Mol Cell Biol, 1999 Feb, 19(2), 1325 - 33 Antagonistic interactions between yeast chaperones Hsp104 and Hsp70 in prion curing; Newnam GP et al.; The maintenance of {PSI}, a prion-like form of the yeast release factor Sup35, requires a specific concentration of the chaperone protein Hsp104: either deletion or overexpression of Hsp104 will cure cells of {PSI} . A major puzzle of these studies was that overexpression of Hsp104 alone, from a heterologous promoter, cures cells of {PSI} very efficiently, yet the natural induction of Hsp104 with heat shock, stationary-phase growth, or sporulation does not . These observations pointed to a mechanism for protecting the genetic information carried by the {PSI} element from vicissitudes of the environment . Here, we show that simultaneous overexpression of Ssa1, a protein of the Hsp70 family, protects {PSI} from curing by overexpression of Hsp104 . Ssa1 protein belongs to the Ssa subfamily, members of which are normally induced with Hsp104 during heat shock, stationary-phase growth, and sporulation . At the molecular level, excess Ssa1 prevents a shift of Sup35 protein from the insoluble (prion) to the soluble (cellular) state in the presence of excess Hsp104 . Overexpression of Ssa1 also increases nonsense suppression by {PSI} when Hsp104 is expressed at its normal level . In contrast, hsp104 deletion strains lose {PSI} even in the presence of overproduced Ssa1 . Overproduction of the unrelated chaperone protein Hsp82 (Hsp90) neither cured {PSI} nor antagonized the {PSI}-curing effect of overproduced Hsp104 . Our results suggest it is the interplay between Hsp104 and Hsp70 that allows the maintenance of {PSI} under natural growth conditions. Mol Cell Biol, 1999 Feb, 19(2), 1271 - 8 Identification of a new sea urchin ets protein, SpEts4, by yeast one-hybrid screening with the hatching enzyme promoter; Wei Z et al.; We report the use of a yeast one-hybrid system to isolate a transcriptional regulator of the sea urchin embryo hatching enzyme gene, SpHE . This gene is asymmetrically expressed along the animal-vegetal axis of sea urchin embryos under the cell-autonomous control of maternal regulatory activities and therefore provides an excellent entry point for understanding the mechanism that establishes animal-vegetal developmental polarity . To search for transcriptional regulators, we used a fragment of the SpHE promoter containing several individual elements instead of the conventional bait that contains a multimerized cis element . This screen yielded a number of positive clones that encode a new member of the Ets family, named SpEts4 . This protein contains transcriptional activation activity, since expression of reporter genes in yeast does not depend on the presence of the yeast GAL4 activation domain . Sequences in the N-terminal region of SpEts4 mediate the activation activity, as shown by deletion or domain-swapping experiments . The newly identified DNA binding protein binds with a high degree of specificity to a SpHE promoter Ets element and forms a complex with a mobility identical to that obtained with 9-h sea urchin embryo nuclear extracts . SpEts4 positively regulates SpHE transcription, since mutation of the SpEts4 site in SpHE promoter transgenes reduces promoter activity in vivo while SpEts4 mRNA coinjection increases its output . As expected for a positive SpHE transcriptional regulator, the timing of SpEts4 gene expression precedes the transient expression of SpHE in the very early sea urchin blastula. Mol Cell Biol, 1999 Feb, 19(2), 1226 - 41 Cyclin B-cdk1 kinase stimulates ORC- and Cdc6-independent steps of semiconservative plasmid replication in yeast nuclear extracts; Duncker BP et al.; Nuclear extracts from Saccharomyces cerevisiae cells synchronized in S phase support the semiconservative replication of supercoiled plasmids in vitro . We examined the dependence of this reaction on the prereplicative complex that assembles at yeast origins and on S-phase kinases that trigger initiation in vivo . We found that replication in nuclear extracts initiates independently of the origin recognition complex (ORC), Cdc6p, and an autonomously replicating sequence (ARS) consensus . Nonetheless, quantitative density gradient analysis showed that S- and M-phase nuclear extracts consistently promote semiconservative DNA replication more efficiently than G1-phase extracts . The observed semiconservative replication is compromised in S-phase nuclear extracts deficient for the Cdk1 kinase (Cdc28p) but not in extracts deficient for the Cdc7p kinase . In a cdc4-1 G1-phase extract, which accumulates high levels of the specific Clb-Cdk1 inhibitor p40(SIC1), very low levels of semiconservative DNA replication were detected . Recombinant Clb5-Cdc28 restores replication in a cdc28-4 S-phase extract yet fails to do so in the cdc4-1 G1-phase extract . In contrast, the addition of recombinant Xenopus CycB-Cdc2, which is not sensitive to inhibition by p40(SIC1), restores efficient replication to both extracts . Our results suggest that in addition to its well-characterized role in regulating the origin-specific prereplication complex, the Clb-Cdk1 complex modulates the efficiency of the replication machinery itself. Mol Cell Biol, 1999 Feb, 19(2), 1144 - 58 Seven novel methylation guide small nucleolar RNAs are processed from a common polycistronic transcript by Rat1p and RNase III in yeast; Qu LH et al.; Through a computer search of the genome of the yeast Saccharomyces cerevisiae, the coding sequences of seven different box C/D antisense small nucleolar RNAs (snoRNAs) with the structural hallmarks of guides for rRNA ribose methylation have been detected clustered over a 1.4-kb tract in an inter-open reading frame region of chromosome XIII . The corresponding snoRNAs have been positively identified in yeast cells . Disruption of the nonessential snoRNA gene cluster specifically suppressed the seven cognate rRNA ribose methylations but did not result in any growth delay under the conditions of yeast culture tested . The seven snoRNAs are processed from a common polycistronic transcript synthesized from an independent promoter, similar to some plant snoRNAs but in marked contrast with their vertebrate functional homologues processed from pre-mRNA introns containing a single snoRNA . Processing of the polycistronic precursor requires nucleases also involved in rRNA processing, i.e., Rnt1p and Rat1p . After disruption of the RNT1 gene, the yeast ortholog of bacterial RNase III, production of the seven mature snoRNAs was abolished, while the polycistronic snoRNA precursor accumulated . In cells lacking functional Rat1p, an exonuclease involved in the processing of both pre-rRNA and intron-encoded snoRNAs, several processing intermediates of the polycistronic precursor accumulated . This allowed for the mapping in the precursor of the presumptive Rnt1p endonucleolytic cuts which provide entry sites for subsequent exonucleolytic trimming of the pre-snoRNAs . In line with known properties of double-stranded RNA-specific RNase III, pairs of Rnt1p cuts map next to each other on opposite strands of long double-helical stems in the secondary structure predicted for the polycistronic snoRNA precursor. Mol Cell Biol, 1999 Feb, 19(2), 1136 - 43 Role of a complex containing Rad17, Mec3, and Ddc1 in the yeast DNA damage checkpoint pathway; Kondo T et al.; Genetic analysis has suggested that RAD17, RAD24, MEC3, and DDC1 play similar roles in the DNA damage checkpoint control in budding yeast . These genes are required for DNA damage-induced Rad53 phosphorylation and considered to function upstream of RAD53 in the DNA damage checkpoint pathway . Here we identify Mec3 as a protein that associates with Rad17 in a two-hybrid screen and demonstrate that Rad17 and Mec3 interact physically in vivo . The amino terminus of Rad17 is required for its interaction with Mec3, and the protein encoded by the rad17-1 allele, containing a missense mutation at the amino terminus, is defective for its interaction with Mec3 in vivo . Ddc1 interacts physically and cosediments with both Rad17 and Mec3, indicating that these three proteins form a complex . On the other hand, Rad24 is not found to associate with Rad17, Mec3, and Ddc1 . DDC1 overexpression can partially suppress the phenotypes of the rad24Delta mutation: sensitivity to DNA damage, defect in the DNA damage checkpoint and decrease in DNA damage-induced phosphorylation of Rad53 . Taken together, our results suggest that Rad17, Mec3, and Ddc1 form a complex which functions downstream of Rad24 in the DNA damage checkpoint pathway. Mol Cell Biol, 1999 Feb, 19(2), 1038 - 48 Fission yeast cdc24 is a replication factor C- and proliferating cell nuclear antigen-interacting factor essential for S-phase completion; Tanaka H et al.; At the nonpermissive temperature the fission yeast cdc24-M38 mutant arrests in the cell cycle with incomplete DNA replication as indicated by pulsed-field gel electrophoresis . The cdc24(+) gene encodes a 501-amino-acid protein with no significant homology to any known proteins . The temperature-sensitive cdc24 mutant is effectively rescued by pcn1(+), rfc1(+) (a fission yeast homologue of RFC1), and hhp1(+), which encode the proliferating cell nuclear antigen (PCNA), the large subunit of replication factor C (RFC), and a casein kinase I involved in DNA damage repair, respectively . The Cdc24 protein binds PCNA and RFC1 in vivo, and the domains essential for Cdc24 function and for RFC1 and PCNA binding colocalize in the N-terminal two-thirds of the molecule . In addition, cdc24(+) genetically interacts with the gene encoding the catalytic subunit of DNA polymerase epsilon, which is stimulated by PCNA and RFC, and with those encoding the fission yeast counterparts of Mcm2, Mcm4, and Mcm10 . These results indicate that Cdc24 is an RFC- and PCNA-interacting factor required for DNA replication and might serve as a target for regulation. Mol Cell Biol, 1999 Feb, 19(2), 979 - 88 Activator-specific requirement of yeast mediator proteins for RNA polymerase II transcriptional activation; Han SJ et al.; The multisubunit Mediator complex of Saccharomyces cerevisiae is required for most RNA polymerase II (Pol II) transcription . The Mediator complex is composed of two subcomplexes, the Rgr1 and Srb4 subcomplexes, which appear to function in the reception of activator signals and the subsequent modulation of Pol II activity, respectively . In order to determine the precise composition of the Mediator complex and to explore the specific role of each Mediator protein, our goal was to identify all of the Mediator components . To this end, we cloned three previously unidentified Mediator subunits, Med9/Cse2, Med10/Nut2, and Med11, and isolated mutant forms of each of them to analyze their transcriptional defects . Differential display and Northern analyses of mRNAs from wild-type and Mediator mutant cells demonstrated an activator-specific requirement for each Mediator subunit . Med9/Cse2 and Med10/Nut2 were required, respectively, for Bas1/Bas2- and Gcn4-mediated transcription of amino acid biosynthetic genes . Gal11 was required for Gal4- and Rap1-mediated transcriptional activation . Med11 was also required specifically for MFalpha1 transcription . On the other hand, Med6 was required for all of these transcriptional activation processes . These results suggest that distinct Mediator proteins in the Rgr1 subcomplex are required for activator-specific transcriptional activation and that the activation signals mediated by these Mediator proteins converge on Med6 (or the Srb4 subcomplex) to modulate Pol II activity. J Biol Chem, 1999 Jan 22, 274(4), 2539 - 48 Domain organization and functional properties of yeast transcription factor IIIA species with different zinc stoichiometries; Pizzi S et al.; Transcription factor IIIA (TFIIIA) binds to the 5 S rRNA gene through its zinc finger domain and directs the assembly of a multiprotein complex that promotes transcription initiation by RNA polymerase III . Limited proteolysis of TFIIIA forms with different zinc stoichiometries, in combination with DNA binding and in vitro transcription analyses, have been used herein to investigate the domain organization and zinc requirements of Saccharomyces cerevisiae TFIIIA . Species containing either nine, six, or three zinc equivalents were produced by reductive resaturation and controlled metal depletion of recombinant TFIIIA . Partial digestion of the metal-saturated, 9 Zn2+-liganded factor yields a stable intermediate comprising the eight N-terminal zinc fingers, and a less stable fragment corresponding to a C-terminal portion including the ninth finger . Proteolyzed TFIIIA has the same 5 S DNA binding ability of the intact protein yet no longer supports in vitro 5 S rRNA synthesis . Both the structural compactness and the 5 S DNA binding ability of the TFIIIA form only containing 3 zinc ions are severely compromised . In contrast, the 6 Zn2+-liganded species was found to be indistinguishable from metal-saturated TFIIIA . By demonstrating the existence of three classes of zinc-binding sites contributing differently to yeast TFIIIA structure and function, the present study provides new evidence for the remarkable flexibility built into this complex transcription factor. J Biol Chem, 1999 Jan 22, 274(4), 1949 - 56 The CDK-activating kinase (Cak1p) from budding yeast has an unusual ATP-binding pocket; Enke DA et al.; Cak1p is an essential protein kinase that phosphorylates and thereby activates the major cyclin-dependent kinase in budding yeast, Cdc28p . The sequence of Cak1p differs from other members of the protein kinase superfamily in several conserved regions . Cak1p lacks the highly conserved glycine loop motif (GXGXXG) that is found in the nucleotide binding fold of virtually all protein kinases and also lacks a number of conserved amino acids found at sites throughout the protein kinase core sequence . We have used kinetic and mutagenic analyses to investigate whether these sequence differences affect the nucleotide-binding properties of Cak1p . Although Cak1p differs dramatically from other protein kinases, it binds ATP with a reasonable affinity, with a KM of 4.8 microM . Mutations of the putative invariant lysine in Cak1p (Lys-31), homologous to a residue required for activity in virtually all protein kinases and that interacts with the ATP phosphates, moderately reduced the ability of Cak1p to bind ATP but did not dramatically affect the catalytic rate of the kinase . Similarly, Cak1p is insensitive to the ATP analog 5'-fluorosulfonylbenzoyladenosine, which inhibits most protein kinases through covalent modification of the invariant lysine . We found that Cak1p is tolerant of mutations within its glycine loop region . Remarkably, Cak1p remains functional even following truncation of its first 31 amino acids, including the glycine loop region and the invariant lysine . We conclude that the Cak1p nucleotide-binding pocket differs significantly from those of most other protein kinases and therefore might provide a specific target for an inhibitory drug. J Biol Chem, 1999 Jan 22, 274(4), 1928 - 33 Heterochromatin organization of a natural yeast telomere . Recruitment of Sir3p through interaction with histone H4 N terminus is required for the establishment of repressive structures; Venditti S et al.; The chromatin organization of eukaryotic telomeres is essential for telomeric function and is currently receiving great attention . In yeast, the structural organization of telomeres involves a complex interplay of telomeric proteins that results in the formation of heterochromatin . This telomeric heterochromatin involves homotypic and heterotypic protein interactions that have been summarized in a general model . Recent analyses have focused on the study of the structural complexity at yeast telomeres to the level of specific nucleosomes and of the distribution of protein complexes in a natural telomeric region (LIII) . In this report, we further analyze the structural complexity of LIII and the implication of this structure on telomeric silencing . It is shown that the establishment of repressive heterochromatin structures at LIII requires the recruitment of Sir3p through interaction with the N terminus of histone H4 . The establishment of such structures does not require acetylation of any of four lysines located in the H4 N terminus (lysines 5, 8, 12, and 16). Biochemistry, 1999 Jan 5, 38(1), 487 - 95 Effect of pH on formation of a nativelike intermediate on the unfolding pathway of a Lys 73 --> His variant of yeast iso-1-cytochrome c; Godbole S et al.; Previous work on a Lys 73 --> His (H73) variant of iso-1-cytochrome c at pH 7.5 {Godbole et al . (1997) Biochemistry 36, 119-126} showed that this variant unfolds through a nativelike intermediate that has properties consistent with replacement of the Met 80 heme ligand by His 73 . Here, the pH dependence of the equilibrium unfolding of the wild type (WT) and H73 proteins have been investigated, since a characteristic pH dependence is expected for the stability of an intermediate stabilized by histidine-heme ligation . Stability has been evaluated using guanidine hydrochloride and pH denaturation methods . Above pH 5, the m-values from guanidine hydrochloride denaturation of the WT and H73 variants remain significantly different, consistent with continued population of this intermediate . At pH 4.5 the m-values for the two proteins are within error the same . To assess stability at lower pH, acid denaturation was carried out . The midpoint is about 3.3 for both proteins but the transition is broader for the H73 protein, suggestive of intermediates again being populated during the unfolding of the H73 protein at this lower pH . Heme ligation by Met 80 was monitored (695 nm absorbance) during gdnHCl (pH 4.5 and 5.0) and acid denaturation, confirming, respectively, the absence and presence of intermediates . A thermodynamic analysis demonstrates that this complex pH dependence for the presence of histidine ligation induced intermediates is expected and implicates a titratable group with a pKa of approximately 6.6 . The analysis also demonstrates when the pH dependences of global stability and stability of an intermediate differ significantly, population of folding intermediates as a function of pH will show novel behavior. Biochemistry, 1999 Jan 5, 38(1), 98 - 104 Calcium binding induces interaction between the N- and C-terminal domains of yeast calmodulin and modulates its overall conformation; Nakashima K et al.; Calmodulin from the yeast Saccharomyces cerevisiae binds 3 mol of Ca2+ cooperatively . We report here lines of evidence supporting the intramolecular interaction between the N- and C-terminal domains which modulates the Ca2+ binding properties of yeast calmodulin . First, the sum of the Ca2+ binding curves of the N-terminal and the C-terminal half-molecule did not yield the Ca2+ binding curve of yeast calmodulin . Second, the mean residue CD of yeast calmodulin at 222 nm (-Delta epsilon222) decreased with increases in the concentration of Ca2+, whereas those of each half-molecule increased . Finally, the C2 proton of His107 in the C-terminal domain of yeast calmodulin showed three resonance peaks with increases in the concentration of Ca2+, each corresponding to the apo, the intermediate, and the Ca2+-saturated state . The intermediate peak could not be observed in the C-terminal half-molecule of yeast calmodulin . Computer simulation considering the macroscopic Ca2+ binding constants assigned this intermediate to a species consisting of the apo C-terminal domain and the N-terminal domain with at least one of the two sites occupied by Ca2+ . Peptide segments spanning the defective fourth Ca2+ binding site may be involved in the interdomain interaction and the yeast-specific function of calmodulin. Nucleic Acids Res, 1999 Feb 1, 27(3), 888 - 94 Genomic detection of new yeast pre-mRNA 3'-end-processing signals; Graber JH et al.; To investigate Saccharomyces cerevisiae 3'-end-processing signals, a set of 1352 unique pre-mRNA 3'-end-processing sites, corresponding to 861 different genes, was identified by alignment of expressed sequence tag sequences with the complete yeast genome . Nucleotide word frequencies in the vicinity of the cleavage sites were analyzed to reveal the signal element features . In addition to previously recognized processing signals, two previously uncharacterized components of the 3'-end-processing signal sequence were discovered, specifically a predominance of U-rich sequences located on either side of the cleavage site . One of these, the downstream U-rich signal, provides a further link between the 3'-end-processing mechanisms of yeast and higher eukaryotes . Analysis of the complete set of 3'-end-processing sites by means of a discrimination function supports a 'contextual' model in which the sum total effectiveness of the signals in all four elements determines whether or not processing occurs. Nucleic Acids Res, 1999 Feb 1, 27(3), 736 - 42 Functional analysis of human MutSalpha and MutSbeta complexes in yeast; Clark AB et al.; Mismatch repair (MMR) is initiated when a heterodimer of hMSH2*hMSH6 or hMSH2*hMSH3 binds to mismatches . Here we perform functional analyses of these human protein complexes in yeast . We use a sensitive genetic system wherein the rate of single-base deletions in a homopolymeric run in the LYS2 gene is 10 000-fold higher in an msh2 mutant than in a wild-type strain . Expression of the human proteins alone or in combination does not reduce the mutation rate of the msh2 strain, and expression of the individual human proteins does not increase the low mutation rate of a wild-type strain . However, co-expression of hMSH2 and hMSH6 in wild-type yeast increases the mutation rate 4000-fold, while co-expression of hMSH2 and hMSH3 elevates the rate 5-fold . Analysis of cell extracts indicates that the proteins are expressed and bind to mismatched DNA . The results suggest that hMutSalpha and hMutSbeta complexes form, bind to and prevent correction of replication slippage errors in yeast . Expression of hMSH6 with hMSH2 containing a proline substituted for a conserved Arg524 eliminates the mutator effect and reduces mismatch binding . The analogous mutation in humans is associated with microsatellite instability, defective MMR and cancer, illustrating the utility of the yeast system for studying human disease alleles. J Clin Microbiol, 1999 Feb, 37(2), 422 - 6 Simple, reliable, and cost-effective yeast identification scheme for the clinical laboratory; Koehler AP et al.; The appearance of colonies on the chromogenic medium CHROMagar Candida combined with observation of morphology on corn meal-Tween 80 agar was used for the identification of 353 clinical yeast isolates . The results were compared with those obtained with API yeast identification kits . The accuracy of identification and the turnaround time were equivalent for each method, and our cultural method was less expensive. EMBO J, 1999 Jan 15, 18(2), 320 - 9 G1 cyclins block the Ime1 pathway to make mitosis and meiosis incompatible in budding yeast; Colomina N et al.; Diploid yeast cells switch from mitosis to meiosis when starved of essential nutrients . While G1 cyclins play a key role in initiating the mitotic cell cycle, entry into meiosis depends on Ime1, a transcriptional activator regulated by both nutritional and cell-type signals . We show here that G1 cyclins downregulate IME1 transcription and prevent the accumulation of the Ime1 protein within the nucleus, which results in repression of early-meiotic gene expression . As G1-cyclin deficient cells do not require nutrient starvation to undergo meiosis, G1 cyclin would exert its role by transmitting essential nutritional signals to Ime1 function . The existence of a negative cross-talk mechanism between mitosis and meiosis may help explain why these two developmental options are incompatible in budding yeast. Genomics, 1999 Jan 1, 55(1), 106 - 12 A yeast artificial chromosome-based physical map of the juvenile amyotrophic lateral sclerosis (ALS2) critical region on human chromosome 2q33-q34; Hadano S et al.; The autosomal recessive form of juvenile amyotrophic lateral sclerosis (ALS2; RFALS Type 3) has previously been mapped to the 8-cM interval flanked by D2S115 and D2S155 on human chromosome 2q33-q34 . We have established a yeast artificial chromosome (YAC) contig spanning an approximately 8-Mb region of the ALS2 candidate region and mapped 52 transcribed DNA sequences including 13 known genes and 39 expressed sequenced tags within this YAC contig . The establishment of a YAC contig and transcript map that spans the region containing the ALS2 mutation is an essential step in the identification of the ALS2 gene . Am J Trop Med Hyg, 1998 Dec, 59(6), 991 - 7 Passive transfer of growth-inhibitory antibodies raised against yeast-expressed recombinant Plasmodium falciparum merozoite surface protein-1(19); Gozalo A et al.; Purified rabbit immunoglobulin raised against yeast-expressed recombinant FVO or 3D7 Plasmodium falciparum merozoite surface protein-1 (MSP-1) 19k-D C terminal fragment (MSP-1(19)) was transfused into malaria-naive Aotus nancymai monkeys that were immediately challenged with FVO asexual stage malaria parasites . Control monkeys received rabbit immunoglobulin raised against the sexual stage antigen Pfs25 or Aotus hyperimmune serum obtained from monkeys immunized by P . falciparum infection and drug cure . Passive transfer of rabbit anti-MSP-1(19) failed to protect against homologous or heterologous challenge and, when compared with negative controls, there were no differences in prepatent periods or time to treatment . Interestingly, rabbit anti-MSP-1(19), but not anti-Pfs25, immunoglobulin, and immune monkey serum prevented the development of antibodies directed against MSP-1(19) fragment by infected monkeys, indicating that the antibodies were reactive with native MSP-1(19) antigen in vivo . The prepatent period and time to treatment was greatly delayed in the two monkeys that received Aotus immune serum, both of which developed a chronic intermittent low level infection . In vitro parasite growth inhibition assays (GIAs) confirmed the presence of inhibitory activity (40% maximum inhibition) in concentrated anti-MSP-1(19) immunoglobulin (4.8 mg/ml), but the peak concentrations we achieved in vivo (1 mg/ml) were not inhibitory in vitro . Subinhibitory levels of anti-MSP-1(19) antibodies achieved by passive transfer were not protective against P . falciparum challenge. Yeast, 1998 Dec, 14(16), 1453 - 69 Expanding yeast knowledge online; Dolinski K et al.; The completion of the Saccharomyces cerevisiae genome sequencing project and the continued development of improved technology for large-scale genome analysis have led to tremendous growth in the amount of new yeast genetics and molecular biology data . Efficient organization, presentation, and dissemination of this information are essential if researchers are to exploit this knowledge . In addition, the development of tools that provide efficient analysis of this information and link it with pertinent information from other systems is becoming increasingly important at a time when the complete genome sequences of other organisms are becoming available . The aim of this review is to familiarize biologists with the type of data resources currently available on the World Wide Web (WWW). J Cell Biol, 1999 Jan 11, 144(1), 83 - 98 Coronin promotes the rapid assembly and cross-linking of actin filaments and may link the actin and microtubule cytoskeletons in yeast; Goode BL et al.; Coronin is a highly conserved actin-associated protein that until now has had unknown biochemical activities . Using microtubule affinity chromatography, we coisolated actin and a homologue of coronin, Crn1p, from Saccharomyces cerevisiae cell extracts . Crn1p is an abundant component of the cortical actin cytoskeleton and binds to F-actin with high affinity (Kd 6 x 10(-9) M) . Crn1p promotes the rapid barbed-end assembly of actin filaments and cross-links filaments into bundles and more complex networks, but does not stabilize them . Genetic analyses with a crn1Delta deletion mutation also are consistent with Crn1p regulating filament assembly rather than stability . Filament cross-linking depends on the coiled coil domain of Crn1p, suggesting a requirement for Crn1p dimerization . Assembly-promoting activity is independent of cross-linking and could be due to nucleation and/or accelerated polymerization . Crn1p also binds to microtubules in vitro, and microtubule binding is enhanced by the presence of actin filaments . Microtubule binding is mediated by a region of Crn1p that contains sequences (not found in other coronins) homologous to the microtubule binding region of MAP1B . These activities, considered with microtubule defects observed in crn1Delta cells and in cells overexpressing Crn1p, suggest that Crn1p may provide a functional link between the actin and microtubule cytoskeletons in yeast. J Cell Biol, 1999 Jan 11, 144(1), 71 - 82 Regulation of the actin cytoskeleton organization in yeast by a novel serine/threonine kinase Prk1p; Zeng G et al.; Normal actin cytoskeleton organization in budding yeast requires the function of the Pan1p/ End3p complex . Mutations in PAN1 and END3 cause defects in the organization of actin cytoskeleton and endocytosis . By screening for mutations that can suppress the temperature sensitivity of a pan1 mutant (pan1-4), a novel serine/threonine kinase Prk1p is now identified as a new factor regulating the actin cytoskeleton organization in yeast . The suppression of pan1-4 by prk1 requires the presence of mutant Pan1p . Although viable, the prk1 mutant is unable to maintain an asymmetric distribution of the actin cytoskeleton at 37 degreesC . Consistent with its role in the regulation of actin cytoskeleton, Prk1p localizes to the regions of cell growth and coincides with the polarized actin patches . Overexpression of the PRK1 gene in wild-type cells leads to lethality and actin cytoskeleton abnormalities similar to those exhibited by the pan1 and end3 mutants . In vitro phosphorylation assays demonstrate that Prk1p is able to phosphorylate regions of Pan1p containing the LxxQxTG repeats, including the region responsible for binding to End3p . Based on these findings, we propose that the Prk1 protein kinase regulates the actin cytoskeleton organization by modulating the activities of some actin cytoskeleton-related proteins such as Pan1p/End3p. Glycobiology, 1999 Jan, 9(1), 93 - 100 A xyloglucan-specific endo-beta-1,4-glucanase from Aspergillus aculeatus: expression cloning in yeast, purification and characterization of the recombinant enzyme; Pauly M et al.; A full-length c-DNA encoding a xyloglucan-specific endo -beta-1, 4-glucanase (XEG) has been isolated from the filamentous fungus Aspergillus aculeatus by expression cloning in yeast . The colonies expressing functional XEG were identified on agar plates containing azurine-dyed cross-linked xyloglucan . The cDNA encoding XEG was isolated, sequenced, cloned into an Aspergillus expression vector, and transformed into Aspergillus oryzae for heterologous expression . The recombinant enzyme was purified to apparent homogeneity by anion-exchange and gel permeation chromatography . The recombinant XEG has a molecular mass of 23,600, an isoelectric point of 3.4, and is optimally stable at a pH of 3.4 and temperature below 30 degreesC . The enzyme hydrolyzes structurally diverse xyloglucans from various sources, but hydrolyzes no other cell wall component and can therefore be considered a xyloglucan-specific endo -beta-1, 4-glucanohydrolase . XEG hydrolyzes its substrates with retention of the anomeric configuration . The Kmof the recombinant enzyme is 3.6 mg/ml, and its specific activity is 260 micromol/min per mg protein . The enzyme was tested for its ability to solubilize xyloglucan oligosaccharides from plant cell walls . It was shown that treatment of plant cell walls with XEG yields only xyloglucan oligosaccharides, indicating that this enzyme can be a powerful tool in the structural elucidation of xyloglucans. Microbiology, 1998 Dec, 144 ( Pt 12), 3463 - 74 Cell integrity and morphogenesis in a budding yeast septin mutant; Cid VJ et al.; The non-sporulating diploid strain V327 of Saccharomyces cerevisiae was previously isolated in a search for thermosensitive autolytic mutants . This strain is very efficient at releasing intracellular proteins into the medium when incubated at high temperatures . The expression of this lytic phenotype depends on a morphogenetic defect, consisting of the appearance of elongated chains of cells . Transmission electron microscopy revealed a mislocalization of septa at semi-permissive temperatures and a total lack of septation together with abnormal cell wall architecture at a non-permissive temperature . The septin-encoding CDC10 gene was cloned by complementation of the pleiotropic phenotype of the V327 mutant . Rescue and sequencing of CDC10 alleles from V327 revealed a point mutation that created a single amino acid change in a region which is well conserved among septins . This new allele was named cdc10-11 . The construction of a cdc10-11 haploid strain by substituting the CDC10 gene with the rescued allele permitted further genetic analyses of the mutation and allowed the construction of new homozygous cdc10-11 diploid strains that showed a reduced ability to sporulate . Fusing both the wild-type and the cdc10-11 alleles to green fluorescent protein (GFP) demonstrated that the mutation does not affect the localization of this septin to the bud neck at the standard growth temperature of 24 degrees C, although the morphogenetic phenotype at 37 degrees C parallels the disappearance of Cdc10-GFP at the ring encircling the septum area. J Pharm Biomed Anal, 1998 Sep 1, 17(6-7), 1037 - 45 Analytical methods and stability assessment of liquid yeast derived sucrase; McIntosh KA et al.; Two independent analytical methods for determining the activity and stability profile of liquid yeast derived sucrase (YS) were established and validated in order to conduct preliminary stability studies as a function of temperature . The methods included a hexokinase-based (HK) enzymatic assay for determining the formation of glucose upon hydrolysis of sucrose by YS, and a direct polarimetric procedure to quantitate YS hydrolysis of sucrose . Both assays were validated with respect to YS dilution, incubation time, sucrose or glucose concentration, linearity of response and within- and between-day variability . A preliminary stability study was conducted over a 24 week period with liquid YS samples stored at -20, 4, 30, 40 and 50 degrees C . Enzymatic activity was monitored as a function of time using both the HK and polarimetric assays . Liquid YS samples stored at -20, 4 and 30 degrees C retained 100% activity after 24 weeks storage, while the samples stored at 40 degrees C lost approximately 70% activity over the same storage period and samples stored at 50 degrees C lost approximately 95% activity after 12 weeks storage . The two methods of analysis gave consistent results over the course of the study. FEBS Lett, 1998 Dec 18, 441(2), 266 - 70 Identification of a human mitochondrial ABC transporter, the functional orthologue of yeast Atm1p; Csere P et al.; We have sequenced the entire coding region of the human ABC transporter ABC7 . The protein represents a 'half-transporter' and displays high sequence similarity to the mouse ABC7 protein and to the mitochondrial ABC transporter Atm1p of Saccharomyces cerevisiae . As shown by immunostaining using a specific antibody, the human ABC7 protein (hABC7) is a constituent of mitochondria . The N-terminus of hABC7 contains the information for targeting and import into the organelles . When synthesised in yeast cells defective in Atm1p (strain delta atm1/hABC7), hABC7 protein can revert the strong growth defect observed for delta atm1 cells to near wild-type behaviour . The known phenotypical consequences of inactivation of the ATM1 gene are almost fully amended by expression of hABC7 protein . delta atm1/hABC7 cells harbour wild-type levels of cytochromes and extra-mitochondrial heme-containing proteins, they contain normal levels of mitochondrial iron, and the cellular content of glutathione is substantially reduced relative to the high levels detected in delta atm1 cells . Our results suggest that hABC7 is a mitochondrial protein, and represents the functional orthologue of yeast Atm1p. FEBS Lett, 1998 Dec 18, 441(2), 177 - 80 Glycan engineering of proteins with whole living yeast cells expressing rat liver alpha2,3-sialytransferase in the porous cell wall; Sievi E et al.; The N-glycans of recombinant proteins produced via the secretory pathway of cultured mammalian cells are often undersialylated, and insect cells lack sialytransferases . Undersialylated glycoproteins are rapidly cleared from the circulation, compromising the effect of pharmaceuticals . We show that incubation with living Saccharomyces cerevisiae cells expressing the catalytic ectodomain of rat liver alpha2,3-sialyltransferase (ST3Ne) in the porous cell wall resulted in sialylation of glycoproteins . The Km values of the yeast enzyme for several substrates were similar to those of recombinant ST3Ne from insect cells and of authentic ST3N . The yeast strain provides an inexpensive self-perpetuating source of ST3N activity for glycan engineering of recombinant proteins. Dev Biol, 1998 Dec 15, 204(2), 451 - 63 Partial rescue of GATA-3 by yeast artificial chromosome transgenes; Lakshmanan G et al.; GATA-3 is essential for murine embryonic development, but elucidating the genetic controls over the complex temporal and tissue-specific transcriptional regulatory pattern of this transcription factor gene has been problematic . Here we report the isolation and characterization of two yeast artificial chromosomes (YACs) bearing the murine GATA-3 gene . Ordered deletions of both YACs show that they define a 1-megabase pair contig spanning the GATA-3 locus . We found that a 120-kb YAC transgene, including 35 kb of 5' as well as 60 kb of 3' flanking sequence, confers normal GATA-3 expression at sites not revealed previously through analysis of plasmid transgenic lines . However, even this 120-kb YAC does not contain sufficient information to recapitulate the complete GATA-3 expression program during embryogenesis . While not complete in its regulatory capacity, the YAC transgene is nonetheless able to complement several homozygous GATA-3 mutant phenotypes and thereby prolong embryonic life . Biol Trace Elem Res, 1998 Nov, 65(2), 143 - 51 Bioavailability of enteral yeast-selenium in preterm infants; Bogye G et al.; There is no data or literature on the effects of supplementing infants with yeast selenium, although its intestinal absorption and bioavailability are higher in adults compared with other selenium compounds . The aim of the present investigation was to study the impact of selenium enriched yeast on the serum selenium concentration of preterm infants living in a low selenium area (Hungary) . Twenty-eight preterm infants with mean+/-SD birth weight of 962+/-129 g and gestational age 27+/-1 wk were randomized into two groups at birth with respect to selenium supplementation . In the supplemented group (n=14) infants received 4.8 mg yeast selenium containing 5 microg selenium daily via nasogastric drip during the first 14 postnatal days . The nonsupplemented infants were used as a reference group . In the supplemented group, the serum selenium concentration increased from 32.1+/-8.5 microg/L to 41.5+/-6.5 microg/L and in the nonsupplemented group it decreased from 25.9+/-6.8 microg/L to 18.2+/-6.4 microg/L from birth in two weeks time . Compared with previous studies, our results suggest that the bioavailability of selenium in the form of yeast selenium is higher than that of other selenium compounds used for preterm infants . We did not observe any complications or side-effects owing to enteral yeast selenium supplementation . We conclude that selenium enriched yeast is a safe and an effective form of short-term enteral selenium supplementation for infants. Plant J, 1998 Nov, 16(4), 453 - 64 The plant PTS1 receptor: similarities and differences to its human and yeast counterparts; Wimmer C et al.; Two targeting signals, PTS1 and PTS2, mediate import of proteins into the peroxisomal matrix . We have cloned and sequenced the watermelon (Citrullus vulgaris) cDNA homologue to the PTS1 receptor gene (PEX5) . Its gene product, CvPex5p, belongs to the family of tetratricopeptide repeat (TPR) containing proteins like the human and yeast counterparts, and exhibits 11 repeats of the sequence W-X2-(E/S)-(Y/F/Q) in its N-terminal half . According to fractionation studies the plant Pex5p is located mainly in the cytosolic fraction and therefore could function as a cycling receptor between the cytosol and glyoxysomes, as has been proposed for the Pex5p of human and some yeast peroxisomes . Transformation of the Hansenula polymorpha peroxisome deficient pex5 mutant with watermelon PEX5 resulted in restoration of peroxisome formation and the synthesis of additional membranes surrounding the peroxisomes . These structures are labeled in immunogold experiments using antibodies against the Hansenula polymorpha integral membrane protein Pex3p, confirming their peroxisomal nature . The plant Pex5p was localized by immunogold labelling mainly in the cytosol of the yeast, but also inside the newly formed peroxisomes . However, import of the PTS1 protein alcohol oxidase is only partially restored by CvPex5p. J Biol Chem, 1999 Jan 15, 274(3), 1835 - 41 A syntaxin homolog encoded by VAM3 mediates down-regulation of a yeast G protein-coupled receptor; Stefan CJ et al.; G protein-coupled receptors that transduce signals for many hormones, neurotransmitters, and inflammatory mediators are internalized and subsequently recycled to the plasma membrane, or down-regulated by targeting to lysosomes for degradation . Here we have characterized yeast alpha-factor receptors tagged with green fluorescent protein (Ste2-GFP) and used them to obtain mutants defective in receptor down-regulation . In wild type cells, Ste2-GFP was functional and localized to the plasma membrane and endocytic compartments . Although GFP was fused to the cytoplasmic tail of the receptor, GFP also accumulated in the lumen of the vacuole, suggesting that the receptor's extracellular and cytoplasmic domains are degraded within the vacuole lumen . Transposon mutagenesis and a visual screen were used to identify mutants displaying aberrant localization of Ste2-GFP . Mutants that accumulated Ste2-GFP in numerous intracellular vesicles carried disruptions of the VAM3/PTH1 gene, which encodes a syntaxin homolog (t-SNARE) required for homotypic vacuole membrane fusion, autophagy and fusion of biosynthetic transport vesicles with the vacuole . We provide evidence that Vam3 is required for the delivery of alpha-factor receptor-ligand complexes to the vacuole . Vam3 homologs in mammalian cells may mediate late steps in the down-regulation and lysosomal degradation pathways of various G protein-coupled receptors. J Biol Chem, 1999 Jan 15, 274(3), 1199 - 202 Rad26, the yeast homolog of the cockayne syndrome B gene product, counteracts inhibition of DNA repair due to RNA polymerase II transcription; Tijsterman M et al.; Transcription-coupled DNA repair (TCR) is responsible for the preferential removal of DNA lesions from the transcribed strands of RNA polymerase II transcribed genes . Saccharomyces cerevisiae rad26 mutants and cells from patients suffering from the hereditary disease Cockayne syndrome display a TCR defective phenotype . Whether this lack of preferential repair has to be explained by a defect in repair or in general transcription is unclear at present . To discriminate between both possibilities, we analyzed repair of UV-induced cyclobutane pyrimidine dimers at single base resolution in yeast cells lacking RAD26, the homolog of the Cockayne syndrome B gene . Disrupting RAD26 affects nucleotide excision repair of transcribed DNA irrespective of the chromatin context, resulting in similar rates of removal for individual cyclobutane pyrimidine dimers throughout the transcribed strand . Notably, repair of transcribed sequences in between core nucleosomal regions is less efficient compared with nontranscribed DNA at these positions, pointing to a nucleotide excision repair impediment caused by blocked RNA polymerase . Our in vivo data demonstrate that the TCR defect in rad26 mutant cells is not due to a general transcription deficiency but results from the inability to release the transcription complex trapped at sites of base damage. Mol Biol Cell, 1999 Jan, 10(1), 63 - 75 Structural and functional analysis of a novel coiled-coil protein involved in Ypt6 GTPase-regulated protein transport in yeast; Tsukada M et al.; The yeast transport GTPase Ypt6p is dispensable for cell growth and secretion, but its lack results in temperature sensitivity and missorting of vacuolar carboxypeptidase Y . We previously identified four yeast genes (SYS1, 2, 3, and 5) that on high expression suppressed these phenotypic alterations . SYS3 encodes a 105-kDa protein with a predicted high alpha-helical content . It is related to a variety of mammalian Golgi-associated proteins and to the yeast Uso1p, an essential protein involved in docking of endoplasmic reticulum-derived vesicles to the cis-Golgi . Like Uso1p, Sys3p is predominatly cytosolic . According to gel chromatographic, two-hybrid, and chemical cross-linking analyses, Sys3p forms dimers and larger protein complexes . Its loss of function results in partial missorting of carboxypeptidase Y . Double disruptions of SYS3 and YPT6 lead to a significant growth inhibition of the mutant cells, to a massive accumulation of 40- to 50-nm vesicles, to an aggravation of vacuolar protein missorting, and to a defect in alpha-pheromone processing apparently attributable to a perturbation of protease Kex2p cycling between the Golgi and a post-Golgi compartment . The results of this study suggest that Sys3p, like Ypt6p, acts in vesicular transport (presumably at a vesicle-docking stage) between an endosomal compartment and the most distal Golgi compartment. J Enzyme Inhib, 1998 Feb, 13(1), 57 - 68 Influence of Tris(hydroxymethyl)aminomethane on kinetic mechanism of yeast alcohol dehydrogenase; Trivic S et al.; Acetaldehyde, propionaldehyde, glyceraldehyde-3-P and 4-dimethylaminocinnamaldehyde form Schiff bases in Tris . HCl buffers; the rates of formation and dissociation of Schiff bases, and equilibrium constants for their formation are very similar for the first three aldehydes . The steady-state kinetic constants for the yeast alcohol dehydrogenase-catalyzed reaction, propan-1-ol + NAD+ reversible propionaldehyde + NADH + H+, have been determined in several Tris . HCl buffers of increasing concentration at pH 8.1 . In the forward direction, oxidation of alcohol, most kinetic constants are increased by increasing concentrations of Tris . In the reverse direction, reduction of aldehyde, substrate, NADH, Tris and Schiff base were equilibrated before enzyme reaction was started . It was found that Schiff base, rather than Tris, binds to free enzyme competitively with respect to NADH . Tris and Schiff base do not influence the binding of aldehyde to enzyme in any way. Biochim Biophys Acta, 1999 Jan 6, 1426(2), 373 - 83 The contribution of cell wall proteins to the organization of the yeast cell wall; Kapteyn JC et al.; Our knowledge of the yeast cell wall has increased rapidly in the past few years, allowing for the first time a description of its structure in molecular terms . Two types of cell wall proteins (CWPs) have been identified that are covalently linked to beta-glucan, namely GPI-CWPs and Pir-CWPs . Both define a characteristic supramolecular complex or structural unit . The GPI building block has the core structure GPI-CWP-->beta1,6-glucan-->beta1,3-glucan, which may become extended with one or more chitin chains . The Pir building block is less well characterized, but preliminary evidence points to the structure, Pir-CWP-->beta1,3-glucan, which probably also may become extended with one or more chitin chains . The molecular architecture of the cell wall is not fixed . The cell can make considerable adjustments to the composition and structure of its wall, for example, during the cell cycle or in response to environmental conditions such as nutrient and oxygen availability, temperature, and pH . When the cell wall is defective, dramatic changes can occur in its molecular architecture, pointing to the existence of cell wall repair mechanisms that compensate for cell damage . Finally, evidence is emerging that at least to a considerable extent the cell wall of Saccharomyces cerevisiae is representative for the cell wall of the Ascomycetes. Biochim Biophys Acta, 1999 Jan 6, 1426(2), 309 - 22 Asparagine-linked glycosylation in the yeast Golgi; Dean N; The Golgi complex is the site where the terminal carbohydrate modification of proteins and lipids occurs . These carbohydrates play a variety of biological roles, ranging from the stabilization of glycoprotein structure to the provision of ligands for cell-cell interactions to the regulation of cell surface properties . Progress in our understanding of the biosynthesis and regulation of glycoconjugates has been accelerating at a rapid pace . Recent advances in the field of yeast glycobiology have been particularly impressive . This review focuses on glycosylation of proteins in the Golgi of the yeast Saccharomyces cerevisiae, with emphasis on the candidate mannosyltransferases that participate in the synthesis of N-linked oligosaccharides . Current views on how these enzymes may be regulated and how glycosylation relates on other cellular processes are also discussed. Biochim Biophys Acta, 1999 Jan 6, 1426(2), 259 - 73 The oligosaccharyltransferase complex from yeast; Knauer R et al.; N-Glycosylation of eukaryotic secretory and membrane-bound proteins is an essential and highly conserved protein modification . The key step of this pathway is the en bloc transfer of the high mannose core oligosaccharide Glc3Man9GlcNAc2 from the lipid carrier dolichyl phosphate to selected Asn-X-Ser/Thr sequences of nascent polypeptide chains during their translocation across the endoplasmic reticulum membrane . The reaction is catalysed by the enzyme oligosaccharyltransferase (OST) . Recent biochemical and molecular genetic studies in yeast have yielded novel insights into this enzyme with multiple tasks . Nine proteins have been shown to be OST components . These are assembled into a heterooligomeric membrane-bound complex and are required for optimal expression of OST activity in vivo in wild type cells . In accord with the evolutionary conservation of core N-glycosylation, there are significant homologies between the protein sequences of OST subunits from yeast and higher eukaryotes, and OST complexes from different sources show a similar organisation as well. Biochim Biophys Acta, 1999 Jan 4, 1426(1), 119 - 25 Effect of astaxanthin rich red yeast (Phaffia rhodozyma) on oxidative stress in rainbow trout; Nakano T et al.; The antioxidative biological effect of dietary red yeast, Phaffia rhodozyma, which is rich in astaxanthin, on rainbow trout, Oncorhynchus mykiss, was examined . The levels of serum transaminase (glutamic-pyruvic transaminase and glutamic-oxaloacetic transaminase) activities and of lipid peroxides (LPO) of fish fed oxidized oil were significantly higher than those of the control fish fed non-oxidized oil . However, the supply of red yeast considerably decreased both enzyme activities and LPO level . Furthermore, the serum lipid (triglycerides, total cholesterol and phospholipids) concentrations were also significantly decreased . Especially, the serum triglyceride level of fish fed the red yeast was as low as that of the control . It was also observed that there were no significant differences in muscle LPO levels between the fish fed red yeast and the control . The present results suggest for the first time that dietary red yeast may effectively suppress the LPO generation of tissue and normalize liver function as well as improving muscle pigmentation of trout . Thus, red yeast should have a reducing effect on oxidized oil-induced oxidative stress in fish. Biochim Biophys Acta, 1999 Jan 5, 1409(3), 113 - 24 Expression of the bovine heart mitochondrial ADP/ATP carrier in yeast mitochondria: significantly enhanced expression by replacement of the N-terminal region of the bovine carrier by the corresponding regions of the yeast carriers; Hashimoto M et al.; To characterize the transport mechanism mediated by the mammalian mitochondrial ADP/ATP carrier (AAC), we tried to express bovine heart mitochondrial AAC (bhAAC) in Saccharomyces cerevisiae . The open reading frame of the bhAAC was introduced into the haploid strain WB-12, in which intrinsic AAC genes were disrupted . Growth of the transformant was very low in glycerol medium, and a little amount of bhAAC was detected in the mitochondrial membrane . For improvement of bhAAC expression in WB-12, we introduced DNA fragments encoding chimeric bhAACs, in which the N-terminal region of the bhAAC extending into the cytosol was replaced by the corresponding regions of the type 1 and type 2 yeast AAC isoforms (yAAC1 and yAAC2) . These transformants grew well, and the amounts of the chimeric bhAACs in their mitochondria were as high as that of yAAC2 . The carriers expressed showed essentially the same ADP transport activities as that of AAC in bovine heart mitochondria. EMBO J, 1999 Jan 4, 18(1), 229 - 40 Mutations in both the structured domain and N-terminus of histone H2B bypass the requirement for Swi-Snf in yeast; Recht J et al.; The chromatin elements targeted by the ATPdependent, Swi-Snf nucleosome-remodeling complex are unknown . To address this question, we generated mutations in yeast histone H2B that suppress phenotypes associated with the absence of Swi-Snf . Sin- (Swi-Snf-independent) mutations occur in residues involved in H2A-H2B dimer formation, dimer- tetramer association, and in the H2B N-terminus . The strongest and most pleiotropic Sin- mutation removed 20 amino acid residues from the H2B N-terminus . This mutation allowed active chromatin to be formed at the SUC2 locus in a snf5Delta mutant and resulted in hyperactivated levels of SUC2 mRNA under inducing conditions . Thus, the H2B N-terminus may be an important target of Swi-Snf in vivo . The GCN5 gene product, the catalytic subunit of several nuclear histone acetytransferase complexes that modify histone N-termini, was also found to act in conjunction with Swi-Snf . The phenotypes of double gcn5Deltasnf5Delta mutants suggest that histone acetylation may play both positive and negative roles in the activity of the Swi-Snf-remodeling factor. Biophys J, 1999 Jan, 76(1 Pt 1), 50 - 64 Molecular dynamics simulations of solvated yeast tRNA(Asp); Auffinger P et al.; Transfer RNA molecules are involved in a variety of biological processes, implying complex recognition events with proteins and other RNAs . From a structural point of view, tRNAs constitute a reference system for studying RNA folding and architecture . A deeper understanding of their structural and functional properties will derive from our ability to model accurately their dynamical behavior . We present the first dynamical model of a fully neutralized and solvated tRNA molecule over a 500-ps time scale . Starting from the crystallographic structure of yeast tRNA(Asp), the 75-nucleotide molecule was modeled with 8055 water molecules and 74 NH4+ counterions, using the AMBER4.1 program and the particle mesh Ewald (PME) method for the treatment of long-range electrostatic interactions . The calculations led to a dynamically stable model of the tRNA molecule . During the simulation, all secondary and tertiary base pairs are maintained while a certain lability of base triples in the tRNA core is observed . This lability was interpreted as resulting from intrinsic factors associated with the "weaker" hydrogen bonding patterns seen in these base triples and from an altered ionic environment of the tRNA molecule . Calculated thermal factors are used to compare the dynamics of the tRNA in solution and in the crystal . The present molecular dynamics simulation of a complex and highly charged nucleic acid molecule attests to the fact that simulation methods are now able to investigate not only the dynamics of proteins, but also that of large RNA molecules . Thus they also provide a basis for further investigations on the structural and functional effects of chemical and posttranscriptionally modified nucleotides as well as on ionic environmental effects. Mutat Res, 1998 Dec 14, 409(3), 181 - 8 Defective Kin28, a subunit of yeast TFIIH, impairs transcription-coupled but not global genome nucleotide excision repair; Tijsterman M et al.; The essential Saccharomyces cerevisiae KIN28 gene encodes a subunit of general transcription factor TFIIH, a multiprotein complex required for RNA polymerase II transcription initiation and nucleotide excision repair (NER) . Kin28 is implicated in the transition from transcription initiation to transcription elongation by phosphorylation of the carboxy-terminal domain (CTD) of the largest subunit of the RNA polymerase II complex . Here, we explore the possibility that Kin28 like the other subunits of TFIIH is involved in NER in vivo, using yeast cells carrying either a wildtype or a thermosensitive KIN28 allele . The removal of UV induced cyclobutane pyrimidine dimers (CPDs) was monitored at base resolution from both strands of the RNA polymerase II transcribed genes RPB2 and URA3 . Cells carrying the thermosensitive KIN28 allele display a transcription-coupled repair (TCR) defect at the non-permissive temperature, which was most pronounced directly downstream of transcription initiation, probably as an indirect result of a general decrease in the level of RNA polymerase II transcription . The fact that CPD removal in non-transcribed DNA is completely unaffected in these cells indicates that Kin28 is not essential for general NER in vivo, providing the first example of a TFIIH subunit that is required for TCR but not for NER in general. Proc Natl Acad Sci U S A, 1999 Jan 5, 96(1), 121 - 6 Calcium influx factor is synthesized by yeast and mammalian cells depleted of organellar calcium stores; Csutora P et al.; Depletion of endoplasmic reticulum Ca2+ stores leads to the entry of extracellular Ca2+ into the cytoplasm, a process termed capacitative or store-operated Ca2+ entry . Partially purified extracts were prepared from the human Jurkat T lymphocyte cell line and yeast in which Ca2+ stores were depleted by chemical and genetic means, respectively . After microinjection into Xenopus laevis oocytes, the extracts elicited a wave of increased cytoplasmic free Ca2+ ({Ca2+}i) that spread from the point of injection across the oocyte . Extracts from cells with replete organellar Ca2+ stores were inactive . The increases depended on extracellular Ca2+, were unaffected by the inositol 1,4,5-trisphosphate (IP3) inhibitor heparin or an anti-IP3 receptor antibody and were unchanged when the endoplasmic reticulum was segregated to the hemisphere opposite the injection site by centrifugation . Confocal microscopy revealed that {Ca2+}i increases were most pronounced at the periphery of the oocyte . The patterns of {Ca2+}i increases were replicated by computer simulations based on a diffusible messenger of about 700 Da that directly activates Ca2+ influx . In addition, ICRAC, a Ca2+ release-activated Ca2+ current monitored in Jurkat cells by whole-cell patch clamp recordings, was more rapidly activated when active extracts were included in the patch pipette than by the inclusion of a Ca2+ chelator or IP3 . These data support the existence in yeast and mammalian cells depleted of Ca2+ stores of a functionally conserved diffusible calcium influx factor that directly activates Ca2+ influx. Eur J Biochem, 1998 Dec 1, 258(2), 430 - 6 Sporulation-specific expression of the yeast DIT1/DIT2 promoter is controlled by a newly identified repressor element and the short form of Rim101p; Bogengruber E et al.; Expression of the yeast genes DIT1 and DIT2 is confined to mid/late sporulation . Transcription of these two divergently arranged genes is controlled by a common 900-bp intergenic region . Random mutagenesis of this promoter and tests with appropriate reporter constructs identified an 11-bp cis-acting palindromic sequence, DIT repressor element (DRE), as a major negative regulatory site during vegetative growth . Repression is exerted by DRE in conjunction with a mid-sporulation element (MSE)-like sequence situated 26 bp away . These sequence elements are both contained within the 76-bp negative regulatory element (NRE) defined previously {Friesen H., Hepworth, S . R . & Segall, J . (1997) Mol . Cell . Biol . 17, 123-134} . The activated form of Rim101p, a transcriptional inducer of the early meiotic gene IME1, enhances expression from the DIT1 promoter both in vegetative and sporulating cells . Activation by Rim101p does not seem to involve binding of Rim101p at either of the two cis-acting sites described here, since reporter constructs with both elements or most of the NRE deleted could still be activated by the short form of Rim101p. Bioorg Med Chem Lett, 1998 Aug 18, 8(16), 2269 - 72 Alkyl side-chain derivatives of sordaricin as potent antifungal agents against yeast; Tse B et al.; Sordarin (1) was converted to 5 and 6, which showed potent antifungal activity against yeast . A series of C1-C9 alkyl side-chain derivatives was prepared, from which it was found that the optimal activity occurred with C5 . A comparison of side chains with different unsaturation showed that the cis-alkene was the most active . This result suggested that the folding of the side chains might be crucial for the optimal activity. Genome Res, 1998 Dec, 8(12), 1259 - 72 Homologs of the yeast longevity gene LAG1 in Caenorhabditis elegans and human; Jiang JC et al.; LAG1 is a longevity gene, the first such gene to be identified and cloned from the yeast Saccharomyces cerevisiae . A close homolog of this gene, which we call LAC1, has been found in the yeast genome . We have cloned the human homolog of LAG1 with the ultimate goal of examining its possible function in human aging . In the process, we have also cloned a homolog from the nematode worm Caenorhabditis elegans . Both of these homologs, LAG1Hs and LAG1Ce-1, functionally complemented the lethality of a lag1delta lac1delta double deletion, despite low overall sequence similarity to the yeast proteins . The proteins shared a short sequence, the Lag1 motif, and a similar transmembrane domain profile . Another, more distant human homolog, TRAM, which lacks this motif, did not complement . LAG1Hs also restored the life span of the double deletion, demonstrating that it functions in establishing the longevity phenotype in yeast . LAG1Hs mapped to 19p12, and it was expressed in only three tissues: brain, skeletal muscle, and testis . This gene possesses a trinucleotide (CTG) repeat within exon 1 . This and its expression profile raise the possibility that it may be involved in neurodegenerative disease . This possibility suggests at least one way in which LAG1Hs might be involved in human aging. Appl Environ Microbiol, 1999 Jan, 65(1), 110 - 6 Threonine overproduction in yeast strains carrying the HOM3-R2 mutant allele under the control of different inducible promoters; Farfan MJ et al.; The HOM3 gene of Saccharomyces cerevisiae codes for aspartate kinase, which plays a crucial role in the regulation of the metabolic flux that leads to threonine biosynthesis . With the aim of obtaining yeast strains able to overproduce threonine in a controlled way, we have placed the HOM3-R2 mutant allele, which causes expression of a feedback-insensitive enzyme, under the control of four distinctive regulatable yeast promoters, namely, PGAL1, PCHA1, PCYC1-HSE2, and PGPH1 . The amino acid contents of strains bearing the different constructs were analyzed both under repression and induction conditions . Although some differences in overall threonine production were found, a maximum of around 400 nmol/mg (dry weight) was observed . Other factors, such as excretion to the medium and activity of the catabolic threonine/serine deaminase, also affect threonine accumulation . Thus, improvement of threonine productivity by yeast cells would probably require manipulation of these and other factors. Curr Genet, 1998 Dec, 34(5), 360 - 7 Genetic analysis of the yeast NUD1 endo-exonuclease: a role in the repair of DNA double-strand breaks; Asefa B et al.; The deoxyribonucleases (DNases) have been shown genetically to be important in the vital processes of DNA repair and recombination . The NUD1 gene, which codes for an endo-exonuclease of Saccharomyces cerevisiae, was analyzed for its role in the DNA double-strand break (DSB) repair processes . While the nud1 strain is only slightly sensitive to ionizing radiation, expression of the HO-endonuclease to introduce a DSB at the MAT locus in that strain results in cell death . Cell survival is inversely proportional to the duration of HO-endonuclease expression . Analysis of the surviving colonies from the nud1 strain indicated that many of the survivors are sterile and that the proportion of these sterile survivors increases with the time of HO-endonuclease expression . On the other hand, the surviving colonies from the isogenic NUD1 strain are mating-proficient . Interestingly, double mutants of nud1 rad52 are more resistant to ionizing irradiation than the rad52 strain and have a cell-survival fraction of 32% for rad52-1 nud1 and 9% for rad52::URA3 nud1 following prolonged HO-endonuclease expression, indicating that nud1 has a suppressor effect on the DSB-induced lethality in rad52 . Polymerase chain reaction analysis showed that many of the nud1 survivors contained small alterations within theMAT locus, suggesting that the survivors arose through the process of non-homologous end-joining . These results suggest that the endo-exonuclease acts at a DSB to promote DNA repair via the homologous recombination pathway. Cell Biochem Biophys, 1998, 29(3), 263 - 79 Ruptured fission yeast walls . Structural discontinuities related to the cell cycle; Piombo S et al.; Distributions of rupture sites of fission yeast cells ruptured by glass beads have been related to a new morphometric analysis . As shown previously (Johnson et al., Cell Biophysics, 1995), ruptures were not randomly distributed nor was their distribution dictated by geometry, rather, ruptures at the extensile end were related to cell length just as the rate of extension is related to cell length . The extension patterns of early log, mid-log, late log, and stationary phase cells from suspension cultures were found to approximate the linear growth patterns of Kubitschek and Clay (1986) . The median length of cells was found to decline through the log phase in an unbalanced manner. Trends Biochem Sci, 1998 Dec, 23(12), 481 - 5 Structural organization of MAP-kinase signaling modules by scaffold proteins in yeast and mammals; Whitmarsh AJ et al.; MAP-kinase signaling pathways are activated by multiple extracellular stimuli . The specificity of activation and function of MAP-kinase signaling modules is determined, in part, by scaffold proteins that create multienzyme complexes . In Saccharomyces cerevisiae, two MAP-kinase-scaffold proteins have been identified . Recent studies of mammalian cells have also led to the identification of putative scaffold proteins . These scaffold proteins appear to facilitate MAP-kinase activation, in response to specific physiological stimuli, and to insulate the bound MAP-kinase module against activation by irrelevant stimuli . Scaffold proteins are therefore critical components of MAP-kinase modules and ensure signaling specificity. J Biol Chem, 1999 Jan 1, 274(1), 543 - 8 Isolation of supernumerary yeast ATP synthase subunits e and i . Characterization of subunit i and disruption of its structural gene ATP18; Vaillier J et al.; Two subunits of the yeast ATP synthase have been isolated . Subunit e was found loosely associated to the complex . Triton X-100 at a 1% concentration removed this subunit from the ATP synthase . The N-terminal sequencing of subunit i has been performed . The data are in agreement with the sequence of the predicted product of a DNA fragment of Saccharomyces cerevisiae chromosome XIII . The ATP18 gene encodes subunit i, which is 59 amino acids long and corresponds to a calculated mass of 6687 Da . Its pI is 9.73 . It is an amphiphilic protein having a hydrophobic N-terminal part and a hydrophilic C-terminal part . It is not apparently related to any subunit described in other ATP synthases . The null mutant showed low growth on nonfermentable medium . Mutant mitochondria display a low ADP/O ratio and a decrease with time in proton pumping after ATP addition . Subunit i is associated with the complex; it is not a structural component of the enzyme but rather is involved in the oxidative phosphorylations . Similar amounts of ATP synthase were measured for wild-type and null mutant mitochondria . Because 2-fold less specific ATPase activity was measured for the null mutant than for the wild-type mitochondria, we make the hypothesis that the observed decrease in the turnover of the mutant enzyme could be linked to a proton translocation defect through F0. J Biol Chem, 1999 Jan 1, 274(1), 354 - 9 In vivo characterization of chimeric phytochromes in yeast; Eichenberg K et al.; Phytochromes are plant photoreceptors that play a major role in photomorphogenesis . Two members of the phytochrome family have been characterized in some detail . Phytochrome A, which controls very low fluence and high irradiance responses, is rapidly degraded in the light, forms sequestered areas of phytochrome (SAPs), and does not exhibit dark reversion in monocotyledonous seedlings . Phytochrome B mediates red/far-red reversible responses, is stable in the light, and does not form SAPs . We report on the behavior in yeast of the phytochrome apoproteins of rice PHYA, tobacco PHYB, and chimeric PHYAB and PHYBA and on the behavior of the respective holoprotein adducts after assembly with phycocyanobilin chromophore (PHY*) . SAP-like formation in yeast was not observed for PHYB, but was detectable for PHYA, PHYAB, and PHYBA . Rice PHYA* did not undergo dark reversion in yeast . Surprisingly, all other tested phytochrome constructs did exhibit dark reversion, including chimeric phytochromes with a short N-terminal part of tobacco PHYB or parsley PHYA fused to rice PHYA . Furthermore, the proportion of phytochrome undergoing dark reversion and the rate of reversion were increased for both the N terminus-swapped constructs and PHYBA* . These results are discussed with respect to structure/function analysis of phytochromes A and B. Anal Biochem, 1998 Dec 1, 265(1), 123 - 8 The yeast mitochondrial intermembrane space: purification and analysis of two distinct fractions; Martin H et al.; We have developed a protocol for the sequential release of the intermembrane space (IMS) content of Saccharomyces cerevisiae mitochondria . Two distinct fractions were obtained: a soluble IMS with cytochrome b2 as key marker and a salt-extractable IMS with cytochrome c as key marker . The identity of several proteins was determined by amino-terminal amino acid sequencing . The IMS fractions were devoid of contaminations from cytosol and mitochondrial outer and inner membranes . By subtraction analysis, the protein profiles of soluble and salt-extractable IMS fractions were depleted of contaminating bands derived from matrix proteins . The fractionation method will provide the basis for the further analysis of IMS proteins and characterization of their functions in bioenergetics, mitochondrial biogenesis, and regulatory processes. Appl Microbiol Biotechnol, 1998 Nov, 50(5), 583 - 8 The influence of carbon source on the level and composition of ceramides of the Candida lipolytica yeast; Rupcic J et al.; Candida lipolytica yeast was grown batchwise on two different carbon sources, glucose and n-hexadecane . Free ceramides were quantitatively isolated from sphingolipid fractions of total lipids by a combination of column chromatography and preparative thin-layer chromatography . Their composition, after acid methanolysis, was analysed by gas-liquid chromatography . The ceramide content accounted for 2.6% of the total cell lipids in hexadecane-grown cells, which was 1.5 times higher than in glucose-grown cells . The fatty acid composition of ceramides was characterized by the predominance of fatty acids shorter than 20 carbon atoms and by high concentrations of fatty acids with 16 carbon atoms after growth on both carbon sources . The dominant fatty acid was hydroxylated 16:0 in the glucose-grown cells and 16:0 in the hexadecane-grown cells . The striking finding was the low degree of fatty acid hydroxylation and relatively high proportion of odd-numbered fatty acids in ceramide of the n-hexadecane-grown cells . The ceramides contained an unusual long-chain base composition . In hexadecane-grown cells more than 60% of the long-chain bases were C19 phytosphingosine . In glucose-grown cells more than one-half of the total long-chain bases were tetrahydroxy bases, 4,5-dihydroxysphinganine and 4,5-dihydroxyeicosasphinganine. Biochemistry (Mosc), 1998 Nov, 63(11), 1307 - 11 Inactivation and conformational changes of yeast alcohol dehydrogenase in trifluoroethanol solutions; Yang Y et al.; Conformational changes of yeast alcohol dehydrogenase in trifluoroethanol solutions have been followed by fluorescence emission and circular dichroism spectroscopy . At low concentration (less than 5%), trifluoroethanol shows a reversible inhibition competitive to ethanol and noncompetitive to NAD+ . The inhibition constants for native and structural-zinc-removed yeast alcohol dehydrogenase were 5.8 and 1.1 mM, respectively, suggesting that the active site becomes more flexible after the structural zinc is removed . At higher trifluoroethanol concentrations the enzyme was irreversibly inactivated . Comparison of inactivation and conformational changes of yeast alcohol dehydrogenase denatured in trifluoroethanol solutions shows that the extent of inactivation is larger than the extent of conformational changes at the same trifluoroethanol concentration . The results obtained from circular dichroism spectra show that the presence of trifluoroethanol can induce the formation of secondary structure of the enzyme. J Cell Biol, 1998 Dec 28, 143(7), 1931 - 45 Tropomyosin-containing actin cables direct the Myo2p-dependent polarized delivery of secretory vesicles in budding yeast; Pruyne DW et al.; The actin cytoskeleton in budding yeast consists of cortical patches and cables, both of which polarize toward regions of cell growth . Tropomyosin localizes specifically to actin cables and not cortical patches . Upon shifting cells with conditionally defective tropomyosin to restrictive temperatures, actin cables disappear within 1 min and both the unconventional class V myosin Myo2p and the secretory vesicle-associated Rab GTPase Sec4p depolarize rapidly . Bud growth ceases and the mother cell grows isotropically . When returned to permissive temperatures, tropomyosin-containing cables reform within 1 min in polarized arrays . Cable reassembly permits rapid enrichment of Myo2p at the focus of nascent cables as well as the Myo2p- dependent recruitment of Sec4p and the exocyst protein Sec8p, and the initiation of bud emergence . With the loss of actin cables, cortical patches slowly assume an isotropic distribution within the cell and will repolarize only after restoration of cables . Therefore, actin cables respond to polarity cues independently of the overall distribution of cortical patches and are able to directly target the Myo2p-dependent delivery of secretory vesicles and polarization of growth. J Cell Biol, 1998 Dec 28, 143(7), 1775 - 87 Fission yeast bub1 is a mitotic centromere protein essential for the spindle checkpoint and the preservation of correct ploidy through mitosis; Bernard P et al.; The spindle checkpoint ensures proper chromosome segregation by delaying anaphase until all chromosomes are correctly attached to the mitotic spindle . We investigated the role of the fission yeast bub1 gene in spindle checkpoint function and in unperturbed mitoses . We find that bub1(+) is essential for the fission yeast spindle checkpoint response to spindle damage and to defects in centromere function . Activation of the checkpoint results in the recruitment of Bub1 to centromeres and a delay in the completion of mitosis . We show that Bub1 also has a crucial role in normal, unperturbed mitoses . Loss of bub1 function causes chromosomes to lag on the anaphase spindle and an increased frequency of chromosome loss . Such genomic instability is even more dramatic in Deltabub1 diploids, leading to massive chromosome missegregation events and loss of the diploid state, demonstrating that bub1(+ )function is essential to maintain correct ploidy through mitosis . As in larger eukaryotes, Bub1 is recruited to kinetochores during the early stages of mitosis . However, unlike its vertebrate counterpart, a pool of Bub1 remains centromere-associated at metaphase and even until telophase . We discuss the possibility of a role for the Bub1 kinase after the metaphase-anaphase transition. Mol Gen Genet, 1998 Nov, 260(2-3), 295 - 9 Mouse homolog of the yeast origin recognition complex subunit ORC1 and chromosomal localization of the cognate mouse gene Orc1; Zisimopoulou P et al.; ORC1 encodes a subunit of the eukaryotic origin recognition complex in yeast, which has been shown to play a crucial role in chromosomal DNA replication and transcriptional silencing . We have identified a mouse protein closely related to Saccharomyces cerevisiae ORC1 as well as to the human, Xenopus and Drosophila homologs . MmORC1 has putative cyclin-dependent phosphorylation sites and a nucleotide triphosphate-binding motif . We have mapped the gene locus of the murine Orc1 gene to chromosome 4, band D . Conservation of structures among members of the ORC1-related proteins suggests that these proteins play a key role in the intiation of DNA replication in all eukaryotes. Biochem Mol Biol Int, 1998 Dec, 46(5), 887 - 94 The effect of heparin and pentosan polysulfate on the thermal stability of yeast alcohol dehydrogenase; Paulikova H et al.; Heparin and pentosan polysulfate as organic polyanions inhibit yeast alcohol dehydrogenase (YADH) . The aim of this study was to determine the effect of heparin and pentosan polysulfate on the thermostability of alcohol dehydrogenase . Spectral and kinetic analyses showed that these compounds increase the thermal stability of the enzyme and eliminate entirely thermal aggregation . The thermostabilizing effect of unfractionated heparin and pentosan polysulfate was accelerated in the presence of NAD+ . The addition of NAD+ (11 microM) to the incubation medium decreased the inhibition of the YADH activity in the presence of pentosan polysulfate (1.32 microM) . Moreover, 38% of the residual activity of YADH was found after a 5-min incubation at 70 degrees C . These findings indicate that heparinoids not only modulate the enzyme activity but also can prevent the protein's thermal denaturation. Cytogenet Cell Genet, 1998, 82(3-4), 147 - 52 Identification and characterization of STK12/Aik2: a human gene related to aurora of Drosophila and yeast IPL1; Kimura M et al.; Mutations in aurora of Drosophila and related Saccharomyces cerevisiae IPL1 protein kinases are known to cause abnormal chromosome segregation . We earlier isolated a cDNA encoding a novel human protein kinase Aik which shares high amino acid identity with the Aurora/Ipl1 protein kinase family . In the present study, a second human cDNA highly homologous to aurora/IPL1 (Aik2) was identified and the nucleotide sequence was determined (gene symbol STK12) . The C-terminal kinase domain of the STK12 encoded protein shares high amino acid sequence identity with those of mouse STK-1 (90%), rat AIM-1 (90%), human Aik (69%), mouse IAK1/Ayk1 (69%), Xenopus pEg2 (68%), Drosophila Aurora (62%), and yeast Ipl1 (45%), whereas the N-terminal domain of the STK12 protein shares little homology with those of Aurora/Ipl1 family members except for AIM-1 and STK-1 . Northern blotting analyses revealed that STK12 expression was high in thymus, while low level expression was detected in small intestine, testis, colon, spleen, and brain . The STK12 protein content in HeLa cells is low in S phase, but it accumulates during M phase . STK12 was mapped to human chromosome 17p13.1 by fluorescence in situ hybridization . The chromosome location of STK12 was further defined using a radiation hybrid panel (Stanford G3), that showed a linkage with marker WI-7901 (LOD Score 7.83) located between D17S938 and D17S786. Mol Cell Biol, 1999 Jan, 19(1), 934 - 40 RNA polymerase II transcription suppresses nucleosomal modulation of UV-induced (6-4) photoproduct and cyclobutane pyrimidine dimer repair in yeast; Tijsterman M et al.; The nucleotide excision repair (NER) pathway is able to remove a wide variety of structurally unrelated lesions from DNA . NER operates throughout the genome, but the efficiencies of lesion removal are not the same for different genomic regions . Even within a single gene or DNA strand repair rates vary, and this intragenic heterogeneity is of considerable interest with respect to the mutagenic potential of carcinogens . In this study, we have analyzed the removal of the two major types of genotoxic DNA adducts induced by UV light, i.e., the pyrimidine (6-4)-pyrimidone photoproduct (6-4PP) and the cyclobutane pyrimidine dimer (CPD), from the Saccharomyces cerevisiae URA3 gene at nucleotide resolution . In contrast to the fast and uniform removal of CPDs from the transcribed strand, removal of lesions from the nontranscribed strand is generally less efficient and is modulated by the chromatin environment of the damage . Removal of 6-4PPs from nontranscribed sequences is also profoundly influenced by positioned nucleosomes, but this type of lesion is repaired at a much higher rate . Still, the transcribed strand is repaired preferentially, indicating that, as in the removal of CPDs, transcription-coupled repair predominates in the removal of 6-4PPs from transcribed DNA . The hypothesis that transcription machinery operates as the rate-determining damage recognition entity in transcription-coupled repair is supported by the observation that this pathway removes both types of UV photoproducts at equal rates without being profoundly influenced by the sequence or chromatin context. Mol Cell Biol, 1999 Jan, 19(1), 585 - 93 The yeast a1 and alpha2 homeodomain proteins do not contribute equally to heterodimeric DNA binding; Jin Y et al.; In diploid cells of the yeast Saccharomyces cerevisiae, the alpha2 and a1 homeodomain proteins bind cooperatively to sites in the promoters of haploid cell-type-specific genes (hsg) to repress their expression . Although both proteins bind to the DNA, in the alpha2 homeodomain substitutions of residues that are involved in contacting the DNA have little or no effect on repression in vivo or cooperative DNA binding with a1 protein in vitro . This result brings up the question of the contribution of each protein in the heterodimer complex to the DNA-binding affinity and specificity . To determine the requirements for the a1-alpha2 homeodomain DNA recognition, we systematically introduced single base-pair substitutions in an a1-alpha2 DNA-binding site and examined their effects on repression in vivo and DNA binding in vitro . Our results show that nearly all substitutions that significantly decrease repression and DNA-binding affinity are at positions which are specifically contacted by either the alpha2 or a1 protein . Interestingly, an alpha2 mutant lacking side chains that make base-specific contacts in the major groove is able to discriminate between the wild-type and mutant DNA sites with the same sequence specificity as the wild-type protein . These results suggest that the specificity of alpha2 DNA binding in complex with a1 does not rely solely on the residues that make base-specific contacts . We have also examined the contribution of the a1 homeodomain to the binding affinity and specificity of the complex . In contrast to the lack of a defective phenotype produced by mutations in the alpha2 homeodomain, many of the alanine substitutions of residues in the a1 homeodomain have large effects on a1-alpha2-mediated repression and DNA binding . This result shows that the two proteins do not make equal contributions to the DNA-binding affinity of the complex. Mol Cell Biol, 1999 Jan, 19(1), 484 - 94 cDNA of the yeast retrotransposon Ty5 preferentially recombines with substrates in silent chromatin; Ke N et al.; The yeast retrotransposon Ty5 preferentially integrates into regions of silent chromatin . Ty5 cDNA also recombines with homologous sequences, generating tandem elements or elements that have exchanged markers between cDNA and substrate . In this study, we demonstrate that Ty5 integration depends upon the conserved DD(35)E domain of integrase and cis-acting sequences at the end of the long terminal repeat (LTR) implicated in integrase binding . cDNA recombination requires Rad52p, which is responsible for homologous recombination . Interestingly, Ty5 cDNA recombines at least three times more frequently with substrates in silent chromatin than with a control substrate at an internal chromosomal locus . This preference depends upon the Ty5 targeting domain that is responsible for integration specificity, suggesting that localization of cDNA to silent chromatin results in the enhanced recombination . Recombination with a telomeric substrate occasionally generates highly reiterated Ty5 arrays, and mechanisms for tandem element formation were explored by using a plasmid-based recombination assay . Point mutations were introduced into plasmid targets, and recombination products were characterized to determine recombination initiation sites . Despite our previous observation of the importance of the LTR in forming tandem elements, recombination cannot simply be explained by crossover events between the LTRs of substrate and cDNA . We propose an alternative model based on single-strand annealing, where single-stranded cDNA initiates tandem element formation and the LTR is required for strand displacement to form a looped intermediate . Retrotransposons are increasingly found associated with chromosome ends, and amplification of Ty5 by both integration and recombination exemplifies how retroelements can contribute to telomere dynamics. Mol Cell Biol, 1999 Jan, 19(1), 384 - 91 Ribosomal protein L3 mutants alter translational fidelity and promote rapid loss of the yeast killer virus; Peltz SW et al.; Programmed -1 ribosomal frameshifting is utilized by a number of RNA viruses as a means of ensuring the correct ratio of viral structural to enzymatic proteins available for viral particle assembly . Altering frameshifting efficiencies upsets this ratio, interfering with virus propagation . We have previously demonstrated that compounds that alter the kinetics of the peptidyl-transfer reaction affect programmed -1 ribosomal frameshift efficiencies and interfere with viral propagation in yeast . Here, the use of a genetic approach lends further support to the hypothesis that alterations affecting the ribosome's peptidyltransferase activity lead to changes in frameshifting efficiency and virus loss . Mutations in the RPL3 gene, which encodes a ribosomal protein located at the peptidyltransferase center, promote approximately three- to fourfold increases in programmed -1 ribosomal frameshift efficiencies and loss of the M1 killer virus of yeast . The mak8-1 allele of RPL3 contains two adjacent missense mutations which are predicted to structurally alter the Mak8-1p . Furthermore, a second allele that encodes the N-terminal 100 amino acids of L3 (called L3Delta) exerts a trans-dominant effect on programmed -1 ribosomal frameshifting and killer virus maintenance . Taken together, these results support the hypothesis that alterations in the peptidyltransferase center affect programmed -1 ribosomal frameshifting. Mol Cell Biol, 1999 Jan, 19(1), 86 - 98 Functional organization of the yeast SAGA complex: distinct components involved in structural integrity, nucleosome acetylation, and TATA-binding protein interaction; Sterner DE et al.; SAGA, a recently described protein complex in Saccharomyces cerevisiae, is important for transcription in vivo and possesses histone acetylation function . Here we report both biochemical and genetic analyses of members of three classes of transcription regulatory factors contained within the SAGA complex . We demonstrate a correlation between the phenotypic severity of SAGA mutants and SAGA structural integrity . Specifically, null mutations in the Gcn5/Ada2/Ada3 or Spt3/Spt8 classes cause moderate phenotypes and subtle structural alterations, while mutations in a third subgroup, Spt7/Spt20, as well as Ada1, disrupt the complex and cause severe phenotypes . Interestingly, double mutants (gcn5Delta spt3Delta and gcn5Delta spt8Delta) causing loss of a member of each of the moderate classes have severe phenotypes, similar to spt7Delta, spt20Delta, or ada1Delta mutants . In addition, we have investigated biochemical functions suggested by the moderate phenotypic classes and find that first, normal nucleosomal acetylation by SAGA requires a specific domain of Gcn5, termed the bromodomain . Deletion of this domain also causes specific transcriptional defects at the HIS3 promoter in vivo . Second, SAGA interacts with TBP, the TATA-binding protein, and this interaction requires Spt8 in vitro . Overall, our data demonstrate that SAGA harbors multiple, distinct transcription-related functions, including direct TBP interaction and nucleosomal histone acetylation . Loss of either of these causes slight impairment in vivo, but loss of both is highly detrimental to growth and transcription. Mol Cell Biol, 1999 Jan, 19(1), 31 - 45 The yeast telomere length counting machinery is sensitive to sequences at the telomere-nontelomere junction; Ray A et al.; Saccharomyces cerevisiae telomeres consist of a continuous 325 +/- 75-bp tract of the heterogeneous repeat TG1-3 which contains irregularly spaced, high-affinity sites for the protein Rap1p . Yeast cells monitor or count the number of telomeric Rap1p molecules in a negative feedback mechanism which modulates telomere length . To investigate the mechanism by which Rap1p molecules are counted, the continuous telomeric TG1-3 sequences were divided into internal TG1-3 sequences and a terminal tract separated by nontelomeric spacers of different lengths . While all of the internal sequences were counted as part of the terminal tract across a 38-bp spacer, a 138-bp disruption completely prevented the internal TG1-3 sequences from being considered part of the telomere and defined the terminal tract as a discrete entity separate from the subtelomeric sequences . We also used regularly spaced arrays of six Rap1p sites internal to the terminal TG1-3 repeats to show that each Rap1p molecule was counted as about 19 bp of TG1-3 in vivo and that cells could count Rap1p molecules with different spacings between tandem sites . As previous in vitro experiments had shown that telomeric Rap1p sites occur about once every 18 bp, all Rap1p molecules at the junction of telomeric and nontelomeric chromatin (the telomere-nontelomere junction) must participate in telomere length measurement . The conserved arrangement of these six Rap1p molecules at the telomere-nontelomere junction in independent transformants also caused the elongated TG1-3 tracts to be maintained at nearly identical lengths, showing that sequences at the telomere-nontelomere junction had an effect on length regulation . These results can be explained by a model in which telomeres beyond a threshold length form a folded structure that links the chromosome terminus to the telomere-nontelomere junction and prevents telomere elongation. Zentralbl Veterinarmed A, 1998 Nov, 45(9), 551 - 7 Selenite and selenium yeast as feed supplements to growing fattening pigs; Ortman K et al.; Twenty-four cross-bred fattening pigs weighing, on average, 24 kg were divided into three equal groups and fed a basic diet containing 0.1 mg selenium/kg . The diet of group A was supplemented with 0.3 mg selenium/kg derived from selenium yeast, group B with 0.1 mg selenium/kg from the same source, and group C with 0.3 mg/kg from sodium selenite . The pigs were slaughtered after 103 days . There was no significant difference between the mean whole blood selenium concentration of the pigs supplemented with selenium yeast neither after five nor after nine weeks of supplementation (group A, 201 and 213 micrograms/litre; group B 192 and 201 micrograms/litre, respectively), but the group supplemented with selenite had significantly lower concentrations both after five and nine weeks (158 and 159 micrograms/litre, respectively) . There were no significant differences between the mean activity of glutathione peroxidase (GSH-Px; in the whole blood of the three groups (group A, 348 and 327 mukat*/litre, group B, 342 and 302 mukat/litre; group C, 332 and 300 mukat/litre, after five and nine weeks, respectively) . The concentration of selenium in the liver of the pigs supplemented with organic selenium was significantly higher than in the pigs supplemented with selenite (group A, 0.54 mg/kg wet weight; group B, 0.54 mg/kg; group C, 0.40 mg/kg) . No deposits of lipofuscin or ceroid pigments were observed in the histological preparations of liver and heart from any of the pigs. EMBO J, 1998 Dec 15, 17(24), 7454 - 68 Control of cleavage site selection during mRNA 3' end formation by a yeast hnRNP; Minvielle-Sebastia L et al.; Endonucleolytic cleavage of pre-mRNAs is the first step during eukaryotic mRNA 3' end formation . It has been proposed that cleavage factors CF IA, CF IB and CF II are required for pre-mRNA 3' end cleavage in yeast . CF IB is composed of a single polypeptide, Nab4p/Hrp1p, which is related to the A/B group of metazoan heterogeneous nuclear ribonucleoproteins (hnRNPs) that function as antagonistic regulators of 5' splice site selection . Here, we provide evidence that Nab4p/Hrp1p is not required for pre-mRNA 3' end endonucleolytic cleavage . We show that CF IA and CF II devoid of Nab4p/Hrp1p are sufficient to cleave a variety of RNA substrates but that cleavage occurs at multiple sites . Addition of Nab4p/Hrp1p prevents these alternative cleavages in a concentration-dependent manner, suggesting an essential and conserved role for some hnRNPs in pre-mRNA cleavage site selection. EMBO J, 1998 Dec 15, 17(24), 7442 - 53 A role for the yeast La protein in U6 snRNP assembly: evidence that the La protein is a molecular chaperone for RNA polymerase III transcripts; Pannone BK et al.; The first protein that binds to all newly synthesized RNA polymerase III transcripts is a highly conserved phosphoprotein known as the La autoantigen . Although binding by the yeast La protein Lhp1p to pre-tRNAs is required for the normal pathway of tRNA maturation, the role of the La protein in the biogenesis of other polymerase III transcripts has been unclear . We identified a mutation in a novel component of the U6 snRNP that causes yeast cells to require Lhp1p for growth . This protein, Lsm8p, is a member of a family of proteins, known as Sm-like proteins, that shares two conserved motifs with the core Sm proteins of the U1, U2, U4 and U5 snRNPs . The lsm8-1 cells have drastically reduced levels of the mature U6 snRNP, consistent with a defect in U6 snRNP assembly . In these cells, Lhp1p stabilizes newly synthesized U6 RNA, thus facilitating assembly of the RNA into the U6 snRNP . These results provide evidence that Lhp1p is a molecular chaperone for polymerase III-transcribed RNAs and implicate Lsm8p as a key component in the very early steps of U6 snRNP assembly. EMBO J, 1998 Dec 15, 17(24), 7416 - 29 Crm1p mediates regulated nuclear export of a yeast AP-1-like transcription factor; Yan C et al.; The yeast AP-1-like transcription factor, Yap1p, activates genes required for the response to oxidative stress . Yap1p is normally cytoplasmic and inactive, but will activate by nuclear translocation if cells are placed in an oxidative environment . Here we show that Yap1p is a target of the beta-karyopherin-like nuclear exporter, Crm1p . Yap1p is constitutively nuclear in a crm1 mutant, and Crm1p binds to a nuclear export sequence (NES)-like sequence in Yap1p in the presence of RanGTP . Recognition of Yap1p by Crm1p is inhibited by oxidation, and this inhibition requires at least one of the three cysteine residues flanking the NES . These results suggest that Yap1p localization is largely regulated at the level of nuclear export, and that the oxidation state affects the accessibility of the Yap1p NES to Crm1p directly . We also show that a mutation in RanGAP (rna1-1) is synthetically lethal with crm1 mutants . Yap1p export is inhibited in both rna1-1 and prp20 (RanGNRF) mutant strains, but Yap1p rapidly accumulates at the nuclear periphery after shifting rna1-1, but not other mutant cells to the non-permissive temperature . Thus, disassembly of export complexes in response to RanGTP hydrolysis may be required for release of substrate from a terminal binding site at the nuclear pore complex (NPC). EMBO J, 1998 Dec 15, 17(24), 7190 - 8 Mutations in the pore regions of the yeast K+ channel YKC1 affect gating by extracellular K+; Vergani P et al.; The product of the Saccharomyces cerevisiae K+-channel gene YKC1 includes two pore-loop sequences that are thought to form the hydrophilic lining of the pore . Gating of the channel is promoted by membrane depolarization and is regulated by extracellular K+ concentration ({K+}o) both in the yeast and when expressed in Xenopus oocytes . Analysis of the wild-type current now shows that: (i) {K+}o suppresses a very slowly relaxing component, accelerating activation; (ii) {K+}o slows deactivation in a dose-dependent fashion; and (iii) Rb+, Cs+ and, to a lesser extent, Na+ substitute for K+ in its action on gating . We have identified single residues, L293 and A428, at equivalent positions within the two pore loops that affect the {K+}o sensitivity . Substitution of these residues gave channels with reduced sensitivity to {K+}o in macroscopic current kinetics and voltage dependence, but had only minor effects on selectivity among alkali cations in gating and on single-channel conductance . In some mutants, activation was slowed sufficiently to confer a sigmoidicity to current rise at low {K+}o . The results indicate that these residues are involved in {K+}o sensing . Their situation close to the permeation pathway points to an interaction between gating and permeation. EMBO J, 1998 Dec 15, 17(24), 7170 - 8 Yeast mitochondrial F1F0-ATP synthase exists as a dimer: identification of three dimer-specific subunits; Arnold I et al.; Using the technique of blue native gel electrophoresis, the oligomeric state of the yeast mitochondrial F1F0-ATP synthase was analysed . Solubilization of mitochondrial membranes with low detergent to protein ratios led to the identification of the dimeric state of the ATP synthase . Analysis of the subunit composition of the dimer, in comparison with the monomer, revealed the presence of three additional small proteins . These dimer-specific subunits of the ATP synthase were identified as the recently described subunit e/Tim11 (Su e/Tim11), the putative subunit g homolog (Su g) and a new component termed subunit k (Su k) . Although, as shown here, these three proteins are not required for the formation of enzymatically active ATP synthase, Su e/Tim11 and Su g are essential for the formation of the dimeric state . Su e/Tim11 appears to play a central role in this dimerization process . The dimer-specific subunits are associated with the membrane bound F0-sector . The F0-sector may thereby be involved in the dimerization of two monomeric F1F0-ATP synthase complexes . We speculate that the F1F0-ATP synthase of yeast, like the other complexes of oxidative phosphorylation, form supracomplexes to optimize transduction of energy and to enhance the stability of the complex in the membrane. Biochem J, 1999 Jan 1, 337 ( Pt 1), 29 - 35 A conserved motif in the yeast nucleolar protein Nop2p contains an essential cysteine residue; King M et al.; Nop2p is an essential nucleolar protein in Saccharomyces cerevisiae that is involved in large ribosomal subunit assembly . It has substantial homology with human p120, the proliferation-associated nucleolar antigen that is overexpressed in many human cancers . A motif containing an invariant Pro-Cys dipeptide is found in Nop2p, p120 and the bacterial Fmu proteins . A total of nine conserved residues, including Pro423 and Cys424, were individually altered in Nop2p by site-directed mutagenesis . Nop2p function was abolished by conversion of Cys424 into either alanine or serine . All of the other Nop2p mutations tested sustained yeast viability, including glycine replacement of Pro423 and the conversion of a second conserved cysteine into alanine . The crucial role of Cys424 in Nop2p is intriguing, due to the critical roles that cysteine residues adjacent to a proline have in a number of nucleotide-modifying enzymes. Biochem J, 1999 Jan 1, 337 ( Pt 1), 13 - 7 Groucho/transducin-like enhancer of split (TLE) family members interact with the yeast transcriptional co-repressor SSN6 and mammalian SSN6-related proteins: implications for evolutionary conservation of transcription repression mechanisms; Grbavec D et al.; The yeast proteins TUP1 and SSN6 form a transcription repressor complex that is recruited to different promoters via pathway-specific DNA-binding proteins and regulates the expression of a variety of genes . TUP1 is functionally related to invertebrate and vertebrate transcriptional repressors of the Groucho/transducin-like Enhancer of split (TLE) family . The aim was to examine whether similar mechanisms underlie the transcription repression functions of TUP1 and Groucho/TLEs by determining whether TLE family members can interact with yeast SSN6 and mammalian SSN6-like proteins . It is shown in the present work that SSN6 binds to TLE1 and mediates transcriptional repression when expressed in mammalian cells . Moreover, TLE1 and TLE2 interact with two mammalian proteins related to SSN6, designated as the products of the ubiquitously transcribed tetratricopeptide-repeat genes on the Y (or X) chromosomes (UTY/X) . These findings suggest that mammalian TLE and UTY/X proteins may mediate repression mechanisms similar to those performed by TUP1-SSN6 in yeast. Nat Biotechnol, 1998 Dec, 16(13), 1334 - 7 Identification of surrogate agonists for the human FPRL-1 receptor by autocrine selection in yeast; Klein C et al.; We describe a procedure for isolating agonists for mammalian G protein-coupled receptors of unknown function . Human formyl peptide receptor like-1 (FPRL-1) receptor, originally identified as an orphan G protein-coupled receptor related to the formyl peptide receptor (FPR1), was expressed in Saccharomyces cells designed to couple receptor activation to histidine prototrophy . Selection for histidine prototrophs among transformants obtained with a plasmid-based library encoding random peptides identified six different agonists, each of whose production yielded autocrine stimulation of the receptor expressed in yeast . A synthetic version of each peptide promoted activation of FPRL-1 expressed in human embryonic kidney (HEK293) cells, and five of the peptides exhibited significant selectivity for activation of FPRL-1 relative to FPR1 . One selective peptide was tested and found to mobilize calcium in isolated human neutrophils . This demonstrates that stimulation of FPRL-1 results in neutrophil activation and suggests that the receptor functions as a component of the inflammatory response . This autocrine selection protocol may be a generally applicable method for providing pharmacological tools to evaluate the physiological roles of the growing number of mammalian orphan G protein-coupled receptors. J Protein Chem, 1998 Oct, 17(7), 591 - 8 Structural dissection of the DNA-binding domain of the yeast transcriptional activator GAL4 reveals an alpha-helical region responsible for dimerization; Gadhavi PL; Limited proteolysis of the DNA-binding domain (residues 1-147) of the yeast transcriptional activator GAL4 has been used to define more precisely the subdomain structure required for DNA binding and dimerization . Two regions of the protein were found to be resistant to proteolysis: the cysteine-rich, zinc-binding region (residues 6-43) and a hydrophobic sequence between residues 52 and 97 . Carboxy-terminal deletion fragments of the DNA-binding domain were generated and assayed by DNase 1 footprinting . This showed that the affinity of DNA binding depends on the sequence between residues 65 and 94 . Structural comparisons by UV circular dichroism (CD) were made and the difference CD spectra indicate that strong alpha-helical content is found specifically in the region between residues 65 and 94, which previous studies have shown to enable dimerization and in this study the formation of a stable protein-DNA complex. J Biol Chem, 1998 Dec 18, 273(51), 34611 - 5 BMCP1, a novel mitochondrial carrier with high expression in the central nervous system of humans and rodents, and respiration uncoupling activity in recombinant yeast; Sanchis D et al.; We report here the cloning and functional analysis of a novel homologue of the mitochondrial carriers predominantly expressed in the central nervous system and referred to as BMCP1 (brain mitochondrial carrier protein-1) . The predicted amino acid sequence of this novel mitochondrial carrier indicates a level of identity of 39, 31, or 30%, toward the mitochondrial oxoglutarate carrier, phosphate carrier, or adenine nucleotide translocator, respectively, and a level of identity of 34, 38, or 39% with the mitochondrial uncoupling proteins UCP1, UCP2, or UCP3, respectively . Northern analysis of mouse, rat, or human tissues demonstrated that mRNA of this novel gene is mainly expressed in brain, although it is 10-30-fold less expressed in other tissues . In situ hybridization analysis of brain showed it is particularly abundant in cortex, hippocampus, thalamus, amygdala, and hypothalamus . Chromosomal mapping indicates that BMCP1 is located on chromosome X of mice and at Xq24 in man . Expression of the protein in yeast strongly impaired growth rate . Analysis of respiration of total recombinant yeast or yeast spheroplasts and in particular of the relationship between respiratory rate and membrane potential of yeast spheroplasts revealed a marked uncoupling activity of respiration, suggesting that although BMCP1 sequence is more distant from the uncoupling proteins (UCPs), this protein could be a fourth member of the UCP family. J Biol Chem, 1998 Dec 18, 273(51), 34328 - 34 Evidence for a salt bridge between transmembrane segments 5 and 6 of the yeast plasma-membrane H+-ATPase; Gupta SS et al.; The plasma-membrane H+-ATPase of Saccharomyces cerevisiae, which belongs to the P2 subgroup of cation-transporting ATPases, is encoded by the PMA1 gene and functions physiologically to pump protons out of the cell . This study has focused on hydrophobic transmembrane segments M5 and M6 of the H+-ATPase . In particular, a conserved aspartate residue near the middle of M6 has been found to play a critical role in the structure and biogenesis of the ATPase . Site-directed mutants in which Asp-730 was replaced by an uncharged residue (Asn or Val) were abnormally sensitive to trypsin, consistent with the idea that the proteins were poorly folded, and immunofluorescence confocal microscopy showed them to be arrested in the endoplasmic reticulum . Similar defects are known to occur when either Arg-695 or His-701 in M5 is replaced by a neutral residue (Dutra, M . B., Ambesi, A., and Slayman, C . W . (1998) J . Biol . Chem . 273, 17411-17417) . To search for possible charge-charge interactions between Asp-730 and Arg-695 or His-701, double mutants were constructed in which positively and negatively charged residues were swapped or eliminated . Strikingly, two of the double mutants (R695D/D730R and R695A/D730A) regained the capacity for normal biogenesis and displayed near-normal rates of ATP hydrolysis and