destabilization of CAG trinucleotide repeat tracts by mismatch repair mutations in yeast; Schweitzer JK et al.; To examine the genetic factors that affect the stability of disease-associated trinucleotide repeats, we have assessed the stability of CAG repeats in yeast strains with mutations in the mismatch repair system . We have found that both pms1 and msh2 mutations destabilize repeat tracts . Destabilization is evidenced both by the increased frequency of repeat length changes and in the pattern of changes that are observed . In wild-type cells repeats are relatively stable when CAG serves as the lagging strand template but relatively unstable when CTG serves as the lagging strand template . Large contractions in repeat length are the most common change . In pms1 and msh2 mutants the relatively stable tracts incur more tract length changes . In addition, many small deletions and some small additions, most often of one repeat unit, are frequent in repeats of the stable orientation . These small changes also are seen as a new class of events that occur in repeats in the unstable orientation . The results show that in yeast the mismatch repair system prevents small changes from occurring but cannot prevent larger changes from occurring.

Hokkaido Igaku Zasshi, 1997 Mar, 72(2), 211 - 24
{Detectability and diagnostic criteria of p53 gene mutations in human oral squamous cell carcinoma using yeast functional assay}; Kashiwazaki H; In yeast functional assay (YF assay), a newly developed screening system for p53 mutation, wild-type p53 gives white yeast colonies and transcriptionally inactive mutant p53 gives red colonies . In the present study, the author applied YF assay to the detection of p53 mutations in human oral squamous cell carcinoma (SCC) . Total RNA was extracted from samples and YF assay was performed . Four SCC cell lines (SAS, HSC-2, HSC-3 and Ca9-22) known to have p53 mutations all gave 100% red colonies, whereas nine oral non-tumor tissues gave 2.9-10% (average 5.2 +/- 2.7%) red colonies . Furthermore, a rat hepatoma cell line, WHp53, which had been transfected with human wild-type p53 expression vector, presented 7.8% red colonies . Thus the functional assays of tissues or cells containing only wild-type p53 give 3-10% red colonies as a background . To assess the detectability of p53 mutations, YF assay was performed on mixtures of wild-type and mutant p53 PCR products at serial ratios . The result showed that the mutation was detectable if 6% population of transcriptionally inactive mutant p53 mRNA were present in the total p53 mRNA . Twenty-two clinical samples of human oral SCC were then tested by YF assay . Fourteen out of 22 cases gave more than 20% red colonies . In these 14 cases, clonal p53 mutations with deletion, nonsense mutation or missense mutation were identified . In a case which gave 17% red colonies, identical p53 mutation was found in 2 out of 6 independent red colonies . However, no identical mutations were found in the cases giving 13, 9 and 8% red colonies . Based on these results, the author proposes that 20% of red colonies is the minimal value for the diagnosis of p53 mutation in YF assay under PCR conditions using Pfu polymerase and hot start method.

Anticancer Res, 1997 Mar-Apr, 17(2A), 1079 - 82
Effect of antineoplastic agents on non proliferating yeast cells: a possible membrane effect of doxorubicin; Karavokyros I et al.; The effect of antineoplastic agents on non proliferating cells of Saccharomyces cerevisiae was investigated . Non growing populations were obtained by suspending cells in saline or H2O and survival rates were determined after exposing the cells to various commercially available agents for five hours . The only agent found to be effective was Doxorubicin which reduced survival rates to less than 5% (p < 0.001) . The action of this drug could be detected in only 4 minutes and was not concentration dependent, therefore it is probable that DNA damage is caused mostly by oxygen free drug radicals . Furthermore, our observations strongly imply the damage of cellular membranes is an alternative reason for cell death, with phosphatidyl-inositol being the most probable candidate target for the drug.

J Biochem (Tokyo), 1997 Mar, 121(3), 568 - 77
Interaction with D-glucose and thermal denaturation of yeast hexokinase B: A DSC study; Catanzano F et al.; DSC measurements have been performed on the monomeric form of yeast hexokinase B in the absence and presence of increasing concentrations of D-glucose . The hexokinase, in the absence of D-glucose, at both pH 8.0 and 8.5, shows reproducible calorimetric profiles characterized by the presence of two partially overlapped peaks . These can be ascribed to the presence of two structural domains in the native conformation of the enzyme, that possess different thermal stabilities and are denatured more or less independently . In the presence of saturating and increasing concentrations of D-glucose, the shape of the DSC profiles dramatically changes, since a single well-shaped peak is present . The binding of D-glucose enhances the interaction between the two lobes, as evidenced by the shrinking of the protein in overall dimensions, and gives rise to DSC profiles resembling those of a single domain protein . To deconvolve the DSC curves we considered a denaturation model consisting of two sequential steps with three macroscopic states of the protein and the binding of D-glucose only to the native state . We carried out two-dimensional nonlinear regression of the excess heat capacity surface constructed with the experimental DSC curves . This approach allows the calculation of a unique set of thermodynamic parameters characterizing both the thermal denaturation of hexokinase, and the binding equilibrium between D-glucose and the enzyme . It was found that the association constant is 9,800+/-1,500 M(-1) at pH 8.0 . The binding of D-glucose is entropy-driven, since the binding enthalpy is zero . This finding is rationalized by a thermodynamic cycle for the association of two molecules in aqueous solution.

Eur J Biochem, 1997 Mar 1, 244(2), 646 - 52
Examination of the thiamin diphosphate binding site in yeast transketolase by site-directed mutagenesis; Meshalkina L et al.; The role of two conserved amino acid residues in the thiamin diphosphate binding site of yeast transketolase has been analyzed by site-directed mutagenesis . Replacement of E162, which is part of a cluster of glutamic acid residues at the subunit interface, by alanine or glutamine results in mutant enzymes with most catalytic properties similar to wild-type enzyme . The two mutant enzymes show, however, significant increases in the K0.5 values for thiamin diphosphate in the absence of substrate and in the lag of the reaction progress curves . This suggests that the interaction of E162 with residue E418, and possibly E167, from the second subunit is important for formation and stabilization of the transketolase dimer . Replacement of the conserved residue D382, which is buried upon binding of thiamin diphosphate, by asparagine and alanine, results in mutant enzymes severely impaired in thiamin diphosphate binding and catalytic efficiency . The 25-80-fold increase in K0.5 for thiamin diphosphate suggests that D382 is involved in cofactor binding, probably by electrostatic compensation of the positive charge of the thiazolium ring and stabilization of a flexible loop at the active site . The decrease in catalytic activities in the D382 mutants indicates that this residue might also be important in subsequent steps in catalysis.

Antonie Van Leeuwenhoek, 1997 Mar, 71(3), 289 - 95
Phaeotheca triangularis, a new meristematic black yeast from a humidifier; de Hoog GS et al.; Two strains of a new species of meristematic fungi, described as Phaeotheca triangularis, were isolated from moisteners of air-conditioning systems . The species is believed to be related to dothideaceous black yeasts . Its morphology, ultrastructure and nutritional physiology are reported.

Mol Biochem Parasitol, 1997 Mar, 85(1), 25 - 40
Analysis in yeast of antimalaria drugs that target the dihydrofolate reductase of Plasmodium falciparum; Wooden JM et al.; Pyrimethamine and cycloguanil are competitive inhibitors of the Plasmodium enzyme dihydrofolate reductase (DHFR) . They have been effective treatments for malaria, but rapid selection of populations of the parasite resistant to these drugs has compromised their effectiveness . Parasites resistant to either drug usually have point mutations in the dhfr gene, but the frequency of these mutations is unknown . To study drug resistance more effectively, we transferred the DHFR domain of the dhfr-thymidylate synthase gene from a drug-sensitive line of P . falciparum to a strain of the budding yeast, Saccharomyces cerevisiae, that lacks endogenous DHFR activity . Expression of the P . falciparum dhfr is controlled by the yeast dhfr 5' and 3' regulatory regions and the heterologous enzyme provided all of the functions of the yeast dhfr gene . These yeast were susceptible to pyrimethamine and cycloguanil at low concentrations that inhibit P . falciparum (IC50 about 10(-8) and 10(-7) M, respectively) . Yeast expressing constructs with dhfr alleles from pyrimethamine-resistant strains were resistant to both pyrimethamine and cycloguanil (IC50 > 10(-6) M); resistance of the yeast depended on the dhfr allele they expressed . The experimental drug WR99210 efficiently killed all three yeast strains (IC50 about 10(-8) M) but the pyrR strains showed collateral hypersensitivity to drug . The yeast transformants carrying the drug-sensitive allele can now be screened quickly and quantitatively to identify new drugs or combinations of drugs and determine which drugs select resistant parasites least efficiently . Such compounds would be excellent candidates for development of treatments with a longer life in clinical practice.

Plant Cell, 1997 Mar, 9(3), 381 - 92
Two cDNAs from potato are able to complement a phosphate uptake-deficient yeast mutant: identification of phosphate transporters from higher plants; Leggewie G et al.; Acquisition as well as translocation of phosphate are essential processes for plant growth . In many plants, phosphate uptake by roots and distribution within the plant are presumed to occur via a phosphate/proton cotransport mechanism . Here, we describe the isolation of two cDNAs, StPT1 and StPT2, from potato (Solanum tuberosum) that show homology to the phosphate/proton cotransporter PHO84 from the yeast Saccharomyces cerevisiae . The predicted products of both cDNAs share 35% identity with the PHO84 sequence . The deduced structure of the encoded proteins revealed 12 membrane-spanning domains with a central hydrophilic region . The molecular mass was calculated to be 59 kD for the StPT1 protein and 58 kD for the StPT2 protein . When expressed in a PHO84-deficient yeast strain, MB192, both cDNAs complemented the mutant . Uptake of radioactive orthophosphate by the yeast mutant expressing either StPT1 or StPT2 was dependent on pH and reduced in the presence of uncouplers of oxidative phosphorylation, such as 2,4-dinitrophenol or carbonyl cyanide m-chlorophenylhydrazone . The K(m) for Pi uptake of the StPT1 and StPT2 proteins was determined to be 280 and 130 microM, respectively . StPT1 is expressed in roots, tubers, and source leaves as well as in floral organs . Deprivation of nitrogen, phosphorus, potassium, and sulfur changed spatial expression as well as the expression level of StPT1 . StPT2 expression was detected mainly in root organs when plants were deprived of Pi and to a lesser extent under sulfur deprivation conditions . No expression was found under optimized nutrition conditions or when other macronutrients were lacking.

Biochem Mol Biol Int, 1997 Mar, 41(3), 481 - 6
Inactivation of aconitase in yeast exposed to oxidative stress; Murakami K et al.; Inactivation of aconitase by oxidative stress was analyzed under the in vivo and in situ conditions of yeast cells . Treatment of yeast cells with paraquat caused a specific inactivation of aconitase without affecting the activity of other citric acid cycle enzymes . Addition of copper plus ascorbic acid to permeabilized yeast cells also inactivated aconitase, but did not affect other TCA cycle-related enzymes . Inactivation of aconitase was suggested to be due to the superoxide and hydroxyl radicals produced from the reaction of O2 with paraquat and by Fenton reaction with copper and ascorbic acid under the in vivo and in situ conditions of yeast, respectively . Citrate the substrate of aconitase effectively protected aconitase from the oxidative inactivation . Toxicity of oxygen to yeast cells can be explained by the specific inactivation of aconitase by oxygen radicals . Increased concentrations of citrate can act as a defense mechanism against oxidative inactivation of aconitase under the exposure of aerobically grown yeast to oxidative stress.

J Anim Sci, 1997 Mar, 75(3), 593 - 7
The effect of dietary active dry yeast supplement on performance of sows during gestation-lactation and their pigs; Jurgens MH et al.; Thirty crossbred sows and their pigs were evaluated through two parities to determine any reproductive or growth performance effects of an active dry yeast supplement added to corn-soybean meal diets . Sow reproductive performance from d 93 of gestation through d 21 of lactation and sow milk composition were evaluated . Pig growth performance was measured from birth to 28 d after weaning . Active dry yeast was added at 0, 1, or .2% of the sow gestation diet, 0, .15, or .3% of the sow lactation diet, 0, .2, or .4% of the pig prestarter diet, 1 wk before and 1 wk after weaning, and 0, .125, or .25% during the last 3 wk in the nursery . The yeast source consisted of a concentrate of live yeast cells of the Saccharomyces cerevisiae strain containing more than 15 x 10(9) live cells/g . Sow body weight at d 93 of gestation, at farrowing, and at d 21 of lactation did not differ (P > .10) among treatment groups . Milk from sows fed active dry yeast contained higher amounts of total solids (P < .05), crude protein (P < .10), and gamma globulin (P < .06) than milk from sows fed the control diet . Sow feed intake during lactation was not affected (P > .10) by treatment, nor were there differences in litter size at birth, litter birth weight, or litter weight at d 21 after farrowing . Active dry yeast supplementation to the sow and pig diets resulted in improved postweaning pig daily gain (P < .05) and gain-to-feed ratio (P < .05) but did not affect (P > .10) feed intake . Based on these data, active dry yeast supplement during late gestation, lactation, and before and after weaning does not alter litter weight at birth or weaning but does increase gamma globulin content of sow's milk and improves postweaning rate and efficiency of weight gain of pigs.

Toxicol Appl Pharmacol, 1997 Mar, 143(1), 205 - 12
Evaluation of chemicals with endocrine modulating activity in a yeast-based steroid hormone receptor gene transcription assay; Gaido KW et al.; There is a concern that chemicals in our environment are affecting human health by disrupting normal endocrine function . Much of the concern has focused on chemicals that can interact directly with steroid hormone receptors . We have used a yeast-based assay to assess chemical interactions with the estrogen, androgen, and progesterone receptors . The yeast transformants used in this study contained the human estrogen, androgen, or progesterone receptor along with the appropriate steroid responsive elements upstream of the beta-galactosidase reporter gene . Chemicals were added to yeast cultures in doses ranging from 10(-12) to 10(-4) M and following incubation, the yeasts were then lysed and assayed for beta-galactosidase activity . Diethylstilbesterol and 17-beta estradiol were most active in the estrogen receptor assay, followed by the phytoestrogen, coumestrol . p-Nonylphenol and bisphenol A were approximately 5000- and 15,000-fold less active, respectively, than estradiol . Methoxychlor, DDT and its metabolites, o,p'-DDD, and o,p'-DDE ranged in potency from 5 to 24 X 10(6) less potent than estradiol . Testosterone and dihydrotestosterone were most potent in the androgen receptor assay, followed by estradiol and progesterone . p,p'-DDE was approximately 10(6)-fold less potent than testosterone . None of the industrial chemicals tested interacted with the progesterone receptor . These data demonstrate the utility of using yeast-based receptor assays for detecting chemical interaction with steroid receptors and these assays should serve as a useful component of an in vitro-in vivo strategy to assess the effects of chemicals on endocrine function.

Methods, 1997 Mar, 11(3), 289 - 99
Yeast metallothionein gene expression in response to metals and oxidative stress; Liu XD et al.; Metals and oxygen are chemically linked in biological systems . Metals and oxygen play important roles in enzymatic reactions, metabolism, and signal transduction; however, metals and oxygen react to form highly toxic oxygen-derived free radical species . In this review we focus on the use of yeast cells, as unicellular eukaryotic model systems, to conduct studies aimed at understanding fundamental mechanisms for the sensation and protective responses to toxic metals and oxygen-derived radicals via the activation of yeast metallothionein gene expression.

J Membr Biol, 1997 Mar 1, 156(1), 37 - 44
Inactivation of the peptide-sensitive channel from the yeast mitochondrial outer membrane: properties, sensitivity to trypsin and modulation by a basic peptide; Pelleschi M et al.; The yeast Peptide Sensitive Channel (PSC), a cationic channel of the mitochondrial outer membrane closes with slow kinetics at potentials of either polarity . The properties of this inactivation closely resemble those of the Voltage-Dependent Anion Channel (VDAC) slow kinetics closures . Addition of trypsin to one compartment suppresses the inactivation observed when this compartment is made positive, but does not affect the inactivation observed at potentials of reverse polarity . Both sides of the channel are sensitive . The reduced form of the Mast Cell Degranulating peptide (rMCD) increases the rate of inactivation, but only when the polarity of the compartment to which it is added is positive . The effect is not reversed by washing the peptide out, but is suppressed by trypsin . The peptide can bind to both sides of the membrane . The effect of rMCD on PSC closely resembles that of the "modulator" on VDAC . The similarities between PSC and VDAC suggest that the former might be a cationic porin of the mitochondrial outer membrane possessing a structure closely related to that of VDAC.

Trends Biochem Sci, 1997 Mar, 22(3), 93 - 7
Transcription factors vs nucleosomes: regulation of the PHO5 promoter in yeast; Svaren J et al.; Activation of the Saccharomyces cerevisiae PHO5 gene is accompanied by the disruption of four positioned nucleosomes at the promoter . The chromatin transition requires a DNA-binding protein, Pho4, and its transactivation domain . The mechanism of nucleosome disruption and the contribution of the nucleosomes to PHO5 regulation are reviewed.

Curr Genet, 1997 Mar, 31(3), 241 - 7
STP1, a gene involved in pre-tRNA processing in yeast, is important for amino-acid uptake and transcription of the permease gene BAP2; Jorgensen MU et al.; The bap1 mutant of Saccharomyces cerevisiae was previously isolated by its reduced uptake of branched-chain amino acids . In the present study, the corresponding wild-type gene was cloned and partial sequencing and subsequent genetic analysis revealed identity to STP1, a gene involved in tRNA maturation . The decrease in amino-acid uptake caused by stp1 mutations is independent of GCN4 . It was previously found that the BAP2 promoter can be activated by the presence of amino acids, notably leucine, in the medium . We found that this activation depends on STP1 . As a simple hypothesis we propose that Stp1p is a transcription factor which activates BAP2, and probably other amino-acid permease genes.

Curr Genet, 1997 Mar, 31(3), 228 - 34
Cloning and characterization of MRP10, a yeast gene coding for a mitochondrial ribosomal protein; Jin C et al.; The nuclear gene MRP10 of Saccharomyces cerevisiae was cloned by complementation of a respiratory deficient mutant N518/L1 . This mutant is defective in mitochondrial translation and shows a tendency to accumulate deletions in mitochondrial DNA (rho-) . Analysis revealed Mrp10p to be a component of the 37 S subunit of the mitochondrial ribosomes . Disruption of MRP10 in a haploid strain of yeast elicits a phenotype identical to that of the original mutant . The respiratory defect of the null mutant is rescued by re-introducing the MRP10 gene in a wild-type mitochondrial DNA background . These results indicate that Mrp10p belongs to the class of yeast mitochondrial ribosomal proteins that are essential for translation . Searches of current databases failed to reveal any homologs among known bacterial or eucaryotic cytoplasmic ribosomal proteins . Some sequence similarity, however, was detected between Mrp10p and Yml37p, previously identified as a component of the yeast mitochondrial 50 S ribosomal subunit.

Mol Endocrinol, 1997 Mar, 11(3), 366 - 78
Analysis of vitamin D analog-induced heterodimerization of vitamin D receptor with retinoid X receptor using the yeast two-hybrid system; Zhao XY et al.; Several synthetic analogs of 1 alpha,25-dihydroxyvitamin D3 {1,25-(OH)2D3} are potent inducers of cellular differentiation and inhibitors of cell growth, yet they are less calcemic than 1,25-(OH)2D3 itself . The mechanisms by which these vitamin D analogs elicit a different profile of cellular activities than 1,25-(OH)2D3 are not fully understood . We propose that the analogs bind to the vitamin D receptor (VDR) to produce a conformational change that is more or less constrained than that induced by 1,25-(OH)2D3 . This conformational change determines the extent of the VDR-retinoid X receptor (RXR) heterodimerization which, in turn, determines the interaction with other factors that specify the selectivity and magnitude of gene transactivation . We used the yeast two-hybrid system to evaluate a series of six vitamin D analogs for their ability to induce VDR-RXR heterodimerization . The VDR-RXR interaction was elicited by the analogs in a concentration-dependent manner . To evaluate how this activity compared with other known steps in 1,25-(OH)2D3 action, we also measured the ability of the same six analogs to bind to VDR, to enhance the binding of VDR-RXR to DNA, to transactivate a vitamin D-response element-reporter construct, and to inhibit proliferation in mammalian cells . Our results indicate that, for most analogs, the level of transcriptional activation correlates well with the strength of VDR-RXR heterodimerization in intact cells . We conclude that the yeast two-hybrid system provides a useful means to investigate heterodimerization potency and that this property contributes significantly to the overall pattern of analog activity . The yeast two-hybrid system, being an intact cell assay and easy to perform, may be a useful supplement to the conventional assays employed to screen vitamin D analogs.

RNA, 1997 Mar, 3(3), 245 - 54
RNA apatamers for yeast ribosomal protein L32 have a conserved purine-rich internal loop; Li H et al.; Two in vitro selection experiments were conducted to determine the RNA sequence requirements for binding ribosomal protein L32 (RPL32) from Saccharomyces cerevisiae . To preserve the wild-type stem-internal loop-stem fold, only a limited portion of the RNA comprising the internal loop region was randomized . Most of the selected RNAs have secondary structures similar to that of the wild-type, and four purines on both sides of the internal loop are highly conserved . Indeed, a pair of 5'-GA-3' dinucleotides is found in all but one of the stem-loop-stem L32 aptamers and these conserved purines may contact the protein directly or form a necessary RNA secondary or tertiary structure . These aptamers have a potential G:U pair bordering the loop adjacent to the conserved GAs, but a cytidine replaces a phylogenetically conserved adenosine at one loop position in many of the selected RNAs . In model RNAs, the cytidine-bearing variant binds protein slightly more strongly than does the wild-type RNA . That the seven-member, 2 + 5 internal loop is important for protein binding is reinforced by the finding that the position, but not the size, of the loop is variable . A minority of the RNA aptamers has three consecutive uridines and may fold into a similar structure, but with the internal loop inverted.

Genetics, 1997 Mar, 145(3), 685 - 96
Epigenetic inheritance of transcriptional silencing and switching competence in fission yeast; Thon G et al.; Epigenetic events allow the inheritance of phenotypic changes that are not caused by an alteration in DNA sequence . Here we characterize an epigenetic phenomenon occurring in the mating-type region of fission yeast . Cells of fission yeast switch between the P and M mating-type by interconverting their expressed mating-type cassette between two allelic forms, mat1-P and mat1-M . The switch results from gene conversions of mat1 by two silent cassettes, mat2-P and mat3-M, which are linked to each other and to mat1 . GREWAL and KLAR observed that the ability to both switch mat1 and repress transcription near mat2-P and mat3-M was maintained epigenetically in a strain with an 8-kb deletion between mat2 and mat3 . Using a strain very similar to theirs, we determined that interconversions between the switching- and silencing-proficient state and the switching and silencing-deficient state occurred less frequently than once per 1000 cell divisions . Although transcriptional silencing was alleviated by the 8-kb deletion, it was not abolished . We performed a mutant search and obtained a class of trans-acting mutations that displayed a strong cumulative effect with the 8-kb deletion . These mutations allow to assess the extent to which silencing is affected by the deletion and provide new insights on the redundancy of the silencing mechanism.

Genetics, 1997 Mar, 145(3), 587 - 94
Suppression of pdc2 regulating pyruvate decarboxylase synthesis in yeast; Velmurugan S et al.; Mutants lacking pyruvate decarboxylase cannot grow on glucose . We have isolated three different complementation groups of extragenic suppressors that suppress mutations in pdc2, a regulatory locus required for the synthesis of the glycolytic enzyme pyruvate decarboxylase . The most frequent of these is a recessive mutation in the structural gene PFK1 of the soluble phosphofructokinase . The other class XSP18 (extragenic suppressor of pdc2) is a dominant temperature-sensitive suppressor that allows the cells to grow on glucose only at 30 degrees but not at 36 degrees . It also affects the normal induction of the glucose-inducible enolase 2, which can be rescued by providing a copy of wild-type xsp18 in trans-heterozygotes . The pyruvate decarboxylase activity in the triple mutant pdc2 pfk1 XSP18 is nearly equal to the sum of the activities in the two double mutants pdc2 pfk1 and pdc2 XSP18, respectively . This implies that the two suppressors act through independent pathways or that there is no cooperativity between them . In the pdc2 pfk1 XSP18, strain, pfk1 suppresses the loss of induction of glucose-inducible enolase 2 brought about by XSP18 but fails to rescue temperature sensitivity . The third class (xsp37) supports the growth of the pdc2 mutant on glucose but fails to support growth on gluconeogenic carbon sources . All the three suppressors suppress pdc2 delta as well and act on PDC1 at the level of transcription.

Mol Cell Biol, 1997 Mar, 17(3), 1580 - 94
Upf1p, Nmd2p, and Upf3p are interacting components of the yeast nonsense-mediated mRNA decay pathway; He F et al.; Rapid turnover of nonsense-containing mRNAs in Saccharomyces cerevisiae is dependent on Upf1p, Nmd2p, and Upf3p, the products of the UPF1, NMD2/UPF2, and UPF3 genes, respectively . We showed previously that Upf1p and Nmd2p interact and that this interaction is required for nonsense-mediated mRNA decay (F . He and A . Jacobson, Genes Dev . 9:437-454, 1995; F . He, A . H . Brown, and A . Jacobson, RNA 2:153-170, 1996) . In this study we have used the yeast two-hybrid system to define other protein-protein interactions among the essential components of this decay pathway . Nmd2p-Upf3p and Upf1p-Upf3p interactions were identified, and the respective domains involved in these interactions were delineated by deletion analysis . The domains of Upf1p and Upf3p putatively involved in their mutual interaction were found to correspond to the domains on the two proteins which interact with Nmd2p, suggesting that Nmd2p bridges Upf1p and Upf3p . This conclusion was reinforced by experiments showing that: (i) deletion of NMD2 completely abolishes interactions between Upf1p and Upf3p and (ii) overexpression of full-length Nmd2p or Nmd2p fragments that retain Upf1p- and Upf3p-interacting domains promotes 10- to 200-fold enhancement of Upf1p-Nmd2p-Upf3p complex formation . These results; the observation that cells harboring either single or multiple deletions of UPF1, NMD2, and UPF3 inhibit nonsense-mediated mRNA decay to the same extent; and an analysis of the possible targets of a dominant-negative NMD2 allele indicate that Upf1p, Nmd2p, Upf3p, and at least one other factor are functionally dependent, interacting components of the yeast nonsense-mediated mRNA decay pathway.

Mol Cell Biol, 1997 Mar, 17(3), 1110 - 7
A basic helix-loop-helix-leucine zipper transcription complex in yeast functions in a signaling pathway from mitochondria to the nucleus; Jia Y et al.; The expression of some nuclear genes in Saccharomyces cerevisiae, such as the CIT2 gene, which encodes a glyoxylate cycle isoform of citrate synthase, is responsive to the functional state of mitochondria . Previous studies identified a basic helix-loop-helix-leucine zipper (bHLH/Zip) transcription factor encoded by the RTG1 gene that is required for both basal expression of the CIT2 gene and its increased expression in respiratory-deficient cells . Here, we describe the cloning and characterization of RTG3, a gene encoding a 54-kDa bHLH/Zip protein that is also required for CIT2 expression . Rtg3p binds together with Rtg1p to two identical sites oriented as inverted repeats 28 bp apart in a regulatory upstream activation sequence element (UASr) in the CIT2 promoter . The core binding site for the Rtg1p-Rtg3p heterodimer is 5'-GGTCAC-3', which differs from the canonical E-box site, CANNTG, to which most other bHLH proteins bind . We demonstrate that both of the Rtg1p-Rtg3p binding sites in the UAS(r) element are required in vivo and act synergistically for CIT2 expression . The basic region of Rtg3p conforms well to the basic region of most bHLH proteins, whereas the basic region of Rtg1p does not . These findings suggest that the Rtg1p-Rtg3p complex interacts in a novel way with its DNA target sites.

Mol Cell Biol, 1997 Mar, 17(3), 1102 - 9
PCF11 encodes a third protein component of yeast cleavage and polyadenylation factor I; Amrani N et al.; Cleavage and polyadenylation factor I (CF I) is one of four factors required in vitro for yeast pre-mRNA 3'-end processing . Two protein components of this factor, encoded by genes RNA14 and RNA15, have already been identified . We describe here another gene, PCF11 (for protein 1 of CF I), that genetically interacts with RNA14 and RNA15 and which presumably codes for a third protein component of CF I . This gene was isolated in a two-hybrid screening designed to identify proteins interacting with Rna14 and Rna15 . PCF11 is an essential gene encoding for a protein of 626 amino acids having an apparent molecular mass of 70 kDa . Thermosensitive mutations in PCF11 are synergistically lethal with thermosensitive alleles of RNA14 and RNA15 . The Pcf11-2 thermosensitive strain shows a shortening of the poly(A) tails and a strong decrease in the steady-state level of actin transcripts after a shift to the nonpermissive temperature as do the thermosensitive alleles of RNA14 and RNA15 . Extracts from the pcf11-1 and pcf11-2 thermosensitive strains and the wild-type strain, when Pcf11 is neutralized by specific antibodies, are deficient in cleavage and polyadenylation . Moreover, fractions obtained by anion-exchange chromatography of extracts from the wild-type strain contain both Pcf11 and Rna15 in the same fractions, as shown by immunoblotting with a Pcf11-specific antibody.

Mol Cell Biol, 1997 Mar, 17(3), 1057 - 64
Effect of association with adenylyl cyclase-associated protein on the interaction of yeast adenylyl cyclase with Ras protein; Shima F et al.; Posttranslational modification of Ras protein has been shown to be critical for interaction with its effector molecules, including Saccharomyces cerevisiae adenylyl cyclase . However, the mechanism of its action was unknown . In this study, we used a reconstituted system with purified adenylyl cyclase and Ras proteins carrying various degrees of the modification to show that the posttranslational modification, especially the farnesylation step, is responsible for 5- to 10-fold increase in Ras-dependent activation of adenylyl cyclase activity even though it has no significant effect on their binding affinity . The stimulatory effect of farnesylation is found to depend on the association of adenylyl cyclase with 70-kDa adenylyl cyclase-associated protein (CAP), which was known to be required for proper in vivo response of adenylyl cyclase to Ras protein, by comparing the levels of Ras-dependent activation of purified adenylyl cyclase with and without bound CAP . The region of CAP required for this effect is mapped to its N-terminal segment of 168 amino acid residues, which coincides with the region required for the in vivo effect . Furthermore, the stimulatory effect is successfully reconstituted by in vitro association of CAP with the purified adenylyl cyclase molecule lacking the bound CAP . These results indicate that the association of adenylyl cyclase with CAP is responsible for the stimulatory effect of posttranslational modification of Ras on its activity and that this may be the mechanism underlying its requirement for the proper in vivo cyclic AMP response.

J Biotechnol, 1997 Feb 28, 53(1), 67 - 74
Purification and characterization of an invertase from Candida utilis: comparison with natural and recombinant yeast invertases; Chavez FP et al.; A periplasmic invertase from the yeast Candida utilis was purified to homogeneity from cells fully derepressed for invertase synthesis . The enzyme was purified by successive Sephacryl S-300, and affinity chromatography and shown to be a dimeric glycoprotein composed of two identical monomer subunits with an apparent molecular mass of 150 kDa . After EndoH treatment, the deglycosylated protein showed an apparent molecular weight of 60 kDa . The apparent K(m) values for sucrose and raffinose were 11 and 150 mM, respectively, similar to those reported in Saccharomyces cerevisiae . The range of optimum temperature was 60-75 degrees C . The optimum pH was 5.5 and the enzyme was stable over pH range 3-6.

J Mol Biol, 1997 Feb 28, 266(3), 559 - 75
Genetic interactions among the transmembrane segments of the G protein coupled receptor encoded by the yeast STE2 gene; Sommers CM et al.; G protein coupled receptors (GPCRs) are integral membrane proteins that mediate cellular responses to a wide variety of extracellular signals . However, the structural basis for activation of this class of receptors by ligand binding is not well understood . We report here the use of a systematic genetic protocol for identifying interactions among the seven transmembrane helices of the GPCR responsible for cellular responses to the alpha-mating pheromone of the yeast Saccharomyces cerevisiae . Random mutations were introduced into the region of the STE2 gene encoding the third transmembrane segment of the alpha-factor receptor, followed by screening for loss of signaling . The limited spectrum of non-conservative mutations recovered, including removal of the only negatively charged side-chain in the transmembrane region, indicates that most substitutions in the third transmembrane segment do not affect receptor function . Three second-site intragenic suppressors of these initial mutations were isolated following mutagenesis of the remaining six transmembrane segments . One of these suppressors, Y266C in the sixth transmembrane segment, is allele specific and shows non-additivity of phenotypes indicative of a physical interaction between the third and sixth transmembrane regions of the receptor . A second suppressor, M218T in the fifth transmembrane segment, exhibits only partial allele specificity . A third suppressor, R58G, in the first transmembrane segment, suppresses a variety of starting alleles and appears to cause global stabilization of the receptor . Analysis of these suppressors and additional alleles can provide a database for modeling GPCR structure.

Biochim Biophys Acta, 1997 Feb 28, 1350(3), 345 - 58
Site-directed mutants of post-translationally modified sites of yeast eEF1A using a shuttle vector containing a chromogenic switch; Cavallius J et al.; Eukaryotic elongation factor 1A (eEF1A, formerly eEF-1 alpha) carries aminoacyl-tRNAs into the A-site of the ribosome in a GTP-dependent manner . In order to probe the structure/function relationships of eEF1A, we have generated site-directed mutants using a modification of a highly versatile yeast shuttle vector, which consists of the insertion of a 66 base long synthetic DNA fragment in the vector's polylinker . Via oligonucleotide-directed mutagenesis, the modification permits the identification of mutant clones based on a chromogenic screen of beta-galactosidase activity . Mutagenesis reactions are performed with two or more oligonucleotides, one introducing the chromogenic shift, and the other(s) introducing the mutation(s) of interest in eEF1A . Several rounds of chromogenic shifts and additional mutations can be performed in succession on the same vector . To address the possible function of the methylated lysines in yeast eEF1A, we have changed the post-translationally modified lysines (residue 30, 79, 316 and 390) to arginines using the above methodology . Yeast with eEF1A mutants that substitute arginine in all four sites do not show any phenotypic change . There is also an apparent equivalency of wild-type and mutant yeast eEF1A in in vitro assays . It is concluded that the post-translational modifications of eEF1A are not of major importance for eEF1A's role in translation.

J Biol Chem, 1997 Feb 28, 272(9), 5995 - 6003
Characterization of two age-induced intracisternal A-particle-related transcripts in the mouse liver . Transcriptional read-through into an open reading frame with similarities to the yeast ccr4 transcription factor; Puech A et al.; Intracisternal A-particle (IAP) sequences are endogenous retrovirus-like elements present at 1,000 copies in the mouse genome . We had previously identified IAP-related transcripts of unusual size (6 and 10 kilobases (kb)), which are observed exclusively in the liver of the aging mouse . In this report, using cDNA libraries that we have constructed from the liver mRNAs of an aged DBA/2 mouse, we have cloned and entirely sequenced the corresponding cDNAs . Both are initiated within the 5' long terminal repeat of a type IDelta1 IAP sequence, and correspond to a read-through into a unique flanking cellular sequence containing a 966-nucleotide open reading frame, located 3' to the IAP sequence . The 6-kb IAP-related transcript corresponds to a post-transcriptional modification of the 10-kb mRNA, and is generated by a splicing event with the donor site in the IAP sequence, and the acceptor site 5' to the open reading frame . This open reading frame is located on chromosome 3, is evolutionarily conserved, and discloses significant similarity to the yeast CCR4 transcription factor at the amino acid level . The specific expression of these age-induced transcripts, which account for more than 50% of the IAP-related transcripts in the liver of old mice, is therefore entirely consistent with the induction of a single genomic locus, thus strengthening the importance of position effects for the expression of transposable elements . Characterization of this locus should now allow studies on its chromatin and methylation status, and on the "molecular factors of senescence" possibly involved in its induction.

J Biol Chem, 1997 Feb 28, 272(9), 5571 - 8
Identification of native complexes containing the yeast coactivator/repressor proteins NGG1/ADA3 and ADA2; Saleh A et al.; NGG1p/ADA3p and ADA2p are dual function regulators that stimulate or inhibit a set of yeast transcriptional activator proteins . In vitro, NGG1p and ADA2p associate in a complex that also contains GCN5p (Horiuchi, J., Silverman, N., Marcus, G . A., and Guarente, L . (1995) Mol . Cell . Biol . 15, 1203-1209) . We have found that NGG1p and ADA2p are coimmunoprecipitated from yeast whole cell extracts . In fact, <2% of cellular ADA2p was not associated with NGG1p . Also in agreement with their association in vivo, the stability of ADA2p and NGG1p depended on the presence of each other . In addition, three NGG1p- and ADA2p-containing peak fractions were resolved by Q-Sepharose Fast Flow ion-exchange chromatography of whole cell extract . The presence of another high molecular mass complex was supported by the separation of one of the NGG1p- and ADA2p-containing peak fractions by gel-filtration chromatography . Together, the combination of ion-exchange and gel-filtration chromatography suggests a total of four complexes, two with sizes of >2 MDa and single complexes of approximately 900 and 200 kDa . At least one of these complexes was found to associate with the TATA-binding protein (TBP) since TBP was present in immunoprecipitates with NGG1p . The association of TBP with the ADA proteins required amino acids 274-307 of NGG1p, a region of NGG1p required for activity . This supports a role for NGG1p in the interaction with TBP and suggests that the interaction with TBP is functionally relevant.

Science, 1997 Feb 28, 275(5304), 1314 - 7
Combinatorial control required for the specificity of yeast MAPK signaling; Madhani HD et al.; In yeast, an overlapping set of mitogen-activated protein kinase (MAPK) signaling components controls mating, haploid invasion, and pseudohyphal development . Paradoxically, a single downstream transcription factor, Ste12, is necessary for the execution of these distinct programs . Developmental specificity was found to require a transcription factor of the TEA/ATTS family, Tec1, which cooperates with Ste12 during filamentous and invasive growth . Purified derivatives of Ste12 and Tec1 bind cooperatively to enhancer elements called filamentation and invasion response elements (FREs), which program transcription that is specifically responsive to the MAPK signaling components required for filamentous growth . An FRE in the TEC1 promoter functions in a positive feedback loop required for pseudohyphal development.

Mol Gen Genet, 1997 Feb 27, 253(6), 766 - 9
The DNA target sequence influences the dependence of the yeast transcriptional activator Gcn4 on co-factors; Tavernarakis N et al.; The yeast transcriptional activator Gcn4 requires the Ada2/Gcn5/Ada3 co-activator complex to exert part of its activation potential . Here we show that the sequence of the DNA target modulates the function of Gcn4 by modifying this requirement . Promoter configurations were generated that rendered Gcn4-induced transcription either completely dependent or completely independent of the Ada2/Gcn5/Ada3 complex . The topological constraints imposed by these configurations suggest that Gcn4 makes multiple contacts with the basic transcription machinery that are subject to modification by the incident DNA target . We propose that these modifications further determine the direction on the chromosome in which an otherwise symmetric, dimeric transcription factor will activate.

FEBS Lett, 1997 Feb 24, 403(3), 249 - 53
Novel tetramer assembly of pyruvate decarboxylase from brewer's yeast observed in a new crystal form; Lu G et al.; A new crystal form of thiamine diphosphate dependent pyruvate decarboxylase from Saccharomyces cerevisiae has been obtained in the presence of the activator pyruvamide . The crystallographic structure analysis reveals differences in the domain packing in the enzyme subunit and a novel assembly of the subunits in the tetramer, when compared to the structure of native PDC . The orientation of the beta domains in the subunit differs by a 6.3 degrees and 8.3 degrees rotation, respectively, whereas the subunit-subunit interface in the dimer, formed by the alpha and gamma domains, is essentially maintained . In the tetramer, one of the dimers rotates relative to the second dimer by approximately 30 degrees creating a new dimer-dimer interface.

J Cell Biol, 1997 Feb 24, 136(4), 789 - 802
COPI-independent anterograde transport: cargo-selective ER to Golgi protein transport in yeast COPI mutants; Gaynor EC et al.; The coatomer (COPI) complex mediates Golgi to ER recycling of membrane proteins containing a dilysine retrieval motif . However, COPI was initially characterized as an anterograde-acting coat complex . To investigate the direct and primary role(s) of COPI in ER/Golgi transport and in the secretory pathway in general, we used PCR-based mutagenesis to generate new temperature-conditional mutant alleles of one COPI gene in Saccharomyces cerevisiae, SEC21 (gamma-COP) . Unexpectedly, all of the new sec21 ts mutants exhibited striking, cargo-selective ER to Golgi transport defects . In these mutants, several proteins (i.e., CPY and alpha-factor) were completely blocked in the ER at nonpermissive temperature; however, other proteins (i.e., invertase and HSP150) in these and other COPI mutants were secreted normally . Nearly identical cargo-specific ER to Golgi transport defects were also induced by Brefeldin A . In contrast, all proteins tested required COPII (ER to Golgi coat complex), Sec18p (NSF), and Sec22p (v-SNARE) for ER to Golgi transport . Together, these data suggest that COPI plays a critical but indirect role in anterograde transport, perhaps by directing retrieval of transport factors required for packaging of certain cargo into ER to Golgi COPII vesicles . Interestingly, CPY-invertase hybrid proteins, like invertase but unlike CPY, escaped the sec21 ts mutant ER block, suggesting that packaging into COPII vesicles may be mediated by cis-acting sorting determinants in the cargo proteins themselves . These hybrid proteins were efficiently targeted to the vacuole, indicating that COPI is also not directly required for regulated Golgi to vacuole transport . Additionally, the sec21 mutants exhibited early Golgi-specific glycosylation defects and structural aberrations in early but not late Golgi compartments at nonpermissive temperature . Together, these studies demonstrate that although COPI plays an important and most likely direct role both in Golgi-ER retrieval and in maintenance/function of the cis-Golgi, COPI does not appear to be directly required for anterograde transport through the secretory pathway.

J Cell Biol, 1997 Feb 24, 136(4), 747 - 59
Dynamics of nuclear pore distribution in nucleoporin mutant yeast cells; Belgareh N et al.; To follow the dynamics of nuclear pore distribution in living yeast cells, we have generated fusion proteins between the green fluorescent protein (GFP) and the yeast nucleoporins Nup49p and Nup133p . In nup133- dividing cells that display a constitutive nuclear pore clustering, in vivo analysis of GFP-Nup49p localization revealed changes in the distribution of nuclear pore complex (NPC) clusters . Furthermore, upon induction of Nup133p expression in a GAL-nup133 strain, a progressive fragmentation of the NPC aggregates was observed that in turn led to a wild-type nuclear pore distribution . To try to uncouple Nup133p-induced NPC redistribution from successive nuclear divisions and nuclear pore biogenesis, we devised an assay based on the formation of heterokaryons between nup133- mutants and cells either expressing or overexpressing Nup133p . Under these conditions, the use of GFP-Nup133p and GFP-Nup49p fusion proteins revealed that Nup133p can be rapidly targeted to the clustered nuclear pores, where its amino-terminal domain is required to promote the redistribution of preexisting NPCs.

Cell, 1997 Feb 21, 88(4), 531 - 42
The yeast phosphatidylinositol kinase homolog TOR2 activates RHO1 and RHO2 via the exchange factor ROM2; Schmidt A et al.; The Saccharomyces cerevisiae phosphatidylinositol kinase homolog TOR2 is required for organization of the actin cytoskeleton . Overexpression of RHO1 or RHO2, encoding Rho-like GTPases, or ROM2, encoding a GDP/GTP exchange factor for RHO1 and RHO2, suppresses a tor2 mutation . Deletion of SAC7, a gene originally identified as a suppressor of an actin mutation, also suppresses a tor2 mutation . SAC7 is a novel GTPase-activating protein for RHO1 . ROM2 exchange activity is reduced in a tor2 mutant, and overexpression of ROM2 lacking its PH domain can no longer suppress a tor2 mutation . Thus, TOR2 signals to the actin cytoskeleton through a GTPase switch composed of RHO1, RHO2, ROM2, and SAC7 . TOR2 activates this switch via ROM2, possibly via the ROM2 PH domain.

J Biol Chem, 1997 Feb 21, 272(8), 5320 - 5
Mft52, an acid-bristle protein in the cytosol that delivers precursor proteins to yeast mitochondria; Cartwright P et al.; We have identified a novel protein, Mft52, in the cytosol of yeast cells . Mft52 has a two-domain structure that includes a receptor-like carboxyl-terminal "acid-bristle" domain, which binds basic, amphipathic mitochondrial targeting sequences . Native Mft52, purified from the cytosol of yeast cells, is found as a large particle eluting in the void volume of a Superose 6 gel filtration column . Fusion proteins, consisting of mitochondrial targeting sequences fused to nonmitochondrial passenger proteins, are targeted to mitochondria in wild-type yeast cells, but defects in the gene encoding Mft52 drastically reduce the delivery of these proteins to the mitochondria . We propose that Mft52 is a subunit of a particle that is part of a system of targeting factors and molecular chaperones mediating the earliest stages of protein targeting to the mitochondria.

Carbohydr Res, 1997 Feb 20, 298(1-2), 117 - 21
The correlation between adhesion of schizophyllan to yeast glucan and its effect on regeneration of yeast protoplast; Hisamatsu M et al.; Schizophyllan, a water-soluble (1-->3)-beta-D-glucan with a triple-helical conformation, adheres to yeast glucan and curdlan gel . As the molecular weight of schizophyllan decreases, both its adhesion to the water-insoluble glucans and its ability to promote the regeneration of yeast protoplasts are reduced . Therefore, we hypothesize that schizophyllan can surround yeast protoplasts by adhering to a fragment of yeast glucan remaining or/and resynthesized on the cell surface and that this encapsulation allows regeneration of the protoplast cells to occur at very high frequency.

Oncogene, 1997 Feb 20, 14(7), 831 - 6
Identification of a dominant-negative mutation in the yeast CDC25 guanine nucleotide exchange factor for Ras; Park W et al.; In previous studies we changed five conserved amino acid residues in the catalytic domain of the yeast Ras-specific guanine nucleotide exchange factor CDC25GEF (Park et al., 1994) . One of the substitutions (R1489E) resulted in a molecule which could bind Ras but was catalytically inactive . These observations suggested that CDC25R1489E might be a dominant-negative mutant . Here we report further experiments which confirm the dominant-negative phenotype of CDC25R1489E . Two lines of evidence indicate that the CDC25R1489E mutant exhibits Ras-specific binding in vivo . First, expression of CDC25R1489E in a wild-type yeast strain caused a partial inhibition of growth which was reversed by overexpression of the wild-type yeast RAS2 protein . Second, expression of CDC25R1489E in a yeast strain containing a temperature-sensitive, dominant-negative RAS2 mutation (RAS2val19ala22) suppressed the temperature-sensitive phenotype . The latter findings suggest that the CDC25R1489E protein bound the mutant RAS2 protein thereby releasing the wild-type CDC25 protein for activation of the wild-type RAS1 protein . Further, using a protein-protein binding assay and guanine nucleotide exchange assay (release of {3H}-GDP) in vitro, we demonstrate that the CDC25R1489E protein can bind wild-type Ras protein but is unable to catalyze GDP-GTP exchange . Thus, the results of genetic and biochemical experiments demonstrate that CDC25R1489E encodes a dominant-negative GEF which blocks the Ras signaling pathway by binding wild-type Ras in a catalytically inactive complex.

EMBO J, 1997 Feb 17, 16(4), 807 - 16
The human homologue of yeast CRM1 is in a dynamic subcomplex with CAN/Nup214 and a novel nuclear pore component Nup88; Fornerod M et al.; The oncogenic nucleoporin CAN/Nup214 is essential in vertebrate cells . Its depletion results in defective nuclear protein import, inhibition of messenger RNA export and cell cycle arrest . We recently found that CAN associates with proteins of 88 and 112 kDa, which we have now cloned and characterized . The 88 kDa protein is a novel nuclear pore complex (NPC) component, which we have named Nup88 . Depletion of CAN from the NPC results in concomitant loss of Nup88, indicating that the localization of Nup88 to the NPC is dependent on CAN binding . The 112 kDa protein is the human homologue of yeast CRM1, a protein known to be required for maintenance of correct chromosome structure . This human CRM1 (hCRM1) localized to the NPC as well as to the nucleoplasm . Nuclear overexpression of the FG-repeat region of CAN, containing its hCRM1-interaction domain, resulted in depletion of hCRM1 from the NPC . In CAN-/- mouse embryos lacking CAN, hCRM1 remained in the nuclear envelope, suggesting that this protein can also bind to other repeat-containing nucleoporins . Lastly, hCRM1 shares a domain of significant homology with importin-beta, a cytoplasmic transport factor that interacts with nucleoporin repeat regions . We propose that hCRM1 is a soluble nuclear transport factor that interacts with the NPC.

Biochim Biophys Acta, 1997 Feb 15, 1318(3), 317 - 21
The ATP-induced K(+)-transport pathway of yeast mitochondria may function as an uncoupling pathway; Manon S et al.; The effect of the presence of K+ during oxidative phosphorylation measured on isolated yeast mitochondria was dependent on phosphate concentration . At 0.5 mM phosphate, K+ did promote an uncoupling of oxidative phosphorylation, which was prevented by decavanadate, a potent inhibitor of the ATP-induced K(+)-transport pathway . AT 5 mM phosphate, no uncoupling effect of K+ could be evidenced . These data suggest that the ATP-induced K(+)-transport pathway may, under certain conditions, function as an uncoupling pathway of oxidative phosphorylation.

Nucleic Acids Res, 1997 Feb 15, 25(4), 843 - 9
Yeast two-hybrid cloning of a novel zinc finger protein that interacts with the multifunctional transcription factor YY1; Kalenik JL et al.; Muscle-restricted transcription of sarcomeric actin genes is negatively controlled by the zinc finger protein YY1, which is down-regulated at the protein level during myogenic differentiation . To identify cellular proteins that might mediate the function/stability of YY1 in muscle cells, we screened an adult human muscle cDNA library using the yeast two-hybrid cloning system . We report the isolation and characterization of a novel protein termed YAF2 (YY1- associated factor 2) that interacts with YY1 . The YAF2 cDNA encodes a 180 amino acid basic protein (pI 10.5) containing a single N-terminal C2-X10-C2 zinc finger . Lysine clusters are present that may function as a nuclear localization signal . Domain mapping analysis shows that the first and second zinc fingers of YY1 are targeted for YAF2 protein interaction . In contrast to the down-regulation of YY1, YAF2 message levels increase during in vitro differentiation of both rat skeletal and cardiac muscle cells . YAF2 appears to have a promyogenic regulatory role, since overexpression of YAF2 in C2 myoblasts stimulates myogenic promoter activity normally restricted by YY1 . Co-transfection of YY1 reverses the stimulatory effect of YAF2 . YAF2 also greatly potentiates proteolytic cleavage of YY1 by the calcium- activated protease m-calpain . The isolation of YAF2 may help in understanding the mechanisms through which inhibitors of myogenic transcription may be antagonized or eliminated by proteolysis during muscle development.

J Mol Biol, 1997 Feb 14, 266(1), 93 - 107
The yeast site-specific recombinase Flp mediates alcoholysis and hydrolysis of the strand cleavage product: mimicking the strand-joining reaction with non-DNA nucleophiles; Knudsen BR et al.; The yeast site-specific recombinase Flp is covalently linked to DNA via a 3'-phosphotyrosyl bond during the strand-breakage step of recombination . We show that this phosphotyrosyl diester bond formed between Flp and DNA can serve as the target for alcoholysis or hydrolysis in an Flp-assisted reaction . Flp does not mediate alcoholysis of the labile phosphodiester bond within the DNA chain under our assay conditions . The body of available evidence supports the notion that the alcoholysis/hydrolysis reaction is mechanistically analogous to the strand-joining step of the recombination pathway . The only difference is that the DNA 5'-hydroxyl group that acts as the nucleophile during recombination is substituted by a non-DNA nucleophile . We find that the alcoholysis reaction occurs only within the normal cleavage complex produced by the "shared active site" assembled at the interface of two Flp monomers . Unlike the strand-joining reaction, alcoholysis does not occur on an activated DNA substrate linked at its 3'-phosphate end to a short tyrosyl peptide (not to the full-length Flp), and bound non-covalently by a Flp monomer . However, even in this substrate that mimics the strand-cleaved state, the joining reaction is competitively inhibited by a polyhydric alcohol such as glycerol.

J Biol Chem, 1997 Feb 14, 272(7), 4412 - 8
The effects of severely decreased hydrophobicity in a subdomain 3/4 loop on the dynamics and stability of yeast G-actin; Kuang B et al.; The hydrophobicity of the subdomain 3/4 hydrophobic loop (262-274) has been implicated to be essential for actin's function . We previously showed (Kuang, B., and Rubenstein, P . A . (1997) J . Biol . Chem . 272, 1237-1247) that a mutant yeast actin (V266G/L267G) with markedly decreased hydrophobicity in this loop conferred severe cold sensitivity to its polymerization . Here we further tested the mutational effect on the conformation and function of G-actin . This GG mutation caused no significant changes in overall secondary structure or in the microenvironment around actin's tryptophan residues, nor did it alter the dissociation constant of G-actin for ATP . However, it lowers the intrinsic ATPase activity and the melting temperature for Mg-GG actin from 51 to 33 degrees C and transforms the conformation of subdomain 2 and the central cleft of G-actin into an F-monomer-like structure . The results suggest that the hydrophobic plug may not only play a role in actin filament stabilization but also may be important for controlling the stability of G-actin and for promoting the conformational change of the monomer needed for addition to a growing actin filament.

J Biol Chem, 1997 Feb 14, 272(7), 3980 - 5
Functional expression of a cDNA to human acyl-coenzyme A:cholesterol acyltransferase in yeast . Species-dependent substrate specificity and inhibitor sensitivity; Yang H et al.; We have identified two yeast genes with similarity to a human cDNA encoding acyl-coenzyme A:cholesterol acyltransferase (ACAT) . Deletion of both yeast genes results in a viable cell with undetectable esterified sterol (Yang, H., Bard, M., Bruner, D . A., Gleeson, A., Deckelbaum, R . J., Aljinovic, G., Pohl, T., Rothstein, R., and Sturley, S . L . (1996) Science 272, 1353-1356) . Here, we expressed the human cDNA in the yeast double mutant, resulting in high level production of ACAT protein, but low in vivo esterification of ergosterol, the predominant yeast sterol . The activity of the human enzyme was increased by incubation of these cells with 25-hydroxy, cholesterol, an established positive regulator of mammalian sterol esterification . In contrast, the yeast enzymes were unaffected by this reagent . In vitro microsomal assays indicated no sterol esterification in extracts from the double mutant . However, significant activity was detected from strains expressing human ACAT when cholesterol was equilibrated with the microsomal membranes . The human enzyme in yeast utilized cholesterol as the preferred sterol and was sensitive to competitive (S58035) and non-competitive (DuP 128) ACAT inhibitors . The yeast esterifying enzymes exhibited a diminished sterol substrate preference and were sensitive only to S58035 . Human ACAT had a broad acyl-CoA substrate specificity, the other substrate for this reaction . By contrast, the yeast enzymes had a marked preference for specific acyl-CoAs, particularly unsaturated C18 forms . These results confirm the yeast genes as functional homologs of the human gene and demonstrate that the enzymes confer substrate specificity to the esterification reaction in both organisms.

Science, 1997 Feb 14, 275(5302), 986 - 90
A protein-counting mechanism for telomere length regulation in yeast; Marcand S et al.; In the yeast Saccharomyces cerevisiae, telomere elongation is negatively regulated by the telomere repeat-binding protein Rap1p, such that a narrow length distribution of telomere repeat tracts is observed . This length regulation was shown to function independently of the orientation of the telomere repeats . The number of repeats at an individual telomere was reduced when hybrid proteins containing the Rap1p carboxyl terminus were targeted there by a heterologous DNA-binding domain . The extent of this telomere tract shortening was proportional to the number of targeted molecules, consistent with a feedback mechanism of telomere length regulation that can discriminate the precise number of Rap1p molecules bound to the chromosome end.

FEBS Lett, 1997 Feb 10, 403(1), 95 - 9
Yeast RAS2 mutations modulating the ras-guanine exchange factor interaction; Hermann-Le Denmat S et al.; We have used a two-hybrid approach to test various forms of Saccharomyces cerevisiae Ras2p for their ability to interact with the human guanine nucleotide exchange factor HGRF55 . We have previously shown that a strong two-hybrid interaction is found between the HGRF55p and the dominant negative Ras2p(G22A) form of ras {Camus et al . (1995) Oncogene 11, 951-959} . We show here that the substitution N123I which weakens the guanine nucleotide binding also promotes ras-GEF interaction . We demonstrate that the R80D substitution alone completely abolishes the interaction of Ras2p(G22A) with GEF, whereas substitutions at positions 81, 82 and 73 have only small effects . Since residue 73 is involved in the response of ras to GEF, we propose that it plays a role in the conformational change induced by the GEF rather than in its binding . Those results emphasize the role of the alpha2 helix of the switch II region in the recognition of the GEF family.

J Cell Biol, 1997 Feb 10, 136(3), 649 - 58
Bee1, a yeast protein with homology to Wiscott-Aldrich syndrome protein, is critical for the assembly of cortical actin cytoskeleton; Li R; Yeast protein, Bee1, exhibits sequence homology to Wiskott-Aldrich syndrome protein (WASP), a human protein that may link signaling pathways to the actin cytoskeleton . Mutations in WASP are the primary cause of Wiskott-Aldrich syndrome, characterized by immuno-deficiencies and defects in blood cell morphogenesis . This report describes the characterization of Bee1 protein function in budding yeast . Disruption of BEE1 causes a striking change in the organization of actin filaments, resulting in defects in budding and cytokinesis . Rather than assemble into cortically associated patches, actin filaments in the buds of delta bee1 cells form aberrant bundles that do not contain most of the cortical cytoskeletal components . It is significant that delta bee1 is the only mutation reported so far that abolishes cortical actin patches in the bud . Bee1 protein is localized to actin patches and interacts with Sla1p, a Src homology 3 domain-containing protein previously implicated in actin assembly and function . Thus, Bee1 protein may be a crucial component of a cytoskeletal complex that controls the assembly and organization of actin filaments at the cell cortex.

J Biol Chem, 1997 Feb 7, 272(6), 3823 - 32
Characterization of putative human homologues of the yeast chromosome transmission fidelity gene, CHL1; Amann J et al.; Helicases are components of numerous protein complexes, including those regulating transcription, translation, DNA replication and repair, splicing, and mitotic chromosome transmission . Helicases unwind double-stranded DNA and RNA homo- and hetero-duplexes . The yeast CHL1 helicase has been linked to maintenance of the high fidelity of chromosome transmission during mitosis . Mutations in this gene result in a 200-fold increase in the rate of aberrant chromosome segregation with a concomitant delay in the cell cycle at G2-M, suggesting that CHL1 is required for the maintenance of proper chromosome transmission . Two highly related human cDNA clones encoding proteins which are homologous to the yeast CHL1 gene product have been isolated . Here we show that these two distinct human CHL1-related mRNAs and proteins (hCHLR1 and hCHLR2) are expressed only in proliferating human cell lines . Quiescent normal human fibroblasts stimulated to re-enter the cell cycle by addition of serum begin to express the CHL1-related proteins as the cells enter S phase, concomitant with the expression of proliferating cell nuclear antigen . Furthermore, expression of the CHL1-related mRNAs is lost when human K562 cells cease to proliferate and terminally differentiate in response to phorbol ester treatments . Human hCHLR expression is not extinguished during hemin-induced differentiation of the same cell line, which produces erythrocyte-like cells that continue to proliferate . These experiments are consistent with the requirement of this putative helicase during either S or G2-M phase but not G1 . In vitro transcribed and translated hCHLR1 protein binds to both single- and double-stranded DNA, supporting the possibility that these proteins are DNA helicases . Finally, affinity-purified hCHLR1 antisera was used to demonstrate the localization of the hCHLR proteins to the nucleolus by indirect immunofluorescence as well as by cell fractionation.

J Biol Chem, 1997 Feb 7, 272(6), 3699 - 706
Oleosin of plant seed oil bodies is correctly targeted to the lipid bodies in transformed yeast; Ting JT et al.; Yeast (Saccharomyces cerevisiae) has been used extensively as a heterologous eukaryotic system to study the intracellular targeting of proteins to different organelles . The lipid bodies in yeast have not been previously subjected to such studies . These organelles are functionally equivalent to the subcellular storage oil bodies in plant seeds . A plant oil body has a matrix of oils (triacylglycerols) surrounded by a layer of phospholipids embedded with abundant structural proteins called oleosins . We tested whether plant oleosin could be correctly targeted to the lipid bodies in transformed yeast . The coding region of a maize (Zea mays L.) oleosin gene was incorporated into yeast high copy and low copy number plasmids in which its expression was under the control of GAL1 promoter . Yeast strains transformed with these plasmids produced oleosin when grown in a medium containing galactose but not glucose . The oleosin produced in yeast had a molecular mass slightly higher than that of the native protein in maize . Oleosin accumulated concomitantly with the storage lipids during growth of the transformed yeast, and it was not secreted . Subcellular fractionation of the cell extracts obtained by two different cell breakage procedures revealed that the oleosin was largely restricted to the lipid bodies . Oleosin apparently did not affect the lipid contents and composition of the transformed yeast lipid bodies but replaced some of the native proteins associated with the organelles . Immunocytochemistry of the transformed yeast cells showed that the oleosin was present mostly on the periphery of the lipid bodies . Oleosin isolated from maize or transformed yeast strain, alone or in the presence of phospholipids or SDS, did not bind to the yeast lipid bodies in vitro . We conclude that plant oleosin is correctly targeted to the lipid bodies in transformed yeast and that yeast may be used as a heterologous system to dissect the intracellular targeting signals in the oleosin.

FEBS Lett, 1997 Feb 3, 402(2-3), 136 - 40
Characterization of dominant lethal mutations in the yeast plasma membrane H+-ATPase gene; Portillo F; Site-directed mutants of yeast ATPase were studied after introduction of mutant alleles into a yeast strain where these alleles were constitutively expressed and the expression of the wild-type chromosomal ATPase gene was turned off . One objection to this constitutive expression system was made apparent recently, as dominant lethal mutations are lost by gene conversion with the wild-type allele during the process . Here, the phenotypes of the mutant alleles, which were studied in a constitutive expression system, are re-evaluated under conditions in which these site-directed mutants are conditionally expressed . We show that 12 of 25 site-directed mutations previously described are actually dominant lethal alleles . In addition, we show that dominant mutant proteins interfere with transport of wild-type ATPase to the plasma membrane.

Mol Biol Cell, 1997 Feb, 8(2), 263 - 77
An essential role of the yeast pheromone-induced Ca2+ signal is to activate calcineurin; Withee JL et al.; Previous studies showed that, in wild-type (MATa) cells, alpha-factor causes an essential rise in cytosolic Ca2+ . We show that calcineurin, the Ca2+/calmodulin-dependent protein phosphatase, is one target of this Ca2+ signal . Calcineurin mutants lose viability when incubated with mating pheromone, and overproduction of constitutively active (Ca(2+)-independent) calcineurin improves the viability of wild-type cells exposed to pheromone in Ca(2+)-deficient medium . Thus, one essential consequence of the pheromone-induced rise in cytosolic Ca2+ is activation of calcineurin . Although calcineurin inhibits intracellular Ca2+ sequestration in yeast cells, neither increased extracellular Ca2+ nor defects in vacuolar Ca2+ transport bypasses the requirement for calcineurin during the pheromone response . These observations suggest that the essential function of calcineurin in the pheromone response may be distinct from its modulation of intracellular Ca2+ levels . Mutants that do not undergo pheromone-induced cell cycle arrest (fus3, far1) show decreased dependence on calcineurin during treatment with pheromone . Thus, calcineurin is essential in yeast cells during prolonged exposure to pheromone and especially under conditions of pheromone-induced growth arrest . Ultrastructural examination of pheromone-treated cells indicates that vacuolar morphology is abnormal in calcineurin-deficient cells, suggesting that calcineurin may be required for maintenance of proper vacuolar structure or function during the pheromone response.

Genes Cells, 1997 Feb, 2(2), 155 - 66
Microtubule-associated coiled-coil protein Ssm4 is involved in the meiotic development in fission yeast; Yamashita A et al.; BACKGROUND: In fission yeast, an RNA species named meiRNA is specifically required for the promotion of the first meiotic division . To dissect the function of this RNA and its partner RNA-binding protein Mei2, we screened for high-copy-number suppressors of the arrest prior to the first meiotic division caused by loss of meiRNA . RESULTS: Analysis of one of the suppressors, named ssm4, suggested that it encodes a coiled-coil protein carrying a microtubule-binding motif at its N-terminus . Expression of ssm4 was restricted to cells undergoing meiosis . Disruption of ssm4 affected neither vegetative growth nor conjugation, but resulted in frequent generation of asci carrying less than four spores . Tagged Ssm4 could colocalize with microtubules in mitotic cells, and was seen to localize at spindles during both the first and the second meiotic division . The microtubule-binding motif was essential for the association of Ssm4 with microtubules and for its function during meiosis, but not for the suppression of loss of meiRNA . Ssm4 appeared to possess a potential to migrate to the nucleus . CONCLUSIONS: Ssm4 is a microtubule-colocalizing protein that plays a role specifically in meiosis . Ssm4 appears to modify the structure or the function of nuclear microtubules in order to promote the meiotic nuclear division.

Biochemistry (Mosc), 1997 Feb, 62(2), 123 - 8
Secretory heat-shock protein of the thermotolerant yeast Hansenula polymorpha . Identification and comparative characteristics; Tsiomenko AB et al.; Thirteen investigated strains of ascomycetous yeasts able to produce secretory heat-shock proteins (sHSPs) do not response equally to a high temperature by induction of the synthesis and secretion of these proteins . In this respect the above yeasts can be divided into three groups having a positive (I), a negative (II), and an indefinite reaction (III) to the heat shock . The thermotolerant yeast Hansenula polymorpha belongs to the first group . In this yeast heat shock induces the synthesis and secretion of sHSP gp280 . This new representative differs from known sHSPs in molecular mass and subunit composition . In other respects (glycosylation, mainly extracellular localization, and the character of export into the culture medium) it displays similar properties.

Genomics, 1997 Feb 1, 39(3), 385 - 92
Construction and characterization of a 10-genome equivalent yeast artificial chromosome library for the laboratory rat, Rattus norvegicus; Cai L et al.; Increasing attention has been focused in recent years on the rat as a model organism for genetic studies, in particular for the investigation of complex traits, but progress has been limited by the lack of availability of large-insert genomic libraries . Here, we report the construction and characterization of an arrayed yeast artificial chromosome (YAC) library for the rat genome containing approximately 40,000 clones in the AB1380 host using the pCGS966 vector . An average size of 736 kb was estimated from 166 randomly chosen clones; thus the library provides 10-fold coverage of the genome, with a 99.99% probability of containing a unique sequence . Eight of 39 YACs analyzed by fluorescence in situ hybridization were found to be chimeric, indicating a proportion of about 20-30% of chimeric clones . The library was spotted on high-density filters to allow the identification of YAC clones by hybridization and was pooled using a 3-dimensional scheme for screening by PCR . Among 48 probes used to screen the library, an average of 9.3 positive clones were found, consistent with the calculated 10-fold genomic coverage of the library . This YAC library represents the first large-insert genomic library for the rat . It will be made available to the research community at large as an important new resource for complex genome analysis in this species.

Nahrung, 1997 Feb, 41(1), 29 - 33
Inactivation of yeast and filamentous fungi by the lactoperoxidase-hydrogen peroxide-thiocyanate-system; Popper L et al.; The antifungal activity of the lactoperoxidase (LPO) system with glucose oxidase (GOD) as source of hydrogen peroxide was determined in salt solution and in apple juice . The test organisms Rhodutorula rubra and Saccharomyces cerevisiae were cultivated aerobically in apple juice, Mucor rouxii was grown on wort agar adjusted to pH 4.5 . Aspergillus niger and Byssochlamys fulva were kept on malt extract agar . Spores of the filamentous fungi were harvested by suspension in salt solution supplemented with Tween 80 and checked microscopically . The antifungal activity of the combined enzyme system was tested with initial counts of approx . 10(5) cfu.ml-1 (yeast cells or spores) suspended in salt solution supplemented with 25 mg.l-1 thiocyanate and 20 g.l-1 glucose or in apple juice supplemented with the same amount of thiocyanate . The tests were performed with 25 ml of the medium in 100 ml Erlenmeyer flasks shaken at 28 degrees C under aerobic conditions . Inactivation was achieved for all test organisms in both media . The yeast strains were found to be least stable while B . fulva was most resistant . A combination of 5 U.ml-1 LPO with 0.5 to 1 U.ml-1 GOD was sufficient for complete inactivation of this mold in salt solution within 2 h . The enzyme system also showed antifungal activity in apple juice at acid pH (3.2), although its effectiveness was reduced . In this medium, B . fulva was inactivated by 20 U.ml-1 LPD and 1 U.ml-1 GOD within 4 h . R . rubra and S . cerevisfiae were unable to survive in apple juice at 5 U.ml-1 LPO combined with 1 U.ml-1 GOD . For inhibition by GOD alone, higher amounts of this enzyme were needed and even then only M . rouxii and R . rubra have been affected within the concentration range tested (maximum 3 U.ml-1).

J Bioenerg Biomembr, 1997 Feb, 29(1), 45 - 54
Assembly of deletion mutants of the Rieske iron-sulfur protein into the cytochrome bc1 complex of yeast mitochondria; Ramabadran RS et al.; The assembly of two deletion mutants of the Rieske iron-sulfur protein into the cytochrome bc1 complex was investigated after import in vitro into mitochondria isolated from a strain of yeast, JPJ1, from which the iron-sulfur protein gene (RIP) had been deleted . The assembly process was investigated by immunoprecipitation of the labeled iron-sulfur protein or the two deletion mutants from detergent-solubilized mitochondria with specific antisera against either the iron-sulfur protein or the bc1 complex (complex III) {Fu and Beattie (1991) . J . Biol . Chem . 266, 16212-16218} . The deletion mutants lacking amino acid residues 55-66 or residues 161-180 were imported into mitochondria in vitro and processed to the mature form via an intermediate form . After import in vitro, the protein lacking residues 161-180 was not assembled into the complex, suggesting that the region of the iron-sulfur protein containing these residues may be involved in the assembly of the protein into the bc1 complex; however, the protein lacking residues 55-66 was assembled in vitro into the bc1 complex as effectively as the wild type iron-sulfur protein . Moreover, this mutant protein was present in the mitochondrial membrane fraction obtained from JPJ1 cells transformed with a single-copy plasmid containing the gene for this protein lacking residues 55-66 . This deletion mutant protein was also assembled into the bc1 complex in vivo, suggesting that the hydrophobic stretch of amino acids, residues 55-66, is not required for assembly of the iron-sulfur protein into the bc1 complex; however, this association did not lead to enzymatic activity of the bc1 complex, as the Rieske FeS cluster was not epr detectable in these mitochondria.

Biosci Biotechnol Biochem, 1997 Feb, 61(2), 396 - 9
Effects of nucleotides on cyanide-resistant respiratory activity in mitochondria isolated from antimycin A-treated yeast Hansenula anomala; Sakajo S et al.; Mitochondria were isolated from Hansenula anomala induced for cyanide-resistant respiration by antimycin A-treatment . Cyanide-resistant respiratory activity in isolated mitochondria was stimulated by AMP, ADP, dAMP, and GMP, but not by ATP, adenosine, cAMP, 2'(3')-AMP, CMP, and UMP . Effects of nucleotides were also observed on cyanide-resistant and salicylhydroxamate-sensitive decylubiquinol oxidase activity . Carbonylcyanide m-chlorophenylhydrazone, oligomycin, and carboxyatractyloside did not affect activation of decylubiquinol oxidase activity by AMP . It is suggested that purine nucleoside 5'-monophosphate or diphosphate stimulates alternative oxidase activity from the outer surface of the mitochondrial inner membrane with a mechanism different from respiratory control . Alternative oxidase activity might be controlled by adenine nucleotides posttranscriptionally in fungi.

Biosci Biotechnol Biochem, 1997 Feb, 61(2), 375 - 7
Amino acid sequence and characterization of aldo-keto reductase from bakers' yeast; Nakamura K et al.; One of the enzymes from bakers' yeast that catalyzes the reduction of alpha- and beta-keto esters has been studied . The N-terminal amino acid sequence of the enzyme shows that the enzyme belongs to the aldo-keto reductase superfamily . The substrate specificity of the enzyme is broad and resembles those of other aldo-keto reductases . The enzyme catalyzes the reduction of keto esters, aldehydes, and aldohexoses.

Trends Genet, 1997 Feb, 13(2), 61 - 6
Production of transgenic mice with yeast artificial chromosomes; Peterson KR et al.; Techniques are now available that allow the transfer of intact yeast artificial chromosome (YAC) DNA into transgenic mice . Coupled with the ability to perform mutagenesis on YAC sequences by homologous recombination in yeast, they enable the analysis of large genes or multigenic loci in vivo . This system has been used to study the developmental regulation of the human beta-globin locus.

Indian J Med Res, 1997 Feb, 105, 93 - 7
Central noradrenergic & dopaminergic modulation of brewer's yeast-induced inflammation & nociception in rats; Hore SK et al.; The possibility of central noradrenergic and dopaminergic modulation of Brewer's yeast-induced peripheral inflammation was investigated in rats . Centrally administered noradrenaline (NA), amphetamine, which liberates NA and dopamine in the central nervous system and L-dopa, the precursor of dopamine significantly suppressed paw oedema . Conversely, the beta-adrenoceptor blocker, propranolol, catecholaminergic neuron degenerator, 6-hydroxydopamine (6-OHDA), dopaminergic antagonist, haloperidol and dopamine synthesis inhibitor, alpha-methyl para tyrosine (AMPT) augmented paw oedema . In addition, 6-OHDA and haloperidol produced significant reduction in pain threshold . The results of this study indicate that central NA and dopamine exert inhibitory effects on Brewer's yeast-induced peripheral inflammation.

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