Mol Cell Biol, 2004 Jul, 24(14), 6184 - 93 mRNA capping enzyme activity is coupled to an early transcription elongation; Kim HJ et al.; One of the temperature-sensitive alleles of CEG1, a guanylyltransferase subunit of the Saccharomyces cerevisiae capping enzyme, showed 6-azauracil (6AU) sensitivity at the permissive growth temperature, which is a phenotype that is correlated with a transcription elongation defect . This temperature-sensitive allele, ceg1-63, has an impaired ability to induce PUR5 in response to 6AU treatment and diminished enzyme-GMP formation activity . However, this cellular and molecular defect is not primarily due to the preferential degradation of the transcript attributed to a lack of cap structure . Our data suggest that the guanylyltransferase subunit of the capping enzyme plays a role in transcription elongation as well as cap formation . First, in addition to the 6AU sensitivity, ceg1-63 is synthetically lethal with elongation-defective mutations in RNA polymerase II . Secondly, it produces a prolonged steady-state level of GAL1 mRNA after glucose shutoff . Third, it decreases the transcription read through a tandem array of promoter-proximal pause sites in an orientation-dependent manner . Taken together, we present direct evidence that suggests a role of capping enzyme in an early transcription . Capping enzyme ensures the early transcription checkpoint by capping of the nascent transcript in time and allowing it to extend further. FEBS Lett, 2004 Jul 2, 569(1-3), 140 - 8 A novel CPx-ATPase from the cadmium hyperaccumulator Thlaspi caerulescens; Bernard C et al.; Thlaspi caerulescens exhibits a unique capacity for cadmium tolerance and accumulation . We investigated the molecular basis of this exceptional Cd(2+) tolerance by screening for T . caerulescens genes, which alleviate Cd(2+) toxicity upon expression in Saccharomyces cerevisiae . This allowed for the isolation of a cDNA encoding a peptide with homology to the C-terminal part of a heavy metal ATPase . The corresponding TcHMA4 full-length sequence was isolated from T . caerulescens and compared to its homolog from Arabidopsis thaliana (AtHMA4) . Expression of TcHMA4 and AtHMA4 cDNAs conferred Cd sensitivity in yeast, while expression of TcHMA4-C and AtHMA4-C cDNAs encoding the C-termini of, respectively, TcHMA4 and AtHMA4 conferred Cd tolerance . Moreover, heterologous expression in yeast suggested a higher Cd binding capacity of TcHMA4-C compared to AtHMA4-C . In planta, both HMA4 genes were expressed at a higher level in roots than in shoots . However, TcHMA4 shows a much higher constitutive expression than AtHMA4 . Our data indicate that HMA4 could be involved in Cd(2+) transport and possibly in the Cd hyperaccumulation character. FEBS Lett, 2004 Jul 2, 569(1-3), 89 - 93 The Arabidopsis thaliana chloroplast inner envelope protein ARTEMIS is a functional member of the Alb3/Oxa1/YidC family of proteins; Funes S et al.; The Arabidopsis thaliana protein ARTEMIS is an integral component of the chloroplast inner envelope required for chloroplast division . It contains a domain of significant homology to members of the Alb3/Oxa1/YidC protein family . Here, we show that upon expression in yeast mitochondria, ARTEMIS can partially take over the function of yeast Oxa1 in the insertion and assembly of mitochondrial membrane proteins . This identifies ARTEMIS as a functional member of the Alb3/Oxa1/YidC protein family and suggests the existence of a novel protein sorting pathway in chloroplasts which integrates polypeptides from the stroma into the inner envelope by an evolutionary conserved process. FEBS Lett, 2004 Jul 2, 569(1-3), 65 - 9 The mitochondrial ABC transporter Atm1p functions as a homodimer; Chloupkova M et al.; The ATP-binding cassette (ABC) transporters constitute one of the largest families of proteins in evolution . The ATM1 gene of the yeast Saccharomyces cerevisiae encodes an ABC protein, which is localized to the mitochondrial inner membrane . A deletion of ATM1 results in the accumulation of up to a 30-fold excess of mitochondrial iron, loss of mitochondrial cytochromes and abnormalities of cytosolic iron metabolism . In this study, we have evaluated the role of conserved sequence elements in Atm1p in its function and dimerization in vivo . We report that conserved residues in the Walker A and B motifs of the nucleotide binding domain, which are required for ATP binding and hydrolysis, are essential for Atm1p function . In addition, we provide evidence that ATP binding is important for Atm1p dimerization. Mol Cell, 2004 Jul 2, 15(1), 107 - 16 The Ras/PKA signaling pathway directly targets the Srb9 protein, a component of the general RNA polymerase II transcription apparatus; Chang YW et al.; RNA polymerase II transcription is a complex process that is controlled at multiple levels . The data presented here add to this repertoire by showing that signal transduction pathways can directly regulate gene expression by targeting components of the general RNA polymerase II apparatus . In particular, this study shows that the Ras/PKA signaling pathway in Saccharomyces cerevisiae regulates the activity of the Srb complex, a regulatory group of proteins that is part of the RNA polymerase II holoenzyme . Genetic and biochemical data indicate that Srb9p is a substrate for PKA and that this phosphorylation modulates the activity of the Srb complex . The Srb complex, like many components of the RNA II polymerase machinery, is responsible for regulating the expression of a relatively large number of genes . Thus, this type of a transcriptional control mechanism would provide the cell with an efficient way of bringing about broad changes in gene expression. Mol Cell, 2004 Jul 2, 15(1), 95 - 105 Cellular toxicity of polyglutamine expansion proteins: mechanism of transcription factor deactivation; Schaffar G et al.; The expression of polyglutamine-expanded mutant proteins in Huntington's disease and other neurodegenerative disorders is associated with the formation of intraneural inclusions . These aggregates could potentially cause cellular toxicity by sequestering essential proteins possessing normal polyQ repeats, including the transcription factors TBP and CBP . We show, in vitro and in cells, that monomers or small soluble oligomers of huntingtin exon1 accumulate in the nucleus and inhibit the function of TBP in a polyQ-dependent manner . FRET experiments indicate that these toxic forms are generated through a conformational rearrangement in huntingtin . Interaction of toxic huntingtin with the benign polyQ repeat of TBP structurally destabilizes the transcription factor, independent of the formation of insoluble coaggregates . Hsp70/Hsp40 chaperones interfere with the conformational change in mutant huntingtin and inhibit the deactivation of TBP . These results outline a molecular mechanism of cellular toxicity in polyQ disease and can explain the beneficial effects of molecular chaperones. Mol Cell, 2004 Jul 2, 15(1), 69 - 81 Precise nucleosome positioning and the TATA box dictate requirements for the histone H4 tail and the bromodomain factor Bdf1; Martinez-Campa C et al.; Acetylation of histone tails plays a key role in chromatin dynamics and is associated with the potential for gene expression . We show here that a 2-3 bp mispositioning of the nucleosome covering the TATA box at PHO5 induces a dependency on the acetylatable lysine residues of the histone H4 N-terminal region and on the TFIID-associated bromodomain factor Bdf1 . This dependency arises either through fusion of the PHO5 promoter to a lacZ reporter or by mutation of the TATA box in the natural gene . The results suggest that promoters in which the TATA box is either absent or poorly accessible on the surface of a nucleosome may compensate by using Bdf1 bromodomains and acetylated H4 tails to anchor TFIID to the promoter during the initial stages of transcription activation . We propose that nucleosome positioning at the nucleotide level provides a subtle, but highly effective, mechanism for gene regulation. Mol Cell, 2004 Jul 2, 15(1), 5 - 15 Targeted mRNA degradation by deadenylation-independent decapping; Badis G et al.; Modulating the rate of mRNA degradation is a fast and efficient way to control gene expression . In a yeast strain deleted of EDC3, a component of the decapping machinery conserved in eukaryotes, the transcript coding the ribosomal protein Rps28b is specifically stabilized, as demonstrated by microarray and time course experiments . This stabilization results from the loss of RPS28B autoregulation, which occurs at the level of mRNA decay . Using mutants of the major deadenylase, we show that this regulation occurs at the level of decapping and bypasses deadenylation . Rps28b interacts with a conserved hairpin structure within the 3'UTR of its own mRNA and with components of the decapping machinery, including Edc3 . We conclude that Rps28b, in the presence of Edc3, directly recruits the decapping machinery on its own mRNA . These findings show that specific modulation of the decapping efficiency on natural transcripts can control mRNA turnover. Mol Cell, 2004 Jul 2, 15(1), 1 - 2 Regulation of mRNA decay: decapping goes solo; Jacobson A; In this issue of Molecular Cell, Badis et al . demonstrate that autoregulated destabilization of the RPS28B mRNA is mediated by Edc3p interaction with an Rps28p/3'UTR mRNP complex . Subsequent recruitment of Dcp1p/Dcp2p to the complex appears to ensure rapid, deadenylation-independent decapping of the mRNA. Mol Microbiol, 2004 Jul, 53(1), 335 - 44 Activation of pleiotropic drug resistance by the J-protein and Hsp70-related proteins, Zuo1 and Ssz1; Eisenman HC et al.; Ssz1 (Pdr13) and Zuo1, members of the Hsp70 and J-protein molecular chaperone families, respectively, form a heterodimer and function on the ribosome with the Hsp70, Ssb, presumably assisting folding of newly synthesized polypeptides . As it has also been reported that Ssz1 induces pleiotropic drug resistance (PDR) when overexpressed, a possible role for Zuo1 in PDR was investigated . The C-terminal domain of Zuo1, which is dispensable for Zuo1's chaperone function on the ribosome, is both necessary and sufficient for PDR induction by Zuo1 . A single domain of Ssz1, the N-terminal ATPase domain, is sufficient for PDR induction as well, indicating that Ssz1 does not function as a chaperone in PDR . No role for Ssb was found in PDR; overexpression did not affect PDR, nor was its presence required for Ssz1's or Zuo1's effect on PDR . As our results also indicate that Ssz1 and Zuo1 must be free of ribosomes to induce PDR, we propose that Ssz1's and Zuo1's function in PDR is distinct from their role as ribosome-associated co-chaperones and may be regulatory in nature. Biochemistry, 2004 Jul 6, 43(26), 8579 - 89 Identification of DNA 3'-phosphatase active site residues and their differential role in DNA binding, Mg2+ coordination, and catalysis; Deshpande RA et al.; DNA 3'-phosphatase (Tpp1) from Saccharomyces cerevisiae, a homologue of human polynucleotide kinase/3'-phosphatase, has been shown to participate in DNA damage repair by removing 3'-phosphate blocking lesions . Tpp1 shows similarity to the l-2-haloacid dehalogenase superfamily of enzymes . By comparison to phosphoserine phosphatase, a well-studied member of this family, we designed conservative and nonconservative substitutions of likely active site residues of Tpp1 and tested them in a variety of assays . From the loss or impairment of activity, we identified D35, D37, T39, S88, K170, D206, and D218 as being involved in Tpp1 catalysis . D35 and K170 were the most critical since maximum inactivation was seen with even conservative mutations . Tpp1 bound DNA through its active site in a Mg(2+)-dependent manner and exhibited a preference for dsDNA . Although Tpp1 bound more strongly to DNA with a free 3' terminus, it also bound well to covalently closed DNA, suggesting a possible lesion scanning mechanism . DNA binding studies further indicated that Tpp1 coordinates Mg(2+) through D35 and D206 and contacts the DNA 3' end through D37 . The removal of 3'-phosphate involved a phospho-Tpp1 intermediate, and our results support D35 as being the point of covalent attachment . On the basis of these similarities in mutant phenotypes of Tpp1 and phosphoserine phosphatase, we propose a reaction mechanism for Tpp1 which explains its strict phosphate specificity. Biochemistry, 2004 Jul 6, 43(26), 8568 - 78 Sml1p is a dimer in solution: characterization of denaturation and renaturation of recombinant Sml1p; Gupta V et al.; Sml1p is a small 104-amino acid protein from Saccharomyces cerevisiae that binds to the large subunit (Rnr1p) of the ribonucleotide reductase complex (RNR) and inhibits its activity . During DNA damage, S phase, or both, RNR activity must be tightly regulated, since failure to control the cellular level of dNTP pools may lead to genetic abnormalities, such as genome rearrangements, or even cell death . Structural characterization of Sml1p is an important step in understanding the regulation of RNR . Until now the oligomeric state of Sml1p was unknown . Mass spectrometric analysis of wild-type Sml1p revealed an intermolecular disulfide bond involving the cysteine residue at position 14 of the primary sequence . To determine whether disulfide bonding is essential for Sml1p oligomerization, we mutated the Cys14 to serine . Sedimentation equilibrium measurements in the analytical ultracentrifuge show that both wild-type and C14S Sml1p exist as dimers in solution, indicating that the dimerization is not a result of a disulfide bond . Further studies of several truncated Sml1p mutants revealed that the N-terminal 8-20 residues are responsible for dimerization . Unfolding/refolding studies of wild-type and C14S Sml1p reveal that both proteins refold reversibly and have almost identical unfolding/refolding profiles . It appears that Sml1p is a two-domain protein where the N-terminus is responsible for dimerization and the C-terminus for binding and inhibiting Rnr1p activity. Biochemistry, 2004 Jul 6, 43(26), 8447 - 58 raf RBD and ubiquitin proteins share similar folds, folding rates and mechanisms despite having unrelated amino acid sequences; Vallee-Belisle A et al.; Recent experimental and theoretical studies in protein folding suggest that the rates and underlying mechanisms by which proteins attain the native state are largely determined by the topological complexity of a specific fold rather than by the fine details of the amino acid sequences . However, such arguments are based upon the examination of a limited number of protein folds . To test this view, we sought to investigate whether proteins belonging to the ubiquitin superfamily display similar folding behavior . To do so, we compared the folding-unfolding transitions of mammalian ubiquitin (mUbi) with those of its close yeast homologue (yUbi), and to those of the structurally related Ras binding domain (RBD) of the serine/threonine kinase raf that displays no apparent sequence homology with the ubiquitin family members . As demonstrated for mUbi {Krantz, B . A., and Sosnick, T . R . (2000) Biochemistry 39, 11696-11701}, we show that a two-state transition model with no burst phase intermediate can describe folding of both yUbi and raf RBD . We further demonstrate that (1) all three proteins refold at rates that are within 1 order of magnitude (1800, 1100, and 370 s(-1) for mUbi, raf RBD, and yUbi, respectively), (2) both mUbi and raf RBD display similar refolding heterogeneity, and (3) the folding free energy barriers of both mUbi and raf RBD display a similar temperature dependence and sensitivity to a stabilizing agent or to mutations of a structurally equivalent central core residue . These findings are consistent with the view that rates and mechanisms for protein folding depend mostly on the complexity of the native structure topology rather than on the fine details of the amino acid sequence. Mol Genet Genomics, 2004 Aug, 272(1), 47 - 56 Epub 2004 Jun 19. DIRS retroelements in arthropods: identification of the recently active TcDirs1 element in the red flour beetle Tribolium castaneum; Goodwin TJ et al.; Members of the DIRS family of retrotransposons differ from most other known retrotransposons in that they encode a tyrosine recombinase (YR), a type of enzyme frequently involved in site-specific recombination . This enzyme is believed to insert the extrachromosomal DNA intermediate of DIRS element retrotransposition into the host genome . DIRS elements have been found in plants, a slime mold, fungi, and a variety of animals including vertebrates, echinoderms and nematodes . They have a somewhat patchy distribution, however, apparently being absent from a number of model organisms such as Saccharomyces cerevisiae, Arabidopsis thaliana and Drosophila melanogaster . In this report we describe the first DIRS retroelement to be identified in an arthropod . This element, TcDirs1, was found in the red flour beetle Tribolium castaneum (Coleoptera) . It is generally similar in sequence and structure to several previously described members of the DIRS group: it is bordered by inverted terminal repeats and it has a similar set of protein-coding domains (Gag, reverse transcriptase/ribonuclease H, and the YR), although these are arranged in a novel fashion . TcDirs1 elements exhibit several features indicative of recent activity, such as intact coding regions, a high level of sequence similarity between distinct elements and polymorphic insertion sites . Given their presence in an experimentally tractable host, these potentially active elements might serve as useful models for the study of DIRS element retrotransposition . An element closely related to TcDirs1 was also detected in sequences from a second arthropod, the honey bee Apis mellifera (Hymenoptera), suggesting that these retrotransposons are long-term residents of arthropod genomes. Mol Genet Genomics, 2004 Jul, 271(6), 651 - 7 Epub 2004 Jun 18. Interaction of FIE, a polycomb protein, with pRb: a possible mechanism regulating endosperm development; Mosquna A et al.; Inactivation of the Arabidopsis protein FERTILIZATION INDEPENDENT ENDOSPERM (FIE) induces division of the central cell of the embryo sac, leading to endosperm development in the absence of fertilization . The mechanism whereby FIE regulates this process is unknown . We postulated that activation of central cell division in fie mutant plants might involve the retinoblastoma protein (pRb), a cell cycle regulatory element . Pull-down and surface plasmon resonance assays demonstrated that FIE interacts in-vitro with the pRb homologues from Arabidopsis (AtRb), maize (ZmRb) and human (HuRb) . The interaction of FIE with ZmRB and HuRb in the yeast two-hybrid system supports the possibility that a FIE-pRb interaction may occur also in planta . Mutational analysis showed that this interaction does not occur via the LxCxE motif of the FIE protein nor via the pocket B domain of pRb . These results suggest that FIE may inhibit premature division of the central cell of the embryo sac, at least partly, through interaction with pRb, and suppression of pRb-regulated genes. Nat Genet, 2004 Jul, 36(7), 707 - 13 Epub 2004 Jun 27. Identification of TFB5, a new component of general transcription and DNA repair factor IIH; Ranish JA et al.; We previously described the use of quantitative proteomics to study macromolecular complexes . Applying the method to analyze a yeast RNA polymerase II preinitiation complex, we identified a new 8-kDa protein, encoded by the uncharacterized open reading frame YDR079c-a, as a potential new component of the preinitiation complex . Here we show that YDR079c-a is a bona fide component of polymerase II preinitiation complexes and investigate its role in transcription . YDR079c-a is recruited to promoters both in vivo and in vitro and is required for efficient transcription in vitro and for normal induction of GAL genes . In addition, YDR079c-a is a core component of general transcription and DNA repair factor IIH and is required for efficient recruitment of TFIIH to a promoter . Yeast lacking YDR079c-a grow slowly, and, like strains carrying mutations in core TFIIH subunits, are sensitive to ultraviolet radiation . YDR079c-a is conserved throughout evolution, and mutations in the human ortholog account for a DNA repair-deficient form of the tricothiodystrophy disorder called TTD-A(2) . The identification of a new, evolutionarily conserved, core TFIIH subunit is essential for our understanding of TFIIH function in transcription, DNA repair and human disease. Methods Mol Biol, 2004, 281, 189 - 98 Functional analysis of APC-Cdh1; Sudo T et al.; To maintain genomic integrity against various kinds of genotoxic stress, cells have multiple checkpoints in the cell cycle . When one of the cell cycle events, such as DNA synthesis, DNA repair, and chromosomal segregation, has not been successfully completed, checkpoints will delay progression until the step is correctly accomplished, and only then will they relieve the arrest to allow the cell to move to the next phase . Cells lacking functional checkpoints display genomic aberrations, resulting in the acquisition of phenotypic changes of cancer cells . Anaphase-promoting complex (APC) is activated by two regulatory proteins: Cdc20 and Cdh1 . In yeast and Drosophila, Cdh1-dependent APC (APC-Cdh1) activity targets mitotic cyclins from the end of mitosis to the G1 phase . Loss of Cdh1 induces unscheduled accumulation of mitotic cyclins in G1, resulting in abrogation of G1 arrest caused by treatment with rapamycin, an inducer of p27Kip1 . Furthermore, Cdh1-deficient DT40 cells fail to maintain DNA damage-induced G2 arrest, and Cdh1-APC is activated by X-irradiation-induced DNA damage . Thus, activation of Cdh1-APC plays a crucial role in both cdk inhibitor-dependent G1 arrest and DNA damage-induced G2 arrest . In light of the differences between normal and cancer cells, checkpoints can be ideal targets for developmental cancer therapeutics . In this chapter, we describe how to analyze three checkpoints (spindle assembly checkpoint, rapamycin-induced G1 checkpoint, and DNA damage-induced G2 checkpoint) in conjunction with cell synchronization. Fungal Genet Biol, 2004 Aug, 41(8), 735 - 53 Interaction of Sclerotinia sclerotiorum with a resistant Brassica napus cultivar: expressed sequence tag analysis identifies genes associated with fungal pathogenesis; Li R et al.; Sclerotinia sclerotiorum is a ubiquitous necrotrophic fungal pathogen capable of infecting a wide range of plants . To identify genes involved in fungal development and pathogenesis we generated 2232 expressed sequence tags (ESTs) from two cDNA libraries constructed using either mycelia grown in pectin medium or tissues from infected Brassica napus stems . A total of 774 individual fungal genes were identified of which 39 were represented only among the infected plant EST collection . Annotation of 534 unigenes was possible following the categories applied to Saccharomyces cerevisiae and the Universal Gene Ontology scheme . cDNAs were identified that encoded potential pathogenicity factors including four endopolygalacturonases, two exopolygalacturonases, and several metabolite transporters . The potential role of these genes, as well as those encoding signal transduction factors, in the infection process is discussed. J Biol Chem, 2004 Sep 3, 279(36), 38047 - 54 Epub 2004 Jun 24. The presequence translocase-associated protein import motor of mitochondria . Pam16 functions in an antagonistic manner to Pam18; Li Y et al.; Transport of preproteins into the mitochondrial matrix requires the presequence translocase of the inner membrane (TIM23 complex) and the presequence translocase-associated motor (PAM) . The motor consists of five essential subunits, the mitochondrial heat shock protein 70 (mtHsp70) and four cochaperones, the nucleotide exchange-factor Mge1, the translocase-associated fulcrum Tim44, the J-protein Pam18, and Pam16 . Pam16 forms a complex with Pam18 and displays similarity to J-proteins but lacks the canonical tripeptide motif His-Pro-Asp (HPD) . We report that Pam16 does not function as a typical J-domain protein but, rather, antagonizes the function of Pam18 . Pam16 specifically inhibits the Pam18-mediated stimulation of the ATPase activity of mtHsp70 . The inclusion of the HPD motif in Pam16 does not confer the ability to stimulate mtHsp70 activity . Pam16-HPD fully substitutes for wild-type Pam16 in vitro and in vivo but is not able to replace Pam18 . Pam16 represents a new type of cochaperone that controls the stimulatory effect of the J-protein Pam18 and regulates the interaction of mtHsp70 with precursor proteins during import into mitochondria. J Anim Sci, 2004 Jun, 82(6), 1882 - 91 Dietary supplementation with phosphorylated mannans improves growth response and modulates immune function of weanling pigs; Davis ME et al.; Phosphorylated mannans derived from the yeast cell wall of Saccharomyces cerevisiae may beneficially modulate immune function in the weanling pig, possibly providing an alternative to the use of dietary growth-promoting antibiotics . Therefore, in this study, 32 pigs averaging 19 d of age and 5.7 +/- 0.2 kg initial BW were randomly assigned to 16 pens in an environmentally controlled nursery to determine the effects of dietary supplementation with phosphorylated mannans on growth and immune function . Average daily gain and G:F ratio increased (P < 0.05) when pigs were fed diets supplemented with mannans from d 0 to 14 after weaning and in the overall experiment . Percentage of neutrophils was lower (P < 0.08) and percentage of lymphocytes was higher (P < 0.05) in blood from pigs fed mannans than when pigs were fed the basal diet . Lamina propria macrophages isolated from pigs fed diets containing mannans phagocytosed a greater (P < 0.05) number of sheep red blood cells (2.63 +/- 0.11) than did lamina propria macrophages isolated from pigs fed the basal diet (2.31 +/- 0.11) . On d 19 after weaning, pigs fed diets supplemented with mannans tended to have a greater (P < 0.10) percentage of CD14+ lamina propria leukocytes than did pigs fed the basal diet . On d 21 following weaning, the percentage of CD14+MHCII+ leukocytes isolated from lamina propria tissue tended (P < 0.10) to be lower when pigs were fed mannans than when pigs were fed the basal diet . Pigs fed diets containing mannans had a lower (P < 0.05) ratio of CD3+CD4+:CD3+CD8+ T lymphocytes isolated from jejunal lamina propria tissue only on d 21 after weaning compared with pigs fed the basal diet . Supplementation of mannans in the diets of weanling pigs improved gain and efficiency, and intermittently affected selected components of the young pigs' immune function both systemically and enterically. Cell Struct Funct, 1999 Oct, 24(5), 365 - 72 Gamma-tubulin at ten: progress and prospects; Oakley BR et al.; The existence of gamma-tubulin was first reported approximately ten years ago, and it is appropriate to review the progress that has been made in gamma-tubulin research and to discuss some of the unanswered questions about gamma-tubulin function . gamma-Tubulin is ubiquitous in eukaryotes and is generally quite conserved . Two highly divergent gamma-tubulins have been discovered, however, one in Saccharomyces cerevisiae and one in Caenorhabditis elegans . Several organisms have two gamma-tubulin genes . In Drosophila melanogaster, the two gamma-tubulins differ significantly in sequence and expression pattern . In other organisms the two gamma-tubulins are almost identical and expression patterns have not been determined . gamma-Tubulin is located at microtubule organizing centers in many organisms, and it is also frequently associated with the mitotic spindle . gamma-Tubulin is essential for the formation of functional mitotic spindles in all organisms that have been examined to date . In animal cells, complexes containing gamma-tubulin are located at microtubule organizing centers where they nucleate the assembly of microtubules . In spite of the considerable progress that has been made in gamma-tubulin research important questions remain to be answered . The exact mechanisms of microtubule nucleation by gamma-tubulin complexes remain to be resolved as do the mechanisms by which microtubule nucleation from gamma-tubulin complexes is regulated . Finally, there is evidence that gamma-tubulin has important functions in addition to microtubule nucleation, and these functions are just beginning to be investigated. EMBO J, 2004 Jul 21, 23(14), 2872 - 81 Epub 2004 Jun 24. Oxygen-induced maturation of SOD1: a key role for disulfide formation by the copper chaperone CCS; Furukawa Y et al.; The antioxidant enzyme Cu,Zn-superoxide dismutase (SOD1) has the distinction of being one of the most abundant disulfide-containing protein known in the eukaryotic cytosol; however, neither catalytic nor physiological roles for the conserved disulfide are known . Here we show that the disulfide status of Saccharomyces cerevisiae SOD1 significantly affects the monomer-dimer equilibrium, the interaction with the copper chaperone CCS, and the activity of the enzyme itself . Disulfide formation in SOD1 by O(2) is slow but is greatly accelerated by the Cu-bound form of CCS (Cu-CCS) in vivo and in vitro even in the presence of excess reductants; once formed, this disulfide is kinetically stable . Biochemical assays reveal that Cu-CCS facilitates Cys oxidation and disulfide isomerization in the stepwise conversion of the immature form of the enzyme to the active state . The immature form of SOD1 is most susceptible to oxidative insult and to aggregation reminiscent of that observed in amyotrophic lateral sclerosis . Thus Cu-CCS mediation of correct disulfide formation in SOD1 is important for regulation of enzyme activity and for prevention of misfolding or aggregation. EMBO J, 2004 Jul 21, 23(14), 2862 - 71 Epub 2004 Jun 24. A complex containing the CCR4 and CAF1 proteins is involved in mRNA deadenylation in Drosophila; Temme C et al.; The CCR4-NOT complex is the major enzyme catalyzing mRNA deadenylation in Saccharomyces cerevisiae . We have identified homologs for almost all subunits of this complex in the Drosophila genome . Biochemical fractionation showed that the two likely catalytic subunits, CCR4 and CAF1, were associated with each other and with a poly(A)-specific 3' exonuclease activity . In Drosophila, the CCR4 and CAF1 proteins were ubiquitously expressed and present in cytoplasmic foci . Individual knock-down of several potential subunits of the Drosophila CCR4-NOT complex by RNAi in tissue culture cells led to a lengthening of bulk mRNA poly(A) tails . Knock-down of two individual subunits also interfered with the rapid deadenylation of Hsp70 mRNA during recovery from heat shock . Similarly, ccr4 mutant flies had elongated bulk poly(A) and a defect in Hsp70 mRNA deadenylation . A minor increase in bulk poly(A) tail length was also observed in Rga mutant flies, which are affected in the NOT2 subunit . The data show that the CCR4-NOT complex is conserved in Drosophila melanogaster and plays a role in general and regulated mRNA deadenylation. Nucleic Acids Res, 2004 Jul 1, 32(Web Server issue), W372 - 4 MITOPRED: a web server for the prediction of mitochondrial proteins; Guda C et al.; MITOPRED web server enables prediction of nucleus-encoded mitochondrial proteins in all eukaryotic species . Predictions are made using a new algorithm based primarily on Pfam domain occurrence patterns in mitochondrial and non-mitochondrial locations . Pre-calculated predictions are instantly accessible for proteomes of Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila, Homo sapiens, Mus musculus and Arabidopsis species as well as all the eukaryotic sequences in the Swiss-Prot and TrEMBL databases . Queries, at different confidence levels, can be made through four distinct options: (i) entering Swiss-Prot/TrEMBL accession numbers; (ii) uploading a local file with such accession numbers; (iii) entering protein sequences; (iv) uploading a local file containing protein sequences in FASTA format . Automated updates are scheduled for the pre-calculated prediction database so as to provide access to the most current data . The server, its documentation and the data are available from http://mitopred.sdsc.edu. Nucleic Acids Res, 2004 Jul 1, 32(Web Server issue), W176 - 80 Gene2Oligo: oligonucleotide design for in vitro gene synthesis; Rouillard JM et al.; There is substantial interest in implementing a bioinformatics tool that allows the design of oligonucleotides to support the development of in vitro gene synthesis . Current protocols to make long synthetic DNA molecules rely on the in vitro assembly of a set of short oligonucleotides, either by ligase chain reaction (LCR) or by assembly PCR . Ideally, such oligonucleotides should represent both strands of the final DNA molecule . They should be adjacent on the same strand and overlap the complementary oligonucleotides from the second strand to ensure good hybridization during assembly . This implies that the thermodynamic properties of each oligonucleotide have to be consistent across the set . Furthermore, any given oligonucleotide has to be totally specific to its target to avoid the creation of incorrectly assembled sequences . We have developed Gene2Oligo , a web-based tool that divides a long input DNA sequence into a set of adjacent oligonucleotides representing both DNA strands . The length of the oligonucleotides is dynamically optimized to ensure both the specificity and the uniform melting temperatures necessary for in vitro gene synthesis . We have successfully designed and used a set of oligonucleotides to synthesize the Saccharomyces cerevisiae cytochrome b5 by using both LCR and assembly PCR. Nucleic Acids Res, 2004 Jul 1, 32(Web Server issue), W83 - 8 PathBLAST: a tool for alignment of protein interaction networks; Kelley BP et al.; PathBLAST is a network alignment and search tool for comparing protein interaction networks across species to identify protein pathways and complexes that have been conserved by evolution . The basic method searches for high-scoring alignments between pairs of protein interaction paths, for which proteins of the first path are paired with putative orthologs occurring in the same order in the second path . This technique discriminates between true- and false-positive interactions and allows for functional annotation of protein interaction pathways based on similarity to the network of another, well-characterized species . PathBLAST is now available at as a web-based query . In this implementation, the user specifies a short protein interaction path for query against a target protein-protein interaction network selected from a network database . PathBLAST returns a ranked list of matching paths from the target network along with a graphical view of these paths and the overlap among them . Target protein-protein interaction networks are currently available for Helicobacter pylori, Saccharomyces cerevisiae, Caenorhabditis elegans and Drosophila melanogaster . Just as BLAST enables rapid comparison of protein sequences between genomes, tools such as PathBLAST are enabling comparative genomics at the network level. Am J Pathol, 2004 Jul, 165(1), 107 - 13 The novel marker, DOG1, is expressed ubiquitously in gastrointestinal stromal tumors irrespective of KIT or PDGFRA mutation status; West RB et al.; We recently characterized gene expression patterns in gastrointestinal stromal tumors (GISTs) using cDNA microarrays, and found that the gene FLJ10261 (DOG1, discovered on GIST-1), encoding a hypothetical protein, was specifically expressed in GISTs . The immunoreactivity of a rabbit antiserum to synthetic DOG1 peptides was assessed on two soft tissue tumor microarrays . The tissue microarrays included 587 soft tissue tumors, with 149 GISTs, including 127 GIST cases for which the KIT and PDGFRA mutation status was known . Immunoreactivity for DOG1 was found in 136 of 139 (97.8%) of scorable GISTs . All seven GIST cases with a PDGFRA mutation were DOG1-positive, while most of these failed to react for KIT . The immunohistochemical findings were confirmed with in situ hybridization probes for DOG1, KIT, and PDGFRA . Other neoplasms in the differential diagnosis of GIST, including desmoid fibromatosis (0 of 17) and Schwannoma (0 of 3), were immunonegative for DOG1 . Only 4 of 438 non-GIST cases were immunoreactive for DOG1 . DOG1, a protein of unknown function, is expressed strongly on the cell surface of GISTs and is rarely expressed in other soft tissue tumors . Reactivity for DOG1 may aid in the diagnosis of GISTs, including PDGFRA mutants that fail to express KIT antigen, and lead to appropriate treatment with imatinib mesylate, an inhibitor of the KIT tyrosine kinase. Antimicrob Agents Chemother, 2004 Jul, 48(7), 2483 - 9 Candida glabrata erg1 mutant with increased sensitivity to azoles and to low oxygen tension; Tsai HF et al.; A Candida glabrata erg1 (Cgerg1) mutant, CgTn201S, was identified by transposon mutagenesis and by increased fluconazole susceptibility . CgERG1 encodes a 489-amino-acid protein which, on the basis of its homology with Saccharomyces cerevisiae ERG1, is a squalene epoxidase essential for ergosterol synthesis . Interruption following codon 475 of CgErg1p decreased the ergosterol content by 50%; caused accumulation of the squalene precursor; increased the levels of susceptibility to fluconazole, itraconazole, and terbinafine; increased the level of resistance to amphotericin B; increased the levels of rhodamine 6G and {(3)H}-fluconazole uptake; reduced the level of growth; and blocked growth under conditions of low oxygen tension . In addition, CgTn201S efficiently took up exogenous cholesterol from cholesterol-containing serum . Cholesterol constituted 34% of the extractable sterols in CgTn201S when it was grown aerobically on serum-containing medium . Under the same conditions, C . albicans contained only 0.1 to 1.2% cholesterol . Exogenous sterols also restored growth under conditions of low oxygen tension . Finally, complementation of the Cgerg1 mutation restored the levels of {(3)H}fluconazole uptake and drug susceptibility to wild-type levels. Antimicrob Agents Chemother, 2004 Jul, 48(7), 2409 - 14 Cloning of S-adenosyl-L-methionine:C-24-Delta-sterol-methyltransferase (ERG6) from Leishmania donovani and characterization of mRNAs in wild-type and amphotericin B-Resistant promastigotes; Pourshafie M et al.; The 24-alkylated sterols have been shown previously to be absent in membranes of amphotericin B (AmB)-resistant Leishmania donovani promastigotes, suggesting that the S- adenosyl-l-methionine:C-24-Delta-sterol-methyltransferase (SCMT or ERG6) was not functional or not expressed in AmB-resistant (AmB-R) parasites . From an L . donovani wild-type clone, we cloned two cDNAs with an identical open reading frame encoding a putative SCMT, the enzyme responsible for a first sterol methylation at the C-24 position . The two cDNAs differed by their 3'-untranslated region (3'-UTR) and 5'-UTR sequences . One transcript (A) had a normal structure with a spliced leader and was highly expressed in normal cells but absent in AmB-R cells . The other (B), which did not possess the spliced leader sequence, was weakly expressed in normal cells but strongly expressed in AmB-R cells . As a functional test, ERG6 null mutant Saccharomyces cerevisiae yeasts were transformed using the pYES2.1 TOPO TA expression vector containing the candidate SCMT1/ERG6 coding sequence cloned from L . donovani . The transformed yeasts exhibited C-24 alkylated sterol expression, mainly ergosterol, within their membranes, proving that the isolated cDNA encodes on a SCMT responsible for sterol methylation . In AmB-R L . donovani promastigotes, the absence of the normal transcript (A) and the expression of an abnormal species (B) devoid of a spliced leader could explain the absence of sterol methylation in these cells . Further studies using a homologous system will allow us to draw conclusions about the relationship between SCMT expression and AmB resistance in Leishmania. Electrophoresis, 2004 Jun, 25(12), 1765 - 71 Chamber with modifiable inner width for performing transversal alternating field electrophoresis in variable numbers of minigels; Riveron AM et al.; We present a transversal alternating field electrophoresis chamber that allows modifiable inner widths to accommodate low- or high-throughput formats, with 7.8 cm opposite electrode separation and 30.4 cm electrode length . Removable slotted sheets divide the chamber into four smaller compartments, each one supporting a minigel of 3.85 cm in length and 7.1 cm in width . Replacements of slotted sheets with solid dielectric blocks with the sizes and shapes of compartments permit to occlude chamber compartments, changing from 4 to 1 the numbers of minigels per run, from 88 to 13 the maximum numbers of samples, and from 1688 to 422 mL the volume of buffer poured into the chamber . Saccharomyces cerevisiae chromosomes gave its characteristic DNA band pattern in all compartments, whereas migrations of DNA molecules are not affected by the occlusion of compartments. Mol Pharmacol, 2004 Jul, 66(1), 153 - 60 Enhanced 7-ethyl-10-hydroxycamptothecin (SN-38) lethality by methylselenocysteine is associated with Chk2 phosphorylation at threonine-68 and down-regulation of Cdc6 expression; Yin MB et al.; Methylselenocysteine (MSC) is an organic selenium compound in preventative clinical trials involving prostate, lung, and colon carcinoma . We found that methioninase-activated MSC potentiates 7-ethyl-10-hydroxycamptothecin (SN-38)-induced cell lethality in vitro in the p53-defective human head and neck carcinoma A253 cells . Activated MSC increases chk2 phosphorylation at threonine-68 induced by SN-38, with no significant effect on chk1 phosphorylation . Cell cycle arrest induced by SN-38, however, was not abrogated or potentiated by MSC . These results suggest that the enhanced cellular lethality of SN-38 by MSC was not associated with cell cycle regulation pathways . Because chk2, in addition to its role in cell cycle arrest, can induce apoptosis by phosphorylation/activation, we examined whether increased chk2 phosphorylation could induce preapoptotic DNA fragmentation . DNA damage analysis showed that megabase DNA fragmentation is decreased, accompanied by the increased 30 to 300 kilobase pairs of DNA fragmentation after exposure to SN-38 with MSC, compared with SN-38 alone . No significant changes in the amount of DNA fragments were observed in cells treated with SN-38 or MSC alone . Moreover, proteolytic destruction of DNA replication-associated proteins cdc6, MCM2, and cdc25A may induce a DNA damage checkpoint response . The observed down-regulation of DNA replication proteins cdc6, MCM2, and cdc25A after exposure to SN-38 with MSC further indicates a relationship between drug response and DNA damage . Exposure to SN-38 with MSC resulted in a significant increase of poly(ADP-ribose) polymerasecleavage and caspase 3 activation . All together, the data support the hypothesis that enhanced lethality of this combination is associated with increased chk2 phosphorylation at Thr68 and down-regulation of specific DNA replication-associated proteins, which result in poly(ADP-ribose) polymerase cleavage, caspase 3 activation, and the induction of 30 to 300 kilobase pairs of DNA fragmentation. J Biochem (Tokyo), 2004 Jun, 135(6), 695 - 700 Human histone deacetylase SIRT2 interacts with the homeobox transcription factor HOXA10; Bae NS et al.; Histone deacetylases are required for transcriptional repression in eukaryotes . Saccharomyces cerevisiae has several histone deacetylases, of which ySir2p is the most conserved throughout evolution . Currently, there is no report on the interacting protein partner of a human Sir2 homolog, SIRT2 . Here we show for the first time that SIRT2 interacts with the homeobox transcription factor, HOXA10, which was identified in a two-hybrid screen . Interactions were confirmed by co-immunoprecipitation from in vitro translations as well as in human cell-free extracts . Taken together with mouse knockout studies, our results raise the intriguing possibility that SIRT2 plays a role in mammalian development. J Biol Chem, 2004 Aug 27, 279(35), 36462 - 9 Epub 2004 Jun 22. DNA repair defects channel interstrand DNA cross-links into alternate recombinational and error-prone repair pathways; Saffran WA et al.; The repair of psoralen interstrand cross-links in the yeast Saccharomyces cerevisiae involves the DNA repair groups nucleotide excision repair (NER), homologous recombination (HR), and post-replication repair (PRR) . In repair-proficient yeast cells cross-links induce double-strand breaks, in an NER-dependent process; the double-strand breaks are then repaired by HR . An alternate error-prone repair pathway generates mutations at cross-link sites . We have characterized the repair of plasmid molecules carrying a single psoralen cross-link, psoralen monoadduct, or double-strand break in yeast cells with deficiencies in NER, HR, or PRR genes, measuring the repair efficiencies and the levels of gene conversions, crossing over, and mutations . Strains with deficiencies in the NER genes RAD1, RAD3, RAD4, and RAD10 had low levels of cross-link-induced recombination but higher mutation frequencies than repair-proficient cells . Deletion of the HR genes RAD51, RAD52, RAD54, RAD55, and RAD57 also decreased induced recombination and increased mutation frequencies above those of NER-deficient yeast . Strains lacking the PRR genes RAD5, RAD6, and RAD18 did not have any cross-link-induced mutations but showed increased levels of recombination; rad5 and rad6 cells also had altered patterns of cross-link-induced gene conversion in comparison with repair-proficient yeast . Our observations suggest that psoralen cross-links can be repaired by three pathways: an error-free recombinational pathway requiring NER and HR and two PRR-dependent error-prone pathways, one NER-dependent and one NER-independent. Proteins, 2004 Aug 1, 56(2), 354 - 66 Predictive reconstruction of the mitochondrial iron-sulfur cluster assembly metabolism: I . The role of the protein pair ferredoxin-ferredoxin reductase (Yah1-Arh1); Alves R et al.; Adrenodoxin reductase homologue (Arh1) and yeast adrenodoxin homologue (Yah1) are essential Saccharomyces cerevisiae mitochondrial proteins involved in heme A biosynthesis and in iron-sulfur cluster (FeSC) assembly . Although the role of Arh1 and Yah1 in heme A biosynthesis is fairly well established, their systemic role on FeSC synthesis is not well understood . Also, while it is thought that the reductase Arh1 provides electrons for the ferredoxin Yah1, two hybrid experiments do not show interaction between the two proteins . In the first part of this article, we use structural bioinformatics methods to evaluate the possibility of interaction between Arh1 and Yah1 . Using protein model building and docking algorithms, we predict a complex between Arh1 and Yah1 that is similar to that of their bovine homologues (adrenodoxin reductase-adrenodoxin), suggesting that Arh1 can indeed reduce Yah1 . The predicted complex allows us to suggest point mutations to either molecule that could hinder Arh1-Yah1 interaction and test the role of Arh1 as the reductase for Yah1 . In the second part of this article, we investigate the physiological role of Arh1-Yah1 on FeSC assembly by deriving alternative mathematical models of the process, based on published information . Comparing the dynamical behavior of each model with that observed in reported experiments emphasizes the importance of Arh1-Yah1 providing electrons for in situ FeSC repair . Only when this mode of action of either of the two proteins in FeSC synthesis is considered can previously reported results be reproduced . Proteins, 2004 Aug 1, 56(2), 188 - 200 Automatic target selection for structural genomics on eukaryotes; Liu J et al.; A central goal of structural genomics is to experimentally determine representative structures for all protein families . At least 14 structural genomics pilot projects are currently investigating the feasibility of high-throughput structure determination; the National Institutes of Health funded nine of these in the United States . Initiatives differ in the particular subset of "all families" on which they focus . At the NorthEast Structural Genomics consortium (NESG), we target eukaryotic protein domain families . The automatic target selection procedure has three aims: 1) identify all protein domain families from currently five entirely sequenced eukaryotic target organisms based on their sequence homology, 2) discard those families that can be modeled on the basis of structural information already present in the PDB, and 3) target representatives of the remaining families for structure determination . To guarantee that all members of one family share a common foldlike region, we had to begin by dissecting proteins into structural domain-like regions before clustering . Our hierarchical approach, CHOP, utilizing homology to PrISM, Pfam-A, and SWISS-PROT chopped the 103,796 eukaryotic proteins/ORFs into 247,222 fragments . Of these fragments, 122,999 appeared suitable targets that were grouped into >27,000 singletons and >18,000 multifragment clusters . Thus, our results suggested that it might be necessary to determine >40,000 structures to minimally cover the subset of five eukaryotic proteomes . J Biol Chem, 2004 Sep 3, 279(36), 37751 - 62 Epub 2004 Jun 21. The bipartite nuclear localization sequence of Rpn2 is required for nuclear import of proteasomal base complexes via karyopherin alphabeta and proteasome functions; Wendler P et al.; 26 S proteasomes fulfill final steps in the ubiquitin-dependent degradation pathway by recognizing and hydrolyzing ubiquitylated proteins . As the 26 S proteasome mainly localizes to the nucleus in yeast, we addressed the question how this 2-MDa multisubunit complex is imported into the nucleus . 26 S proteasomes consist of a 20 S proteolytically active core and 19 S regulatory particles, the latter composed of two subcomplexes, namely the base and lid complexes . We have shown that 20 S core particles are translocated into the nucleus as inactive precursor complexes via the classic karyopherin alphabeta import pathway . Here, we provide evidence that nuclear import of base and lid complexes also depends on karyopherin alphabeta . Potential classic nuclear localization sequences (NLSs) of base subunits were analyzed . Rpn2 and Rpt2, a non-ATPase subunit and an ATPase subunit of the base complex, harbor functional NLSs . The Rpt2 NLS deletion yielded wild type localization . However, the deletion of the Rpn2 NLS resulted in improper nuclear proteasome localization and impaired proteasome function . Our data support the model by which nuclear 26 S proteasomes are assembled from subcomplexes imported by karyopherin alphabeta. Cell, 2004 Jun 25, 117(7), 887 - 98 Cln3 activates G1-specific transcription via phosphorylation of the SBF bound repressor Whi5; de Bruin RA et al.; G1-specific transcriptional activation by Cln3/CDK initiates the budding yeast cell cycle . To identify targets of Cln3/CDK, we analyzed the SBF and MBF transcription factor complexes by multidimensional protein interaction technology (MudPIT) . Whi5 was identified as a stably bound component of SBF but not MBF . Inactivation of Whi5 leads to premature expression of G1-specific genes and budding, whereas overexpression retards those processes . Whi5 inactivation bypasses the requirement for Cln3 both for transcriptional activation and cell cycle initiation . Whi5 associates with G1-specific promoters via SBF during early G1 phase, then dissociates coincident with transcriptional activation . Dissociation of Whi5 is promoted by Cln3 in vivo . Cln/CDK phosphorylation of Whi5 in vitro promotes its dissociation from SBF complexes . Mutation of putative CDK phosphorylation sites, at least five of which are phosphorylated in vivo, strongly reduces SBF-dependent transcription and delays cell cycle initiation . Like mammalian Rb, Whi5 is a G1-specific transcriptional repressor antagonized by CDK. Cell, 2004 Jun 25, 117(7), 849 - 50 RB from a bud's eye view; Schaefer JB et al.; RB and related proteins block transcriptional activation of genes critical to initiation of the cell cycle and suppress unwanted cell division . The circuitry controlling this response is generally conserved from humans to yeast, but no negative regulator like RB has been found in yeast . In this issue of Cell, two studies reveal that Whi5 appears to play the role of RB in preventing precocious cell cycle entry in budding yeast. Nat Cell Biol, 2004 Jul, 6(7), 634 - 41 Epub 2004 Jun 20. Proteolysis-independent regulation of the transcription factor Met4 by a single Lys 48-linked ubiquitin chain; Flick K et al.; The ubiquitin ligase SCF(Met30) is required for cell cycle progression in budding yeast . The critical function of SCF(Met30) is inactivation of the transcriptional activator Met4 . Here we show that a single ubiquitin chain is attached to Met4 through lysine at position 163 . Inhibition of Met4 ubiquitination by mutating lysine to arginine at this position constitutively activates, but does not stabilize, Met4 . This supports a proteolysis-independent role of Cdc34-SCF(Met30)-catalysed Met4 ubiquitination . Surprisingly, the ubiquitin chain attached to Met4 is linked through Lys 48 in ubiquitin, a ubiquitin chain structure that is usually required for substrate targeting to the 26S proteasome . These results suggest that Lys 48-linked ubiquitin chains can have a regulatory role independent of proteolysis. Ann N Y Acad Sci, 2004 May, 1020, 212 - 26 Bayesian decomposition: analyzing microarray data within a biological context; Ochs MF et al.; The detection and correct identification of cancer, especially at an early stage, are vitally important for patient survival and quality of life . Since signaling pathways play critical roles in cancer development and metastasis, methods that reliably assess the activity of these pathways are critical to understand cancer and the response to therapy . Bayesian Decomposition (BD) identifies signatures of expression that can be linked directly to signaling pathway activity, allowing the changes in mRNA levels to be used as downstream indicators of pathway activity . Here, we demonstrate this ability by identifying the downstream expression signal associated with the mating response in Saccharomyces cerevisiae and showing that this signal disappears in deletion mutants of genes critical to the MAPK signaling cascade used to trigger the response . We also show the use of BD in the context of supervised learning, by analyzing the Mus musculus tissue-specific data set provided by Project Normal . The algorithm correctly removes routine metabolic processes, allowing tissue-specific signatures of expression to be identified . Gene ontology is used to interpret these signatures . Since a number of modern therapeutics specifically target signaling proteins, it is important to be able to identify changes in signaling pathways in order to use microarray data to interpret cancer response . By removing routine metabolic signatures and linking specific signatures to signaling pathway activity, BD makes it possible to link changes in microarray results to signaling pathways. Biochem Biophys Res Commun, 2004 Jul 16, 320(1), 197 - 204 In silico characterization of the INO80 subfamily of SWI2/SNF2 chromatin remodeling proteins; Bakshi R et al.; Proteins belonging to SNF2 family of DNA dependent ATPases are important members of the chromatin remodeling complexes that are implicated in epigenetic control of gene expression . The yeast Ino80, the catalytic ATPase subunit of the INO80 complex, is the most recently described member of the SNF2 family . Outside the conserved ATPase domain, it has very little similarity with other well-characterized SNF2 proteins hence it is believed to represent a new subfamily . We have identified new members of this subfamily in different organisms and have detected characteristic features of this subfamily . Using various data mining tools we have identified a new, previously undetected domain in all members of this subfamily . This domain designated DBINO is characteristic of the INO80 subfamily and is predicted to have DNA-binding function . The presence of this domain in all the INO80 subfamily proteins from different organisms suggests its conserved function in evolution. Curr Biol, 2004 Jun 22, 14(12), R483 - 5 Membrane targeting: getting Arl to the Golgi; Graham TR; Post-translational modification with myristoyl or prenyl groups is essential for membrane association of many small GTPases in the Ras superfamily . Two recent papers show that, rather than myristoylation, amino-terminal acetylation of the Arf-like protein Arl3p is required for Golgi targeting via an interaction with an integral membrane protein called Sys1. Neurochem Res, 2004 Jul, 29(7), 1405 - 9 Syntaxin 1A and receptor for activated C kinase interact with the N-terminal region of human dopamine transporter; Lee KH et al.; The dopamine transporter (DAT) regulates the extent and duration of dopamine receptor activation through sodium-dependant reuptake of dopamine into presynaptic neurons, resulting in termination of dopaminergic neurotransmission . Using the yeast two-hybrid system, we have identified novel interactions between DAT, the SNARE protein syntaxin 1A, and the receptor for activated C kinases (RACK1) . This association involves the intracellular N-terminal domain of human DAT (hDAT) . Our data suggest that hDAT may exist as dimers or oligomers and that its protein-protein interactions with syntaxin 1A and RACK1 form functional regulatory complexes that may mediate DAT trafficking through modulation of hDAT phosphorylation by PKC. Nature, 2004 Jun 17, 429(6993), 724 - 30 Structural analysis of a eukaryotic sliding DNA clamp-clamp loader complex; Bowman GD et al.; Sliding clamps are ring-shaped proteins that encircle DNA and confer high processivity on DNA polymerases . Here we report the crystal structure of the five-protein clamp loader complex (replication factor-C, RFC) of the yeast Saccharomyces cerevisiae, bound to the sliding clamp (proliferating cell nuclear antigen, PCNA) . Tight interfacial coordination of the ATP analogue ATP-gammaS by RFC results in a spiral arrangement of the ATPase domains of the clamp loader above the PCNA ring . Placement of a model for primed DNA within the central hole of PCNA reveals a striking correspondence between the RFC spiral and the grooves of the DNA double helix . This model, in which the clamp loader complex locks onto primed DNA in a screw-cap-like arrangement, provides a simple explanation for the process by which the engagement of primer-template junctions by the RFC:PCNA complex results in ATP hydrolysis and release of the sliding clamp on DNA. Toxicol Sci, 2004 Sep, 81(1), 133 - 8 Epub 2004 Jun 16. TCDD affects DNA double strand-break repair; Chan CY et al.; 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), an environmental toxicant, elicits a spectrum of deleterious biological responses including carcinogenesis . We hypothesize that TCDD exposure exerts its carcinogenicity, in part, by affecting the repair of DNA double strand breaks (DSBs) through homologous recombination (HR), mediated by the AhR signaling pathway . To investigate this hypothesis we used a Chinese hamster ovary (CHO) cell line (CHO 33) containing a neo direct repeat recombination reporter substrate to determine whether TCDD affects DNA DSB repair . The Saccharomyces cerevisiae mitochondrial endonuclease I-SceI was used to induce a site specific DSB within the upstream neo recombination substrate in the CHO 33 cells . The cells were then exposed to 500 pM of TCDD in the presence or absence of the AhR antagonist alpha-naphthoflavone (0.1 microM) for 24 h . Two weeks later HR frequencies were determined by counting the number of functional neo expressing, G418-resistant colonies per live cells plated . TCDD significantly increased HR frequency, demonstrating that it does in fact modulate the repair of DNA DSBs . Southern blot analysis of G418-resistant colonies using a cDNA neo probe determined that both gene conversion and gene deletion HR events occurred as a result of DNA DSB repair and TCDD exposure . Exposure of cells to alpha-naphthoflavone resulted in a significant decrease in TCDD-induced HR frequency . These results demonstrate that TCDD, potentially acting via the AhR, can modulate HR repair of DNA DSBs in CHO 33 cells. Proc Natl Acad Sci U S A, 2004 Jun 22, 101(25), 9315 - 20 Epub 2004 Jun 15. Heteroduplex rejection during single-strand annealing requires Sgs1 helicase and mismatch repair proteins Msh2 and Msh6 but not Pms1; Sugawara N et al.; Recombination between moderately divergent DNA sequences is impaired compared with identical sequences . In yeast, an HO endonuclease-induced double-strand break can be repaired by single-strand annealing (SSA) between flanking homologous sequences . A 3% sequence divergence between 205-bp sequences flanking the double-strand break caused a 6-fold reduction in repair compared with identical sequences . This reduction in heteroduplex rejection was suppressed in a mismatch repair-defective msh6 Delta strain and partially suppressed in an msh2 separation-of-function mutant . In mlh1 Delta strains, heteroduplex rejection was greater than in msh6 Delta strains but less than in wild type . Deleting PMS1, MLH2,or MLH3 had no effect on heteroduplex rejection, but a pms1 Delta mlh2 Delta mlh3 Delta triple mutant resembled mlh1 Delta . However, correction of the mismatches within heteroduplex SSA intermediates required PMS1 and MLH1 to the same extent as MSH2 and MSH6 . An SSA competition assay in which either diverged or identical repeats can be used for repair showed that heteroduplex DNA is likely to be unwound rather than degraded . This conclusion is supported by the finding that deleting the SGS1 helicase also suppressed heteroduplex rejection. Mol Cell Biol, 2004 Jul, 24(13), 6029 - 39 Molecular requirements for gene expression mediated by targeted histone acetyltransferases; Jacobson S et al.; Histone acetyltransferases (HATs) play fundamental roles in regulating gene expression . HAT complexes with distinct subunit composition and substrate specificity act on chromatin-embedded genes with different promoter architecture and chromosomal locations . Because requirements for HAT complexes vary, a central question in transcriptional regulation is how different HAT complexes function in different chromosomal contexts . Here, we have tested the ability of targeted yeast HATs to regulate gene expression of an epigenetically silenced locus . Of a panel of HAT fusion proteins targeted to a telomeric reporter gene, Sas3p and Gcn5p selectively increased expression of the silenced gene . Reporter gene expression was not solely dependent on acetyltransferase activity of the targeted HAT . Further analysis of Gcn5p-mediated gene expression revealed collateral requirements for HAT complex subunits Spt8p and Spt3p, which interact with TATA-binding protein, and for a gene-specific transcription factor . These data demonstrate plasticity of gene expression mediated by HATs upon encountering novel promoter architecture and chromatin context . The telomeric location of the reporter gene used in these studies also provides insight into the molecular requirements for heterochromatin boundary formation and for overcoming transcriptional silencing. Mol Cell Biol, 2004 Jul, 24(13), 5639 - 49 Leukemia proto-oncoprotein MLL forms a SET1-like histone methyltransferase complex with menin to regulate Hox gene expression; Yokoyama A et al.; MLL (for mixed-lineage leukemia) is a proto-oncogene that is mutated in a variety of human leukemias . Its product, a homolog of Drosophila melanogaster trithorax, displays intrinsic histone methyltransferase activity and functions genetically to maintain embryonic Hox gene expression . Here we report the biochemical purification of MLL and demonstrate that it associates with a cohort of proteins shared with the yeast and human SET1 histone methyltransferase complexes, including a homolog of Ash2, another Trx-G group protein . Two other members of the novel MLL complex identified here are host cell factor 1 (HCF-1), a transcriptional coregulator, and the related HCF-2, both of which specifically interact with a conserved binding motif in the MLL(N) (p300) subunit of MLL and provide a potential mechanism for regulating its antagonistic transcriptional properties . Menin, a product of the MEN1 tumor suppressor gene, is also a component of the 1-MDa MLL complex . Abrogation of menin expression phenocopies loss of MLL and reveals a critical role for menin in the maintenance of Hox gene expression . Oncogenic mutant forms of MLL retain an ability to interact with menin but not other identified complex components . These studies link the menin tumor suppressor protein with the MLL histone methyltransferase machinery, with implications for Hox gene expression in development and leukemia pathogenesis. Extremophiles, 2004 Jun, 8(3), 253 - 8 Epub 2004 Apr 09. Identification of replication origins in the genome of the methanogenic archaeon, Methanocaldococcus jannaschii; Zhang R et al.; Methanocaldococcus jannaschii has been notorious as an archaeon in which the replication origins are difficult to identify . Although extensive efforts have been exerted on this issue, the locations of replication origins still remain elusive 7 years after the publication of its complete genome sequence in 1996 . Ambiguous results were obtained in identifying the replication origins of M . jannaschii based on all theoretical and experimental approaches . In the genome of M . jannaschii, we found that an ORF (MJ0774), annotated as a hypothetical protein, is a homologue of the Cdc6 protein . The position of the gene is at a global minimum of the x component of the Z curve, i.e., RY disparity curve, which has been used to identify replication origins in other Archaea . In addition, an intergenic region (694,540-695,226 bp) that is between the cdc6 gene and an adjacent ORF shows almost all the characteristics of known replication origins, i.e., it is highly rich in AT composition (80%) and contains multiple copies of repeat elements and AT stretches . Therefore, these lines of evidence strongly suggest that the identified region is a replication origin, which is designated as oriC1 . The analysis of the y component of the Z curve, i.e., MK disparity curve, suggests the presence of another replication origin corresponding to one of the peaks in the MK disparity curve at around 1,388 kb of the genome . Planta, 2004 Sep, 219(5), 856 - 66 Epub 2004 Jun 10. Localization to the rhizoid tip implicates a Fucus distichus Rho family GTPase in a conserved cell polarity pathway; Fowler JE et al.; Generation and expression of cell polarity in brown algal zygotes of the Fucales involve regulation of the actin cytoskeleton and localized secretion . We used degenerate PCR to isolate cDNAs that encode two small GTPases, FdRac1 and FdRab8, from zygotes of Fucus distichus (L.) Powell . Sequence analysis placed FdRac1 in the Rho family, which regulates actin, and FdRab8 in the Rab family, which regulates vesicle transport . As expected, bacterially expressed forms of both proteins bound GTP in vitro . When expressed in budding yeast, FdRac1 showed some functional overlap with CDC42, the Saccharomyces cerevisiae Rho family gene required for yeast cell polarity . Immunolocalization revealed an asymmetric distribution of FdRac1 in polarized zygotes and embryos, with FdRac1 concentrated at or near the growing tip of the algal rhizoid . Our data support the hypothesis that FdRac1 regulates algal cell polarity, possibly via the actin cytoskeleton . Because brown algae belong to the heterokont group, which diverged from other groups early in eukaryotic evolution, we argue that the Rho family function of regulating cell polarity is ancient and may extend throughout the eukaryotes. Cell Mol Life Sci, 2004 Jun, 61(12), 1485 - 97 Molecular characterization of Arabidopsis PHO80-like proteins, a novel class of CDKA;1-interacting cyclins; Torres Acosta JA et al.; Cyclins are regulatory proteins that interact with cyclin-dependent kinases (CDKs) to control progression through the cell cycle . In Arabidopsis thaliana, 34 cyclin genes have been described, grouped into five different types (A, B, D, H, and T) . A novel class of seven cyclins was isolated and characterized in Arabidopsis, designated P-type cyclins (CYCPs) . They all share a conserved central region of 100 amino acids ("cyclin box") displaying homology to the corresponding region of the PHO80 cyclin from Saccharomyces cerevisiae and the related G1 cyclins from Trypanosoma cruzi and T . brucei . The CYCP4;2 gene was able to partially re-establish the phosphate-dependent expression of the PHO5 gene in a pho80 mutant strain of yeast . The CYCPs interact preferentially with CDKA;1 in vivo and in vitro as shown by yeast two-hybrid analysis and co-immunoprecipitation experiments . P-type cyclins were mostly expressed in proliferating cells, albeit also in differentiating and mature tissues . The possible role of CYCPs in linking cell division, cell differentiation, and the nutritional status of the cell is discussed. Genome Res, 2004 Jul, 14(7), 1232 - 47 Epub 2004 Jun 14. Local definition of Ty1 target preference by long terminal repeats and clustered tRNA genes; Bachman N et al.; LTR-containing retrotransposons reverse transcribe their RNA genomes, and the resulting cDNAs are integrated into the genome by the element-encoded integrase protein . The yeast LTR retrotransposon Ty1 preferentially integrates into a target window upstream of tDNAs (tRNA genes) in the yeast genome . We investigated the nature of these insertions and the target window on a genomic scale by analyzing several hundred de novo insertions upstream of tDNAs in two different multicopy gene families . The pattern of insertion upstream of tDNAs was nonrandom and periodic, with peaks separated by approximately 80 bp . Insertions were not distributed equally throughout the genome, as certain tDNAs within a given family received higher frequencies of upstream Ty1 insertions than others . We showed that the presence and relative position of additional tDNAs and LTRs surrounding the target tDNA dramatically influenced the frequency of insertion events upstream of that target . Curr Opin Genet Dev, 2004 Apr, 14(2), 218 - 26 RNA polymerase II structure: from core to functional complexes; Cramer P; New structural studies of RNA polymerase II (Pol II) complexes mark the beginning of a detailed mechanistic analysis of the eukaryotic mRNA transcription cycle . Crystallographic models of the complete Pol II, together with new biochemical and electron microscopic data, give insights into transcription initiation . The first X-ray analysis of a Pol II complex with a transcription factor, the elongation factor TFIIS, supports the idea that the polymerase has a 'tunable' active site that switches between mRNA synthesis and cleavage . The new studies also show that domains of transcription factors can enter polymerase openings, to modulate function during transcription. Curr Opin Genet Dev, 2004 Apr, 14(2), 147 - 54 The highly conserved and multifunctional NuA4 HAT complex; Doyon Y et al.; Histone acetyltransferase complexes have been shown to be key regulators of gene expression . Among these, the NuA4 complex, first characterized in yeast, stands out as it controls multiple key nuclear functions in eukaryotic cells . Many subunits of this protein assembly have been directly linked to global and targeted acetylation of histone H4 tails in vivo, regulation of transcription, cell-cycle progression as well as to the process of DNA repair . Recent studies presented here have established its remarkable structural conservation from yeast to human cells and contributed to the understanding of its diverse functions. Curr Opin Genet Dev, 2004 Apr, 14(2), 139 - 46 Facts about FACT and transcript elongation through chromatin; Belotserkovskaya R et al.; The regulation of transcription elongation within the context of chromatin is a topic of great interest . Even though chromatin presents a barrier to transcription by the PolII machinery in vitro, this process is rather efficient in vivo . Importantly, the chromatin structure of the actively transcribed genes is altered as part of this process . A large number of factors implicated in the control of transcript elongation have been identified through genetics, biochemistry and targeted proteomics approaches . However the precise roles and mechanisms of action of these factors remain obscure . A significant advance came about this past year with the elucidation of the roles of FACT and Spt6 in transcription elongation . These factors facilitate PolII passage through chromatin by destabilizing the nucleosome structure as well as reassemble nucleosomes traversed by PolII. J Biol Chem, 2004 Sep 3, 279(36), 37741 - 50 Epub 2004 Jun 11. Atg21 is required for effective recruitment of Atg8 to the preautophagosomal structure during the Cvt pathway; Meiling-Wesse K et al.; Atg21 and Atg18 are homologue yeast proteins . Whereas Atg18 is essential for the Cvt pathway and autophagy, a lack of Atg21 only blocks the Cvt pathway . Our proteinase protection experiments now demonstrate that growing atg21Delta cells fail to form proaminopeptidase I-containing Cvt vesicles . Quantitative measurement of autophagy in starving atg21Delta cells showed only 35% of the wild-type rate . This suggests that Atg21 plays a nonessential role in improving the fidelity of autophagy . The intracellular localization of Atg21 is unique among the Atg proteins . In cells containing multiple vacuoles, Atg21-yellow fluorescent protein clearly localizes to the vertices of the vacuole junctions . Cells with a single vacuole show most of the protein at few perivacuolar punctae . This distribution pattern is reminiscent to the Vps class C(HOPS) (homotypic fusion and vacuolar protein sorting) protein complex . In growing cells, Atg21 is required for effective recruitment of Atg8 to the preautophagosomal structure . Consistently, the covalent linkage of Atg8 to the lipid phosphatidylethanolamine is significantly retarded . Lipidated Atg8 is supposed to act during the elongation of autophagosome precursors . However, despite the reduced autophagic rate and the retardation of Atg8 lipidation, electron microscopy of starved atg21Delta ypt7Delta double mutant cells demonstrates the formation of normally sized autophagosomes with an average diameter of 450 nm. Cancer Cell, 2004 Jun, 5(6), 519 - 23 Lost in translation: dysregulation of cap-dependent translation and cancer; Bjornsti MA et al.; Activation of the phosphatidylinositol 3' kinase-Akt pathway has long been associated with malignant transformation and antiapoptotic signaling . Mutations downstream of Akt that activate the TOR kinase are found in tumor-prone syndromes, while overexpression of translation initiation complex components, such as eIF4E, occurs frequently in human cancer . However, direct roles for TOR signaling or eIF4E overexpression, in the genesis of cancer, have been lacking . Recent papers, including one by in this issue of Cancer Cell, clearly establish that dysregulation of cap-dependent translation confers malignant characteristics and induces cancer by suppressing apoptosis, underscoring the potential of therapeutics that selectively target the Akt-TOR-eIF4E pathway. Science, 2004 Jun 11, 304(5677), 1644 - 7 Phospholipid metabolism regulated by a transcription factor sensing phosphatidic acid; Loewen CJ et al.; Cells regulate the biophysical properties of their membranes by coordinated synthesis of different classes of lipids . Here, we identified a highly dynamic feedback mechanism by which the budding yeast Saccharomyces cerevisiae can regulate phospholipid biosynthesis . Phosphatidic acid on the endoplasmic reticulum directly bound to the soluble transcriptional repressor Opi1p to maintain it as inactive outside the nucleus . After the addition of the lipid precursor inositol, this phosphatidic acid was rapidly consumed, releasing Opi1p from the endoplasmic reticulum and allowing its nuclear translocation and repression of target genes . Thus, phosphatidic acid appears to be both an essential ubiquitous metabolic intermediate and a signaling lipid. Boll Chim Farm, 2004 Mar, 143(2), 62 - 4 Pharmacokinetics of selenium following oral administration selenium preparation in rabbits; Szulc-Musiol B et al.; The aim of study was to investigate the bioavailability of selenium after oral administration of selenium yeast . As a reference preparation was used sodium selenite . The preparations were investigated in rabbits, according to a randomized two way crossover design in the fasted state . Each animal was given selenium preparation in the form of the single oral dose 0.5 mg Se/kg body weight . A washout period of one week separated both treatment periods . The selenium concentration was determined in serum spectrofluorometry . The divalent equation of one-compartment model was the simplest formula describing the course of selenium changes in serum of rabbits and giving the pharmacokinetic parameters . Pharmacokinetic variables (mean maximum plasma concentration, mean time to reach maximum plasma concentration, and the mean area under the plasma concentration-time curve) were not statistically different for the two preparations . It can be concluded that the two selenium preparations are likely to be bioequivalent. J Biol Chem, 2004 Jul 30, 279(31), 32401 - 6 Epub 2004 Jun 09. Inhibition of TATA binding protein dimerization by RNA polymerase III transcription initiation factor Brf1; Alexander DE et al.; The Brf1 subunit of TFIIIB plays an important role in recruiting the TATA-binding protein (TBP) to the up-stream region of genes transcribed by RNA polymerase III . When TBP is not bound to promoters, it sequesters its DNA binding domain through dimerization . Promoter assembly factors therefore might be required to dissociate TBP into productively binding monomers . Here we show that Saccharomyces cerevisiae Brf1 induces TBP dimers to dissociate . The high affinity TBP binding domain of Brf1 is not sufficient to promote TBP dimer dissociation but in addition requires the TFIIB homology domain of Brf1 . A model is proposed to explain how two distinct functional domains of Brf1 work in concert to dissociate TBP into monomers. Eukaryot Cell, 2004 Jun, 3(3), 598 - 609 The signal from the initiation of meiotic recombination to the first division of meiosis; Malone RE et al.; Two of the unique events that occur in meiosis are high levels of genetic recombination and the reductional division . Our previous work demonstrated that the REC102, REC104, REC114, and RAD50 genes, required to initiate meiotic recombination in Saccharomyces cerevisiae, are needed for the proper timing of the first meiotic (MI) division . If these genes are absent, the MI division actually begins at an earlier time . This paper demonstrates that the meiotic recombination genes MER2/REC107, SPO11, and MRE2 and the synaptonemal complex genes HOP1 and RED1 are also required for the normal delay of the MI division . A rec103/ski8 mutant starts the MI division at the same time as in wild-type cells . Our data indicate no obvious correlation between the timing of premeiotic S phase and the timing of the first division in Rec- mutants . Cells with rec102 or rec104 mutations form MI spindles before wild-type cells, suggesting that the initiation signal acts prior to spindle formation . Neither RAD9 nor RAD24 is needed to transduce the signal, which delays the first division . The timing of the MI division in RAD24 mutants indicates that the pachytene checkpoint is not active in Rec+ cells and suggests that the coordination between recombination and the MI division in wild-type cells may occur primarily due to the initiation signal . Finally, at least one of the targets of the recombination initiation signal is the NDT80 gene, a transcriptional regulator of middle meiotic gene expression required for the first division . Annu Rev Biochem, 2004, 73, 177 - 208 Regulation of telomerase by telomeric proteins; Smogorzewska A et al.; Telomeres are essential for genome stability in all eukaryotes . Changes in telomere functions and the associated chromosomal abnormalities have been implicated in human aging and cancer . Telomeres are composed of repetitive sequences that can be maintained by telomerase, a complex containing a reverse transcriptase (hTERT in humans and Est2 in budding yeast), a template RNA (hTERC in humans and Tlc1 in yeast), and accessory factors (the Est1 proteins and dyskerin in humans and Est1, Est3, and Sm proteins in budding yeast) . Telomerase is regulated in cis by proteins that bind to telomeric DNA . This regulation can take place at the telomere terminus, involving single-stranded DNA-binding proteins (POT1 in humans and Cdc13 in budding yeast), which have been proposed to contribute to the recruitment of telomerase and may also regulate the extent or frequency of elongation . In addition, proteins that bind along the length of the telomere (TRF1/TIN2/tankyrase in humans and Rap1/Rif1/Rif2 in budding yeast) are part of a negative feedback loop that regulates telomere length . Here we discuss the details of telomerase and its regulation by the telomere. Curr Protein Pept Sci, 2004 Jun, 5(3), 201 - 11 Deubiquitinating enzymes are IN/(trinsic to proteasome function); Guterman A et al.; Covalent conjugation of the ubiquitin tag to cellular proteins plays a central role in a number of processes, the most notable among them being degradation by the 26S proteasome . A fundamental property of this process is that ubiquitination, in contrast to subsequent degradation, is reversible due to a number of deubiquitinating enzymes that mediate the disassembly of ubiquitin-protein conjugates . The uniqueness of ubiquitin as a reversible tag necessitates mechanisms to guarantee its efficiency . Interestingly, some deubiquitinating enzymes are associated with the 26S proteasome itself . We include a brief overview of the key proteasome-associated deubiquitinating enzymes such as Rpn11/POH1, UCH37/Uch2, Ubp6/Usp14 and Doa4/Ubp4 . We go on to discuss how these enzymes may contribute to, or possibly counteract, proteolysis by the proteasome . For example, cumulative evidence points to a partitioning of proteasome action between proteolysis and deubiquitination . On the one hand, inhibition of proteolysis promotes deubiquitination, while on the other hand, inhibition of deubiquitination can promote proteolysis . The plethora of deubiquitinating enzymes may serve as proof reading devices altering the equilibrium between these two processes and allowing for reversal of fortune at various stages of the process . To promote degradation over deubiquitination, certain polyubiquitin conformations could be stabilized or protected from deubiquitinating enzymes in order that they can serve as efficient targeting signals leading to the proteasome . We hypothesize that polvubiquitin chains could also serve as "timers": by slowing down chain disassembly, longer chains allow ample time for unfolding and proteolysis of the substrate. Proteomics, 2004 May, 4(5), 1433 - 8 Differential phosphoproteome profiling by affinity capture and tandem matrix-assisted laser desorption/ionization mass spectrometry; Metodiev MV et al.; Protein phosphorylation is a ubiquitous post-translational modification that affects a significant subset of the proteome and plays an especially important role in signal transduction and cell cycle control in eukaryotic organisms . Recently developed methods that couple multidimensional liquid chromatography to electrospray mass spectrometers can be used to analyze entire phosphoproteomes . However, they require considerable investments and technical skills that are only available in a few highly specialized laboratories . These methods also appear to be biased . Statistical analyses show that peptides from abundant proteins and multiply phosphorylated peptides are disproportionately identified . We describe an economic alternative that utilizes a phospho-affinity step to isolate the intact phosphoproteins . These are subsequently characterized by electrophoresis and identified by direct de novo sequencing using tandem mass spectrometry . We applied this technique to probe signal-induced changes in the phosphoproteome of human U937 cells, and found that the pools of two cancer-related phosphoproteins implicated in intracellular hormones signaling are dramatically altered in the course of monocyte to macrophage differentiation. J Immunol, 2004 Jun 15, 172(12), 7548 - 55 Bap29/31 influences the intracellular traffic of MHC class I molecules; Paquet ME et al.; In this study, we examine the role of the putative cargo receptor B cell-associated protein (Bap)29/31 in the export of MHC class I molecules out of the endoplasmic reticulum (ER) . We show that Bap31 binds to two allotypes of mouse class I molecules, with the interaction initiated at the time of H chain association with beta(2)-microglobulin and maintained until the class I molecule has left the ER . We also show that Bap31 is part of the peptide-loading complex, although is not required for its formation . Bap31 binds not only to class I molecules, but can bind to tapasin in the absence of class I . Consistent with an important role in recruiting class I molecules to transport vesicles, we show that in the absence of Bap29/31, there is a loss of class I colocalization with mSec31 (p137), a component of mammalian coat protein complex II coats . This observation is also associated with a delay in class I traffic from ER to Golgi . Our results are consistent with the view that class I molecules are largely recruited to ER exit sites by Bap29/31, and that Bap29/31 is a cargo receptor for MHC class I molecules. Cell, 2004 Jun 11, 117(6), 721 - 33 Mapping global histone acetylation patterns to gene expression; Kurdistani SK et al.; Histone acetyltransferases and deacetylases with specificities for different sites of acetylation affect common chromatin regions . This could generate unique patterns of acetylation that may specify downstream biological processes . To search for existence of these patterns and their relationship to gene activity, we analyzed the genome-wide acetylation profiles for eleven lysines in the four core histones of Saccharomyces cerevisiae . We find that both hyper- and hypoacetylation of individual lysines are associated with transcription, generating distinct patterns of acetylation that define groups of biologically related genes . The genes within these groups are significantly coexpressed, mediate similar physiological processes, share unique cis-regulatory DNA motifs, and are enriched for binding of specific transcription factors . Our data also indicate that the in vivo binding of the transcription factor Bdf1 is associated with acetylation on most lysines but relative deacetylation on H4 lysine 16 . Thus, certain acetylation patterns may be used as surfaces for specific protein-histone interactions, providing one mechanism for coordinate regulation of chromatin processes that are biologically related. Curr Biol, 2004 May 25, 14(10), R397 - 9 Peroxisome membrane biogenesis: the stage is set; Schliebs W et al.; Pex3p and Pex19p are key players in the post-translational import of peroxisomal membrane proteins . New data suggest that these peroxins act in tandem, Pex19p as a cytosolic chaperone and import receptor for peroxisomal membrane proteins, and Pex3p as docking factor at the peroxisomal membrane. Nat Biotechnol, 2004 Jul, 22(7), 871 - 6 Epub 2004 Jun 06. Temperature-sensitive control of protein activity by conditionally splicing inteins; Zeidler MP et al.; Conditional or temperature-sensitive (TS) alleles represent useful tools with which to investigate gene function . Indeed, much of our understanding of yeast has relied on temperature-sensitive mutations which, when available, also provide important insights into other model systems . However, the rarity of temperature-sensitive alleles and difficulty in identifying them has limited their use . Here we describe a system to generate temperature-sensitive alleles based on conditionally active inteins . We have identified temperature-sensitive splicing variants of the yeast Saccharomyces cerevisiae vacuolar ATPase subunit (VMA) intein inserted within Gal4 and transferred these into Gal80 . We show that Gal80-intein(TS) is able to efficiently provide temporal regulation of the Gal4/upstream activation sequence (UAS) system in a temperature-dependent manner in Drosophila melanogaster . Given the minimal host requirements necessary for temperature-sensitive intein splicing, this technique has the potential to allow the generation and use of conditionally active inteins in multiple host proteins and model systems, thereby widening the use of temperature-sensitive alleles for functional protein analysis. Free Radic Biol Med, 2004 Jul 1, 37(1), 23 - 35 Genome-wide transcriptional responses to a lipid hydroperoxide: adaptation occurs without induction of oxidant defenses; Alic N et al.; Free radicals can initiate the oxidation of polyunsaturated fatty acids in cells through the process of lipid peroxidation . The genome-wide transcriptional changes in Saccharomyces cerevisiae after treatment with the toxic lipid peroxidation product linoleic acid hydroperoxide (LoaOOH) were identified . High-dose treatment led to a switch in transcription from biosynthetic to protective functions . This response encompassed a set of genes stimulated predominantly by LoaOOH, and not by other oxidants or heat shock, which contained components of the pleiotropic drug resistance system . The dose dependence of the transcriptional response revealed that large and widespread changes occur only in response to higher doses . Pretreatment of cells with sublethal doses of LoaOOH induces resistance to an otherwise lethal dose through the process of adaptation . Adaptive doses elicited a more subtle transcriptional response affecting metabolic functions, including an increase in the capacity for detoxification and downregulation of the rate of protein synthesis . Surprisingly, the cellular response to adaptive doses did not include induction of oxidative-stress defense enzymes nor of transcripts involved in general cellular defense systems . Biol Cell, 2004 Apr, 96(3), 193 - 9 An overview of the KIN1/PAR-1/MARK kinase family; Tassan JP et al.; Members of the KIN1/PAR-1/MARK kinase family are conserved from yeast to humans and share a similar primary structural organization . Several kinases of this family appear to be at the crossroads of various biological functions including cell polarity, cell cycle control, intracellular signalisation, microtubules stability and protein stability . Here we present an overview of known roles of KIN1/PAR-1/MARK kinases including pEg3 a newly identified member which is regulated during the cell cycle and is a potential regulator of the cell cycle progression . Some common modes of action can be deciphered for this protein kinase family. Curr Biol, 2004 Jun 8, 14(11), 1002 - 6 A barrier to lateral diffusion in the cleavage furrow of dividing mammalian cells; Schmidt K et al.; Barriers to diffusion of proteins and lipids play an important role in generating functionally specialized regions of the plasma membrane . Such barriers have been reported at the base of axons, at the bud neck in Saccharomyces cerevisiae, as well as at the tight junctions of epithelia . How diffusion barriers are formed and how they effect behavior of both inner and outer leaflets of the bilayer are not fully understood . Here, we provide evidence for a cortical barrier to diffusion within the cleavage furrow of mammalian cells . Photobleaching-based assays were used to measure diffusion of three membrane proteins with differing topologies and putative lipid raft association, as well as the lipid analog dialkylindocarbocyanine (DiI C18, ), across the cleavage furrow . There was a block in diffusion of proteins with a cytosolic domain, but not of proteins anchored in the outer leaflet of the PM or of DiI . Diffusion of lipid raft proteins in the inner and outer leaflets of the membrane was not directly coupled . The distribution of Septin proteins, as opposed to cortical actin, was consistent with a functional role in limiting diffusion . Plant Physiol, 2004 Jun, 135(2), 1027 - 39 Epub 2004 Jun 04. A novel family of cys-rich membrane proteins mediates cadmium resistance in Arabidopsis; Song WY et al.; Cadmium (Cd) is a widespread pollutant that is toxic to plant growth . However, only a few genes that contribute to Cd resistance in plants have been identified . To identify additional Cd(II) resistance genes, we screened an Arabidopsis cDNA library using a yeast (Saccharomyces cerevisiae) expression system employing the Cd(II)-sensitive yeast mutant ycf1 . This screening process yielded a small Cys-rich membrane protein (Arabidopsis plant cadmium resistance, AtPcrs) . Database searches revealed that there are nine close homologs in Arabidopsis . Homologs were also found in other plants . Four of the five homologs that were tested also increased resistance to Cd(II) when expressed in ycf1 . AtPcr1 localizes at the plasma membrane in both yeast and Arabidopsis . Arabidopsis plants overexpressing AtPcr1 exhibited increased Cd(II) resistance, whereas antisense plants that showed reduced AtPcr1 expression were more sensitive to Cd(II) . AtPcr1 overexpression reduced Cd uptake by yeast cells and also reduced the Cd contents of both yeast and Arabidopsis protoplasts treated with Cd . Thus, it appears that the Pcr family members may play an important role in the Cd resistance of plants. Bioinformatics, 2004 Nov 22, 20(17), 3013 - 20 Epub 2004 Nov 22. Protein complex prediction via cost-based clustering; King AD et al.; MOTIVATION: Understanding principles of cellular organization and function can be enhanced if we detect known and predict still undiscovered protein complexes within the cell's protein-protein interaction (PPI) network . Such predictions may be used as an inexpensive tool to direct biological experiments . The increasing amount of available PPI data necessitates an accurate and scalable approach to protein complex identification . RESULTS: We have developed the Restricted Neighborhood Search Clustering Algorithm (RNSC) to efficiently partition networks into clusters using a cost function . We applied this cost-based clustering algorithm to PPI networks of Saccharomyces cerevisiae, Drosophila melanogaster and Caenorhabditis elegans to identify and predict protein complexes . We have determined functional and graph-theoretic properties of true protein complexes from the MIPS database . Based on these properties, we defined filters to distinguish between identified network clusters and true protein complexes . Conclusions: Our application of the cost-based clustering algorithm provides an accurate and scalable method of detecting and predicting protein complexes within a PPI network. Curr Protein Pept Sci, 2004 Jun, 5(3), 177 - 84 Cullin-based ubiquitin ligase and its control by NEDD8-conjugating system; Chiba T et al.; Several studies have examined the importance of ubiquitin-like posttranslational modifiers (which consist of an unexpectedly large family) . Of these, NEDD8 (also called Rub1, related to ubiquitin 1) with a high homology to ubiquitin is covalently linked to all members of cullin (Cul)-family proteins through an enzymatic cascade analogous to ubiquitylation . Cul-family proteins are scaffold proteins for a wide series of ubiquitin-protein ligase complexes, such as SCFs (Skp1, Cul-1, Roc1, and F-box proteins), which regulate the degradation of broad range of cellular proteins . Unlike ubiquitin, which mostly acts as a degradation signal for the target proteins, NEDD8 acts as an activation signal for Cul-family proteins; i.e., Cul-based ubiquitin-protein ligases . Accordingly, the NEDD8 conjugation pathway regulating Cul-protein function is responsible for a diverse array of biologically important processes, such as the cell cycle progression, signalling cascades and developmental programs . Furthermore, recent studies have revealed that the COP9/Signalosome complex interacts physically and genetically with Cul-family proteins, and catalyzes deconjugation of NEDD8 ligated to Cul-family proteins . This review summarizes recent advances in biochemical and genetic studies on how the NEDD8-modifying system regulates Cul-family proteins and their physiology. Mol Cells, 2004 Apr 30, 17(2), 223 - 9 Interaction of tomato mosaic virus movement protein with tobacco RIO kinase; Yoshioka K et al.; Tomato mosaic virus (ToMV) has a regulatory gene encoding a movement protein (MP) that is involved in the cell-to-cell movement of viral RNA through plasmodesmata . To identify the host cell factors interacting with ToMV MP, we used a recombinant MP probe to isolate cDNA clones from a phage expression library of Nicotiana tabacum by a far-Western screening method . One of the cDNA clones encoded an MP-interacting protein, MIP-T7, homologous to the yeast novel protein kinase, Rio1p . We isolated a full-length cDNA by RT-PCR . The putative gene product was designated NtRIO, and shared 33 and 73% amino acid identity with yeast and Arabidopsis RIO kinases, respectively . In vitro analyses using recombinant proteins showed that NtRIO also interacted with a different MP derived from Cucumber mosaic virus . NtRIO had autophosphorylation activity and phosphorylated ToMV MP . Addition of recombinant tobacco casein kinase 2 resulted in a marked increase in the phosphorylation of NtRIO . The interaction between NtRIO and ToMV MP was inhibited by phosphorylation of NtRIO. Proc Natl Acad Sci U S A, 2004 Jun 15, 101(24), 8987 - 92 Epub 2004 Jun 03. Spatiotemporal retinoid-X receptor activation detected in live vertebrate embryos; Luria A et al.; Most studies on the nuclear retinoid-X receptor (RXR) have focused on its role as a heterodimeric partner but less about its own activation pattern during development and the distribution of potential endogenous ligands . The aim of this study is to visualize the distribution of activated RXRalpha in live transgenic Xenopus laevis embryos across a wide range of developmental stages . We adopted a nuclear receptor-Gal4 fusion/upstream activation sequence-based reporter system for our assay . Strong activation of the RXRalpha ligand-binding domain was observed in a segment of the spinal cord just posterior to the hindbrain . This activation is first detected in neurula stage embryos and persists up to swimming tadpole stages, after which activation strongly declines . Addition of exogenous ligands, such as 9-cis retinoic acid or all-trans retinoic acid, expands the activation of RXR throughout the spinal cord but not in the brain, whereas the RXR-specific ligand LG268 expanded the Gal4-RXR activation into the brain and olfactory epithelia . Treatment with the RAR-specific ligand 4-(E-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl)benzoic acid or thyroid hormone had no effect on Gal4-RXR activation, whereas these compounds significantly increased their corresponding Gal4/receptor fusion proteins under similar conditions . Embryos expressing a Gal4-RXR fusion protein with a deletion in the ligand-dependent activation domain (AF2) show no reporter gene activation . The results shown in this paper reveal a specific activation pattern for Gal4-RXRalpha specifically in the developing spinal cord and suggest the existence of RXR ligand "hot-spots" in this region. J Biol Chem, 2004 Aug 6, 279(32), 33456 - 62 Epub 2004 Jun 03. Isohumulones, bitter acids derived from hops, activate both peroxisome proliferator-activated receptor alpha and gamma and reduce insulin resistance; Yajima H et al.; The peroxisome proliferator-activated receptors (PPARs) are dietary lipid sensors that regulate fatty acid and carbohydrate metabolism . The hypolipidemic effects of fibrate drugs and the therapeutic benefits of the thiazolidinedione drugs are due to their activation of PPARalpha and -gamma, respectively . In this study, isohumulones, the bitter compounds derived from hops that are present in beer, were found to activate PPARalpha and -gamma in transient co-transfection studies . Among the three major isohumulone homologs, isohumulone and isocohumulone were found to activate PPARalpha and -gamma . Diabetic KK-Ay mice that were treated with isohumulones (isohumulone and isocohumulone) showed reduced plasma glucose, triglyceride, and free fatty acid levels (65.3, 62.6, and 73.1%, respectively, for isohumulone); similar reductions were found following treatment with the thiazolidinedione drug, pioglitazone . Isohumulone treatment did not result in significant body weight gain, although pioglitazone treatment did increase body weight (10.6% increase versus control group) . C57BL/6N mice fed a high fat diet that were treated with isohumulones showed improved glucose tolerance and reduced insulin resistance . Furthermore, these animals showed increased liver fatty acid oxidation and a decrease in size and an increase in apoptosis of their hypertrophic adipocytes . A double-blind, placebo-controlled pilot study for studying the effect of isohumulones on diabetes suggested that isohumulones significantly decreased blood glucose and hemoglobin A1c levels after 8 weeks (by 10.1 and 6.4%, respectively, versus week 0) . These results suggest that isohumulones can improve insulin sensitivity in high fat diet-fed mice with insulin resistance and in patients with type 2 diabetes. J Biol Chem, 2004 Jul 30, 279(31), 32373 - 84 Epub 2004 Jun 03. PINA is essential for growth and positively influences NIMA function in Aspergillus nidulans; Joseph JD et al.; The phospho-Ser/Thr-directed prolyl-isomerase Pin1 was originally identified in vertebrate systems as a negative regulator of NIMA, a Ser/Thr protein kinase that regulates the G(2)/M transition in Aspergillus nidulans . Here we explore the physiological roles of the Pin1 orthologue, PINA, in A . nidulans and evaluate the relevance of the interaction of PINA with NIMA in this fungus . We find pinA to be an essential gene in A . nidulans . In addition, when PINA levels are reduced 50-fold the cells grow at a reduced rate . Upon germination under conditions that repress PINA expression, the cells are delayed in the interphase activation of NIMX(cdc2), whereas they traverse the other phases of the cell cycle at a similar rate to controls . These results indicate that a marked reduction of PINA results in a lengthening of G(1) . Additionally, PINA repression increases the rate at which the cells enter mitosis following release from a hydroxyurea arrest without altering the sensitivity of the fungus to agents that activate the replication or DNA damage checkpoints . In contrast to predictions based on Pin1, the physical interaction between PINA and NIMA is primarily dependent upon the prolylisomerase domain of PINA and the C-terminal 303 amino acids of NIMA . Finally, reduction of PINA levels exacerbates the nimA5 temperature-sensitive mutant, whereas overexpression of PINA decreases the severity of this mutation, results that are consistent with a positive genetic interaction between PINA and NIMA . Thus, although PINA is essential and positively regulates NIMA function, A . nidulans is most sensitive to a reduction in PINA concentration in G(1) rather than in G(2)/M. Biochem Biophys Res Commun, 2004 Jun 25, 319(2), 456 - 63 Dibasic amino acid residues at the carboxy-terminal end of kinase homology domain participate in the plasma membrane localization and function of phosphatidylinositol 5-kinase gamma; Arioka M et al.; Type I phosphatidylinositol 4-phosphate (PI(4)P) 5-kinases (PIP5Ks) catalyze the synthesis of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)), an essential lipid molecule involved in various cellular processes such as regulation of actin cytoskeleton and membrane traffic . The protein localizes to the plasma membrane where its activity has been shown to be regulated by small GTPase ARFs and/or phosphatidic acid . Deletion analysis of amino- or carboxy-terminal sequences of PIP5Kgamma fused with EGFP demonstrated that the presence of central kinase homology domain (KHD), a 380 amino acid-long region highly conserved among PIP5K family, was necessary and sufficient for the plasma membrane localization of PIP5Kgamma . Particularly, the dibasic Arg-Lys sequence located at the carboxy-terminal end of KHD was shown to be crucial for the plasma membrane targeting of PIP5Kgamma, since the deletion or charge-reversal mutation of this dibasic sequence resulted in the mislocalization of the protein to the cytoplasm . Mislocalized mutants also failed to complement the temperature-sensitive growth of Saccharomyces cerevisiae mss4-1 mutant defective in PIP5K function . The presence of dibasic residues at the C-terminal end of KHD was conserved among mammalian as well as invertebrate PIP5K family members, but not in the type II PIPKs that are not targeted to the plasma membrane, suggesting that the conserved dibasic motif provides a mechanism essential for the proper localization and cellular function of PIP5Ks. Carbohydr Res, 2004 Jun 1, 339(8), 1453 - 7 Synthesis, (1-->3)-beta-D-glucanase-binding ability, and phytoalexin-elicitor activity of a mixture of 3,4-epoxybutyl (1-->3)-beta-D-oligoglucosides; Huang GL et al.; We describe a approach for the synthesis of a mixture of 3,4-epoxybutyl (1-->3)-beta-D-oligoglucosides . The particular (1-->3)-beta-D-glucan isolated from the cell walls of Saccharomyces cerevisiae was recovered from the aqueous medium as water-insoluble particles by the spray drying (GS) method, and it was characterized by FTIR spectroscopy . The acid-solubilized (1-->3)-beta-D-oligoglucosides were prepared by partial acid hydrolysis of glucan particles, which were qualitatively analyzed by fluorophore-assisted carbohydrate electrophoresis (FACE) . The peracetylated 3-butenyl (1-->3)-beta-D-oligoglucosides were synthesized by treating peracetylated (1-->3)-beta-D-oligoglucosides with the 3-butenyl alcohols and a Lewis acid (SnCl4) catalyst . Epoxidation of the peracetylated 3-butenyl oligoglucosides took place with m-chloroperoxybenzoic acid (m-CPBA) . NaOMe in dry methanol was used for the deacetylation of the blocked derivatives, to give the 3,4-epoxybutyl (1-->3)-beta-D-oligoglucoside mixture in an overall yield of 21% . The sample was analyzed by positive-ion electrospray ionization mass spectrometry (ESIMS) . In a 3,4-epoxybutyl (1-->3)-beta-D-oligoglucoside-binding (1-->3)-beta-D-glucanase assay, we found that the (1-->3)-beta-D-glucanase was obviously inactivated by the 3,4-epoxybutyl (1-->3)-beta-D-oligoglucosides . At the same time, we found the 3,4-epoxybutyl (1-->3)-beta-D-oligoglucoside mixture was more active as compared to the underivatized oligoglucoside mixture in eliciting phytoalexin accumulation in tobacco cotyledon tissue . Furthermore, it could be kept for a longer time than a (1-->3)-beta-D-oligoglucoside mixture, which indicated it is much more stable than (1-->3)-beta-D-oligoglucosides . FEBS Lett, 2004 Jun 4, 567(2-3), 302 - 6 Arabidopsis homologues of the autophagy protein Atg8 are a novel family of microtubule binding proteins; Ketelaar T et al.; Autophagy is the non-selective transport of proteins and superfluous organelles destined for degradation to the vacuole in fungae, or the lysosome in animal cells . Some of the genes encoding components of the autophagy pathway are conserved in plants, and here we show that Arabidopsis homologues of yeast Atg8 (Apg8/Aut7) and Atg4 (Apg4/Aut2) partially complement the yeast deletion strains . The yeast double mutant, a deletion strain with respect to both Atg8 and Atg4, could not be complemented by Arabidopsis Atg8, indicating that Arabidopsis Atg8 requires Atg4 for its function . Moreover, Arabidopsis Atg8 and Arabidopsis Atg4 interact directly in a two-hybrid assay . Interestingly, Atg8 shows significant homology with the microtubule binding light chain 3 of MAP1A and B, and here we show that Arabidopsis Atg8 binds microtubules . Our results demonstrate that a principle component of the autophagic pathway in plants is similar to that in yeast and we suggest that microtubule binding plays a role in this process. FEBS Lett, 2004 Jun 4, 567(2-3), 259 - 64 Regulatory mechanisms controlling biogenesis of ubiquitin and the proteasome; London MK et al.; Analysis of several Saccharomyces cerevisiae ump mutants with defects in ubiquitin (Ub)-mediated proteolysis yielded insights into the regulation of the polyubiquitin gene UBI4 and of proteasome genes . High-molecular weight Ub-protein conjugates accumulated in ump mutants with impaired proteasome function with a concomitant decrease in the amount of free Ub . In these mutants, transcriptional induction of UBI4 was depending in part on the transcription factor Rpn4 . Deletion of UBI4 partially suppressed the growth defects of ump1 mutants, indicating that accumulation of polyubiquitylated proteins is deleterious to cell growth . Transcription of proteasome subunit genes was induced in ump mutants affecting the proteasome, as well as under conditions that mediate DNA damage or the formation of abnormal proteins . This induction required the transcriptional activator Rpn4 . Elevated Rpn4 levels in proteasome-deficient mutants or as a response to abnormal proteins were due to increased metabolic stability . Up-regulation of proteasome genes in response to DNA damage, in contrast, is shown to operate via induction of RPN4 transcription. Gene, 2004 May 26, 333, 135 - 41 Representing GC variation along eukaryotic chromosomes; Paces J et al.; Genome sequencing now permits direct visual representation, at any scale, of GC heterogeneity along the chromosomes of several higher eukaryotes . Plots can be easily obtained from the chromosomal sequences, yet sequence releases of mammalian or plant chromosomes still tend to use small scales or window sizes that obscure important large-scale compositional features . To faithfully reveal, at one glance, the compositional variation at a given scale, we have devised a simple scheme that combines line plots with color-coded shading of the regions underneath the plots . The scheme can be applied to different eukaryotic genomes to facilitate their comparison, as illustrated here for a sample of chromosomes chosen from seven selected species . As a complement to a previously published compact view of isochores in the human genome sequence, we include here an analogous map for the recently sequenced mouse genome, and discuss the contribution of repetitive DNA to the GC variation along the plots . Supplementary information, including a database of color-coded GC profiles for all recently sequenced eukaryotes and the program draw_chromosomes_gc.pl used to obtain them, are available at. DNA Repair (Amst), 2004 Jul 2, 3(7), 769 - 76 The Hog1 MAP kinase pathway and the Mec1 DNA damage checkpoint pathway independently control the cellular responses to hydrogen peroxide; Haghnazari E et al.; The DNA damage checkpoint pathway and the MAP kinase pathway respond to various forms of environmental as well as endogenous stresses through signal transduction mechanisms involving protein kinases . Both pathways are intertwined in mammalian cells, but potential crosstalk between these two pathways in budding yeast has not been examined yet . We show that the Rad53 checkpoint kinase and the Hog1 MAP kinase of Saccharomyces cerevisiae become phosphorylated upon exposure to hydrogen peroxide, indicative of activation of the DNA damage checkpoint and MAP kinase pathways in response to oxidative stress . Rad53 kinase is equally activated in wild type and in hog1-Delta cells . Likewise, the activation of Hog1 MAP kinase is not dependent on Mec1 kinase, the central checkpoint kinase in budding yeast . Mutants in either pathway are sensitive to hydrogen peroxide and the double mutants exhibit a near perfectly additive phenotype . These data demonstrate that the DNA damage checkpoint pathway and the MAP kinase pathway respond to oxidative stress independently of each other and suggest that these two stress signaling pathways are activated by different types of insults induced by hydrogen peroxide. DNA Repair (Amst), 2004 Jul 2, 3(7), 729 - 42 Mutations in the nucleotide-binding domain of MutS homologs uncouple cell death from cell survival; Drotschmann K et al.; After genotoxic insult, the decision to repair or undergo cell death is pivotal for undamaged cell survival, and requires a highly controlled coordination of both pathways . Disruption of this regulation results in tumorigenesis and failure of cancer therapy . Mismatch repair (MMR) proteins have a unique role by contributing to both pathways, though direct evidence for their function in the DNA damage response is ambiguous . We report separation of function mutants in the ATPase domains of yeast MutS homologous (MSH) proteins that uncouple MMR-dependent DNA repair from damage response to cisplatin . While mutations in the ATPase domain have devastating effects on the mutation rate of the cell, ATPase processing is mostly dispensable for the cell death phenotype; only limited processing by the MSH6 subunit is required in DNA damage response . Different DNA binding patterns and nucleotide sensitivity of Msh2/Msh6-DNA adduct and protein-mismatch complexes, respectively, suggest that the presence of different DNA lesions influences the requirement for ATP . Limited proteolysis of purified protein gives first indications for differences in nucleo