Chin J Biotechnol, 1994, 10(3), 163 - 8 Expression and secretion of glucoamylase of Aspergillus niger in Saccharomyces cerevisiae; Tang G et al.; Glucoamylase cDNA synthesized from A . niger mutant T21 was modified at 5' and 3' ends in order to clone it into yeast shuttle plasmid YFD18 and to cause fusion between cDNA and the leader region of the yeast-mating pheromone alpha-factor . The modified cDNA was then inserted into YFD18 at HindIII site . Saccharomyces cerevisiae Y33 was transformed with the resultant recombinant plasmid YFD18HH6 . Analysis of transformants including halo formation on starch medium plate, SDS-PAGE of culture filtration and determination of glucoamylase activity showed that the yeast transformed with plasmid containing glucoamylase cDNA efficiently secreted glucoamylase into the medium . This fact indicated that yeast alpha-factor was able to direct the synthesis and secretion of functional glucoamylase of Aspergillus . In addition the protolytic cleavage site involved in the maturation of glucoamylase in A . niger also worked in S . cerevisiae. Biochimie, 1994, 76(7), 674 - 92 The cytochrome b5-fold: an adaptable module; Lederer F; The family of b5-like cytochromes encompasses, besides cytochrome b5 itself, hemoprotein domains covalently associated with other redox proteins, in flavocytochrome b2 (L-lactate dehydrogenase), sulfite oxidase and assimilatory nitrate reductase . A comparison of about 40 amino acid sequences deposited in data banks shows that eight residues are invariant and about 15 positions carry strongly conservative substitutions . Examination of the location of these invariant and conserved positions in the light of the three-dimensional structures of beef cytochrome b5 and S cerevisiae flavocytochrome b2 suggests a strongly conserved protein structure for the b5-like heme-binding domain throughout evolution . Numerous NMR studies have demonstrated the existence of a positional isomerism for the heme, which involves both a 180 degree-rotation around the heme alpha,gamma-meso carbon atoms and a rotation through an axis normal to the heme plane at the iron . NMR studies did not detect significant differences in protein structure between reduced and oxidized states, or between species . The role of a number of side chains was probed by site-directed mutagenesis . Studies of complex formation and of electron transfer rates between cytochrome b5 and redox partners have led to the idea that complexation is driven by electrostatic forces, that it is generally the exposed heme edge which makes contact with electron donors and acceptors, but that there are multiple overlapping sites within this general area . For the bi- and trifunctional members of the family, extrapolation of available data would suggest a mobile heme-binding domain within a complex structure . In these cases the existence of a single interaction area for both electron donor and acceptor, or of two different ones, remains open to discussion. Biochimie, 1994, 76(6), 501 - 14 Structural studies on recombinant and point mutants of flavocytochrome b2; Tegoni M et al.; Flavocytochrome b2 from S cerevisiae is a homotetramer with a molecular mass of 4 x 58 kDa . It catalyses the oxidation of L-lactate into pyruvate and the electron transfer to cytochrome c in the mitochondrial intermembrane space . Each monomer is composed of a flavinmononucleotide (FMN) carrying domain and a 'b5-like' heme domain . The wild type structure has been described at a resolution of 2.4 A . We report here on the refined structure of the E . coli native recombinant flavocytochrome b2 from S cerevisiae inhibited by sulphite and that of two point mutants, Y143F and Y254F, in which pyruvate is bound to the active site . The crystals, obtained under very different conditions from those of the native enzyme, are isostructural (P 3(2) 2 1, a=b=164.5 A, c=114.0 A) . In line with the similarities found to exist in the kinetic behaviour of the native and recombinant protein, few structural differences were observed here, and the crystallographic data further confirm the intrinsic mobility of the heme domain . The superimposable position of the aromatic rings of Phe 143 in the mutant Y143F and Tyr 143 in the native protein makes it seem unlikely that the aromatic ring may be directly involved in the intramolecular electron transfer . The fact that a very restricted number of domain interactions was observed in Y143F shows that Tyr 143 is one of the amino acids essential to the formation of the productive complex . In the Y143F mutant, the number of catalytically efficient complexes is probably drastically decreased, which will severely limit the rate of intramolecular election transfer . The structure of Y254F shows a reorientation of the substrate at the active site . Together with the kinetic results, this finding definitely excludes the possibility that Tyr 254 may act as general base and that the substrate may interact directly with Phe 254 in the mutant . The model between flavocytochrome b2 and cytochrome c will serve as a basis for designing suitable mutants of the amino acids involved either in the interaction or the electron transfer. Cell Mol Biol Res, 1994, 40(4), 273 - 84 The expression and processing of human beta-amyloid peptide precursors in Saccharomyces cerevisiae: evidence for a novel endopeptidase in the yeast secretory system; Hines V et al.; In mammalian cells, the transmembrane beta-amyloid peptide precursor (beta-APP) undergoes a complex series of alternative proteolytic processing steps that result in the secretion of varying proportions of its extra-cellular domain (protease nexin II) and beta-amyloid peptide . The protein is also reinternalized and degraded in the endosomal-lysosomal system . The relative efficiencies of these competing processes determine the yield of beta-amyloid peptide . Several proteases have been implicated in this complex processing pathway, although none has been identified to date . The yeast secretory system contains proteases homologous to mammalian pro-hormone convertases and is susceptible to genetic manipulation . We therefore investigated the expression and processing of the beta-amyloid peptide precursors (beta-APP-695 and beta-APP-751) in Saccharomyces cerevisiae transformed with human beta-APP cDNA's . beta-APP (695 or 751) cDNA either with its authentic signal sequence or the yeast-derived prepro-alpha-factor leader, was inserted into a glucose-regulated expression vector and transfected into a protease-deficient yeast strain . In all instances, expression of beta-APP was about 1% of total protein . Protease protection studies indicated that either the natural human signal sequence or the alpha-factor leader sequence targetted beta-APP to the endoplasmic reticulum and inserted it with the amino-terminal domain in the lumen . All of the beta-APP fused to the alpha-factor leader proceeded to the trans-Golgi, where Kex2 endopeptidase removed the leader and released the normal amino-terminus of beta-APP . About one-half of the beta-APP was also cleaved at the "alpha-secretase" site in the middle of the beta-peptide sequence, 12 residues before the membrane-spanning sequence . A fraction of the alpha-secretase-cleaved beta-APP appeared in the culture medium; however, most of it associated with the exterior of the cells . The carboxyl-terminal fragments formed by cleavage at the alpha-secretase site accumulated in the membranes . Other proteolytic processes generated membrane-associated carboxyl-terminal fragments that also resembled those found in mammalian cells . These results indicate that the secretory system of S . cerevisiae possesses proteases with specificities similar to the mammalian enzymes that process beta-APP. Antonie Van Leeuwenhoek, 1994, 65(3), 211 - 6 Protein targeting and secretion in filamentous fungi . A progress report; Punt PJ et al.; Although the application of filamentous fungi, such as Aspergillus niger for the production of extracellular proteins is well established for several decades, hardly any information is available about the molecular mechanisms of the process of protein secretion in these organisms . Two lines of research initiated towards a systematic analysis of the mechanism of protein targeting and secretion are presented in this paper . 1--To study routing and targeting of proteins in filamentous fungi the availability of a versatile reporter/carrier protein will be of considerable importance . Experiments towards the identification of such a protein are presented . 2--In analogy to the situation in Saccharomyces cerevisiae, the availability of defined (conditional) mutations in the secretion pathway will provide very important information about the organisation of the pathway . Therefore, based on results obtained in S . cerevisiae, the cloning of several fungal 'secretion' genes was started . The results of the cloning and characterisation of one of these genes is presented. Biosci Biotechnol Biochem, 1994 Jan, 58(1), 208 - 10 Primary structure of the ADE1 gene from Candida utilis; Nishiya Y; The ADE1 gene from Candida utilis CA(u)-37, a strain used for commercially producing enzymes, was cloned by complementation of the ade1 mutation of Saccharomyces cerevisiae . It was composed of 903 bp, and the deduced amino acid sequence was 70% homologous to those of the ADE1 genes of S . cerevisiae and Candida maltosa . The highly preserved region of SAICAR synthetase, the ADE1 gene product, was also found by a homology search. Biotechnology (N Y), 1994 Jan, 12(1), 47 - 50 Current methods for manipulating baculoviruses; Davies AH; Recombinant baculoviruses have become popular expression vectors for heterologous proteins . Recently, several novel strategies for manipulating the baculovirus genome have been developed . These include linearizing the viral genome at the point of insertion of the foreign gene to be expressed, reconstituting a replicating baculovirus genome in S . cerevisiae and E . coli, and in vitro enzymatically mediated recombination into the genome . Each of these techniques constitutes a distinct approach to the problem of manipulating this complex DNA molecule, and each has distinct advantages for the various purposes to which the recombinant viruses may be applied. Cell Mol Biol Res, 1994, 40(5-6), 481 - 8 Structure and function of Saccharomyces cerevisiae casein kinase II; Glover CV et al.; Analysis of casein kinase II in organisms amenable to genetic manipulation is essential to elucidating the physiological function(s) of this ubiquitous protein kinase . This paper summarizes work from our laboratory on the enzyme from Saccharomyces cerevisiae . The biochemistry, molecular biology, and genetics of S . cerevisiae casein kinase II are reviewed and discussed. Folia Microbiol (Praha), 1994, 39(4), 287 - 90 Regulation of sterol biosynthesis in Saccharomyces cerevisiae; Behalova B et al.; Sterol synthesis in Saccharomyces cerevisiae was primarily controlled by the growth rate . At low specific growth rates the intermediates of ergosterol biosynthesis prevailed in cells . At the same time, the total sterol content reached about 6% of dry matter whereas the content of ergosterol was only 2-2.5%, which seems to be the maximum value for S . cerevisiae . After esterification with fatty acids these sterol intermediates are stored in lipid globules together with reserve triacylglycerols . The sporulating S . cerevisiae cells contained 3.5% sterols and 1.5% ergosterol of dry matter. Mol Biol Rep, 1994-95, 20(3), 135 - 41 Isolation and characterization of a multienzyme complex containing DNA replicative enzymes from mitochondria of S . cerevisiae . Multienzyme complex from yeast mitochondria; Murthy V et al.; A 40 S multienzyme complex containing mtDNA polymerase was isolated from mitochondria of S . cerevisiae by density gradient centrifugation and by gel filtration chromatography . Besides DNA polymerase, RNA polymerase, primase, 3'-->5' exonuclease and an ATPase activities were found to be associated with it . The presence of some of these enzymes were confirmed by Western blot . This high molecular weight multienzyme complex containing DNA has most of the attributes of a putative replisome. Gene, 1993 Dec 31, 137(2), 265 - 70 Isolation and nucleotide sequences of the genes encoding killer toxins from Hansenula mrakii and H . saturnus; Kimura T et al.; The HMK gene, encoding a killer toxin (HMK) of Hansenula mrakii strain IFO 0895, and the HSK gene, encoding a killer toxin (HSK) of H . saturnus strain IFO 0117, were cloned and sequenced . The HMK and HSK genes encode precursors to killer toxins of 125 amino acids (aa) and 124 aa, respectively . Both precursors have an N-terminal signal sequence of 37 aa which may be removed by a signal peptidase, and a propeptide which may be cleaved off by a KEX2-like protease . There is extensive homology between the aa sequences of HMK and HSK with the exception of the addition of one aa residue in HMK . The HMK and HSK genes were placed, separately, downstream from the yeast GAL10 promoter and introduced into a mutant of Saccharomyces cerevisiae that was resistant to the HMK . The transformants were capable of killing sensitive yeasts in medium that contained galactose with killing spectra similar to those of the donor strains of the toxins . These observations suggest that both killer toxins were synthesized and secreted from S . cerevisiae cells and killed sensitive yeasts, perhaps by the same mechanism as that associated with the donor strains and, moreover, that the difference in primary structure between the two toxins is responsible for the difference in their killing spectra. Gene, 1993 Dec 31, 137(2), 223 - 6 Construction of glucose-repressible yeast expression vectors; Yao B et al.; A set of two episomal yeast expression vectors, pYME1 and pYME2, were constructed . These Saccharomyces cerevisiae-Escherichia coli shuttle vectors each contain a modified yeast MAL6S (encoding maltase) promoter that is expressed constitutively, but is subject to carbon catabolite repression by glucose . Expression from this promoter is still dependent upon the presence of active MALR (regulatory) protein . These expression vectors are particularly useful because most S . cerevisiae strains are MAL+, thereby exhibiting a wider host range than GAL-based vector systems . These pYME1 and pYME2 vectors are capable of expression to levels comparable to GAL-based expression plasmids and much higher than a variety of other repressible promoter vectors . The vectors are identical, except that their multiple cloning sites (MCS) are in opposite orientations, making them convenient for inserting heterologous genes. Gene, 1993 Dec 31, 137(2), 179 - 85 Cloning of the dihydroxyacid dehydratase-encoding gene (ILV3) from Saccharomyces cerevisiae; Velasco JA et al.; The biosynthesis of branched-chain amino acids (aa) involves three shared pathways through which pyruvate or alpha-ketobutyrate are converted into alpha-keto acids, precursors of valine, leucine or isoleucine . In eukaryotes, few of these common enzymes have been purified to homogeneity, and the whole complement of biosynthetic genes has not been cloned from a single species . In yeasts, most of these genes (ILV genes) have been cloned and sequenced, with the exception of that coding for dihydroxyacid dehydratase (DAD, EC 4.2.1.9), the third enzyme in the common pathways . We have isolated Saccharomyces cerevisiae genomic sequences by hybridization to an oligodeoxyribonucleotide (oligo) probe designed from a highly conserved domain among bacterial DAD-encoding genes . The cloned sequences have been located to S . cerevisiae chromosome X, mapped within 0.4 centiMorgans (cM) of the ilv3 locus, and found to complement the ilv3 mutations of various yeast strains . Nucleotide (nt) and aa sequence analyses of the longest open reading frame (ORF) located within the cloned sequences identified them as the ILV3 gene, which codes for the yeast DAD . With our cloning of ILV3, yeast becomes the only eukaryotic system from which all ILV genes have been cloned, thus allowing direct molecular analyses of their regulation. Cell, 1993 Dec 31, 75(7), 1379 - 87 Dual roles of a multiprotein complex from S . cerevisiae in transcription and DNA repair; Feaver WJ et al.; Yeast RNA polymerase II initiation factor b, homolog of human TFIIH, is a protein kinase capable of phosphorylating the C-terminal repeat domain of the polymerase; it possesses a DNA-dependent ATPase activity as well . The 85 kd and 50 kd subunits of factor b are now identified as RAD3 and SSL1 proteins, respectively; both are known to be involved in DNA repair . Factor b interacts specifically with another DNA repair protein, SSL2 . The ATPase activity of factor b may be due entirely to that associated with a helicase function of RAD3 . Factor b transcriptional activity was unaffected, however, by amino acid substitution at a conserved residue in the RAD3 nucleotide-binding domain, suggesting that the ATPase/helicase function is not required for transcription . These results identify factor b as a core repairosome, which may be responsible for the preferential repair of actively transcribed genes in eukaryotes. Biochem Biophys Res Commun, 1993 Dec 30, 197(3), 1173 - 8 Identification of a hyperactive mating pheromone of Saccharomyces cerevisiae; Caldwell GA et al.; The yeast mating pheromone a-factor is a farnesylated peptide {YIIKGVFWDPAC(Farnesyl)-OCH3} involved in the signal transduction cascade which leads to sexual conjugation of haploid cells . We have identified a synthetic analog of the a-factor, {D-Ala5} a-factor, which exhibits 4-6 fold greater biological activity than that of a-factor as judged by two different assay systems . In contrast, {L-Ala5} a-factor has 4-16 fold lower activity than wild-type a-factor . {D-Ala5} a-factor remains susceptible to cleavage by the yeast alpha-cell specific protease a-factorase, thereby ruling out increased activity due to greater stability . This report describes the first example of a hyperactive S . cerevisiae pheromone and may suggest a conformationally preferred form of this lipopeptide ligand. Nucleic Acids Res, 1993 Dec 25, 21(25), 5945 - 9 Identification of a chicken RAD52 homologue suggests conservation of the RAD52 recombination pathway throughout the evolution of higher eukaryotes; Bezzubova OY et al.; Degenerate oligonucleotides encoding conserved regions of the Rad52 protein of S . cerevisiae and its homologue, the Rad22 protein of S . pombe, were used to clone a chicken RAD52 counterpart by the polymerase chain reaction . Sequence comparison of the chicken and yeast proteins reveals a strongly conserved region between positions 40 and 178 of the chicken Rad52 sequence indicating that this part of the protein is under strong evolutionary pressure . The first 39 amino acids and the 3' end of the chicken Rad52 homologue does not share significant similarity with the yeast proteins . High abundance of the mRNA in testis makes it likely that the chicken Rad52 protein plays a role in meiotic recombination. Nucleic Acids Res, 1993 Dec 25, 21(25), 5803 - 16 Analysis of the RNA-recognition motif and RS and RGG domains: conservation in metazoan pre-mRNA splicing factors; Birney E et al.; We present a systematic analysis of sequence motifs found in metazoan protein factors involved in constitutive pre-mRNA splicing and in alternative splicing regulation . Using profile analysis we constructed a database enriched in protein sequences containing one or more presumptive copies of the RNA-recognition motif (RRM) . We provide an accurate alignment of RRMs and structure-based criteria for identifying new RRMs, including many that lack the prototype RNP-1 submotif . We present a comprehensive table of 125 sequences containing 252 RRMs, including 22 previously unreported RRMs in 17 proteins . The presence of a putative RRM in these proteins, which are implicated in a variety of cellular processes, strongly suggests that their function involves binding to RNA . Unreported homologies in the RRM-enriched database to the metazoan SR family of splicing factors are described for an Arg-rich human nuclear protein and two yeast proteins (S . pombe mei2 and S . cerevisiae Npl3) . We have rigorously tested the phylogenetic relationships of a large sample of RRMs . This analysis indicates that the RRM is an ancient conserved region (ACR) that has diversified by duplication of genes and intragenic domains . Statistical analyses and classification of repeated Arg-Ser (RS) and RGG domains in various protein splicing factors are presented. J Biol Chem, 1993 Dec 25, 268(36), 27148 - 53 Purification and characterization of a new DNA polymerase from budding yeast Saccharomyces cerevisiae . A probable homolog of mammalian DNA polymerase beta; Shimizu K et al.; A new DNA polymerase activity was identified and purified to near homogeneity from extracts of mitotic and meiotic cells of the yeast Saccharomyces cerevisiae . This activity increased at least 5-fold during meiosis, and it was shown to be associated with a 68-kDa polypeptide as determined by SDS-polyacrylamide gel electrophoresis . This new DNA polymerase did not have any detectable 3'-->5' exonuclease activity and preferred small gapped DNA as a template-primer . The activity was inhibited by dideoxyribonucleoside 5'-triphosphates and N-ethylmaleimide but not by concentrations of aphidicolin which completely inhibit either DNA polymerases I (alpha), II (epsilon), or III (delta) . Since no polypeptide(s) in the extensively purified DNA polymerase fractions cross-reacted with antibodies raised against yeast DNA polymerases I, II, and III, we called this enzyme DNA polymerase IV . The DNA polymerase IV activity increased at least 10-fold in a yeast strain overexpressing the gene product predicted from the YCR14C open-reading frame (identified on S . cerevisiae chromosome III and provisionally called POLX), while no activity was detected in a strain where POLX was deleted . These results strongly suggest that DNA polymerase IV is encoded by the POLX gene and is a probable homolog of mammalian DNA polymerase beta. Gene, 1993 Dec 22, 136(1-2), 295 - 300 Cloning and sequence analysis of an H(+)-ATPase-encoding gene from the human dimorphic pathogen Histoplasma capsulatum; Schafer MP et al.; A gene related to the PMA1 gene from Saccharomyces cerevisiae was isolated from the pathogenic human dimorphic fungus, Histoplasma capsulatum, using fungal-specific oligodeoxyribonucleotide (oligo) probes . This gene has been given the name Hc-PMA1 . The structural organization of Hc-PMA1 consists of three exons (375, 2329 and 44 bp) and two introns (115 and 116 bp) . The nucleotide sequence predicts an H(+)-ATPase-related protein of 916 amino acids (aa) . Comparison of the deduced aa sequence to that of Neurospora crassa and S . cerevisiae (PMA1) plasma membrane H(+)-ATPases showed a greater similarity to that from N . crassa (85% identity) . Furthermore, the two introns in the Hc-PMA1 gene interrupt the coding region in the precise locations determined for two of the four N . crassa Nc-PMA introns . H . capsulatum intron 1 contains two repeat motifs, d(TA)16 and d(TG)10, each potentially capable of forming non-B DNA structures . Northern analysis of H . capsulatum total RNA indicated that the Hc-PMA1-specific mRNA is approx . 3.3 kb in size, in agreement with the predicted size of the gene. Science, 1993 Dec 17, 262(5141), 1838 - 44 Origin recognition complex (ORC) in transcriptional silencing and DNA replication in S . cerevisiae; Foss M et al.; In Saccharomyces cerevisiae, the HMR-E silencer blocks site-specific interactions between proteins and their recognition sequences in the vicinity of the silencer . Silencer function is correlated with the firing of an origin of replication at HMR-E . An essential gene with a role in transcriptional silencing was identified by means of a screen for mutations affecting expression of HMR . This gene, known as ORC2, was shown to encode a component of the origin recognition complex that binds yeast origins of replication . A temperature-sensitive mutation in ORC2 disrupted silencing in cells grown at the permissive temperature . At the restrictive temperature, the orc2-1 mutation caused cell cycle arrest at a point in the cell cycle indicative of blocks in DNA replication . The orc2-1 mutation also resulted in the enhanced mitotic loss of a plasmid, suggestive of a defect in replication . These results provide strong evidence for an in vivo role of ORC in both chromosomal replication and silencing, and provide a link between the mechanism of silencing and DNA replication. Biochem Biophys Res Commun, 1993 Dec 15, 197(2), 372 - 9 Detection of purine cytosine permease of S . cerevisiae: use of antibodies against a synthetic peptide corresponding to a predicted sequence in the N-terminal domain of the protein; Grandier-Vazeille X et al.; A synthetic peptide, selected in the predicted N-terminal amino-acid sequence of the purine cytosine permease (gene FCY2), linked to albumins proved a remarkably good immunogen in rabbits . In ELISA, sera reacted with the synthetic peptide and with specific proteins of plasma-membrane-enriched fractions of mutant Saccharomyces cerevisiae pAB strains (amplified FCY2 gene) with high titers and high avidity . Western blots of plasma membrane proteins of pAB strain probed with antisera showed two bands: a major (45 kDa) and minor band (50 kDa) . On the contrary, plasma-membrane-enriched fractions of mutant S . cerevisiae pJDB strain (deficient in FCY2 gene) gave no signal when probed in the same conditions . These results demonstrate the specificity of the antisera and also suggest that the 45 kDa and 50 kDa proteins are both products of the FCY2 gene. Proc Natl Acad Sci U S A, 1993 Dec 15, 90(24), 11855 - 9 Protection against chloroethylnitrosourea cytotoxicity by eukaryotic 3-methyladenine DNA glycosylase; Matijasevic Z et al.; A eukaryotic 3-methyladenine DNA glycosylase gene, the Saccharomyces cerevisiae MAG gene, was shown to prevent N-(2-chloroethyl)-N-nitrosourea toxicity . Disruption of the MAG gene by insertion of the URA3 gene increased the sensitivity of S . cerevisiae cells to N-(2-chloroethyl)-N-nitrosourea, and the expression of MAG in glycosylase-deficient Escherichia coli cells protected against the cytotoxic effects of N-(2-chloroethyl)-N-nitrosourea . Extracts of E . coli cells that contain and express the MAG gene released 7-hydroxyethylguanine and 7-chloroethylguanine from N-(2-chloroethyl)-N-nitrosourea-modified DNA in a protein- and time-dependent manner . The ability of a eukaryotic glycosylase to protect cells from the cytotoxic effects of a haloethylnitrosourea and to release N-(2-chloroethyl)-N-nitrosourea-induced DNA modifications suggests that mammalian glycosylases may play a role in the resistance of tumor cells to the antitumor effects of the haloethylnitrosoureas. Proc Natl Acad Sci U S A, 1993 Dec 15, 90(24), 11782 - 6 PAS10 is a tetratricopeptide-repeat protein that is essential for the import of most matrix proteins into peroxisomes of Saccharomyces cerevisiae; Van der Leij I et al.; pas mutants of Saccharomyces cerevisiae are disturbed in peroxisome assembly (pas) and proliferation . Here we report the characterization of the PAS10 gene and its product (PAS10) that is essential for the import of a large subset of proteins into the peroxisomal matrix . PAS10, a protein of 69 kDa, is a member of the tetratricopeptide repeat, or snap helix, protein family, characterized by several direct repeats of a degenerate 34-amino acid motif (Sikorski, R . S., Boguski, M . S., Goebl, M . & Hieter, P . (1990) Cell 60, 307-317) . Other members of this family are MAS70 (S . cerevisiae) and MOM72 (Neurospora crassa), which are mitochondrial receptors for protein import . A pas10 null mutant accumulates peroxisomal, leaflet-like membrane structures and exhibits deficient import of a number of peroxisomal matrix enzymes, particularly of proteins with an SKL-like import signal . In contrast, 3-ketoacyl-CoA thiolase associated with these membranes is resistant in vitro to degradation by proteinase K, indicating true protein import . These results suggest that PAS10 is an essential component of a peroxisomal import machinery which mediates the translocation of a specific subset of proteins to the peroxisomal matrix with an SKL-like import signal. EMBO J, 1993 Dec 15, 12(13), 5051 - 6 MAC1, a nuclear regulatory protein related to Cu-dependent transcription factors is involved in Cu/Fe utilization and stress resistance in yeast; Jungmann J et al.; The related transcription factors ACE1 of Saccharomyces cerevisiae and AMT1 of Candida glabrata are involved in copper metabolism by activating the transcription of copper metallothionein genes . ACE1 and AMT1 are 'copper-fist' transcription factors which possess a conserved cysteine-rich copper binding domain required for DNA binding . Here we report the identification of a nuclear protein from S . cerevisiae, MAC1, whose N-terminal region is highly similar to the copper and DNA binding domains of ACE1 and AMT1 . Loss-of-function mutants of MAC1 have a defect in the plasma membrane Cu(II) and Fe(III) reductase activity, are slow growing, respiratory deficient, and hypersensitive to heat and exposure to cadmium, zinc, lead and H2O2 . Conversely, a dominant gain-of-function mutant of MAC1 shows an elevated reductase activity and is hypersensitive to copper . We have identified two target genes of MAC1 whose altered expression in mutants of MAC1 can account for some of the observed mutant phenotypes . First, MAC1 is involved in basal level transcription of FRE1, encoding a plasma membrane component associated with both Cu(II) and Fe(III) reduction . Second, MAC1 is involved in the H2O2-induced transcription of CTT1, encoding the cytosolic catalase . This suggests that MAC1 may encode a novel metal-fist transcription factor required for both basal and regulated transcription of genes involved in Cu/Fe utilization and the stress response. FEBS Lett, 1993 Dec 13, 335(3), 380 - 5 Molecular analysis of SAR1-related cDNAs from a mouse pituitary cell line; Shen KA et al.; Vesicular transport between the endoplasmic reticulum (ER) and the Golgi in the yeast Saccharomyces cerevisiae requires a Ras-like, small GTP-binding protein, Sar1p {1-3} . Whether a functional homologue operates in export from the ER in mammalian cells is unknown, nor is it clear if transport in other branches of the secretory pathway requires member(s) of a gene family . In this study, we used a PCR approach to examine the complexity of SAR1-related sequences expressed in mammalian cells that possess multiple secretory pathways . Amplification of cDNA sequences from rodent pituitary cells with primers corresponding to two conserved GTP binding domains of Sar1p yielded several clones with sequences homologous to Sar1 and/or the closely related ADP-ribosylation factor (ARF) family . Of these, only two showed closer homologies to S . cerevisiae Sar1 than members of the ARF family and are designated as mSARa and mSARb . Northern blot analysis shows that mSARa is expressed in most tissues including liver, heart, brain, skeletal muscle and kidney . In contrast, mSARb is preferentially expressed in skeletal muscle and liver . The full-length cDNA of mSARa isolated from a mouse pituitary AtT-20 cDNA library encodes a protein of 198 amino acids, and is 61.6% identical to Sar1p from S . cerevisiae . Thus in contrast to the large rab family of GTP-binding proteins, vesicular transport in mammalian cells appears to be mediated by a relatively small number of Sar1-related proteins. FEBS Lett, 1993 Dec 6, 335(2), 273 - 6 Internal ribosome entry site of encephalomyocarditis virus RNA is unable to direct translation in Saccharomyces cerevisiae; Evstafieva AG et al.; To evaluate the potential of the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) to promote efficient expression of foreign genes in the yeast, S . cerevisiae, we have constructed E . coli-yeast shuttle vectors in which the EMCV 5' non-coding region was fused to the reporter gene, human prothymosin alpha . Efficiency of translation of corresponding RNA transcripts in mammalian cell-free systems was highly dependent on the sequence context and/or position of the initiation codon . No translation of these IRES-dependent mRNAs occurred in S . cerevisiae. Cell, 1993 Dec 3, 75(5), 1027 - 38 The human mutator gene homolog MSH2 and its association with hereditary nonpolyposis colon cancer; Fishel R et al.; We have identified a human homolog of the bacterial MutS and S . cerevisiae MSH proteins, called hMSH2 . Expression of hMSH2 in E . coli causes a dominant mutator phenotype, suggesting that hMSH2, like other divergent MutS homologs, interferes with the normal bacterial mismatch repair pathway . hMSH2 maps to human chromosome 2p22-21 near a locus implicated in hereditary nonpolyposis colon cancer (HNPCC) . A T to C transition mutation has been detected in the -6 position of a splice acceptor site in sporadic colon tumors and in affected individuals of two small HNPCC kindreds . These data and reports indicating that S . cerevisiae msh2 mutations cause an instability of dinucleotide repeats like those associated with HNPCC suggest that hMSH2 is the HNPCC gene. Anat Rec, 1993 Dec, 237(4), 441 - 52 Modulation of the Golgi apparatus in Saccharomyces cerevisiae sec7 mutants as seen by three-dimensional electron microscopy; Rambourg A et al.; The three-dimensional configuration of the Golgi apparatus has been examined with the electron microscope in thick Golgi sections of Saccharomyces cerevisiae prepared from a wild-type strain and from sec7 mutants maintained for various periods of time at the nonpermissive temperature of 37 degrees C and then returned to the permissive temperature of 24 degrees C . Reduced osmium postfixation of glutaraldehyde fixed specimens stained intensely the content of Golgi elements and thus facilitated their three-dimensional characterization . In wild-type S . cerevisiae, the Golgi elements usually appeared as isolated networks of membranous tubules dispersed throughout the cytoplasm . Along such networks, distensions filled with stained material were similar in size to nearby secretory granules, suggesting that the latter formed by fragmentation of the Golgi elements . In sec7 mutants maintained at 37 degrees C in low (0.1%) glucose medium, secretion granules progressively decreased in number and soon disappeared . Concomitantly the networks of Golgi tubules increased in size and complexity, lost their distensions, and then transformed into flattened saccules forming stacks of up to seven or eight saccules that were similar to the Golgi stacks seen in mammalian cells . However in contrast to the latter, connections between the saccules were evident and Golgi-associated small vesicles were generally absent . Following return to the permissive temperature (24 degrees C), secretion granules reappeared, the Golgi stacks progressively decreased in size, and resumed their initial state of separated small tubular networks . Thus in sec7 mutant, grown at 37 degrees C in low glucose medium, segregation of secretory granules is blocked . As a result, Golgi membranes accumulate to form a continuous system of stacked and interconnected saccules. Curr Genet, 1993 Dec, 24(6), 487 - 90 Transport properties of a C . albicans amino-acid permease whose putative gene was cloned and expressed in S . cerevisiae; Sychrova H et al.; Using a gene bank of C . albicans, the lysine-permease deficiency in a strain of S . cerevisiae was complemented, and the restriction map of the corresponding C . albicans DNA fragment was constructed . Its expression in S . cerevisiae showed that the permease of C . albicans actively transports arginine (KT = 18 mumol/l, Jmax = 26 nmol/min per mg dry weight), lysine (KT = 12 mumol/l, Jmax = 18 nmol/min per mg dry weight), histidine (KT = 37 mumol/l, Jmax = 9.7 nmol/min per mg dry weight), as well as their toxic analogues canavanine and thialysine, with high affinity . The intracellular concentration of basic amino acids transported into S . cerevisiae by the C . albicans permease reaches more than a thousand-times-higher value compared to the external concentration in the medium . Accumulated amino acids do not leave the cells . The uptake is strongly reduced by the protonophores and inhibitors of plasma membrane H(+)-ATPase. Curr Genet, 1993 Dec, 24(6), 472 - 80 Regulation of the ADE2 gene from Saccharomyces cerevisiae; Stotz A et al.; Regulation of ADE2 gene expression was investigated in the yeast S . cerevisiae using translational fusions between this gene and the lacZ gene from E . coli . Expression was repressed in the presence of adenine and slightly increased under amino-acid starvation conditions . The promoters of the ADE2 gene, and of other genes involved in adenine biosynthesis, contain the hexanucleotide sequence TGACTC . A search for the hexanucleotide TGACTC in yeast promoter sequences revealed that many genes not related to amino-acid biosynthesis contain such sequences . We show here that these elements play a crucial role in ADE2 regulation since mutations in two such elements drastically reduced gene expression . Maximal expression required the transcriptional activators Bas1, Bas2 and Gcn4, whereas Yap1 had only minor effects. Mol Gen Genet, 1993 Dec, 241(5-6), 680 - 4 Co-regulation with genes of phospholipid biosynthesis of the CTR/HNM1-encoded choline/nitrogen mustard permease in Saccharomyces cerevisiae; Li Z et al.; An 815 bp region of the promoter of the Saccharomyces cerevisiae gene CTR/HNM1, encoding choline permease was sequenced and its regulatory function analysed by deletion studies in an in-frame promoter-lacZ construct . In addition to the TATA box, a 10 bp motif (consensus 5'-CATGTGAAAT-3') was found to be mandatory for CTR/HNM1 expression . This 'decamer' motif is located between nucleotides -262 and -271 and is identical in 9 of 10 bp with the regulatory motif found in the S . cerevisiae INO1 and CHO1 genes . Constructs with the 10 bp sequence show high constitutive expression, while elimination or alterations at three nucleotide positions, of the decamer motif in the context of an otherwise unchaged promoter leads to total loss of beta-galactosidase production . Expression of the CTR/HNM1 gene in wild-type cells is regulated by the phospholipid precursors inositol and choline; no such influence is seen in cells bearing mutations in the phospholipid regulatory genes INO2, INO4, and OPI1 . There is no regulation by INO2 and OPI1 in the absence of the decamer motif . However constructs not containing this sequence (promoter intact to positions -213 or -152) are still controlled by INO4 . Other substrates of the choline permease, i.e . ethanolamine, nitrogen mustard and nitrogen half mustard do not regulate expression of CTR/HNM1. Mol Cell Biol, 1993 Dec, 13(12), 7718 - 24 Regulated and constitutive activity by CDC25Mm (GRF), a Ras-specific exchange factor; Cen H et al.; Serum stimulates cells to increase their proportion of Ras protein in the active GTP-bound state . We have recently identified four types (I to IV) of apparently full-length cDNAs from a single mammalian gene, called CDC25Mm or GRF, which is homologous to the Ras-specific exchange factor CDC25 of S . cerevisiae . The largest cDNA (type IV) is brain specific, with the other three classes, although they have distinct 5' ends, essentially representing progressive N-terminal deletions of this cDNA . When placed in a retroviral expression vector, all four types of cDNAs induced morphologic transformation of NIH 3T3 cells and an increase in the basal level of GTP.Ras . Serum stimulation of these transformants lead to a further increase in GTP.Ras only in cells expressing the type IV cDNA . Each type of GRF protein was found in cytosolic and membrane fractions, and the protein in each fraction could stimulate guanine nucleotide release from GDP.Ras in vitro . When NIH 3T3 cells and cells expressing the type IV protein were transfected with two versions of a mutant ras gene, one encoding membrane-associated Ras protein and the other encoding a cytosolic Ras protein, the basal levels of GTP bound to both forms of the mutant Ras protein were significantly higher in the cells expressing the type IV protein . However, serum increased the level of GTP bound to the membrane-associated mutant Ras protein in NIH 3T3 cells and in cells expressing the type IV protein but not in cells expressing the cytosolic version of the Ras protein . We conclude that each type of CDC25Mm induces cell transformation via the ability of its C terminus to stimulate guanine nucleotide exchange on Ras, the presence of N-terminal sequences is associated with a serum-dependent change in GTP.Ras, and the serum-dependent increase in GTP.Ras by exogenous CDC25Mm or by endogenous exchange factors probably requires membrane association of both Ras and the exchange factor. Mol Cell Biol, 1993 Dec, 13(12), 7213 - 21 A common element involved in transcriptional regulation of two DNA alkylation repair genes (MAG and MGT1) of Saccharomyces cerevisiae; Xiao W et al.; The Saccharomyces cerevisiae MAG gene encodes a 3-methyladenine DNA glycosylase that protects cells from killing by alkylating agents . MAG mRNA levels are induced not only by alkylating agents but also by DNA-damaging agents that do not produce alkylated DNA . We constructed a MAG-lacZ gene fusion to help identify the cis-acting promoter elements involved in regulating MAG expression . Deletion analysis defined the presence of one upstream activating sequence and one upstream repressing sequence (URS) and suggested the presence of a second URS . One of the MAG URS elements matches a decamer consensus sequence present in the promoters of 11 other S . cerevisiae DNA repair and metabolism genes, including the MGT1 gene, which encodes an O6-methylguanine DNA repair methyltransferase . Two proteins of 26 and 39 kDa bind specifically to the MAG and MGT1 URS elements . We suggest that the URS-binding proteins may play an important role in the coordinate regulation of these S . cerevisiae DNA repair genes. J Bacteriol, 1993 Dec, 175(23), 7689 - 96 Physiological characterization of putative high-affinity glucose transport protein Hxt2 of Saccharomyces cerevisiae by use of anti-synthetic peptide antibodies; Wendell DL et al.; Characterization and quantification of the Hxt2 (hexose transport) protein of Saccharomyces cerevisiae indicate that it is one of a set of differentially expressed high-affinity glucose transporters . The protein product of the HXT2 gene was specifically detected by antibodies raised against a synthetic peptide encompassing the 13 carboxyl-terminal amino acids predicted by the HXT2 gene sequence . Hxt2 migrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a broad band or closely spaced doublet with an average M(r) of 47,000 . Hxt2 cofractionated with the plasma membrane ATPase, Pma1, indicating that it is a plasma membrane protein . Hxt2 was not solubilized by high pH or urea but was solublized by detergents, which is characteristic of an integral membrane protein . Expression of the Hxt2 protein was measured under two different conditions that produce expression of high-affinity glucose transport: a medium shift from a high (2.0%) to a low (0.05%) glucose concentration (referred to below as high and low glucose) and growth from high to low glucose . Hxt2 as measured by immunoblotting increased 20-fold upon a shift from high-glucose to low-glucose medium, and the high-affinity glucose transport expressed had a strong HXT2-dependent component . Surprisingly, Hxt2 was not detectable when S . cerevisiae growing in high glucose approached glucose exhaustion, and the high-affinity glucose transport expressed under these conditions did not have an HXT2-dependent component . The role of Hxt2 in growth during aerobic batch culture in low-glucose medium was examined . An hxt2 null mutant grew and consumed glucose significantly more slowly than the wild type, and this phenotype correlated directly with appearance of the Hxt2 protein. J Virol, 1993 Dec, 67(12), 7472 - 81 Comparing regions of the Epstein-Barr virus ZEBRA protein which function as transcriptional activating sequences in Saccharomyces cerevisiae and in B cells; Miller G et al.; The ZEBRA protein activates expression of Epstein-Barr virus early-lytic-cycle genes in human B lymphocytes . Here it is shown that ZEBRA also behaves as a sequence-specific transcriptional activator in Saccharomyces cerevisiae . Deletional mutagenesis defined three regions of ZEBRA that participate in activation in S . cerevisiae . These regions are designated YI (amino acids {aa} 1 to 25), YII (aa 51 to 102), and YIII (aa 228 to 245) . Two of the three regions of the native ZEBRA protein act together to mediate activation when assayed on ZEBRA binding sites . However, when fused to the DNA binding domain of GAL4 and assayed on GAL4 binding sites, regions YII and YIII were each sufficient to confer activation in S . cerevisiae . Regions of ZEBRA which affected activation in S . cerevisiae were also required in human B lymphocytes . The amino-terminal region of ZEBRA (aa 1 to 98) was required for activation both in S . cerevisiae and in human B cells; deletion of the carboxy-terminal 18 aa also significantly reduced activation in both cell types . Thus, the behavior of ZEBRA in human B cells and S . cerevisiae suggests that the protein contains universal activation motifs that interact with conserved components of the transcription machinery . However, certain deletion mutants of ZEBRA containing mutations in the N-terminal region exhibited discordant behaviors in S . cerevisiae and in B cells . For example, deletion of ZEBRA aa 26 to 51 impaired activation to a great extent in B cells but had little or no effect in S . cerevisiae . The discordant mutants may reflect interactions with a variable domain of a conserved component or unique interactions with specialized components of the basal transcription apparatus in different cells. EMBO J, 1993 Dec, 12(12), 4833 - 42 Drosophila protein phosphatase V functionally complements a SIT4 mutant in Saccharomyces cerevisiae and its amino-terminal region can confer this complementation to a heterologous phosphatase catalytic domain; Mann DJ et al.; The sequence of a Drosophila melanogaster cDNA encoding a novel 35 kDa protein serine/threonine phosphatase, termed PPV, is presented . PPV is 40-41% identical to Drosophila PP1, 53% identical to Drosophila PP2A and 63% identical to Saccharomyces cerevisiae SIT4 . Complementation studies demonstrated that PPV can functionally rescue a temperature sensitive mutant of SIT4, a protein phosphatase required for the G1 to S transition of the cell cycle . When placed under the SIT4 promoter, PPV cDNA is able to replace the SIT4 gene in S . cerevisiae . The amino-terminal domain of PPV fused to another phosphatase catalytic region (PP1) also rescues the temperature sensitive SIT4 mutant and the SIT4 deletion mutant, implicating this region in binding to regulatory subunits and/or altering specificity . In Drosophila, a substantial transient increase in both PPV mRNA and protein occurs in late syncytial and early cellular blastoderm embryos . At the latter stage PPV is localized to the cytoplasm of cells at the cortex . This increase in PPV correlates with introduction of the G2 phase of the cell cycle, elevated zygotic transcription and cellularization, indicating that PPV may play a role in one or more of these processes. Biochem Mol Biol Int, 1993 Dec, 31(6), 1081 - 90 Trehalose metabolism during sporulation in Saccharomyces cerevisiae; Ferreira JC et al.; S . cerevisiae has at least two enzymes for trehalose synthesis . In this paper we report some characteristics of the ADPG-dependent trehalose-6-phosphate synthase . As opposed to the UDPG-dependent enzyme, the ADPG-dependent activity did not respond to a glucose signal nor to a heat shock . Mutant strains, deficient in the UDPG-dependent enzyme but possessing the ADPG-dependent activity, did not accumulate trehalose either during growth or during sporulation . A diploid, homozygous for the deficiency in the UDPG-dependent synthase activity, was constructed . During sporulation the ADPG-dependent activity remained constant . However, when mutant spores were subjected to a glucose signal, trehalose was accumulated to the same extent as in control spores. Yeast, 1993 Dec, 9(12), 1309 - 18 A series of yeast/Escherichia coli lambda expression vectors designed for directional cloning of cDNAs and cre/lox-mediated plasmid excision; Brunelli JP et al.; A series of Saccharomyces cerevisiae/Escherichia coli lambda/plasmid expression vectors have been constructed which allow easy excision of the plasmid sequences from lambda . Features of six are described, and two designated lambda PG15 and lambda AD5, are characterized in detail . Transcription of cloned sequences is controlled by the alternative promoters, ADH2, PGK, GAL10 and SV40 early, and by the CYC1 transcriptional terminator . Unique EcoRI and XhoI restriction sites in the intervening polylinker make these lambda vectors compatible for directional cloning of 'ZAP'-synthesized cDNAs . Inserted DNAs have been previously shown to have high levels of the genetic activity in both S . cerevisiae and E . coli, allowing these vectors to be used for genetic complementation in both species . Plasmid recovery from the lambda vector is mediated by the activity of the cre-encoded enzyme upon lox sequences flanking the plasmid and adjoining the lambda arms . The plasmids contain the yeast 2 microns origin and E . coli pBR322 origin, the URA3 or TRP1 yeast selectable markers, and ampicillin-resistance marker in E . coli . The usefulness of the lambda PG15 and the lambda AD5 cloning vectors was demonstrated by constructing large Neurospora crassa cDNA libraries . The lambda PG15-N . crassa library was used to infect purE, purC and trpC mutants of E . coli, and complemented and/or suppressed prototrophic colonies were selected . The flexibility and power of this system for cloning of cDNAs is discussed. Cell Growth Differ, 1993 Dec, 4(12), 965 - 73 Evolutionary conservation of Xenopus laevis mitogen-activated protein kinase activation and function; Waskiewicz AJ et al.; Saccharomyces cerevisiae possesses at least four mitogen-activated protein (MAP) kinase family members, encoded by the FUS3, KSS1, HOG1, and MPK1 genes, that participate in three distinct signaling pathways . We have tested whether a MAP kinase from Xenopus laevis (Xp42) can function in budding yeast, by expressing wild-type and mutant forms of Xp42 in different strains of S . cerevisiae . In Xenopus cells, Xp42 is phosphorylated on threonine188 and tyrosine190 when activated by a MAP kinase kinase (MAPKK) . In S . cerevisiae, Xp42 is constitutively phosphorylated on tyrosine190 . Since a kinase-inactive mutant of Xp42 is also phosphorylated, this phosphorylation is presumably due to activation by an endogenous MAPKK . Xp42 phosphorylation and kinase activity are dependent on yeast Bck1p, a putative MAPKK kinase (MAPKKK) and indirect upstream activator of Mpk1p . The loss of either Ste7p or Pbs2p, the upstream activators of Fus3p, Kss1p, and Hog1p, does not decrease the phosphorylation stoichiometry of Xp42 . We also show that expression of Xenopus MAP kinase permits an mpk1::TRP1 deletion strain to grow at 37 degrees C . We conclude that S . cerevisiae and X . laevis possess evolutionarily conserved cascades, where biochemical activation and substrate specificity of MAP kinase have been maintained. Mol Microbiol, 1993 Dec, 10(5), 1101 - 11 A gene encoding sn-glycerol 3-phosphate dehydrogenase (NAD+) complements an osmosensitive mutant of Saccharomyces cerevisiae; Larsson K et al.; Osmoregulatory mutants of Saccharomyces cerevisiae with a defect in their capacity to readjust the cell volume/buoyant density after osmotically induced dehydration were enriched by density gradient centrifugation . Colonies derived from cells that remained dense after dehydration were screened for sensitivity to high concentrations of NaCl and defects in their osmotically induced production and intracellular accumulation of glycerol . The isolated osg (osmosensitive glycerol defective) mutants were recessive in heterozygous diploids and fell into four complementation groups (osg1-osg4) . The osg1-1 mutant, described in this work, is unable to grow at low water potential and shows a decreased capacity for glycerol production and a strongly reduced activity of NAD(+)-dependent sn-glycerol 3-phosphate dehydrogenase (GPD), an enzyme in the glycerol-producing pathway . Complementation of the osg1-1 salt sensitivity defect with a low copy yeast genomic library led to the cloning of GPD1, encoding an S . cerevisiae GPD consisting of 391 amino acids and sharing 47-50% identity with GPD from other sources . Micro-sequencing of the N-terminus of purified S . cerevisiae GPD revealed a 20-amino-acid sequence that was identical to a nucleotide-deduced amino acid sequence in GPD1, but indicated that the enzyme is produced with an N-terminal extension that is removed from the functional enzyme . Subcellular fractionation does not indicate, however, that the putative pre-sequence targets GPD to any organelle; the enzyme appears to be located in the cytoplasm . Chromoblot and tetrad analysis were used to position the GPD1 gene to chromosome IV, with a distance of about 18 cM from trp1. J Med Chem, 1993 Nov 26, 36(24), 3859 - 62 R2 C-terminal peptide inhibition of mammalian and yeast ribonucleotide reductase; Fisher A et al.; Eucaryotic ribonucleotide reductases (RR) catalyze the reduction of ribonucleoside diphosphates to 2'-deoxyribonucleoside diphosphates . Each has an R1(2)R2(2) quaternary structure with each subunit playing a critical role in catalysis . Separation of the subunits results in loss of activity . Previous studies have demonstrated that peptides corresponding to the C-terminus of R2 disrupt subunit association by competion with R2 and have potential usefulness as therapeutics . Extensive structure-function studies have been carried out on peptide inhibition of herpes simplex RR in an effort to develop antiviral agents based on the observation that the herpes simplex R2 C-terminus, YAGAVVNDL, is quite different from the corresponding mammalian sequence . In this work we report a detailed structure-function analysis of peptide inhibition of mammalian and, to a more limited extent, Saccharomyces cerevisiae RRs . Our results for mammalian RR support the following conclusions with regard to the effect of substitution on inhibitory potency: (a) the N-acetylated R2 C-terminal heptapeptide N-AcPhe384Thr385Leu386Asp387Ala388Asp389Phe390 (N-AcF7TLDADF1) is the minimal core peptide length required; deletion of the N-terminus or of middle positions (resulting in penta- and hexapeptides) results in large losses in inhibitory potency; (b) a free carboxylate is required on the C-terminal Phe; (c) Phe is strongly preferred to Leu in positions 1 and 7 and a bulky aliphatic group is preferred in position 5; (d) neither negative charge in positions 2 or 4 nor a polar side chain in position 6 are required for peptide binding, contrary to what evolutionary patterns in the R2 C-terminus of RR would suggest . S . cerevisiae RR displays a similar length dependence on the corresponding N-acetylated R2 C-terminal heptapeptide, N-AcFTFNEDF . This peptide has a 4-fold higher inhibitory potency toward S . cerevisiae RR than toward mammalian RR . Such selectivity raises the possibility that peptide analogs related to R2 C-termini can be developed as therapeutic agents even against organisms having R2 C-terminal sequences similar to that of mammalian RR. Nucleic Acids Res, 1993 Nov 25, 21(23), 5500 - 8 Functional analysis of mRNA 3' end formation signals in the convergent and overlapping transcription units of the S . cerevisiae genes RHO1 and MRP2; Peterson JA et al.; The Saccharomyces cerevisiae genes RHO1 and MRP2 are convergently transcribed, with 281 base pairs separating their termination codons . Transcript mapping revealed at least 111 base pairs within the RHO1-MRP2 intercoding region are transcribed in both directions . Transplacement experiments showed distinct sequences of 70 nt for MRP2 and 179 nt for RHO1 were sufficient for normal mRNA 3' end formation . The MRP2 signal functioned in either orientation, although relatively inefficiently in the non-native orientation . This element contains a polyAT sequence essential for 3' end formation in both orientations . RHO1 or MRP2 3' end formation was not affected by overproduction or elimination of the complementary, natural antisense transcript . In contrast, insertion of a strong promoter that extended antisense transcripts beyond their normal 3' ends inactivated either MRP2 or RHO1 . These data suggest that transcript termination in the compact yeast genome can be important to prevent inactivation of downstream genes as a result of antisense transcription. Nucleic Acids Res, 1993 Nov 25, 21(23), 5391 - 7 SnR31, snR32, and snR33: three novel, non-essential snRNAs from Saccharomyces cerevisiae; Balakin AG et al.; Genes for three novel yeast snRNAs have been identified and tested for essentiality . Partial sequence information was developed for RNA extracted from isolated nuclei and the respective gene sequences were discovered by screening a DNA sequence database . The three RNAs contain 222, 188 and 183 nucleotides and are designated snR31, snR32 and snR33, respectively . Each RNA is derived from a single copy gene . The SNR31 gene is adjacent to a gene for an unnamed protein associated with the cap-binding protein eIF-4E . The SNR32 gene is next to a gene for ribosomal protein L41 and the gene for SNR33 is on chromosome III, between two open reading frames with no known function . Genetic disruption analyses showed that none of the three snRNAs is required for growth . The new RNAs bring the number of non-spliceosomal snRNAs characterized thus far in S . cerevisiae to 14, of which only three are essential. Nature, 1993 Nov 25, 366(6453), 365 - 8 Yeast excision repair gene RAD2 encodes a single-stranded DNA endonuclease; Habraken Y et al.; In eukaryotes nucleotide excision repair of DNA damaged by ultraviolet radiation requires several gene products; defects in this process result in the cancer-prone syndrome xeroderma pigmentosum (XP) in humans . The RAD2 gene is one of at least seven genes indispensable for excision repair in the yeast Saccharomyces cerevisiae, and its encoded protein shares remarkable homology with the XP group-G gene product . Here we overproduce the RAD2-encoded protein in S . cerevisiae, purify it to near homogeneity, and show that RAD2 protein in the presence of magnesium degrades circular single-stranded DNA . The RAD2 endonuclease is specific for single-stranded DNA as it does not act on double-stranded DNA . Given the absolute requirement for RAD2 in the incision step of excision repair, our findings directly implicate RAD2 protein and its human homologue XPG protein as a catalytic component that incises the damaged DNA strand during excision repair . Furthermore, our results indicate that eukaryotes probably employ two distinct endonuclease activities to mediate the dual incision at the damage site. Nature, 1993 Nov 25, 366(6453), 358 - 62 S . cerevisiae 26S protease mutants arrest cell division in G2/metaphase; Ghislain M et al.; We isolated two mutants from the yeast Saccharomyces cerevisiae, cim3-1 and cim5-1, that arrest cell division in G2/metaphase at 37 degrees C . CIM3 (identical to SUG1; ref . 1) and CIM5 are similar to each other and are members of a family of putative ATPases that have been proposed to be 26S protease subunits . We show here that CIM5 is the functional yeast homologue of the human MSS1 protein and that homologues of CIM3 and CIM5 are present in a highly purified preparation of the Drosophila 26S protease . The short-lived ubiquitin-proline-beta-galactosidase fusion protein is stabilized in cim mutants, but Leu-beta-galactosidase is not . The CLB2 and CLB3 cyclins also accumulate in the cim mutants . Thus the 26S protease is required in vivo for the degradation of ubiquitinated substrates and for anaphase chromosome separation. J Biol Chem, 1993 Nov 25, 268(33), 24699 - 706 How do "Zn2 cys6" proteins distinguish between similar upstream activation sites? Comparison of the DNA-binding specificity of the GAL4 protein in vitro and in vivo; Vashee S et al.; The GAL4 protein of Saccharomyces cerevisiae is the prototype of a family of transcription factors that contain a "Zn2Cys6" coordination complex in the DNA-binding domain . GAL4 activates the transcription of genes involved in galactose and melibiose metabolism by binding to sites that contain one or more copies of a sequence 5'-CGGN5TN5CCG-3' . Other Zn2Cys6 proteins in S . cerevisiae also recognize sequences containing two CGG triplets, but with different spacings between them . In this report we investigate the mechanism by which GAL4 distinguishes its bona fide binding site from similar sequences as well as from bulk genomic DNA . In vitro, GAL4 recognizes with moderate to high affinity a variety of sites of the general formula (A/C)GGN10-12CCG . This level of specificity is apparently insufficient for the activator to carry out its biological role . However, many of the sites to which GAL4 binds in vitro do not support GAL4-activated transcription in vivo . In most cases there is not a quantitative correlation between the relative affinity of a site for GAL4 in vitro and the level of GAL4-dependent transcription supported by it in vivo . These data imply that there is some mechanism in vivo by which the intrinsic binding specificity of GAL4 is modified. J Biol Chem, 1993 Nov 25, 268(33), 24629 - 34 Biochemical comparisons of the Saccharomyces cerevisiae Bem2 and Bem3 proteins . Delineation of a limit Cdc42 GTPase-activating protein domain; Zheng Y et al.; The Bem2 and Bem3 proteins, which appear to play roles in the regulation of bud site formation in Saccharomyces cerevisiae, show striking homology to a number of proteins that compose a family of GTPase-activating proteins (GAPs) for the rho-subgroup of ras-related GTP-binding proteins . These members include human platelet GAP for Cdc42Hs (the human homolog of a S . cerevisiae GTP-binding protein that regulates bud site assembly), the break point cluster region protein, the brain protein chimerin, the 85-kDa regulatory subunit (p85) of the phosphatidylinositol 3-kinase, and the ras-GAP-binding protein (p190) . A fusion protein composed of the glutathione S-transferase protein and the rho-GAP homology region of Bem3 (designated GST-Bem3) stimulates the GTPase activity of the wild-type Cdc42Hs protein (Cdc42HsGly-12), but has no stimulatory effect on a GTPase-defective mutant (Cdc42HsVal-12), whereas a GST-Bem2 fusion protein does not stimulate the GTPase activity of either form of Cdc42Hs . We have compared the ability of GST-Bem3 to serve as a GAP for Cdc42Hs relative to other members of the rho-GAP subfamily and found the following order of potency: human platelet Cdc42Hs GAP > p190 > Bem3 > break point cluster region protein, whereas p85, like Bem2, shows no GAP activity or any ability to bind to the GTP-bound form of Cdc42Hs . We have taken advantage of the functional specificity exhibited by Bem3 (versus Bem2) in using Bem2/Bem3 chimeras, as well as different deletion mutant versions of the Bem3 protein, to delineate the limits of a functional Cdc42 GAP domain . The results of this study indicate that the carboxyl-terminal approximately 224 amino acids (which contain three regions of homology to the other members of the rho-GAP family) represent a "limit GAP." The first two appear to be important for binding to Cdc42Hs and for partial GAP activity. Proc Natl Acad Sci U S A, 1993 Nov 15, 90(22), 10514 - 8 Identification of barriers to rotation of DNA segments in yeast from the topology of DNA rings excised by an inducible site-specific recombinase; Gartenberg MR et al.; Controlled excision of DNA segments to yield intracellular DNA rings of well-defined sequences was utilized to study the determinants of transcriptional supercoiling of closed circular DNA in the yeast Saccharomyces cerevisiae . In delta top1 top2ts strains of S . cerevisiae expressing Escherichia coli DNA topoisomerase I, accumulation of positive supercoils in intracellular DNA normally occurs upon thermal inactivation of DNA topoisomerase II because of the simultaneous generation of positively and negatively supercoiled domains by transcription and the preferential relaxation of the latter by the bacterial enzyme . Positive supercoil accumulation in DNA rings is shown to depend on the presence of specific sequence elements; one likely cause of this dependence is that the persistence of oppositely supercoiled domains in an intracellular DNA ring requires the presence of barriers to rotation of the DNA segments connecting the domains . Analysis of the S . cerevisiae 2-microns plasmid partition system by this approach suggests that the plasmid-encoded REP1 and REP2 proteins are involved in forming such a barrier in DNA containing the REP3 sequence. J Biol Chem, 1993 Nov 15, 268(32), 24083 - 8 Photoaffinity labeling of the 45-kDa and 55-kDa forms of phosphatidylinositol 4-kinase from the yeast Saccharomyces cerevisiae; Nickels JT Jr et al.; The membrane-associated 45- and 55-kDa forms of phosphatidylinositol (PI) 4-kinase (ATP:phosphatidylinositol 4-phosphotransferase, EC 2.7.1.67) from Saccharomyces cerevisiae are inhibited by ADP by a competitive mechanism with respect to ATP . We initiated studies toward defining the ATP and ADP sites on the PI 4-kinases using azidonucleotide photoaffinity labeling probes . The photoprobe 8-azido-ATP fulfilled the criteria of a specific photoaffinity label for the 45- and 55-kDa PI 4-kinases . 8-Azido-ATP was a substrate and a competitive inhibitor of the PI 4-kinases with Ki values similar to the Km for ATP . 8-Azido-ATP photoinactivated the enzymes and was photoincorporated into the enzymes in a dose-dependent manner at concentrations similar to the Ki values for the photoprobe . ATP, the true substrate, provided specific protection against photoinactivation and photoincorporation of the PI 4-kinases with 8-azido-ATP, whereas GTP, a nonspecific nucleotide, provided no protection against photoinactivation and photoincorporation . Photoaffinity labeling of the PI 4-kinases with 8-azido-ATP was specifically prevented with ADP . The photoprobe 8-azido-ADP also fulfilled the criteria needed to validate its use as a specific photoprobe for the PI 4-kinases . Photoinactivation of the PI 4-kinases with 8-azido-ADP was prevented specifically with ATP . Taken together, these data supported the conclusion that the ATP and ADP sites on the membrane-associated 45- and 55-kDa PI 4-kinases from S . cerevisiae were the same. FEBS Lett, 1993 Nov 15, 334(2), 233 - 6 Cloning and expression in yeast of a higher plant chorismate mutase . Molecular cloning, sequencing of the cDNA and characterization of the Arabidopsis thaliana enzyme expressed in yeast; Eberhard J et al.; Chorismate mutase (EC 5.4.99.5) catalyzes the first step in the branch of the shikimate pathway which leads to the aromatic amino acids, phenylalanine and tyrosine . We have isolated a cDNA for this enzyme from the higher plant, Arabidopsis thaliana, by complementing a yeast strain (aro7) with a cDNA library from A . thaliana . This is the first chorismate mutase cDNA isolated from a plant . It encodes a protein of 334 amino acids . The identity of the deduced amino acid sequence is 41% to the chorismate mutase sequence from Saccharomyces cerevisiae . The N-terminal portion of the deduced amino acid sequence has no homology to the S . cerevisiae sequence but resembles known plastid-specific transit peptides . The A . thaliana chorismate mutase expressed in yeast revealed allosteric control by the three aromatic amino acids, as previously described for plastidic chorismate mutase isozymes. Cell, 1993 Nov 5, 75(3), 543 - 55 SIR3 and SIR4 proteins are required for the positioning and integrity of yeast telomeres; Palladino F et al.; Heritable inactivation of genes occurs in specific chromosomal domains located at the silent mating type loci and at telomeres of S . cerevisiae . The SIR genes (for silent information regulators) are trans-acting factors required for this repression mechanism . We show here that the SIR3 and SIR4 gene products have a sub-nuclear localization similar to the telomere-associated RAP1 protein, which is found primarily in foci at the nuclear periphery of fixed yeast spheroplasts . In strains deficient for either SIR3 or SIR4, telomeres lose their perinuclear localization, as monitored by RAP1 immunofluorescence . The length of the telomeric repeat shortens in sir3 and sir4 mutant strains, and the mitotic stability of chromosome V is reduced . These data suggest that SIR3 and SIR4 are required for both the integrity and subnuclear localization of yeast telomeres, the loss of which correlates with loss of telomere-associated gene repression. Mol Cell Biol, 1993 Nov, 13(11), 7112 - 21 The SH2 domain is required for stable phosphorylation of p56lck at tyrosine 505, the negative regulatory site; Gervais FG et al.; The catalytic function of Src-related tyrosine protein kinases is repressed by phosphorylation of a conserved carboxy-terminal tyrosine residue . Recent studies suggest that this inhibitory event is not the result of autophosphorylation but that it is mediated by another cytoplasmic tyrosine protein kinase, termed p50csk . In this report, we have evaluated the processes regulating the extent of phosphorylation of the inhibitory carboxy-terminal tyrosine residue of p56lck, a lymphocyte-specific member of the Src family . By analyzing kinase-defective variants of p56lck expressed in mouse NIH 3T3 cells, we have found that the noncatalytic Src homology 2 (SH2) domain, but not the SH3 sequence or the sites of Lck myristylation and autophosphorylation, is necessary for stable phosphorylation at the carboxy-terminal tyrosine 505 . Further studies in which Lck and Csk were coexpressed in S . cerevisiae indicated that the absence of the SH2 domain did not affect the ability of Csk to phosphorylate p56lck at tyrosine 505 . However, we observed that incubation of cells with the tyrosine phosphatase inhibitor pervanadate restored the tyrosine 505 phosphorylation of Lck polypeptides devoid of the SH2 motif . Additionally, the presence of the SH2 sequence protected tyrosine 505 from in vitro dephosphorylation by the hemopoietic tyrosine protein phosphatase CD45 . Taken together, these findings raised the possibility that the SH2 motif contributes to the physiological suppression of the catalytic function of p56lck at least in part through its ability to stabilize phosphorylation at the inhibitory site. Curr Genet, 1993 Nov, 24(5), 388 - 93 Regulation of Saccharomyces cerevisiae catalase gene expression by copper; Lapinskas P et al.; Treatment of Saccharomyces cerevisiae cells with copper induces the activity of Cu/Zn superoxide dismutase (SOD) and catalase . To understand the level at which Cu regulates catalase, the expression of the S . cerevisiae CTA1 (encoding the peroxisomal catalase A) and CTT1 (encoding the cytosolic catalase T) genes was monitored as a function of Cu treatment . Copper was found to specifically induce transcription of CTT1, but not CTA1, mRNA . Moreover, genetic and biochemical studies demonstrate that this induction is independent of the ACE1 Cu trans-activator controlling the expression of yeast Cu/Zn SOD and metallothionein genes . Copper regulation of CTT1 thus appears to represent a novel metal regulatory pathway in S . cerevisiae cells. Curr Genet, 1993 Nov, 24(5), 377 - 81 The ogd1 and kgd1 mutants lacking 2-oxoglutarate dehydrogenase activity in yeast are allelic and can be differentiated by the cloned amber suppressor; Mockovciakova D et al.; The activity of mitochondrial 2-oxoglutarate dehydrogenase in S . cerevisiae can be impaired either by the ogd1 or the kgd1 mutation . The OGD1 gene and two suppressor genes were isolated by complementation of the ogd1 mutant . The complementation of the kdg1 mutant by the OGD1 gene, an allelism test, and meiotic mapping, revealed that the ogd1 and kgd1 mutations are allelic . The two mutations were differentiated by the cloned suppressor gene which was able to partially complement ogd1, but not kgd1 . The molecular analysis of the suppressor gene revealed its identity with the natural tRNA(GlnCAG) gene found in the upstream region of URA10. Hum Mol Genet, 1993 Nov, 2(11), 1835 - 40 Expression and processing of human ornithine-delta-aminotransferase in Saccharomyces cerevisiae; Dougherty KM et al.; Ornithine-delta-aminotransferase catalyzes the conversion of ornithine to glutamate-gamma-semialdehyde . In humans, deficiency of this mitochondrial matrix enzyme results in the progressive blinding disorder, gyrate atrophy of the choroid and retina . To explore yeast as an expression system, we introduced a cDNA encoding human ornithine-delta-aminotransferase into an ornithine aminotransferase-deficient strain of Saccharomyces cerevisiae . The human enzyme was expressed at high levels, with activity 20-fold greater than that of wild-type yeast and 10-fold higher than in human fibroblasts . Although the normal location of ornithine-delta-aminotransferase in S . cerevisiae is cytosolic, human ornithine-delta-aminotransferase expressed in S . cerevisiae was localized to the mitochondrial matrix with correct proteolytic processing of its mitochondrial leader sequence . Despite this anomalous location in yeast, human ornithine-delta-aminotransferase complemented the phenotype of the mutant strain, restoring its ability to utilize ornithine as a sole nitrogen source . We also expressed a vitamin B6-responsive missense allele of ornithine-delta-aminotransferase (V332M) and showed that the biochemical phenotype of this allele is easily demonstrated confirming the usefulness of this system for examining mutations causing gyrate atrophy. J Gen Microbiol, 1993 Nov, 139 ( Pt 11), 2783 - 92 Activities of the enzymes of the Ehrlich pathway and formation of branched-chain alcohols in Saccharomyces cerevisiae and Candida utilis grown in continuous culture on valine or ammonium as sole nitrogen source; Derrick S et al.; Valine aminotransferase, a key enzyme in both biosynthesis and breakdown of branched-chain amino acids, showed consistently higher activity in Candida utilis grown in continuous culture than in Saccharomyces cerevisiae, while pyruvate decarboxylase and alcohol dehydrogenase, the other two enzymes of the Ehrlich pathway of branched-chain alcohol formation, were lower in activity . By spheroplast lysis, it was shown that valine aminotransferase followed the distribution of pyruvate decarboxylase in being located in the cytosol . Replacement of ammonium as nitrogen source by valine during conditions of carbon or nitrogen limitation caused increased specific activities of these three enzymes in S . cerevisiae, but (with one exception) decreased those of C . utilis . Of the metabolites accumulating in the culture medium, little or no ethanol or branched-chain alcohols were present during carbon-limited growth of either organism, but the change to nitrogen limitation resulted in increases in concentration of 20- to 100-fold in pyruvate, acetate and non-pyruvate keto acids as well as the accumulation of branched-chain alcohols in both organisms, and of ethanol, ethyl acetate and glycerol in S . cerevisiae . When valine was the limiting nitrogen source, there was an increase in non-pyruvate keto acids and a 10- to 16-fold increase in 2-methylpropanol . Total branched-chain alcohols formed under nitrogen limitation were 2-fold higher in S . cerevisiae than in C . utilis, irrespective of nitrogen source . Accumulation of branched-chain alcohols, ethanol, acetate and glycerol was also observed during carbon-limited growth of S . cerevisiae with valine as nitrogen source at dilution rates above the critical rate for transition to respirofermentative growth . Less than 70% of the valine carbon metabolized during growth of S . cerevisiae and only 15% of that used during growth of C . utilis was recovered in identified metabolic products . Even allowing for losses by volatilization during aeration, this suggests that a significant amount of the valine is being metabolized by a route or routes other than the Ehrlich pathway, possibly via the action of branched-chain 2-keto acid dehydrogenase . The molar growth yield for the nitrogen source under either carbon or nitrogen limitation was significantly lower for growth on valine than for growth on ammonium, suggesting that breakdown of valine requires more energy . It is evident that not all the enzymes involved in branched-chain amino acid metabolism in yeasts have yet been identified, nor are their interactions properly understood. FEBS Lett, 1993 Nov 1, 333(3), 280 - 2 Expression of Saccharomyces cerevisiae inorganic pyrophosphatase in Escherichia coli; Kurilova SA et al.; A segment of DNA encoding Saccharomyces cerevisiae inorganic pyrophosphatase (ppa gene) was amplified by the polymerase chain reaction . The pSCH1 and pSCB6 plasmids containing the ppa gene were obtained . Transformation of the E . coli BL21 strain with the resulting recombinant plasmids and selection of clones having extremely high expression of inorganic pyrophosphatase (PPase) were carried out . Superproduction of recombinant S . cerevisiae PPase up to 50% of the total bacterial protein was achieved . The enzyme was readily obtained and purified to homogeneity with the use of a simple purification technique . This work is the first description of S . cerevisiae PPase superproducer creation. EMBO J, 1993 Nov, 12(11), 4425 - 31 dUTP pyrophosphatase is an essential enzyme in Saccharomyces cerevisiae; Gadsden MH et al.; dUTP pyrophosphatase (dUTPase; EC 3.6.1.23) catalyses the hydrolysis of dUTP to dUMP and PPi and thereby prevents the incorporation of uracil into DNA during replication . Although it is widely believed that dUTPase is essential for cell viability because of this role, direct evidence supporting this assumption has not been presented for any eukaryotic system . We have analysed the role of dUTPase (DUT1) in the life cycle of yeast . Using gene disruption and tetrad analysis, we find that DUT1 is necessary for the viability of S . cerevisiae; however, under certain conditions dut1 null mutants survive if supplied with exogenous thymidylate (dTMP) . Analyses with isogenic uracil-DNA-glycosylase (UNG1) deficient or proficient strains indicate that in the absence of dUTPase, cell death results from the incorporation of uracil into DNA and the attempted repair of this damage by UNG1-mediated excision repair . However, in dut1 ung1 double mutants, starvation for dTMP causes dividing cells to arrest and die in all phases of the cell cycle . This latter effect suggests that the extensive stable substitution of uracil for thymine in DNA leads to a general failure in macromolecular synthesis . These results are in general agreement with previous models in thymine-less death that implicate dUTP metabolism . They also suggest an alternative approach for chemotherapeutic drug design. Int J Hyperthermia, 1993 Nov-Dec, 9(6), 783 - 97 Thermal response of yeast cells overexpressing hsp70 genes; Weitzel G et al.; We have performed experiments to examine whether the overexpression of different members of the hsp70 family (SSA1, SSA2 and SSA4) in yeast Saccharomyces cerevisiae protects cells from thermal stress . Yeast cells were transformed with plasmids containing the SSA1 or SSA4 gene which was placed under the control of a galactose-inducible GAL1 promoter . In galactose, transformed yeast cells successfully overexpressed these hsp70 proteins at 23 degrees C, their normal growth temperature . When cell survival after heat shock at 50 or 56 degrees C was examined, our results showed that overexpression of SSA1 or SSA4 protein did not protect yeast cells against thermal stress, nor affect the cells' ability to develop thermotolerance . In contrast, thermal sensitivity was modified significantly by growing cells in galactose . Cellular survival of these cells after a 50 degrees C, 30-min heat treatment was 10(4)-fold higher than that of the same strains grown in glucose . In order to study the effects of overexpression of hsp70 on the thermal response of yeast cells independent of the carbon source, we eliminated the glucose-galactose-associated differences in thermal sensitivity by cloning the aforementioned hsp70 genes under the control of a glyceraldehyde phosphatase GAP promoter . S . cerevisiae transformed with these plasmids overexpressed the appropriate hsp70 gene in glucose as well as in galactose at 23 degrees C . Again, the overexpression of hsp70 neither protected cells from thermal stress, nor had a significant effect on the development of thermotolerance. Biochemistry, 1993 Oct 26, 32(42), 11293 - 301 Purification and characterization of a novel metalloendopeptidase from Saccharomyces cerevisiae; Hrycyna CA et al.; We previously identified an activity in the soluble fraction of the yeast Saccharomyces cerevisiae that is a candidate for catalyzing the proteolytic maturation of farnesylated-CXXX precursor polypeptides . We describe here a 1259-fold purification of this activity by chromatography on DEAE-cellulose, hydroxylapatite, phenyl-Sepharose, and Sephacryl S-200 . Sodium dodecyl sulfate gel electrophoresis of this preparation demonstrated a single 68-kDa polypeptide chain . The experimentally determined N-terminal amino acid sequence was identical at all 20 positions with residues 28-47 of the deduced sequence of the S . cerevisiae YCL57w gene product . This analysis suggests that the YCL57w gene encodes this enzyme and that the initial translation product may contain a leader peptide . Its complete deduced amino acid sequence shows significant homology to a number of zinc metallopeptidases and is most closely related to rat metalloendopeptidase 24.15 (E.C . 3.4.24.15), an enzyme that preferentially cleaves after hydrophobic residues . Using the purified yeast enzyme, we show a unique cleavage site in the peptides bradykinin and beta-neoendorphin four residues from the C-terminus on the carboxyl side of a hydrophobic amino acid . The cleavage pattern for neurotensin revealed a major site three residues from the C-terminus also on the carboxyl side of a hydrophobic residue and a minor site four residues from the C-terminus of the peptide . This specificity is similar to that of rat endopeptidase 24.15 and may explain why the farnesylated peptide employed in our studies is a good substrate for the yeast enzyme.(ABSTRACT TRUNCATED AT 250 WORDS) Proc Natl Acad Sci U S A, 1993 Oct 15, 90(20), 9538 - 41 An accuracy center in the ribosome conserved over 2 billion years; Alksne LE et al.; The accuracy of translation in Escherichia coli is profoundly influenced by three interacting ribosomal proteins, S12, S4, and S5 . Mutations at lysine-42 of S12, originally isolated as causing resistance to streptomycin, increase accuracy . Countervailing "ribosomal ambiguity mutations" (ram) in S4 or S5 decrease accuracy . In the eukaryotic ribosome of Saccharomyces cerevisiae, mutations in SUP46 and SUP44, encoding the proteins equivalent to S4 and S5, lead to omnipotent suppression--i.e., to less accurate translation . The evolution of ribosomal protein S12 can be traced, by comparison with archaebacteria and Tetrahymena, to S28 of S . cerevisiae, even though the two proteins share only very limited regions of homology . However, one region that has been conserved contains a lysine residue whose mutation leads to increased accuracy in E . coli . We have introduced into S28 of yeast the same amino acid substitutions that led to the original streptomycin-resistant mutations in E . coli . We find that they have a profound effect on the accuracy of translation and interact with SUP44 and SUP46, just as predicted from the E . coli model . Thus, the interplay of these three proteins to provide the optimal level of accuracy of translation has been conserved during the 2 billion years of evolution that separate E . coli from S . cerevisiae. Nucleic Acids Res, 1993 Oct 11, 21(20), 4726 - 33 Cpf1 protein induced bending of yeast centromere DNA element I; Niedenthal RK et al.; The centromere complex is a multicomponent structure essential for faithful chromosome transmission . Here we show that the S . cerevisiae centromere protein Cpf1 bends centromere DNA element I (CDEI) with the bend angle ranging from 66 degrees to 71 degrees . CDEI DNA sequences that carry point mutations which lead to reduced Cpf1 binding affinity and in vivo centromere activity are still able to show bending . The Cpf1 induced bend is directed towards the major groove with the bend centre located in CDEI . An intrinsic bend cannot replace the Cpf1 induced DNA bend for in vivo centromere function . An in vivo phasing experiment suggests that both the distance and the correct spatial arrangement of the CDEI/Cpf1 complex to CDEII and CDEIII are important for optimal centromere function. Poult Sci, 1993 Oct, 72(10), 1867 - 72 The use of Saccharomyces cerevisiae to suppress the effects of aflatoxicosis in broiler chicks; Stanley VG et al.; Three hundred and sixty day-old commercial broiler chicks were assigned to a 3 x 2 factorial arrangement of treatments to examine the effects of three levels of Saccharomyces cerevisiae 0, .05, and .1% of feed, and two levels of aflatoxin, 0 and 5 ppm, singly and in combination on aflatoxicosis . Each treatment group was replicated three times . The S . cerevisiae and aflatoxin were incorporated in a standard commercial broiler ration and fed to chicks for 4 wk . Data were collected weekly on body weights, and at the end of the experiment on the relative weights of the liver, proventriculus, pancreas, and heart . Serum concentrations of the albumin, total protein, cholesterol, uric acid, triglycerides, and enzyme activities of alanine transaminase, aspartate aminotransferase, lactase dehydrogenase, and creatine phosphokinase were also evaluated . Results showed that chicks receiving aflatoxin-contaminated feed had suppressed body weight (456 g), which significantly improved (516 g) with the inclusion of .1% S . cerevisiae . The relative weights of liver (3.58%), heart (.916%), and proventriculus (.770%), which increased significantly with the addition of 5 ppm of aflatoxin, were restored to 3.00, .783, and .680%, respectively, with the dietary inclusion of .1% S . cerevisiae . The serum concentrations of albumin and total protein (.66 and 1.62 g/100 mL), which were significantly decreased by aflatoxin, were elevated to .88 and 2.24 g/100 mL, respectively, with the inclusion of .1% S . cerevisiae.(ABSTRACT TRUNCATED AT 250 WORDS) Mol Cell Biol, 1993 Oct, 13(10), 6114 - 23 PUB1: a major yeast poly(A)+ RNA-binding protein; Matunis MJ et al.; The expression of RNA polymerase II transcripts can be regulated at the posttranscriptional level by RNA-binding proteins . Although extensively characterized in metazoans, relatively few RNA-binding proteins have been characterized in the yeast Saccharomyces cerevisiae . Three major proteins are cross-linked by UV light to poly(A)+ RNA in living S . cerevisiae cells . These are the 72-kDa poly(A)-binding protein and proteins of 60 and 50 kDa (S.A . Adam, T.Y . Nakagawa, M.S . Swanson, T . Woodruff, and G . Dreyfuss, Mol . Cell . Biol . 6:2932-2943, 1986) . Here, we describe the 60-kDa protein, one of the major poly(A)+ RNA-binding proteins in S . cerevisiae . This protein, PUB1 {for poly(U)-binding protein 1}, was purified by affinity chromatography on immobilized poly(rU), and specific monoclonal antibodies to it were produced . UV cross-linking demonstrated that PUB1 is bound to poly(A)+ RNA (mRNA or pre-mRNA) in living cells, and it was detected primarily in the cytoplasm by indirect immunofluorescence . The gene for PUB1 was cloned and sequenced, and the sequence was found to predict a 51-kDa protein with three ribonucleoprotein consensus RNA-binding domains and three glutamine- and asparagine-rich auxiliary domains . This overall structure is remarkably similar to the structures of the Drosophila melanogaster elav gene product, the human neuronal antigen HuD, and the cytolytic lymphocyte protein TIA-1 . Each of these proteins has an important role in development and differentiation, potentially by affecting RNA processing . PUB1 was found to be nonessential in S . cerevisiae by gene replacement; however, further genetic analysis should reveal important features of this class of RNA-binding proteins. J Bacteriol, 1993 Oct, 175(20), 6484 - 91 Genetic evidence for a functional relationship between Hsp104 and Hsp70; Sanchez Y et al.; The phenotypes of single Hsp104 and Hsp70 mutants of the budding yeast Saccharomyces cerevisiae provide no clue that these proteins are functionally related . Mutation of the HSP104 gene severely reduces the ability of cells to survive short exposures to extreme temperatures (thermotolerance) but has no effect on growth rates . On the other hand, mutations in the genes that encode Hsp70 proteins have significant effects on growth rates but do not reduce thermotolerance . The absence of a thermotolerance defect in S . cerevisiae Hsp70 mutants is puzzling, since the protein clearly plays an important role in thermotolerance in a variety of other organisms . In this report, examination of the phenotypes of combined Hsp104 and Hsp70 mutants uncovers similarities in the functions of Hsp104 and Hsp70 not previously apparent . In the absence of the Hsp104 protein, Hsp70 is very important for thermotolerance in S . cerevisiae, particularly at very early times after a temperature upshift . Similarly, Hsp104 plays a substantial role in vegetative growth under conditions of decreased Hsp70 protein levels . These results suggest a close functional relationship between Hsp104 and Hsp70. Neuron, 1993 Oct, 11(4), 703 - 11 The C . elegans unc-18 gene encodes a protein expressed in motor neurons; Gengyo-Ando K et al.; The C . elegans unc-18 gene is required to maintain normal acetylcholine levels . We determined the complete structure of an unc-18 cDNA that encodes a protein of 591 highly charged and hydrophilic amino acids . The protein shows sequence similarity with elements of the secretory pathway in the yeast S . cerevisiae . Antibodies raised against a portion of the unc-18-encoded protein (UNC-18) detected a 68 kd soluble antigen on immunoblots and intensely stained all vertical cord motor neurons in situ . These findings suggest that UNC-18 participates in the axonal transport system and influences the acetylcholine flow in motor neurons. Yeast, 1993 Oct, 9(10), 1131 - 7 The complete sequence of a 6794 bp segment located on the right arm of chromosome II of Saccharomyces cerevisiae . Finding of a putative dUTPase in a yeast; Doignon F et al.; The DNA sequence of a 6794 bp fragment located at about 100 kb from the right telomere of chromosome II from Saccharomyces cerevisiae has been determined . Sequence analysis reveals five open reading frames . One is the ARO4 gene encoding the 3-deoxy-D-arabinoheptulosonate 7-phosphate synthase . Another presents strong homology with the S5 ribosomal protein from bacteria . The open reading frame YBR1705 shows significant homology with dUTPase, suggesting for the first time the existence of such an enzyme in S . cerevisiae. Genetics, 1993 Oct, 135(2), 321 - 6 Responsiveness to exogenous cAMP of a Saccharomyces cerevisiae strain conferred by naturally occurring alleles of PDE1 and PDE2; Mitsuzawa H; The Saccharomyces cerevisiae strain P-28-24C, from which cAMP requiring mutants derived, responded to exogenously added cAMP . Upon the addition of cAMP, this strain showed phenotypes shared by mutants with elevated activity of the cAMP pathway . Genetic analysis involving serial crosses of this strain to a strain with another genetic background revealed that the responsiveness to cAMP results from naturally occurring loss-of-function alleles of PDE1 and PDE2, which encode low and high affinity cAMP phosphodiesterases, respectively . In addition, P-28-24C was found to carry a mutation conferring slow growth that lies in CYR1, which encodes adenylate cyclase, and the slow growth phenotype caused by the cyr1 mutation was suppressed by the pde2 mutation . Therefore P-28-24C is fortuitously a pde1 pde2 cyr1 triple mutant . Responsiveness to cAMP conferred by pde mutations suggests that S . cerevisiae cells are permeable to cAMP to some extent and that the apparent absence of effect of exogenously added cAMP on wild-type cells is due to immediate degradation by cAMP phosphodiesterases. Mol Gen Genet, 1993 Oct, 241(1-2), 177 - 84 Activity of the yeast MAP kinase homologue Slt2 is critically required for cell integrity at 37 degrees C; Martin H et al.; Deletion of the SLT2 gene of Saccharomyces cerevisiae, which codes for a homologue of MAP (mitogen-activated) protein kinases, causes an autolytic lethal phenotype in cells grown at 37 degrees C . The gene encodes domains characteristic of protein kinases, which include a lysine (at position 54) that lies 19 residues from a glycine-rich cluster, considered to be the putative ATP binding site . The ability of three mutant alleles of SLT2 generated by site-directed mutagenesis, namely E54 (glutamic acid), R54 (arginine) and F54 (phenylalanine), to complement slt2 mutants was tested . All three failed to complement the autolytic phenotype and were unable to restore growth and viability of cells . A strain obtained by transplacement of slt2-F54 also behaved as a thermosensitive autolytic mutant . By immunoprecipitation with polyclonal antibodies raised against Slt2 protein expressed in Escherichia coli, it was possible to confirm that alteration of the lysine-54 residue did not affect the stability of the protein, thus allowing us to conclude that activity of the Slt2 protein kinase is critically required for growth and morphogenesis of S . cerevisiae at 37 degrees C . A significant fraction of the mutant cell population lysed at 24 degrees C and the cells displayed a characteristic alteration of the surface consisting of a typical depression in an area of the cell wall . At 37 degrees C, the cell surface was clearly disorganized. Biochem J, 1993 Oct 1, 295 ( Pt 1), 195 - 201 A novel mono-branched lipid phosphate acts as a substrate for dolichyl phosphate mannose synthetase; Wilson IB et al.; Dolichyl phosphate mannose synthetase (GDP-mannose: dolichyl-phosphate O-beta-D-mannosyltransferase; EC 2.4.1.83) is an enzyme that is involved in glycoconjugate biosynthesis and possesses a putatively conserved dolichol binding site . In order to probe the interaction between the enzyme and the dolichol chain, lipid phosphates varying in length and extent of branching have been tested as substrates in crude microsomal preparations from Saccharomyces cerevisiae . It was found that phytanyl (3,7,11,15-tetramethylhexadecanyl) phosphate was utilized at 60-70% of the efficiency of the natural dolichyl lipid in transfer of {3,4,-3H}mannose from GDP-Man to organic soluble material, whereas addition of S-3-methyloctadecanyl phosphate, which is of similar length to the phytanyl analogue but with only one branch, resulted in approximately 25% of the incorporation of the natural substrate . Incubations with the unbranched tetradecanyl phosphate and with the short, doubly branched R- and S-dihydrocitronellyl (3,7-dimethyloctanyl) phosphates exhibited levels of activity similar to incubations with no exogenous acceptor . These results were qualitatively confirmed with experiments on Escherichia coli harbouring the S . cerevisiae DPM1 gene . The {3H}mannosylated lipid-linked material from microsomal incubations was purified by anion-exchange chromatography . The major saccharide component recovered after hydrolysis was determined to be mannose, but a mannose-containing disaccharide was also present . It is concluded that branching of lipid phosphates is essential for substrates of dolichyl phosphate mannose synthetase and that significant transfer of mannose occurs even if only branching at C-3 is present. Biosci Biotechnol Biochem, 1993 Oct, 57(10), 1731 - 5 Protein kinase activity associated with the IME2 gene product, a meiotic inducer in the yeast Saccharomyces cerevisiae; Kominami K et al.; The IME2 gene product (Ime2) is required for entry into meiosis and sporulation in S . cerevisiae . It has been predicted to be composed of two domains, an amino-terminal domain with homology to protein kinases and a carboxy-terminal acidic domain . The Ime2 was identified in extracts of meiotic cells carrying multi- but not low-copy IME2 in immunoblot analysis using an Ime2-specific antibody . Immune complexes were found to phosphorylate Ime2 and several exogenous proteins . Low-copy plasmids expressing truncated Ime2 proteins that lack part of or the entire carboxy-terminal domain enabled cells to undergo sporulation even under a certain repressive nutritional condition . These cells contained increased levels of protein kinase activity compared with control cells . These results suggest that the amino-terminal domain has a protein kinase activity and that the acidic tail is not essential for either the kinase activity or sporulation but serves in a negative role . An Ime2-beta-galactosidase fusion was shown by immunofluorescence microscopy to be localized predominantly to the nucleus, suggesting a nuclear function of Ime2. Gene, 1993 Sep 30, 132(1), 49 - 55 A Saccharomyces cerevisiae upstream activating sequence mediates induction of peroxisome proliferation by fatty acids; Filipits M et al.; Peroxisome proliferation in Saccharomyces cerevisiae is induced by fatty acids via as yet unknown mechanisms . We have initiated a study of these mechanisms by identifying control sequences sufficient for fatty acid control of the CTA1 gene (encoding the peroxisomal catalase A) . Promoter regions previously shown to be necessary for control were tested for their potential to mediate induction by oleic acid to a CYC1::lacZ fusion gene . A region previously demonstrated to control CTA1 via the ADR1 transcription activator (bp -156 to -184) does not mediate induction by oleic acid . In contrast, an adjacent sequence (-184 to -198) is sufficient for oleic acid induction, and a neighbouring element (-197 to -215) has marginal inducing activity . These two elements are characterized by a consensus sequence, 5'-CGGNNNTNA ('peroxisome box'), which is found in a number of S . cerevisiae peroxisomal protein-encoding genes . Mutation of either the CGG or the TNA block in the box has a dramatic down-regulating effect on the gene expression in oleic acid medium. Cell, 1993 Sep 10, 74(5), 899 - 908 The essential function of yeast protein disulfide isomerase does not reside in its isomerase activity; LaMantia ML et al.; Protein disulfide isomerase (PDI) is believed to function in vivo by catalyzing the isomerization of disulfide bonds in proteins and thereby facilitating their folding . In S . cerevisiae PDI is encoded by an essential gene . Deletion of nearly one-third of the C-terminal residues of PDI altered PDI's cellular localization but not cell viability . Further deletions resulted in lethality, but these truncated proteins still exhibited PDI activity in vitro . Cells carrying a variant PDI in which both-CGHC-active sites were disrupted were viable . However, these cells exhibited a delay in the disulfide bond formation and transport of carboxypeptidase Y . In vitro enzyme assays revealed that disruption of both sites abolished catalytic activity . These results indicate that PDI catalyzes disulfide bond formation both in vivo and in vitro and that the integrity of the active sites is required for catalysis . However, this catalytic activity is not essential for yeast viability. Cell, 1993 Sep 10, 74(5), 855 - 61 Homologs of the synaptobrevin/VAMP family of synaptic vesicle proteins function on the late secretory pathway in S . cerevisiae; Protopopov V et al.; The yeast S . cerevisiae possesses two genes, SNC1 and SNC2, that encode homologs of vertebrate synaptic vesicle-associated membrane proteins, also known as synaptobrevins . Here we describe the cloning of SNC2 and demonstrate that yeast lacking both SNC genes are deficient in normal bulk secretion, accumulate large numbers of post-Golgi vesicles, and display a variety of conditional lethal phenotypes . In addition, we show that yeast Snc proteins localize to post-Golgi transport vesicles that accumulate in a late-acting sec mutant . Our findings clearly place the Snc proteins on the late secretory pathway in S . cerevisiae and provide direct in vivo evidence that synaptobrevin-like proteins mediate the targeting and transport of secretory proteins. J Biol Chem, 1993 Sep 5, 268(25), 18513 - 8 Protein phosphatases 1, 2A, and 2C are protein histidine phosphatases; Kim Y et al.; Eukaryotic cellular proteins contain phosphohistidine . To search for protein histidine phosphatases, protein histidine kinase from Saccharomyces cerevisiae was used to phosphorylate histone H4 on histidine at position 75 in the H4 amino acid sequence . Incubation of the phosphorylated histone H4 with either protein phosphatase 1, 2A, or 2C resulted in extensive removal of phosphate from the phosphorylated histone . Thus, protein phosphatases 1, 2A, and 2C are histidine phosphatases as well as serine/threonine phosphatases . Calcium/calmodulin-regulated protein phosphatase (protein phosphatase 2B) did not remove phosphate from phosphohistidine . The histidine phosphatase reaction was tested for a magnesium requirement and effects of inhibitor-1 and okadaic acid . In all cases, the protein phosphatases behaved as they do in their serine/threonine phosphatase activity . Extracts of the yeast, S . cerevisiae, contain protein histidine phosphatase activity . Quantitative measurement of phosphatase activity shows that the activity against phosphohistidine is a major activity of protein phosphatases 1, 2A, and 2C. Mol Cell Biol, 1993 Sep, 13(9), 5861 - 76 Genetic and biochemical characterization of a phosphatidylinositol-specific phospholipase C in Saccharomyces cerevisiae; Flick JS et al.; Hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) by phosphatidylinositol-specific phospholipase C (PI-PLC) generates two second messengers, inositol 1,4,5-trisphosphate and 1,2-diacylglycerol . The polymerase chain reaction was used to isolate a Saccharomyces cerevisiae gene (PLC1) that encodes a protein of 869 amino acids (designated Plc1p) that bears greatest resemblance to the delta isoforms of mammalian PI-PLC in terms of overall sequence similarity and domain arrangement . Plc1p contains the conserved X and Y domains found in all higher eukaryotic PI-PLCs (51 and 29% identity, respectively, to the corresponding domains of rat delta 1 PI-PLC) and also contains a presumptive Ca(2+)-binding site (an E-F hand motif) . Plc1p, modified by in-frame insertion of a His6 tract and a c-myc epitope near its amino terminus, was overexpressed from the GAL1 promoter, partially purified by nickel chelate affinity chromatography, and shown to be an active PLC enzyme in vitro with properties similar to those of its mammalian counterparts . Plc1p activity was strictly Ca2+ dependent: at a high Ca2+ concentration (0.1 mM), the enzyme hydrolyzed PIP2 at a faster rate than phosphatidylinositol, and at a low Ca2+ concentration (0.5 microM), it hydrolyzed PIP2 exclusively . Cells carrying either of two different deletion-insertion mutations (plc1 delta 1::HIS3 and plc1 delta 2::LEU2) were viable but displayed several distinctive phenotypes, including temperature-sensitive growth (inviable above 35 degrees C), osmotic sensitivity, and defects in the utilization of galactose, raffinose, and glycerol at permissive temperatures (23 to 30 degrees C) . The findings reported here suggest that hydrolysis of PIP2 in S . cerevisiae is required for a number of nutritional and stress-related responses. Mol Cell Biol, 1993 Sep, 13(9), 5567 - 81 Regulation of dimorphism in Saccharomyces cerevisiae: involvement of the novel protein kinase homolog Elm1p and protein phosphatase 2A; Blacketer MJ et al.; The Saccharomyces cerevisiae genes ELM1, ELM2, and ELM3 were identified on the basis of the phenotype of constitutive cell elongation . Mutations in any of these genes cause a dimorphic transition to a pseudohyphal growth state characterized by formation of expanded, branched chains of elongated cells . Furthermore, elm1, elm2, and elm3 mutations cause cells to grow invasively under the surface of agar medium . S . cerevisiae is known to be a dimorphic organism that grows either as a unicellular yeast or as filamentous cells termed pseudohyphae; although the yeast-like form usually prevails, pseudohyphal growth may occur during conditions of nitrogen starvation . The morphologic and physiological properties caused by elm1, elm2, and elm3 mutations closely mimic pseudohyphal growth occurring in conditions of nitrogen starvation . Therefore, we propose that absence of ELM1, ELM2, or ELM3 function causes constitutive execution of the pseudohyphal differentiation pathway that occurs normally in conditions of nitrogen starvation . Supporting this hypothesis, heterozygosity at the ELM2 or ELM3 locus significantly stimulated the ability to form pseudohyphae in response to nitrogen starvation . ELM1 was isolated and shown to code for a novel protein kinase homolog . Gene dosage experiments also showed that pseudohyphal differentiation in response to nitrogen starvation is dependent on the product of CDC55, a putative B regulatory subunit of protein phosphatase 2A, and a synthetic phenotype was observed in elm1 cdc55 double mutants . Thus, protein phosphorylation is likely to regulate differentiation into the pseudohyphal state. Biochem J, 1993 Sep 1, 294 ( Pt 2), 557 - 61 Selective use of H4 acetylation sites in the yeast Saccharomyces cerevisiae; Clarke DJ et al.; The acetylation of specific lysine residues in the histone H4 may play a role in regulating various genes in the yeast Saccharomyces cerevisiae {Grunstein (1990) Annu . Rev . Cell Biol . 6, 643-678} . The detailed consideration of this possibility has been hampered by the lack of information on the frequency with which different H4 lysine residues are acetylated in yeast . In this paper, we use Western blotting from acid/urea/Triton gels and immunostaining with antisera specific for H4 molecules acetylated at particular lysine residues to show that 70-80% of H4 molecules in S . cerevisiae contain one or more acetylated lysines, and that lysines-5, -8, -12 and -16 are acetylated in an ordered, non-random fashion . The monoacetylated isoform (H4Ac1) is acetylated predominantly at lysine-16 (rarely at lysine-12), H4Ac2 is acetylated at lysine-16 and at either lysine-12 or at -8, while lysine-5 is acetylated frequently only in H4Ac3 and in H4Ac4. Oncogene, 1993 Sep, 8(9), 2335 - 42 The carboxy-terminal domain of c-Myb activates reporter gene expression in yeast; Seneca S et al.; We have shown previously that c-myb expressed in the yeast S . cerevisiae mediated efficient transcriptional activation of reporter genes designed with specific Myb Recognition Elements (MRE's), confirming that this proto-oncogene is able to function as a regulator of transcription in that heterologous context . Here we show that in yeast, as in higher eucaryotic cells, the central domain of c-Myb displays transactivating capacity . In yeast, however, the carboxy-terminal region, defined as a negative regulatory domain in higher cells, activates transcription as well and appears to be a more potent transactivating domain than the central domain itself . Within this region two domains, namely C1 and C2, have been defined that contribute about equally to the activity of the carboxy-terminal region . C1 spans the sequences missing in AMV v-myb while C2, which contains the leucine-zipper motif is specifically absent in the E26 v-myb in addition to C1 . The c-Myb DNA-binding domain itself has no effect on the level of transcription in yeast . We also show that AMV v-Myb stimulates gene expression in yeast with about half the efficiency of full length c-Myb . The fact that the carboxy-terminal region either stimulates or inhibits transactivation properties of c-Myb, depending on the cellular context, stresses the participation of putative c-Myb partner proteins in Myb regulated processes and reopens the question of whether the oncogenic activation of c-myb is indeed due to the increased transactivation capacity of its onco derivatives. Mol Cell Biol, 1993 Sep, 13(9), 5469 - 78 Genetic transfer and expression of reconstructed yeast artificial chromosomes containing normal and translocated BCL2 proto-oncogenes; Silverman GA et al.; The goal of this study was to determine whether it will be feasible to study the expression of a large, human gene, such as the BCL2 proto-oncogene, by DNA transfection . The BCL2 proto-oncogene is 230 kb in size and is deregulated in tumor cells by translocation into the immunoglobulin heavy-chain locus . Yeast artificial chromosomes (YACs) containing the human BCL2 gene were altered by homologous recombination in Saccharomyces cerevisiae to yield replicas of the normal and translocated alleles . Constructions containing either allele and ranging in size from 360 to 800 kb were integrated stably into a mouse tumor line . Fifty-eight percent of the clones contained a copy of the entire YAC insert . Over 50% of these clones expressed appropriate levels of human BCL2 RNA and protein . These studies suggested that the expression of large human genes and their pathologic rearrangements can be studied by transfection techniques employing YACs propagated in S . cerevisiae. Mol Cell Biol, 1993 Sep, 13(9), 5377 - 82 The phylogenetically invariant ACAGAGA and AGC sequences of U6 small nuclear RNA are more tolerant of mutation in human cells than in Saccharomyces cerevisiae; Datta B et al.; U6 small nuclear RNA (snRNA) is the most highly conserved of the five spliceosomal snRNAs that participate in nuclear mRNA splicing . The proposal that U6 snRNA plays a key catalytic role in splicing {D . Brow and C . Guthrie, Nature (London) 337:14-15, 1989} is supported by the phylogenetic conservation of U6, the sensitivity of U6 to mutation, cross-linking of U6 to the vicinity of the 5' splice site, and genetic evidence for extensive base pairing between U2 and U6 snRNAs . We chose to mutate the phylogenetically invariant 41-ACAGAGA-47 and 53-AGC-55 sequences of human U6 because certain point mutations within the homologous regions of Saccharomyces cerevisiae U6 selectively block the first or second step of mRNA splicing . We found that both sequences are more tolerant to mutation in human cells (assayed by transient expression in vivo) than in S . cerevisiae (assayed by effects on growth or in vitro splicing) . These differences may reflect different rate-limiting steps in the particular assays used or differential reliance on redundant RNA-RNA or RNA-protein interactions . The ability of mutations in U6 nucleotides A-45 and A-53 to selectively block step 2 of splicing in S . cerevisiae had previously been construed as evidence that these residues might participate directly in the second chemical step of splicing; an indirect, structural role seems more likely because the equivalent mutations have no obvious phenotype in the human transient expression assay. EMBO J, 1993 Sep, 12(9), 3685 - 92 Co-correction of the ERCC1, ERCC4 and xeroderma pigmentosum group F DNA repair defects in vitro; Biggerstaff M et al.; The mammalian ERCC1-encoded polypeptide is required for nucleotide excision repair of damaged DNA and is homologous to Saccharomyces cerevisiae RAD10, which functions in repair and mitotic intrachromosomal recombination . Rodent cells representing repair complementation group 1 have nonfunct