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 Sac