J Biol Chem, 2004 Jun 25, 279(26), 27607 - 12 Epub 2004 Apr 21. A novel mode of chaperone action: heme activation of Hap1 by enhanced association of Hsp90 with the repressed Hsp70-Hap1 complex; Lan C et al.; Molecular chaperones Hsp90 and Hsp70 control many signal transducers, including cyclin-dependent kinases and steroid receptors . The yeast heme-responsive transcriptional activator Hap1 is a native substrate of both Hsp90 and Hsp70 . Hsp90 and Hsp70 are critical for the precise regulation of Hap1 activity by heme . Here, to decipher the molecular events underlying the actions of Hsp90 and Hsp70 in heme regulation, we purified various multichaperone-Hap1 complexes and characterized the complexes linked to Hap1 repression and activation by two-dimensional electrophoresis analysis . Notably, we found that in vitro Hap1 is associated continuously with Ssa and its co-chaperones, and this association is not weakened by heme . Heme enhances the interaction between Hap1 and Hsp90 . In vivo, defective Ssa, Ydj1, or Sro9 function causes Hap1 derepression in the absence of heme, whereas defective Hsp90 function causes reduced Hap1 activity at high heme concentrations . These results show that continuous association of Hap1 with Ssa, Ydj1, and Sro9 confers Hap1 repression, whereas enhanced association of Hsp90 with the repressed Hap1-Ssa-Ydj1-Sro9 complex by heme causes Hap1 activation . This novel mechanism of chaperone action may operate to control the activity of other important signal transducers. Mol Microbiol, 2004 May, 52(3), 785 - 99 The Aspergillus fumigatus transcriptional activator CpcA contributes significantly to the virulence of this fungal pathogen; Krappmann S et al.; We have cloned and characterized the Aspergillus fumigatus cpcA gene encoding the transcriptional activator of the cross-pathway control system of amino acid biosynthesis . cpcA encodes a functional orthologue of Saccharomyces cerevisiae Gcn4p . The coding sequence of the 2.2 kb transcript is preceded by two short upstream open reading frames, the larger one being well conserved among Aspergilli . Deletion strains in which either the coding sequence or the entire locus are replaced by a bifunctional dominant marker are impaired in their cross-pathway control response upon amino acid starvation, as demonstrated by analyses of selected reporter genes and specific enzymatic activities . In a murine model of pulmonary aspergillosis, cpcAdelta strains display attenuated virulence . Pathogenicity is restored to wild-type levels in strains with reconstitution of the genomic locus . Competitive mixed infection experiments additionally demonstrate that cpcAdelta strains are less able to survive in vivo than their wild-type progenitor . Our data suggest that specific stress conditions are encountered by A . fumigatus within the mammalian host and that the fungal cross-pathway control system plays a significant role in pulmonary aspergillosis. RNA, 2004 May, 10(5), 828 - 40 Comparative analysis detects dependencies among the 5' splice-site positions; Carmel I et al.; Human-mouse comparative genomics is an informative tool to assess sequence functionality as inferred from its conservation level . We used this approach to examine dependency among different positions of the 5' splice site . We compiled a data set of 50,493 homologous human-mouse internal exons and analyzed the frequency of changes among different positions of homologous human-mouse 5' splice-site pairs . We found mutual relationships between positions +4 and +5, +5 and +6, -2 and +5, and -1 and +5 . We also demonstrated the association between the exonic and the intronic positions of the 5' splice site, in which a stronger interaction of U1 snRNA and the intronic portion of the 5' splice site compensates for weak interaction of U1 snRNA and the exonic portion of the 5' splice site, and vice versa . By using an ex vivo system that mimics the effect of mutation in the 5' splice site leading to familial dysautonomia, we demonstrated that U1 snRNA base-pairing with positions +6 and -1 is the only functional requirement for mRNA splicing of this 5' splice site . Our findings indicate the importance of U1 snRNA base-pairing to the exonic portion of the 5' splice site. J Biol Chem, 2004 Jul 2, 279(27), 28585 - 91 Epub 2004 Apr 20. The human Rap1 protein complex and modulation of telomere length; O'Connor MS et al.; Proper maintenance of telomere length and structure is necessary for normal proliferation of mammalian cells . Mammalian telomere length is regulated by a number of proteins including human repressor activator protein (hRap1), a known association factor of TRF2 . To further delineate hRap1 function and its associated proteins, we affinity-purified and identified the hRap1 protein complex through mass spectrometry analysis . In addition to TRF2, we found DNA repair proteins Rad50, Mre11, PARP1 (poly(ADP-ribose) polymerase), and Ku86/Ku70 to be in this telomeric complex . We demonstrated by deletional analysis that Rad-50/Mre-11 and Ku86 were recruited to hRap1 independent of TRF2 . PARP1, however, most likely interacted with hRap1 through TRF2 . Interestingly, knockdown of endogenous hRap1 expression by small hairpin interference RNA resulted in longer telomeres . In addition, overexpression of full-length and mutant hRap1 that lacked the BRCA1 C-terminal domain functioned as dominant negatives and extended telomeres . Deletion of a novel linker domain of hRap1 (residues 199-223), however, abolished the dominant negative effect of hRap1 overexpression . These results indicate that hRap1 negatively regulates telomere length in vivo and suggest that the linker region of hRap1 may modulate the recruitment of negative regulators of telomere length. J Cell Biol, 2004 Apr 26, 165(2), 181 - 90 Epub 2004 Apr 19. The role of Cdc6 in ensuring complete genome licensing and S phase checkpoint activation; Oehlmann M et al.; Before S phase, cells license replication origins for initiation by loading them with Mcm2-7 heterohexamers . This process is dependent on Cdc6, which is recruited to unlicensed origins . Using Xenopus egg extracts we show that although each origin can load many Mcm2-7 hexamers, the affinity of Cdc6 for each origins drops once it has been licensed by loading the first hexamers . This encourages the distribution of at least one Mcm2-7 hexamer to each origin, and thereby helps to ensure that all origins are licensed . Although Cdc6 is not essential for DNA replication once licensing is complete, Cdc6 regains a high affinity for origins once replication forks are initiated and Mcm2-7 has been displaced from the origin DNA . We show that the presence of Cdc6 during S phase is essential for the checkpoint kinase Chk1 to become activated in response to replication inhibition . These results show that Cdc6 plays multiple roles in ensuring precise chromosome duplication. J Cell Biol, 2004 Apr 26, 165(2), 167 - 73 Epub 2004 Apr 19. Alternative topogenesis of Mgm1 and mitochondrial morphology depend on ATP and a functional import motor; Herlan M et al.; Mitochondrial morphology and inheritance of mitochondrial DNA in yeast depend on the dynamin-like GTPase Mgm1 . It is present in two isoforms in the intermembrane space of mitochondria both of which are required for Mgm1 function . Limited proteolysis of the large isoform by the mitochondrial rhomboid protease Pcp1/Rbd1 generates the short isoform of Mgm1 but how this is regulated is unclear . We show that near its NH2 terminus Mgm1 contains two conserved hydrophobic segments of which the more COOH-terminal one is cleaved by Pcp1 . Changing the hydrophobicity of the NH2-terminal segment modulated the ratio of the isoforms and led to fragmentation of mitochondria . Formation of the short isoform of Mgm1 and mitochondrial morphology further depend on a functional protein import motor and on the ATP level in the matrix . Our data show that a novel pathway, to which we refer as alternative topogenesis, represents a key regulatory mechanism ensuring the balanced formation of both Mgm1 isoforms . Through this process the mitochondrial ATP level might control mitochondrial morphology. J Biol Chem, 2004 Jun 25, 279(26), 27567 - 74 Epub 2004 Apr 19. Identification and characterization of AGS4: a protein containing three G-protein regulatory motifs that regulate the activation state of Gialpha; Cao X et al.; Activators of G-protein signaling 1-3 (AGS1-3) were identified in a functional screen of mammalian cDNAs that activated G-protein signaling in the absence of a receptor . We report the isolation and characterization of an additional AGS protein (AGS4) from a human prostate leiomyosarcoma cDNA library . AGS4 is identical to G18.1b, which is encoded by a gene within the major histocompatibility class III region of chromosome 6 . The activity of AGS4 in the yeast-based functional screen was selective for G(i2)/G(i3) and independent of guanine-nucleotide exchange by G(i)alpha . RNA blots indicated enrichment of AGS4/G18.1b mRNA in heart, placenta, lung, and liver . Immunocytochemistry with AGS4/G18.1b-specific antisera indicated a predominant nonhomogeneous, extranuclear distribution within the cell following expression in COS7 or Chinese hamster ovary cells . AGS4/G18.1b contains three G-protein regulatory motifs downstream of an amino terminus domain with multiple prolines . Glutathione S-transferase (GST)-AGS4/G18.1b fusion proteins interacted with purified G(i)alpha, and peptides derived from each of the G-protein regulatory motifs inhibited guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) binding to purified G(i)alpha(1) . AGS4/G18.1b was also complexed with G(i)alpha(3) in COS7 cell lysates following cell transfection . However, AGS4/G18.1b did not alter the generation of inositol phosphates in COS7 cells cotransfected with the Gbetagamma-regulated effector phospholipase C-beta2 . These data suggest either that an additional signal is required to position AGS4/G18.1b in the proper cellular location where it can access heterotrimer and promote subunit dissociation or that AGS4 serves as an alternative binding partner for G(i)alpha independent of Gbetagamma participating in G-protein signaling events that are independent of classical G-protein-coupled receptors at the cell surface. Eur J Biochem, 2004 May, 271(9), 1615 - 22 Searching for folded proteins in vitro and in silico; Watters AL et al.; Understanding the sequence determinants of protein structure, stability and folding is critical for understanding how natural proteins have evolved and how proteins can be engineered to perform novel functions . The complexity of the protein folding problem requires the ability to search large volumes of sequence space for proteins with specific structural or functional characteristics . Here we describe our efforts to identify novel proteins using a phage-display selection strategy from a 'mini-exon' shuffling library generated from the yeast genome and from completely random sequence libraries, and compare the results to recent successes in generating novel proteins using in silico protein design. Biochemistry, 2004 Apr 27, 43(16), 4844 - 54 Identification of a novel 29-linked polyubiquitin binding protein, Ufd3, using polyubiquitin chain analogues; Russell NS et al.; Lysine 48-linked polyubiquitin chains are the best understood form of polyubiquitin and are necessary for the function of the ubiquitin-proteasome system . However, other forms of polyubiquitin (e.g., K29- and K63-linked chains) are also present in vivo . Less is known about the functional roles of these linkages or the proteins specifically interacting with these forms of polyubiquitin . Use of native polyubiquitin chains to identify binding proteins is complicated by the difficulties of synthesis and stability . Here, we report the synthesis of a nonhydrolyzable analogue of 29-linked polyubiquitin chains on an affinity support and its use in identifying proteins that bind 29-linked polyubiquitin chains . The 29-linked Ub4 resin was stable and tightly bound recombinant human Isopeptidase T (USP5), a deubiquitinating enzyme known to bind the 29-linked polyubiquitin chains . Two high affinity interactors of the 29-linked polyubiquitin analogues were identified from Saccharomyces cerevisiae lysates . They were identified as Ubp14, the yeast ortholog of Isopeptidase T, and Ufd3, a member of the ubiquitin-fusion degradation pathway with unknown function . Purified recombinant Ufd3 bound to the resin as well, confirming that Ufd3 is a novel binding partner of polyubiquitin . These results demonstrate the efficacy of using polyubiquitin analogue affinity supports to identify novel binding partners of specifically linked polyubiquitin chains . Identification of these proteins will lead to a greater understanding of the physiological relevance of different polyubiquitin linkages. Biochemistry, 2004 Apr 27, 43(16), 4781 - 90 Purified human SUV3p exhibits multiple-substrate unwinding activity upon conformational change; Shu Z et al.; Suv3 of Saccharomyces cerevisiae has been classified as a mitochondrial RNA helicase . However, the helicase domain in both yeast and human SUV3 varies considerably from the typical RNA helicase motifs . To investigate its enzymatic activities, a homogeneously purified preparation of SUV3 is required . Expression of a processed form of human SUV3 carrying an N-terminal deletion of 46 amino acids (SUV3DeltaN46) in a yeast suv3 null mutant, which otherwise fails to grow in a nonfermentable carbon source and forms petite colonies in glucose medium, rescues the null phenotype . Through a five-step chromatographic procedure, an 83 kDa SUV3DeltaN46 protein (SUV3-83) and a partially degraded 70 kDa product (SUV3-70) containing amino acids 68-685 were purified to homogeneity . Single- or double-stranded DNA and RNA stimulated ATPase activity of both proteins . SUV3-70, which retains core catalytic domains, can bind and unwind multiple duplex substrates of RNA and DNA with a 5'-3' directionality over a wide range of pH, while SUV3-83 has helicase activity at only acidic pH . ATP, but not nonhydrolyzable ATP, is essential for the unwinding activity, suggesting the requirement of the energy derived from ATP hydrolysis . Consistent with this notion, suv3 mutants containing alanine (A) or arginine (R) substitutions at the conserved lysine residue in the ATP binding site (K213) lost ATPase activity and also failed to unwind the substrates . Importantly, circular dichroism (CD) spectral analysis showed that SUV3-83, at pH 5.0, adopts a conformation similar to that of SUV3-70, suggesting a conformational change in SUV3-83 is required for its helicase activity . The physiological relevance of the multiple-substrate helicase activity of human SUV3 is discussed. Biochemistry, 2004 Apr 27, 43(16), 4731 - 40 Lys691 and Asp714 of the Na+/K+-ATPase alpha subunit are essential for phosphorylation, dephosphorylation, and enzyme turnover; Su P et al.; P-type ATPases such as the sodium pump appear to be members of a superfamily of hydrolases structurally typified by the L-2-haloacid dehalogenases . In the dehalogenase L-DEX-ps, Lys151 serves to stabilize the excess negative charge in the substrate/reaction intermediates and Asp180 coordinates a water molecule that is directly involved in ester intermediate hydrolysis . To investigate the importance of the corresponding Lys691 and Asp714 of the sodium pump alpha subunit, sodium pump mutants were expressed in yeast and analyzed for their properties . Lys691Ala, Lys691Asp, Asp714Ala, and Asp714Arg mutants were inactive, not only with respect to ATPase activity but also to interaction with the highly sodium pump-specific inhibitors ouabain or palytoxin (PTX) . In contrast, conservative mutants Lys691Arg and Asp714Glu retained some of the partial activities of the wild-type enzyme, although they completely failed to display any ATPase activity . Yeast cells expressing Lys691Arg and Asp714Glu mutants are sensitive to the sodium pump-specific inhibitor PTX and lose intracellular K+ . Their sensitivity to PTX, with EC50 values of 118 +/- 24 and 76.5 +/- 3.6 nM, respectively, was clearly reduced by almost 7- or 4-fold below that of the native sodium pump (17.8 +/- 2.7 nM) . Ouabain was recognized under these conditions with low affinity by the mutants and inhibited the PTX-induced K+ efflux from the yeast cells . The EC50 for the ouabain effect was 183 +/- 20 microM for Lys691Arg and 2.3 +/- 0.08 mM for the Asp714Glu mutant . The corresponding value obtained with cells expressing the native sodium pump was 69 +/- 18 microM . In the presence of Pi and Mg2+, none of the mutant sodium pumps were able to bind ouabain . When Mg2+ was omitted, however, both Lys691Asp and Asp714Glu mutants displayed ouabain binding that was reduced by Mg2+ with an EC50 of 0.76 +/- 0.11 and 2.3 +/- 0.2 mM, respectively . In the absence of Mg2+, ouabain binding was also reduced by K+ . The EC50 values were 1.33 +/- 0.23 mM for the wild-type enzyme, 0.93 +/- 0.2 mM for the Lys691Arg mutant, and 1.02 +/- 0.24 mM for the Asp714Glu enzyme . None of the neutral or nonconservative mutants displayed any ouabain-sensitive ATPase activity . Ouabain-sensitive phosphatase activity, however, was present in membranes containing either the wild-type (1105 +/- 100 micromol of p-nitrophenol phosphate hydrolyzed min(-1) mg of protein(-1)) or the Asp714Glu mutant (575 +/- 75 micromol min(-1) mg(-1)) sodium pump . Some phosphatase activity was also associated with the Lys691Arg mutant (195 +/- 63 micromol min(-1) mg(-1)) . The results are consistent with Lys691 and Asp714 being essential for the phosphorylation/dephosphorylation process that allows the sodium pump to accomplish the catalytic cycle. Biochem Biophys Res Commun, 2004 May 14, 317(4), 1030 - 6 The nucleocapsid protein of the SARS coronavirus is capable of self-association through a C-terminal 209 amino acid interaction domain; Surjit M et al.; Severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) caused a severe outbreak in several regions of the world in 2003 . The virus is a novel coronavirus isolated from patients exhibiting atypical pneumonia and may have originated from wild animals such as civet cats in southern China . The genome of SARS-CoV is a positive-sense, single-stranded RNA whose sequence is distantly related to all known coronaviruses that infect humans and animals . Like other known coronaviruses, SARS-CoV is an enveloped virus containing three outer structural proteins, namely the membrane (M), envelope (E), and spike (S) proteins . The nucleocapsid (N) protein together with the viral RNA genome presumably form a helical core located within the viral envelope . The SARS-CoV nucleocapsid (N) protein is a 423 amino-acid, predicted phospho-protein of 46 kDa that shares little homology with other members of the coronavirus family . A short serine-rich stretch, and a putative bipartite nuclear localization signal are unique to it, thus suggesting its involvement in many important functions during the viral life cycle . In this report we have cloned the N gene of the SARS coronavirus, and studied its property of self-association to form dimers . We expressed the N protein as a fusion protein in the yeast two-hybrid system to demonstrate self-association and confirmed dimerization of the N protein from mammalian cell lysates by coimmunoprecipitation . Furthermore, via deletion analysis, we have shown that the C-terminal 209 amino-acid region constitutes the interaction domain responsible for self-association of the N protein to form dimers. FEBS Lett, 2004 Apr 23, 564(1-2), 41 - 6 The composition of the RNA polymerase I transcription machinery switches from initiation to elongation mode; Bier M et al.; The amounts of RNA polymerase I (Pol I) and basal rDNA transcription factors were determined in yeast whole cell extracts . A 17-fold excess of Pol I was found compared to the Pol I-specific initiation factors upstream activating factor (UAF) and core factor (CF) which underlines that both initiation factors interact with a minor fraction of Pol I when rDNA transcription is active . Surprisingly, Rrn3p, another Pol I-specific initiation factor, is more abundant in cell lysates than UAF and CF . Our analyses revealed that a large fraction of cellular Rrn3p is not associated with Pol I . However, the amount of initiation-active Rrn3p which forms a stable complex with Pol I corresponds to the levels of UAF and CF which have been shown to bind the promoter . Initiation-active Rrn3p dissociates from the template during or immediately after Pol I has switched from initiation to elongation . Our data support a model in which the elongating Pol I leaves the initiation factors UAF, CF and Rrn3p close by the promoter. FEMS Yeast Res, 2004 May, 4(7), 721 - 9 Der1p, a protein required for degradation of malfolded soluble proteins of the endoplasmic reticulum: topology and Der1-like proteins; Hitt R et al.; The endoplasmic reticulum (ER) contains a highly effective protein quality control system eliminating malfolded proteins by a mechanism called ER-associated protein degradation (ERAD) . Here, we unravel the topology of Der1p, a previously identified component of the ERAD system . Der1p contains four transmembrane domains, its N- and C-terminus protrude into the cytoplasm and contribute to its function . Additionally, we describe a yeast homologue of Der1p, Dfm1p, which does not seem to be involved in ERAD . In contrast, a Caenorhabditis elegans orthologue of Der1p, R151.6, is capable of complementing der1-defective phenotypes in yeast. FEMS Yeast Res, 2004 May, 4(7), 673 - 82 Hansenula polymorpha Swi1p and Snf2p are essential for methanol utilisation; Ozimek P et al.; We have cloned the Hansenula polymorpha SWI1 and SNF2 genes by functional complementation of mutants that are defective in methanol utilisation . These genes encode proteins similar to Saccharomyces cerevisiae Swi1p and Snf2p, which are subunits of the SWI/SNF complex . This complex belongs to the family of nucleosome-remodeling complexes that play a role in transcriptional control of gene expression . Analysis of the phenotypes of constructed H . polymorpha SWI1 and SNF2 disruption strains indicated that these genes are not necessary for growth of cells on glucose, sucrose, or various organic nitrogen sources which involve the activity of peroxisomal oxidases . Both disruption strains showed a moderate growth defect on glycerol and ethanol, but were fully blocked in methanol utilisation . In methanol-induced cells of both disruption strains, two peroxisomal enzymes involved in methanol metabolism, alcohol oxidase and dihydroxyacetone synthase, were hardly detectable, whereas in wild-type cells these proteins were present at very high levels . We show that the reduction in alcohol oxidase protein levels in H . polymorpha SWI1 and SNF2 disruption strains is due to strongly reduced expression of the alcohol oxidase gene . The level of Pex5p, the receptor involved in import of alcohol oxidase and dihydroxyacetone synthase into peroxisomes, was also reduced in both disruption strains compared to that in wild-type cells. Mol Biol Cell, 2004 Jul, 15(7), 3366 - 78 Epub 2004 Apr 16. Sgt1p and Skp1p modulate the assembly and turnover of CBF3 complexes required for proper kinetochore function; Rodrigo-Brenni MC et al.; Kinetochores are composed of a large number of protein complexes that must be properly assembled on DNA to attach chromosomes to the mitotic spindle and to coordinate their segregation with the advance of the cell cycle . CBF3 is an inner kinetochore complex in the budding yeast Saccharomyces cerevisiae that nucleates the recruitment of all other kinetochore proteins to centromeric DNA . Skp1p and Sgt1p act through the core CBF3 subunit, Ctf13p, and are required for CBF3 to associate with centromeric DNA . To investigate the contribution of Skp1p and Sgt1p to CBF3 function, we have used a combination of in vitro binding assays and a unique protocol for synchronizing the assembly of kinetochores in cells . We have found that the interaction between Skp1p and Sgt1p is critical for the assembly of CBF3 complexes . CBF3 assembly is not restricted during the cell cycle and occurs in discrete steps; Skp1p and Sgt1p contribute to a final, rate-limiting step in assembly, the binding of the core CBF3 subunit Ctf13p to Ndc10p . The assembly of CBF3 is opposed by its turnover and disruption of this balance compromises kinetochore function without affecting kinetochore formation on centromeric DNA. BMC Bioinformatics . 2004 Apr 16;5(1):38. Predicting co-complexed protein pairs using genomic and proteomic data integration; Zhang LV et al.; BACKGROUND: Identifying all protein-protein interactions in an organism is a major objective of proteomics . A related goal is to know which protein pairs are present in the same protein complex . High-throughput methods such as yeast two-hybrid (Y2H) and affinity purification coupled with mass spectrometry (APMS) have been used to detect interacting proteins on a genomic scale . However, both Y2H and APMS methods have substantial false-positive rates . Aside from high-throughput interaction screens, other gene- or protein-pair characteristics may also be informative of physical interaction . Therefore it is desirable to integrate multiple datasets and utilize their different predictive value for more accurate prediction of co-complexed relationship . RESULTS: Using a supervised machine learning approach--probabilistic decision tree, we integrated high-throughput protein interaction datasets and other gene- and protein-pair characteristics to predict co-complexed pairs (CCP) of proteins . Our predictions proved more sensitive and specific than predictions based on Y2H or APMS methods alone or in combination . Among the top predictions not annotated as CCPs in our reference set (obtained from the MIPS complex catalogue), a significant fraction was found to physically interact according to a separate database (YPD, Yeast Proteome Database), and the remaining predictions may potentially represent unknown CCPs . CONCLUSIONS: We demonstrated that the probabilistic decision tree approach can be successfully used to predict co-complexed protein (CCP) pairs from other characteristics . Our top-scoring CCP predictions provide testable hypotheses for experimental validation. Proc Natl Acad Sci U S A, 2004 Apr 27, 101(17), 6564 - 9 Epub 2004 Apr 15. Cells have distinct mechanisms to maintain protection against different reactive oxygen species: oxidative-stress-response genes; Thorpe GW et al.; The complete set of viable deletion strains in Saccharomyces cerevisiae was screened for sensitivity of mutants to five oxidants to identify cell functions involved in resistance to oxidative stress . This screen identified a unique set of mainly constitutive functions providing the first line of defense against a particular oxidant; these functions are very dependent on the nature of the oxidant . Most of these functions are distinct from those involved in repair and recovery from damage, which are generally induced in response to stress, because there was little correlation between mutant sensitivity and the reported transcriptional response to oxidants of the relevant gene . The screen identified 456 mutants sensitive to at least one of five different types of oxidant, and these were ranked in order of sensitivity . Many genes identified were not previously known to have a role in resistance to reactive oxygen species . These encode functions including protein sorting, ergosterol metabolism, autophagy, and vacuolar acidification . Only two mutants were sensitive to all oxidants examined, only 12 were sensitive to at least four, and different oxidants had very different spectra of deletants that were sensitive . These findings highlight the specificity of cellular responses to different oxidants: No single oxidant is representative of general oxidative stress . Mitochondrial respiratory functions were overrepresented in mutants sensitive to H(2)O(2), and vacuolar protein-sorting mutants were enriched in mutants sensitive to diamide . Core functions required for a broad range of oxidative-stress resistance include transcription, protein trafficking, and vacuolar function. J Biol Chem, 2004 Jul 2, 279(27), 28298 - 303 Epub 2004 Apr 14. Ugo1p links the Fzo1p and Mgm1p GTPases for mitochondrial fusion; Sesaki H et al.; In yeast, mitochondrial fusion requires Ugo1p and two GTPases, Fzo1p and Mgm1p . Ugo1p is anchored in the mitochondrial outer membrane with its N terminus facing the cytosol and C terminus in the intermembrane space . Fzo1p is also an outer membrane protein, whereas Mgm1p is located in the intermembrane space . Recent studies suggest that these three proteins form protein complexes that mediate mitochondrial fusion . Here, we show that the cytoplasmic domain of Ugo1p directly interacts with Fzo1p, whereas its intermembrane space domain binds to Mgm1p . We identified the Ugo1p-binding site in Fzo1p and demonstrated that Ugo1p-Fzo1p interaction is essential for the formation of mitochondrial shape, maintenance of mitochondrial DNA, and fusion of mitochondria . Although the GTPase domains of Fzo1p and Mgm1p regulate mitochondrial fusion, they were not required for association with Ugo1p . Furthermore, we found that Ugo1p bridges the interaction between Fzo1p and Mgm1p in mitochondria . Our data indicate that distinct regions of Ugo1p bind directly to Fzo1p and Mgm1p and thereby link these two GTPases during mitochondrial fusion. Traffic, 2004 May, 5(5), 383 - 92 The ubiquitin ligase Rsp5p is required for modification and sorting of membrane proteins into multivesicular bodies; Morvan J et al.; Precursor forms of vacuolar proteins with transmembrane domains, such as the carboxypeptidase S Cps1p and the polyphosphatase Phm5p, are selectively sorted in endosomal compartments to vesicles that invaginate, budding into the lumen of the late endosomes, resulting in the formation of multivesicular bodies (MVBs) . These proteins are then delivered to the vacuolar lumen following fusion of the MVBs with the vacuole . The sorting of Cps1p and Phm5p to these structures is mediated by ubiquitylation, and in doa4 mutant cells, which have reduced level of free ubiquitin, these proteins are missorted to the vacuolar membrane . A RING-finger ubiquitin ligase Tul1p has been shown to participate in the ubiquitylation of Cps1p and Phm5p . We show here that the HECT-ubiquitin ligase Rsp5p is also required for the ubiquitylation of these proteins, and therefore for their sorting to MVBs . Rsp5p is an essential ubiquitin ligase containing an N-terminal C2 domain followed by three WW domains, and a C-terminal catalytic HECT domain . In cells with low levels of Rsp5p (npi1 mutant cells), vacuolar hydrolases do not reach the vacuolar lumen and are instead missorted to the vacuolar membrane . The C2 domain and both the second and third WW domains of Rsp5p are important determinants for sorting to MVBs . Ubiquitylation of Cps1p was strongly reduced in the npi1 mutant strain and ubiquitylation was completely abolished in the npi1 tul1Delta double mutant . These data demonstrate that Rsp5p plays a novel and key role in intracellular trafficking, and extend the currently very short list of substrates ubiquitylated in vivo by several different ubiquitin ligases acting cooperatively. DNA Repair (Amst), 2004 May 4, 3(5), 483 - 94 Effect of 8-oxoguanine on transcription elongation by T7 RNA polymerase and mammalian RNA polymerase II; Tornaletti S et al.; 8-Oxoguanine (8-oxoG) is a major oxidative lesion produced in DNA by normal cellular metabolism or after exposure to exogenous sources such as ionizing radiation . Persistence of this lesion in DNA causes G to T transversions, with deleterious consequences for the cell . As a result, several repair processes have evolved to remove this lesion from the genome . It has been reported that 8-oxoG is subject to transcription-coupled repair (TCR), a process dedicated to removal of lesions from transcribed strands of expressed genes . A current model assumes that RNA polymerase arrest at the site of the lesion is required for initiation of TCR . As a first step to understand how TCR of 8-oxoG occurs, we have studied the effect of 8-oxoG on transcription elongation by T7 RNA polymerase (T7 RNAP) and rat liver RNA polymerase II (RNAPII) . We have utilized an in vitro transcription system with purified RNA polymerase and initiation factors, and substrates containing a single 8-oxoG in the transcribed or in the non-transcribed strand downstream of the T7 promoter or the Adenovirus major late promoter . We found that 8-oxoG only slightly inhibited T7 RNAP transcription, with a readthrough frequency of up to 95% . Similarly, this lesion only transiently blocked transcription by RNAPII . However, changes in nucleotide concentration affected the extent of RNAPII blockage at the 8-oxoG . When this lesion was positioned in the non-transcribed strand, complete lesion bypass was observed with either polymerase . Binding of the Saccharomyces cerevisiae MSH2-MSH6 complex to 8-oxoG containing substrates did not increase the frequency of RNAPII arrest at the site of the lesion, suggesting that this complex was displaced by the elongating polymerase . These results are discussed in the context of possible models for TCR. Cell, 2004 Apr 16, 117(2), 185 - 98 Predicting gene expression from sequence; Beer MA et al.; We describe a systematic genome-wide approach for learning the complex combinatorial code underlying gene expression . Our probabilistic approach identifies local DNA-sequence elements and the positional and combinatorial constraints that determine their context-dependent role in transcriptional regulation . The inferred regulatory rules correctly predict expression patterns for 73% of genes in Saccharomyces cerevisiae, utilizing microarray expression data and sequences in the 800 bp upstream of genes . Application to Caenorhabditis elegans identifies predictive regulatory elements and combinatorial rules that control the phased temporal expression of transcription factors, histones, and germline specific genes . Successful prediction requires diverse and complex rules utilizing AND, OR, and NOT logic, with significant constraints on motif strength, orientation, and relative position . This system generates a large number of mechanistic hypotheses for focused experimental validation, and establishes a predictive dynamical framework for understanding cellular behavior from genomic sequence. Behav Genet, 2004 Jul, 34(4), 395 - 406 The cAMP transduction cascade mediates olfactory reception in Drosophila melanogaster; Gomez-Diaz C et al.; Two main second messenger systems depending on IP3 and cAMP have been related to olfaction in vertebrates as well as invertebrates . In Drosophila melanogaster, the availability of mutations affecting one or the other pathway (rdgB and norpA or rut and dnc, respectively) allowed showing of abnormal olfactory behavior phenotypes associated with olfactory transduction in complete living animals . However, because rut and dnc genes showed ubiquitous expression at olfactory receptor organs and some brain locations, the mutant behavior cannot be assigned exclusively to olfactory reception . In this report, overexpression of the dnc gene directed specifically to different olfactory receptor neuron subsets was used to produce dominant mutants . Abnormal olfactory behavior was found in 62.5% of the 8 lines studied in response to some odorants, depending on the affected neuronal subset . These results suggest that even for a small number of tested odorants (5), cAMP cascade is involved in olfactory reception to an important extent . Mol Cell Biol, 2004 May, 24(9), 3983 - 91 Functional interaction between TFIIB and the Rpb2 subunit of RNA polymerase II: implications for the mechanism of transcription initiation; Chen BS et al.; The general transcription factor TFIIB is required for accurate initiation, although the mechanism by which RNA polymerase II (RNAP II) identifies initiation sites is not well understood . Here we describe results from genetic and biochemical analyses of an altered form of yeast TFIIB containing an arginine-78 --> cysteine (R78C) replacement in the "B-finger" domain . TFIIB R78C shifts start site selection downstream of normal and confers a cold-sensitive growth defect (Csm(-)) . Suppression of the R78C Csm(-) phenotype identified a functional interaction between TFIIB and the Rpb2 subunit of RNAP II and defined a novel role for Rpb2 in start site selection . The rpb2 suppressor encodes a glycine-369 --> serine (G369S) replacement, located in the "lobe" domain of Rpb2 and near the Rpb9 subunit, which was identified previously as an effector of start site selection . The Rpb2-Rpb9 "lobe-jaw" region of RNAP II is downstream of the catalytic center and distal to the site of RNAP II-TFIIB interaction . A TFIIB R78C mutant extract was defective for promoter-specific run-on transcription but yielded an altered pattern of abortive initiation products, indicating that the R78C defect does not preclude initiation . The sua7-3 rpb2-101 double mutant was sensitive to 6-azauracil in vivo and to nucleoside triphosphate substrate depletion in vitro . In the context of the recent X-ray structure of the yeast RNAP II-TFIIB complex, these results define a functional interaction between the B-finger domain of TFIIB and the distal lobe-jaw region of RNAP II and provide insight into the mechanism of start site selection. Mol Cell Biol, 2004 May, 24(9), 3815 - 26 The GTPase-activating enzyme Gyp1p is required for recycling of internalized membrane material by inactivation of the Rab/Ypt GTPase Ypt1p; Lafourcade C et al.; Rab/Ypt GTPases are key regulators of membrane trafficking and together with SNARE proteins mediate selective fusion of vesicles with target compartments . A family of GTPase-activating enzymes (GAPs) specific for Rab/Ypt GTPases has been discovered, but little is known about their function and substrate specificity in vivo . Here we show that the GAP activity of Gyp1p, a yeast member of this family, is specifically required for recycling of the SNARE Snc1p and the membrane dye FM4-64, implying that inactivation of a Rab/Ypt GTPase may be necessary for recycling of membrane material . Interestingly, recycling of GFP-Snc1p in gyp1 Delta cells is partially restored by reducing the activity of Ypt1p . Moreover, GFP-Snc1p accumulated intracellularly in wild-type cells expressing a GTP-locked, mutant form of Ypt1p (Ypt1p-Q67L), suggesting that GTP hydrolysis of Ypt1p is essential for recycling . Ypt6p is known to be required for the fusion of recycling vesicles to the late Golgi compartment . Interestingly, the deletions of GYP1 and YPT6 were synthetic lethal, raising the possibility that at least two distinct pathways are involved in recycling of membrane material. Mol Cell Biol, 2004 May, 24(9), 3670 - 81 Posttranscriptional regulation of HO expression by the Mkt1-Pbp1 complex; Tadauchi T et al.; Cells of budding yeast give rise to mother and daughter cells, which differ in that only mother cells express the HO endonuclease gene and are thereby able to switch mating types . In this study, we identified the MKT1 gene as a positive regulator of HO expression . The MKT1 gene encodes a protein with two domains, XPG-N and XPG-I, which are conserved among a family of nucleases, including human XPG endonuclease . Loss of MKT1 had little effect on HO mRNA levels but resulted in decreased protein levels . This decrease was dependent on the 3' untranslated region of the HO transcript . We screened for proteins that associate with Mkt1 and isolated Pbp1, a protein that is known to associate with Pab1, a poly(A)-binding protein . Loss of PBP1 resembles an mkt1 Delta deletion, causing decreased expression of HO at the posttranscriptional level . Mkt1 and Pbp1 cosedimented with polysomes in sucrose gradients, with Mkt1 distribution in the polysomes dependent on Pbp1, but not vice versa . These observations suggest that a complex of Mkt1 and Pbp1 regulates the translation of HO mRNA. Mol Cell Biol, 2004 May, 24(9), 3577 - 87 The Apc5 subunit of the anaphase-promoting complex/cyclosome interacts with poly(A) binding protein and represses internal ribosome entry site-mediated translation; Koloteva-Levine N et al.; The anaphase-promoting complex/cyclosome (APC/C) is a multisubunit ubiquitin ligase that mediates the proteolysis of cell cycle proteins in mitosis and G(1) . We used a yeast three-hybrid screen to identify proteins that interact with the internal ribosome entry site (IRES) of platelet-derived growth factor 2 mRNA . Surprisingly, this screen identified Apc5, although it does not harbor a classical RNA binding domain . We found that Apc5 binds the poly(A) binding protein (PABP), which directly binds the IRES element . PABP was found to enhance IRES-mediated translation, whereas Apc5 overexpression counteracted this effect . In addition to its association with the APC/C complex, Apc5 binds much heavier complexes and cosediments with the ribosomal fraction . In contrast to Apc3, which is associated only with the APC/C and remains intact during differentiation, Apc5 is degraded upon megakaryocytic differentiation in correlation with IRES activation . Expression of Apc5 in differentiated cells abolished IRES activation . This is the first report implying an additional role for an APC/C subunit, apart from its being part of the APC/C complex. Genetics, 2004 Mar, 166(3), 1141 - 54 Neurological proteins are not enriched for repetitive sequences; Huntley MA et al.; Proteins associated with disease and development of the nervous system are thought to contain repetitive, simple sequences . However, genome-wide surveys for simple sequences within proteins have revealed that repetitive peptide sequences are the most frequent shared peptide segments among eukaryotic proteins, including those of Saccharomyces cerevisiae, which has few to no specialized developmental and neurological proteins . It is therefore of interest to determine if these specialized proteins have an excess of simple sequences when compared to other sets of compositionally similar proteins . We have determined the relative abundance of simple sequences within neurological proteins and find no excess of repetitive simple sequence within this class . In fact, polyglutamine repeats that are associated with many neurodegenerative diseases are no more abundant within neurological specialized proteins than within nonneurological collections of proteins . We also examined the codon composition of serine homopolymers to determine what forces may play a role in the evolution of extended homopolymers . Codon type homogeneity tends to be favored, suggesting replicative slippage instead of selection as the main force responsible for producing these homopolymers. Genes Dev, 2004 Apr 1, 18(7), 816 - 29 Wnt-1 signal induces phosphorylation and degradation of c-Myb protein via TAK1, HIPK2, and NLK; Kanei-Ishii C et al.; The c-myb proto-oncogene product (c-Myb) regulates both the proliferation and apoptosis of hematopoietic cells by inducing the transcription of a group of target genes . However, the biologically relevant molecular mechanisms that regulate c-Myb activity remain unclear . Here we report that c-Myb protein is phosphorylated and degraded by Wnt-1 signal via the pathway involving TAK1 (TGF-beta-activated kinase), HIPK2 (homeodomain-interacting protein kinase 2), and NLK (Nemo-like kinase) . Wnt-1 signal causes the nuclear entry of TAK1, which then activates HIPK2 and the mitogen-activated protein (MAP) kinase-like kinase NLK . NLK binds directly to c-Myb together with HIPK2, which results in the phosphorylation of c-Myb at multiple sites, followed by its ubiquitination and proteasome-dependent degradation . Furthermore, overexpression of NLK in M1 cells abrogates the ability of c-Myb to maintain the undifferentiated state of these cells . The down-regulation of Myb by Wnt-1 signal may play an important role in a variety of developmental steps. Genes Dev, 2004 Apr 1, 18(7), 782 - 93 BRU1, a novel link between responses to DNA damage and epigenetic gene silencing in Arabidopsis; Takeda S et al.; DNA repair associated with DNA replication is important for the conservation of genomic sequence information, whereas reconstitution of chromatin after replication sustains epigenetic information . We have isolated and characterized mutations in the BRU1 gene of Arabidopsis that suggest a novel link between these underlying maintenance mechanisms . Bru1 plants are highly sensitive to genotoxic stress and show stochastic release of transcriptional gene silencing . They also show increased intrachromosomal homologous recombination and constitutively activated expression of poly (ADP-ribose) polymerase-2 (AtPARP-2), the induction of which is associated with elevated DNA damage . Bru1 mutations affect the stability of heterochromatin organization but do not interfere with genome-wide DNA methylation . BRU1 encodes a novel nuclear protein with two predicted protein-protein interaction domains . The developmental abnormalities characteristic of bru1 mutant plants resemble those triggered by mutations in genes encoding subunits of chromatin assembly factor (CAF-1), the condensin complex, or MRE11 . Comparison of bru1 with these mutants indicates cooperative roles in the replication and stabilization of chromatin structure, providing a novel link between chromatin replication, epigenetic inheritance, S-phase DNA damage checkpoints, and the regulation of meristem development. Genes Dev, 2004 Apr 1, 18(7), 769 - 81 The NAD(+)-dependent Sir2p histone deacetylase is a negative regulator of chromosomal DNA replication; Pappas DL Jr et al.; The establishment of DNA synthesis during the S phase is a multistep process that occurs in several stages beginning in late mitosis . The first step is the formation of a large prereplicative complex (pre-RC) at individual replication origins and occurs during exit from mitosis and entry into G1 phase . To better understand the genetic requirements for pre-RC formation, we selected chromosomal suppressors of a temperature-sensitive cdc6-4 mutant defective for pre-RC assembly . Loss-of-function mutations in the chromatin-modifying genes SIR2, and to a lesser extent in SIR3 and SIR4, suppressed the cdc6-4 temperature-sensitive lethality . This suppression was independent of the well-known silencing roles for the SIR proteins at the HM loci, at telomeres, or at the rDNA locus . A deletion of SIR2 uniquely rescued both the DNA synthesis defect of the cdc6-4 mutant and its severe plasmid instability phenotype for many origins . A SIR2 deletion suppressed additional initiation mutants affecting pre-RC assembly but not mutants that act subsequently . These findings suggest that Sir2p negatively regulates the initiation of DNA replication through a novel mechanism and reveal another connection between proteins that initiate DNA synthesis and those that establish silent heterochromatin in budding yeast. Anal Biochem, 2004 May 1, 328(1), 22 - 8 Thiol-tolerant assay for quantitative colorimetric determination of chloride released from whole-cell biodehalogenations; Jorg G et al.; Determination of inorganic chloride released from a chloro-organic compound by whole-cell or enzymatic dehalogenation can be affected by free thiols, phosphate, sugars, pH, the chloro-organic substrate, and its dehalogenation product . For these reasons a highly sensitive, colorimetric chloride assay on the basis of {FeCl}2+ (lambda(max) = 340 nm) formed in highly acidic medium which is insensitive to composition of culture medium, free thiols, substrate, and dehalogenation product was developed . It is applicable to both fungal and bacterial high-cell-density cultures . The {FeCl}2+ method provides reliable data and is convenient for the rapid and facile determination of dehalogenation kinetics. Arch Biochem Biophys, 2004 May 1, 425(1), 87 - 94 Evaluation of phage display system and leech-derived tryptase inhibitor as a tool for understanding the serine proteinase specificities; Campos IT et al.; A small combinatorial library of LDTI mutants (5.2 x 10(4)) restricted to the P1-P4' positions of the reactive site was displayed on the pCANTAB 5E phagemid, and LDTI fusion phages were produced and selected for potent neutrophil elastase and plasmin inhibitors . Strong fusion phage binders were analyzed by ELISA on enzyme-coated microtiter plates and the positive phages had their DNA sequenced . The LDTI variants: 29E (K8A, I9A, L10F, and K11F) and 19E (K8A, K11Q, and P12Y) for elastase and 2Pl (K11W and P12N), 8Pl (I9V, K11W, and P12E), and 10Pl (I9T, K11L, and P12L) for plasmin were produced with a Saccharomyces cerevisiae expression system . New strong elastase and plasmin inhibitors were 29E and 2Pl, respectively . LDTI-29E was a potent and specific neutrophil elastase inhibitor K(i) =0.5 nM), affecting no other tested enzymes . LDTI-2Pl was the strongest plasmin inhibitor ( K(i) =1.7nM) in the LDTI mutant library . This approach allowed selection of new specific serine proteinase inhibitors for neutrophil elastase and plasmin (a thrombin inhibitor variant was previously described), from a unique template molecule, LDTI, a Kazal type one domain inhibitor, by only 2-4 amino acid replacements . Our data validate this small LDTI combinatorial library as a tool to generate specific serine proteinase inhibitors suitable for drug design and enzyme-inhibitor interaction studies. Life Sci, 2004 May 7, 74(25), 3065 - 74 Anti-estrogenic activity of fifty chemicals evaluated by in vitro assays; Jung J et al.; We examined the anti-estrogenic activity of 50 chemicals by the yeast two-hybrid assay and detected the activity of hexachlorophene, pentachlorophenol, and vitamin K3 (menadione), in that order . These chemicals were also observed to inhibit the transcriptional activity of 17beta-estradiol in a reporter gene assay system using MCF-7 cells, estrogen receptor-positive breast cancer cells, and to bind directly to estrogen receptor alpha in a competitive binding assay system, although the order of the activity was slightly different among the 3 assays . These findings suggested that three of fifty chemicals could inhibit estrogen activity by competitive binding with 17beta-estradiol to the estrogen receptor. Proc Natl Acad Sci U S A, 2004 Apr 20, 101(16), 5934 - 9 Epub 2004 Apr 12. Network motifs in integrated cellular networks of transcription-regulation and protein-protein interaction; Yeger-Lotem E et al.; Genes and proteins generate molecular circuitry that enables the cell to process information and respond to stimuli . A major challenge is to identify characteristic patterns in this network of interactions that may shed light on basic cellular mechanisms . Previous studies have analyzed aspects of this network, concentrating on either transcription-regulation or protein-protein interactions . Here we search for composite network motifs: characteristic network patterns consisting of both transcription-regulation and protein-protein interactions that recur significantly more often than in random networks . To this end we developed algorithms for detecting motifs in networks with two or more types of interactions and applied them to an integrated data set of protein-protein interactions and transcription regulation in Saccharomyces cerevisiae . We found a two-protein mixed-feedback loop motif, five types of three-protein motifs exhibiting coregulation and complex formation, and many motifs involving four proteins . Virtually all four-protein motifs consisted of combinations of smaller motifs . This study presents a basic framework for detecting the building blocks of networks with multiple types of interactions. Mol Cancer Ther, 2004 Apr, 3(4), 393 - 402 Homologous recombination is a highly conserved determinant of the synergistic cytotoxicity between cisplatin and DNA topoisomerase I poisons; van Waardenburg RC et al.; Phase I and II clinical trails are currently investigating the antitumor activity of cisplatin and camptothecins (CPTs; DNA topoisomerase I poisons), based on the dramatic synergistic cytotoxicity of these agents in some preclinical models . However, the mechanistic basis for this synergism is poorly understood . By exploiting the evolutionary conservation of DNA repair pathways from genetically tractable organisms such as budding and fission yeasts to mammalian cells, we demonstrate that the synergism of CPT and cisplatin requires homologous recombination . In yeast and mammalian cell lines defective for RAD52 and XRCC2/3, respectively, the combination of these agents proved antagonistic, while greater than additive activity was evident in isogenic wild-type cells . Homologous recombination appears to mediate a similar interaction of X-rays and CPT, but antagonizes the synergism of cytarabine (Ara-C) with CPT . These findings suggest that homologous recombination comprises an evolutionarily conserved determinant of cellular sensitivity when CPTs are used in combination with other therapeutics. J Virol, 2004 May, 78(9), 4684 - 99 Herpes simplex virus virion host shutoff protein is stimulated by translation initiation factors eIF4B and eIF4H; Doepker RC et al.; The virion host shutoff protein (vhs) of herpes simplex virus triggers accelerated degradation of cellular and viral mRNAs while sparing other cytoplasmic RNA species . Previous work has shown that vhs forms a complex with translation initiation factor eIF4H, which displays detectable RNase activity in the absence of other viral or host proteins . However, the contributions of eIF4H and other host factors to the activity and mRNA targeting properties of vhs have not yet been directly examined . An earlier report from our laboratory demonstrated that rabbit reticulocyte lysate (RRL) contains one or more factors that strongly stimulate the RNase activity of vhs produced in Saccharomyces cerevisiae . We report here that such yeast extracts display significant vhs-dependent RNase activity in the absence of mammalian factors . This activity differs from that displayed by vhs generated in RRL in that it is not targeted to the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) . Activity was strongly enhanced by the addition of RRL, eIF4H, or the related translation factor eIF4B . RRL also reconstituted strong targeting to the EMCV IRES, resulting in a major change in the RNA cleavage pattern . In contrast, eIF4H and eIF4B did not reconstitute IRES-directed targeting . These data indicate that eIF4B and 4H stimulate the nuclease activity of vhs, and they provide evidence that additional mammalian factors are required for targeting to the EMCV IRES. J Virol, 2004 May, 78(9), 4617 - 27 Hepatitis C virus quasispecies variability modulates nonstructural protein 5A transcriptional activation, pointing to cellular compartmentalization of virus-host interactions; Pellerin M et al.; Hepatitis C virus (HCV) behaves in infected patients as a complex mixture of genetically distinct but closely related variants referred to as a "quasispecies." By using quasispecies analysis strategies, we showed that HCV nonstructural protein 5A (NS5A) has a quasispecies distribution in infected humans and that NS5A quasispecies undergo significant genetic evolution over time, as a result of random accumulation of nucleotide mutations during replication . Genetic evolution of the NS5A quasispecies results in sporadic amino acid changes in the protein sequence . By using the functional in vitro model of HCV NS5A transcriptional activation in Saccharomyces cerevisiae, we showed that natural NS5A quasispecies variants induce different levels of transcriptional activation, according to the charge of the residues (and possibly minor conformational changes) in the quasispecies variant sequence . These findings show that the accumulation of mutations on HCV genomes during replication randomly generates variant proteins with quantitatively different functional properties . The fact that each new variant protein is initially produced in a single infected hepatocyte and may or may not subsequently spread throughout the liver (depending on the replication capacities of the variant virus) points to cellular compartmentalization of virus-host interactions during chronic infection . This feature of quasispecies-distributed viruses could play an important role in various aspects of the viral life cycle and related disease. J Cell Biol, 2004 Apr, 165(1), 135 - 44 The C2 domain of the Rsp5 ubiquitin ligase binds membrane phosphoinositides and directs ubiquitination of endosomal cargo; Dunn R et al.; Ubiquitin ligases of the Nedd4 family regulate membrane protein trafficking by modifying both cargo proteins and the transport machinery with ubiquitin . Here, we investigate the role of the yeast Nedd4 homologue, Rsp5, in protein sorting into vesicles that bud into the multivesicular endosome (MVE) en route to the vacuole . A mutant lacking the Rsp5 C2 domain is unable to ubiquitinate or sort biosynthetic cargo into MVE vesicles, whereas endocytic cargo is ubiquitinated and sorted efficiently . The C2 domain binds specifically to phosphoinositides in vitro and is sufficient for localization to membranes in intact cells . Mutation of a lysine-rich patch on the surface of the C2 domain abolishes membrane interaction and disrupts sorting of biosynthetic cargo . Translational fusion of ubiquitin to a biosynthetic cargo protein alleviates the requirement for the C2 domain in its MVE sorting . These results demonstrate that the C2 domain specifies Rsp5-dependent ubiquitination of endosomal cargo and suggest that Rsp5 function is regulated by membrane phosphoinositides. J Cell Biol, 2004 Apr, 165(1), 41 - 52 Misfolded proteins are sorted by a sequential checkpoint mechanism of ER quality control; Vashist S et al.; Misfolded proteins retained in the endoplasmic reticulum (ER) are degraded by the ER-associated degradation pathway . The mechanisms used to sort them from correctly folded proteins remain unclear . Analysis of substrates with defined folded and misfolded domains has revealed a system of sequential checkpoints that recognize topologically distinct domains of polypeptides . The first checkpoint examines the cytoplasmic domains of membrane proteins . If a lesion is detected, it is retained statically in the ER and rapidly degraded without regard to the state of its other domains . Proteins passing this test face a second checkpoint that monitors domains localized in the ER lumen . Proteins detected by this pathway are sorted from folded proteins and degraded by a quality control mechanism that requires ER-to-Golgi transport . Although the first checkpoint is obligatorily directed at membrane proteins, the second monitors both soluble and membrane proteins . Our data support a model whereby "properly folded" proteins are defined biologically as survivors that endure a series of distinct checkpoints. Curr Med Chem, 2004 Apr, 11(7), 807 - 26 SIR2: the biochemical mechanism of NAD(+)-dependent protein deacetylation and ADP-ribosyl enzyme intermediates; Sauve AA et al.; The Sir2 family of enzymes is a recently described class of NAD(+)-dependent protein deacetylases that use NAD+ as a reactant to deacetylate acetyllysine residues of protein substrates to form the aminolysine sidechain and a novel product 2'-O-acetyl-ADP-ribose . The founding member of the Sir2 proteins, the yeast Sir2p, has been identified as a key member of SIR complexes responsible for the long-term silencing of genes in the yeast Saccharomyces cerevisiae . Increase of Sir2 activity by caloric restriction or osmotic stress increases genome stability and lifespan in this organism . The Sir2 reaction mechanism couples ADP-ribosyltransfer and hydrolysis reactions via the formation of a stabilized ADPR-peptidyl intermediate . Principles of the chemistry of stabilized ADPR intermediates are examined for Sir2 and the mechanistically related ADP-ribosylcyclase CD38 . An examination of the crystal structures of Sir2 family members is presented with a view to the chemical requirements of the Sir2 reaction . The present review describes the current knowledge of the Sir2 reaction, the reaction mechanism and the regulation of Sir2. Biochemistry, 2004 Apr 20, 43(15), 4592 - 600 The tRNA-interacting factor p43 associates with mammalian arginyl-tRNA synthetase but does not modify its tRNA aminoacylation properties; Guigou L et al.; Arginyl-tRNA synthetase (ArgRS) is one of the nine synthetase components of a multienzyme complex containing three auxiliary proteins as well . We previously established that the N-terminal moiety of the auxiliary protein p43 associates with the N-terminal, eukaryotic-specific polypeptide extension of ArgRS . Because p43 is homologous to Arc1p, a yeast general RNA-binding protein that associates with MetRS and GluRS and plays the role of tRNA-binding cofactor in the aminoacylation reaction, we analyzed the functional significance of p43-ArgRS association . We had previously showed that full-length ArgRS, corresponding to the ArgRS species associated within the multisynthetase complex, and ArgRS with a deletion of 73 N-terminal amino acid residues, corresponding to a free species of ArgRS, both produced in yeast, have similar catalytic parameters (Lazard, M., Kerjan, P., Agou, F., and Mirande, M . (2000) J . Mol . Biol . 302, 991-1004) . However, a recent study had suggested that association of p43 to ArgRS reduces the apparent K(M) of ArgRS to tRNA (Park, S . G., Jung, K . H., Lee, J . S., Jo, Y . J., Motegi, H., Kim, S., and Shiba, K . (1999) J . Biol . Chem . 274, 16673-16676) . In this study, we analyzed in detail, by gel retardation assays and enzyme kinetics, the putative role of p43 as a tRNA-binding cofactor of ArgRS . The association of p43 with ArgRS neither strengthened tRNA-binding nor changed kinetic parameters in the amino acid activation or in the tRNA aminoacylation reaction . Furthermore, selective removal of the C-terminal RNA-binding domain of p43 from the multisynthetase complex did not affect kinetic parameters for ArgRS . Therefore, p43 has a dual function . It promotes association of ArgRS to the complex via its N-terminal domain, but its C-terminal RNA-binding domain may act as a tRNA-interacting factor for an as yet unidentified component of the complex. Dig Liver Dis, 2004 Feb, 36 Suppl 1, S2 - 7 Somatostatin receptors and regulation of cell proliferation; Bousquet C et al.; Somatostatin is an inhibitory neuropeptide, which acts on various targets throughout the body to regulate a variety of physiological functions including inhibition of endocrine and exocrine secretions, modulation of neurotransmission, motor and cognitive functions, inhibition of intestinal motility, absorption of nutrients and ions, vascular contractility and inhibition of normal and tumour cell proliferation . It exerts its effects through interaction with five somatostatin receptors (sst1-sst5), which belong to the family of G-protein-coupled receptors with seven transmembrane spanning domains and are variably expressed in a variety of tumours such as gastroenteropancreatic tumours, pituitary tumours, and carcinoid tumours . This review covers the present knowledge regarding the molecular mechanisms involved in somatostatin antineoplastic activity . Evidence that sst2 receptor acts as a tumour suppressor is also discussed. Nat Cell Biol, 2004 May, 6(5), 414 - 9 Epub 2004 Apr 11. Golgi targeting of ARF-like GTPase Arl3p requires its Nalpha-acetylation and the integral membrane protein Sys1p; Setty SR et al.; Myristoylation of ARF family GTPases is required for their association with Golgi and endosomal membranes, where they regulate protein sorting and the lipid composition of these organelles . The Golgi-localized ARF-like GTPase Arl3p/ARP lacks a myristoylation signal, indicating that its targeting mechanism is distinct from myristoylated ARFs . We demonstrate that acetylation of the N-terminal methionine of Arl3p requires the NatC N(alpha)-acetyltransferase and that this modification is required for its Golgi localization . Chemical crosslinking and fluorescence microscopy experiments demonstrate that localization of Arl3p also requires Sys1p, a Golgi-localized integral membrane protein, which may serve as a receptor for acetylated Arl3p. Nat Cell Biol, 2004 May, 6(5), 405 - 13 Epub 2004 Apr 11. Targeting of the Arf-like GTPase Arl3p to the Golgi requires N-terminal acetylation and the membrane protein Sys1p; Behnia R et al.; The GTPase Arl3p is required to recruit a second GTPase, Arl1p, to the Golgi in Saccharomyces cerevisiae . Arl1p binds to the GRIP domain, which is present in a number of long coiled-coil proteins or 'golgins' . Here we show that Arl3p is not myristoylated like most members of the Arf family, but is instead amino-terminally acetylated by the NatC complex . Targeting of Arl3p also requires a Golgi membrane protein Sys1p . The human homologues of Arl3p (Arf-related protein 1 (ARFRP1)) and Sys1p (hSys1) can be isolated in a complex after chemical cross-linking . This suggests that the targeting of ARFRP1/Arl3p to the Golgi is mediated by a direct interaction between its acetylated N terminus and Sys1p/hSys1. Plant Physiol, 2004 Apr, 134(4), 1763 - 74 Epub 2004 Apr 09. Characterization of three functional high-affinity ammonium transporters in Lotus japonicus with differential transcriptional regulation and spatial expression; D'Apuzzo E et al.; Ammonium is a primary source of nitrogen for plants . In legume plants ammonium can also be obtained by symbiotic nitrogen fixation, and NH(4)(+) is also a regulator of early and late symbiotic interaction steps . Ammonium transporters are likely to play important roles in the control of nodule formation as well as in nitrogen assimilation . Two new genes, LjAMT1;2 and LjAMT1;3, were cloned from Lotus japonicus . Both were able to complement the growth defect of a yeast (Saccharomyces cerevisiae) ammonium transport mutant . Measurement of {(14)C}methylammonium uptake rates and competition experiments revealed that each transporter had a high affinity for NH(4)(+) . The K(i) for ammonium was 1.7, 3, and 15 microm for LjAMT1;1, 1;2, and 1;3, respectively . Real-time PCR revealed higher expression of LjAMT1;1, 1;2, and 1;3 genes in leaves than in roots and nodule, with expression levels decreasing in the order LjAMT1;1 > 1;2 > 1;3 except in flowers, in which LjAMT1;3 was expressed at higher level than in leaves, and LjAMT1;1 showed the lowest level of expression . Expression of LjAMT1;1 and 1;2 in roots was induced by nitrogen deprivation . Expression of LjAMT1;1 was repressed in leaves exposed to elevated CO(2) concentrations, which also suppress photorespiration . Tissue and cellular localization of LjAMT1 genes expression, using promoter-beta-glucuronidase and in situ RNA hybridization approaches, revealed distinct cellular spatial localization in different organs, including nodules, suggesting differential roles in the nitrogen metabolism of these organs. Mol Biol Cell, 2004 Jun, 15(6), 2758 - 70 Epub 2004 Apr 09. Deficiencies in the endoplasmic reticulum (ER)-membrane protein Gab1p perturb transfer of glycosylphosphatidylinositol to proteins and cause perinuclear ER-associated actin bar formation; Grimme SJ et al.; The essential GAB1 gene, which encodes an endoplasmic reticulum (ER)-membrane protein, was identified in a screen for mutants defective in cellular morphogenesis . A temperature-sensitive gab1 mutant accumulates complete glycosylphosphatidylinositol (GPI) precursors, and its temperature sensitivity is suppressed differentially by overexpression of different subunits of the GPI transamidase, from strong suppression by Gpi8p and Gpi17p, to weak suppression by Gaa1p, and to no suppression by Gpi16p . In addition, both Gab1p and Gpi17p localize to the ER and are in the same protein complex in vivo . These findings suggest that Gab1p is a subunit of the GPI transamidase with distinct relationships to other subunits in the same complex . We also show that depletion of Gab1p or Gpi8p, but not Gpi17p, Gpi16p, or Gaa1p causes accumulation of cofilin-decorated actin bars that are closely associated with the perinuclear ER, which highlights a functional interaction between the ER network and the actin cytoskeleton. Eukaryot Cell, 2004 Apr, 3(2), 546 - 52 Inactivation of transcription factor gene ACE2 in the fungal pathogen Candida glabrata results in hypervirulence; Kamran M et al.; During an infection, the coordinated orchestration of many factors by the invading organism is required for disease to be initiated and to progress . The elucidation of the processes involved is critical to the development of a clear understanding of host-pathogen interactions . For Candida species, the inactivation of many fungal attributes has been shown to result in attenuation . Here we demonstrate that the Candida glabrata homolog of the Saccharomyces cerevisiae transcription factor gene ACE2 encodes a function that mediates virulence in a novel way . Inactivation of C . glabrata ACE2 does not result in attenuation but, conversely, in a strain that is hypervirulent in a murine model of invasive candidiasis . C . glabrata ace2 null mutants cause systemic infections characterized by fungal escape from the vasculature, tissue penetration, proliferation in vivo, and considerable overstimulation of the proinflammatory arm of the innate immune response . Compared to the case with wild-type fungi, mortality occurs much earlier in mice infected with C . glabrata ace2 cells, and furthermore, 200-fold lower doses are required to induce uniformly fatal infections . These data demonstrate that C . glabrata ACE2 encodes a function that plays a critical role in mediating the host-Candida interaction . It is the first virulence-moderating gene to be described for a Candida species. Eukaryot Cell, 2004 Apr, 3(2), 311 - 7 Nrg1 and nrg2 transcriptional repressors are differently regulated in response to carbon source; Berkey CD et al.; The Nrg1 and Nrg2 repressors of Saccharomyces cerevisiae have highly similar zinc fingers and closely related functions in the regulation of glucose-repressed genes . We show that NRG1 and NRG2 are differently regulated in response to carbon source at both the RNA and protein levels . Expression of NRG1 RNA is glucose repressed, whereas NRG2 RNA levels are nearly constant . Nrg1 protein levels are elevated in response to glucose limitation or growth in nonfermentable carbon sources, whereas Nrg2 levels are diminished . Chromatin immunoprecipitation assays showed that Nrg1 and Nrg2 bind DNA both in the presence and absence of glucose . In mutant cells lacking the corepressor Ssn6(Cyc8)-Tup1, promoter-bound Nrg1, but not Nrg2, functions as an activator in a reporter assay, providing evidence that the two Nrg proteins have distinct properties . We suggest that the differences in expression and function of these two repressors, in combination with their similar DNA-binding domains, contribute to the complex regulation of the large set of glucose-repressed genes. J Cell Sci, 2004 Apr 1, 117(Pt 9), 1665 - 73 Epub 2004 Mar 09. Active and specific recruitment of a soluble cargo protein for endoplasmic reticulum exit in the absence of functional COPII component Sec24p; Fatal N et al.; Exit of proteins from the yeast endoplasmic reticulum (ER) is thought to occur in vesicles coated by four proteins, Sec13p, Sec31p, Sec23p and Sec24p, which assemble at ER exit sites to form the COPII coat . Sec13p may serve a structural function, whereas Sec24p has been suggested to operate in selection of cargo proteins into COPII vesicles . We showed recently that the soluble glycoprotein Hsp150 exited the ER in the absence of Sec13p function . Here we show that its ER exit did not require functional Sec24p . Hsp150 was secreted to the medium in a sec24-1 mutant at restrictive temperature 37 degrees C, while cell wall invertase and vacuolar carboxypeptidase Y remained in the ER . The determinant guiding Hsp150 to this transport route was mapped to the C-terminal domain of 114 amino acids by deletion analysis, and by an HRP fusion protein-based EM technology adapted here for yeast . This domain actively mediated ER exit of Sec24p-dependent invertase in the absence of Sec24p function . However, the domain was entirely dispensable for ER exit when Sec24p was functional . The Sec24p homolog Sfb2p was shown not to compensate for nonfunctional Sec24p in ER exit of Hsp150 . Our data show that a soluble cargo protein, Hsp150, is selected actively and specifically to budding sites lacking normal Sec24p by a signature residing in its C-terminal domain. Science, 2004 Apr 9, 304(5668), 286 - 9 Tip20p prohibits back-fusion of COPII vesicles with the endoplasmic reticulum; Kamena F et al.; Directionality in intracellular trafficking is essential to ensure the correct localization of proteins along the secretory pathway . Here, we found evidence for an active mechanism that prohibited back-fusion of de novo-generated vesicles with their donor compartment . Tip20p is a peripheral membrane protein implicated in consumption of COPI vesicles at the endoplasmic reticulum . However, a specific mutant of TIP20 did not interfere with COPII vesicle generation but allowed these vesicles to fuse back to the endoplasmic reticulum, a process that does not occur normally in the cell. Proc Natl Acad Sci U S A, 2004 Apr 20, 101(16), 6068 - 73 Epub 2004 Apr 08. Transcriptional termination by RNA polymerase I requires the small subunit Rpa12p; Prescott EM et al.; We identify Rpa12p of RNA polymerase I (Pol I) as a termination factor . Combined analyses using transcription run-on, electron microscopy-visualized chromatin spreading and RT-PCR have been applied to the rRNA-encoding genes of Saccharomyces cerevisiae . These confirm that Pol I termination occurs close to the Reb1p-dependent terminator in wild-type strains . However, deletion mutants for the 3' end-processing enzyme Rnt1p or the Rpa12p subunit of Pol I both show Pol I transcription in the spacer . For Deltarpa12, these spacer polymerases are devoid of nascent transcripts, suggesting they are immediately degraded . The homology of Rpa12p to the small subunit Rpb9p of Pol II and Rpc11p of Pol III, both implicated in transcriptional termination, points to a common termination mechanism for all three classes of RNA polymerase. Bioinformatics, 2004 Oct 12, 20(15), 2370 - 9 Epub 2004 Apr 08. Combining pattern discovery and discriminant analysis to predict gene co-regulation; Simonis N et al.; MOTIVATION: Several pattern discovery methods have been proposed to detect over-represented motifs in upstream sequences of co-regulated genes, and are for example used to predict cis-acting elements from clusters of co-expressed genes . The clusters to be analyzed are often noisy, containing a mixture of co-regulated and non-co-regulated genes . We propose a method to discriminate co-regulated from non-co-regulated genes on the basis of counts of pattern occurrences in their non-coding sequences . METHODS: String-based pattern discovery is combined with discriminant analysis to classify genes on the basis of putative regulatory motifs . RESULTS: The approach is evaluated by comparing the significance of patterns detected in annotated regulons (positive control), random gene selections (negative control) and high-throughput regulons (noisy data) from the yeast Saccharomyces cerevisiae . The classification is evaluated on the annotated regulons, and the robustness and rejection power is assessed with mixtures of co-regulated and random genes. EMBO J, 2004 May 5, 23(9), 1957 - 67 Epub 2004 Apr 08. Set1 is required for meiotic S-phase onset, double-strand break formation and middle gene expression; Sollier J et al.; The Set1 protein of Saccharomyces cerevisiae is a histone methyltransferase (HMTase) acting on lysine 4 of histone H3 . Inactivation of the SET1 gene in a diploid leads to a sporulation defect . We have studied various processes that take place during meiotic differentiation in set1Delta diploid cells . The absence of Set1 leads to a delay of meiotic S-phase onset, which reflects a defect in DNA replication initiation . The timely induction of meiotic DNA replication does not require the Set1 HMTase activity, but depends on the SET domain . In addition, set1Delta displays a severe impairment of the DNA double-strand break formation, which is not only the consequence of the replication delay . Transcriptional profiling experiments show that the induction of middle meiotic genes, but not of early meiotic genes, is affected by the loss of Set1 . In contrast to meiotic replication, the transcriptional induction of the middle meiotic genes appears to depend on the methylation of H3-K4 . Our results unveil multiple roles of Set1 in meiotic differentiation and distinguish between HMTase-dependent and -independent Set1 functions. Mol Endocrinol, 2004 Jul, 18(7), 1620 - 30 Epub 2004 Apr 07. The heat shock protein 70 cochaperone hip enhances functional maturation of glucocorticoid receptor; Nelson GM et al.; Multiple molecular chaperones interact with steroid receptors to promote functional maturation and stability of receptor complexes . The heat shock protein (Hsp)70 cochaperone Hip has been identified in conjunction with Hsp70, Hsp90, and the Hsp70/Hsp90 cochaperone Hop/Sti1p in receptor complexes during an intermediate stage of receptor assembly, but a functional requirement for Hip in the receptor assembly process has not been established . Because the budding yeast Saccharomyces cerevisiae contains orthologs for most of the receptor-associated chaperones yet lacks an orthologous Hip gene, we exploited the well-established yeast model for steroid receptor function to ask whether Hip can alter steroid receptor function in vivo . Introducing human Hip into yeast enhances hormone-dependent activation of a reporter gene by glucocorticoid receptor (GR) . Because Hip does not similarly enhance signaling by mineralocorticoid, progesterone, or estrogen receptors, a general effect on transcription can be excluded . Instead, Hip promotes functional maturation of GR without increasing steady-state levels of GR protein . Unexpectedly, Hip binding to Hsp70 is not critical for boosting GR responsiveness to hormone . In conclusion, Hip functions by a previously unrecognized mechanism to promote the efficiency of GR maturation in cells. Proc Natl Acad Sci U S A, 2004 Mar 30, 101(13), 4519 - 24 Epub 2004 Mar 18. Tying synaptonemal complex initiation to the formation and programmed repair of DNA double-strand breaks; Henderson KA et al.; During meiosis, homologous chromosomes recombine and become closely apposed along their lengths within the synaptonemal complex (SC) . In part because Spo11 is required both to make the double-strand breaks (DSBs) that initiate recombination and to promote normal SC formation in many organisms, it is clear that these two processes are intimately coupled . The molecular nature of this linkage is not well understood, but it has been proposed that SC formation initiates locally at the sites of ongoing recombination and in particular at the subset of sites that will eventually give rise to crossovers . To test this hypothesis, we examined further the relationship between DSBs and SC formation in Saccharomyces cerevisiae . SCs were monitored in a series of spo11 missense mutants with varying DSB frequencies . Alleles that blocked DSB formation gave SC phenotypes indistinguishable from a deletion mutant, and partial loss-of-function mutations with progressively more severe DSB defects caused corresponding defects in SC formation . These results strongly correlate SC formation with Spo11 catalytic activity per se . Numbers of Zip3 complexes on chromosomes, thought to represent the sites of SC initiation, also declined when Spo11 activity decreased, but in a markedly nonlinear fashion: hypomorphic spo11 alleles caused larger defects in DSB formation than in Zip3 complex formation . This nonlinear response of Zip3 closely paralleled the response of crossover recombination products . The quantitative relationship between Zip3 foci, SC formation, and crossing over strongly implicates crossover-designated recombination intermediates as the sites of SC initiation. Proc Nutr Soc, 2004 Feb, 63(1), 21 - 9 Intestinal and placental zinc transport pathways; Ford D; Mammalian members of the cation diffusion facilitator (CDF) and zrt-, irt-like protein (ZIP) families of Zn transporters, initially identified in Saccharomyces cerevisiae and Arabidopsis thalania spp., have been cloned during the last 8 years and have been classified as families SLC30 and SLC39 respectively . The cloning of human Zn transporters ZnT-like transporter 1 (hZTL1)/ZnT5 (SLC30A5) and hZIP4 (SLC39A4) were major advances in the understanding of the molecular mechanisms of dietary Zn absorption . Both transporters are localised at the enterocyte apical membrane and are, therefore, potentially of fundamental importance in dietary Zn uptake . hZTL1 mediates Zn uptake when expressed in Xenopus laevis oocytes and hZIP4 is mutated in most cases of the inherited Zn deficiency disease acrodermatitis enteropathica . Localisation of hZTL1/ZnT5 at the apical membrane of the placental syncytiotrophoblast indicates a fundamental role in the transfer of Slc30 Zn to the foetus . Observations in rodent models indicate that in the intestine increased Zn availability increases expression of Zn transporters . Human intestinal Caco-2 cells show a similar response to increasing the Zn2+ concentration of the nutrient medium in relation to the expression of mRNA corresponding to several Zn transporters and that of ZnT1 (SLC30A1) and hZTL1/ZnT5 proteins . In the human placental cell line JAR, however, expression at the mRNA level of a number of Zn transporters is not modified by Zn availability, whilst ZnT1 and hZTL1/ZnT5 proteins are reduced under Zn-supplemented conditions . These differences between Caco-2 and JAR cells in Zn transporter gene responses to Zn supply may reflect the different extracellular Zn concentrations encountered by the corresponding cell types in vitro. Biochem J, 2004 Jul 1, 381(Pt 1), 35 - 42 Identification of a dehydrogenase acting on D-2-hydroxyglutarate; Achouri Y et al.; Extracts of frozen rat liver were found to catalyse the formation of 3H2O from DL-2-hydroxy{2-3H}glutarate . Three peaks of enzyme activities were observed on separation by chromatography on DEAE-Sepharose . The first and second peaks corresponded to an enzyme acting on L-2-hydroxyglutarate and the third peak corresponded to an enzyme acting on D-2-hydroxyglutarate, as indicated by competitive inhibition of the detritiation of the racemic radioactive compound by the unlabelled L- and D-isomers respectively . The enzyme acting on the D-form was further characterized . It was independent of NAD or NADP and it converted D-2-hydroxyglutarate into a-ketoglutarate, transferring electrons to artificial electron acceptors . It also oxidized D-lactate, D-malate and meso-tartrate and was stimulated by Zn2+, Co2+ and Mn2+, but not by Mg2+ or Ca2+ . Subcellular fractionation indicated that it was present in the mitochondrial fraction . The enzyme was further purified by chromatography on Blue Trisacryl and phenyl-Sepharose, up to a stage where only a few bands were still visible by SDS/PAGE . Among the four candidate polypeptides that were identified by MS, one corresponded to a predicted mitochondrial protein homologous with FAD-dependent D-lactate dehydrogenase . The corresponding human protein was expressed in HEK-293 cells and it was shown to catalyse the detritiation of DL-2-hydroxy{2-3H}glutarate with similar properties as the purified rat enzyme. J Am Chem Soc, 2004 Apr 14, 126(14), 4682 - 8 Inelastic incoherent neutron scattering measurements of intact cells and tissues and detection of interfacial water; Ford RC et al.; We have previously used inelastic incoherent neutron scattering spectroscopy to investigate the properties of aqueous suspensions of biomolecules as a function of hydration . These experiments led to the identification of signals corresponding to interfacial (hydration) water at low water content . A prediction from these studies was that in the crowded environment inside living cells, a significant proportion of the water would be interfacial, with profound implications for biological function . Here we describe the first inelastic incoherent neutron scattering spectroscopy studies of living cells and tissues . We find that the interfacial water signal is similar to that observed for water interacting with purified biomolecules and other solutes, i.e., it is strongly perturbed in the librational and translational intermolecular optical regions of the spectrum at 20-150 meV . The ratio of interfacial water compared to total water in cells (approximately 30%) is in line with previous experimental data for hydration water and calculations based on simple assumptions. J Psychiatry Neurosci, 2004 Mar, 29(2), 134 - 7 Eating disorders with binge-eating behaviour are associated with the s allele of the 3'-UTR VNTR polymorphism of the dopamine transporter gene; Shinohara M et al.; OBJECTIVE: The dopaminergic system is associated with feelings of pleasure and reward and with positive hedonic processes related to food, sexual activity and certain substances . Because it is recognized that patients who have eating disorders with binge-eating behaviour have a high comorbidity of substance dependence, we examined the association between the variable number of tandem repeats (VNTR) polymorphism in the 3; untranslated region of the dopamine transporter gene (DAT1) and eating disorders with binge-eating behaviour . METHODS: The subjects were 90 female Japanese patients with eating disorders diagnosed using DSM-IV; they were compared with 115 healthy female controls . Genomic DNA was extracted from whole blood, and standard polymerase chain reaction testing was performed . We compared the frequencies of a short allele (7 or 9 repeats) and a long allele (10 or 11 repeats) in both groups . RESULTS: In the group who had an eating disorder with binge-eating behaviour, the frequency of a short allele was significantly higher compared with the control group . CONCLUSION: It seems plausible that the association between the DAT1 VNTR and binge-eating behaviour indicates that dysregulation of dopamine reuptake may act as a common pathophysiologic mechanism in eating disorders with binge-eating behaviour and in disorders related to substance use. Proc Natl Acad Sci U S A, 2004 Apr 20, 101(16), 5964 - 9 Epub 2004 Apr 06. Mechanisms for activating Cu- and Zn-containing superoxide dismutase in the absence of the CCS Cu chaperone; Carroll MC et al.; The Cu- and Zn-containing superoxide dismutase 1 (SOD1) largely obtains Cu in vivo by means of the action of the Cu chaperone CCS . Yet, in the case of mammalian SOD1, a secondary pathway of activation is apparent . Specifically, when human SOD1 is expressed in either yeast or mammalian cells that are null for CCS, the SOD1 enzyme retains a certain degree of activity . This CCS-independent activity is evident with both wild-type and mutant variants of SOD1 that have been associated with familial amyotrophic lateral sclerosis . We demonstrate here that the CCS-independent activation of mammalian SOD1 involves glutathione, particularly the reduced form, or GSH . A role for glutathione in CCS-independent activation was seen with human SOD1 molecules that were expressed in either yeast cells or immortalized fibroblasts . Compared with mammalian SOD1, the Saccharomyces cerevisiae enzyme cannot obtain Cu without CCS in vivo, and this total dependence on CCS involves the presence of dual prolines near the C terminus of the SOD1 polypeptide . Indeed, the insertion of such prolines into human SOD1 rendered this molecule refractory to CCS-independent activation . The possible implications of multiple pathways for SOD1 activation are discussed in the context of SOD1 evolutionary biology and familial amyotrophic lateral sclerosis. Mol Cell, 2004 Apr 9, 14(1), 1 - 15 Mitochondrial signaling: the retrograde response; Butow RA et al.; Mitochondrial retrograde signaling is a pathway of communication from mitochondria to the nucleus that influences many cellular and organismal activities under both normal and pathophysiological conditions . In yeast it is used as a sensor of mitochondrial dysfunction that initiates readjustments of carbohydrate and nitrogen metabolism . In both yeast and animal cells, retrograde signaling is linked to TOR signaling, but the precise connections are unclear . In mammalian cells, mitochondrial dysfunction sets off signaling cascades through altered Ca(2+) dynamics, which activate factors such as NFkappaB, NFAT, and ATF . Retrograde signaling also induces invasive behavior in otherwise nontumorigenic cells implying a role in tumor progression. J Eukaryot Microbiol, 2004 Jan-Feb, 51(1), 30 - 7 Comparative genomics of Pneumocystis carinii with other protists: implications for life style; Cushion MT; Three protistan genomes were analyzed for differential genetic traits that may be associated with biological adaptations to their unique life styles . The microsporidian, Encephalitozoon cuniculi, an obligate intracellular parasite; the ascomycetes, Pneumocystis carinii, considered an opportunistic pathogen; and Saccharomyces cerevisiae, a model organism exhibiting a free-living life style, were used in comparisons of genomic architecture, reproductive strategies, and metabolic capacity predicted by the presence of signature genes . Genome size, gene number, and metabolic function decreased as the organisms became more dependent on their hosts . In contrast, gene density and the percentage of genes dedicated to cell growth and division were substantially increased in the genome of E . cuniculi . The obligate life style was associated with reductions in gene number, genome size, and reduced metabolic capacity while the free-living life style was coincident with gene duplications and duplication of large portions of the genome . The genomic characteristics and metabolic capacity of P . carinii were usually intermediate between those of the other two protistan genomes, but unique characteristics such as the presence of a single rDNA locus may indicate that these organisms could be in the process of becoming more host dependent. J Cell Biol, 2004 Apr, 165(1), 99 - 109 Epub 2004 Apr 05. Pheromone-induced polarization is dependent on the Fus3p MAPK acting through the formin Bni1p; Matheos D et al.; During mating, budding yeast cells reorient growth toward the highest concentration of pheromone . Bni1p, a formin homologue, is required for this polarized growth by facilitating cortical actin cable assembly . Fus3p, a pheromone-activated MAP kinase, is required for pheromone signaling and cell fusion . We show that Fus3p phosphorylates Bni1p in vitro, and phosphorylation of Bni1p in vivo during the pheromone response is dependent on Fus3p . fus3 mutants exhibited multiple phenotypes similar to bni1 mutants, including defects in actin and cell polarization, as well as Kar9p and cytoplasmic microtubule localization . Disruption of the interaction between Fus3p and the receptor-associated Galpha subunit caused similar mutant phenotypes . After pheromone treatment, Bni1p-GFP and Spa2p failed to localize to the cortex of fus3 mutants, and cell wall growth became completely unpolarized . Bni1p overexpression suppressed the actin assembly, cell polarization, and cell fusion defects . These data suggest a model wherein activated Fus3p is recruited back to the cortex, where it activates Bni1p to promote polarization and cell fusion. J Biol Chem, 2004 May 21, 279(21), 22781 - 5 Epub 2004 Apr 02. Sam35 of the mitochondrial protein sorting and assembly machinery is a peripheral outer membrane protein essential for cell viability; Milenkovic D et al.; The mitochondrial outer membrane contains two integral proteins essential for cell viability, Tom40 of the translocase of the outer membrane (TOM complex) and Sam50 of the sorting and assembly machinery (SAM complex) . Here we report the identification of Sam35, the first peripheral mitochondrial outer membrane protein that is essential for cell viability . Sam35 (encoded by the Saccharomyces cerevisiae ORF YHR083w) is a novel subunit of the SAM complex and is crucial for the assembly pathway of outer membrane beta-barrel proteins, such as the precursors of Tom40 and porin . Sam35 is not required for the import of inner membrane or matrix targeted proteins . The presence of two essential proteins in the SAM complex, Sam35 and Sam50, indicates that it plays a central role in mitochondrial biogenesis. J Biol Chem, 2004 Jun 4, 279(23), 24444 - 51 Epub 2004 Apr 02. Human Mob proteins regulate the NDR1 and NDR2 serine-threonine kinases; Devroe E et al.; Human NDR1 (nuclear Dbf2-related) is a widely expressed nuclear serine-threonine kinase that has been implicated in cell proliferation and/or tumor progression . Here we present molecular characterization of the human NDR2 serine-threonine kinase, which shares approximately 87% sequence identity with NDR1 . NDR2 is expressed in most human tissues with the highest expression in the thymus . In contrast to NDR1, NDR2 is excluded from the nucleus and exhibits a punctate cytoplasmic distribution . The differential localization of NDR1 and NDR2 suggests that each kinase may serve distinct functions . Thus, to identify proteins that interact with NDR1 or NDR2, epitope-tagged kinases were immunoprecipitated from Jurkat T-cells . Two uncharacterized proteins that are homologous to the Saccharomyces cerevisiae kinase regulators Mob1 and Mob2 were identified . We demonstrate that NDR1 and NDR2 partially colocalize with human Mob2 in HeLa cells and confirm the NDR-Mob interactions in cell extracts . Interestingly, NDR1 and NDR2 form stable complexes with Mob2, and this association dramatically stimulates NDR1 and NDR2 catalytic activity . In summary, this work identifies a unique class of human kinase-activating subunits that may be functionally analagous to cyclins. Appl Environ Microbiol, 2004 Apr, 70(4), 1931 - 4 Fum3p, a 2-ketoglutarate-dependent dioxygenase required for C-5 hydroxylation of fumonisins in Fusarium verticillioides; Ding Y et al.; Fumonisins are polyketide-derived mycotoxins produced by several agriculturally important Fusarium species . The B series fumonisins, FB(1), FB(2), FB(3), and FB(4), are fumonisins produced by wild-type Fusarium verticillioides strains, differing in the number and location of hydroxyl groups attached to the carbon backbone . We characterized the protein encoded by FUM3, a gene in the fumonisin biosynthetic gene cluster . The 33-kDa FUM3 protein (Fum3p) was heterologously expressed and purified from Saccharomyces cerevisiae . Yeast cells expressing the Fum3p converted FB(3) to FB(1), indicating that Fum3p catalyzes the C-5 hydroxylation of fumonisins . This result was verified by assaying the activity of Fum3p purified from yeast cells . The C-5 hydroxylase activity of purified Fum3p required 2-ketoglutarate, Fe(2+), ascorbic acid, and catalase, all of which are required for 2-ketoglutarate-dependent dioxygenases . The protein also contains two His motifs that are highly conserved in this family of dioxygenases . Thus, Fum3p is a 2-ketoglutarate-dependent dioxygenase required for the addition of the C-5 hydroxyl group of fumonisins. Cell, 2004 Apr 2, 117(1), 9 - 15 Early decision; meiotic crossover interference prior to stable strand exchange and synapsis; Bishop DK et al.; During meiosis, DNA double-strand breaks ultimately yield two types of recombinants: crossovers (CO) and noncrossovers (NCO) . Recent studies in budding yeast show the CO/NCO designation occurs before stable strand exchange and thus well before Holliday junction resolution . Chromosome synapsis occurs after CO/NCO designation and is not required for the regulated distribution of COs along chromosomes manifested as CO interference. Cell, 2004 Apr 2, 117(1), 5 - 7 SWRred not shaken; mixing the histones; Korber P et al.; The recently isolated SWR1 complex catalyzes an ATP-dependent histone exchange with specificity for the histone variant H2A.Z . This provides a new theme in chromatin remodeling mechanisms and an explanation of how histone variants are incorporated into the nucleosome outside of S phase . In connection with the isolation of histone variant specific chaperone complexes, we are beginning to see the mechanisms that mix the histone octamer with intriguing implications for epigenetic inheritance. Genes Cells, 2004 Apr, 9(4), 305 - 15 Transcription-mediated hyper-recombination in HOT1; Serizawa N et al.; Recombination hotspots are DNA sequences which enhance recombination around that region . HOT1 is one of the best-studied mitotic hotspots in yeast . HOT1 includes a RNA polymerase I (PolI) transcription promoter which is responsible for 35S ribosomal rRNA gene (rDNA) transcription . In a PolI defective mutant the HOT1 hotspot activity is abolished, therefore transcription of HOT1 is thought to be an important factor for the recombination stimulation . However, it is not clear whether the transcription itself or other pleiotropic phenotypes stimulates recombination . To investigate the role of transcription, we made a highly activated PolI transcription system in HOT1 by using a strain whose rDNA repeats are deleted (rdnDeltaDelta) . In the rdnDeltaDelta strain, HOT1 transcription was increased about 14 times compared to wild-type . Recombination activity stimulated by HOT1 in this strain was also elevated, about 15 times, compared to wild-type . These results indicate that the level of PolI transcription in HOT1 determines efficiency of the recombination . Moreover, Fob1p, which is essential for both the recombination stimulation activity and transcription of HOT1, was dispensable in the rdnDeltaDelta strains . This suggests that Fob1p is functioning as a PolI transcriptional activator in the wild-type strain. Philos Trans R Soc Lond B Biol Sci, 2004 Jan 29, 359(1441), 79 - 86 Repairing a double-strand chromosome break by homologous recombination: revisiting Robin Holliday's model; Haber JE et al.; Since the pioneering model for homologous recombination proposed by Robin Holliday in 1964, there has been great progress in understanding how recombination occurs at a molecular level . In the budding yeast Saccharomyces cerevisiae, one can follow recombination by physically monitoring DNA after the synchronous induction of a double-strand break (DSB) in both wild-type and mutant cells . A particularly well-studied system has been the switching of yeast mating-type (MAT) genes, where a DSB can be induced synchronously by expression of the site-specific HO endonuclease . Similar studies can be performed in meiotic cells, where DSBs are created by the Spo11 nuclease . There appear to be at least two competing mechanisms of homologous recombination: a synthesis-dependent strand annealing pathway leading to noncrossovers and a two-end strand invasion mechanism leading to formation and resolution of Holliday junctions (HJs), leading to crossovers . The establishment of a modified replication fork during DSB repair links gene conversion to another important repair process, break-induced replication . Despite recent revelations, almost 40 years after Holliday's model was published, the essential ideas he proposed of strand invasion and heteroduplex DNA formation, the formation and resolution of HJs, and mismatch repair, remain the basis of our thinking. Philos Trans R Soc Lond B Biol Sci, 2004 Jan 29, 359(1441), 31 - 8 Mechanisms involved in regulating DNA replication origins during the cell cycle and in response to DNA damage; Early A et al.; Replication origins in eukaryotic cells never fire more than once in a given S phase . Here, we summarize the role of cyclin-dependent kinases in limiting DNA replication origin usage to once per cell cycle in the budding yeast Saccharomyces cerevisiae . We have examined the role of different cyclins in the phosphorylation and regulation of several replication/regulatory factors including Cdc6, Sic1, ORC and DNA polymerase alpha-primase . In addition to being regulated by the cell cycle machinery, replication origins are also regulated by the genome integrity checkpoint kinases, Mec1 and Rad53 . In response to DNA damage or drugs which interfere with the progression of replication forks, the activation of late-firing replication origins is inhibited . There is evidence indicating that the temporal programme of origin firing depends upon the local histone acetylation state . We have attempted to test the possibility that checkpoint regulation of late-origin firing operates through the regulation of the acetylation state . We found that overexpression of the essential histone acetylase, Esal, cannot override checkpoint regulation of origin firing . We have also constructed a temperature-sensitive esa1 mutant . This mutant is unable to resume cell cycle progression after alpha-factor arrest . This can be overcome by overexpression of the G1 cyclin, Cln2, revealing a novel role for Esal in regulating Start. Philos Trans R Soc Lond B Biol Sci, 2004 Jan 29, 359(1441), 7 - 16 Dynamics of pre-replication complex proteins during the cell division cycle; Prasanth SG et al.; Replication of the human genome every time a cell divides is a highly coordinated process that ensures accurate and efficient inheritance of the genetic information . The molecular mechanism that guarantees that many origins of replication fire only once per cell-cycle has been the area of intense research . The origin recognition complex (ORC) marks the position of replication origins in the genome and serves as the landing pad for the assembly of a multiprotein, pre-replicative complex (pre-RC) at the origins, consisting of ORC, cell division cycle 6 (Cdc6), Cdc10-dependent transcript (Cdt1) and mini-chromosome maintenance (MCM) proteins . The MCM proteins serve as key participants in the mechanism that limits eukaryotic DNA replication to once-per-cell-cycle and its binding to the chromatin marks the final step of pre-RC formation, a process referred to as 'replication licensing' . We present data demonstrating how the MCM proteins associate with the chromatin during the G1 phase, probably defining pre-RCs and then anticipate replication fork movement in a precisely coordinated manner during the S phase of the cell cycle . The process of DNA replication must also be carefully coordinated with other cell-cycle processes including mitosis and cytokinesis . Some of the proteins that control initiation of DNA replication are likely to interact with the pathways that control these important cell-cycle transitions . Herein, we discuss the participation of human ORC proteins in other vital functions, in addition to their bona fide roles in replication. Proc Natl Acad Sci U S A, 2004 Apr 13, 101(15), 5518 - 23 Epub 2004 Apr 02. Oxygen and the copper chaperone CCS regulate posttranslational activation of Cu,Zn superoxide dismutase; Brown NM et al.; Oxidative stress leads to the up-regulation of many antioxidant enzymes including Cu,Zn superoxide dismutase (SOD1) via transcriptional mechanisms; however, few examples of posttranslational regulation are known . The copper chaperone for SOD1 (CCS) is involved in physiological SOD1 activation, and its primary function is thought to be delivery of copper to the enzyme . Data presented here are consistent with a previously uncharacterized function for CCS in the SOD1 pathway, namely mediating enzyme activation in response to increases in oxygen tension . Activity assays with pure proteins and cell extracts reveal that O(2) (or superoxide) is required for activation of SOD1 by CCS . Dose-response studies with a translational blocking agent demonstrate that the cellular oxidative response to O(2) is multitiered: existing apo-pools of SOD1 are activated by CCS in the early response, followed by increasing expression of SOD1 protein with persistent oxidative stress . This CCS function provides oxidant-responsive posttranslational regulation of SOD1 activity and may be relevant to a wide array of physiological stresses that involve a sudden elevation of oxygen availability. Plant Physiol, 2004 Apr, 134(4), 1586 - 97 Epub 2004 Apr 02. Molecular analyses of the Arabidopsis TUBBY-like protein gene family; Lai CP et al.; In mammals, TUBBY-like proteins play an important role in maintenance and function of neuronal cells during postdifferentiation and development . We have identified a TUBBY-like protein gene family with 11 members in Arabidopsis, named AtTLP1-11 . Although seven of the AtTLP genes are located on chromosome I, no local tandem repeats or gene clusters are identified . Except for AtTLP4, reverse transcription-PCR analysis indicates that all these genes are expressed in various organs in 6-week-old Arabidopsis . AtTLP1, 2, 3, 6, 7, 9, 10, and 11 are expressed ubiquitously in all the organs tested, but the expression of AtTLP5 and 8 shows dramatic organ specificity . These 11 family members share 30% to 80% amino acid similarities across their conserved C-terminal tubby domains . Unlike the highly diverse N-terminal region of animal TUBBY-like proteins, all AtTLP members except AtTLP8 contain a conserved F-box domain (51-57 residues) . The interaction between AtTLP9 and ASK1 (Arabidopsis Skp1-like 1) is confirmed via yeast (Saccharomyces cerevisiae) two-hybrid assays . Abscisic acid (ABA)-insensitive phenotypes are observed for two independent AtTLP9 mutant lines, whereas transgenic plants overexpressing AtTLP9 are hypersensitive to ABA . These results suggest that AtTLP9 may participate in the ABA signaling pathway. J Androl, 2004 May-Jun, 25(3), 363 - 8 Expression of a novel RAD23B mRNA splice variant in the human testis; Huang X et al.; A gene coding a novel human RAD23B protein named RAD23-like protein B, presumably involved in spermatogenesis, was identified and characterized using a complementary DNA (cDNA) microarray . In the human testis, its expression was 2.33 times higher than it was in the embryo testis, with a particularly high expression in ejaculated human spermatozoa . The full length of this gene is 1548 bp, and the putative protein is 338 amino acids long . This protein is homologous to RAD23B, which is one of two human homologs of Saccharomyces cerevisiae RAD23, and is involved in both nucleotide excision repair (NER) and ubiquitin (Ub)-dependent proteolysis . However, RAD23-like protein B lacks the Ub-like (UbL) domain that functions as a proteasome localization signal . Multiple-tissue expression profile of the messenger RNA (mRNA) that encodes the RAD23-like protein B also showed that it is highly expressed in the human testis and in ejaculated spermatozoa . Our present study indicates that this novel alternative splicing form of RAD23B is correlated with human spermatogenesis. Biochem Biophys Res Commun, 2004 Apr 30, 317(2), 648 - 53 A tRNA(TRP) gene mediates the suppression of cbs2-223 previously attributed to ABC1/COQ8; Hsieh EJ et al.; The Saccharomyces cerevisiae gene ABC1 was originally isolated as a multicopy suppressor of a yeast strain harboring a mutation in a cytochrome b translational activator (cbs2-223) . Based on this identification, Abc1p was postulated to activate the bc1 complex and function as a chaperone of cytochrome b . ABC1 was subsequently identified as COQ8 and found to be necessary for yeast coenzyme Q synthesis . In this work we show that a segment of yeast genomic DNA containing ABC1/COQ8 and neighboring genes suppresses the respiratory and Q-deficient phenotypes of the coq6 mutant, coq6-1 . COQ6 is essential for yeast coenzyme Q biosynthesis . We show that a tRNA(TRP) gene located downstream of ABC1/COQ8 mediates suppression of the cbs2-223 and coq6-1 mutations, and each is identified here as containing UGA nonsense codons . The inability of ABC1/COQ8 to suppress the cbs2-223 allele in multicopy indicates it may not be a chaperone as previously reported. FEBS Lett, 2004 Apr 9, 563(1-3), 28 - 34 Identification of a fatty acid Delta11-desaturase from the microalga Thalassiosira pseudonana; Tonon T et al.; A set of genomic DNA sequences putatively encoding front-end desaturases were identified by in silico analysis of the draft genome of the marine microalga Thalassiosira pseudonana . Among these candidate genes, an open reading frame named TpdesN was found to be full-length, intronless, and constitutively expressed during cell cultivation . The predicted amino acid sequence of the corresponding protein, TpDESN, exhibited typical features of desaturases involved in the production of polyunsaturated fatty acids (PUFAs) in algae, i.e . a cytochrome b5-like domain at the N-terminus and three conserved histidine-rich motifs in the desaturase domain . Expression of TpDESN in Saccharomyces cerevisiae revealed that this enzyme was not involved in PUFA synthesis, but specifically desaturated palmitic acid 16:0 to 16:1Delta11 . To our knowledge, until this report, Delta11-desaturase activity had only been detected in insect cells. Mol Cell Biol, 2004 Apr, 24(8), 3562 - 76 Swm1/Apc13 is an evolutionarily conserved subunit of the anaphase-promoting complex stabilizing the association of Cdc16 and Cdc27; Schwickart M et al.; The anaphase-promoting complex (APC/C) is a large ubiquitin-protein ligase which controls progression through anaphase by triggering the degradation of cell cycle regulators such as securin and B-type cyclins . The APC/C is an unusually complex ligase containing at least 10 different, evolutionarily conserved components . In contrast to APC/C's role in cell cycle regulation little is known about the functions of individual subunits and how they might interact with each other . Here, we have analyzed Swm1/Apc13, a small subunit recently identified in the budding yeast complex . Database searches revealed proteins related to Swm1/Apc13 in various organisms including humans . Both the human and the fission yeast homologues are associated with APC/C subunits, and they complement the phenotype of an SWM1 deletion mutant of budding yeast . Swm1/Apc13 promotes the stable association with the APC/C of the essential subunits Cdc16 and Cdc27 . Accordingly, Swm1/Apc13 is required for ubiquitin ligase activity in vitro and for the timely execution of APC/C-dependent cell cycle events in vivo. Mol Cell Biol, 2004 Apr, 24(8), 3324 - 36 Human Spt6 stimulates transcription elongation by RNA polymerase II in vitro; Endoh M et al.; Recent studies have suggested that Spt6 participates in the regulation of transcription by RNA polymerase II (RNAPII) . However, its underlying mechanism remains largely unknown . One possibility, which is supported by genetic and biochemical studies of Saccharomyces cerevisiae, is that Spt6 affects chromatin structure . Alternatively, Spt6 directly controls transcription by binding to the transcription machinery . In this study, we establish that human Spt6 (hSpt6) is a classic transcription elongation factor that enhances the rate of RNAPII elongation . hSpt6 is capable of stimulating transcription elongation both individually and in concert with DRB sensitivity-inducing factor (DSIF), comprising human Spt5 and human Spt4 . We also provide evidence showing that hSpt6 interacts with RNAPII and DSIF in human cells . Thus, in vivo, hSpt6 may regulate multiple steps of mRNA synthesis through its interaction with histones, elongating RNAPII, and possibly other components of the transcription machinery. Mol Cell Biol, 2004 Apr, 24(8), 3277 - 85 Association of Rad9 with double-strand breaks through a Mec1-dependent mechanism; Naiki T et al.; Rad9 is required for the activation of DNA damage checkpoint pathways in budding yeast . Rad9 is phosphorylated after DNA damage in a Mec1- and Tel1-dependent manner and subsequently interacts with Rad53 . This Rad9-Rad53 interaction has been suggested to trigger the activation and phosphorylation of Rad53 . Here we show that Mec1 controls the Rad9 accumulation at double-strand breaks (DSBs) . Rad9 was phosphorylated after DSB induction and associated with DSBs . However, its phosphorylation and association with DSBs were significantly decreased in cells carrying a mec1Delta or kinase-negative mec1 mutation . Mec1 phosphorylated the S/TQ motifs of Rad9 in vitro, the same motifs that are phosphorylated after DNA damage in vivo . In addition, multiple mutations in the Rad9 S/TQ motifs resulted in its defective association with DSBs . Phosphorylation of Rad9 was partially defective in cells carrying a weak mec1 allele (mec1-81), whereas its association with DSBs occurred efficiently in the mec1-81 mutants, as found in wild-type cells . However, the Rad9-Rad53 interaction after DSB induction was significantly decreased in mec1-81 mutants, as it was in mec1Delta mutants . Deletion mutation in RAD53 did not affect the association of Rad9 with DSBs . Our results suggest that Mec1 promotes association of Rad9 with sites of DNA damage, thereby leading to full phosphorylation of Rad9 and its interaction with Rad53. Mol Cell Biol, 2004 Apr, 24(8), 3213 - 26 Requirement of Rrm3 helicase for repair of spontaneous DNA lesions in cells lacking Srs2 or Sgs1 helicase; Schmidt KH et al.; The Rrm3 DNA helicase of Saccharomyces cerevisiae interacts with proliferating cell nuclear antigen and is required for replication fork progression through ribosomal DNA repeats and subtelomeric and telomeric DNA . Here, we show that rrm3 srs2 and rrm3 sgs1 mutants, in which two different DNA helicases have been inactivated, exhibit a severe growth defect and undergo frequent cell death . Cells lacking Rrm3 and Srs2 arrest in the G(2)/M phase of the cell cycle with 2N DNA content and frequently contain only a single nucleus . The phenotypes of rrm3 srs2 and rrm3 sgs1 mutants were suppressed by disrupting early steps of homologous recombination . These observations identify Rrm3 as a new member of a network of pathways, involving Sgs1 and Srs2 helicases and Mus81 endonuclease, suggested to act