EMBO J, 1998 May 1, 17(9), 2482 - 93 A dileucine-like sorting signal directs transport into an AP-3-dependent, clathrin-independent pathway to the yeast vacuole; Vowels JJ et al.; Transport of yeast alkaline phosphatase (ALP) to the vacuole depends on the clathrin adaptor-like complex AP-3, but does not depend on proteins necessary for transport through pre-vacuolar endosomes . We have identified ALP sequences that direct sorting into the AP-3-dependent pathway using chimeric proteins containing residues from the ALP cytoplasmic domain fused to sequences from a Golgi-localized membrane protein, guanosine diphosphatase (GDPase) . The full-length ALP cytoplasmic domain, or ALP amino acids 1-16 separated from the transmembrane domain by a spacer, directed GDPase chimeric proteins from the Golgi complex to the vacuole via the AP-3 pathway . Mutation of residues Leu13 and Val14 within the ALP cytoplasmic domain prevented AP-3-dependent vacuolar transport of both chimeric proteins and full-length ALP . This Leucine-Valine (LV)-based sorting signal targeted chimeric proteins and native ALP to the vacuole in cells lacking clathrin function . These results identify an LV-based sorting signal in the ALP cytoplasmic domain that directs transport into a clathrin-independent, AP-3-dependent pathway to the vacuole . The similarity of the ALP sorting signal to mammalian dileucine sorting motifs, and the evolutionary conservation of AP-3 subunits, suggests that dileucine-like signals constitute a core element for AP-3-dependent transport to lysosomal compartments in all eukaryotic cells. Nucleic Acids Res, 1998 May 1, 26(9), 2063 - 8 Cloning and characterization of a human DEAH-box RNA helicase, a functional homolog of fission yeast Cdc28/Prp8; Imamura O et al.; During the splicing process, spliceosomal snRNAs undergo numerous conformational rearrangements that appear to be catalyzed by proteins belonging to the DEAD/H-box superfamily of RNA helicases . We have cloned a new RNA helicase gene, designated DBP2 (DEAH-boxprotein), homologous to the Schizosaccaromyces pombe cdc28(+)/prp8(+) gene involved in pre-mRNA splicing and cell cycle progression . The full-length DBP2 contains 3400 nucleotides and codes for a protein of 1041 amino acids with a calculated mol . wt of 119 037 Da . Transfection experiments demonstrated that the GFP-DBP2 gene product, transiently expressed in HeLa cells, was localized in the nucleus . The DBP2 gene was mapped by FISH to the MHC region on human chromosome 6p21.3, a region where many malignant, genetic and autoimmune disease genes are linked . Because the expression of DBP2 gene in S.pombe prp8 mutant cells partially rescued the temperature-sensitive phenotype, we conclude that DBP2 is a functional human homolog of the fission yeast Cdc28/Prp8 protein. Nucleic Acids Res, 1998 May 1, 26(9), 2050 - 7 Accelerated mRNA decay in conditional mutants of yeast mRNA capping enzyme; Schwer B et al.; Current models of mRNA decay in yeast posit that 3' deadenylation precedes enzymatic removal of the 5' cap, which then exposes the naked end to 5' exonuclease action . Here, we analyzed gene expression in Saccharomyces cerevisiae cells bearing conditional mutations of Ceg1 (capping enzyme), a 52 kDa protein that transfers GMP from GTP to the 5' end of mRNA to form the GpppN cap structure . Shift of ceg1 mutants to restrictive temperature elicited a rapid decline in the rate of protein synthesis, which correlated with a sharp reduction in the steady-state levels of multiple individual mRNAs . ceg1 mutations prevented the accumulation of SSA1 and SSA4 mRNAs that were newly synthesized at the restrictive temperature . Uncapped poly(A)+ SSA4 mRNA accumulated in cells lacking the 5' exoribonuclease Xrn1 . These findings provide genetic evidence for the long-held idea that the cap guanylate is critical for mRNA stability . The deadenylation-decapping-degradation pathway appears to be short-circuited when Ceg1 is inactivated. Protein Expr Purif, 1998 Jun, 13(1), 30 - 5 Purification and characterization of human procolipase expressed in yeast cells; Cordle RA et al.; We report the successful, efficient, and large-scale expression of recombinant human procolipase in yeast . Using the full-length cDNA of human procolipase, constructs were made using either the native human procolipase signal peptide sequence or the signal peptide sequence of yeast . These constructs were used to transform yeast cells, and expression was followed . Only minimal expression was seen with the procolipase using the native human signal peptide . Robust secretion of the procolipase occurred when the yeast signal peptide was exchanged for the native signal peptide . Expression yielded more than 30 mg/liter . The recombinant protein was purified from the medium by immunoaffinity chromatography . The highly purified procolipase was free of proteolytic degradation and displayed activity and binding characteristics that were indistinguishable from those of tissue-purified human pancreatic colipase . Expression in yeast cells provides a useful tool for expressing intact, unprocessed recombinant wild-type and mutated procolipase. Nat Biotechnol, 1996 Jul, 14(7), 884 - 7 FACS-based isolation of slowly growing cells: double encapsulation of yeast in gel microdrops; Gift EA et al.; Isolating hyperproducing cells is important in biotechnology, but these cells usually grow slowly and can be overgrown by poorly producing cells . We describe a new method of isolating slowly growing cells from among rapidly growing cells, which has the potential for automation and high throughput (e.g., 100,000 cells/h) . A model system is presented consisting of a mixed population of slowly growing mutant and rapidly growing wild-type yeast, which were encapsulated in double agarose gel microdrops (dGMDs); with most dGMDs initially containing single cells . Double encapsulation locates parent cells near dGMD centers, making microcolony measurement more accurate . After a 15-h incubation, fluorescent activated cell sorting was used to analyze and sort dGMDs with small microcolonies (slow growers) from dGMDs with large microcolonies (rapid growers) . Successful isolation of slow growers from a mixed population of predominantly rapidly growing Saccharomyces cerevisiae cells was achieved. Nat Biotechnol, 1996 Jul, 14(7), 880 - 3 Use of microphysiometry for analysis of heterologous ion channels expressed in yeast; Hahnenberger KM et al.; Measurement of extracellular acidification rates by microphysiometry provides a means to analyze the function of ion channels expressed in yeast cells . These measurements depend on the proton pumping action of the H(+)-ATPase, a central component of the yeast plasma membrane . We used microphysiometry to analyze the activity of two ion channels expressed in yeast . In one example, an inwardly rectifying K+ channel, gpIRK1, provides a potassium uptake function when expressed in a potassium transporter-defective yeast strain . Rates of acidification in gpIRK1-expressing cells directly reflect channel function . Addition of cesium, an inhibitor of gpIRK1 activity, results in an immediate reduction in acidification rates . In a second example, expression of a nonselective cation channel, the influenza virus M2 protein, is believed to interfere with the maintenance of the electrochemical proton gradient by the H(+)-ATPase . In cells expressing the M2 channel, addition of inhibitors increases the rate of proton extrusion . Moreover, functional differences between two M2 inhibitors, amantadine and BL-1743, are distinguished by the microphysiometer . This application demonstrates the utility of the microphysiometer for functional studies of ion channels; it is adaptable to a screening process for compounds that modulate ion channel activity. Biochim Biophys Acta, 1998 Jun 22, 1403(2), 158 - 68 Protein-protein interactions between keratin polypeptides expressed in the yeast two-hybrid system; Schnabel J et al.; Keratin filaments are obligatory heteropolymers of type I and type II keratin polypeptides . Specific type I/type II pairs are coexpressed in vivo . In contrast, all type I/type II pairs assemble into filaments in vitro, but the different pairs have different stabilities as demonstrated by treatment with increasing concentrations of urea . We have used the yeast two-hybrid system to analyse type I/type II interactions in a cellular context . We measured interactions between two different keratin pairs and we confirm the findings that K6+K17 form very stable heterodimers whereas K8+K18 interactions were weaker . The deletion of head domains did not reduce the strength of type I/type II interactions . Rather, the affinities were increased and the differences between the two pairs were retained in headless mutants . These findings argue against a major role of the head domains in directing heterodimer interactions and in defining heterodimer stabilities. Biochim Biophys Acta, 1998 May 28, 1371(2), 157 - 62 The assembly of yeast mitochondrial ATP synthase: subunit depletion in vivo suggests ordered assembly of the stalk subunits b, OSCP and d; Straffon AF et al.; The abundance in vivo of each of three subunits b, OSCP and d, components of the stalk region of the yeast mitochondrial ATP synthase complex, was manipulated by a controlled depletion strategy . Western blots of whole cell lysates were used to study the effect of depletion of each of these subunits on the cellular levels of other subunits of the enzyme complex . A hierarchy of subunit stability was determined and interpreted to indicate the order of assembly of these three subunits of the stalk region . Thus, subunit b is assembled first, followed by OSCP and then by subunit d . RNA, 1998 Apr, 4(4), 374 - 93 A comprehensive biochemical and genetic analysis of the yeast U1 snRNP reveals five novel proteins; Gottschalk A et al.; The U1 snRNP is essential for recognition of the pre-mRNA 5'-splice site and the subsequent assembly of the spliceosome . Yeast U1 snRNP is considerably more complex than its metazoan counterpart, which suggests possible differences between yeast and metazoa in early splicing events . We have comprehensively analyzed the composition of yeast U1 snRNPs using a combination of biochemical, mass spectrometric, and genetic methods . We demonstrate the specific association of four novel U1 snRNP proteins, Snu71p, Snu65p, Nam8p, and Snu56p, that have no known metazoan homologues . A fifth protein, Npl3p, is an abundant cellular component that reproducibly co-purifies with the U1 snRNP, but its association is salt-sensitive . Therefore, we are unable to establish conclusively whether it binds specifically to the U1 snRNP . Interestingly, Nam8p and Npl3p were previously assigned functions in (pre-m)RNA-metabolism; however, so far, no association with U1 snRNP has been demonstrated or proposed . We also show that the yeast SmB protein is a U1 snRNP component . Yeast U1 snRNP therefore contains 16 different proteins, including seven snRNP core proteins, three homologues of the metazoan U1 snRNP-specific proteins, and six yeast-specific U1 snRNP proteins . We have simultaneously continued the characterization of additional mutants isolated in a synthetic lethal (MUD) screen for genes that functionally cooperate with U1 snRNA . Consistent with the biochemical results, mud10, mud15, and mud16 are alleles of SNU56, NAM8, and SNU65, respectively . mud10 and mud15 affect the in vivo splicing efficiency of noncanonical introns . Moreover, mud10p strongly affects the in vitro formation of splicing complexes, and extracts from the mud15 strain contain a U1 snRNP that migrates aberrantly on native gels . Finally, we show that Nam8p/Mud15p contributes to the stability of U1 snRNP. Oncogene, 1998 May 14, 16(19), 2527 - 39 Human tumor-derived p53 proteins exhibit binding site selectivity and temperature sensitivity for transactivation in a yeast-based assay; Di Como CJ et al.; p53 is a sequence-specific transcriptional activator with a number of known target genes which contain p53-responsive elements . Mutations in p53 have been identified within its sequence-specific DNA binding domain in more than half of all human tumors, although a subset of tumor-derived p53 mutants have retained the ability to bind DNA and activate transcription under certain conditions . In order to broaden our understanding of this transactivating ability, we examined the efficacy by which p53 mutants bind to and activate reporters in an Saccharomyces cerevisiae-based assay . Analysis of 19 human tumor-derived p53 mutants, spanning the DNA binding domain of p53 and including the 'hot-spot' class, revealed a broad array of transcriptional transactivation abilities at 24 degrees C, 30 degrees C and 37 degrees C, despite the fact that each mutant had originally been identified as being inactive for transactivation in yeast against a single p53-responsive RGC site-containing reporter . One class of mutants (P177L, R267W, C277Y and R283H) retained wild-type or near wild-type activity that is binding site-selective, even at physiological temperature (37 degrees C) . Another class of mutants (V143A, M1601/A161T, H193R, Y220C and 1254F), all positioned for maintaining the beta-scaffold of p53, also retained selective activity, but preferentially at sub-physiological temperatures (24 degrees and 30 degrees C) . Strikingly, however, in contrast to the other tumor derived mutants, all of the previously identified 'hot-spot' mutants were completely inactive with all sites tested . Moreover, a double mutant, L22E/W23S, located within the activation region and previously shown to be transcriptionally inactive in fibroblasts, retained wild-type or near wild-type binding site-selective activity in yeast . Finally, we found that transcriptional activity in vivo does not necessarily correlate with DNA binding in vitro. J Cell Sci, 1998 Jul, 111 ( Pt 13), 1779 - 89 Localisation and interaction of the protein components of the yeast 2 mu circle plasmid partitioning system suggest a mechanism for plasmid inheritance; Scott-Drew S et al.; Replicating plasmids are highly unstable in yeast, because they are retained in mother cells . The 2 mu circle plasmid overcomes this maternal inheritance bias by using a partitioning system that involves the plasmid encoded proteins Rep1p and Rep2p, and the cis-acting locus STB . It is thus widely exploited as a cloning vehicle in yeast . However, little is known about the cellular or molecular mechanisms by which effective partitioning is achieved, and models of both free diffusion and plasmid localisation have been proposed . Here we show that Rep1p and Rep2p proteins interact to form homo- and hetero-complexes in vitro . In vivo, Rep1p and Rep2p are shown to be nuclear proteins, exhibiting sub-nuclear concentration in distinct foci . The number of foci appears constant regardless of plasmid copy number and cell ploidy level . Before cell division, the number of foci increases, and we observe approximately equal allocation of foci to mother and daughter cell nuclei . We show that whereas Rep2p expressed alone is found exclusively in the nucleus, Rep1p requires the presence of Rep2p for effective nuclear localisation . High levels of 2 mu plasmid induce a multiple-budded elongated cell phenotype, which we show can be phenocopied by overexpression of both REP1 and REP2 together but not alone . Taken together, these results suggest that Rep1p and Rep2p interact in vivo, and occupy defined nuclear sites that are allocated to both mother and daughter nuclei during division . We propose a model for 2 mum plasmid partitioning based on these results, involving the association of plasmid DNA with specific, segregated subnuclear sites. J Virol, 1998 Jul, 72(7), 5638 - 47 A small yeast RNA blocks hepatitis C virus internal ribosome entry site (HCV IRES)-mediated translation and inhibits replication of a chimeric poliovirus under translational control of the HCV IRES element; Das S et al.; Hepatitis C virus (HCV) infection frequently leads to chronic hepatitis and cirrhosis of the liver and has been linked to development of hepatocellular carcinoma . We previously identified a small yeast RNA (IRNA) capable of specifically inhibiting poliovirus (PV) internal ribosome entry site (IRES)-mediated translation . Here we report that IRNA specifically inhibits HCV IRES-mediated translation both in vivo and in vitro . A number of human hepatoma (Huh-7) cell lines expressing IRNA were prepared and characterized . Constitutive expression of IRNA was not detrimental to cell growth . HCV IRES-mediated cap-independent translation was markedly inhibited in cells constitutively expressing IRNA compared to control hepatoma cells . However, cap-dependent translation was not significantly affected in these cell lines . Additionally, Huh-7 cells constitutively expressing IRNA became refractory to infection by a PV-HCV chimera in which the PV IRES is replaced by the HCV IRES . In contrast, replication of a PV-encephalomyocarditis virus (EMCV) chimera containing the EMCV IRES element was not affected significantly in the IRNA-producing cell line . Finally, the binding of the La autoantigen to the HCV IRES element was specifically and efficiently competed by IRNA . These results provide a basis for development of novel drugs effective against HCV infection. Proc Natl Acad Sci U S A, 1998 Jun 9, 95(12), 6915 - 8 A yeast model for the study of Batten disease; Pearce DA et al.; Although the CLN3 gene for Batten disease, the most common inherited neurovisceral storage disease of childhood, was identified in 1995, the function of the corresponding protein still remains elusive . We previously cloned the Saccharomyces cerevisiae homologue to the human CLN3 gene, designated BTN1, which is not essential and whose product is 39% identical and 59% similar to Cln3p . We report that btn1-Delta deletion yeast strains are more resistant to D-(-)-threo-2-amino-1-{p-nitrophenyl}-1,3-propanediol (denoted ANP), a phenotype that is complemented in yeast by the human CLN3 gene . Furthermore, the severity of Batten disease in humans and the degree of ANP resistance in yeast are related when the equivalent amino acid replacements in Cln3p and Btn1p are compared . These results indicate that yeast can be used as a model for the study of Batten disease. Proc Natl Acad Sci U S A, 1998 Jun 9, 95(12), 6699 - 704 Control of pre-mRNA accumulation by the essential yeast protein Nrd1 requires high-affinity transcript binding and a domain implicated in RNA polymerase II association; Steinmetz EJ et al.; Nrd1 is an essential yeast protein of unknown function that has an RNA recognition motif (RRM) in its carboxyl half and a putative RNA polymerase II-binding domain, the CTD-binding motif, at its amino terminus . Nrd1 mediates a severe reduction in pre-mRNA production from a reporter gene bearing an exogenous sequence element in its intron . The effect of the inserted element is highly sequence-specific and is accompanied by the appearance of 3'-truncated transcripts . We have proposed that Nrd1 binds to the exogenous sequence element in the nascent pre-mRNA during transcription, aided by the CTD-binding motif, and directs 3'-end formation a short distance downstream . Here we show that highly purified Nrd1 carboxyl half binds tightly to the RNA element in vitro with sequence specificity that correlates with the efficiency of cis-element-directed down-regulation in vivo . A large deletion in the CTD-binding motif blocks down-regulation but does not affect the essential function of Nrd1 . Furthermore, a nonsense mutant allele that produces truncated Nrd1 protein lacking the RRM has a dominant-negative effect on down-regulation but not on cell growth . Viability of this and several other nonsense alleles of Nrd1 appears to require translational readthrough, which in one case is extremely efficient . Thus the CTD-binding motif of Nrd1 is important for pre-mRNA down-regulation but is not required for the essential function of Nrd1 . In contrast, the RNA-binding activity of Nrd1 appears to be required both for down-regulation and for its essential function. Proc Natl Acad Sci U S A, 1998 Jun 9, 95(12), 6693 - 8 Deposition-related sites K5/K12 in histone H4 are not required for nucleosome deposition in yeast; Ma XJ et al.; Histone H4 can be acetylated at N-terminal lysines K5, K8, K12, and K16, but newly synthesized H4 is diacetylated at K5/K12 in diverse organisms . This pattern is widely thought to be important for histone deposition onto replicating DNA . To investigate the importance of K5/K12 we have mutagenized these lysines in yeast and assayed for nucleosome assembly . Assaying was done in the absence of the histone H3 N terminus, which has functions redundant with those of H4 in histone deposition . Nucleosome assembly was assayed by three methods . Because nucleosome depletion may be lethal, we examined cell viability . We also analyzed nucleosome assembly in vivo and in vitro by examining plasmid superhelicity density in whole cells and supercoiling in yeast cell extracts . All three approaches demonstrate that mutagenizing K5 and K12 together does not prevent cell growth and histone deposition in vivo or in vitro . Therefore, K5/K12 cannot be required for nucleosome assembly in yeast . It is only when the first three sites of acetylation-K5, K8, and K12-are mutagenized simultaneously that lethality occurs and assembly is most strongly decreased both in vivo and in vitro . These data argue for the redundancy of sites K5, K8, and K12 in the deposition of yeast histone H4. Mol Biol Cell, 1998 Jun, 9(6), 1339 - 49 Heat stress activates fission yeast Spc1/StyI MAPK by a MEKK-independent mechanism; Shiozaki K et al.; Fission yeast Spc1/StyI MAPK is activated by many environmental insults including high osmolarity, oxidative stress, and heat shock . Spc1/StyI is activated by Wis1, a MAPK kinase (MEK), which is itself activated by Wik1/Wak1/Wis4, a MEK kinase (MEKK) . Spc1/StyI is inactivated by the tyrosine phosphatases Pyp1 and Pyp2 . Inhibition of Pyp1 was recently reported to play a crucial role in the oxidative stress and heat shock responses . These conclusions were based on three findings: 1) osmotic, oxidative, and heat stresses activate Spc1/StyI in wis4 cells; 2) oxidative stress and heat shock activate Spc1/StyI in cells that express Wis1AA, in which MEKK consensus phosphorylation sites were replaced with alanine; and 3) Spc1/StyI is maximally activated in Deltapyp1 cells . Contrary to these findings, we report: 1) Spc1/StyI activation by osmotic stress is greatly reduced in wis4 cells; 2) wis1-AA and Deltawis1 cells have identical phenotypes; and 3) all forms of stress activate Spc1/StyI in Deltapyp1 cells . We also report that heat shock, but not osmotic or oxidative stress, activate Spc1 in wis1-DD cells, which express Wis1 protein that has the MEKK consensus phosphorylation sites replaced with aspartic acid . Thus osmotic and oxidative stress activate Spc1/StyI by a MEKK-dependent process, whereas heat shock activates Spc1/StyI by a novel mechanism that does not require MEKK activation or Pyp1 inhibition. Mol Biol Cell, 1998 Jun, 9(6), 1309 - 21 Cyclin B proteolysis and the cyclin-dependent kinase inhibitor rum1p are required for pheromone-induced G1 arrest in fission yeast; Stern B et al.; The blocking of G1 progression by fission yeast pheromones requires inhibition of the cyclin-dependent kinase cdc2p associated with the B-cyclins cdc13p and cig2p . We show that cyclosome-mediated degradation of cdc13p and cig2p is necessary for down-regulation of B-cyclin-associated cdc2p kinase activity and for phermone-induced G1 arrest . The cyclin-dependent kinase inhibitor rum1p is also required to maintain this G1 arrest; it binds both cdc13p and cig2p and is specifically required for cdc13p proteolysis . We propose that rum1p acts as an adaptor targeting cdc13p for degradation by the cyclosome . In contrast, the cig2p-cdc2p kinase can be down-regulated, and the cyclin cig2p can be proteolyzed independently of rum1p . We suggest that pheromone signaling inhibits the cig2p-cdc2p kinase, bringing about a transient G1 arrest . As a consequence, rum1p levels increase, thus inhibiting and inducing proteolysis of the cdc13p-cdc2p kinase; this is necessary to maintain G1 arrest . We have also shown that pheromone-induced transcription occurs only in G1 and is independent of rum1p. J Biol Chem, 1998 Jun 12, 273(24), 15110 - 8 Golgi localization and functionally important domains in the NH2 and COOH terminus of the yeast CLC putative chloride channel Gef1p; Schwappach B et al.; GEF1 encodes the single CLC putative chloride channel in yeast . Its disruption leads to a defect in iron metabolism (Greene, J . R., Brown, N . H., DiDomenico, B . J., Kaplan, J., and Eide, D . (1993) Mol . Gen . Genet . 241, 542-553) . Since disruption of GEF2, a subunit of the vacuolar H+-ATPase, leads to a similar phenotype, it was previously suggested that the chloride conductance provided by Gef1p is necessary for vacuolar acidification . We now show that gef1 cells indeed grow less well at less acidic pH . However, no defect in vacuolar acidification is apparent from quinacrine staining, and Gef1p co-localizes with Mnt1p in the medial Golgi . Thus, Gef1p may be important in determining Golgi pH . Systematic alanine scanning of the amino and the carboxyl terminus revealed several regions essential for Gef1p localization and function . One sequence (FVTID) in the amino terminus conforms to a class of sorting signals containing aromatic amino acids . This was further supported by point mutations . Alanine scanning of the carboxyl terminus identified a stretch of roughly 25 amino acids which coincides with the second CBS domain, a conserved protein motif recently identified . Mutations in the first CBS domain also destroyed proper function and localization . The second CBS domain can be transplanted to the amino terminus without loss of function, but could not be replaced by the corresponding domain of the homologous mammalian channel ClC-2. J Cell Sci, 1998 Apr, 111 ( Pt 8), 1031 - 7 Endocytic internalization in yeast and animal cells: similar and different; Geli MI et al.; The internalization step of endocytosis has been the focus of several laboratories during the last forty years . Unlike some other budding events in the cell, many fundamental questions regarding the molecular machinery involved in the mechanism of budding itself still remain unsolved . Over the last few years the general picture of the field has quickly evolved from the originally simplistic view which postulated that clathrin polymerization is the major force driving budding at the plasma membrane . Refinement of the assays and molecular markers to measure endocytosis in animal cells has shown that other factors in addition to the clathrin coat are required and that endocytosis can also take place through clathrin-independent mechanisms . At the same time, recent introduction of genetic approaches to study endocytosis has accelerated the identification of molecules required for this process . The isolation of endocytosis mutants in budding yeast has been especially fruitful in this respect . Preliminary comparison of the results obtained in yeast and animal cells did not seem to coincide, but further progress in both systems now suggests that part of the divergence originally seen may be due to the particular experimental approaches used rather than fundamental differences in endocytic mechanisms . In this review we present a short historical overview on the advances made in yeast and animal cells regarding the study of endocytosis, underlining both emerging similarities and still interesting differences. Br J Biomed Sci, 1997 Dec, 54(4), 237 - 9 Evaluation of CHROMagar Candida medium for the isolation and direct identification of yeast species from the female genital tract; Anson JJ et al.; The performance and cost-effectiveness of CHROMagar Candida in the isolation and identification of Candida species from female genital tract specimens were investigated . Eight hundred and forty-three specimens were inoculated in parallel onto malt extract agar and CHROMagar Candida . Yeasts isolated on CHROMagar were provisionally identified by morphology and pigmentation . Those isolated on malt extract agar were further identified by a germ tube test, and negative isolates were identified biochemically . A cost comparison was performed, detailing labour and material costs . Of 119 yeast isolates, seven were detected only on CHROMagar . Mixed Candida spp . from three specimens were only differentiated on CHROMagar . There was a 100% correlation between the results of the germ tube test and provisional identity on CHROMagar . CHROMagar Candida facilitated the presumptive identification of yeast isolates from the female genital tract, and the detection of mixed populations . The cost of the media was higher than malt extract agar and germ tube testing, but the rapid identification of isolates to species level and ease of use may be of sufficient advantage to justify the extra cost. J Cell Biol, 1998 May 4, 141(3), 689 - 701 Characterization of the human homologue of the yeast spc98p and its association with gamma-tubulin; Tassin AM et al.; A trimeric complex formed by Tub4p, the budding yeast gamma-tubulin, and the two spindle pole body components, Spc98p and Spc97p, has recently been characterized in Saccharomyces cerevisiae . We reasoned that crucial functions, such as the control of microtubule nucleation, could be maintained among divergent species . SPC98-related sequences were searched in dbEST using the BLASTN program . Primers derived from the human expressed sequence tag matching SPC98 were used to clone the 5' and 3' cDNA ends by rapid amplification of cDNA ends (RACE)-PCR . The human Spc98 cDNA presents an alternative splicing at the 3' end . The deduced protein possesses 22% identity and 45% similarity with the yeast homologue . We further report that the human Spc98p, like gamma-tubulin, is concentrated at the centrosome, although a large fraction is found in cytosolic complexes . Sucrose gradient sedimentation of the cytosolic fraction and immunoprecipitation experiments demonstrate that both gamma-tubulin and HsSpc98p are in the same complex . Interestingly, Xenopus sperm centrosomes, which are incompetent for microtubule nucleation before their activation in the egg cytoplasm, were found to contain similar amounts of both Spc98p and gamma-tubulin to human somatic centrosomes, which are competent for microtubule nucleation . Finally, affinity-purified antibodies against Spc98p inhibit microtubule nucleation on isolated centrosomes, as well as in microinjected cells, suggesting that this novel protein is indeed required for the nucleation reaction. J Cell Biol, 1998 May 4, 141(3), 663 - 74 The mammalian gamma-tubulin complex contains homologues of the yeast spindle pole body components spc97p and spc98p; Murphy SM et al.; gamma-Tubulin is a universal component of microtubule organizing centers where it is believed to play an important role in the nucleation of microtubule polymerization . gamma-Tubulin also exists as part of a cytoplasmic complex whose size and complexity varies in different organisms . To investigate the composition of the cytoplasmic gamma-tubulin complex in mammalian cells, cell lines stably expressing epitope-tagged versions of human gamma-tubulin were made . The epitope-tagged gamma-tubulins expressed in these cells localize to the centrosome and are incorporated into the cytoplasmic gamma-tubulin complex . Immunoprecipitation of this complex identifies at least seven proteins, with calculated molecular weights of 48, 71, 76, 100, 101, 128, and 211 kD . We have identified the 100- and 101-kD components of the gamma-tubulin complex as homologues of the yeast spindle pole body proteins Spc97p and Spc98p, and named the corresponding human proteins hGCP2 and hGCP3 . Sequence analysis revealed that these proteins are not only related to their respective homologues, but are also related to each other . GCP2 and GCP3 colocalize with gamma-tubulin at the centrosome, cosediment with gamma-tubulin in sucrose gradients, and coimmunoprecipitate with gamma-tubulin, indicating that they are part of the gamma-tubulin complex . The conservation of a complex involving gamma-tubulin, GCP2, and GCP3 from yeast to mammals suggests that structurally diverse microtubule organizing centers such as the yeast spindle pole body and the animal centrosome share a common molecular mechanism for microtubule nucleation. J Biol Chem, 1998 May 8, 273(19), 11917 - 22 The role of charged amino acids in the alpha1-beta4 loop of the iron-sulfur protein of the cytochrome bc1 complex of yeast mitochondria; Obungu VH et al.; Previous experiments using deletion mutants of the iron-sulfur protein had indicated that amino acid residues 138-153 might be involved in the assembly of this protein into the cytochrome bc1 complex . To determine which specific residues might be involved in the assembly process, charged amino acids located in the alpha1-beta4 loop of the iron-sulfur protein were mutated to uncharged residues and tryptophan 152 to phenylalanine . The mutant genes were used to transform yeast cells (JPJ1) lacking the iron-sulfur protein gene . Mutants R146I and W152F had almost undetectable growth in medium containing glycerol/ethanol, whereas mutants D143A, K148I, and D149A grew more slowly than the wild type . Activity of the cytochrome bc1 complex was decreased 50, 90, 67, 89, and 90% in mutants D143A, R146I, K148I, D149A, and W152F, respectively, but unchanged in mutants D139A, Q141I, D145L, and V147S . In all of these mutants except W152F, the cytochrome c1 content, determined by immunoblotting, was comparable with that of wild-type cells . However, immunoblotting revealed that the content of the iron-sulfur protein was decreased proportionately in the five mutants with lowered enzymatic activity and growth suggesting that these amino acids are critical for maintaining the stability of the iron-sulfur protein . The efficiency of assembly in vitro compared with the wild type determined by selective immunoprecipitation was unchanged in the mutants with the exception of R146I, D149A, and W152F where decreases of 80, 60, and 60%, respectively, were observed suggesting that these amino acids are critical for the proper assembly of the iron-sulfur protein into the bc1 complex. J Biol Chem, 1998 May 8, 273(19), 11852 - 61 INP51, a yeast inositol polyphosphate 5-phosphatase required for phosphatidylinositol 4,5-bisphosphate homeostasis and whose absence confers a cold-resistant phenotype; Stolz LE et al.; Sequence analysis of Saccharomyces cerevisiae chromosome IX identified a 946 amino acid open reading frame (YIL002C), designated here as INP51, that has carboxyl- and amino-terminal regions similar to mammalian inositol polyphosphate 5-phosphatases and to yeast SAC1 . This two-domain primary structure resembles the mammalian 5-phosphatase, synaptojanin . We report that Inp51p is associated with a particulate fraction and that recombinant Inp51p exhibits intrinsic phosphatidylinositol 4,5-bisphosphate 5-phosphatase activity . Deletion of INP51 (inp51) results in a "cold-tolerant" phenotype, enabling significantly faster growth at temperatures below 15 degreesC as compared with a parental strain . Complementation analysis of an inp51 mutant strain demonstrates that the cold tolerance is strictly due to loss of 5-phosphatase catalytic activity . Furthermore, deletion of PLC1 in an inp51 mutant does not abrogate cold tolerance, indicating that Plc1p-mediated production of soluble inositol phosphates is not required . Cells lacking INP51 have a 2-4-fold increase in levels of phosphatidylinositol 4,5-bisphosphate and inositol 1,4, 5-trisphosphate, whereas cells overexpressing Inp51p exhibit a 35% decrease in levels of phosphatidylinositol 4,5-bisphosphate . We conclude that INP51 function is critical for proper phosphatidylinositol 4,5-bisphosphate homeostasis . In addition, we define a novel role for a 5-phosphatase loss of function mutant that improves the growth of cells at colder temperatures without alteration of growth at normal temperatures, which may have useful commercial applications. J Biol Chem, 1998 May 8, 273(19), 11719 - 27 Tlg2p, a yeast syntaxin homolog that resides on the Golgi and endocytic structures; Abeliovich H et al.; Intracellular membrane fusion events in eukaryotic cells are thought to be mediated by protein-protein interactions between soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex proteins . We have identified and analyzed a new yeast syntaxin homolog, Tlg2p . Tlg2p is unique among known syntaxin family proteins in possessing a sizeable hydrophilic domain of 63 amino acids that is C-terminal to the membrane spanning region and nonessential for Tlg2p function . Tlg2p resides on the endosome and late Golgi by co-localization with an endocytic intermediate and co-fractionation with markers for both endosomes and late Golgi . Cells depleted for Tlg2p missort a portion of carboxypeptidase Y and are defective in endocytosis . In addition, we report that Tlg2p forms a SEC18-dependent SNARE complex with Snc2p, a vesicle SNARE known to function in Golgi to plasma membrane trafficking . Based on these findings we propose that Tlg2p is a t-SNARE that functions in transport from the endosome to the late Golgi and on the endocytic pathway. Nature, 1998 Jun 4, 393(6684), 487 - 91 A cytokinesis checkpoint requiring the yeast homologue of an APC-binding protein; Muhua L et al.; Checkpoint controls ensure that events of the cell-division cycle are completed with fidelity and in the correct order . In budding yeast with a mutation in the motor protein dynein, the mitotic spindle is often misaligned and therefore slow to enter the neck between mother cell and budding daughter cell . When this occurs, cytokinesis (division of the cytoplasm into two) is delayed until the spindle is properly positioned . Here we describe mutations that abolish this delay, indicating the existence of a new checkpoint mechanism . One mutation lies in the gene encoding the yeast homologue of EB1, a human protein that binds the adenomatous polyposis coli (APC) protein, a tumour suppressor . EB1 is located on microtubules of the mitotic spindle and is important in spindle assembly . EB1 may therefore, by associating with microtubules, contribute to the sensor mechanism that activates the checkpoint . Another mutation affects Stt4, a phosphatidylinositol-4-OH kinase . Cold temperature is an environmental stimulus that causes misalignment of the mitotic spindle in yeast and appears to activate this checkpoint mechanism. J Cell Biol, 1998 Jun 1, 141(5), 1169 - 79 The yeast spindle pole body component Spc72p interacts with Stu2p and is required for proper microtubule assembly; Chen XP et al.; We have previously shown that Stu2p is a microtubule-binding protein and a component of the Saccharomyces cerevisiae spindle pole body (SPB) . Here we report the identification of Spc72p, a protein that interacts with Stu2p . Stu2p and Spc72p associate in the two-hybrid system and can be coimmunoprecipitated from yeast extracts . Stu2p and Spc72p also interact with themselves, suggesting the possibility of a multimeric Stu2p-Spc72p complex . Spc72p is an essential component of the SPB and is able to associate with a preexisting SPB, indicating that there is a dynamic exchange between soluble and SPB forms of Spc72p . Unlike Stu2p, Spc72p does not bind microtubules in vitro, and was not observed to localize along microtubules in vivo . A temperature-sensitive spc72 mutation causes defects in SPB morphology . In addition, most spc72 mutant cells lack cytoplasmic microtubules; the few cytoplasmic microtubules that are observed are excessively long, and some of these are unattached to the SPB . spc72 cells are able to duplicate and separate their SPBs to form a bipolar spindle, but spindle elongation and chromosome segregation rarely occur . The chromosome segregation block does not arrest the cell cycle; instead, spc72 cells undergo cytokinesis, producing aploid cells and polyploid cells that contain multiple SPBs. RNA, 1998 Jun, 4(6), 647 - 57 The RNA sequence context defines the mechanistic routes by which yeast arginyl-tRNA synthetase charges tRNA; Sissler M et al.; Arginylation of tRNA transcripts by yeast arginyl-tRNA synthetase can be triggered by two alternate recognition sets in anticodon loops: C35 and U36 or G36 in tRNA(Arg) and C36 and G37 in tRNA(Asp) (Sissler M, Giege R, Florentz C, 1996, EMBO J 15:5069-5076) . Kinetic studies on tRNA variants were done to explore the mechanisms by which these sets are expressed . Although the synthetase interacts in a similar manner with tRNA(Arg) and tRNA(Asp), the details of the interaction patterns are idiosyncratic, especially in anticodon loops (Sissler M, Eriani G, Martin F, Giege R, Florentz C, 1997, Nucleic Acids Res 25:4899-4906) . Exchange of individual recognition elements between arginine and aspartate tRNA frameworks strongly blocks arginylation of the mutated tRNAs, whereas full exchange of the recognition sets leads to efficient arginine acceptance of the transplanted tRNAs . Unpredictably, the similar catalytic efficiencies of native and transplanted tRNAs originate from different k(cat) and Km combinations . A closer analysis reveals that efficient arginylation results from strong anticooperative effects between individual recognition elements . Nonrecognition nucleotides as well as the tRNA architecture are additional factors that tune efficiency . Altogether, arginyl-tRNA synthetase is able to utilize different context-dependent mechanistic routes to be activated . This confers biological advantages to the arginine aminoacylation system and sheds light on its evolutionary relationship with the aspartate system. J Dairy Sci, 1998 May, 81(5), 1345 - 52 Effect of yeast on feed intake and performance of cows fed diets based on corn silage during early lactation; Wohlt JE et al.; Thirty-six multiparous Holstein cows were fed a mixture of corn silage and concentrate {1:1; dry matter (DM) basis} and long hay (0.9 kg/d) through wk 18 of lactation . Beginning at 30 d prepartum through wk 4 of lactation, the total mixed rations of 18 of these cows were top-dressed daily with 10 g of Biomate Yeast Plus (Chr . Hansen's, Inc., Milwaukee, WI) . The other 18 cows served as controls . At wk 5, both control and treated cows were divided into three groups and fed 0, 10, or 20 g/d of yeast . Yeast supplementation during early lactation significantly improved DM intake, milk yield, and the digestibility of crude protein and acid detergent fiber . Least squares means for DM intake, fat-corrected milk yield, crude protein digestibility, and acid detergent fiber digestibility for cows fed 0, 10, 20 g/d of yeast during wk 5 to 18 of lactation were 23.8, 24.7, and 25.0 kg/d; 37.7, 40.7, and 41.4 kg/d; 78.5, 80.8, and 79.5%; and 54.4, 60.2, and 56.8%, respectively . Although numerical responses in DM intake and milk yield were greater for cows fed 20 g/d of yeast than for cows fed 10 g/d of yeast, the response was not significant. Genetics, 1998 Jun, 149(2), 857 - 64 Modulation of tubulin polypeptide ratios by the yeast protein Pac10p; Alvarez P et al.; Normal assembly and function of microtubules require maintenance of the proper levels of several proteins, including the tubulin polypeptides themselves . For example, in yeast a significant excess of beta-tubulin causes rapid microtubule disassembly and subsequent cell death . Even the modest excess of beta-tubulin produced by genetic alterations such as deletion of the minor alpha-tubulin gene TUB3 affects cell growth and can confer microtubule phenotypes . We show here that the levels of the yeast protein Pac10p affect the relative levels of the tubulin polypeptides . Cells deleted for PAC10 have the same phenotypes as do cells that express reduced levels of alpha-tubulin or Rbl2p, two proteins that bind beta-tubulin . Conversely, overexpression of Pac10p enhances the ability of alpha-tubulin or Rbl2p to suppress the lethality associated with excess beta-tubulin . However, Pac10p is itself not a beta-tubulin binding protein . Pac10 null cells show a 30% decrease in the ratio of alpha-tubulin to beta-tubulin . The results suggest that Pac10p modulates the level of alpha-tubulin in the cell, and so influences microtubule morphogenesis and tubulin metabolism. Genetics, 1998 Jun, 149(2), 807 - 15 Mutational analysis of the yeast DEAH-box splicing factor Prp16; Hotz HR et al.; Prp16 is an essential yeast splicing factor that catalyzes RNA-dependent hydrolysis of nucleoside triphosphates . Prp16 is a member of the DEAH-box protein family, which is defined by six collinear sequence motifs . The importance of residues within four of the conserved motifs was assessed by alanine-scanning mutagenesis . Mutant alleles of PRP16 were tested for in vivo function by complementation of a Deltaprp16 null strain . In motif I (GETGSGKT), alanine substitutions at Gly-378, Lys-379, and Thr-380 were lethal, whereas replacement of the amino acids in positions 373-377 were viable . In the signature DEAH-box (motif II), Asp-473 and Glu-474 were essential, whereas the H476A mutant was viable . The S505A and T507A mutants in motif III (SAT) were viable . In motif VI (QRSGRAGRTAPG), mutants Q685A, R686A, G688A, R689A, and R692A were lethal, whereas G691A, P695A, and G696A supported growth . Instructive structure-function relationships were established by conservative substitutions at essential residues identified by alanine scan . Overexpression of nonviable alleles impaired the growth of wild-type PRP16 cells . Deletion analysis of the 1071-amino-acid Prp16 protein revealed that the N-terminal 204 amino acids and the C-terminal 100 residues were dispensable for PRP16 function in vivo . These studies provide an instructive framework for functional analysis of other DEAH-box splicing factors. Plant Mol Biol, 1998 May, 37(1), 141 - 54 Maize contains a Lon protease gene that can partially complement a yeast pim1-deletion mutant; Barakat S et al.; We have identified a gene in maize that encodes a product belonging to the Lon protease family . In yeast and mammals, Lon-type proteases catalyze the ATP-dependent degradation of mitochondrial matrix proteins . The maize gene, which we have designated LON1, is predicted to encode a protein with a molecular mass of 97.7 kDa . Lon1p is more similar in sequence to bacterial Lon proteases than to the yeast and human mitochondrial Lon proteases . LON1 transcripts are present in shoots of 4-day-old etiolated maize seedlings, and transcript levels decrease when these seedlings are heat-shocked . LON1 transcripts are also present at comparable levels in leaves and roots of 2-week-old greenhouse-grown seedlings . In yeast, the mitochondrial Lon-type protease, Pim1p, has been implicated in mitochondrial protein turnover, the assembly of mitochondrial enzyme complexes, and mitochondrial DNA maintenance, and it is essential for respiratory function . We show that maize Lon1p can replace the Pim1p function in yeast for maintaining mitochondrial DNA integrity, but not in the assembly of cytochrome a x a3 complexes. Microbiol Mol Biol Rev, 1998 Jun, 62(2), 334 - 61 Yeast carbon catabolite repression; Gancedo JM; Glucose and related sugars repress the transcription of genes encoding enzymes required for the utilization of alternative carbon sources; some of these genes are also repressed by other sugars such as galactose, and the process is known as catabolite repression . The different sugars produce signals which modify the conformation of certain proteins that, in turn, directly or through a regulatory cascade affect the expression of the genes subject to catabolite repression . These genes are not all controlled by a single set of regulatory proteins, but there are different circuits of repression for different groups of genes . However, the protein kinase Snf1/Cat1 is shared by the various circuits and is therefore a central element in the regulatory process . Snf1 is not operative in the presence of glucose, and preliminary evidence suggests that Snf1 is in a dephosphorylated state under these conditions . However, the enzymes that phosphorylate and dephosphorylate Snf1 have not been identified, and it is not known how the presence of glucose may affect their activity . What has been established is that Snf1 remains active in mutants lacking either the proteins Grr1/Cat80 or Hxk2 or the Glc7 complex, which functions as a protein phosphatase . One of the main roles of Snf1 is to relieve repression by the Mig1 complex, but it is also required for the operation of transcription factors such as Adr1 and possibly other factors that are still unidentified . Although our knowledge of catabolite repression is still very incomplete, it is possible in certain cases to propose a partial model of the way in which the different elements involved in catabolite repression may be integrated. EMBO J, 1998 May 15, 17(10), 2914 - 25 A single amino acid substitution in yeast eIF-5A results in mRNA stabilization; Zuk D et al.; Most factors known to function in mRNA turnover are not essential for cell viability . To identify essential factors, approximately 4000 temperature-sensitive yeast strains were screened for an increase in the level of the unstable CYH2 pre-mRNA . At the non-permissive temperature, five mutants exhibited decreased decay rates of the CYH2 pre-mRNA and mRNA, and the STE2, URA5 and PAB1 mRNAs . Of these, the mutant ts1159 had the most extensive phenotype . Expression of the TIF51A gene (encoding eIF-5A) complemented the temperature-sensitive growth and mRNA decay phenotypes of ts1159 . The tif51A allele was rescued from these cells and shown to encode a serine to proline change within a predicted alpha-helical segment of the protein . ts1159 also exhibited an approximately 30% decrease in protein synthesis at the restrictive temperature . Measurement of amino acid incorporation in wild-type cells incubated with increasing amounts of cycloheximide demonstrated that a decrease in protein synthesis of this magnitude could not account for the full extent of the mRNA decay defects observed in ts1159 . Interestingly, the ts1159 cells accumulated uncapped mRNAs at the non-permissive temperature . These results suggest that eIF-5A plays a role in mRNA turnover, perhaps acting downstream of decapping. Nucleic Acids Res, 1998 May 15, 26(10), 2433 - 41 Upf1 and Upf2 proteins mediate normal yeast mRNA degradation when translation initiation is limited; Barnes CA; mRNA degradation is coupled with the process of mRNA translation . For example, an mRNA molecule, on which translation is prematurely terminated because of a nonsense codon, may be rapidly degraded . This nonsense-mediated mRNA decay in the yeast Saccharomyces cerevisiae is mediated by the Upf1 and Upf2 proteins . Yeast mRNAs can also be selectively destabilized by limiting the rate of translation initiation . Two such destabilized mRNAs, from the SSA1 and SSA2 genes, have been identified using temperature-sensitive mutations affecting the Prt1 component of eukaryotic initiation factor 3 . For SSA1 and SSA2 mRNAs, and for structurally modified SSA mRNA derivatives, I show here that degradation is triggered when translation initiation is limited but ongoing . This initiation-dependent mRNA degradation is limited to a subset of mRNAs that includes at least those from the SSA1 and SSA2 genes, and occurs through Upf1- and Upf2-mediated processes, although sequence elements characteristic of nonsense-mediated decay are not evident in these mRNAs. Nucleic Acids Res, 1998 May 15, 26(10), 2415 - 9 Meiotic double-strand breaks in yeast artificial chromosomes containing human DNA; Ira G et al.; Meiotic recombination in the yeast Saccharomyces cerevisiae is initiated by double-strand breaks (DSB) in chromosomal DNA . These DSB, which can be mapped in the rad 50S mutant yeast strain, are caused by a topoisomerase II-like enzyme, the protein Spo11 . Evidence suggests that this protein is located in the axial element of the meiotic chromosome which implies that the DSB are located in these chromosomes in the vicinity of the bases of the DNA loops . We have found that in the yeast artificial chromosomes carrying human DNA, at the level of resolution obtained by pulsed field gel electrophoresis (PFGE), the meiotic DSB in the diploid yeast are co-localized with the DNase I hypersensitive sites (HS) in a haploid strain of yeast . These HS are located close to sequences which, under stress, have the potential to form secondary structures containing unpaired nucleotides . Clusters of such sequences could be a hallmark of the bases of the chromatin loops. Nucleic Acids Res, 1998 May 15, 26(10), 2329 - 36 Genomic footprinting of the yeast zinc finger protein Rme1p and its roles in repression of the meiotic activator IME1; Shimizu M et al.; The zinc finger protein Rme1p is a negative regulator of the meiotic activator IME1 in Saccharomyces cerevisiae . Prior studies have shown that Rme1p binds in vitro to a site near nt -2030 in the IME1 upstream region, but a genomic mutation in that site has little effect on repression of IME1 . To identify Rme1p binding sites in vivo , we have examined the binding of Rme1p to genomic sites through in vivo footprinting . We show that Rme1p binds to two sites in the IME1 upstream region, near nt -1950 and -2030 . Mutations in both binding sites abolish repression of chromosomal IME1 by Rme1p, whereas a mutation in either single site causes partial derepression . Therefore, both Rme1p binding sites are essential for repression of IME1 . Prior studies have shown that repression by Rme1p depends upon RGR1 and SIN4 , which specify RNA polymerase II mediator subunits that are required for normal nucleosome density . We find that RGR1 and SIN4 are not simply required for Rme1p to bind to DNA in vivo . These results suggest that Rme1p functions directly as a repressor of IME1 and that Rgr1p and Sin4p are required for DNA-bound Rme1p to exert repression. Gene Ther, 1998 Mar, 5(3), 339 - 44 Monitoring adenoviral p53 transduction efficiency by yeast functional assay; Tada M et al.; Monitoring the transduction efficiency is of paramount importance in gene therapy . To monitor adenovirus-mediated wild-type p53 gene transfer, we have used a quantitative assay which tests the ability of human p53 to activate transcription in yeast . Selective amplification of cellular and viral p53 transcripts followed by quantitative assessment of mutant p53 content with the assay permits measurement of the wild-type p53 transduction efficiency into SF-188, U251MG and HUG31 glioblastoma cells . One reverse transcription primer tracks the wild-type/mutant ratio of endogenous p53 mRNA (P2), and the other the wild-type/mutant ratio of both endogenous and exogenous p53 mRNA (P1) . Following infection of cell lines homozygous for mutant p53, the apparent transduction efficiency calculated (tau 0 = {P1-P2}/{1 + P2}) correlated with the level of p21 expression . Transduction efficiency in heterozygous wild-type/mutant HUG31 cells increased linearly with multiplicity of infection (MOI) for tau 0 values between 0.5 and 5.9, and admixture of normal cell-derived RNA produced only a modest reduction in tau 0 value, in keeping with theoretical predictions . These results suggest that the yeast p53 functional assay may be a useful tool for monitoring p53 gene therapy. Biochemistry, 1998 May 12, 37(19), 6967 - 74 Conformational changes in yeast pyruvate kinase studied by 205Tl+ NMR; Loria JP et al.; The interaction of the monovalent cation with yeast pyruvate kinase (yPK) has been investigated by 205Tl+ NMR . TlNO3 activates yPK to 80-90% activity compared to KCl with an apparent Ka of 1.00 +/- 0.03 mM in the presence of 4 mM Mn(NO3)2 as the activating divalent cation . At higher concentrations of Tl+, enzyme inhibition is observed with an apparent KI of 180 +/- 10 mM . The extent of inhibition is dependent on the nature and concentration of the divalent cation . The effect of Mn2+ on the 1/T1 and 1/T2 values of 205Tl+ in the presence of yPK was determined at 173.02 MHz (300 MHz, 1H) and 346.03 MHz (600 MHz, 1H) . The temperature dependence of the relaxation rates indicates that fast exchange conditions prevail for 205Tl+ longitudinal relaxation rates . The correlation time, tauc, for the Mn2+-205Tl+ interaction was estimated by a frequency dependence of 1/T1m for several enzyme complexes, and an average value of tauc was determined to be 0.91 ns . The distance between Tl+ and Mn2+ at the active site of yPK was calculated from the paramagnetic contribution of Mn2+ to the longitudinal (1/T1m) relaxation rates of Tl+ bound to yPK . For the apo yPK complex, the Tl+ to Mn2+ distance is 6.7 +/- 0.2 A . Upon addition of phosphoenolpyruvate (PEP) to form the yPK-Tl-Mn-PEP complex, the inter-cation distance decreases to 6.1 +/- 0.3 A . The addition of the allosteric activator fructose 1,6-bisphosphate (FBP) to form the yPK-Tl+-Mn2+-PEP-FBP complex gives an intermetal distance of 6.2 +/- 0.2 A . In the yPK-Tl-Mn-FBP complex, a Tl+-Mn2+ distance of 6.0 +/- 0.1 A is observed, indicating that FBP causes a conformational change at the active site in the absence of PEP . Analogous 205Tl NMR experiments with competitive inhibitors of PEP (oxalate, BrPEP) indicate that these ligands do not induce the same conformational changes as do the physiological substrates and activators . Similar experiments with the nonallosteric rabbit muscle PK were also performed and analyzed. Genes Dev, 1998 May 1, 12(9), 1278 - 89 The yeast Ada complex mediates the ligand-dependent activation function AF-2 of retinoid X and estrogen receptors; vom Baur E et al.; Nuclear receptors can function as ligand-inducible transregulators in both mammalian and yeast cells, indicating that important features of control of transcription have been conserved throughout evolution . Here, we report the isolation and characterization of a yeast protein that exhibits properties expected for a coactivator/mediator of the ligand-dependent activation function AF-2 present in the ligand-binding domain (LBD, region E) of the retinoid X (RXRalpha) and estrogen (ERalpha) receptors . This protein is identical to Ada3, a component of the yeast Ada coactivator complex . We demonstrate that: (1) the region encompassing residues 347-702 of Ada3 interacts with the LBD of RXRalpha and ERalpha in a ligand-dependent manner in yeast; (2) this interaction corresponds to a direct binding and requires the integrity of the core of the AF-2 activating domain (AF-2 AD) of both RXRalpha and ERalpha; (3) Ada3 as well as Ada2 and Gcn5, two other components of the Ada complex, are required for maximal AF-2 activity in yeast; and (4) Ada3 is able to enhance the AF-2 activity of RXRalpha and ERalpha when overexpressed in yeast and mammalian cells . Taken together, these data indicate that ligand-dependent transactivation by RXRalpha and ERalpha in yeast is mediated at least in part by the Ada complex, in which the Ada3 subunit directly binds to the holoreceptor LBD. Biochim Biophys Acta, 1998 Apr 29, 1397(2), 131 - 6 A novel DEAD-box RNA helicase exhibits high sequence conservation from yeast to humans; Eisen A et al.; We have identified a novel Drosophila protein, DBP80, that exhibits significant similarity to mouse mDEAD5, yeast TIF1/2, and mammalian eIF-4A . DBP80 is a member of a subclass of DEAD-box proteins that contains a distinct domain, PX(I/R)ILLKR(E/D)EETLEGIKQ(F/Y)(F/Y), in addition to the seven canonical helicase domains . J Biol Chem, 1998 May 1, 273(18), 10827 - 30 Rpb3, stoichiometry and sequence determinants of the assembly into yeast RNA polymerase II in vivo; Svetlov V et al.; Stoichiometry of the third largest subunit (Rpb3) of the yeast RNA polymerase II is a subject of continuing controversy . In this work we utilized immunoaffinity and nickel-chelate chromatographic techniques to isolate the RNA polymerase II species assembled in vivo in the presence of the His6-tagged and untagged Rpb3 . The distribution pattern of tagged and untagged subunits among the RNA polymerase II molecules is consistent with a stoichiometry of 1 Rpb3 polypeptide per molecule of RNA polymerase . Deletion of either alpha-homology region (amino acids 29-55 or 226-267) from the Rpb3 sequence abolished its ability to assemble into RNA polymerase II in vivo. J Biol Chem, 1998 May 1, 273(18), 10811 - 4 pICln binds to a mammalian homolog of a yeast protein involved in regulation of cell morphology; Krapivinsky G et al.; Since its cloning and tentative identification as a chloride channel, the function of the pICln protein has been debated . Although there is no consensus regarding the specific function of pICln, it was suggested to play a role, directly or indirectly, in the function of a swelling-induced chloride conductance . Previously, the protein was shown to exist in several discrete protein complexes . To determine the function of the protein, we have begun the systematic identification of all proteins to which it binds . Here we show that four proteins firmly bind to pICln and identify the 72-kDa pICln-binding protein by affinity purification and peptide microsequencing . The interaction between this protein and pICln was verified several ways, including the extraction of several pICln clones from a cDNA library using the 72-kDa protein as a bait in a yeast two-hybrid screen . The protein is homologous to the yeast Skb1 protein . Skb1 interacts with Shk1, a homolog of the p21(Cdc42/Rac)-activated protein kinases (PAKs) . The known involvement of PAKs in cytoskeletal rearrangement suggests that pICln may be linked to a system regulating cell morphology. EMBO J, 1998 Apr 1, 17(7), 2095 - 106 Human homologs of yeast prp16 and prp17 reveal conservation of the mechanism for catalytic step II of pre-mRNA splicing; Zhou Z et al.; Pre-mRNA splicing takes place in two catalytic steps . The second step is poorly understood, especially in mammals . In yeast, the splicing factors, Prps 16, 17, 18 and Slu7 function exclusively in step II . Here we report the isolation of cDNAs encoding human Prps 16 and 17 which are 41 and 36% identical to their yeast counterparts . The Prp16 gene is essential in yeast, and we show that a chimeric yeast-human Prp16 protein rescues a yeast Prp16 knockout strain . Immunodepletion of hPrp16 from splicing extracts specifically blocks step II, and the activity can be fully restored with recombinant hPrp16 . Moreover, both hPrps 16 and 17 associate with the spliceosome late in the splicing pathway . Mutations at the 3' splice site that specifically block step II do not affect the association of hPrps 16 and 17 with the spliceosome, indicating that these factors may function at a stage of step II prior to recognition of the 3' splice site . Recently, the human homologs of Prp18 and Slu7 were identified . The observation that humans contain homologs of all four known step II proteins in yeast indicates that the mechanism for catalytic step II is highly conserved. EMBO J, 1998 Apr 1, 17(7), 2086 - 94 Prp22, a DExH-box RNA helicase, plays two distinct roles in yeast pre-mRNA splicing; Schwer B et al.; In order to assess the role of Prp22 in yeast pre-mRNA splicing, we have purified the 130 kDa Prp22 protein and developed an in vitro depletion/reconstitution assay . We show that Prp22 is required for the second step of actin pre-mRNA splicing . Prp22 can act on pre-assembled spliceosomes that are arrested after step 1 in an ATP-independent fashion . The requirement for Prp22 during step 2 depends on the distance between the branchpoint and the 3' splice site, suggesting a previously unrecognized role for Prp22 in splice site selection . We characterize the biochemical activities of Prp22, a member of the DExH-box family of proteins, and we show that purified recombinant Prp22 protein is an RNA-dependent ATPase and an ATP-dependent RNA helicase . Prp22 uses the energy of ATP hydrolysis to effect the release of mRNA from the spliceosome . Thus, Prp22 has two distinct functions in yeast pre-mRNA splicing: an ATP-independent role during the second catalytic step and an ATP-requiring function in disassembly of the spliceosome. EMBO J, 1998 Apr 1, 17(7), 2055 - 66 Hus1p, a conserved fission yeast checkpoint protein, interacts with Rad1p and is phosphorylated in response to DNA damage; Kostrub CF et al.; The hus1+ gene is one of six fission yeast genes, termed the checkpoint rad genes, which are essential for both the S-M and DNA damage checkpoints . Classical genetics suggests that these genes are required for activation of the PI-3 kinase-related (PIK-R) protein, Rad3p . Using a dominant negative allele of hus1+, we have demonstrated a genetic interaction between hus1+ and another checkpoint rad gene, rad1+ . Hus1p and Rad1p form a stable complex in wild-type fission yeast, and the formation of this complex is dependent on a third checkpoint rad gene, rad9+, suggesting that these three proteins may exist in a discrete complex in the absence of checkpoint activation . Hus1p is phosphorylated in response to DNA damage, and this requires rad3+ and each of the other checkpoint rad genes . Although there is no gene related to hus1+ in the Saccharomyces cerevisiae genome, we have identified closely related mouse and human genes, suggesting that aspects of the checkpoint control mechanism are conserved between fission yeast and higher eukaryotes. J Mol Biol, 1998 Mar 27, 277(2), 179 - 97 Role of the human homolog of the yeast transcription factor SPT5 in HIV-1 Tat-activation; Wu-Baer F et al.; The transactivator protein Tat stimulates transcriptional elongation from the HIV-1 LTR . One mechanism by which Tat increases HIV-1 transcription is by interacting with RNA polymerase II and TFIIH to increase phosphorylation of the polymerase C-terminal domain . Recent studies indicate that specific elongation factors may also be required to modulate Tat function . Here, we used biochemical analysis and in vitro transcription assays to identify cellular factors required for Tat activation . This analysis resulted in the purification of a cellular factor Tat-CT1 which is a human homolog of the yeast transcription factor SPT5 . Immunodepletion of Tat-CTl from HeLa extract demonstrated that this factor was involved in transcriptional activation by Tat . However, the absence of this factor from HeLa extract did not prevent transcriptional activation by VP16 . These findings are consistent with a model in which Tat-mediated effects on transcriptional elongation are mediated in part by the action of the human homolog of the yeast transcription factor SPT5 . Mol Gen Genet, 1998 Apr, 258(1-2), 139 - 47 Phosphoinositide-specific phospholipase C forms a complex with 14-3-3 proteins and is involved in expression of UV resistance in fission yeast; Andoh T et al.; The fission yeast plc1+ gene encodes phosphoinositide-specific phospholipase C . The two- hybrid interaction assay with plexA-plc1+ as a bait revealed that Plc1p interacted with the 14-3-3 proteins Rad24p and Rad25p . Formation of a complex containing Plc1p and Rad24p in vivo was confirmed by an immunological method . As predicted from the fact that rad24 null mutant cells are hypersensitive to UV irradiation, plc1 null mutant cells were almost as sensitive to UV irradiation as rad24 null mutant cells . In addition, deletion of rad24 in the plc1 null mutant cells did not enhance the UV sensitivity, indicating that plc1+ and rad24+ belong to the same epistasis group with respect to UV sensitivity . Whereas Rad24p has been reported to be involved in the DNA damage checkpoint pathway, the delay to mitosis after UV irradiation was not defective either in rad24 null mutant cells or in plcl null mutant cells in our analysis . Thus, Plc1p is responsible for resistance to UV irradiation, but not for the DNA damage checkpoint pathway, in cooperation with 14-3-3 proteins. Mol Gen Genet, 1998 Apr, 258(1-2), 148 - 55 A screen for upstream components of the yeast protein kinase C signal transduction pathway identifies the product of the SLG1 gene; Jacoby JJ et al.; We employed the constitutive BCK1-20 allele of the gene for the MAP kinase kinase kinase (MAP-KKK) in the yeast Pkc signal transduction pathway to develop a genetic screen for mutants in genes encoding upstream components . Transposon mutagenesis yielded a mutant that was completely dependent on the active allele in the absence of osmotic stabilization . The transposon had integrated at the yeast SLG1 (HCS77) locus . This gene encodes a putative membrane protein . Haploid slg1 deletion strains are sensitive to caffeine, as expected for mutants in the Pkc pathway, as well as a variety of other drugs . The response to elevated temperatures and the dependence on osmotic stabilization depends on the genetic background . Thus, in the strain used for mutagenesis, disruption of SLG1 causes the cells to become non-viable in the absence of osmotic stabilization at both 30 degrees C and 37 degrees C . In a different genetic background this phenotype was not observed . Sensitivity of the haploid deletion mutants to caffeine can be partially suppressed by overexpression of genes for other components of the Pkc pathway, such as PKC1, SLT2, ROM2, and STE20 . In addition, a SLG1-lacZ reporter construct shows higher expression in the presence of caffeine or magnesium chloride in a wild-type diploid background. Mol Gen Genet, 1998 Apr, 258(1-2), 95 - 103 Datin, a yeast poly(dA:dT)-binding protein, behaves as an activator of the wild-type ILV1 promoter and interacts synergistically with Reb1p; Moreira JM et al.; A cis-acting element required for GCN4-independent basal-level transcription of ILV1 was previously identified in our laboratories as a binding site for the REB1 protein (Reb1p) . Further deletion analysis of the ILV1 promoter region identified a second element also required for GCN4-independent basal-level ILV1 expression . This second element is an A.T-rich tract (26 As out of 32 nucleotides) situated 15 bp downstream of the Reb1p-binding site . Deletion of both the Reblp site and the poly(dA:dT) element totally eliminates basal activity of the ILV1 promoter . We show that the two elements act synergistically to control ILV1 expression and that the synergistic effect is distance dependent . We demonstrate that (i) datin (Dat1p), the only known poly (dA:dT)-binding protein in yeast, specifically binds to the ILV1 poly(dA:dT) element in vitro; (ii) Dat1p functions as a trans-activating factor in the ILV1 context; and (iii) the synergistic activation observed in vivo between the Reb1p site and the poly(dA:dT) element depends on the presence of the structural gene for Dat1p, DAT1. Mol Plant Microbe Interact, 1998 Jun, 11(6), 458 - 65 The plasma membrane H(+)-ATPase from the biotrophic rust fungus Uromyces fabae: molecular characterization of the gene (PMA1) and functional expression of the enzyme in yeast; Struck C et al.; To study the molecular basis of biotrophic nutrient uptake by plant parasitic rust fungi, the gene (Uf-PMA1) encoding the plasma membrane H(+)-ATPase from Uromyces fabae was isolated . Uf-PMA1 exists probably as a single gene . However, two nearly identical sequences were identified; the similarity apparently is due to two Uf-PMA1 alleles in the dikaryotic hyphae . Multiple Uf-PMA1 transcripts were observed during early rust development, and reduced amounts of a single Uf-PMA1 mRNA were observed in haustoria and rust-infected leaves . This is in contrast to elevated enzyme activity in haustoria compared to germinated spores (C . Struck, M . Hahn, and K . Mendgen . Fungal Genet . Biol . 20:30-35, 1996) . Unexpectedly, the PMA1-encoded rust protein is more similar to H(+)-ATPases from plants (55% identity) than from ascomycetous fungi (36% identity) . When the rust PMA1 cDNA was expressed in Saccharomyces cerevisiae, both the wild-type enzyme and a mutant derivative (delta 76) deleted for the 76 C-terminal amino acids were able to support growth of a yeast strain lacking its own H(+)-ATPases . Compared to the wild-type, the delta 76 mutant enzyme displayed increased affinity to ATP, a higher vanadate sensitivity, and a more alkaline pH optimum . These results indicate that the C-terminal region of the rust enzyme exhibits auto-regulatory properties. Gene, 1998 Jun 8, 212(2), 305 - 14 Usage of non-canonical promoter sequence by the yeast mitochondrial RNA polymerase; Biswas TK; Prior work has demonstrated that a conserved nonanucleotide {5'-TATAAGTAA(+2)} promoter sequence is used by the mitochondrial {mt}1 RNA polymerase in Saccharomyces cerevisiae . However, the highly AT-rich yeast mt genome carries many other promoter-like sequences, but only a fraction of them are involved in gene-specific transcription . To examine the sequence variability of this nonanucleotide promoter motif, single or multiple nt substitutions were introduced into the canonical promoter sequence . The transcriptional activity of these altered promoter sequences was examined under the in-vitro reaction conditions . The results presented here determined that several variant promoter sequences (i . e . TAAAAGTAA, TATAAGAAA, TATAAGTAG, TATAAGAAG, TATAAGAGA, TATAAGGGA, TATAAGTGG, TAAAAGTAG) were efficiently used by the mtRNA polymerase . However, a single (i.e . AATAAGTAA, TTTAAGTAA, TATTAGTAA, TATAACTAA, TATAAGGAA, TATAAGTAT) or multiple (TATAGGAAA, TAAAAGGAA, TATAGGGAA, TAAAGGAAA, TAAAGGGAA) nt substitution(s) in other locations drastically reduced mt promoter function . Interestingly, some of these poorly or partially active promoter variants (i.e . TATAAGGAA, TATAAGTAT, TATAAGTCA) became fully functional in the presence of sequence-specific dinucleotide primer . Since dinucleotide primer bypasses the first phosphodiester bond formation in transcription, it is suggested that the -1T-->G, +1A-->C and +2A-->T mutations affect mt transcription at the level of initiation rather than polymerase binding. Gene, 1998 Jun 8, 212(2), 197 - 202 YIpDCE1 - an integrating plasmid for dual constitutive expression in yeast; Stearman R et al.; YIpDCE1 (Dual Constitutive Expression), a novel Saccharomyces cerevisiae integrating plasmid, constitutively expresses two genes under the control of separate phosphoglycerol kinase promoters . YIpDCE1 contains the complete ADE2 gene which can be used as a marker for selecting integrants at mutant ade2 loci commonly present in laboratory yeast strains . The YIpDCE1 plasmid can be inserted into the ade2-101 locus of the HF7c strain used in two hybrid screens . Thus it could be useful for analysis of two hybrid interactions that occur in the context of additional protein components (e.g . modifying enzymes such as kinases or phosphatases, or multimeric complexes consisting of three or four distinct protein components) . YIpDCE1 has been used to create strains simultaneously overexpressing the permease (FTR1) and oxidase (FET3) components of the yeast high-affinity iron uptake system . This confers constitutive high-affinity iron uptake on the transformed strains, bypassing the normal regulatory mechanisms. J Nat Prod, 1998 May, 61(5), 696 - 8 Three new metabolites from the marine yeast aureobasidium pullulans Shigemori H, Tenma M, Shimazaki K, Kobayashi J. Two new diketopiperazines (1 and 2) with a d-cis-4-hydroxyproline residue, and orcinotriol (3), a new 1,3-dihydroxyphenol derivative, were isolated from cultured broth of the yeast Aureobasidium pullulans, which was isolated from an Okinawan marine sponge . The structures of the compounds, including their absolute stereochemistries, were determined by spectroscopic data and chemical means. Curr Opin Genet Dev, 1998 Apr, 8(2), 194 - 9 Replication origins in yeast versus metazoa: separation of the haves and the have nots; Gilbert DM; The recent flood of information concerning Saccharomyces cerevisiae replication origins and the proteins that interact with them contrasts alarmingly to the trickle of progress in our understanding of metazoan origins . In mammalian cells, origins are complex and heterogeneous, and appear to be selected by features of nuclear architecture that are re-established after each mitosis . Studies in Xenopus egg extracts have shown that once per cell cycle replication does not require specific origin sequences, despite the identification of functional homologues to yeast origin-binding proteins . These observations suggest that initiation of DNA replication in higher eukaryotes is focused to specific genomic regions by features of chromosome structure. Biochemistry, 1998 May 19, 37(20), 7444 - 55 Steady state and time-resolved fluorescence study of residual structures in an unfolded form of yeast phosphoglycerate kinase; Garcia P et al.; A previous study performed using steady state fluorescence has revealed the existence of residual structures surrounding the two tryptophan residues in an unfolded form of yeast phosphoglycerate kinase {Garcia, P., et al . (1995) Biochemistry 34, 397-404} . In this paper, we present a more detailed characterization of these residual structures, through the study of two single tryptophan-containing mutants of yPGK, W333F and W308Y . Denaturation experiments have first been performed at low temperatures to assess the nature of the interactions stabilizing these residual structures . On the other hand, the compactness and dynamics of the protein matrix were probed using tryptophan fluorescence quenching by acrylamide at various denaturant concentrations . Taking into consideration the changes in sample viscosity induced by addition of guanidinium chloride made feasible the use of this technique during the denaturation process . These different approaches have shown that the residual structures around tryptophan 308 are mainly stabilized by hydrophobic interactions and are more compact and less fluctuant than the ones surrounding tryptophan 333 . Native and denatured yPGK have also been studied by time-resolved fluorescence spectroscopy . In the native protein, tryptophan buried in the core, W333, is mainly associated with a lifetime close to 0.1 ns, whereas tryptophan that is partially accessible to the solvent, W308, has a lifetime close to 0 . 5 ns . The time-resolved tryptophan fluorescence emission of wild-type yPGK can be accounted for quantitatively by the summed emissions of its two single tryptophan mutants . The significance of minor long lifetime components is discussed for the two tryptophan residues . This new assignment of fluorescent decay times has allowed for the detection of a folding intermediate in which the environment of tryptophan 333 is modified for denaturant concentrations lower than those for tryptophan 308. Biochemistry (Mosc), 1998 Mar, 63(3), 259 - 75 Denatured states of yeast phosphoglycerate kinase; Damaschun G et al.; Structures of proteins in unfolded states have important implications for the protein folding problem and for the translocation of polypeptide chains . Acid-denatured, cold-denatured, and 6 M guanidine hydrochloride (GuHCl) denatured yeast phosphoglycerate kinase (PGK) are ensembles of flexible unfolded molecules with rapidly interconverting structures of the individual polypeptide chains . They differ, however, in their physical properties, such as in coil size and in stiffness over a short distance along the chain . These properties of polypeptide chains can be described well by persistence statistics . A solution containing 0.7 M GuHCl at 4.5 degrees C is nearly a Theta-solvent for PGK . By contrast, 6 M GuHCl is a good solvent for PGK . Acid-denatured PGK at low ionic strength has the most expanded and stiffest chains . The conformation of heat-denatured PGK should be more compact than that of random walk chains at the Theta-point, as can be inferred from measurements on other proteins . Investigations of heat-denatured PGK by scattering methods are unfeasible due to aggregation of the protein . The persistence length as a measure of chain stiffness varies between a = 1.74 nm for cold-denatured PGK and a = 3.0 nm for acid-denatured PGK . The distribution functions of the gyration radii were calculated from the X-ray scattering data for all unfolded states and compared with the radius of gyration of the natively folded molecule. Appl Environ Microbiol, 1998 Jun, 64(6), 2215 - 9 Isolation and characterization of a dibenzofuran-degrading yeast: identification of oxidation and ring cleavage products; Hammer E et al.; We characterized the ability of a yeast to cleave the aromatic structure of the dioxin-like compound dibenzofuran . The yeast strain was isolated from a dioxin-contaminated soil sample and identified as Trichosporon mucoides . During incubation of glucose-pregrown cells with dibenzofuran, six major metabolites were detected by high-performance liquid chromatography . The formation of four different monohydroxylated dibenzofurans was proven by comparison of analytical data (gas chromatography-mass spectrometry) with that for authentic standards . Further oxidation produced 2, 3-dihydroxydibenzofuran and its ring cleavage product 2-(1-carboxy methylidene)-2,3-dihydrobenzo{b}furanylidene glycolic acid, which were characterized by mass spectrometry and 1H nuclear magnetic resonance spectroscopy . These two metabolites are derived from 2-hydroxydibenzofuran and 3-hydroxydibenzofuran, as shown by incubation experiments using these monohydroxylated dibenzofurans as substrates. FEBS Lett, 1998 May 8, 427(2), 213 - 9 Retinol-induced secretion of human retinol-binding protein in yeast; Reppe S et al.; Retinol-binding protein (RBP) functions as a transporter for retinol (vitamin A) in plasma in higher eukaryotes . We have successfully expressed human RBP in Saccharomyces cerevisiae, and its secretion was found to be induced by retinol also in this lower eukaryote . Reduced induction of secretion by retinol in a temperature-sensitive sec18-1 mutant that is blocked in secretion at the restricted temperature suggests that as in mammalian cells, RBP can be released from the endoplasmic reticulum upon addition of retinol . Thus, the molecular mechanism involved in retinol-dependent secretion of RBP appears to be conserved in yeast, and this points to yeast as a putative model system for studying retinol-regulated secretion of RBP . RBP purified from yeast was found to be indistinguishable from RBP purified from human plasma in several functional assays. Vaccine, 1998 Feb, 16(4), 329 - 34 Major adverse reactions to yeast-derived hepatitis B vaccines--a review; Grotto I et al.; Yeast-derived recombinant DNA hepatitis B vaccines usage became widely accepted since the early 1990s . Severe adverse events have been reported infrequently in adults and rarely in infants and children given hepatitis B vaccine in the ten years which have passed since the introduction of the vaccine . Some of the data were summarized in previous review articles . Our review of the literature revealed reports of serious adverse reactions which included immediate reactions (anaphylaxis and urticaria) as well as delayed reactions, including skin, rheumatic, vasculitic (including Systemic Lupus Erythematosus and glumerulonephritis), hematologic, ophthalmologic and neurologic reactions . These cases were summarized and a pathogenetic mechanism is offered. Yeast, 1998 Apr 30, 14(6), 573 - 81 Cloning and sequencing of a cDNA encoding a copper-zinc superoxide dismutase enzyme from the marine yeast Debaryomyces hansenii; Hernandez-Saavedra NY et al.; Cu-Zn superoxide dismutase (SOD-1) is a ubiquitously occurring eukaryotic enzyme with a variety of important effects on respiring organisms . A gene (dhsod-1) encoding a Cu-Zn superoxide dismutase of the marine yeast Debaryomyces hansenii was cloned using mRNA by the RT-PCR technique . The deduced amino-acid sequence shows approximately 70% homology with that of cytosolic superoxide dismutase from Saccharomyces cerevisiae and Neurospora crassa, as well as lower homologies (between 55 and 65%) with the corresponding enzyme of other eukaryotic organisms, including human . The gene sequence encodes a protein of 153 amino acids with a calculated molecular mass of 15-92 kDa, in agreement with the observed characteristics of the purified protein from D . hansenii. Yeast, 1998 Apr 30, 14(6), 501 - 15 Influence of monovalent cations on yeast cytoplasmic and vacuolar pH; Calahorra M et al.; The effects of monovalent cations on the internal pH of yeast were studied . Our former procedure was modified, inducing maximal alkalinization of the cells with 100 mM-NH4OH instead of Tris base . The pH values were lower than reported before (Pena et al., J . Baceteriol . 1995 177, 1017-1022) . With glucose as substrate, the internal cytoplasmic pH reached higher values when incubating at an external pH of 6.0, as compared to pH 4.0 . Monovalent cations added approximately 5 min after glucose produced a further increase in the internal pH, which was higher at a previous incubation pH of 4.0 than that observed at pH 6.0 . The selectivity of the changes followed a similar order to that of the transport system for monovalent cations . When incubating cells with glucose for more than 30 min, the initial changes of the internal pH appeared to be regulated by the cell . However, under the fluorescence microscope, it was observed that pyranine, which was confined to the cytoplasm during the first 15 min, was progressively concentrated in the vacuole . By studying the fluorescence changes of cells electroporated and then incubated with glucose or glucose plus potassium, we could follow the internal pH of this organelle, obtaining values within the range reported by other authors . Also, in cells preincubated with glucose for 60 min, and electroporated afterwards, the fluorescence of pyranine, which only entered the cytoplasm, allowed us to measure the pH of this compartment, showing that it was more alkaline than the vacuole . Moreover, the cytoplasmic pH increased upon addition of glucose or potassium . The vacuolar pH, on the other hand, increased upon addition of potassium after glucose, but decreased upon addition of glucose . In addition, incubation of the cells with glucose with or without pyranine produced vesiculation of the vacuole. Proc Natl Acad Sci U S A, 1998 May 26, 95(11), 5906 - 12 Precise sequence complementarity between yeast chromosome ends and two classes of just-subtelomeric sequences; Britten RJ; The terminal regions (last 20 kb) of Saccharomyces cerevisiae chromosomes universally contain blocks of precise sequence similarity to other chromosome terminal regions . The left and right terminal regions are distinct in the sense that the sequence similarities between them are reverse complements . Direct sequence similarity occurs between the left terminal regions and also between the right terminal regions, but not between any left ends and right ends . With minor exceptions the relationships range from 80% to 100% match within blocks . The regions of similarity are composites of familiar and unfamiliar repeated sequences as well as what could be considered "single-copy" (or better "two-copy") sequences . All terminal regions were compared with all other chromosomes, forward and reverse complement, and 768 comparisons are diagrammed . It appears there has been an extensive history of sequence exchange or copying between terminal regions . The subtelomeric sequences fall into two classes . Seventeen of the chromosome ends terminate with the Y' repeat, while 15 end with the 800-nt "X2" repeats just adjacent to the telomerase simple repeats . The just-subterminal repeats are very similar to each other except that chromosome 1 right end is more divergent. Biochim Biophys Acta, 1998 Apr 22, 1371(1), 96 - 100 Stimulation of the yeast mitochondrial calcium uniporter by hypotonicity and by ruthenium red; Bazhenova EN et al.; The Ca2+ uptake by mitochondria from the yeast Endomyces magnusii has earlier been found to be driven by the membrane potential and to be stimulated by spermine . It thus functions in a similar fashion as the animal mitochondrial calcium uniporter . Here, it is shown that the uptake is stimulated, i.e., Ca2+ can be accumulated from lower {Ca2+}, under hypotonic conditions . Ruthenium Red, an inhibitor of the animal uniporter, under certain conditions, stimulates the yeast uniporter . The mechanism of the stimulation by hypotonicity and Ruthenium Red is discussed . Acta Neuropathol (Berl), 1998 Mar, 95(3), 291 - 6 Rare occurrence of inactivating p53 gene mutations in primary non-astrocytic tumors of the central nervous system: reappraisal by yeast functional assay; Nozaki M et al.; While it is established that p53 mutation plays a critical role in the carcinogenesis of astrocytic brain tumors, its role remains to be clarified for other types of tumors in the central nervous system (CNS) . Using a yeast-based assay which tests the ability of human p53 to activate transcription, we analyzed p53 mutations in 85 non-astrocytic CNS tumors, including 4 benign neuronal tumors (3 central neurocytomas and 1 pineocytoma), 12 primitive neuroectodermal tumors, 14 germ cell tumors (7 germinomas, 7 non-germinomatous tumors), 4 craniopharyngiomas, 14 ependymomas, 22 schwannomas, 10 primary brain lymphomas in immunocompetent patients, and 5 bone tumors of the skull . The only tumors found to contain p53 mutations were 3 malignant lymphomas . The presence of mutations in these cases was confirmed by DNA sequencing . Given the high accuracy and sensitivity of the yeast assay and previous negative results using conventional techniques, this indicates that p53 mutation is a rare event in non-astrocytic CNS tumor types examined here. Nucleic Acids Res, 1998 Apr 1, 26(7), 1826 - 33 Position-independent expression of a human nerve growth factor-luciferase reporter gene cloned on a yeast artificial chromosome vector; Asselbergs FA et al.; Two yeast artificial chromosomes containing the entire human nerve growth factor gene were isolated and mapped . By homologous recombination a luciferase gene was precisely engineered into the coding portion of the NGF gene and a neomycin selection marker was placed adjacent to one of the YAC telomeres . Expression of the YAC-based NGF reporter gene and a plasmid-based NGF reporter gene were compared with the regulation of endogenous mouse NGF protein in mouse L929 fibroblasts . In contrast to the plasmid-based reporter gene, expression and regulation of the YAC-based reporter gene was independent of the site of integration of the transgene . Basic fibroblast growth factor and okadaic acid stimulated expression of the YAC transgene, whereas transforming growth factor-beta and dexamethasone inhibited it . Although cyclic AMP strongly stimulated production of the endogenous mouse NGF, no effect was seen on the human NGF reporter genes . Downregulation of the secretion of endogenous mouse NGF already occurred at an EC50 of 1-2 nM dexamethasone, but downregulation of the expression of NGF reporter genes occurred only at EC50 of 10 nM . This higher concentration was also required for upregulation of luciferase genes driven by the dexamethasone-inducible promoter of the mouse mammary tumor virus in L929 fibroblasts. Dev Dyn, 1998 May, 212(1), 38 - 48 Differential preimplantation regulation of two mouse homologues of the yeast SWI2 protein; LeGouy E et al.; Epigenetic regulation of gene expression through modification of chromatin organization is an important mechanism in the development of eucaryotic organisms . We investigated the developmentally regulated expression of the mouse mBRG1 and mbrm genes, which are homologous to the yeast SWI2 gene . Both proteins are involved in chromatin remodeling as components of the mammalian SWI/SNF complex . The analysis was performed at a time in mouse development when the formation of a functional zygotic nucleus is closely linked to extensive chromatin modifications . Reverse transcription-polymerase chain reaction (RT-PCR) analysis in mature oocytes and through the first cleavage stages showed that both genes were highly expressed as maternal products but that they subsequently exhibited considerable differences in their level of expression when the transition to zygotic transcription occurred . Immunodetection of the two proteins with specific antibodies paralleled the RT-PCR analysis . The mBRG1 protein was present throughout preimplantation development, whereas zygotic mbrm was clearly detectable only when differentiation first occurs at the blastocyst stage . At this stage, mbrm was restricted to the inner cell mass . Cell type-specific expression of mbrm was also observed after in vitro differentiation of embryonic stem cells . These results indicate that the two murine homologues of SWI2 have substantially different roles in chromatin organization during the onset of embryonic development. Curr Opin Immunol, 1998 Apr, 10(2), 131 - 6 The yeast two-hybrid screening technique and its use in the study of protein-protein interactions in apoptosis; Wallach D et al.; The yeast two-hybrid technique provides a general approach for cloning cDNAs merely by exploiting the ability of their encoded proteins to bind to a protein of interest . The technique therefore offered a useful access to the analysis of the mechanisms of cell death at the initial stage of their study, when only a few of the proteins involved and very little about their mode of action were known . Conversely, the knowledge of cell death mechanisms gained by this technique provided a useful insight into both the potential and the limitations of this technique. Biochim Biophys Acta, 1998 Apr 2, 1383(2), 351 - 5 Significance of the enzymatic properties of yeast S39A enolase to the catalytic mechanism; Brewer JM et al.; The S39A mutant of yeast enolase (isozyme 1), prepared by site-directed mutagenesis, has a relative Vmax of 0.01% and an activation constant for Mg2+ ca . 10-fold higher, compared with native enzyme . It is correctly folded . There is little effect of solvent viscosity on activity . We think that the loop Ser36-His43 fails to move to the 'closed' position upon catalytic Mg2+ binding, weakening several electrostatic interactions involved in the mechanism. Genes Dev, 1998 May 15, 12(10), 1464 - 73 Phosphorylation and association with the transcription factor Atf1 regulate localization of Spc1/Sty1 stress-activated kinase in fission yeast; Gaits F et al.; Control of gene expression by stress-activated protein kinase (SAPK) cascades is crucial for combating cytotoxic stress . Elements of these cascades have been investigated in detail, but regulation of stress signal transduction from the cytoplasm to the nucleus is poorly understood . Herein are reported subcellular localization studies of fission yeast Spc1, a homolog of human p38 and budding yeast Hog1p SAPKs . Stress induces transient nuclear localization of Spc1 . Nuclear translocation of Spc1 is coupled with disassociation from its activator kinase Wis1 . However, Spc1 does not concentrate in the nucleus of Deltawis1 cells; therefore Wis1 does not tether Spc1 in the cytoplasm . Unphosphorylatable forms of Spc1 are dispersed in the cytoplasm and nucleus, even in cells that also produce wild-type Spc1 . Thus, Spc1 must be phosphorylated by Wis1 to localize in the nucleus . Nuclear retention of Spc1 requires Atf1, a transcription factor that is the key nuclear substrate of Spc1 . Nuclear localization of Atf1 requires Pcr1, a heterodimerization partner of Atf1 . These studies show that phosphorylation and association with Atf1 are required for nuclear localization of Spc1. Gene, 1998 Apr 28, 211(1), 133 - 40 A human homolog of the yeast CDC7 gene is overexpressed in some tumors and transformed cell lines; Hess GF et al.; The Cdc7 protein kinase of Saccharomyces cerevisiae is a critical regulator of several aspects of DNA metabolism and cell cycle progression . We describe the isolation of a human gene encoding a Cdc7 homolog . The Cdc7Hs protein sequence is 27% identical to that of the yeast protein, includes features unique to yeast Cdc7, and contains all conserved catalytic residues of protein kinases . The human sequence also shows significant similarity to the cyclin-dependent kinases, in accordance with evidence that yeast Cdc7 is related to the cdks . CDC7Hs is expressed in many normal tissues, but overexpressed in certain tumor types and all transformed cell lines examined . In some of the tumors tested, CDC7Hs expression correlates with expression of a proliferation marker, the histone H3 gene . In other cases, no such correlation was observed . This suggests that CDC7Hs expression may be associated hyperproliferation in some tumors and neoplastic transformation in others. Genetics, 1998 May, 149(1), 57 - 72 Regulation of yeast glycogen metabolism and sporulation by Glc7p protein phosphatase; Ramaswamy NT et al.; Glc7p is an essential serine/threonine type 1 protein phosphatase (PP1) from the yeast Saccharomyces cerevisiae, which has a role in many processes including cell cycle progression, sporulation, glycogen accumulation, translation initiation, and glucose repression . Two hallmarks of PP1 enzymes are very high amino acid sequence conservation and association of the catalytic subunit with a variety of noncatalytic, regulatory subunits . We tested the hypothesis that PP1 sequence conservation was the result of each PP1 residue playing a role in multiple intermolecular interactions . Analysis of 24 glc7 mutants, isolated primarily by their glycogen accumulation traits, revealed that every mutated Glc7p residue altered many noncatalytic subunit affinities and conferred unselected sporulation traits to various degrees . Furthermore, quantitative analysis showed that Glc7p affinity for the glycogen-binding noncatalytic subunit Gac1p was not the only parameter that determines the glycogen accumulation by a glc7 mutant . Sds22p is one Glc7p noncatalytic subunit that is essential for mitotic growth . Surprisingly, several mutant Glc7p proteins had undetectable affinity for Sds22p, yet grew apparently normally . The characterization of glc7 diploid sporulation revealed that Glc7p has at least two meiotic roles . Premeiotic DNA synthesis was undetectable in glc7 mutants with the poorest sporulation . In the glc7 diploids examined, expression of the meiotic inducer IME1 was proportional to the glc7 diploid sporulation frequency . Moreover, IME1 hyperexpression could not suppress glc7 sporulation traits . The Glc7p/Gip1p holoenzyme may participate in completion of meiotic divisions or spore packaging because meiotic dyads predominate when some glc7 diploids sporulate. Genetics, 1998 May, 149(1), 45 - 56 Yeast dom34 mutants are defective in multiple developmental pathways and exhibit decreased levels of polyribosomes; Davis L et al.; The DOM34 gene of Saccharomyces cerevisiae is similar to genes found in diverse eukaryotes and archaebacteria . Analysis of dom34 strains shows that progression through the G1 phase of the cell cycle is delayed, mutant cells enter meiosis aberrantly, and their ability to form pseudohyphae is significantly diminisehd . RPS30A, which encodes ribosomal protein S30, was identified in a screen for high-copy suppressors of the dom34delta growth defect . dom34delta mutants display an altered polyribosome profile that is rescued by expression of RPS30A . Taken together, these data indicate that Dom34p functions in protein translation to promote G1 progression and differentiation . A Drosophila homolog of Dom34p, pelota, is required for the proper coordination of meiosis and spermatogenesis . Heterologous expression of pelota in dom34delata mutants restores wild-type growth and differentiation, suggesting conservation of function between the eukaryotic members of the gene family. J Biol Chem, 1998 May 22, 273(21), 12909 - 13 Complex interactions of the protein L-isoaspartyl methyltransferase and calmodulin revealed with the yeast two-hybrid system; O'Connor MB et al.; The widely distributed protein-L-isoaspartyl, D-aspartyl carboxylmethyltransferase (EC 2.1.1.77) is hypothesized to play a role in the repair or metabolism of deamidated and isomerized proteins that are spontaneously generated during the aging of proteins in cells . The yeast two-hybrid system was used to identify proteins that potentially interact with the methyltransferase in a cellular processing pathway . Two cDNAs, both encoding calmodulin, were isolated from a human fetal brain cDNA library using the human methyltransferase as the bait . Enzymatic assays with purified components revealed a complex set of interactions between the methyltransferase and calmodulin . Calmodulin weakly stimulated protein carboxylmethyltransferase activity in vitro at concentrations of the two proteins reflecting their representation in mammalian brain . Calmodulin stimulation of methyltransferase was observed in both the presence and absence of calcium, although the effect was greater in the presence of calcium . Native calmodulin was not a substrate for the carboxylmethyltransferase, but deamidated variants of calmodulin act as substrates for the methyltransferase, with calculated Km values of 3.6 and 8.6 microM for calcium-liganded and unliganded calmodulin, respectively . Both the effector and substrate interactions of calmodulin with the protein isoaspartyl methyltransferase likely contributed to the positive results obtained with the two-hybrid system. Novartis Found Symp, 1998, 214, 87 - 99; discussion 99-103 Multiple epigenetic events regulate mating-type switching of fission yeast; Klar AJ et al.; Two epigenetic events at mat1, one of which is DNA strand specific, are required to initiate recombination during mating-type switching . The third, a chromosomally borne imprinted event at the mat2/3 interval regulates silencing and directionality of switching, and prohibits interchromosomal recombination . We speculate that the unit of inheritance in the mat2/3 interval is both DNA plus its associated chromatin structure . Such a control is likely to be essential in maintaining particular states of gene expression during development. Semin Cell Dev Biol, 1998 Apr, 9(2), 135 - 41 Regulation of G protein signalling in yeast; Dohlman HG et al.; A common property of cell signaling systems is the ability to adapt to chronic stimulation . A genetic analysis of receptor/G protein signaling in yeast has led to the identification of a new class of regulators of G protein signaling (RGS proteins), as well as to new insights about the regulatory role of G protein modifications (myristoylation, palmitoylation) . Similar modes of regulation are now known to exist in humans . These discoveries fill some important gaps in our understanding of signal transduction, and provide an instructive example of how model organisms, like yeast, can provide new insights relevant to signal regulation in higher eukaryotes. Semin Cell Dev Biol, 1998 Apr, 9(2), 129 - 34 Down regulation of yeast G protein-coupled receptors; Riezman H; G protein-coupled receptors are essential components of the pheromone signaling pathway in Saccharomyces cerevisiae . When ligands bind to these receptors, their down regulation is stimulated . The sequence of events in this process is proposed to be the following: ligand-induced conformational change, receptor hyperphosphorylation and ubiquitination, internalization by an actin-dependent mechanism and subsequent delivery through at least two endocytic intermediates to the vacuole where the receptors are degraded. Ann N Y Acad Sci, 1998 Apr 15, 842, 195 - 8 Molecular dissection of the genetic targets of ALG7 in the serpentine receptor-mediated signal transduction pathway in yeast; Lennon K et al.; These initial studies show that deregulated expression of ALG7 affects diverse cellular functions crucial to development, including proliferation, differentiation, and morphogenesis . Furthermore, the data suggest multiple genetic targets for ALG7 and provide the basis for future dissection of these developmentally relevant pathways. J Mol Biol, 1998 Apr 24, 278(1), 67 - 78 The role of the 3' external transcribed spacer in yeast pre-rRNA processing; Allmang C et al.; We have undertaken a deletion analysis of the 3' external transcribed spacer (3' ETS) in the pre-rRNA of Saccharomyces cerevisiae . A stem loop structure immediately 3' to the 25 S rRNA region is necessary and sufficient for processing of the 3' ETS . This is believed to be by cotranscriptional cleavage by Rnt1p, the yeast homologue of RNase III . In addition, this stem-loop is required for cleavage of site A3 by RNase MRP and for processing at site B1L, in the 3' region of ITS1 . Processing at an upstream site in ITS1, site A2, and at sites in the 5' external transcribed spacer are not affected, even by complete deletion of the 3' ETS . We conclude that processing in the 3' ETS and in ITS1 is coupled . This would constitute a quality control that prevents synthesis of the 5 . 8 S rRNA and 5' end maturation of the 25 S rRNA in transcripts which are incomplete due to premature transcription termination . Curr Genet, 1998 Apr, 33(4), 276 - 83 Cloning and characterization of the yeast RAD1 homolog gene (mus-38) from Neurospora crassa: evidence for involvement in nucleotide excision repair; Hatakeyama S et al.; A Neurospora crassa gene encoding a product with homology to the Saccharomyces cerevisiae Rad1 nucleotide excision repair (NER) protein was isolated by degenerate PCR . The predicted protein consists of 892 amino acids with a molecular weight of 100.4 kDa, and 32-37% identity to the XPF/ERCC4 protein family . The homolog was mapped to the left arm of linkage group I, the location of the mus-38 gene . Subsequently, gene inactivation and complementation studies identified the RAD1 homolog as mus-38 . Immunological assays showed that the mus-18 (UV-specific endonuclease) and mus-38 strains have partial and normal UV-damage excision activities, respectively, but removal of thymine dimers and TC (6-4) photoproducts is abolished in the mus-18 mus-38 double mutant . The double mutant also was synergistically more sensitive to UV than either single mutant . The data suggest that mus-38 may participate in a different NER pathway from that involving the mus-18 gene. EMBO J, 1998 Apr 15, 17(8), 2353 - 8 Structure of wild-type yeast RNA polymerase II and location of Rpb4 and Rpb7; Jensen GJ et al.; The three-dimensional structure of wild-type yeast RNA polymerase II has been determined at a nominal resolution of 24 A . A difference map between this structure and that of the polymerase lacking subunits Rpb4 and Rpb7 showed these two subunits forming part of the floor of the DNA-binding (active center) cleft, and revealed a slight inward movement of the protein domain surrounding the cleft . Surface plasmon resonance measurements showed that Rpb4 and Rpb7 stabilize a minimal pre-initiation complex containing promoter DNA, TATA box-binding protein (TBP), transcription factor TFIIB and the polymerase . These findings suggest that Rpb4 and Rpb7 play a role in coupling the entry of DNA into the active center cleft to closure of the cleft . Such a role can explain why these subunits are necessary for promoter-specific transcription in vitro and for a normal stress response in vivo. Science, 1998 May 1, 280(5364), 741 - 4 Yeast Ku as a regulator of chromosomal DNA end structure; Gravel S et al.; During telomere replication in yeast, chromosome ends acquire an S-phase-specific overhang of the guanosine-rich strand . Here it is shown that in cells lacking Ku, a heterodimeric protein involved in nonhomologous DNA end joining, these overhangs are present throughout the cell cycle . In vivo cross-linking experiments demonstrated that Ku is bound to telomeric DNA . These results show that Ku plays a direct role in establishing a normal DNA end structure on yeast chromosomes, conceivably by funct