Nucleic Acids Res, 1989 Nov 25, 17(22), 9039 - 50 Vaccinia virus encodes a polypeptide with DNA ligase activity; Kerr SM et al.; Vaccinia virus gene SalF 15R potentially encodes a polypeptide of 63 kD which shares 30% amino acid identity with S . pombe and S . cerevisiae DNA ligases . DNA ligase proteins can be identified by incubation with alpha-(32P)ATP, resulting in the formation of a covalent DNA ligase-AMP adduct, an intermediate in the enzyme reaction . A novel radio-labelled polypeptide of approximately 61 kD appears in extracts from vaccinia virus infected cells after incubation with alpha-(32P)ATP . This protein is present throughout infection and is a DNA ligase as the radioactivity is discharged in the presence of either DNA substrate or pyrophosphate . DNA ligase assays show an increase in enzyme activity in cell extracts after vaccinia virus infection . A rabbit antiserum, raised against a bacterial fusion protein of beta-galactosidase and a portion of SalF 15R, immune-precipitates polypeptides of 61 and 54 kD from extracts of vaccinia virus-infected cells . This antiserum also immune-precipitates the novel DNA ligase-AMP adduct, thus proving that the observed DNA ligase is encoded by SalF 15R. Cell, 1989 Nov 17, 59(4), 681 - 6 Genetic analysis of mammalian GAP expressed in yeast; Ballester R et al.; We have designed a vector to express the mammalian GAP protein in the yeast S . cerevisiae . When expressed in yeast, GAP inhibits the function of the human H-rasgly12 protein, but not that of the H-rasval12 protein, and complements the loss of IRA1 . IRA1 is a yeast gene that encodes a protein with homology to GAP and acts upstream of RAS . Mammalian GAP can therefore function in yeast and interact with yeast RAS . Because expression of GAP complements ira1-mutants, we propose that GAP shares some biochemical functions with IRA1 . Other studies indicate that IRA1 controls the level of RAS activity, presumably by regulating GTP hydrolysis . By analogy, we propose that GAP may play a similar role. Cell, 1989 Nov 17, 59(4), 637 - 47 Epigenetic inheritance of transcriptional states in S . cerevisiae; Pillus L et al.; SIR1, one of several genes required for repression of yeast silent mating type loci, has a unique role in repression of the HML alpha locus . Single-cell assays revealed that cells with mutant alleles of SIR1, including presumptive null alleles, existed as populations of genetically identical cells whose members were in one of two different regulatory states . A minority of cells had a repressed HML alpha locus whereas the majority had a derepressed HML alpha locus . The two states were mitotically stable, although rare changes in state were observed during mitotic growth, possibly reflecting heritable changes to the HML alpha locus at or before replication . Analysis of changes in state suggests that SIR1 protein has a role in the establishment but not the maintenance of repression of silent mating type genes, whereas SIR2, SIR3, and SIR4 are required for maintenance. J Biol Chem, 1989 Nov 15, 264(32), 19161 - 8 A covalently constrained congener of the Saccharomyces cerevisiae tridecapeptide mating pheromone is an agonist; Xue CB et al.; An analog of alpha-factor, the Saccharomyces cerevisiae tridecapeptide mating pheromone (Trp-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr), in which the side chains of Lys7 and Gln10 were covalently linked, was synthesized using solid phase methodologies . The yield of the purified cyclic analog cyclo7,10{Nle12}alpha-factor was 30%, and its structure was verified by amino acid analysis, peptide sequencing, fast atom bombardment-mass spectrometry, and proton nuclear magnetic resonance spectroscopy . Cyclo7,10{Nle12}alpha-factor caused growth arrest and morphological alterations in S . cerevisiae MATa cells qualitatively identical to those induced by linear pheromone and was one-fourth to one-twentieth as active as the linear alpha-factor depending upon the S . cerevisiae strain tested . Consistent with the relative activities of the linear and cyclic peptides, binding competition studies indicated that cyclo7,10{Nle12}alpha-factor had approximately 20-40-fold less affinity for the alpha-factor receptor . Hydrolysis of the cyclic peptide by the target cells did not lead to opening of the ring and was less rapid than that of linear alpha-factor . The alpha-factor antagonist des-Trp1-{Ala3,Nle12}alpha-factor reversed the activity of the cyclic analog, and cyclo7,10{Nle12}alpha-factor was not active at the restrictive temperature in a temperature-sensitive receptor mutant . These results support the conclusion that the cyclic alpha-factor occupies the same binding site within the receptor as is occupied by the natural pheromone . The cyclic alpha-factor represents a rare example of an agonist among covalently constrained congeners of small linear peptide messengers. Biochem Int, 1989 Nov, 19(5), 1019 - 30 Relationship between fluidity and L-alanine transport in a fatty acid auxotroph of Saccharomyces cerevisiae; Mishra P et al.; The influence of the physical state of membrane on L-alanine uptake has been investigated in Saccharomyces cerevisiae KD115, an unsaturated fatty acid auxotrophic mutant . By monitoring the unsaturation index and steady state fluorescence polarization of 1,6 diphenyl hexatriene (DPH), it was observed that at mid log phase the membrane fluidity increased with an increase in the number of double bonds of supplemented fatty acid . Arrhenius plots of the velocities for L-alanine transport in cells grown on palmitoleate, oleate, linoleate and linolenate were biphasic and dependent on supplemented unsaturated fatty acid . Results illustrate a correlation between membrane fluidity and shift in transition points . Further, results confirm the role of fatty acyl milieu in regulation of transport activity of S . cerevisiae. J Cell Sci, 1989 Nov, 94 ( Pt 3), 511 - 6 Induction of repressible acid phosphatase by unsaturated fatty acid in Saccharomyces cerevisiae; Doi S et al.; We studied the induction of acid phosphatase (APase) by fatty acids in Saccharomyces cerevisiae . S . cerevisiae has two types of APase: constitutive and repressible enzymes . The synthesis of the latter APase is normally derepressed by depletion of inorganic phosphate (Pi) in the incubation medium . Of the saturated and unsaturated fatty acids tested, linoleic, linolenic and arachidonic acids induced the synthesis of APase even in the presence of a high concentration of Pi, whereas palmitic, stearic and oleic acids did not . De novo protein synthesis but not stimulation of secretion of the enzyme was required for the induction . Genetic analyses using plasmids carrying the genes, PHO5 and PHO3, that code for repressible APase and constitutive APase, respectively, showed that linolenic acid induced the formation of repressible APase . Linolenic acid inhibited the uptake of exogenous 32Pi and simultaneously lowered the intracellular level of Pi . These circumstances indicate that linolenic acid-induced derepression of repressible APase is primarily caused by a fall in the intracellular level of Pi . However, cells that had been preincubated in the presence of a high concentration of Pi produced APase shortly after the addition of linolenic acid . It is, therefore, suggested that, as well as a normal regulatory mechanism for derepression of repressible APase, a mechanism independent of the external level of Pi participates in the induction of repressible APase by linolenic acid. Mol Gen Genet, 1989 Nov, 219(3), 495 - 8 Plasmid multimerization is dependent on RAD52 activity in Saccharomyces cerevisiae; Harashima S et al.; A mutant plasmid, pX, derived from the 1453 base pair small plasmid, YARp1 (or TRP1 RI circle), consists of 849 base pairs of DNA bearing the TRP1 gene and the ARS1 sequence of Saccharomyces cerevisiae and, unlike YARp1 and other commonly used yeast plasmids, highly multimerizes in a S . cerevisiae host . The multimerization of pX was dependent on RAD52, which is known to be necessary for homologous recombination in S . cerevisiae . Based upon this observation, a regulated system of multimerization of pX with GAL1 promoter-driven RAD52 has been developed . We conclude that the regulated multimerization of pX could provide a useful model system to study genetic recombination in the eukaryotic cell, in particular to investigate recombination intermediates and the effects of various trans-acting mutations on the multimerization and recombination of plasmids. Yeast, 1989 Nov-Dec, 5(6), 509 - 24 Isolation, DNA sequence and regulation of a new cell division cycle gene from the yeast Saccharomyces cerevisiae; Hasegawa H et al.; A new complementation group of temperature-sensitive mutants of the yeast Saccharomyces cerevisiae (ts26-1 and ts26-2) has been isolated and characterized . This mutation maps at 40.7 cM from arg8 and 48.9 cM from arg1 on the left arm of chromosome XV of yeast, providing that it is a newly identified gene . The dumbbell-shape terminal morphology of the mutant cells at the restrictive temperatures is a characteristic of mutants defective in DNA replication . To study the defect of macromolecule synthesis in the mutant cells, DNA, RNA, and protein synthesis were measured at both permissive and restrictive temperatures . The data suggest that the primary defect of this mutation is at the initiation step of DNA synthesis . The gene has been cloned from an S . cerevisiae genomic library by rescue of the conditional lethality of the mutants . It is present as a single copy in the haploid genome . DNA-RNA hybridization of the gene has identified 1 kb RNA, which is under cell-division-cycle control . DNA sequence analysis of the gene has identified an open reading frame capable of encoding a protein of molecular weight 25,055 (214 amino acids). Mol Cell Biol, 1989 Nov, 9(11), 4789 - 98 Basal-level expression of the yeast HSP82 gene requires a heat shock regulatory element; McDaniel D et al.; Previous studies have shown that heat shock factor is constitutively bound to heat shock elements in Saccharomyces cerevisiae . We demonstrate that mutation of the heat shock element closest to the TATA box of the yeast HSP82 promoter abolishes basal-level transcription without markedly affecting inducibility . The mutated heat shock element no longer bound putative heat shock factor, either in vitro or in vivo, but still resided within a nuclease-hypersensitive site in the chromatin . Thus, constitutive binding of heat shock factor to heat shock elements in S . cerevisiae appears to functionally direct basal-level transcription. Mol Cell Biol, 1989 Nov, 9(11), 4777 - 88 Photolyases from Saccharomyces cerevisiae and Escherichia coli recognize common binding determinants in DNA containing pyrimidine dimers; Baer M et al.; DNA photolyases catalyze the light-dependent repair of pyrimidine dimers in DNA . The results of nucleotide sequence analysis and spectroscopic studies demonstrated that photolyases from Saccharomyces cerevisiae and Escherichia coli share 37% amino acid sequence homology and contain identical chromophores . Do the similarities between these two enzymes extend to their interactions with DNA containing pyrimidine dimers, or does the organization of DNA into nucleosomes in S . cerevisiae necessitate alternative or additional recognition determinants? To answer this question, we used chemical and enzymatic techniques to identify the contacts made on DNA by S . cerevisiae photolyase when it is bound to a pyrimidine dimer and compared these contacts with those made by E . coli photolyase and by a truncated derivative of the yeast enzyme when bound to the same substrate . We found evidence for a common set of interactions between the photolyases and specific phosphates in the backbones of both strands as well as for interactions with bases in both the major and minor grooves of dimer-containing DNA . Superimposed on this common pattern were significant differences in the contributions of specific contacts to the overall binding energy, in the interactions of the enzymes with groups on the complementary strand, and in the extent to which other DNA-binding proteins were excluded from the region around the dimer . These results provide strong evidence both for a conserved dimer-binding motif and for the evolution of new interactions that permit photolyases to also act as accessory proteins in nucleotide excision repair . The locations of the specific contacts made by the yeast enzyme indicate that the mechanism of nucleotide excision repair in this organism involves incision(s) at a distance from the pyrimidine dimer. Mol Cell Biol, 1989 Nov, 9(11), 4696 - 705 Isolation of constitutive mutations affecting the proline utilization pathway in Saccharomyces cerevisiae and molecular analysis of the PUT3 transcriptional activator; Marczak JE et al.; The enzymes of the proline utilization pathway (the products of the PUT1 and PUT2 genes) in Saccharomyces cerevisiae are coordinately regulated by proline and the PUT3 transcriptional activator . To learn more about the control of this pathway, constitutive mutations in PUT3 as well as in other regulators were sought . A scheme using a gene fusion between PUT1 (S . cerevisiae proline oxidase) and galK (Escherichia coli galactokinase) was developed to select directly for constitutive mutations affecting the PUT1 promoter . These mutations were secondarily screened for their effects in trans on the promoter of the PUT2 (delta 1-pyrroline-5-carboxylate dehydrogenase) gene by using a PUT2-lacZ (E . coli beta-galactosidase) gene fusion . Three different classes of mutations were isolated . The major class consisted of semidominant constitutive PUT3 mutations that caused PUT2-lacZ expression to vary from 2 to 22 times the uninduced level . A single dominant mutation in a new locus called PUT5 resulted in low-level constitutive expression of PUT2-lacZ; this mutation was epistatic to the recessive, noninducible put3-75 allele . Recessive constitutive mutations were isolated that had pleiotropic growth defects; it is possible that these mutations are not specific to the proline utilization pathway but may be in genes that control several pathways . Since the PUT3 gene appears to have a major role in the regulation of this pathway, a molecular analysis was undertaken . This gene was cloned by functional complementation of the put3-75 mutation . Strains carrying a complete deletion of this gene are viable, proline nonutilizing, and indistinguishable in phenotype from the original put3-75 allele . The PUT3 gene encodes a 2.8-kilobase-pair transcript that is not regulated by proline at the level of RNA accumulation . The presence of the gene on a high-copy-number plasmid did not alter the regulation of one of its target genes, PUT2-lacZ, suggesting that the PUT3 gene product is not limiting and that a titratable repressor is not involved in the regulation of this pathway. Biochem J, 1989 Nov 1, 263(3), 973 - 6 Structural basis for the kinetic differences between flavocytochromes b2 from the yeasts Hansenula anomala and Saccharomyces cerevisiae; Black MT et al.; To understand the structural basis for the different catalytic behaviour of the flavocytochromes b2 from Saccharomyces cerevisiae and Hansenula anomala we have cloned and sequenced the gene encoding the latter . We have compared the amino acid sequences of the mature proteins in the context of the known crystal structure of S . cerevisiae flavocytochrome b2 . Overall there is 60% sequence identity, but two surface loops in particular are strikingly different in primary structure and net charge. J Bacteriol, 1989 Nov, 171(11), 6349 - 52 Arsenic oxide-induced thermotolerance in Saccharomyces cerevisiae; Chang EC et al.; The growth response of Saccharomyces cerevisiae to arsenite and arsenate and the relationship between the enhancement of heat shock protein (hsp) synthesis caused by these arsenic oxides and thermotolerance are reported . Arsenite and arsenate transiently inhibited cell growth and overall protein synthesis; arsenate enhanced the synthesis of the 42-, 74-, 84-, and 100-kilodalton hsps, whereas arsenite enhanced synthesis of only the 74-kilodalton hsp . The induction of these hsps reached a maximum 45 min following metal oxide treatment and then declined . A delayed thermotolerance peaked 4 h after metal oxide addition, at which time cell growth and protein synthesis were recovering . These data show that the arsenate- and arsenite-induced thermotolerance in S . cerevisiae cells does not appear to be causally related to either hsp synthesis or cell cycle arrest. Proc Natl Acad Sci U S A, 1989 Nov, 86(22), 8778 - 82 MSI1, a negative regulator of the RAS-cAMP pathway in Saccharomyces cerevisiae; Ruggieri R et al.; We have previously demonstrated that the IRA1-encoded protein inhibits the function of the RAS protein in a fashion antagonistic to the function of the CDC25 protein in the RAS-cAMP pathway in Saccharomyces cerevisiae . In an attempt to identify genes involved in the regulation of this pathway, high-copy-number plasmid suppressors of the heat shock sensitivity of the ira1 mutation were isolated . One such suppressor, MSI1, was found to encode a putative protein of 422 amino acids that shows homology to the beta subunit of the mammalian guanine nucleotide-binding regulatory proteins . Overexpression of the MSI1 gene could suppress the heat shock sensitivity and the defect in sporulation caused by the ira1 and RAS2Val19 mutations but not those of the bcy1 mutation . Furthermore, the high level of intracellular cAMP in ira1 and RAS2Val19 cells was reduced by the MSI1 gene carried on a YEp-based plasmid . These results suggest that the MSI1 protein is a negative regulator of the RAS-mediated induction of cAMP in S . cerevisiae. Yeast, 1989 Nov-Dec, 5(6), 459 - 67 Similar and different domains of chitin synthases 1 and 2 of S . cerevisiae: two isozymes with distinct functions; Silverman SJ; Saccharomyces cerevisiae produces two chitin synthases (Chs1 and Chs2) encoded by separate genes . Although these enzymes catalyze the same reaction, Chs2 is essential for septum formation whereas Chs1 has a repair function . To determine if these physiological differences are reflected in the enzyme structures, the CHS2 gene was sequenced and compared to that of CHS1 . The predicted amino acid sequence of Chs2 shares substantial similarity with that of Chs1 in the carboxyl two-thirds of the protein . The amino one-third segments differ in predicted isoelectric point by almost 5 pH units . It is suggested that the similar regions are related to common catalytic function . The unrelated regions may be involved in regulation or localization of the respective enzymes . CHS1 and CHS2 are unlinked but may have arisen from the duplication of an ancestral gene. Biochimie, 1989 Nov-Dec, 71(11-12), 1145 - 55 Studies on the CoQH2-cytochrome c reductase segment of the respiratory chain of yeast mitochondria, using mutants of the cytochrome b split gene; Lemesle-Meunier D; Our work relating to the role of cytochrome b in the CoQH2-cytochrome c reductase segment of the respiratory chain of S . cerevisiae mitochondria is reviewed here and new results are reported . The results concerning the structure-function relationship of cytochrome b in this complex, analyzed within the framework of the eight transmembrane alpha helice cytochrome b folding model, agree with the following features of the proton motive Q cycle (or SQ cycle): i) the antimycin A and myxothiazol binding domains are located on opposite sides of the inner mitochondrial membrane; and ii) the antimycin A binding domain is associated with the b562 domain, the myxothiazol domain with the b565 domain . These results were obtained from structural data derived from amino-acid sequence studies on mit- mutants and from biochemical studies of these mutants . However, functional studies are reported here that are not in agreement with the following features of the above models: i) the serial arrangement of the two hemes of cytochrome b and ii) the isolation of cytochrome b from redox changes with the couple fumarate/succinate in the presence of antimycin A and myxothiazol. J Gen Microbiol, 1989 Nov, 135 ( Pt 11), 2791 - 8 D-xylose utilization by Saccharomyces cerevisiae; van Zyl C et al.; Although it is generally accepted that Saccharomyces cerevisiae is unable to assimilate D-xylose, four strains were found to utilize xylose aerobically at different efficiencies in the presence of a mixture of substrates . The degree of D-xylose utilization by S . cerevisiae ATCC 26602 depended upon the presence of other substrates or yeast extract . The greatest amount of xylose (up to 69% over 7 d) was utilized when sugar substrates such as D-ribose were co-metabolized . Much lower degrees of utilization occurred with co-metabolism of organic acids, polyols or ethanol . A mixture of D-glucose, D-ribose, D-raffinose, glycerol and D-xylose resulted in greater xylose utilization than the presence of a single substrate and xylose . The absence of growth on a co-substrate alone did not prevent the utilization of xylose in its presence . Xylose was co-metabolized with ribose under anaerobic conditions but at a much slower rate than under aerobic conditions . When {14C}xylose was utilized in the presence of ribose under anaerobic conditions, the radioactive label was detected mainly in xylitol and not in the small amounts of ethanol produced . Under aerobic conditions the radioactive label was distributed between xylitol (91.3 +/- 0.8%), CO2 (2.6 +/- 2.3%) and biomass (1.7 +/- 0.6%) . No other metabolic products were detected . Whereas most xylose was dissimilated rather than assimilated by S . cerevisiae, the organism apparently possesses a pathway which completely oxidizes xylose in the presence of another substrate. Biochem Biophys Res Commun, 1989 Oct 16, 164(1), 362 - 9 Mitochondrial rRNA-containing petite strains of yeast (Saccharomyces cerevisiae) show a normal nuclear-mitochondrial stringent response; McEntee CM et al.; The nuclear-mitochondrial stringent response was examined in isonuclear rho+, 21S rRNA-containing rho-, and rho o strains of S . cerevisiae . By 30 min after nutritional downshift, nuclear rDNA transcription falls to 15% of control levels congruently in all strains, as assayed via whole-cell RNA or by hybrid selection of specific double-labeled transcripts . Both in vivo and in vitro, the mitochondrial stringent response is identical between the rho- strain and its parental rho+ strain, and in both, the kinetics and magnitude of the organellar response mirror those of the nuclear response . The data show that mitochondrial transcription and protein synthesis are not required for stringent regulation of either nuclear or mitochondrial rDNA transcription. J Biol Chem, 1989 Oct 5, 264(28), 16862 - 6 Secretion by yeast of the zymogen form of Mucor rennin, an aspartic proteinase of Mucor pusillus, and its conversion to the mature form; Hiramatsu R et al.; The Mucor rennin gene encoding a prepro-form of the fungal aspartic proteinase from Mucor pusillus was expressed under the control of the yeast GAL7 promoter in Saccharomyces cerevisiae . An inactive zymogen of the enzyme with the 44-amino-acid pro-sequence was identified in the medium during the initial stage of cultivation . Processing of the purified zymogen to the mature enzyme proceeded autocatalytically under the acidic conditions . The rate of processing was accelerated by an increase in the concentration of the zymogen or addition of the mature enzyme . The in vitro processing was inhibited by inhibitors for the aspartic proteinases . The zymogen with no proteinase activity due to a mutation at the active site residue, Asp, was still processed at a relatively slower rate in a wild-type strain of yeast, but no processing occurred in the pep4-3 mutant strain of S . cerevisiae deficient in yeast proteinase A . Thus, Mucor rennin is excreted in a form of zymogen, which is then processed in the yeast secretion pathway mainly by the autocatalytic proteolysis but, alternatively, by a proteinase of yeast. Curr Genet, 1989 Oct, 16(4), 267 - 72 Organization of ribosomal RNA genes in Alternaria alternata Japanese pear pathotype, a host-selective AK-toxin-producing fungus; Tsuge T et al.; DNA encoding ribosomal RNA (rRNA) of Alternaria alternata Japanese pear pathotype has been cloned in lambda replacement vector, lambda Fix . Restriction endonuclease mapping and Southern hybridization with the 18S and 28S rRNAs of Saccharomyces cerevisiae revealed the A . alternata rDNA to be tandemly repeating 8.15-kilobase pair unit . The restriction fragments of the unit were then subcloned in the plasmid vector Bluescribe M13- and partially sequenced . The determined sequences were compared with previously reported sequences of S . cerevisiae rRNAs and their genes . The locations of DNA sequences encoding the 5.8S, 18S, and 28S rRNAs were determined by homology search using reported sequences . The complete DNA sequence for 5.8S rRNA of the fungus was found to be highly conserved at more than 90% homology in the fungi analyzed . However, sequence diversities were observed in limited regions involved in a helix structure, the helix (e), found at position 116-137. Curr Genet, 1989 Oct, 16(4), 261 - 6 An improved host-vector system for Candida maltosa using a gene isolated from its genome that complements the his5 mutation of Saccharomyces cerevisiae; Hikiji T et al.; The host-vector system of an n-alkane-assimilating-yeast, Candida maltosa, which we previously constructed using an autonomously replicating sequence (ARS) region isolated from the genome of this yeast, utilizes C . maltosa J288 (leu2-) as a host . As this host had a serious growth defect on n-alkane, we developed an improved host-vector system using C . maltosa CH1 (his-) as host . The vectors were constructed with the Candida ARS region and a DNA fragment isolated from the genome of C . maltosa . Since this DNA fragment could complement histidine auxotrophy of both C . maltosa CH1 and S . cerevisiae (his5-), we termed the gene contained in this DNA fragment C-HIS5 . The vectors were characterized in terms of transformation frequency and stability, and the nucleotide sequence of C-HIS5 was determined . The deduced amino acid sequence (389 residues) shared 51% homology with that of HIS5 of S . cerevisiae (384 residues; Nishiwaki et al . 1987). Curr Genet, 1989 Oct, 16(4), 219 - 23 Interactions of the RAD7 and RAD23 excision repair genes of Saccharomyces cerevisiae with DNA repair genes in different epistasis groups; Schiestl RH et al.; The RAD7 and RAD23 genes of S . cerevisiae affect the efficiency of excision repair of UV-damaged DNA . We have examined the UV survival of strains carrying the rad7 and rad23 deletion mutation in combination with deletion mutations in genes affecting different DNA repair pathways . As expected, the rad7 delta and rad23 delta mutations interact epistatically with the excision repair defective rad1 delta mutation, and synergistically with the rad6 delta and rad52 delta mutations that affect the postreplication repair and recombinational repair pathways, respectively . However, the rad7 delta rad6 delta and the rad23 delta rad6 delta mutants exhibit the same level of UV sensitivity as the rad1 delta rad6 delta mutant . This observation is of interest since, in contrast to the rad7 delta or the rad23 delta mutations, the rad1 delta mutant is very UV sensitive and highly excision defective . This observation suggest that RAD6 and RAD7 and RAD23 genes complete for the same substrate during DNA repair. Mol Cell Biol, 1989 Oct, 9(10), 4467 - 72 Translation in Saccharomyces cerevisiae: initiation factor 4E-dependent cell-free system; Altmann M et al.; The gene encoding translation initiation factor 4E (eIF-4E) from Saccharomyces cerevisiae was randomly mutagenized in vitro . The mutagenized gene was reintroduced on a plasmid into S . cerevisiae cells having their only wild-type eIF-4E gene on a plasmid under the control of the regulatable GAL1 promoter . Transcription from the GAL1 promoter (and consequently the production of wild-type eIF-4E) was then shut off by plating these cells on glucose-containing medium . Under these conditions, the phenotype conferred upon the cells by the mutated eIF-4E gene became apparent . Temperature-sensitive S . cerevisiae strains were identified by replica plating . The properties of one strain, 4-2, were further analyzed . Strain 4-2 has two point mutations in the eIF-4E gene . Upon incubation at 37 degrees C, incorporation of {35S}methionine was reduced to 15% of the wild-type level . Cell-free translation systems derived from strain 4-2 were dependent on exogenous eIF-4E for efficient translation of certain mRNAs, and this dependence was enhanced by preincubation of the extract at 37 degrees C . Not all mRNAs tested required exogenous eIF-4E for translation. Proc Natl Acad Sci U S A, 1989 Oct, 86(20), 7961 - 5 Cloning a eukaryotic DNA glycosylase repair gene by the suppression of a DNA repair defect in Escherichia coli; Chen J et al.; If eukaryotic genes could protect bacteria with defects in DNA repair, this effect could be exploited for the isolation of eukaryotic DNA repair genes . We have thus cloned a DNA repair gene from Saccharomyces cerevisiae that directs the synthesis of a DNA glycosylase that specifically releases 3-methyladenine from alkylated DNA and in so doing protects alkylation-sensitive Escherichia coli from killing by methylating agents . The cloned yeast gene was then used to generate a mutant strain of S . cerevisiae that carries a defect in the glycosylase gene and is extremely sensitive to DNA methylation . This approach may allow the isolation of a large number of eukaryotic DNA repair genes. Proc Natl Acad Sci U S A, 1989 Oct, 86(20), 7785 - 9 Yeast TATA-box transcription factor gene; Schmidt MC et al.; The first step in the transcription of most protein-encoding genes in eukaryotes is the binding of a transcription factor to the TATA-box promoter element . This TATA-box transcription factor was purified from extracts of the yeast Saccharomyces cerevisiae by using reconstitution of in vitro transcription reactions as an assay . The activity copurified with a protein whose sodium dodecyl sulfate/polyacrylamide gel mobility is 25 kDa . The sequence of the amino-terminal 21 residues of this protein was determined by sequential Edman degradation . A yeast genomic library was screened with mixed oligonucleotides encoding six residues of the protein sequence . The yeast TATA-box factor gene was cloned, and DNA sequencing revealed a 720-base-pair open reading frame encoding a 27,016-Da protein . The identity of the clone was confirmed by expressing the gene in Escherichia coli and detecting TATA-box factor DNA binding and transcriptional activities in extracts of the recombinant E . coli . The TATA-box factor gene was mapped to chromosome five of S . cerevisiae . RNA blot hybridization and nuclease S1 analysis indicated that the major TATA-box factor mRNA is 1.3 kilobases, including an unusually long 5' untranslated region of 188 +/- 5 nucleotides . Homology searches showed a region of distant similarity to the calcium-binding structures of calpains, a structure that has a conformation similar to the helix-turn-helix motif of DNA binding proteins. J Bacteriol, 1989 Oct, 171(10), 5659 - 67 REV3, a Saccharomyces cerevisiae gene whose function is required for induced mutagenesis, is predicted to encode a nonessential DNA polymerase; Morrison A et al.; We have cloned the REV3 gene of Saccharomyces cerevisiae by complementation of the rev3 defect in UV-induced mutagenesis . The nucleotide sequence of this gene encodes a predicted protein of Mr 172,956 showing significant sequence similarity to Epstein-Barr virus DNA polymerase and to other members of a class of DNA polymerases including human DNA polymerase alpha and yeast DNA polymerase I . REV3 protein shows less sequence identity, and presumably a more distant evolutionary relationship, to the latter two enzymes than they do to each other . Haploids carrying a complete deletion of REV3 are viable . We suggest that induced mutagenesis in S . cerevisiae depends on a specialized DNA polymerase that is not required for other replicative processes . REV3 is located 2.8 centimorgans from CDC60, on chromosome XVI. J Appl Bacteriol, 1989 Oct, 67(4), 401 - 10 Molecular cloning and expression in Saccharomyces cerevisiae and Neurospora crassa of the invertase gene from Neurospora crassa; Caru M et al.; A plasmid (named pCN2) carrying a 7.6 kb BamHI DNA insert was isolated from a Neurospora crassa genomic library raised in the yeast vector YRp7 . Saccharomyces cerevisiae suco and N . crassa inv strains transformed with pNC2 were able to grow on sucrose-based media and expressed invertase activity . Saccharomyces cerevisiae suco (pNC2) expressed a product which immunoreacted with antibody raised against purified invertase from wild type N . crassa, although S . cerevisiae suc+ did not . The cloned DNA hybridized with a 7.6 kb DNA fragment from BamHI-restricted wild type N . crassa DNA . Plasmid pNC2 transformed N . crassa Inv- to Inv+ by integration either near to the endogenous inv locus (40% events) or at other genomic sites (60% events) . It appears therefore that the cloned DNA piece encodes the N . crassa invertase enzyme . A 3.8 kb XhoI DNA fragment, derived from pNC2, inserted in YRp7, in both orientation, was able to express invertase activity in yeast, suggesting that it contains an intact invertase gene which is not expressed from a vector promoter. FEBS Lett, 1989 Sep 25, 255(2), 455 - 9 Isolation and expression in Saccharomyces cerevisiae of a gene encoding an alpha-amylase from Schwanniomyces castellii; Abarca D et al.; A gene (SWA1) encoding an alpha-amylase activity from Schwanniomyces castellii has been cloned and expressed, via yeast cloning vector YEp13, in Saccharomyces cerevisiae . By using a riboprobe which is internal to the SWA1 gene, a 1.55 kb transcript was detected in the poly(A)+ RNA from both Sw . castellii and a S . cerevisiae clone harboring the SWA1 gene . This transcript should, therefore, correspond to the SWA1 gene . In addition, the DNA strand determining the alpha-amylase activity has been defined . Transcription of the SWA1 gene appears to be highly regulated in Sw . castellii, whereas it is constitutive in the S . cerevisiae harboring this gene. FEBS Lett, 1989 Sep 25, 255(2), 290 - 4 Purification of the inducible alpha-agglutinin of S . cerevisiae and molecular cloning of the gene; Hauser K et al.; The alpha-agglutinin responsible for mating type-specific agglutination of S . cerevisiae alpha-cells has been purified to homogeneity . The glycoprotein released from the cell surface under mild conditions has a relative molecular mass of 200 to 300 kDa as determined by SDS-gel electrophoresis . The protein moiety corresponds to 68.2 kDa . With an oligonucleotide corresponding to the N-terminal amino acid sequence, the alpha-agglutinin gene has been cloned and sequenced . From the DNA sequence, a protein of 631 amino acids with 12 potential N-glycosylation sites is predicted . The carboxy terminal one-third of the protein is not required for agglutination activity. Nucleic Acids Res, 1989 Sep 25, 17(18), 7211 - 9 RADH, a gene of Saccharomyces cerevisiae encoding a putative DNA helicase involved in DNA repair . Characteristics of radH mutants and sequence of the gene; Aboussekhra A et al.; A new type of radiation-sensitive mutant of S . cerevisiae is described . The recessive radH mutation sensitizes to the lethal effect of UV radiations haploids in the G1 but not in the G2 mitotic phase . Homozygous diploids are as sensitive as G1 haploids . The UV-induced mutagenesis is depressed, while the induction of gene conversion is increased . The mutation is believed to channel the repair of lesions engaged in the mutagenic pathway into a recombination process, successful if the events involve sister-chromatids but lethal if they involve homologous chromosomes . The sequence of the RADH gene reveals that it may code for a DNA helicase, with a Mr of 134 kDa . All the consensus domains of known DNA helicases are present . Besides these consensus regions, strong homologies with the Rep and UvrD helicases of E . coli were found . The RadH putative helicase appears to belong to the set of proteins involved in the error-prone repair mechanism, at least for UV-induced lesions, and could act in coordination with the Rev3 error-prone DNA polymerase. Cell, 1989 Sep 22, 58(6), 1183 - 91 SPT15, the gene encoding the yeast TATA binding factor TFIID, is required for normal transcription initiation in vivo; Eisenmann DM et al.; Mutations in the S . cerevisiae SPT15 gene were isolated as suppressors of insertion mutations that alter the transcription of adjacent genes . Molecular and genetic analysis of the cloned SPT15 gene has shown that it is the same as the gene that encodes the TATA binding factor TFIID . Analysis of spt15 mutants has demonstrated that alterations in TFIID can change transcription initiation in vivo . In addition, we demonstrate that TFIID is essential for growth and that spt15 mutations are pleiotropic, as spt15 mutants grow slowly and have defects in both mating and sporulation . Therefore, TFIID is an essential transcription factor in vivo, likely to be required for normal expression of a large number of genes. Cell, 1989 Sep 22, 58(6), 1107 - 19 A putative protein kinase overcomes pheromone-induced arrest of cell cycling in S . cerevisiae; Courchesne WE et al.; MATa cells carrying an sst2 mutation are unable to recover from the G1-specific cell cycle arrest induced by the mating pheromone alpha factor . The KSS1 gene, when overexpressed, suppresses this adaptation defect . KSS1 overexpression also suppresses the recovery defect manifested by cells expressing an alpha factor receptor lacking its 136 amino acid cytoplasmic tail . Because SST2 product and the receptor tail contribute independently to events that allow recovery from pheromone-induced growth arrest, KSS1 function defines a third independent process that promotes desensitization . The KSS1 gene encodes an apparent protein kinase homologous to the CDC28 (S . cerevisiae) and cdc2+ (S . pombe) gene products . The recovery-promoting activity of the KSS1 gene requires a functional WHI1 gene, which encodes a yeast homolog to animal cyclins, suggesting that the KSS1 and WHI1 proteins act in the same growth control pathway. J Biol Chem, 1989 Sep 15, 264(26), 15628 - 33 Energy-linked anion transport . Cloning, sequencing, and characterization of a full length cDNA encoding the rat liver mitochondrial proton/phosphate symporter; Ferreira GC et al.; A full length cDNA clone encoding the precursor of the rat liver mitochondrial phosphate transporter (H+/Pi symporter) has been isolated from a cDNA library using a bovine heart partial length phosphate transporter clone as a hybridization probe . The entire clone is 1263 base pairs in length with 5'- and 3'-untranslated regions of 16 and 168 base pairs, respectively . The open reading frame encodes for the mature protein (312 amino acids) preceded by a presequence of 44 amino acids enriched in basic residues . The polypeptide sequence predicted from the DNA sequence was confirmed by analyzing the first 17 amino-terminal amino acids of the pure phosphate transporter protein . The rat liver phosphate transporter differs from the bovine heart transporter in 32 amino acids (i.e . approximately 10%) . It contains a region from amino acid 139 to 159 which is 37% identical with the beta-subunit of the liver mitochondrial ATP synthase . Amino acid sequence comparisons of the Pi transporter with Pi binding proteins, other H+-linked symporters, and the human glucose transporter did not reveal significant sequence homology . Analysis of genomic DNA from both rat and S . cerevisiae by Southern blots using the rat liver mitochondrial Pi carrier cDNA as a probe revealed remarkably similar restriction patterns, a finding consistent with the presence in lower and higher eukaryotes of homologous Pi carrier proteins . This is the first report of the isolation, sequencing, and characterization of a full length cDNA coding for a protein involved in energy-coupled Pi transport. FEBS Lett, 1989 Sep 11, 255(1), 5 - 9 Electron-transfer restoration by vitamin K3 in a complex III-deficient mutant of S . cerevisiae and sequence of the corresponding cytochrome b mutation; Brivet-Chevillotte P et al.; The yeast box-mutant W7 exhibits deficiencies in cytochrome b and in nuclear coded complex III subunits, a phenotype observed previously in a patient with mitochondrial myopathy . DNA sequence analysis of mutant W7 revealed a single base transition in the cytochrome b gene; the mutated residue Gly 131 is perfectly conserved in all known cytochromes b and belongs to the Qo domain . Mutant W7 provides a model system for evaluating the action of therapeutic agents, such as vitamin K3 which restored NADH-oxidase activity in the mutant as well as in the antimycin-inhibited wild type . However, with the mutant, a greater quantity of menadione was necessary due to a decrease in other complex activities, and a much lower electron-flow fraction passed through cytochrome oxidase. Cell, 1989 Sep 8, 58(5), 857 - 67 The poly(A) binding protein is required for poly(A) shortening and 60S ribosomal subunit-dependent translation initiation; Sachs AB et al.; Depletion of the essential poly(A) binding protein (PAB) in S . cerevisiae by promoter inactivation or by the utilization of a temperature-sensitive mutation (pab1-F364L) results in the inhibition of translation initiation and poly(A) tail shortening . Reversion analysis of pab1-F364L yielded seven independent, extragenic cold-sensitive mutations (spb1-spb7) that also suppress a PAB1 deletion . These mutations allow translation initiation without significantly changing poly(A) tail lengths in the absence of PAB, and they affect the amount of 60S ribosomal subunit . Consistent with this, SPB2 encodes the ribosomal protein L46 . These data suggest that the 60S subunit mediates the PAB requirement of translation initiation, thereby ensuring that only intact poly(A)+ mRNA will be translated efficiently in vivo. Biofizika, 1989 Sep-Oct, 34(5), 808 - 13 {Analysis of distribution of S . cerevisiae cells for protein content using cell cycle models}; Ivanitskaia IuG et al.; Histograms of cell distributions according to protein content obtained by means of flow cytofluorometry characterize the physiological state of the population as a whole and permit to calculate the velocity of protein accumulation in the cell in the course of the cell cycle . Dependence of population heterogeneity on culturing conditions is considered . Mathematical analysis of histograms of continuous cultures of S . cerevisiae is carried out at dilution rates 0.4 hours-1 and 0.05 hours-1 . Calculations are carried out on condition that the protein content in the cell rises a) exponentially and b) linearly in the course of the cell cycle . At low growth rate (0.05 hours-1) the distribution is bimodal and therefore it is highly informative . The assumption concerning linear accumulation of the protein allows good approximation of the experimental distributions by the theoretical ones. Izv Akad Nauk SSSR Biol, 1989 Sep-Oct, (5), 786 - 91 {A limited turbidostat yeast culture under heat shock conditions}; Kaliuzhin VA; A response of limited turbidostat S . cerevisiae 14 culture on rapid increase in temperature from optimal 30 degrees to supraoptimal 37.5 degrees C . The temporal thermotolerance was absent in glucose and phosphate-limited cultures . Limitation of nitrogen, Mg, betaalanine, biotin, and, to a certain extent, potassium did not decrease the thermotolerance . The pattern of changes in age composition and specific optical density was found to be similar in limited and unlimited cultures . The glucose and phosphate expenditures at 37.5 degrees C were estimated . The response to heat shock was found to depend on the nature of the limiting factor and the extent of limiting. Mol Gen Genet, 1989 Sep, 218(3), 465 - 74 DNA damage activates transcription and transposition of yeast Ty retrotransposons; Bradshaw VA et al.; A set of genes isolated from Saccharomyces cerevisiae showed increased transcript levels after yeast had been exposed to ultraviolet (UV) light or 4-nitroquinoline-1-oxide (4NQO) . Included among these DNA damage responsive (DDR) genes were members of the Ty retrotransposon family of yeast . Northern hybridization analysis indicated that maximal levels of a 5.6 kb transcript encoded by the Ty elements accumulated in cells after 4 to 6 h of exposure to 4NQO . The induced levels of transcripts varied from two- to tenfold for different Ty probes although similar kinetics and dose responses were observed for transcripts hybridizing to the different Ty family members . Pulse labeling experiments suggested that the accumulation of Ty transcripts was due, in part, to an increased rate of Ty message synthesis . Transposition of Ty elements to two target loci encoding distinct alcohol dehydrogenase enzymes, ADH2 and ADH4, was examined in cells exposed to increasing doses of UV light or 4NQO . The frequency of Ty insertion into these genetic regions following DNA damaging treatments increased by as much as 17-fold compared with untreated cells . These results provide direct evidence that transposable elements can be activated by physical and chemical mutagens/carcinogens and that transpositional mutagenesis is induced by these agents in S . cerevisiae. Mol Cell Biol, 1989 Sep, 9(9), 3992 - 8 Regulation of STA1 gene expression by MAT during the life cycle of Saccharomyces cerevisiae; Dranginis AM; STA1 encodes a secreted glucoamylase of the yeast Saccharomyces cerevisiae var . diastaticus . Glucoamylase secretion is controlled by the mating type locus MAT; a and alpha haploid yeast cells secrete high levels of the enzyme, but a/alpha diploid cells produce undetectable amounts . It has been suggested that STA1 is regulated by MATa2 (I . Yamashita, Y . Takano, and S . Fukui, J . Bacteriol . 164:769-773, 1985), which is a MAT transcript of previously unknown function . In contrast, this work shows that deletion of the entire MATa2 gene had no effect on STA1 regulation but that deletion of MATa1 sequences completely abolished mating-type control . In all cases, glucoamylase activity levels reflected STA1 mRNA levels . It appears that STA1 is a haploid-specific gene that is regulated by MATa1 and a product of the MAT alpha locus and that this regulation occurs at the level of RNA accumulation . STA1 expression was also shown to be glucose repressible . STA1 mRNA was induced in diploids during sporulation along with SGA, a closely linked gene that encodes an intracellular sporulation-specific glucoamylase of S . cerevisiae . A diploid strain with a MATa1 deletion showed normal induction of STA1 in sporulation medium, but SGA expression was abolished . Therefore, these two homologous and closely linked glucoamylase genes are induced by different mechanisms during sporulation . STA1 induction may be a response to the starvation conditions necessary for sporulation, while SGA induction is governed by the pathway by which MAT regulates sporulation . The strain containing a complete deletion of MATa2 grew, mated, and sporulated normally. Biochem Biophys Res Commun, 1989 Aug 15, 162(3), 1409 - 16 Biochemical and genetic characterization of the structure of yeast ornithine decarboxylase; Fonzi WA; The ornithine decarboxylase gene of S . cerevisiae encodes a predicted protein of approximately 53 kD highly homologous with the ornithine decarboxylase of other species . However, the native enzyme has been reported as an 86 kD protein . Our molecular sieve analysis indicated a Mr = 110,000 for the native enzyme . SDS-PAGE analysis of {H3}-alpha-difluoromethylornithine labelled enzyme demonstrated a subunit Mr of approximately 50 kD and suggested the native enzyme is a dimer . Genetic analyses support this conclusion . The complementary, ornithine decarboxylase deficient mutations spe 1A and spe 1B were mapped to the enzyme structural gene by linkage analysis and gene conversion mapping . This demonstrated that the mutations exhibit intragenic complementation which suggests protein-protein interactions and an oligomeric structure for the yeast enzyme . We conclude that yeast ornithine decarboxylase is a dimeric enzyme of 53 kD subunits. Nucleic Acids Res, 1989 Aug 11, 17(15), 5923 - 31 Altered mRNA cap recognition activity of initiation factor 4E in the yeast cell cycle division mutant cdc33; Altmann M et al.; The mutation in the S . cerevisiae cell cycle division mutant cdc33 consists of a single G to A transition in the open reading frame encoding translation initiation factor 4E (eIF-4E) . This leads to the substitution of glycine 113 by aspartic acid close to tryptophane 115 in the protein . This mutation reduces cap binding activity of eIF-4E as measured by binding of eIF-4E to m7GDP agarose columns and slows down overall protein synthesis at the non-permissive temperature . Comparison of the cdc33 mutation with other mutations affecting eIF-4E function supports the view that tryptophane residues and their flanking regions are involved in cap binding activity of eIF-4E. Nucleic Acids Res, 1989 Aug 11, 17(15), 6109 - 27 Isolation and characterization of a human telomere; Cheng JF et al.; A method is described that allows cloning of human telomeres in S . cerevisiae by joining human telomeric restriction fragments to yeast artificial chromosome halves . The resulting chimeric yeast-human chromosomes propagate as true linear chromosomes, demonstrating that the human telomere structure is capable of functioning in yeast and suggesting that telomere functions are evolutionarily conserved between yeast and human . One cloned human telomere, yHT1, contains 4 kb of human genomic DNA sequence next to the tandemly repeating TTAGGG hexanucleotide . Genomic hybridizations using both cloned DNA and TTAGGG repeats have revealed a common structural organization of human telomeres . This 4 kb of genomic DNA sequence is present in most, but not all, human telomeres, suggesting that the region is not involved in crucial chromosome-specific functions . However, the extent of common features among the human telomeres and possible similarities in organization with yeast telomeres suggest that this region may play a role in general chromosome behavior such as telomere-telomere interactions . Unlike the simple telomeric TTAGGG repeats, our cloned human genomic DNA sequence does not cross-hybridize with rodent DNA . Thus, this clone allows the identifications of the terminal restriction fragments of specific human chromosomes in human-rodent hybrid cells. J Gen Microbiol, 1989 Aug, 135 ( Pt 8), 2173 - 9 Rise in intracellular pH is concurrent with 'start' progression of Saccharomyces cerevisiae; Anand S et al.; Intracellular pH (pHi) was determined during arrest and recovery of temperature sensitive-cell division cycle mutants of Saccharomyces cerevisiae . In all mutants, pHi decreased during arrest; but when the mutants were released from arrest a rapid increase in pHi ensued in only cdc28- and cdc37-arrested cells . Both of these mutations cause arrest at 'start', the sole regulatory point in the S . cerevisiae cell cycle . In cells with cdc4 or cdc7 mutations, which arrest past start, pHi remained constant and exhibited a decrease, respectively, upon recovery of growth . The activity of plasma membrane ATPase decreased during the first 30 min of recovery of cdc28-arrested cells, concomitant with the rise in pHi . During the same period, there was no significant change in activity in cdc4-bearing cells, whereas an increase was observed for cdc7-bearing cells . Increase in pHi may be used as a specific signal by S . cerevisiae for start traversal and commitment to a new cycle. Mol Microbiol, 1989 Aug, 3(8), 1003 - 10 Yeast 2 micron vectors replicate and undergo recombination in Torulaspora delbrueckii; Compagno C et al.; In order to develop a procedure for transformation of the industrial yeast Torulaspora delbrueckii, we have constructed a set of recombinant plasmids carrying Saccharomyces cerevisiae ARS and 2 microns origin of replication and kanamycin-G418 resistance gene of Tn903(601) as a selective marker . In this paper we show that S . cerevisiae ARS vectors can replicate autonomously and that vectors bearing the whole S . cerevisiae 2 microns sequence yield stable transformants . We also present evidence to show that 2 microns vectors undergo an FLP-mediated inter- and intramolecular recombination, which suggests that T . delbrueckii can support the amplification and partition mechanisms of these plasmids. Genetika, 1989 Aug, 25(8), 1373 - 83 {Features of integration of recombinant cosmids, containing Aspergillus terreus DNA, in the Saccharomyces cerevisiae genome}; Shubochkina EA et al.; A genome clonotheque of 25-40 kb Sau3A fragments of Aspergillus terreus DNA was constructed in the episomal cosmid vector pES33 containing the ARG4 gene of yeast . 23 independently originated stable Arg+ transformants were selected after transformation of the cir0 yeast strain ESH-O with pooled cosmid molecules . Both genetic and Southern analysis showed that 39% of these stable transformants occurred due to recombination between DNA sequences from A . terreus and Saccharomyces cerevisiae chromosome XII which took place most likely in the regions of homology within the ribosomal clusters . The data present the first evidence of in vivo recombination between foreign sequences and their S . cerevisiae counterparts. Mol Cell Biol, 1989 Aug, 9(8), 3447 - 56 The yeast gene ERG6 is required for normal membrane function but is not essential for biosynthesis of the cell-cycle-sparking sterol; Gaber RF et al.; In Saccharomyces cerevisiae, methylation of the principal membrane sterol at C-24 produces the C-28 methyl group specific to ergosterol and represents one of the few structural differences between ergosterol and cholesterol . C-28 in S . cerevisiae has been suggested to be essential for the sparking function (W . J . Pinto and W . R . Nes, J . Biol . Chem . 258:4472-4476, 1983), a cell cycle event that may be required to enter G1 (C . Dahl, H.-P . Biemann, and J . Dahl, Proc . Natl . Acad . Sci . USA 84:4012-4016, 1987) . The sterol biosynthetic pathway in S . cerevisiae was genetically altered to assess the functional role of the C-28 methyl group of ergosterol . ERG6, the putative structural gene for S-adenosylmethionine: delta 24-methyltransferase, which catalyzes C-24 methylation, was cloned, and haploid strains containing erg6 null alleles (erg6 delta 1 and erg6 delta ::LEU2) were generated . Although erg6 delta cells are unable to methylate ergosterol precursors at C-24, they exhibit normal vegatative growth, suggesting that C-28 sterols are not essential in S . cerevisiae . However, erg6 delta cells exhibit pleiotropic phenotypes that include defective conjugation, hypersensitivity to cycloheximide, resistance to nystatin, a severely diminished capacity for genetic transformation, and defective tryptophan uptake . These phenotypes reflect the role of ergosterol as a regulator of membrane permeability and fluidity . Genetic mapping experiments revealed that ERG6 is located on chromosome XIII, tightly linked to sec59. J Bacteriol, 1989 Aug, 171(8), 4486 - 93 Sequence and structure of the yeast galactose transporter; Szkutnicka K et al.; The previously cloned GAL2 gene of the Saccharomyces cerevisiae galactose transporter has been sequenced . The nucleotide sequence predicts a protein with 574 amino acids (Mr, 63,789) . Hydropathy plots suggest that there are 12 membrane-spanning segments . The galactose transporter shows both sequence and structural homology with a superfamily of sugar transporters which includes the human HepG2-erythrocyte and fetal muscle glucose transporters, the rat brain and liver glucose transporters, the Escherichia coli xylose and arabinose permeases, and the S . cerevisiae glucose, maltose, and galactose transporters . Sequence and structural motifs at the N-terminal and C-terminal regions of the proteins support the view that the genes of this superfamily arose by duplication of a common ancestral gene . In addition to the sequence homology and the presence of the 12 membrane-spanning segments, the members of the superfamily show characteristic lengths and distributions of the charged, hydrophilic connecting loops . There is indirect evidence that the transporter is an N-glycoprotein . However, its only N-glycosylation site occurs in a charged, hydrophilic segment . This could mean that this segment is part of a hydrophilic channel in the membrane . The transporter has a substrate site for the cyclic AMP-dependent protein kinase which may be a target of catabolite inactivation . The transporter lacks a strong sequence enriched for proline (P), glutamate (E), aspartate, serine (S), and threonine (T) and flanked by basic amino acids (PEST sequence) even though it has a short half-life . Mechanisms for converting the poor PEST to a possible PEST sequence are considered . Like the other members of the superfamily, the galactose transporter lacks a signal sequence. Mol Cell Biol, 1989 Aug, 9(8), 3314 - 22 Failure to induce a DNA repair gene, RAD54, in Saccharomyces cerevisiae does not affect DNA repair or recombination phenotypes; Cole GM et al.; The Saccharomyces cerevisiae RAD54 gene is transcriptionally regulated by a broad spectrum of DNA-damaging agents . Induction of RAD54 by DNA-damaging agents is under positive control . Sequences responsible for DNA damage induction (the DRS element) lie within a 29-base-pair region from -99 to -70 from the most proximal transcription start site . This inducible promoter element is functionally separable from a poly(dA-dT) region immediately downstream which is required for constitutive expression . Deletions which eliminate induction of RAD54 transcription by DNA damage but do not affect constitutive expression have no effect on growth or survival of noninducible strains relative to wild-type strains in the presence of DNA-damaging agents . The DRS element is also not required for homothallic mating type switching, transcriptional induction of RAD54 during meiosis, meiotic recombination, or spontaneous or X-ray-induced mitotic recombination . We find no phenotype for a lack of induction of RAD54 message via the damage-inducible DRS, which raises significant questions about the physiology of DNA damage induction in S . cerevisiae. Cell, 1989 Jul 28, 58(2), 409 - 19 A hyper-recombination mutation in S . cerevisiae identifies a novel eukaryotic topoisomerase; Wallis JW et al.; A hyper-recombination mutation was isolated that causes an increase in recombination between short repeated delta sequences surrounding the SUP4-omicron gene in S . cerevisiae . The wild-type copy of this gene was cloned by complementation of one of its pleiotropic phenotypes, slow growth . DNA sequence of the clone revealed a 656 amino acid open reading frame capable of encoding a protein homologous to the bacterial type I topoisomerase . No homology was detected with previously identified eukaryotic topoisomerases . Construction of double mutants with either of the two known yeast topoisomerase genes revealed synergistic effects on growth suggesting overlapping functions . Expression of bacterial topoisomerase I in yeast can fully complement the slow growth defect of a null mutation . We have named this locus TOP3 and suggest that it defines a novel eukaryotic topoisomerase gene. Nucleic Acids Res, 1989 Jul 25, 17(14), 5751 - 64 A point mutation in a mitochondrial tRNA gene abolishes its 3' end processing; Zennaro E et al.; A temperature sensitive mutation mapping in the tRNA region of the mitochondrial genome of S . cerevisiae has been found to abolish 3' processing of tRNA(asp) . Mutant cells grown for a few generations at the non-permissive temperature were found to specifically lack mature tRNA(asp) and to accumulate 3' unprocessed precursors of this tRNA . The accumulation of precursors of other mitochondrial tRNAs was also observed under the same conditions . After longer incubation times, a generalized decrease of mitochondrial transcripts could be observed . The mutation was genetically mapped in a limited region surrounding the tRNA(asp) gene and found, by sequencing, to consist of a C- greater than T transition at position 61 of the tRNA(asp) gene. Nucleic Acids Res, 1989 Jul 11, 17(13), 4993 - 5001 Ty4, a novel low-copy number element in Saccharomyces cerevisiae: one copy is located in a cluster of Ty elements and tRNA genes; Stucka R et al.; We have identified a composite element, Ty4, in S . cerevisiae that is ca 6.3 kb in length and contains two tau sequences as long terminal repeats . According to hybridization analyses, Ty4 occurs in low but varying copy number (one to four copies) in different yeast strains . By several criteria, Ty4 is a novel type of retroelement which is similar but not related to the other Ty elements in yeast . Two cosmid clones from strain C836 (c90 and c476) carrying individual copies of Ty4 were isolated . By restriction analysis and nucleotide sequence we show that c476 derives from the 'transposition right arm hot spot' of chromosome III {1} . The analysis of c476 revealed that an initiator tRNA(Met) gene is present at this locus and that an unusual concentration of different Ty elements has occurred: in addition to the Ty4, a Ty1 and a Ty2 element were detected in this region, confirming its highly polymorphic character. FEBS Lett, 1989 Jul 3, 250(2), 311 - 6 Processing of transcripts of a dimeric tRNA gene in yeast uses the nuclease responsible for maturation of the 3' termini upon 5 S and 37 S precursor rRNAs; Piper PW et al.; The rna82 mutation of Saccharomyces cerevisiae inactivates an RNA processing activity responsible for maturation of 3'-terminal sequences upon 5 S and 37 S ribosomal RNA precursors . This study describes a difference in the processing of transcripts of an S . cerevisiae dimeric tRNA gene (tRNA(arg)-tRNA(Asp) in RNA polymerase III in vitro transcription extracts prepared from rna82 and wild-type cells . The mutant extract accumulated additional processing intermediates containing tRNA(Arg) sequences as compared to the extract from wild-type cells . The structure of these intermediates revealed a defect in removal of the 10 nucleotides left 3' to the tRNA(Arg) sequence by the RNase P cleavage immediately 5' to tRNA(Asp) . This is the first demonstration of a mutational defect affecting maturation of 3' sequences upon a eukaryotic tRNA precursor. Semin Thromb Hemost, 1989 Jul, 15(3), 288 - 92 Production and evaluation of recombinant hirudin; Courtney M et al.; Hirudin, a 65 amino acid polypeptide form the medicinal leech, is an extremely efficient and specific thrombin inhibitor whose therapeutic potential has been demonstrated in a number of animal models . We have developed protocols for the production of recombinant hirudin by secretion from S . cerevisiae and carried out a full biologic evaluation of the purified product . These studies showed that natural and recombinant hirudin was similar in structure and in biologic function in vitro . Moreover, the recombinant protein displayed strong antithrombotic activity in several experimental thrombosis models in vivo, confirming the molecule's promise in the therapy of thrombotic disorders. Biokhimiia, 1989 Jul, 54(7), 1090 - 7 {The relation of glutathione reductase and diaphorase activity of glutathione reductase from Saccharomyces cerevisiae}; Chenas NK et al.; Glutathione reductase from S . cerevisiae (EC 1.6.4.2) catalyzes the NADPH oxidation by glutathione in accordance with a "ping-pong" scheme . The catalytic constant kcat) is 240 s-1 (pH 7.0, 25 degrees C); kcat for the diaphorase reaction is 4-5 s-1 . The enzyme activity does not change markedly at pH 5.5-8.0 . At pH less than or equal to 7.0, NADP+ acts as a competitive inhibitor towards NADPH and as a noncompetitive inhibitor towards glutathione . NADP+ increases the diaphorase activity of the enzyme . The maximal activity is observed, when the NADP+/NADPH ratio exceeds 100 . At pH 8.0, NADP+ acts as a mixed type inhibitor during the reduction of glutathione . High concentrations of NADP+ also inhibit the diaphorase activity due to the reoxidation of the reduced enzyme by NADP+ at pH 8.0 . The redox potential of glutathione reductase calculated from the inhibition data is--306 mV (pH 8.0) . Glutathione reductase reduces quinoidal compounds in an one-electron way . The hyperbolic dependence of the logarithm of the oxidation constant on the one electron reduction potential of quinone is observed . It is assumed that quinones oxidize the equilibtium fraction of the two-electron reduced enzyme containing reduced FAD. FEMS Microbiol Lett, 1989 Jul 1, 51(1), 55 - 9 A possible role for acid phosphatase with thiamin-binding activity encoded by PHO3 in yeast; Nosaka K et al.; Periplasmic soluble thiamin-binding protein in Saccharomyces cerevisiae (Iwashima, A . et al . (1979) Biochim . Biophys . Acta 577, 217-220) was demonstrated to be encoded by PHO3 gene that codes for thiamin repressible acid phosphatase (Schweingruber, M.E . et al . (1986) J . Biol . Chem . 261, 15877-15882) by genetic analysis . The pho3 mutant cells of S . cerevisiae in contrast to the parent cells have markedly reduced activity of the uptake of {14C}thiamin phosphates, suggesting that thiamin repressible acid phosphatase plays a role in the hydrolysis of thiamin phosphates in the periplasmic space prior to the uptake of their thiamin moieties by S . cerevisiae. DNA, 1989 Jul-Aug, 8(6), 409 - 18 Expression of bovine cytochrome P450c17 cDNA in Saccharomyces cerevisiae; Sakaki T et al.; We constructed expression plasmids for bovine adrenal cytochrome P450c17 (P450c17) by inserting the corresponding cDNA between the yeast alcohol dehydrogenase I promoter and terminator of the expression vector pAAH5 . Plasmids pA alpha 1 and pA alpha 2 contained the entire coding region for bovine P450c17, whereas pAC alpha 1 included the cDNA coding for chimeric P450c alpha consisting of the amino-terminal 45 amino acid residues of rat P450c and the carboxy-terminal 482 amino acid residues of bovine P450c17 . The transformed Saccharomyces cerevisiae AH22/pA alpha 1, AH22/pA alpha 2, and AH22/pAC alpha 1 cells produced about 1 x 10(5), 1 x 10(5), and 2 x 10(4) molecules per cell of the corresponding P450 hemoproteins, respectively . On incubation with the cultures of each of the three strains, progesterone was specifically converted into 17 alpha-hydroxyprogesterone, which was not further converted into androstenedione, indicating that the three strains showed 17 alpha-hydroxylase activity, but almost no C17,20-lyase activity . The microsomal fraction prepared from the AH22/pA alpha 1 cells showed 17 alpha-hydroxylase activity toward progesterone and pregnenolone to higher extents, and exhibited C17,20-lyase activity toward 17 alpha-hydroxypregnenolone to a lesser extent and almost no C17,20-lyase activity toward 17 alpha-hydroxyprogesterone . These results indicated that bovine P450c17 synthesized in S . cerevisiae cells manifests the 17 alpha-hydroxylase activity, but not the C17,20-lyase activity. J Clin Microbiol, 1989 Jul, 27(7), 1689 - 91 Saccharomyces cerevisiae pneumonia in a patient with acquired immune deficiency syndrome; Tawfik OW et al.; The clinical course of a patient with a polymicrobial pneumonia that included Saccharomyces cerevisiae infection is described . S . cerevisiae was recovered from autopsy cultures of the lungs, spleen, oral mucosa, and small intestine, and organisms morphologically consistent with S . cerevisiae were visualized in histologic sections of the lung . The role of this organism as a human pathogen is reviewed. Cell, 1989 Jun 30, 57(7), 1223 - 36 S . cerevisiae encodes an essential protein homologous in sequence and function to mammalian BiP; Normington K et al.; The endoplasmic reticulum (ER) of mammalian cells contains a 78 kd protein (BiP) that is believed to assist in the folding of secretory and transmembrane proteins . We have used a cDNA encoding mouse BiP to isolate the homologous gene from S . cerevisiae, which encodes a sequence of 682 amino acids, 431 of which are identical to mouse BiP . Like its mammalian counterpart, yeast BiP is encoded by an HSP70-like gene whose transcription is stimulated by the presence of unfolded polypeptides in the ER . The gene encoding yeast BiP is essential for cell growth and, unexpectedly, is identical to the recently cloned KAR2 gene . Expression of mammalian BiP in S . cerevisiae can complement a mutant allele of KAR2 that is temperature sensitive for growth and nonconditionally defective for karyogamy . These results suggest that deficiencies in BiP may cause generalized failure of protein folding in the ER, leading to pleiotropic effects on cellular metabolism. Cell, 1989 Jun 16, 57(6), 997 - 1007 The fission yeast dis2+ gene required for chromosome disjoining encodes one of two putative type 1 protein phosphatases; Ohkura H et al.; S . pombe dis mutants block mitotic chromosome disjunction in a manner reminiscent of aneuploidy formation, and belong to three distinct genes, dis1-dis3 . We cloned two independent genomic DNAs that complemented both the cold-sensitive and caffeine-hypersensitive phenotype of dis2-11 . These genes, dis2+ and a suppressor sds21+, encode proteins (calculated MW 37,000) with similar predicted amino acid sequences . dis2+ and sds21+ have overlapping functions, and disruptants are lethal only when both genes are disrupted . The gene products identified by anti-dis2 serum are enriched in nuclei . By hybridization, we obtained two cDNA clones from mouse and one genomic clone from S . cerevisiae; the latter complements S . pombe dis2-11 . These dis2+ and similar polypeptides of yeasts and mouse are found to be highly homologous (75%-90% identical) to rabbit protein phosphatase 1 . The implications of these findings are discussed with regard to mitotic control. Wei Sheng Wu Xue Bao, 1989 Jun, 29(3), 174 - 9 {Cloning and expression of tryptophan synthetase gene (TRP5) in yeast}; Yun DF et al.; TRP5, one of five genes required for tryptophan synthetase in S . cerevisiae, has been isolated on recombinant plasmids . A genomic DNA bank, containing the entire yeast genome was constructed by complete digestion of yeast 1412-4D DNA with restriction endonuclease BamH1, size fractionated by sucrose gradient (2-4 kb), and insertion of the fragments into the yeast shuttle vector pCN60 . 9 recombinants plasmids capable of complementing trp5 mutations were isolated by transformation of yeast cell C9 (alpha, trp5, adel, ade6) . The recombinant plasmids, containing 3.2 kb DNA fragments located TRP5 gene, were named pCN60 (TRP5) . Tryptophan synthetase activity of transformants was 3-fold higher than that of original strain 1412-4D. Curr Genet, 1989 Jun, 15(6), 399 - 401 A DNA sequence in Saccharomyces exiguus is homologous with the HO gene of Saccharomyces cerevisiae; Hisatomi T et al.; The DNA of Saccharomyces exiguus was analyzed by Southern hybridization using cloned MATa, MAT alpha, and HO genes of Saccharomyces cerevisiae as probes . It was shown that S . exiguus has a DNA sequence homologous with the HO gene of S . cerevisiae and that this DNA sequence is on a chromosome of about 940 kb of DNA in S . exiguus . However, there is no DNA sequence in S . exiguus that is homologous with the MAT genes of S . cerevisiae. Curr Genet, 1989 Jun, 15(6), 385 - 92 Meiotic segregation of circular plasmid-minichromosomes from intact chromosomes in Saccharomyces cerevisiae; Kaback DB; Distributive disjunction is defined by first meiotic division segregation of either two nonhomologous chromosomes that lack homologous pairing partners, or of two homologous chromosomes that have failed to undergo crossing-over . In the yeast Saccharomyces cerevisiae, plasmid minichromosomes, synthetic linear chromosomes and a fragment of a real chromosome have been observed to segregate from nonhomologous DNA species at the first meiotic divisions . Suggesting that this organism may have a distributive mechanism for chromosome segregation . However, it is not known whether intact chromosomes also participate in a distributive process . To determine whether intact, full length, S . cerevisiae chromosomes could segregate from nonhomologous chromosomal species, the meiotic behavior of an unpaired intact copy of chromosome I has been analyzed with respect to several centromere-containing circular plasmid minichromosomes . Strains monosomic or trisomic for chromosome I were transformed with centromere plasmids containing either homologous or nonhomologous inserts, sporulated, and analyzed genetically both for the presence of plasmid and for the number of copies of chromosome I . Each plasmid segregated from an intact unpaired copy of chromosome I at the first meiotic division in a significant majority (63-93%) of the asci examined . These results suggest that intact chromosomes from S . cerevisiae are capable of distributive disjunction. EMBO J, 1989 Jun, 8(6), 1867 - 77 A DNA binding protein that recognizes oligo(dA).oligo(dT) tracts; Winter E et al.; Oligo(dA).oligo(dT) tracts are common intergenic sequences in many organisms . In the yeast Saccharomyces cerevisiae, these sequences have been shown to influence transcription of adjacent genes . We have purified an oligo(dA).oligo(dT)-binding protein from S . cerevisiae and cloned its gene . This protein, which has been named datin, requires at least 9-11 bp of oligo(dA).oligo(dT) DNA for high affinity binding . The gene for datin (the DAT gene) encodes a 248-residue protein which contains a number of repeated sequence motifs . Datin purified from yeast corresponds to the N-terminal half of the DAT gene product . Null mutants in the DAT gene are viable but phenotypically distinguishable from congenic wild-type strains . We discuss unusual structural features and biochemical properties of datin in relation to its possible functions. Mol Cell Biol, 1989 Jun, 9(6), 2695 - 705 Structure and regulation of KGD1, the structural gene for yeast alpha-ketoglutarate dehydrogenase; Repetto B et al.; Nuclear respiratory-defective mutants of Saccharomyces cerevisiae have been screened for lesions in the mitochondrial alpha-ketoglutarate dehydrogenase complex . Strains assigned to complementation group G70 were ascertained to be deficient in enzyme activity due to mutations in the KGD1 gene coding for the alpha-ketoglutarate dehydrogenase component of the complex . The KGD1 gene has been cloned by transformation of a representative kgd1 mutant, C225/U1, with a recombinant plasmid library of wild-type yeast nuclear DNA . Transformants containing the gene on a multicopy plasmid had three- to four-times-higher alpha-ketoglutarate dehydrogenase activity than did wild-type S . cerevisiae . Substitution of the chromosomal copy of KGD1 with a disrupted allele (kgd1::URA3) induced a deficiency in alpha-ketoglutarate dehydrogenase . The sequence of the cloned region of DNA which complements kgd1 mutants was found to have an open reading frame of 3,042 nucleotides capable of coding for a protein of Mw 114,470 . The encoded protein had 38% identical residues with the reported sequence of alpha-ketoglutarate dehydrogenase from Escherichia coli . Two lines of evidence indicated that transcription of KGD1 is catabolite repressed . Higher steady-state levels of KGD1 mRNA were detected in wild-type yeast grown on the nonrepressible sugar galactose than in yeast grown on high glucose . Regulation of KGD1 was also studied by fusing different 5'-flanking regions of KGD1 to the lacZ gene of E . coli and measuring the expression of beta-galactosidase in yeast . Transformants harboring a fusion of 693 nucleotides of the 5'-flanking sequence expressed 10 times more beta-galactosidase activity when grown under derepressed conditions . The response to the carbon source was reduced dramatically when the same lacZ fusion was present in a hap2 or hap3 mutant . The promoter element(s) responsible for the regulated expression of KGD1 has been mapped to the -354 to -143 region . This region contained several putative activation sites with sequences matching the core element proposed to be essential for binding of the HAP2 and HAP3 regulatory proteins. J Biol Chem, 1989 May 25, 264(15), 9022 - 9 Molecular cloning of Saccharomyces cerevisiae CDC6 gene . Isolation, identification, and sequence analysis; Zhou C et al.; The CDC6 gene product is required for entering the S phase of the cell cycle in Saccharomyces cerevisiae . It has been isolated on recombinant plasmids by selection for complementation of temperature-sensitive alleles with a yeast genomic library . The entire complementing activity is carried on a 1.8-kilobase chromosomal DNA fragment, as revealed by deletion mapping . Northern blotting shows that the size of the CDC6 mRNA is about 1.7 kilobases . A Southern blot of yeast chromosomes which were separated by the field inversion gel electrophoresis method indicates that the isolated DNA fragment is derived from chromosome X . The locus from which the clone was derived was marked by integration with a nutritional marker and found by meiotic mapping to cosegregate with CDC6 . Thus, we conclude that we have isolated the authentic CDC6 gene . Nucleotide sequence analysis of the CDC6 gene has revealed an open reading frame that encodes a protein with Mr = 57,969 . There are five potential Asn-X-(Ser/Thr) glycosylation sites and a highly conserved nucleotide-binding site in the CDC6 sequence . Although computer surveys indicate overall sequence homology between S . cerevisiae CDC6 protein and Saccharomyces pombe CDC10 START protein, they may not be functionally equivalent as evaluated by the complementation assay. J Biol Chem, 1989 May 25, 264(15), 8753 - 8 Cloning of the glutamine:fructose-6-phosphate amidotransferase gene from yeast . Pheromonal regulation of its transcription; Watzele G et al.; The activity of the amino sugar-synthesizing enzyme L-glutamine:D-fructose-6-phosphate aminotransferase (EC 2.6.1.16) in haploid a cells of Saccharomyces cerevisiae increases 1.7-fold after alpha factor addition . The gene (the gene should be called GFA1 for glutamine:fructose-6-phosphate amidotransferase) for the enzyme has been cloned by complementing the gcn1 mutation (Whelan, W . L., and Ballou, C . E . (1975) J . Bacteriol . 125, 1545-1557) . Its expression is increased 2-3 times within 15 min when the mating pheromone is added . The gene codes for a protein of 716 amino acids in length . It is highly homologous (64%) to the corresponding gene of Escherichia coli, except for a sequence coding for 83 amino acids (numbers 204-286), which is lacking in E . coli . The amino-terminal region of the coding sequence also shows a high degree of homology to the corresponding sequence of the E . coli and S . cerevisiae L-glutamine:phosphoribosylpyrophosphate amidotransferase . In the promotor region of the S . cerevisiae L-glutamine:D-fructose-6-phosphate amidotransferase gene the heptanucleotide "TGAAACA," shown to be required for pheromone control of transcription (Kronstad, J . W., Holly, J . A., and MacKay, V . L . (1987) Cell 50, 369-377), is present six times. Cell, 1989 May 19, 57(4), 675 - 81 Estrogen can regulate the cell cycle in the early G1 phase of yeast by increasing the amount of adenylate cyclase mRNA; Tanaka S et al.; The effects of beta-estradiol (estrogen; a minor component of yeast cells) on S . cerevisiae cells in the G0 and G1 phases were examined . Results showed that estrogen stimulated the recovery of growth from G0 arrest induced by nutrient limitation or ts mutation of cdc35 (adenylate cyclase) in the early G1 phase, and inhibited entry into the resting G0 phase by increasing the intracellular cAMP level . However, estrogen had no effect on late G1 arrest induced by the alpha factor or ts mutation of cdc36 . Estrogen was found to lead to higher steady-state levels of adenylate cyclase mRNA but not to affect the expression of the RAS1 and RAS2 genes, although these can also alter the intracellular cAMP level . These results suggest that estrogen influences the cell cycle of yeast in the early G1 phase by controlling the level of cAMP through the increase of adenylate cyclase mRNA. Nucleic Acids Res, 1989 May 11, 17(9), 3435 - 46 Expression of pGKL killer 28K subunit in Saccharomyces cerevisiae: identification of 28K subunit as a killer protein; Tokunaga M et al.; Saccharomyces cerevisiae and other yeast cells harboring the linear double stranded (ds) DNA plasmids pGKL1 and pGKL2 secrete a killer toxin consisting of 97K, 31K and 28K subunits into the culture medium (EMBO J . 5, 1995-2002 (1986), Nucleic Acids Res., 15, 1031-1046 (1987} . The 28K subunit of the killer toxin was successfully expressed in S . cerevisiae when it was cloned on a circular plasmid with its putative promoter region replaced with that of S . cerevisiae chromosomal genes . The expression of the 28K subunit of the killer toxin in killer-sensitive cells resulted in the death of the host cells . This killing activity by the 28K subunit was prevented by the expression of the killer immunity, indicating that the killing activity of the killer toxin complex was carried out by the 28K subunit . Although the 28K subunit was synthesized as a intact precursor protein with its own signal sequence, it was not secreted into the culture medium but remained in the host cells . This indicated that 28K subunit killed host cells from inside of the cells rather than from outside . We further suggested that 28K killer subunit without 97K and 31K subunits did not kill the killer-sensitive cells from outside. FEBS Lett, 1989 May 8, 248(1-2), 111 - 4 O-mannosylation of recombinant human insulin-like growth factor I (IGF-I) produced in Saccharomyces cerevisiae; Hard K et al.; A glycosylated form of recombinant human insulin-like growth factor I (IGF-I) expressed in Saccharomyces cerevisiae was shown to contain mannose as the only carbohydrate constituent . All oligosaccharide chains of the glycoprotein could be released by mild alkaline treatment, and separated from the protein by gel-permeation chromatography on Bio-Gel P-2 . The structures of these O-linked carbohydrate chains were determined by 500-MHz 1H-NMR spectroscopy, affording the disaccharide Man alpha 1-2Man as the major component and the tetrasaccharide Man alpha 1-3Man alpha 1-2Man alpha 1-2Man as a minor component . Reference oligosaccharides were prepared from mannoproteins released from the cell wall of S . cerevisiae X2180 (alpha-wild type) . In addition to previously reported structures, ranging from mannose to mannotetraose, the pentasaccharide Man alpha 1-3Man alpha 1-3Man alpha 1-2Man alpha 1-2Man was identified in the cell wall mannoprotein. Cell, 1989 May 5, 57(3), 459 - 67 Coconversion of flanking sequences with homothallic switching; McGill C et al.; Homothallic switching in S . cerevisiae involves replacing the DNA of the expressed allele at the mating type locus (MAT) with a duplicate of sequences from the unexpressed loci HML or HMR . The MATa and MAT alpha alleles differ by a DNA substitution that is flanked by sequences in common to MAT, and the donor loci HML and HMR . Using restriction site polymorphisms between MAT and the donor loci, we demonstrate that the extent of MAT DNA that is replaced during switching is variable and that there is a gradient of coconversion across the X region . Coconversion events occur on both sides of the double-strand cleavage by the HO gene product . The two cells produced after a switch often differ at the flanking site, indicating a DNA heteroduplex intermediate. Curr Genet, 1989 May, 15(5), 319 - 25 Regulated overproduction of alpha-amylase by transformation of the amylolytic yeast Schwanniomyces occidentalis; Dohmen RJ et al.; High frequency transformation of a Schwanniomyces occidentalis mutant defective in the last step of tryptophan synthesis was achieved with plasmids containing the tryptophan synthetase gene (TRP5) of Saccharomyces cerevisiae and an autonomous replication sequence from S . occidentalis, which we called "SwARS1" . The SwARS1 fragment is also functional in S . cerevisiae . The average copy number of the plasmids in both yeast species was 5-10 per cell under selective conditions . S . occidentalis cells that were transformed with an autonomously replicating plasmid carrying the cloned alpha-amylase gene from S . occidentalis secreted about five times more alpha-amylase than cells without additional copies of the alpha-amylase gene . Both the chromosomal copy and the plasmid-carried copies of the alpha-amylase gene were repressed in the presence of glucose . This transformation system provides a possibility to improve starch degradation by S . occidentalis. Electrophoresis, 1989 May-Jun, 10(5-6), 296 - 302 Transverse alternating field electrophoresis and applications to mammalian genome mapping; Gardiner K et al.; The transverse alternating field electrophoresis system is a pulsed field gel apparatus that has been used to separate DNA molecules that range in size from a few thousand to approximately 7 million base pairs . This apparatus uses a vertical gel and a simple electrode arrangement to produce electric fields that are uniform across all lanes of the gel . The velocity of identical molecules does not vary from lane to lane, and hence there is no distortion in the paths of the DNA . The performance of this system is illustrated here using the chromosomes from S . pombe and S . cerevisiae, and restriction enzyme digested mammalian DNA . The mobility of molecules up to 1100 kilobase pairs is linear with size and can be accomplished in overnight runs . Resolution of very large molecules requires electrophoresis for several days, but molecules from 200 to 7000 kilobase pairs can be separated on a single gel . This electrophoresis system has been used extensively in the construction of a physical map of human chromosome 21, and examples of this application are discussed. Proc Natl Acad Sci U S A, 1989 May, 86(10), 3694 - 8 Enhanced meiotic recombination on the smallest chromosome of Saccharomyces cerevisiae; Kaback DB et al.; Chromosome I is the smallest chromosome in Saccharomyces cerevisiae and contains a DNA molecule that is only 250 kilobases (kb) . Approximately 75% of this DNA molecule has been cloned . A restriction map for the entire DNA molecule from chromosome I was determined and most of its genetically mapped genes were located on this physical map . Based on the average rate of recombination (centimorgans/kb) found for other S . cerevisiae chromosomes, the outermost markers on the genetic map of chromosome I were expected to be close to the ends of the DNA molecule . While the rightmost genetic marker was 3 kb from the end, the leftmost marker, CDC24, was located near the middle of the left arm, suggesting that the genetic map would be much longer . To extend the genetic map, a copy of the S . cerevisiae URA3 gene was integrated in the outermost cloned region located 32 kb centromere distal to CDC24, and the genetic map distance between these two genes was determined . The new marker substantially increased the genetic map length of chromosome I . In addition, we determined the relationship between physical and genetic map distance along most of the length of the chromosome . Consistent with the longer genetic map, the average rate of recombination between markers on chromosome I was greater than 50% higher than the average found on other yeast chromosomes . Owing to its small size, it had been estimated that approximately 5% of the chromosome I homologues failed to undergo meiotic recombination . New measurements of the zero-crossover class indicated that the enhanced rate of recombination ensures at least one genetic exchange between virtually every pair of chromosome I homologues. Cell, 1989 Apr 21, 57(2), 295 - 303 Conservation of mitotic controls in fission and budding yeasts; Russell P et al.; In fission yeast, the initiation of mitosis is regulated by a control network that integrates the opposing activities of mitotic inducers and inhibitors . To evaluate whether this control system is likely to be conserved among eukaryotes, we have investigated whether a similar mitotic control operates in the distantly related budding yeast S . cerevisiae . We have found that the protein kinase encoded by the mitotic inhibitor gene wee1+ of fission yeast, which acts to delay mitosis, is able also to delay the initiation of mitosis when expressed in S . cerevisiae . The wee1+ activity is counteracted in S . cerevisiae by the gene product of MIH1, a newly identified gene capable of encoding a protein of MW 54,000, which is a structural and functional homolog of the cdc25+ mitotic inducer of fission yeast . Expression of wee1+ in a mih1- strain prevents the initiation of mitosis . These data indicate that important features of the cdc25+-wee1+ mitotic control network identified in S . pombe are conserved in S . cerevisiae, and therefore are also likely to be generally conserved among eukaryotic organisms. Gene, 1989 Apr 15, 77(1), 21 - 30 The C-terminal part of a gene partially homologous to CDC 25 gene suppresses the cdc25-5 mutation in Saccharomyces cerevisiae; Boy-Marcotte E et al.; In Saccharomyces cerevisiae, the product of the CDC25 gene is required for progression in the cell division cycle . It is necessary for cAMP production . It has been suggested that the CDC25 gene product acts through Ras proteins . We report the cloning of a DNA fragment from a new gene able to suppress the thermosensitive phenotype of the cdc25-5 mutation . It is unable to suppress the defect of a mutant of the adenylate cyclase gene or of the ras1, ras2ts double mutant . This DNA fragment prevents the drop in cAMP level in cdc25-5 mutant cells shifted to restrictive temperature . The complementing part of this fragment contains a truncated open reading frame (ORF) corresponding to the 3' end of a gene we named SCD25 . The 584-amino acid sequence deduced from this ORF shares 45% identity with the 592-aa C-terminal part of the CDC25 ORF which is sufficient for complementation of cdc25 mutations . Some of the common sequences between these two genes are also partially homologous with the amino acid sequence of LTE1, another gene of S . cerevisiae . The capacity of the SCD25 fragment to suppress a cdc25 mutation and its homology to the C-terminal part of the CDC25 led us to propose that the CDC25 and the SCD25 C-terminal fragments each encode a protein domain which is capable in itself to support a similar biochemical function. Biochemistry, 1989 Apr 4, 28(7), 2941 - 9 Purification and biochemical characterization of recombinant hirudin produced by Saccharomyces cerevisiae; Riehl-Bellon N et al.; Recombinant hirudin was produced by the yeast Saccharomyces cerevisiae using the alpha-pheromone prepro sequence to direct its secretion into the culture medium . The secreted hirudin was isolated to greater than or equal to 95% purity as measured by 205-nm absorbance integration from a reverse-phase chromatogram . One major activity peak corresponding to the complete, correctly processed molecule and two minor activity peaks corresponding to C-terminally truncated forms were identified . The primary structure of the major peak, determined by N-terminal sequencing of tryptic peptides, was that predicted from the cDNA sequence, and the molecular mass analyzed by fast atom bombardment mass spectrometry (FAB-MS) was 6892.6 (calculated 6892.5) . UV spectral analysis suggested that, in contrast to the natural molecule, recombinant hirudin produced by S . cerevisiae is not sulfated. Curr Genet, 1989 Apr, 15(4), 277 - 82 An optional group I intron between the chloroplast small subunit rRNA genes of Chlamydomonas moewusii and C . eugametos; Durocher V et al.; We report the presence of a 402 bp group I intron in the chloroplast small subunit (SSU) rRNA gene of Chlamydomonas moewusii . The intron in inserted within the highly conserved '530 loop', at a site corresponding to positions 531-532 of the E . coli 16rRNA . Residues surrounding the insertion site almost certainly play an important role in ribosomal proofreading function as they proved to be protected by tRNAs in E . coli 16S rRNA (Moazed and Noller 1986; Stern et al . 1986) . The C . moewusii intron revealed a secondary structure model which differs substantially from those of the typical subgroup IA and IB introns . This model, however, shows striking similarities with the structures of the C . reinhardtii chloroplast 23S rRNA gene intron (Rochaix et al . 1985), the S . cerevisiae mitochondrial COB3 intron (Holl et al . 1985) and the three introns of phage T4 in the nrdB, td and sunY genes (Shub et al . 1988) . The SSU rRNA gene intron is absent from C . eugametos, an alga that is interfertile with C . moewusii . The presence/absence of the intron account for a 390 bp restriction fragment length polymorphism between the two algal SSU rRNA genes, a polymorphic locus that is strictly co-inherited with a tightly linked streptomycin resistance mutation (sr-2) in interspecific hybrids between the two algae. Antibiot Khimioter, 1989 Apr, 34(4), 276 - 9 {Study of immunoregulating properties of an interferon inducer in animals with various reactions to the antigen}; Sysoeva GM et al.; Influence of dsRNA isolated from killer yeast of S . cerevisiae on humoral and cellular immune responses in mice CBA/CaY and C57Bl/6Y with opposing reaction to the antigen was studied . It was shown that after administration of the yeast dsRNA preparation to the animals simultaneously with the antigen there was an increase in the number of the antibody forming cells in the spleen and the titer of hemolytic antibodies in blood serum of the animals with high and low reactions to the antigen . After sensitization with different doses of sheep red blood cells (10(7) and 10(8)) the preparation had immunomodulating action on development of DTH in mice CBA/CaY . The effect of the dsRNA preparation on the immunity system depended on the preparation dose, antigen loading and animal genotype and was the most marked in mice CBA/CaY with interferon levels in blood serum 2-3 times higher than those in mice C57Bl/6Y. Yeast, 1989 Apr, 5 Spec No, S459 - 63 Sterol uptake by anaerobically grown Saccharomyces cerevisiae; Youings A et al.; Wild-type Saccharomyces cerevisiae Y185 grown anaerobically was used to investigate the uptake and subsequent utilisation of sterol in S . cerevisiae . Results confirmed a minimum free sterol concentration below which growth was retarded . The yeast does, however, appear capable of taking up sterol well in excess of that required for growth . Evidence is presented indicating the ability of the yeast cell wall to bind cholesterol both in vivo and with isolated cell walls . There appears to be a role for low density vesicles in the movement of sterol once within the cell. Mol Cell Biol, 1989 Apr, 9(4), 1488 - 97 Introduction of extra telomeric DNA sequences into Saccharomyces cerevisiae results in telomere elongation; Runge KW et al.; The termini of Saccharomyces cerevisiae chromosomes consist of tracts of C1-3A (one to three cytosine and one adenine residue) sequences of approximately 450 base pairs in length . To gain insights into trans-acting factors at telomeres, high-copy-number linear and circular plasmids containing tracts of C1-3A sequences were introduced into S . cerevisiae . We devised a novel system to distinguish by color colonies that maintained the vector at 1 to 5, 20 to 50, and 100 to 400 copies per cell and used it to change the amount of telomeric DNA sequences per cell . An increase in the number of C1-3A sequences caused an increase in the length of telomeric C1-3A repeats that was proportional to plasmid copy number . Our data suggest that telomere growth is inhibited by a limiting factor(s) that specifically recognizes C1-3A sequences and that this factor can be effectively competed for by long tracts of C1-3A sequences at telomeres or on circular plasmids . Telomeres without this factor are exposed to processes that serve to lengthen chromosome ends. J Bacteriol, 1989 Apr, 171(4), 1862 - 9 Nucleotide sequence of the wild-type RAD4 gene of Saccharomyces cerevisiae and characterization of mutant rad4 alleles; Couto LB et al.; Shuttle plasmids carrying the wild-type RAD4 gene of Saccharomyces cerevisiae cannot be propagated in Escherichia coli (R . Fleer, W . Siede, and E . C . Friedberg, J . Bacteriol . 169:4884-4892, 1987) . In order to determine the nucleotide sequence of the cloned gene, we used a plasmid carrying a mutant allele that allows plasmid propagation in E . coli . The wild-type sequence in the region of this mutation was determined from a second plasmid carrying a different mutant rad4 allele . We established the locations and characteristics of a number of spontaneously generated plasmid-borne RAD4 mutations that alleviate the toxicity of the wild-type gene in E . coli and of several mutagen-induced chromosomal mutations that inactivate the excision repair function of RAD4 . These mutations are situated in very close proximity to each other, and all are expected to result in the expression of truncated polypeptides missing the carboxy-terminal one-third of the Rad4 polypeptide . This region of the gene may be important both for the toxic effect of the Rad4 protein in E . coli and for its role in DNA repair in S . cerevisiae. Curr Genet, 1989 Apr, 15(4), 239 - 46 A mitochondrial frameshift suppressor maps in the tRNASer-var1 region of the mitochondrial genome of the yeast S . cerevisiae; Weiss-Brummer B et al.; A polypeptide chain-terminating mutation (M5631) previously has been shown to be a +1T insertion in the yeast mitochondrial gene oxi1, coding for subunit II of the cytochrome c oxidase . A spontaneously arisen frameshift suppressor (mfs-1) that is mitochondrially inherited suppresses this mutation to a considerable extent . The suppressor mutation was mapped by genetic and molecular analyses in the mitochondrial tRNASer-var1 region of the mitochondrial genome of the yeast S . cerevisiae . Genetic analyses show that the suppressor mfs-1 does not suppress other known mitochondrial frameshift mutations, or missense and nonsense mutations. Yeast, 1989 Apr, 5 Spec No, S313 - 20 Increased extracellular secretion in fragile mutants of Saccharomyces cerevisiae; Waltschewa L et al.; Evidences are presented for a generally increased extracellular secretion capability of the fragile mutants of S . cerevisiae . Proteins secreted in wild type yeasts to the periplasmic space can not be retained by the defective cell wall of the fragile cells and are released into the medium . Changes in the structure of non-mannan components of the cell wall might be the reason for the extracellular release of the periplasmic proteins. Mol Cell Biol, 1989 Apr, 9(4), 1794 - 8 Conserved pattern of antisense overlapping transcription in the homologous human ERCC-1 and yeast RAD10 DNA repair gene regions; van Duin M et al.; We report that the genes for the homologous Saccharomyces cerevisiae RAD10 and human ERCC-1 DNA excision repair proteins harbor overlapping antisense transcription units in their 3' regions . Since naturally occurring antisense transcription is rare in S . cerevisiae and humans (this is the first example in human cells), our findings indicate that antisense transcription in the ERCC-1-RAD10 gene regions represents an evolutionarily conserved feature. FEBS Lett, 1989 Mar 27, 246(1-2), 49 - 53 Characterisation of the reactivity of autoantibodies in primary biliary cirrhosis; Fussey SP et al.; Autoantibodies in the sera of patients with primary biliary cirrhosis, shown previously to recognise the E2 polypeptide of the mammalian pyruvate dehydrogenase complex (PDC), have been demonstrated to react with the E2 component of PDC from bacteria (E . coli) and yeast (S . cerevisiae) . Limited tryptic digestion, which cleaves E2 into well-characterised domains, followed by Western blotting indicates that the main immunodominant region of PDC E2 lies within the lipoic acid-containing domains of the polypeptide. Cell, 1989 Mar 24, 56(6), 1019 - 32 The degradation signal in a short-lived protein; Bachmair A et al.; Our previous work has shown that the amino-terminal residue of a short-lived protein is a distinct component of the protein's degradation signal . To define the complete signal, otherwise identical dihydrofolate reductase test proteins bearing different extensions and either a "stabilizing" or a "destabilizing" amino-terminal residue were expressed in the yeast S . cerevisiae and their in vivo half-lives compared . The amino-terminal degradation signal is shown to comprise two distinct determinants . One, discovered previously, is the protein's amino-terminal residue . The second determinant, identified in the present work, is a specific lysine residue whose function in the degradation signal is not dependent on the unique amino acid sequences in the vicinity of the residue . The mechanistic significance of the second determinant is illuminated by the finding that in a targeted, short-lived protein, a chain of branched ubiquitin-ubiquitin conjugates is confined to a lysine residue that has been identified in the present work as the second determinant of the degradation signal. Biochemistry, 1989 Mar 7, 28(5), 2099 - 106 Site-directed mutagenesis of yeast C1-tetrahydrofolate synthase: analysis of an overlapping active site in a multifunctional enzyme; Barlowe CK et al.; C1-tetrahydrofolate (THF) synthase is a trifunctional protein possessing the activities 10-formyl-THF synthetase, 5,10-methenyl-THF cyclohydrolase, and 5,10-methylene-THF dehydrogenase . The current model divides this protein into two functionally independent domains with dehydrogenase/cyclohydrolase activities sharing an overlapping active site on the N-terminal domain and synthetase activity associated with the C-terminal domain . Previous chemical modification studies on C1-THF synthase from the yeast Saccharomyces cerevisiae indicated at least two cysteinyl residues involved in the dehydrogenase/cyclohydrolase reactions {Appling, D . R., & Rabinowitz, J . C . (1985) Biochemistry 24, 3540-3547} . In the present work, site-directed mutagenesis of the S . cerevisiae ADE3 gene, which encodes C1-THF synthase, was used to individually change each cysteine contained within the dehydrogenase/cyclohydrolase domain (Cys-11, Cys-144, and Cys-257) to serine . The resulting proteins were overexpressed in yeast and purified for kinetic analysis . Site-specific mutations in the dehydrogenase/cyclohydrolase domain did not affect synthetase activity, consistent with the proposed domain structure . The C144S and C257S mutations result in 7- and 2-fold increases, respectively, in the dehydrogenase Km for NADP+ . C144S lowers the dehydrogenase maximal velocity roughly 50% while C257S has a maximal velocity similar to that of the wild type . Cyclohydrolase catalytic activity is reduced 20-fold by the C144S mutation but increased 2-fold by the C257S mutation . Conversion of Cys-11 to serine has a negligible effect on dehydrogenase/cyclohydrolase activity . A double mutant, C144S/C257S, results in catalytic properties roughly multiplicative of the individual mutations.(ABSTRACT TRUNCATED AT 250 WORDS) Curr Genet, 1989 Mar, 15(3), 171 - 5 Identification, cloning and characterisation of a new gene required for full pyruvate decarboxylase activity in Saccharomyces cerevisiae; Wright AP et al.; Biochemical evidence that pyruvate decarboxylase in S . cerevisiae might be constituted from two independently encoded subunits led us to question genetic evidence for a single structural gene . The main evidence for this was that three "structural" mutations appeared to be alleles of the same gene, PDC1 (Schmitt and Zimmermann 1982) . We report that one of these mutations (pdc1-30) is not allelic either to other pdc1 alleles or to pdc2 mutations and therefore is has been renamed pdc3-30 thus identifying a new gene, PDC3 . We have cloned the PDC3 gene, it represents a unique sequence in the genome and targeted integration shows tight linkage to the PDC3 locus . However, the size, abundance and regulation of the PDC3 transcript suggest that it does not encode a second structural gene . Possible functions for the PDC3 gene product are discussed. Yeast, 1989 Mar-Apr, 5(2), 131 - 9 Two pathways of DNA double-strand break repair in G1 cells of Saccharomyces cerevisiae; Glasunov AV et al.; G1 cells of the diploid yeast Saccharomyces cerevisiae are known to be capable of a slow repair of DNA double-strand breaks (DSB) during holding the cells in a non-nutrient medium (Luchnik et al., 1977; Frankenberg-Schwager et al., 1980) . In the present paper, S . cerevisiae cells gamma-irradiated in the G1 phase of the cell cycle are shown to be capable of fast repair of DNA DSB; this process is completed within 30-40 min of holding the cells in water at 28 degrees C . For this reason, the kinetics of DNA DSB repair during holding the cells in a non-nutrient medium are biphasic, i.e., the first, 'fast' phase is completed within 30-40 min, whereas the second, 'slow' phase is completed within 48 h . Mutations rad51, rad52, rad54 and rad55 inhibit the fast repair of DNA DSB, whereas mutations rad50, rad53 and rad57 do not significantly influence this process . It has been shown that the observed fast and slow repair of DNA DSB in the G1 diploid cells of S . cerevisiae are separate pathways of DNA DSB repair in yeast. Mol Cell Biol, 1989 Mar, 9(3), 1309 - 15 Control of Saccharomyces cerevisiae catalase T gene (CTT1) expression by nutrient supply via the RAS-cyclic AMP pathway; Bissinger PH et al.; In Saccharomyces cerevisiae, lack of nutrients triggers a pleiotropic response characterized by accumulation of storage carbohydrates, early G1 arrest, and sporulation of a/alpha diploids . This response is thought to be mediated by RAS proteins, adenylate cyclase, and cyclic AMP (cAMP)-dependent protein kinases . This study shows that expression of the S . cerevisiae gene coding for a cytoplasmic catalase T (CTT1) is controlled by this pathway: it is regulated by the availability of nutrients . Lack of a nitrogen, sulfur, or phosphorus source causes a high-level expression of the gene . Studies with strains with mutations in the RAS-cAMP pathway and supplementation of a rca1 mutant with cAMP show that CTT1 expression is under negative control by a cAMP-dependent protein kinase and that nutrient control of CTT1 gene expression is mediated by this pathway . Strains containing a CTT1-Escherichia coli lacZ fusion gene have been used to isolate mutants with mutations in the pathway . Mutants characterized in this investigation fall into five complementation groups . Both cdc25 and ras2 alleles were identified among these mutants. Plasmid, 1989 Mar, 21(2), 147 - 50 Versatile cassettes designed for the copper inducible expression of proteins in yeast; Macreadie IG et al.; A series of yeast expression vectors and cassettes utilizing the CUP1 gene of Saccharomyces cerevisiae have been constructed . The cassettes contain multiple cloning sites for gene fusions and were created by inserting a 27-bp polylinker at the +14 position of the CUP1 gene . The cassettes are portable as restriction fragments and enable copper-regulated expression of foreign proteins in S . cerevisiae . In copper sensitive yeast, multiple copies of the CUP1 cassettes confer copper resistance due to the production of the copper metallothionein . Genes cloned into the CUP1 cassettes, however, usually prevent translation of the metallothionein leading to a loss of resistance . This could be useful for one-step cloning into yeast. Nucleic Acids Res, 1989 Feb 25, 17(4), 1353 - 69 The acid phosphatase genes PHO10 and PHO11 in S . cerevisiae are located at the telomeres of chromosomes VIII and I; Venter U et al.; Of the three regulated acid phosphatase genes in S . cerevisiae (PHO5, PHO10 and PHO11) two have previously been cloned (PHO5 and PHO11) . We have now identified PHO10 and show by restriction mapping that it is highly homologous to PHO11 . This homology includes not only the coding sequence but also a stretch of about 2 kb upstream and 2.2 kb downstream of the genes . Analysis of strains in which either gene had been disrupted shows that the two genes are located at the telomeres of two different chromosomes . PHO10 3.6 kb from the end of a chromosome I . This makes PHO11 the gene closest to the end of a chromosome that has been physically mapped so far in S . cerevisiae . The organization of the two genes varies strongly from strain to strain consistent with a high incidence of telomere rearrangement . In one of twenty transformants examined a conversion event could be directly demonstrated that resulted in a chromosome VIII which had acquired a copy of the telomere from chromosome I. Cell, 1989 Feb 24, 56(4), 619 - 30 Elevated recombination rates in transcriptionally active DNA; Thomas BJ et al.; We have examined the effect of RNA polymerase II-dependent transcription on recombination between directly repeated sequences of the GAL10 gene in S . cerevisiae . Direct repeat recombination leading either to plasmid loss or conversion was examined in isogenic strains containing null mutations in the positive activator, GAL4, or the repressor, GAL80 . A 15-fold increase in the rate of plasmid loss is observed in cells constitutively expressing the construct compared with cells that are not . Conversion events that retain the integrated plasmid are not stimulated by expression of the repeats . Northern analysis of strains containing plasmid inserts with various promoter mutations suggests that the stimulation in recombination is mediated by events initiating within the integrated plasmid sequences. Cell, 1989 Feb 24, 56(4), 599 - 605 DNA polymerase III, a second essential DNA polymerase, is encoded by the S . cerevisiae CDC2 gene; Sitney KC et al.; Three nuclear DNA polymerases have been described in yeast: DNA polymerases I, II, and III . DNA polymerase I is encoded by the POL1 gene and is essential for DNA replication . Since the S . cerevisiae CDC2 gene has recently been shown to have DNA sequence similarity to the active site regions of other known DNA polymerases, but to nevertheless be different from DNA polymerase I, we examined cdc2 mutants for the presence of DNA polymerases II and III . DNA polymerase II was not affected by the cdc2 mutation . DNA polymerase III activity was significantly reduced in the cdc2-1 cell extracts . We conclude that the CDC2 gene encodes yeast DNA polymerase III and that DNA polymerase III, therefore, represents a second essential DNA polymerase in yeast. Nucleic Acids Res, 1989 Feb 11, 17(3), 1019 - 34 A structural analysis of P . polycephalum U1 RNA at the RNA and gene levels . Are ther differentially expressed U1 RNA genes in P . polycephalum? U1 RNA evolution; Myslinski E et al.; U1 RNAs were isolated from P . polycephalum microplasmodia nuclei and sequenced . A P . polycephalum gene coding for U1 RNA was also isolated . The coding region of this gene differs at 3 positions compared to the isolated U1 RNA species . Both isolated RNAs and the gene encoded RNA can be folded according to the secondary structure model previously proposed for U1 RNA . Putative regulatory elements very similar to those required for efficient transcription of U RNA genes from vertebrates, in particular, the -200 distal enhancer element, are present in the flanking regions of this gene . The presence of several U1 RNA genes in P . polycephalum was confirmed by Southern b