Mol Cell Biol, 2005 Feb, 25(3), 896 - 906 Rejoining of DNA double-strand breaks as a function of overhang length; Daley JM et al.; The ends of spontaneously occurring double-strand breaks (DSBs) may contain various lengths of single-stranded DNA, blocking lesions, and gaps and flaps generated by end annealing . To investigate the processing of such structures, we developed an assay in which annealed oligonucleotides are ligated onto the ends of a linearized plasmid which is then transformed into Saccharomyces cerevisiae . Reconstitution of a marker occurs only when the oligonucleotides are incorporated and repair is in frame, permitting rapid analysis of complex DSB ends . Here, we created DSBs with compatible overhangs of various lengths and asked which pathways are required for their precise repair . Three mechanisms of rejoining were observed, regardless of overhang polarity: nonhomologous end joining (NHEJ), a Rad52-dependent single-strand annealing-like pathway, and a third mechanism independent of the first two mechanisms . DSBs with overhangs of less than 4 bases were mainly repaired by NHEJ . Repair became less dependent on NHEJ when the overhangs were longer or had a higher GC content . Repair of overhangs greater than 8 nucleotides was as much as 150-fold more efficient, impaired 10-fold by rad52 mutation, and highly accurate . Reducing the microhomology extent between long overhangs reduced their repair dramatically, to less than NHEJ of comparable short overhangs . These data support a model in which annealing energy is a primary determinant of the rejoining efficiency and mechanism. J Cell Biol, 2005 Jan 17, 168(2), 315 - 28 Effects of Arp2 and Arp3 nucleotide-binding pocket mutations on Arp2/3 complex function; Martin AC et al.; Contributions of actin-related proteins (Arp) 2 and 3 nucleotide state to Arp2/3 complex function were tested using nucleotide-binding pocket (NBP) mutants in Saccharomyces cerevisiae . ATP binding by Arp2 and Arp3 was required for full Arp2/3 complex nucleation activity in vitro . Analysis of actin dynamics and endocytosis in mutants demonstrated that nucleotide-bound Arp3 is particularly important for Arp2/3 complex function in vivo . Severity of endocytic defects did not correlate with effects on in vitro nucleation activity, suggesting that a critical Arp2/3 complex function during endocytosis may be structural rather than catalytic . A separate class of Arp2 and Arp3 NBP mutants suppressed phenotypes of mutants defective for actin nucleation . An Arp2 suppressor mutant increased Arp2/3 nucleation activity . Electron microscopy of Arp2/3 complex containing this Arp2 suppressor identified a structural change that also occurs upon Arp2/3 activation by nucleation promoting factors . These data demonstrate the importance of Arp2 and Arp3 nucleotide binding for nucleating activity, and Arp3 nucleotide binding for maintenance of cortical actin cytoskeleton cytoarchitecture. Genes Dev, 2005 Jan 15, 19(2), 255 - 69 The control of Spo11's interaction with meiotic recombination hotspots; Prieler S et al.; Programmed double-strand breaks (DSBs), which initiate meiotic recombination, arise through the activity of the evolutionary conserved topoisomerase homolog Spo11 . Spo11 is believed to catalyze the DNA cleavage reaction in the initial step of DSB formation, while at least a further 11 factors assist in Saccharomyces cerevisiae . Using chromatin-immunoprecipitation (ChIP), we detected the transient, noncovalent association of Spo11 with meiotic hotspots in wild-type cells . The establishment of this association requires Rec102, Rec104, and Rec114, while the timely removal of Spo11 from chromatin depends on several factors, including Mei4 and Ndt80 . In addition, at least one further component, namely, Red1, is responsible for locally restricting Spo11's interaction to the core region of the hotspot . In chromosome spreads, we observed meiosis-specific Spo11-Myc foci, independent of DSB formation, from leptotene until pachytene . In both rad50S and com1Delta/sae2Delta mutants, we observed a novel reaction intermediate between Spo11 and hotspots, which leads to the detection of full-length hotspot DNA by ChIP in the absence of artificial cross-linking . Although this DNA does not contain a break, its recovery requires Spo11's catalytic residue Y135 . We propose that detection of uncross-linked full-length hotspot DNA is only possible during the reversible stage of the Spo11 cleavage reaction, in which rad50S and com1Delta/sae2Delta mutants transiently arrest. Mol Genet Genomics . 2005 Jan 15; {Epub ahead of print} Characterisation of Aspergillus niger prolyl aminopeptidase; Basten DE et al.; We have cloned a gene (papA) that encodes a prolyl aminopeptidase from Aspergillus niger . Homologous genes are present in the genomes of the Eurotiales A . nidulans, A . fumigatus and Talaromyces emersonii, but the gene is not present in the genome of the yeast Saccharomyces cerevisiae . Cell extracts of strains overexpressing the gene under the control of its own promoter showed a fourfold to sixfold increase in prolyl aminopeptidase activity, but no change in phenylalanine or leucine aminopeptidase activity . The overexpressed enzyme was subsequently purified and characterised . The enzyme specifically removes N-terminal proline and hydroxyproline residues from peptides . It is the first enzyme of its kind from a eukaryotic organism that has been characterised. J Biol Chem . 2005 Jan 18; {Epub ahead of print} Silencing near tRNA genes requires nucleolar localization; Wang L et al.; Transcription by RNA polymerase II is antagonized by the presence of a nearby tRNA gene in Saccharomyces cerevisiae . To test hypotheses concerning the mechanism of this tRNA gene-mediated (tgm) silencing, the effects of specific gene deletions were determined . The results show that the mechanism of silencing near tRNA genes is fundamentally different from other forms of transcriptional silencing in yeast . Rather, tgm silencing is dependent on the ability to cluster the dispersed tRNA genes in or near the nucleolus, constituting a form of three-dimensional gene control. Nucleic Acids Res, 2005 Jan 14, 33(1), 388 - 99 Print 2005. Plant tRNA ligases are multifunctional enzymes that have diverged in sequence and substrate specificity from RNA ligases of other phylogenetic origins; Englert M et al.; Pre-tRNA splicing is an essential process in all eukaryotes . It requires the concerted action of an endonuclease to remove the intron and a ligase for joining the resulting tRNA halves as studied best in the yeast Saccharomyces cerevisiae . Here, we report the first characterization of an RNA ligase protein and its gene from a higher eukaryotic organism that is an essential component of the pre-tRNA splicing process . Purification of tRNA ligase from wheat germ by successive column chromatographic steps has identified a protein of 125 kDa by its potentiality to covalently bind AMP, and by its ability to catalyse the ligation of tRNA halves and the circularization of linear introns . Peptide sequences obtained from the purified protein led to the elucidation of the corresponding proteins and their genes in Arabidopsis and Oryza databases . The plant tRNA ligases exhibit no overall sequence homologies to any known RNA ligases, however, they harbour a number of conserved motifs that indicate the presence of three intrinsic enzyme activities: an adenylyltransferase/ligase domain in the N-terminal region, a polynucleotide kinase in the centre and a cyclic phosphodiesterase domain at the C-terminal end . In vitro expression of the recombinant Arabidopsis tRNA ligase and functional analyses revealed all expected individual activities . Plant RNA ligases are active on a variety of substrates in vitro and are capable of inter- and intramolecular RNA joining . Hence, we conclude that their role in vivo might comprise yet unknown essential functions besides their involvement in pre-tRNA splicing. Trends Biochem Sci, 2005 Jan, 30(1), 7 - 10 SAGA unveiled; Timmers HT et al.; Transcriptional regulation in eukaryotes is intimately coupled to chromatin dynamics . The SAGA (Spt-Ada-Gcn5) histone acetyltransferase (HAT) complex of Saccharomyces cerevisiae is a multi-subunit co-factor for RNA polymerase II transcription . However, not all gene activation events require its intrinsic HAT activity . In addition, SAGA subunits can also restrict gene transcription . The recently published structural model from the laboratories of Fred Winston and Patrick Schultz of the SAGA complex provides a framework to rationalize these findings and to direct further investigation of this crucial transcriptional co-factor. Cell, 2005 Jan 14, 120(1), 99 - 110 Coordinated Remodeling of Cellular Metabolism during Iron Deficiency through Targeted mRNA Degradation; Puig S et al.; Iron (Fe) is an essential micronutrient for virtually all organisms and serves as a cofactor for a wide variety of vital cellular processes . Although Fe deficiency is the primary nutritional disorder in the world, cellular responses to Fe deprivation are poorly understood . We have discovered a posttranscriptional regulatory process controlled by Fe deficiency, which coordinately drives widespread metabolic reprogramming . We demonstrate that, in response to Fe deficiency, the Saccharomyces cerevisiae Cth2 protein specifically downregulates mRNAs encoding proteins that participate in many Fe-dependent processes . mRNA turnover requires the binding of Cth2, an RNA binding protein conserved in plants and mammals, to specific AU-rich elements in the 3' untranslated region of mRNAs targeted for degradation . These studies elucidate coordinated global metabolic reprogramming in response to Fe deficiency and identify a mechanism for achieving this by targeting specific mRNA molecules for degradation, thereby facilitating the utilization of limited cellular Fe levels. Cancer Gene Ther . 2005 Jan 14; {Epub ahead of print} Delta24-hyCD adenovirus suppresses glioma growth in vivo by combining oncolysis and chemosensitization; Conrad C et al.; Replication-competent adenoviruses could provide an efficient method for delivering therapeutic genes to tumors . The most promising strategies among adenovirus-based oncolytic systems are designed to exploit free E2F-1 activity in cancer cells, which in the absence of pRb activates transcription and regulates the expression of genes involved in differentiation, proliferation, and apoptosis . We previously developed Delta24, an E1A-mutant, conditionally replicative oncolytic adenovirus . Here, we examine the ability of a second-generation Delta24 (Delta24-hyCD) engineered to express a humanized form of the Saccharomyces cerevisiae cytosine deaminase gene (hyCD) . Real-time quantitative PCR, Western blotting, thin-layer chromatography, and radioisotope quantitative enzymatic assays confirmed the production of a catalytically active hyCD enzyme in the setting of an oncolytic infection in vitro; other experiments assessing local production of 5-fluorouracil and a concomitant bystander effect showed improved cytotoxicity . The IC(50) dose of 5-fluorocytosine (5-FC) required for a complete cytopathic effect by the Delta24-hyCD virus was fivefold lower than with Delta24 alone in U251MG and U87MG malignant glioma (MG) cell lines . Intratumoral treatment of mice bearing intracranial U87MG xenografts with Delta24-hyCD+5-FC significantly improved survival, confirming that Delta24-hyCD with 5-FC is a more efficient anticancer tool than Delta24 alone . Histopathologically, Delta24-hyCD replication was accompanied by progressively augmented oncolysis and drug-induced necrosis . These findings demonstrate that Delta24-hyCD with concomitant systemic 5-FC is a significant improvement over the earlier Delta24 oncolytic tumor-selective strategy for therapy of experimental gliomas.Cancer Gene Therapy advance online publication, 14 January 2005; doi:10.1038/sj.cgt.7700750. Nature, 2005 Jan 13, 433(7022), 171 - 6 Stabilization of microtubule dynamics at anaphase onset promotes chromosome segregation; Higuchi T et al.; Microtubules of the mitotic spindle form the structural basis for chromosome segregation . In metaphase, microtubules show high dynamic instability, which is thought to aid the 'search and capture' of chromosomes for bipolar alignment on the spindle . Microtubules suddenly become more stable at the onset of anaphase, but how this change in microtubule behaviour is regulated and how important it is for the ensuing chromosome segregation are unknown . Here we show that in the budding yeast Saccharomyces cerevisiae, activation of the phosphatase Cdc14 at anaphase onset is both necessary and sufficient for silencing microtubule dynamics . Cdc14 is activated by separase, the protease that triggers sister chromatid separation, linking the onset of anaphase to microtubule stabilization . If sister chromatids separate in the absence of Cdc14 activity, microtubules maintain high dynamic instability; this correlates with defects in both the movement of chromosomes to the spindle poles (anaphase A) and the elongation of the anaphase spindle (anaphase B) . Cdc14 promotes localization of microtubule-stabilizing proteins to the anaphase spindle, and dephosphorylation of the kinetochore component Ask1 contributes to both the silencing of microtubule turnover and successful anaphase A. J Virol, 2005 Feb, 79(3), 1438 - 51 Mutual Interference between Genomic RNA Replication and Subgenomic mRNA Transcription in Brome Mosaic Virus; Grdzelishvili VZ et al.; Replication by many positive-strand RNA viruses includes genomic RNA amplification and subgenomic mRNA (sgRNA) transcription . For brome mosaic virus (BMV), both processes occur in virus-induced, membrane-associated compartments, require BMV replication factors 1a and 2a, and use negative-strand RNA3 as a template for genomic RNA3 and sgRNA syntheses . To begin elucidating their relations, we examined the interaction of RNA3 replication and sgRNA transcription in Saccharomyces cerevisiae expressing 1a and 2a, which support the full RNA3 replication cycle . Blocking sgRNA transcription stimulated RNA3 replication by up to 350%, implying that sgRNA transcription inhibits RNA3 replication . Such inhibition was independent of the sgRNA-encoded coat protein and operated in cis . We further found that sgRNA transcription inhibited RNA3 replication at a step or steps after negative-strand RNA3 synthesis, implying competition with positive-strand RNA3 synthesis for negative-strand RNA3 templates, viral replication factors, or common host components . Consistent with this, sgRNA transcription was stimulated by up to 400% when mutations inhibiting positive-strand RNA3 synthesis were introduced into the RNA3 5'-untranslated region . Thus, BMV subgenomic and genomic RNA syntheses mutually interfered with each other, apparently by competition for one or more common factors . In plant protoplasts replicating all three BMV genomic RNAs, mutations blocking sgRNA transcription often had lesser effects on RNA3 accumulation, possibly because RNA3 also competed with RNA1 and RNA2 replication templates and because any increase in RNA3 replication at the expense of RNA1 and RNA2 would be self-limited by decreased 1a and 2a expression from RNA1 and RNA2. Nucleic Acids Res, 2005 Jan 12, 33(1), 171 - 81 Print 2005. Functional roles of carboxylate residues comprising the DNA polymerase active site triad of Ty3 reverse transcriptase; Bibillo A et al.; Aspartic acid residues comprising the -D-(aa) n -Y-L-D-D- DNA polymerase active site triad of reverse transcriptase from the Saccharomyces cerevisiae long terminal repeat-retrotransposon Ty3 (Asp151, Asp213 and Asp214) were evaluated via site-directed mutagenesis . An Asp151-->Glu substitution showed a dramatic decrease in catalytic efficiency and a severe translocation defect following initiation of DNA synthesis . In contrast, enzymes harboring the equivalent alteration at Asp213 and Asp214 retained DNA polymerase activity . Asp151-->Asn and Asp213-->Asn substitutions eliminated both polymerase activities . However, while Asp214 of the triad could be replaced by either Asn or Glu, introducing Gln seriously affected processivity . Mutants of the carboxylate triad at positions 151 and 213 also failed to catalyze pyrophosphorolysis . Finally, alterations to the DNA polymerase active site affected RNase H activity, suggesting a close spatial relationship between these N- and C-terminal catalytic centers . Taken together, our data reveal a critical role for Asp151 and Asp213 in catalysis . In contrast, the second carboxylate of the Y-L-D-D motif (Asp214) is not essential for catalysis, and possibly fulfills a structural role . Although Asp214 was most insensitive to substitution with respect to activity of the recombinant enzyme, all alterations at this position were lethal for Ty3 transposition. J Biol Chem . 2005 Jan 11; {Epub ahead of print} The mammalian YL1 protein is a shared subunit of the TRRAP/TIP60 histone acetyltransferase and SRCAP complexes; Cai Y et al.; The multiprotein mammalian TRRAP/TIP60-containing histone acetyltransferase (HAT) complex performs critical functions in a variety of cellular processes including transcriptional activation, double strand DNA break repair, and apoptosis . We previously isolated the TRRAP/TIP60 complex from HeLa cells (Cai, Y., Jin, J., Tomomori-Sato, C., Sato, S., Sorokina, I., Parmely, T.J., Conaway, R.C., and Conaway, J.W . (2003) J . Biol . Chem . 278, 42733-42736) . Analysis of proteins present in preparations of the TRRAP/TIP60 complex led to the identification of several new subunits, as well as several potential subunits including the YL1 protein . Here we present evidence that the YL1 protein is a previously unrecognized subunit of the TRRAP/TIP60 HAT complex . In addition, we present evidence that YL1 is also a component of a novel mammalian multiprotein complex that includes the SNF2-related helicase SRCAP and resembles the recently described Saccharomyces cerevisiae SWR1 chromatin remodeling complex . Taken together, our findings identify the YL1 protein as a new subunit of the TRRAP/TIP60 HAT complex, and they suggest that YL1 plays multiple roles in chromatin modification and remodeling in cells. Yeast . 2005 Jan 11;22(2):141-155 {Epub ahead of print} SKG1, a suppressor gene of synthetic lethality of kex2Deltagas1Delta mutations, encodes a novel membrane protein that affects cell wall composition; Tomishige N et al.; The fungal GAS1-related genes encode GPI-anchored beta-1,3-glucanosyltransferase, and their loss causes a defect in the assembly of the cell wall . The KEX2 gene encodes a processing protease in the late Golgi compartment and its loss also results in defects in the cell wall . Simultaneous mutations of these genes are lethal in Saccharomyces cerevisiae . To understand the basis of this synthetic lethality, we screened for multicopy suppressors and identified 13 SKG (suppressor of kex2 gas1 synthetic lethality) genes . SKG1 encodes a transmembrane protein that localizes on the inner surface of the plasma membrane at the bud and in the daughter cell . The multicopy SKG1 increases the sensitivity of cells to zymolyase, and the skg1Delta null mutation increases resistance to it . This zymolyase susceptibility corresponds to an increase of alkali-soluble beta-1,3-glucan and a decrease of chitin in the cell wall . Thus SKG1 encodes a novel protein that affects the cell wall polymer composition in the growing region of the cell . Copyright (c) 2005 John Wiley & Sons, Ltd. J Biol Chem . 2005 Jan 11; {Epub ahead of print} The 1.1 A structure of the spindle checkpoint protein Bub3p reveals functional regions; Wilson DK et al.; Bub3p is a protein that mediates the spindle checkpoint, a signaling pathway that ensures correct chromosome segregation in organisms ranging from yeast to mammals . It is known to function by co-localizing at least two other proteins, Mad3p and the protein kinase Bub1p to the kinetochore of chromosomes which are not properly attached to mitotic spindles, ultimately resulting in cell cycle arrest . Prior sequence analysis suggested that Bub3p was composed of three or four WD repeats (also known as WD40 and ss-transducin repeats), short sequence motifs appearing in clusters of 4 to 16 found in many hundreds of eukaryotic proteins which fold into four stranded blade-like sheets . We have determined the crystal structure of Bub3p from Saccharomyces cerevisiae at 1.1 A and a crystallographic R-factor of 15.3%, revealing seven authentic repeats . In light of this, it appears that many of these repeats therefore remain hidden in sequences of other proteins . Analysis of random and site-directed mutants identifies the surface of Bub3p involved in checkpoint function through binding of Bub1p and Mad3p . Sequence alignments indicate that these surfaces are mostly conserved across Bub3 proteins from diverse species . A structural comparison with other proteins containing WD repeats suggests that these folds may bind partner proteins using similar surface areas on the top and sides of the propeller . The sequences composing these regions are the most divergent within the repeat across all WD repeat proteins and could potentially be modulated to provide specificity in partner protein binding without perturbation of the core structure. Eukaryot Cell, 2005 Jan, 4(1), 103 - 10 Proposed Function of the Accumulation of Plasma Membrane-Type Ca2+-ATPase mRNA in Resting Cysts of the Ciliate Sterkiella histriomuscorum; Lescasse R et al.; From an mRNA differential-display analysis of the encystment-excystment cycle of the ciliate Sterkiella histriomuscorum, we have isolated an expressed sequence tag encoding a plasma membrane-type Ca(2+)-ATPase (PMCA) . PMCAs are located either in the plasma membranes or in the membranes of intracellular organelles, and their function is to pump calcium either out of the cell or into the intracellular calcium stores, respectively . The S . histriomuscorum macronuclear PMCA gene (ShPMCA) and its corresponding cDNA were cloned; it is the first member of the Ca(2+)-ATPase family identified in Sterkiella . The predicted protein of 1,065 amino acids exhibits 37% identity with PMCAs of diverse organisms . A phylogenetic analysis showed its relatedness to homologs of two alveolates: the ciliate Paramecium tetraurelia and the apicomplexan Toxoplasma gondii . Overexpression of the protein ShPMCA failed to rescue the wild-type phenotype of three Ca(2+)-ATPase-defective mutant strains of Saccharomyces cerevisiae; this failure contrasts with the reported ability of the PMCAs of parasites to complement defects in yeast . ShPMCA mRNA is markedly accumulated during encystment and in resting cysts, suggesting a function during excystment . To address the possibility of a signaling role for calcium at excystment, the capacity of calcium to induce excystment was examined. Eukaryot Cell, 2005 Jan, 4(1), 63 - 71 Tor and Cyclic AMP-Protein Kinase A: Two Parallel Pathways Regulating Expression of Genes Required for Cell Growth; Zurita-Martinez SA et al.; In the budding yeast Saccharomyces cerevisiae, the Tor and cyclic AMP-protein kinase A (cAMP-PKA) signaling cascades respond to nutrients and regulate coordinately the expression of genes required for cell growth, including ribosomal protein (RP) and stress-responsive (STRE) genes . The inhibition of Tor signaling by rapamycin results in repression of the RP genes and induction of the STRE genes . Mutations that hyperactivate PKA signaling confer resistance to rapamycin and suppress the repression of RP genes imposed by rapamycin . By contrast, partial inactivation of PKA confers rapamycin hypersensitivity but only modestly affects RP gene expression . Complete inactivation of PKA impairs RP gene expression and concomitantly enhances STRE gene expression; remarkably, this altered transcriptional pattern is still sensitive to rapamycin and thus subject to Tor control . These findings illustrate how the Tor and cAMP-PKA signaling pathways respond to nutrient signals to govern gene expression required for cell growth via two parallel routes, and they have broad implication for our understanding of analogous regulatory networks in normal and neoplastic mammalian cells. Structure (Camb), 2005 Jan, 13(1), 143 - 53 Crystal Structure and Functional Implications of Pyrococcus furiosus Hef Helicase Domain Involved in Branched DNA Processing; Nishino T et al.; DNA and RNA frequently form various branched intermediates that are important for the transmission of genetic information . Helicases play pivotal roles in the processing of these transient intermediates during nucleic acid metabolism . The archaeal Hef helicase/ nuclease is a representative protein that processes flap- or fork-DNA structures, and, intriguingly, its C-terminal half belongs to the XPF/Mus81 nuclease family . Here, we report the crystal structure of the helicase domain of the Hef protein from Pyrococcus furiosus . The structure reveals a novel helical insertion between the two conserved helicase core domains . This positively charged extra region, structurally similar to the "thumb" domain of DNA polymerase, plays critical roles in fork recognition . The Hef helicase/nuclease exhibits sequence similarity to the Mph1 helicase from Saccharomyces cerevisiae; XPF/Rad1, involved in DNA repair; and a putative Hef homolog identified in mammals . Hence, our findings provide a structural basis for the functional mechanisms of this helicase/nuclease family. Yi Chuan, 2004 Nov, 26(6), 929 - 33 {Analysis of the mRNA expression similarity of genes in the same gene expression regulatory pathway.}; Li CX et al.; In this work we analysed the relationship of gene expression from the point of view of gene expression regulatory pathway . Using seven sets of Saccharomyces cerevisiae gene chip expression profile data, and information from two pathway database (KEGG and CYGD), we analysed the mRNA expression similarity of genes in the same gene expression regulatory pathway by Genehub software, that involved totally 16 pathways with more than 495 genes . From the calculation of two different similarity measures-Pearson correlation coefficient and Spearman correlation coefficient, we found that about 94 percent of gene expression regulatory pathways are correlatively expressed in more than 4(including 4) sets of expression profile data, and it presents additional evidence for the correlation between gene function and its expression in the view of gene expression regulatory pathways. Science, 2005 Jan 7, 307(5706), 130 - 3 The centromeric protein Sgo1 is required to sense lack of tension on mitotic chromosomes; Indjeian VB et al.; Chromosome alignment on the mitotic spindle is monitored by the spindle checkpoint . We identify Sgo1, a protein involved in meiotic chromosome cohesion, as a spindle checkpoint component . Budding yeast cells with mutations in SGO1 respond normally to microtubule depolymerization but not to lack of tension at the kinetochore, and they have difficulty attaching sister chromatids to opposite poles of the spindle . Sgo1 is thus required for sensing tension between sister chromatids during mitosis, and its degradation when they separate may prevent cell cycle arrest and chromosome loss in anaphase, a time when sister chromatids are no longer under tension. FEBS J, 2005 Jan, 272(1), 47 - 58 Structural and functional analysis of the interaction of the AAA-peroxins Pex1p and Pex6p; Birschmann I et al.; The AAA-peroxins Pex1p and Pex6p play a critical role in peroxisome biogenesis but their precise function remains to be established . These two peroxins consist of three distinct regions (N, D1, D2), two of which (D1, D2) contain a conserved approximately 230 amino acid cassette, which is common to all ATPases associated with various cellular activities (AAA) . Here we show that Pex1p and Pex6p from Saccharomyces cerevisiae do interact in vivo . We assigned their corresponding binding sites and elucidated the importance of ATP-binding and -hydrolysis of Pex1p and Pex6p for their interaction . We show that the interaction of Pex1p and Pex6p involves their first AAA-cassettes and demonstrate that ATP-binding but not ATP-hydrolysis in the second AAA-cassette (D2) of Pex1p is required for the Pex1p-Pex6p interaction . Furthermore, we could prove that the second AAA-cassettes (D2) of both Pex1p and Pex6p were essential for peroxisomal biogenesis and thus probably comprise the overall activity of the proteins. J Food Prot, 2004 Dec, 67(12), 2741 - 6 Influence of pH on complexing of model beta-d-glucans with zearalenone; Yiannikouris A et al.; Previous studies have shown that isolated beta-(1,3 and 1,6)-D-glucans and related alkali-extracted fractions from the cell wall of Saccharomyces cerevisiae are able to complex with zearalenone in vitro (affinity up to 50%) and thus may reduce the bioavailability of toxins in the digestive tract . The complexation mechanisms involve cooperative interaction between the two chemical entities that can be computed by Hill's model . Various linear or branched soluble or insoluble beta-D-glucans were evaluated to elucidate their roles in the adsorption mechanisms under three pH conditions (3.0, 6.0, and 8.0) found in the digestive tract . A constant quantity of each beta-D-glucans (1 mg/ml) was mixed at 39 degrees C with increasing amounts of zearalenone (2 to 100 microg/ml), and the amount of bound toxin was measured . Acidic and neutral conditions gave the highest affinity rates (64 to 77%) by beta-(1,3)-D-glucans, whereas alkaline conditions decreased adsorption except when beta-(1,6)-D-glucan side chains were branched on beta-(1,3)-D-glucans . Alkaline conditions appear to impede the active three dimensional conformation of beta-D-glucans and favor single helix and/or random coil structures . Study of the equilibrium between beta-D-glucan-bound and free toxins revealed that two types of chemical interactions occur during toxin complexation with beta-D-glucans, identified as weak chemical linkages such as hydrogen and van der Waals bonds. Mol Cell Proteomics . 2005 Jan 4; {Epub ahead of print} Double standards in quantitative proteomics: Direct comparative assessment of difference in Gel electrophoresis (DiGE)and metabolic stable isotope labeling; Kolkman A et al.; Quantitative protein expression profiling is a crucial part of proteomics and requires methods that are able to efficiently provide accurate and reproducible differential expression values for proteins in two or more biological samples . In this report we evaluate in a direct comparative assessment two state-of-the-art quantitative proteomics approaches, namely difference in gel electrophoresis (DiGE) and metabolic stable isotope labeling . Therefore, Saccharomyces cerevisiae was grown under well-defined experimental conditions in chemostats under two single-nutrient limited growth conditions, using 14N or 15N ammonium sulfate as single nitrogen source . Following lysis and protein extraction from the two yeast samples, the proteins were fluorescently labeled using different fluorescent CyDyes . Subsequently, the yeast samples were mixed and the proteins separated by 2D gel electrophoresis . Following in-gel digestion the resulting peptides were analyzed by mass spectrometry using a MALDI-TOF mass spectrometer . Relative ratios in protein expression between these two yeast samples were determined using both DiGE and metabolic stable isotope labeling . Focusing on a small, albeit representative set of proteins covering the whole gel-range, including some protein isoforms, and ranging from low- to high abundance, we observe that the correlation between these two methods of quantification is good, with the differential ratios determined following the equation RMet.Lab . = 0.98 RDiGE, with r2 = 0.89 . Although the correlation between DiGE and metabolic stable isotope labeling is exceptionally good, we do observe and discuss (dis)advantages of both methods as well in relation to other (quantitative) approaches. J Biol Chem . 2005 Jan 4; {Epub ahead of print} Functional comparison of human and Drosophila hop reveals novel role in steroid receptor maturation; Carrigan PE et al.; Hsp70/Hsp90 organizing protein (Hop) coordinates Hsp70 and Hsp90 interactions during assembly of steroid receptor complexes . Hop is composed of three tetratricopeptide repeat (TPR) domains (TPR1, TPR2a, and TPR2b) and two DP-repeat domains (DP1 and DP2); Hsp70 interacts directly with TPR1 and Hsp90 with TPR2a, but the function of other domains is less clear . Human Hop (hHop) and the Saccharomyces cerevisiae ortholog Sti1p, which share a common domain arrangement, are functionally interchangeable in a yeast growth assay and in supporting efficient maturation of glucocorticoid receptor (GR) function . To gain a better understanding of Hop structure/ function relationships, we have extended comparisons to the Hop ortholog from Drosophila melanogaster (dHop), which lacks DP1 . Although dHop binds Hsp70 and Hsp90 and can rescue the growth defect in yeast lacking Sti1p, dHop fails to support GR function in yeast, which suggests a novel role for Hop in GR maturation that goes beyond Hsp binding . Chimeric Hop constructs combining human and Drosophila domains demonstrate that the C-terminal domain DP2 is critical for this previously unrecognized role in steroid receptor function. J Biol Chem . 2005 Jan 4; {Epub ahead of print} The DNA damage checkpoint response requires histone H2B ubiquitination by Rad6-Bre1 and H3 methylation by Dot1; Giannattasio M et al.; The cellular response to DNA lesions entails the recruitment of several checkpoint and repair factors to damaged DNA, and chromatin modifications may play a role in this process . Here we show that in Saccharomyces cerevisiae epigenetic modification of histones is required for checkpoint activity in response to a variety of genotoxic stresses . We demonstrate that ubiquitination of histone H2B on lysine 123, by the Rad6-Bre1 complex, is necessary for activation of Rad53 kinase and cell cycle arrest . We found a similar requirement for Dot1-dependent methylation of histone H3 . Loss of H3-K79 methylation does not affect Mec1 activation, while it renders cells checkpoint defective by preventing phosphorylation of Rad9 . Such results suggest that histone modifications may have a role in checkpoint function by modulating the interactions of Rad9 with chromatin and active Mec1 kinase. Mol Cell Biol, 2005 Jan, 25(2), 637 - 51 Histone H2B Ubiquitylation Is Associated with Elongating RNA Polymerase II; Xiao T et al.; Rad6-mediated ubiquitylation of histone H2B at lysine 123 has been linked to transcriptional activation and the regulation of lysine methylation on histone H3 . However, how Rad6 and H2B ubiquitylation contribute to the transcription and histone methylation processes is poorly understood . Here, we show that the Paf1 transcription elongation complex and the E3 ligase for Rad6, Bre1, mediate an association of Rad6 with the hyperphosphorylated (elongating) form of RNA polymerase II (Pol II) . This association appears to be necessary for the transcriptional activities of Rad6, as deletion of various Paf1 complex members or Bre1 abolishes H2B ubiquitylation (ubH2B) and reduces the recruitment of Rad6 to the promoters and transcribed regions of active genes . Using the inducible GAL1 gene as a model, we find that the recruitment of Rad6 upon activation occurs rapidly and transiently across the gene and coincides precisely with the appearance of Pol II . Significantly, during GAL1 activation in an rtf1 deletion mutant, Rad6 accumulates at the promoter but is absent from the transcribed region . This fact suggests that Rad6 is recruited to promoters independently of the Paf1 complex but then requires this complex for entrance into the coding region of genes in a Pol II-associated manner . In support of a role for Rad6-dependent H2B ubiquitylation in transcription elongation, we find that ubH2B levels are dramatically reduced in strains bearing mutations of the Pol II C-terminal domain (CTD) and abolished by inactivation of Kin28, the serine 5 CTD kinase that promotes the transition from initiation to elongation . Furthermore, synthetic genetic array analysis reveals that the Rad6 complex interacts genetically with a number of known or suspected transcription elongation factors . Finally, we show that Saccharomyces cerevisiae mutants bearing defects in the pathway to H2B ubiquitylation display transcription elongation defects as assayed by 6-azauracil sensitivity . Collectively, our results indicate a role for Rad6 and H2B ubiquitylation during the elongation cycle of transcription and suggest a mechanism by which H3 methylation may be regulated. Mol Cell Biol, 2005 Jan, 25(2), 575 - 89 Phosphatidylinositol 3-Kinase-Mediated Effects of Glucose on Vacuolar H+-ATPase Assembly, Translocation, and Acidification of Intracellular Compartments in Renal Epithelial Cells; Sautin YY et al.; Vacuolar H(+)-ATPases (V-ATPases) are a family of ATP-driven proton pumps . They maintain pH gradients between intracellular compartments and are required for proton secretion out of the cytoplasm . Mechanisms of extrinsic control of V-ATPase are poorly understood . Previous studies showed that glucose is an important regulator of V-ATPase assembly in Saccharomyces cerevisiae . Human V-ATPase directly interacts with aldolase, providing a coupling mechanism for glucose metabolism and V-ATPase function . Here we show that glucose is a crucial regulator of V-ATPase in renal epithelial cells and that the effect of glucose is mediated by phosphatidylinositol 3-kinase (PI3K) . Glucose stimulates V-ATPase-dependent acidification of the intracellular compartments in human proximal tubular cells HK-2 and porcine renal epithelial cells LLC-PK(1) . Glucose induces rapid ATP-independent assembly of the V(1) and V(o) domains of V-ATPase and extensive translocation of the V-ATPase V(1) and V(o) domains between different membrane pools and between membranes and the cytoplasm . In HK-2 cells, glucose stimulates polarized translocation of V-ATPase to the apical plasma membrane . The effects of glucose on V-ATPase trafficking and assembly can be abolished by pretreatment with the PI3K inhibitor LY294002 and can be reproduced in glucose-deprived cells by adenoviral expression of the constitutively active catalytic subunit p110alpha of PI3K . Taken together these data provide evidence that, in renal epithelial cells, glucose plays an important role in the control of V-ATPase-dependent acidification of intracellular compartments and V-ATPase assembly and trafficking and that the effects of glucose are mediated by PI3K-dependent signaling. Mol Cell, 2005 Jan 7, 17(1), 145 - 51 A kinase-independent function of cks1 and cdk1 in regulation of transcription; Yu VP et al.; We describe a function in transcription for the Saccharomyces cerevisiae cell cycle regulatory cyclin-dependent kinase Cdc28 (Cdk1) and its interacting protein, Cks1 . The Cks1/Cdc28 complex is recruited to multiple coding regions in the genome and is necessary for efficient expression of a significant subset of genes . This transcriptional role is mediated through a requirement of Cdc28/Cks1 for recruiting proteasomes to coding regions . However, it is independent of the protein kinase activity of Cdc28 . In the absence of Cks1, neither Cdc28 nor the proteasome can be recruited . Consequently, there is a failure to maintain efficient transcription. Mol Cell, 2005 Jan 7, 17(1), 61 - 8 An Extended Signal Involved in Eukaryotic -1 Frameshifting Operates through Modification of the E Site tRNA; Bekaert M et al.; By using a sensitive search program based on hidden Markov models (HMM), we identified 74 viruses carrying frameshift sites among 1500 fully sequenced virus genomes . These viruses are clustered in specific families or genera . Sequence analysis of the frameshift sites identified here, along with previously characterized sites, identified a strong bias toward the two nucleotides 5' of the shifty heptamer signal . Functional analysis in the yeast Saccharomyces cerevisiae demonstrated that high frameshifting efficiency is correlated with the presence of a Psi39 modification in the tRNA present in the E site of the ribosome at the time of frameshifting . These results demonstrate that an extended signal is involved in eukaryotic frameshifting and suggest additional interactions between tRNAs and the ribosome during decoding. Biochim Biophys Acta, 2005 Jan 5, 1686(3), 181 - 9 Specific formation of arachidonic acid and eicosapentaenoic acid by a front-end Delta5-desaturase from Phytophthora megasperma; Hornung E et al.; The biosynthesis of arachidonic acid (20:4(Delta5Z,8Z,11Z,14Z)) from linoleic acid in plants by transgenic means requires the sequential and specific action of two desaturation reactions and one elongation reaction . Here, we describe the isolation of a specific acyl-lipid-desaturase catalyzing the formation of the double bond at position 5 from a cDNA library from Phytophthora megasperma . The isolated full-length cDNA harbors a sequence of 1740 bp encoding a protein of 477 amino acids with a calculated molecular weight of 53.5 kDa . The desaturase sequence contained a predicted N-terminal cytochrome b(5)-like domain, as well as three histidine-rich domains . For functional identification, the cDNA was expressed in Saccharomyces cerevisiae, and the formation of newly formed fatty acids was analyzed . The expression of the heterologous enzyme resulted in the formation of arachidonic acid after di-homo-gamma-linolenic acid supplementation and in the formation of eicosapentaenoic acid synthesis from omega3-arachidonic acid . Results presented here on the substrate specificity identify this expressed protein as a classical Delta5-acyl-lipid-desaturase, capable of specifically introducing a double bond at the Delta5 position solely in 20-carbon-atom chain length fatty acids containing a double bond at position Delta8 . Detailed analysis of the different lipid species showed a preferential occurrence of the desaturation reaction for fatty acids esterified to phosphatidylcholine. Vaccine, 2005 Jan 19, 23(9), 1170 - 7 Development of a recombinant Leishmania major strain sensitive to ganciclovir and 5-fluorocytosine for use as a live vaccine challenge in clinical trials; Davoudi N et al.; To provide a safer live challenge strain for use in clinical vaccine trials, a double drug sensitive strain of Leishmania major was derived using advances in gene targeting technology by stably introducing into the chromosome a modified HSV-1 thymidine kinase gene (tk), conferring increased sensitivity to ganciclovir (GCV), and a Saccharomyces cerevisiae cytosine deaminase gene (cd), conferring sensitivity to 5-fluorocytosine (5-FC) . In vitro studies showed that the homozygous L . major (tk-cd(+/+)) promastigotes were killed by either drug alone, and together the drugs acted synergistically . In vivo infection studies showed that progressively growing lesions in BALB/c mice, caused by L . major (tk-cd(+/+)), were completely cured by 2 weeks of treatment with either drug alone or in combination . Treated animals showed no signs of reoccurrence of infection for at least 4 months when the experiments were terminated. Biochemistry, 2005 Jan 11, 44(1), 321 - 328 Is the Prion Domain of Soluble Ure2p Unstructured? Pierce MM, Baxa U, Steven AC, Bax A, Wickner RB. The {URE3} prion is a self-propagating amyloid form of the Ure2 protein of Saccharomyces cerevisiae . Deletions in the C-terminal nitrogen regulation domain of Ure2p increase the frequency with which the N-terminal prion domain polymerizes into the prion form, suggesting that the C-terminus stabilizes the prion domain or that the structured C-terminal region sterically impairs amyloid formation . We find by in vivo two-hybrid analysis no evidence of interaction of prion domain and C-terminal domain . Furthermore, surface plasmon resonance spectrometry shows no evidence of interaction of prion domain and C-terminal domain, and cleavage at a specific site between the domains frees the two fragments . Our NMR analysis indicates that most residues of the prion domain are in fact disordered in the soluble form of Ure2p . Deleting the tether holding the C-terminal structured region to the amyloid core does not impair prion formation, arguing against steric impairment of amyloid formation . These results suggest that the N-terminal prion domain is unstructured in the soluble protein and does not have a specific interaction with the C-terminus. Biochemistry, 2005 Jan 11, 44(1), 213 - 24 Reaching for Mechanistic Consensus Across Life Kingdoms: Structure and Insights into Catalysis of the myo-Inositol-1-phosphate Synthase (mIPS) from Archaeoglobus fulgidus(,); Stieglitz KA et al.; myo-Inositol-1-phosphate synthase (mIPS) catalyzes the first step in the synthesis of l-myo-inositol-1-phosphate . We have solved and refined the structure of the mIPS from the hyperthermophilic sulfate reducer Archaeoglobus fulgidus at 1.9 A resolution . The enzyme crystallized from poly(ethylene glycol) in the P1 space group with one tetramer in the asymmetric unit and provided a view of the entire biologically active oligomer . Despite significant changes in sequence length and amino acid composition, the general architecture of the archaeal enzyme is similar to that of the eukaryotic mIPS from Saccharomyces cerevisiae and bacterial mIPS from Mycobacterium tuberculosis . The enhanced thermostability of the archaeal enzyme as compared to that from yeast is consistent with deletion of a number of surface loops that results in a significantly smaller protein . In the structure of the A . fulgidus mIPS, the active sites of all four subunits were fully ordered and contained NAD(+) and inorganic phosphate . The structure also contained a single metal ion (identified as K(+)) in two of the four subunits . The analysis of the electrostatic potential maps of the protein suggested the presence of a second metal-ion-binding site in close proximity to the first metal ion and NAD(+) . The modeling of the substrate and known inhibitors suggests a critical role for the second metal ion in catalysis and provides insights into the common elements of the catalytic cycle in enzymes from different life kingdoms. J Biol Chem . 2005 Jan 4; {Epub ahead of print} PIG-V involved in transferring the second mannose in glycosylphosphatidylinositol; Young Kang J et al.; Glycosylphosphatidylinositol (GPI) is a glycolipid that anchors many proteins to the eukaryotic cell surface . The biosynthetic pathway of GPI is mediated by sequential additions of sugars and other components to phosphatidylinositol (PI) . Four mannoses in the GPI are transferred from dolichol-phosphate-mannose (Dol-P-Man) and are linked through different glycosidic linkages . Therefore, four Dol-P-Man-dependent mannosyltransferases, GPI-MT-I, -MT-II, -MT-III and -MT-IV for the first, second, third and fourth mannoses, respectively, are required for generation of GPI . GPI-MT-I (PIG-M), GPI-MT-III (PIG-B) and GPI-MT-IV (SMP3) were previously reported but GPI-MT-II has remained to be identified . Here we report the cloning of PIG-V involved in transferring the second mannose in the GPI anchor . Human PIG-V encodes a 493-amino-acid, ER resident protein with eight putative transmembrane regions . Saccharomyces cerevisiae protein encoded in ORF YBR004c, which we termed GPI18, has 25% amino acid identity to human PIG-V . Viability of the yeast gpi18 deletion mutant was restored by human PIG-V cDNA . PIG-V has two functionally important conserved regions facing the ER lumen . Taken together, we suggest that PIG-V is the second mannosyltransferase in GPI anchor biosynthesis. Biopolymers . 2004 Dec 27; {Epub ahead of print} Synthetic peptides as probes for conformational preferences of domains of membrane receptors; Naider F et al.; Peptide models have been widely used to investigate conformational aspects of domains of proteins since the early 1950s . A pioneer in this field was Dr . Murray Goodman, who applied a battery of methodologies to study the onset of structure in homooligopeptides . This article reviews some of Dr . Goodman's contributions, and reports recent studies using linear and constrained peptides corresponding to the first extracellular loop and linear peptides corresponding to the sixth transmembrane domain of a G-protein coupled receptor from the yeast Saccharomyces cerevisiae . Peptides containing 30-40 residues were synthesized using solid-phase methods and purified to near homogeneity by reversed phase high performance liquid chromatography . CD and NMR analyses indicated that the first extracellular loop peptides were mostly flexible in water, and assumed some helical structure near the N-terminus in trifluoroethanol and in the presence of micelles . Comparison of oligolysines with native loop residues revealed that three lysines at each terminus of a peptide corresponding to the sixth transmembrane domain of the alpha-factor receptor resulted in better aqueous solubility and greater helicity than the native loop residues . (c) 2004 Wiley Periodicals, Inc . Biopolymers (Pept Sci), 2005. Plant Physiol, 2005 Jan, 137(1), 117 - 26 Epub 2004 Dec 23. The AtProT Family . Compatible Solute Transporters with Similar Substrate Specificity But Differential Expression Patterns; Grallath S et al.; Proline transporters (ProTs) mediate transport of the compatible solutes Pro, glycine betaine, and the stress-induced compound gamma-aminobutyric acid . A new member of this gene family, AtProT3, was isolated from Arabidopsis (Arabidopsis thaliana), and its properties were compared to AtProT1 and AtProT2 . Transient expression of fusions of AtProT and the green fluorescent protein in tobacco (Nicotiana tabacum) protoplasts revealed that all three AtProTs were localized at the plasma membrane . Expression in a yeast (Saccharomyces cerevisiae) mutant demonstrated that the affinity of all three AtProTs was highest for glycine betaine (K(m) = 0.1-0.3 mm), lower for Pro (K(m) = 0.4-1 mm), and lowest for gamma-aminobutyric acid (K(m) = 4-5 mm) . Relative quantification of the mRNA level using real-time PCR and analyses of transgenic plants expressing the beta-glucuronidase (uidA) gene under control of individual AtProT promoters showed that the expression pattern of AtProTs are complementary . AtProT1 expression was found in the phloem or phloem parenchyma cells throughout the whole plant, indicative of a role in long-distance transport of compatible solutes . beta-Glucuronidase activity under the control of the AtProT2 promoter was restricted to the epidermis and the cortex cells in roots, whereas in leaves, staining could be demonstrated only after wounding . In contrast, AtProT3 expression was restricted to the above-ground parts of the plant and could be localized to the epidermal cells in leaves . These results showed that, although intracellular localization, substrate specificity, and affinity are very similar, the transporters fulfill different roles in planta. J Cell Sci, 2005 Jan 15, 118(Pt 2), 323 - 9 Epub 2004 Dec 22. Functional analysis of CLPT1, a Rab/GTPase required for protein secretion and pathogenesis in the plant fungal pathogen Colletotrichum lindemuthianum; Siriputthaiwan P et al.; In eukaryotic cells, Rab/GTPases are major regulators of vesicular trafficking and are involved in essential processes including exocytosis, endocytosis and cellular differentiation . To investigate the role of these proteins in fungal pathogenicity, a dominant-negative mutant allele of CLPT1, a Rab/GTPase of the bean pathogen Colletotrichum lindemuthianum, was expressed in transgenic strains . This mutated gene encodes the amino-acid substitution N123I analogous to the N133I substitution in a known trans-dominant inhibitor of the Sec4 Rab/GTPase from Saccharomyces cerevisiae . A pectinase gene promoter was used to drive the CLPT1(N123I) allele in C . lindemuthianum, allowing the expression of the foreign gene on pectin medium and during pathogenesis, but not on glucose . The same strategy was used to overexpress the wild-type CLPT1 allele . During growth on pectin medium, production of extracellular pectinases was strongly impaired only in CLPT1(N123I)-expressing strains . Cytological analysis revealed that CLPT1(N123I) strains accumulated intracellular aggregates only on pectin, resulting from the fusion of vesicles containing polysaccharides or glycoproteins . Moreover, these strains showed a severe reduction of pathogenesis and were unable to penetrate the host cells . These results indicated that the Rab/GTPase CLPT1 is essential for fungal pathogenesis by regulating the intracellular transport of secretory vesicles involved in the delivery of proteins to the extracellular medium and differentiation of infectious structures. Curr Genet . 2004 Dec 21; {Epub ahead of print} Analysis of the functional domains of the mismatch repair homologue Msh1p and its role in mitochondrial genome maintenance; Mookerjee SA et al.; Mitochondrial DNA (mtDNA) repair occurs in all eukaryotic organisms and is essential for the maintenance of mitochondrial function . Evidence from both humans and yeast suggests that mismatch repair is one of the pathways that functions in overall mtDNA stability . In the mitochondria of the yeast Saccharomyces cerevisiae, the presence of a homologue to the bacterial MutS mismatch repair protein, MSH1, has long been known to be essential for mitochondrial function . The mechanisms for which it is essential are unclear, however . Here, we analyze the effects of two point mutations, msh1-F105A and msh1-G776D, both predicted to be defective in mismatch repair; and we show that they are both able to maintain partial mitochondrial function . Moreover, there are significant differences in the severity of mitochondrial disruption between the two mutants that suggest multiple roles for Msh1p in addition to mismatch repair . Our overall findings suggest that these additional predicted functions of Msh1p, including recombination surveillance and heteroduplex rejection, may be primarily responsible for its essential role in mtDNA stability. Cell Cycle, 2004 Nov, 3(11), 1433 - 9 Epub 2004 Nov 06. Cell size and Cln-Cdc28 complexes mediate entry into meiosis by modulating cell growth; Day A et al.; In the yeast Saccharomyces cerevisiae, mitotic cell cycle progression depends upon the G(1)-phase cyclin-dependent kinase Cln-Cdc28 and cell growth to a minimum cell size . In contrast, Cln-Cdc28 inhibits entry into meiosis, and a cell growth requirement for sporulation has not been established . Here, we report that entry into meiosis also depends upon cell growth . Moreover, sporulation and cell growth rates were proportional to cell size; large cells grew rapidly and sporulated sooner while smaller cells grew slowly and sporulated later . In addition, Cln2 protein levels were higher in smaller cells suggesting that Cln-Cdc28 activity represses meiosis in smaller cells by preventing cell growth . In support of this hypothesis, loss of Clns, or the presence of a cdc28 mutation increased cell growth specifically in smaller cells and accelerated meiosis in these cells . Finally, overexpression of CLNs repressed meiosis in smaller cells, but not in large cells . Taken together, these results demonstrate that Cln-Cdc28 represses entry into meiosis in part by inhibiting cell growth. Cell Cycle . 2005 Jan 17;4(1) {Epub ahead of print} Two-Faced Cyclins with Eyes on the Targets; Archambault V et al.; We recently reported that the 'hydrophobic patch' (HP) of theSaccharomyces cerevisiae S-phase cyclin Clb5 facilitates its interaction with Orc6 (via its Cy or RXL motif), providing a mechanism that helps prevent rereplication from individual origins.(1) This is the first finding of a biological function for an interaction between a cyclin and a cyclin-binding motif (Cy or RXL motif) in a target protein in Saccharomyces cerevisiae . It is also the first such example involving a B-type cyclin in any organism . Yet, some of our observations as well as work from other groups suggest that HP-RXL interactions are functionally important for cyclin-Cdk signaling to other targets . The evolutionary conservation of the HP motif suggests that it allows cyclins to carry out important and specialized functions. Plant Cell, 2005 Jan, 17(1), 311 - 25 Epub 2004 Dec 17. A NAC Domain Protein Interacts with Tomato leaf curl virus Replication Accessory Protein and Enhances Viral Replication; Selth LA et al.; Geminivirus replication enhancer (REn) proteins dramatically increase the accumulation of viral DNA species by an unknown mechanism . In this study, we present evidence implicating SlNAC1, a new member of the NAC domain protein family from tomato (Solanum lycopersicum), in Tomato leaf curl virus (TLCV) REn function . We isolated SlNAC1 using yeast (Saccharomyces cerevisiae) two-hybrid technology and TLCV REn as bait, and confirmed the interaction between these proteins in vitro . TLCV induces SlNAC1 expression specifically in infected cells, and this upregulation requires REn . In a transient TLCV replication system, overexpression of SlNAC1 resulted in a substantial increase in viral DNA accumulation . SlNAC1 colocalized with REn to the nucleus and activated transcription of a reporter gene in yeast, suggesting that in healthy cells it functions as a transcription factor . Together, these results imply that SlNAC1 plays an important role in the process by which REn enhances TLCV replication. Nucleic Acids Res, 2005 Jan 1, 33 Database Issue, D369 - 73 PROPHECY--a database for high-resolution phenomics; Fernandez-Ricaud L et al.; The rapid recent evolution of the field phenomics--the genome-wide study of gene dispensability by quantitative analysis of phenotypes--has resulted in an increasing demand for new data analysis and visualization tools . Following the introduction of a novel approach for precise, genome-wide quantification of gene dispensability in Saccharomyces cerevisiae we here announce a public resource for mining, filtering and visualizing phenotypic data--the PROPHECY database . PROPHECY is designed to allow easy and flexible access to physiologically relevant quantitative data for the growth behaviour of mutant strains in the yeast deletion collection during conditions of environmental challenges . PROPHECY is publicly accessible at http://prophecy.lundberg.gu.se. Eur J Biochem, 2004 Dec, 271(23-24), 4629 - 37 NARG2 encodes a novel nuclear protein with (S/T)PXX motifs that is expressed during development; Sugiura N et al.; We previously identified a partial expressed sequence tag clone corresponding to NARG2 in a screen for genes that are expressed in developing neurons and misexpressed in transgenic mice that lack functional N-methyl-d-aspartate receptors . Here we report the first characterization of the mouse and human NARG2 genes, cDNAs and the proteins that they encode . Mouse and human NARG2 consist of 988 and 982 amino acids, respectively, and share 74% identity . NARG2 does not display significant homology to other known genes, and lower organisms such as Saccharomyces cerevisiae, Drosophila melanogaster and Fugu rubripes appear to lack NARG2 orthologs . In vitro translation of the mouse cDNA yields a 150 kDa protein . NARG2 localizes to the nucleus in transfected cells, and deletion of a canonical basic nuclear localization signal suggests that this and other sequences in the protein cooperate for nuclear targeting . NARG2 consists of 16 exons in both mice and humans, 11 of which are identical in length, and alternative splicing is evident in both species . Exon 10 is the largest, and exhibits a much higher rate of nonsynonymous nucleotide substitution than the others . In addition, NARG2 contains (S/T)PXX motifs (11 in mouse NARG2, six in human NARG2) . Northern blot analysis and RNase protection demonstrated that NARG2 is expressed at relatively high levels in dividing and immature cells, and that it is down-regulated upon terminal differentiation . The results indicate that NARG2 encodes a novel (S/T)PXX motif-containing nuclear protein, and suggest that NARG2 may play an important role in the early development of a number of different cell types. Plant Mol Biol, 2004 Oct, 56(3), 413 - 21 Cloning and functional characterization of the high-affinity K+ transporter HAK1 of pepper; Martinez-Cordero MA et al.; High-affinity K+ uptake in plants plays a crucial role in K+ nutrition and different systems have been postulated to contribute to the high-affinity K+ uptake . The results presented here with pepper (Capsicum annum) demonstrate that a HAK1-type transporter greatly contributes to the high-affinity K+ uptake observed in roots . Pepper plants starved of K+ for 3 d showed high-affinity K+ uptake (Km of 6 microM K+) that was very sensitive to NH and their roots expressed a high-affinity K+ transporter, CaHAK1, which clusters in group I of the KT/HAK/KUP family of transporters . When expressed in yeast ( Saccharomyces cerevisiae ), CaHAK1 mediated high-affinity K+ and Rb+ uptake with Km values of 3.3 and 1.9 microM, respectively . Rb+ uptake was competitively inhibited by micromolar concentrations of NH and Cs+, and by millimolar concentrations of Na+. Plant Mol Biol, 2004 May, 55(3), 389 - 98 ENHANCER of TRY and CPC 2 (ETC2) reveals redundancy in the region-specific control of trichome development of Arabidopsis; Kirik V et al.; An evolutionarily conserved set of proteins consisting of MYB and bHLH transcription factors and a WD40 domain protein is known to act in concert to control various developmental processes including trichome and root hair development . Their function is difficult to assess because most of them belong to multigene families and appear to act in a redundant fashion . In this study we identified an enhancer of the two root hair and trichome patterning mutants triptychon (try) and caprice (cpc), enhancer of try and cpc2 (etc2) . The ETC2 gene shows high sequence similarity to the single-repeat MYB genes CPC and TRY . Overexpression results in the suppression of trichomes and overproduction of root hairs similarly as observed for TRY and CPC suggesting that ETC2 has similar biochemical properties . The etc2 single mutant shows an increase in trichome number on leaves and petioles . Double and triple mutant analysis indicates that the ETC2 gene acts redundant with TRY and CPC in trichome patterning. Plant Mol Biol, 2004 May, 55(1), 45 - 59 Defining subdomains of the K domain important for protein-protein interactions of plant MADS proteins; Yang Y et al.; The MADS proteins APETALA3 (AP3), PISTILLATA (PI), SEPALLATAI (SEPI), SEP2, SEP3, AGAMOUS, and APETALA are required for proper floral organ identity in Arabidopsis flowers . All of these floral MADS proteins conserve two domains: the MADS domain that mediates DNA binding and dimerization, and the K domain that mediates protein protein interaction . The K domain is postulated to form a several amphipathic c-helices referred to as K1, K2, and K3 . The K1 and K2 helicies are located entirely within the K domain while the K3 helix spans the K domain-C domain boundary . Here we report on our studies on the interactions of the B class MADS proteins AP3 and PI with the E class MADS proteins SEP1, SEP2, and SEP3 . A comparative analysis of mutants in the K domain reveals that the subdomains mediating the PI/AP3 interaction are different from the subdomains mediating the PI/SEP3 (or PI/SEP1) interaction . The strong PI/SEP3 (or PI/SEP1) interaction requires K2, part of K3, and the interhelical region between K1 and K2 . By contrast, K1, K2 and the region between K1 and K2 are important for strong AP3/PI interaction . Most of the K3 helix does not appear to be important for either the PI/AP3 or the PI/SEP3 (or PI/SEP1) interaction . Conserved hydrophobic positions are most important for the strength of both PI/AP3 and PI/SEP3 dimerization, though ionic and/or polar interactions appear to play a secondary role. Plant Mol Biol, 2004 Apr, 54(6), 805 - 15 Arabidopsis metallothioneins 2a and 3 enhance resistance to cadmium when expressed in Vicia faba guard cells; Lee J et al.; The Arabidopsis metallothionein genes AtMT1 and AtMT2 confer Cd(II) resistance to Cd(II)-sensitive yeast, but it has not been directly shown whether they or other metallothioneins provide the same protection to plants . We tested whether AtMT2a and AtMT3 can confer Cd(II) resistance to plant cells by introducing GFP- or RFP-fused forms into guard cells of Vicia faba by biolistic bombardment . AtMT2a and AtMT3 protected guard cell chloroplasts from degradation upon exposure to Cd(II), an effect that was confirmed using an FDA assay to test the viability of the exposed guard cells . AtMT2a- and AtMT3-GFP were localized in the cytoplasm both before and after treatment of V . faba guard cells or Arabidopsis protoplasts with Cd(II), and the levels of reactive oxygen species were lower in transformed guard cells than in non-transformed cells after Cd(II)-treatment . These results suggest that the Cd(II)-detoxification mechanism of AtMT2a and AtMT3 may not include sequestration into vacuoles or other organelles, but does involve reduction of the level of reactive oxygen species in Cd(II)-treated cells . Increased expression of AtMT2a and AtMT3 was observed in Arabidopsis seedlings exposed to Cd(II) . Together, these data support a role for the metallothioneins AtMT2a and AtMT3 in Cd(II) resistance in intact plant cells. Oncogene, 2004 Dec 16, 23(58), 9314 - 25 WIPI-1alpha (WIPI49), a member of the novel 7-bladed WIPI protein family, is aberrantly expressed in human cancer and is linked to starvation-induced autophagy; Proikas-Cezanne T et al.; WD-repeat proteins are regulatory beta-propeller platforms that enable the assembly of multiprotein complexes . Here, we report the functional and bioinformatic analysis of human WD-repeat protein Interacting with PhosphoInosides (WIPI)-1alpha (WIPI49/Atg18), a member of a novel WD-repeat protein family with autophagic capacity in Saccharomyces cerevisiae and Caenorhabditis elegans, recently identified as phospholipid-binding effectors . Our phylogenetic analysis divides the WIPI protein family into two paralogous groups that fold into 7-bladed beta-propellers . Structural modeling identified two evolutionary conserved interaction sites in WIPI propellers, one of which may bind phospholipids . Human WIPI-1alpha has LXXLL signature motifs for nuclear receptor interactions and binds androgen and estrogen receptors in vitro . Strikingly, human WIPI genes were found aberrantly expressed in a variety of matched tumor tissues including kidney, pancreatic and skin cancer . We found that endogenous hWIPI-1 protein colocalizes in part with the autophagosomal marker LC3 at punctate cytoplasmic structures in human melanoma cells . In addition, hWIPI-1 accumulated in large vesicular and cup-shaped structures in the cytoplasm when autophagy was induced by amino-acid deprivation . These cytoplasmic formations were blocked by wortmannin, a classic inhibitor of PI-3 kinase-mediated autophagy . Our data suggest that WIPI proteins share an evolutionary conserved function in autophagy and that autophagic capacity may be compromised in human cancers. Nature, 2004 Dec 16, 432(7019), 872 - 7 Structural basis for the assembly of a nuclear export complex; Matsuura Y et al.; The nuclear import and export of macromolecular cargoes through nuclear pore complexes is mediated primarily by carriers such as importin-beta . Importins carry cargoes into the nucleus, whereas exportins carry cargoes to the cytoplasm . Transport is orchestrated by nuclear RanGTP, which dissociates cargoes from importins, but conversely is required for cargo binding to exportins . Here we present the 2.0 A crystal structure of the nuclear export complex formed by exportin Cse1p complexed with its cargo (Kap60p) and RanGTP, thereby providing a structural framework for understanding nuclear protein export and the different functions of RanGTP in export and import . In the complex, Cse1p coils around both RanGTP and Kap60p, stabilizing the RanGTP-state and clamping the Kap60p importin-beta-binding domain, ensuring that only cargo-free Kap60p is exported . Mutagenesis indicated that conformational changes in exportins couple cargo binding to high affinity for RanGTP, generating a spring-loaded molecule to facilitate disassembly of the export complex following GTP hydrolysis in the cytoplasm. Mol Cell Biol, 2005 Jan, 25(1), 488 - 98 The Ccr4-Not complex independently controls both Msn2-dependent transcriptional activation--via a newly identified Glc7/Bud14 type I protein phosphatase module--and TFIID promoter distribution; Lenssen E et al.; The Ccr4-Not complex is a conserved global regulator of gene expression, which serves as a regulatory platform that senses and/or transmits nutrient and stress signals to various downstream effectors . Presumed effectors of this complex in yeast are TFIID, a general transcription factor that associates with the core promoter, and Msn2, a key transcription factor that regulates expression of stress-responsive element (STRE)-controlled genes . Here we show that the constitutively high level of STRE-driven expression in ccr4-not mutants results from two independent effects . Accordingly, loss of Ccr4-Not function causes a dramatic Msn2-independent redistribution of TFIID on promoters with a particular bias for STRE-controlled over ribosomal protein gene promoters . In parallel, loss of Ccr4-Not complex function results in an alteration of the posttranslational modification status of Msn2, which depends on the type 1 protein phosphatase Glc7 and its newly identified subunit Bud14 . Tests of epistasis as well as transcriptional analyses of Bud14-dependent transcription support a model in which the Ccr4-Not complex prevents activation of Msn2 via inhibition of the Bud14/Glc7 module in exponentially growing cells . Thus, increased activity of STRE genes in ccr4-not mutants may result from both altered general distribution of TFIID and unscheduled activation of Msn2. Mol Cell Biol, 2005 Jan, 25(1), 451 - 60 Structural and functional analysis of essential pre-mRNA splicing factor Prp19p; Ohi MD et al.; U-box-containing Prp19p is an integral component of the Prp19p-associated complex (the nineteen complex, or NTC) that is essential for activation of the spliceosome . Prp19p makes numerous protein-protein contacts with other NTC components and is required for NTC stability . Here we show that Prp19p forms a tetramer in vitro and in vivo and we map the domain required for its oligomerization to a central tetrameric coiled-coil . Biochemical and in vivo analyses are consistent with Prp19p tetramerization providing an interaction surface for a single copy of its binding partner, Cef1p . Electron microscopy showed that the isolated Prp19p tetramer is an elongated particle consisting of four globular WD40 domains held together by a central stalk consisting of four N-terminal U-boxes and four coiled-coils . These structural and functional data provide a basis for understanding the role of Prp19p as a key architectural component of the NTC. Mol Cell Biol, 2005 Jan, 25(1), 440 - 50 Glucose inhibits meiotic DNA replication through SCFGrr1p-dependent destruction of Ime2p kinase; Purnapatre K et al.; In the budding yeast Saccharomyces cerevisiae, the cell division cycle and sporulation are mutually exclusive cell fates; glucose, which stimulates the cell division cycle, is a potent inhibitor of sporulation . Addition of moderate concentrations of glucose (0.5%) to sporulation medium did not inhibit transcription of two key activators of sporulation, IME1 and IME2, but did increase levels of Sic1p, a cyclin-dependent kinase inhibitor, resulting in a block to meiotic DNA replication . The effects of glucose on Sic1p levels and DNA replication required Grr1p, a component of the SCF(Grr1p) ubiquitin ligase . Sic1p is negatively regulated by Ime2p kinase, and several observations indicate that glucose inhibits meiotic DNA replication through SCF(Grr1p)-mediated destruction of this kinase . First, Ime2p was destabilized in the presence of glucose, and this turnover required Grr1p, a second component of SCF(Grr1p), Cdc53p, and an SCF(Grr1p)-associated E2 enzyme, Cdc34p . Second, Ime2p-ubiquitin conjugates were detected under conditions of rapid Ime2p turnover, and conjugation of Ime2p to ubiquitin required GRR1 . Third, a mutant form of Ime2p (Ime2(DeltaPEST)), in which a putative Grr1p-interacting sequence was deleted, was more stable than wild-type Ime2p . Finally, expression of the IME2(DeltaPEST) allele bypassed the block to meiotic DNA replication caused by 0.5% glucose . In addition, Grr1p is required for later events in sporulation independently of its role in Ime2p turnover. Mol Cell Biol, 2005 Jan, 25(1), 403 - 13 Proteasome-mediated degradation of cotranslationally damaged proteins involves translation elongation factor 1A; Chuang SM et al.; Rad23 and Rpn10 play synergistic roles in the recognition of ubiquitinated proteins by the proteasome, and loss of both proteins causes growth and proteolytic defects . However, the physiological targets of Rad23 and Rpn10 have not been well defined . We report that rad23Delta rpn10Delta is unable to grow in the presence of translation inhibitors, and this sensitivity was suppressed by translation elongation factor 1A (eEF1A) . This discovery suggested that Rad23 and Rpn10 perform a role in translation quality control . Certain inhibitors increase translation errors during protein synthesis and cause the release of truncated polypeptide chains . This effect can also be mimicked by ATP depletion . We determined that eEF1A interacted with ubiquitinated proteins and the proteasome following ATP depletion . eEF1A interacted with the proteasome subunit Rpt1, and the turnover of nascent damaged proteins was deficient in rpt1 . An eEF1A mutant (eEF1A(D156N)) that conferred hyperresistance to translation inhibitors was much more effective at eliminating damaged proteins and was detected in proteasomes in untreated cells . We propose that eEF1A is well suited to detect and promote degradation of damaged proteins because of its central role in translation elongation . Our findings provide a mechanistic foundation for defining how cellular proteins are degraded cotranslationally. Phys Rev Lett . 2004 Nov 12;93(20):208105 . Epub 2004 Nov 12. Protein folding rates correlate with heterogeneity of folding mechanism; Oztop B et al.; By observing trends in the folding kinetics of experimental 2-state proteins at their transition midpoints, and by observing trends in the barrier heights of numerous simulations of coarse-grained, C(alpha) model Go proteins, we show that folding rates correlate with the degree of heterogeneity in the formation of native contacts . Statistically significant correlations are observed between folding rates and measures of heterogeneity inherent in the native topology, as well as between rates and the variance in the distribution of either experimentally measured or simulated phi values. Bioinformatics . 2004 Dec 14; {Epub ahead of print} Dynamic simulation of protein complex formation on a genomic scale; Beyer A et al.; MOTIVATION: One of the central questions in the post-genomic era is the understanding of protein-protein interactions and of protein complex formation . It has been observed that protein complex size distributions of the yeast Saccharomyces cerevisiae decay exponentially . The shape of these size distributions reflects mechanisms of protein complex association and dissociation . RESULTS: We present the most simple dynamical model that is able to reproduce the observed protein complex size distribution for yeast . This protein association-dissociation model (PAD-model) simulates the dynamics of protein complex formation on a genomic scale for about 50 million protein molecules . By ruling out different model variants it is possible to elucidate fundamental features of the protein complex dynamics, e.g . complex association is independent of complex size . In addition, the PAD-model provides information about the complexity of the yeast proteome and it gives an idea of how many complexes could not be identified during the measurements . AVAILABILITY: All programs used for this publication are available on request from the authors . SUPPLEMENTARY INFORMATION: Supplementary information about the model and its interpretation can be downloaded from http://www.imb-jena.de/tsb/pad. Nucleic Acids Res, 2004 Dec 14, 32(22), 6519 - 30 Print 2004. Coordinated functions of WSS1, PSY2 and TOF1 in the DNA damage response; O'Neill BM et al.; The stabilization and processing of stalled replication forks is required to maintain genome integrity in all organisms . In an effort to identify novel proteins that might be involved in stabilizing stalled replication forks, Saccharomyces cerevisiae mutant wss1Delta was isolated from a high-throughput screening of approximately 5000 deletion strains for genes involved in the response to continuous, low-intensity UV irradiation . Disruption of WSS1 resulted in synergistic increases in UV sensitivity with null mutants of genes involved in recombination (RAD52) and cell cycle control (RAD9 and RAD24) . WSS1 was also found to interact genetically with SGS1, TOP3, SRS2 and CTF4, which are involved in recombination, repair of replication forks and the establishment of sister chromatid cohesion . A yeast two-hybrid screen identified a potential physical interaction between Wss1 and both Psy2 and Tof1 . Genetic interactions were also detected between PSY2 and TOF1, as well as between each gene and RAD52 and SRS2, and between WSS1 and TOF1 . Tof1 is known to be involved in stabilizing stalled replication forks and our data suggest that Wss1 and Psy2 similarly function to stabilize or process stalled or collapsed replication forks. Plant Cell, 2005 Jan, 17(1), 149 - 63 Epub 2004 Dec 14. Conserved ERAD-Like Quality Control of a Plant Polytopic Membrane Protein; Muller J et al.; The endoplasmic reticulum (ER) of eukaryotic cells serves as a checkpoint tightly monitoring protein integrity and channeling malformed proteins into different rescue and degradation routes . The degradation of several ER lumenal and membrane-localized proteins is mediated by ER-associated protein degradation (ERAD) in yeast (Saccharomyces cerevisiae) and mammalian cells . To date, evidence for the existence of ERAD-like mechanisms in plants is indirect and based on heterologous or artificial substrate proteins . Here, we show that an allelic series of single amino acid substitution mutants of the plant-specific barley (Hordeum vulgare) seven-transmembrane domain mildew resistance o (MLO) protein generates substrates for a postinsertional quality control process in plant, yeast, and human cells, suggesting conservation of the underlying mechanism across kingdoms . Specific stabilization of mutant MLO proteins in yeast strains carrying defined defects in protein quality control demonstrates that MLO degradation is mediated by HRD pathway-dependent ERAD . In plants, individual aberrant MLO proteins exhibit markedly reduced half-lives, are polyubiquitinated, and can be stabilized through inhibition of proteasome activity . This and a dependence on homologs of the AAA ATPase CDC48/p97 to eliminate the aberrant variants strongly suggest that MLO proteins are endogenous substrates of an ERAD-related plant quality control mechanism. Proc Natl Acad Sci U S A, 2004 Dec 21, 101(51), 17616 - 21 Epub 2004 Dec 14. Potent inhibition of huntingtin aggregation and cytotoxicity by a disulfide bond-free single-domain intracellular antibody; Colby DW et al.; Huntington's disease (HD) is a progressive neurodegenerative disorder caused by an expansion in the number of polyglutamine-encoding CAG repeats in the gene that encodes the huntingtin (htt) protein . A property of the mutant protein that is intimately involved in the development of the disease is the propensity of the glutamine-expanded protein to misfold and generate an N-terminal proteolytic htt fragment that is toxic and prone to aggregation . Intracellular antibodies (intrabodies) against htt have been shown to reduce htt aggregation by binding to the toxic fragment and inactivating it or preventing its misfolding . Intrabodies may therefore be a useful gene-therapy approach to treatment of the disease . However, high levels of intrabody expression have been required to obtain even limited reductions in aggregation . We have engineered a single-domain intracellular antibody against htt for robust aggregation inhibition at low expression levels by increasing its affinity in the absence of a disulfide bond . Furthermore, the engineered intrabody variable light-chain (V(L))12.3, rescued toxicity in a neuronal model of HD . We also found that V(L)12.3 inhibited aggregation and toxicity in a Saccharomyces cerevisiae model of HD . V(L)12.3 is significantly more potent than earlier anti-htt intrabodies and is a potential candidate for gene therapy treatment for HD . To our knowledge, this is the first attempt to improve affinity in the absence of a disulfide bond to improve intrabody function . The demonstrated importance of disulfide bond-independent binding for intrabody potency suggests a generally applicable approach to the development of effective intrabodies against other intracellular targets. Arch Neurol, 2004 Dec, 61(12), 1935 - 7 Studies of COX16, COX19, and PET191 in human cytochrome-c oxidase deficiency; Tay SK et al.; BACKGROUND: Cytochrome-c oxidase (COX) is the terminal enzyme of the mitochondrial electron transport chain, and COX deficiency is a common cause of mitochondrial diseases . Cytochrome-c oxidase is composed of 13 subunits, of which 3 are encoded by mitochondrial DNA and 10 by nuclear DNA . Mutations have been identified in each of the 3 mitochondrial DNA genes but in none of the nuclear DNA genes . However, COX deficiency has been attributed to mutations in several nuclear DNA-encoded ancillary proteins needed for COX assembly and function . Despite this progress, the molecular basis of COX deficiency remains elusive in many patients, justifying the identification and screening of additional COX assembly genes, such as COX16, COX19, and PET191 . OBJECTIVE: To determine if COX16, COX19, and PET191 are implicated in human COX deficiency . METHODS: Mutation screening was performed on 53 patients with isolated COX deficiency by direct sequencing of COX19 and by single-strand conformational polymorphism analysis for COX16 and PET191 . RESULTS: No mutations were found in COX16, COX19, or PET191 in these patients . CONCLUSIONS: The COX16, COX19, and PET191 genes are either not involved or very rarely involved in human COX deficiency . Mutations in additional COX assembly genes remain to be identified. J Cell Biol, 2004 Dec 20, 167(6), 1099 - 112 Epub 2004 Dec 13. Quantitative mass spectrometry reveals a role for the GTPase Rho1p in actin organization on the peroxisome membrane; Marelli M et al.; We have combined classical subcellular fractionation with large-scale quantitative mass spectrometry to identify proteins that enrich specifically with peroxisomes of Saccharomyces cerevisiae . In two complementary experiments, isotope-coded affinity tags and tandem mass spectrometry were used to quantify the relative enrichment of proteins during the purification of peroxisomes . Mathematical modeling of the data from 306 quantified proteins led to a prioritized list of 70 candidates whose enrichment scores indicated a high likelihood of them being peroxisomal . Among these proteins, eight novel peroxisome-associated proteins were identified . The top novel peroxisomal candidate was the small GTPase Rho1p . Although Rho1p has been shown to be tethered to membranes of the secretory pathway, we show that it is specifically recruited to peroxisomes upon their induction in a process dependent on its interaction with the peroxisome membrane protein Pex25p . Rho1p regulates the assembly state of actin on the peroxisome membrane, thereby controlling peroxisome membrane dynamics and biogenesis. Bioinformatics, 2003 Sep 1, 19(13), 1710 - 1 Detecting putative orthologs; Wall DP et al.; We developed an algorithm that improves upon the common procedure of taking reciprocal best blast hits(rbh) in the identification of orthologs . The method-reciprocal smallest distance algorithm (rsd)-relies on global sequence alignment and maximum likelihood estimation of evolutionary distances to detect orthologs between two genomes . rsd finds many putative orthologs missed by rbh because it is less likely than rbh to be misled by the presence of a close paralog. Eukaryot Cell, 2004 Dec, 3(6), 1619 - 26 The small-subunit processome is a ribosome assembly intermediate; Bernstein KA et al.; The small-subunit (SSU) processome is a large ribonucleoprotein required for the biogenesis of the 18S rRNA and likely corresponds to the terminal knobs visualized by electron microscopy on the 5' end of nascent rRNAs . The original purification of the SSU processome of Saccharomyces cerevisiae resulted in the identification of 28 proteins . Here, we characterize 12 additional protein components, including five small-ribosomal-subunit proteins (Rps4, Rps6, Rps7, Rps9, and Rps14) that had previously been copurified . Our multiple criteria for including a component as a bona fide SSU processome component included coimmunoprecipitation with Mpp10 (an SSU processome component), the U3 snoRNA, and the anticipated pre-rRNAs . Importantly, the association of specific ribosomal proteins with the SSU processome suggests that the SSU processome has roles in both pre-rRNA processing and ribosome assembly . These ribosomal proteins may be analogous to the primary or secondary RNA binding proteins first described in bacterial in vitro ribosome assembly maps . In addition to the ribosomal proteins and based on the same experimental approach, we found seven other proteins (Utp18, Noc4, Utp20, Utp21, Utp22, Emg1, and Krr1) to be bona fide SSU processome proteins. J Biol Chem . 2004 Dec 8; {Epub ahead of print} Two steps in Maf1-dependent repression of transcription by RNA polymerase III; Desai N et al.; In Saccharomyces cerevisiae, Maf1 is essential for mediating the repression of transcription by RNA polymerase (pol) III in response to diverse cellular conditions . These conditions activate distinct signaling pathways that converge at or above Maf1 . Thus, Maf1-dependent repression is thought to involve a common set of downstream inhibitory effects on the pol III machinery . Here we provide support for this view and define two steps in Maf1-dependent transcriptional repression . We show that chlorpromazine (CPZ)-induced repression of pol III transcription is achieved by inhibiting de novo assembly of the initiation factor TFIIIB onto DNA as well as the recruitment of pol III to pre-assembled TFIIIB-DNA complexes . Additionally, Brf1 was identified as a target of repression in extracts of CPZ-treated cells . Maf1-Brf1 and Maf1-pol III interactions were implicated in the inhibition of TFIIIB-DNA complex assembly and polymerase recruitment by recombinant Maf1 . Co-immunoprecipitation experiments confirmed these interactions in yeast extracts and demonstrated that Maf1 does not differentially sequester Brf1 or pol III under repressing conditions . The results suggest that Maf1 functions by a non-stoichiometric mechanism to repress pol III transcription. FEBS Lett, 2004 Dec 17, 578(3), 297 - 304 Learning module networks from genome-wide location and expression data; Xu X et al.; We develop a systematic algorithm for discovering network of regulatory modules, which identifies regulatory modules and their regulation program by integrating genome-wide location and expression data . Unlike previous approaches {Eisen, M.B., Spellman, P.T., Brown, P.O . and Botstein, D . (1998) Proc . Natl . Acad . Sci . USA 95, 14863-14868; Tavazoie, S., Hughes, J.D., Campbell, M.J., Cho, R.J . and Church, G.M . (1999) Nat . Genet . 22, 281-285; Ihmels, J., Friedlander, G., Bergmann, S., Sarig, O., Ziv, Y . and Barkai, N . (2002) Nat . Genet . 31, 370-377; Segal, E., Shapira, M., Regev, A., Pe'er, D., Botstein, D., Koller, D . and Friedman, N . (2003) Nat . Genet . 34, 166-176} that relied primarily on gene expression data, our algorithm regards the regulator binding data as prior knowledge that provide direct evidence of physical regulatory interactions . We applied the method to a Saccharomyces cerevisiae genome-wide location data {Lee, T.I., Rinaldi, N.J., Robert, F., Odom, D.T., Bar-Joseph, Z., Gerber, G.K., Hannett, N.M., Harbison, C.T., Thompson, C.M., Simon, I., Zeitlinger, J., Jennings, E.G., Murray, H.L . Gordon, D.B., Ren, B., Wyrick, J.J., Tagne, J.B., Volkert, T.L., Fraenkel, E., Gifford, D.K . and Young, R.A . (2002) Science 298, 799-804} for 106 DNA-binding transcription factors and 250 gene expression experiments under the conditions from the cell cycle to responses to various stress conditions . The results show that our method is able to identify functionally coherent modules and their proper regulators . Supplementary materials are available at http://compbio.sibnet.org/projects/module-network/. Fungal Genet Biol, 2005 Jan, 42(1), 1 - 8 A rapid method for promoter exchange in Aspergillus nidulans using recombinant PCR; Zarrin M et al.; Recombinant PCR has been used to generate linear fragments for promoter replacement by transformation in Aspergillus nidulans . A cassette vector carrying the pyr-4 non-homologous selectable marker and conditional promoter Pr-alcA was constructed for use as a template for PCR, and is suitable for testing the function of essential genes . Two genes involved in polar growth, cotA and bemA, were used to assess the system . Efficient targeting was possible with both genes using approximately 500bp of flanking homologous sequence . Depending on yield, the linear PCR product could be used directly for transformation, or after first cloning into a suitable vector . bemA, a putative homologue of the Saccharomyces cerevisiae BEM1 gene was identified through sequence comparison . In A . nidulans, this protein appears to have a similar role to the yeast Bem1p, which acts as a scaffold protein involved in the establishment of cell polarity. J Cell Biol, 2004 Dec 6, 167(5), 889 - 901 Vesicles carry most exocyst subunits to exocytic sites marked by the remaining two subunits, Sec3p and Exo70p; Boyd C et al.; Exocytosis in the budding yeast Saccharomyces cerevisiae occurs at discrete domains of the plasma membrane . The protein complex that tethers incoming vesicles to sites of secretion is known as the exocyst . We have used photobleaching recovery experiments to characterize the dynamic behavior of the eight subunits that make up the exocyst . One subset (Sec5p, Sec6p, Sec8p, Sec10p, Sec15p, and Exo84p) exhibits mobility similar to that of the vesicle-bound Rab family protein Sec4p, whereas Sec3p and Exo70p exhibit substantially more stability . Disruption of actin assembly abolishes the ability of the first subset of subunits to recover after photobleaching, whereas Sec3p and Exo70p are resistant . Immunogold electron microscopy and epifluorescence video microscopy indicate that all exocyst subunits, except for Sec3p, are associated with secretory vesicles as they arrive at exocytic sites . Assembly of the exocyst occurs when the first subset of subunits, delivered on vesicles, joins Sec3p and Exo70p on the plasma membrane . Exocyst assembly serves to both target and tether vesicles to sites of exocytosis. Virology, 2005 Jan 5, 331(1), 181 - 9 HIV-1 incorporates and proteolytically processes human NDR1 and NDR2 serine-threonine kinases; Devroe E et al.; Mammalian genomes encode two related serine-threonine kinases, nuclear Dbf2 related (NDR)1 and NDR2, which are homologous to the Saccharomyces cerevisiae Dbf2 kinase . Recently, a yeast genetic screen implicated the Dbf2 kinase in Ty1 retrotransposition . Since several virion-incorporated kinases regulate the infectivity of human immunodeficiency virus type 1 (HIV-1), we speculated that the human NDR1 and NDR2 kinases might play a role in the HIV-1 life cycle . Here we show that the NDR1 and NDR2 kinases were incorporated into HIV-1 particles . Furthermore, NDR1 and NDR2 were cleaved by the HIV-1 protease (PR), both within virions and within producer cells . Truncation at the PR cleavage site altered NDR2 subcellular localization and inhibited NDR1 and NDR2 enzymatic activity . These studies identify two new virion-associated host cell enzymes and suggest a novel mechanism by which HIV-1 alters the intracellular environment of human cells. Biochim Biophys Acta, 2004 Dec 15, 1667(2), 167 - 73 PcMtr, an aromatic and neutral aliphatic amino acid permease of Penicillium chrysogenum; Trip H et al.; The gene encoding an aromatic and neutral aliphatic amino acid permease of Penicillium chrysogenum was cloned, functionally expressed and characterized in Saccharomyces cerevisiae M4276 . The permease, designated PcMtr, is structurally and functionally homologous to Mtr of Neurospora crassa, and unrelated to the Amino Acid Permease (AAP) family which includes most amino acid permeases in fungi . Database searches of completed fungal genome sequences reveal that Mtr type permeases are not widely distributed among fungi, suggesting a specialized function. Rev Gastroenterol Disord, 2004 Fall, 4(4), 167 - 74 Serologic markers in inflammatory bowel disease: state of the art; Sandborn WJ; A variety of serologic tests are emerging that are relevant to the diagnosis and treatment of Crohn's disease and ulcerative colitis . These laboratory tests include: anti-neutrophil cytoplasmic antibody with perinuclear staining (pANCA); anti-Saccharomyces cerevisiae antibody (ASCA); outer membrane porin C (Omp C); and I2 antibody (novel homologue of the bacterial transcription-factor families) . The potential roles for serologic testing for inflammatory bowel disease (IBD) include adjunctive diagnostic testing in patients with known IBD, screening testing for IBD in patients with compatible gastrointestinal symptoms, and serving as a marker of unique disease course or prediction of response to specific treatments . This article reviews the use of pANCA, ASCA, I2, and Omp C in patients with IBD. J Comput Biol, 2004, 11(4), 695 - 713 A statistically driven approach for image segmentation and signal extraction in cDNA microarrays; Bergemann TL et al.; The increasing use of cDNA microarrays necessitates the development of methods for extracting quality data . Here, we set forth hurdles to overcome in image analysis of microarrays . We emphasize the importance of objective data extraction methods resulting in reliable signal estimates . Based on statistical principles, we describe a method for automated grid alignment, spot detection, background estimation, flagging, and signal extraction . A software application that we call SignalViewer has been implemented for this method . We identify areas where we improved upon current methods used for array image analysis at each step in the process . Finally, we give examples to illustrate the performance of our algorithms on raw data. J Comput Biol, 2004, 11(4), 642 - 59 Closed-loop learning control of bio-networks; Ku J et al.; A general goal of systems biology is to acquire a detailed quantitative understanding of the life-sustaining interactions between genes and proteins . There arises an interesting question of whether these network dynamics can be controlled externally . In the open-loop approach to experimental biology, a control design would be chosen based on a desired target response and modeling with all the available knowledge about the system . If the system is not completely understood or disturbances occur, then unexpected deviations from the desired response can arise . A means to circumvent this difficulty is to optimize the controls in a closed-loop operation by modifying successive input controls based on the performance of previous controls . This paper presents a simulation of closed-loop learning control applied to biological systems in order to generate a desired response . The most powerful advantage of this technique is that the controls are deduced based on experimental results and the process can operate without a model for the underlying biochemical network . This feature eliminates the problem of faulty predictions as well as the need for a detailed understanding of the underlying molecular pathways, suggesting that biological systems can be controlled even before the post-systems biology era. Nucleic Acids Res, 2004, 32(21), 6276 - 83 Print 2004. Analysis of transcription asymmetries along the tRNAE-COB operon: evidence for transcription attenuation and rapid RNA degradation between coding sequences; Krause K et al.; Mitochondrial gene expression in yeast is believed to be regulated predominantly at the post-transcriptional level . However, the contribution of mitochondrial transcription and RNA-turnover rates to differential gene regulation is still largely unknown . Mitochondrial run-on transcription and hybrid selection assays showed that some of the multigenic transcription units of the mitochondrial genome are transcribed evenly, whereas others are transcribed asymmetrically, with higher transcription rates for promoter-proximal genes, than for promoter-distal genes . The tRNA(E)-cytochrome b (COB) operon was analyzed in detail to investigate the mechanisms underlying transcription rate asymmetries in yeast mitochondria . We showed that a drop in transcription rates occurs in a particular region between the coding sequences and is independent of the coding sequence of the downstream COB gene . Deletion of the region between tRNA(E) and COB coding sequences decreases the drop in transcription rates . Deletion of the nuclear gene encoding the Pet 127 protein, which is involved in mitochondrial RNA 5' processing and degradation, also partially relieves transcriptional asymmetry . Therefore, asymmetry is probably due to a combination of attenuated transcription at specific sites between the coding sequences and very rapid RNA degradation. Genome Biol . 2004;5(12):R95 . Epub 2004. Integrating phenotypic and expression profiles to map arsenic-response networks; Haugen AC et al.; BACKGROUND: Arsenic is a nonmutagenic carcinogen affecting millions of people . The cellular impact of this metalloid in Saccharomyces cerevisiae was determined by profiling global gene expression and sensitivity phenotypes . These data were then mapped to a metabolic network composed of all known biochemical reactions in yeast, as well as the yeast network of 20,985 protein-protein/protein-DNA interactions . RESULTS: While the expression data unveiled no significant nodes in the metabolic network, the regulatory network revealed several important nodes as centers of arsenic-induced activity . The highest-scoring proteins included Fhl1, Msn2, Msn4, Yap1, Cad1 (Yap2), Pre1, Hsf1 and Met31 . Contrary to the gene-expression analyses, the phenotypic-profiling data mapped to the metabolic network . The two significant metabolic networks unveiled were shikimate, and serine, threonine and glutamate biosynthesis . We also carried out transcriptional profiling of specific deletion strains, confirming that the transcription factors Yap1, Arr1 (Yap8), and Rpn4 strongly mediate the cell's adaptation to arsenic-induced stress but that Cad1 has negligible impact . CONCLUSIONS: By integrating phenotypic and transcriptional profiling and mapping the data onto the metabolic and regulatory networks, we have shown that arsenic is likely to channel sulfur into glutathione for detoxification, leads to indirect oxidative stress by depleting glutathione pools, and alters protein turnover via arsenation of sulfhydryl groups on proteins . Furthermore, we show that phenotypically sensitive pathways are upstream of differentially expressed ones, indicating that transcriptional and phenotypic profiling implicate distinct, but related, pathways. Genome Biol . 2004;5(12):R100 . Epub 2004. Estimating genomic coexpression networks using first-order conditional independence; Magwene PM et al.; We describe a computationally efficient statistical framework for estimating networks of coexpressed genes . This framework exploits first-order conditional independence relationships among gene-expression measurements to estimate patterns of association . We use this approach to estimate a coexpression network from microarray gene-expression measurements from Saccharomyces cerevisiae . We demonstrate the biological utility of this approach by showing that a large number of metabolic pathways are coherently represented in the estimated network . We describe a complementary unsupervised graph search algorithm for discovering locally distinct subgraphs of a large weighted graph . We apply this algorithm to our coexpression network model and show that subgraphs found using this approach correspond to particular biological processes or contain representatives of distinct gene families. Mol Cell, 2004 Dec 3, 16(5), 749 - 60 The N-terminal domain of Nup159 forms a beta-propeller that functions in mRNA export by tethering the helicase Dbp5 to the nuclear pore; Weirich CS et al.; Nuclear export of mRNA in eukaryotic cells is mediated by soluble transport factors and components of the nuclear pore complex (NPC) . The cytoplasmically oriented nuclear pore protein Nup159 plays a critical role in mRNA export through its conserved N-terminal domain (NTD) . Here, we report the crystal structure of the Nup159 NTD, refined to 2.5 A . The structure reveals an unusually asymmetric seven-bladed beta-propeller that is structurally conserved throughout eukarya . Using structure-based conservation analysis, we have targeted specific surface residues for mutagenesis . Residue substitutions in a conserved loop of the NTD abolish in vitro binding to Dbp5, a DEAD box helicase required for mRNA export . In vivo, these mutations cause Dbp5 mislocalization and block mRNA export . These findings suggest that the Nup159 NTD functions in mRNA export as a binding platform, tethering shuttling Dbp5 molecules at the nuclear periphery and locally concentrating this mRNA remodeling factor at the cytoplasmic face of the NPC. Mol Cell, 2004 Dec 3, 16(5), 687 - 700 DNA replication checkpoint prevents precocious chromosome segregation by regulating spindle behavior; Krishnan V et al.; The DNA replication checkpoint maintains replication fork integrity and prevents chromosome segregation during replication stresses . Mec1 and Rad53 (human ATM/ATR- and Chk2-like kinases, respectively) are critical effectors of this pathway in yeast . When treated with replication inhibitors, checkpoint-deficient mec1 or rad53 mutant fails to maintain replication fork integrity and proceeds to partition unreplicated chromosomes . We show that this unnatural chromosome segregation requires neither the onset of mitosis nor APC activation, cohesin cleavage, or biorientation of kinetochores . Instead, the checkpoint deficiency leads to deregulation of microtubule-associated proteins Cin8 and Stu2, which, in the absence of both chromosome cohesion and bipolar attachment of kinetochores to microtubules, induce untimely spindle elongation, causing premature chromosome separation . The checkpoint's ability to prevent nuclear division is abolished by combined deficiency of microtubule-destabilizing motor Kip3 and Mad2 functions . Thus, the DNA replication checkpoint prevents precocious chromosome segregation, not by inhibiting entry into mitosis as widely believed, but by directly regulating spindle dynamics. Mol Cell Biol, 2004 Dec, 24(24), 10802 - 13 Growth rate and cell size modulate the synthesis of, and requirement for, G1-phase cyclins at start; Schneider BL et al.; In Saccharomyces cerevisiae, commitment to cell cycle progression occurs at Start . Progression past Start requires cell growth and protein synthesis, a minimum cell size, and G(1)-phase cyclins . We examined the relationships among these factors . Rapidly growing cells expressed, and required, dramatically more Cln protein than did slowly growing cells . To clarify the role of cell size, we expressed defined amounts of CLN mRNA in cells of different sizes . When Cln was expressed at nearly physiological levels, a critical threshold of Cln expression was required for cell cycle progression, and this critical threshold varied with both cell size and growth rate: as cells grew larger, they needed less CLN mRNA, but as cells grew faster, they needed more Cln protein . At least in part, large cells had a reduced requirement for CLN mRNA because large cells generated more Cln protein per unit of mRNA than did small cells . When Cln was overexpressed, it was capable of promoting Start rapidly, regardless of cell size or growth rate . In summary, the amount of Cln required for Start depends dramatically on both cell size and growth rate . Large cells generate more Cln1 or Cln2 protein for a given amount of CLN mRNA, suggesting the existence of a novel posttranscriptional size control mechanism. Mol Cell Biol, 2004 Dec, 24(24), 10766 - 76 5-fluorouracil enhances exosome-dependent accumulation of polyadenylated rRNAs; Fang F et al.; The antimetabolite 5-fluorouracil (5FU) is a widely used chemotherapeutic for the treatment of solid tumors . Although 5FU slows DNA synthesis by inhibiting the ability of thymidylate synthetase to produce dTMP, the drug also has significant effects on RNA metabolism . Recent genome-wide assays for 5FU-induced haploinsufficiency in Saccharomyces cerevisiae identified genes encoding components of the RNA processing exosome as potential targets of the drug . In this report, we used DNA microarrays to analyze the effect of 5FU on the yeast transcriptome and found that the drug causes the accumulation of polyadenylated fragments of the 27S rRNA precursor and that defects in the nuclear exoribonuclease Rrp6p enhance this effect . The size distribution of these RNAs and their sensitivity to Rrp6p suggest that they are normally degraded by the nuclear exosome and a 5'-3' exoribonuclease . Consistent with this hypothesis, 5FU inhibits the growth of RRP6 mutants with defects in the degradation function of the enzyme and it interferes with the degradation of an rRNA precursor . The detection of poly(A)(+) pre-RNAs in strains defective in various steps in ribosome biogenesis suggests that the production of poly(A)(+) pre-rRNAs may be a general result of defects in rRNA processing . These findings suggest that 5FU inhibits an exosome-dependent surveillance pathway that degrades polyadenylated precursor rRNAs. Mol Cell Biol, 2004 Dec, 24(24), 10733 - 41 DNA cross-link repair protein SNM1A interacts with PIAS1 in nuclear focus formation; Ishiai M et al.; The yeast SNM1/PSO2 gene specifically functions in DNA interstrand cross-link (ICL) repair, and its role has been suggested to be separate from other DNA repair pathways . In vertebrates, there are three homologs of SNM1 (SNM1A, SNM1B, and SNM1C/Artemis; SNM1 family proteins) whose functions are largely unknown . We disrupted each of the SNM1 family genes in the chicken B-cell line DT40 . Both SNM1A- and SNM1B-deficient cells were sensitive to cisplatin but not to X-rays, whereas SNM1C/Artemis-deficient cells exhibited sensitivity to X-rays but not to cisplatin . SNM1A was nonepistatic with XRCC3 (homologous recombination), RAD18 (translesion synthesis), FANCC (Fanconi anemia), and SNM1B in ICL repair . SNM1A protein formed punctate nuclear foci depending on the conserved SNM1 (metallo-beta-lactamase) domain . PIAS1 was found to physically interact with SNM1A, and they colocalized at nuclear foci . Point mutations in the SNM1 domain, which disrupted the interaction with PIAS1, led to mislocalization of SNM1A in the nucleus and loss of complementation of snm1a cells . These results suggest that interaction between SNM1A and PIAS1 is required for ICL repair. Dev Biol, 2005 Jan 1, 277(1), 92 - 101 Belle is a Drosophila DEAD-box protein required for viability and in the germ line; Johnstone O et al.; DEAD-box proteins are ATP-dependent RNA helicases that function in various stages of RNA processing and in RNP remodeling . Here, we report identification and characterization of the Drosophila protein Belle (Bel), which belongs to a highly conserved subfamily of DEAD-box proteins including yeast Ded1p, Xenopus An3, mouse PL10, human DDX3/DBX, and human DBY . Mutations in DBY are a frequent cause of male infertility in humans . Bel can substitute in vivo for Ded1p, an essential yeast translation factor, suggesting a requirement for Bel in translation initiation . Consistent with an essential cellular function, strong loss of function mutations in bel are recessive lethal with a larval growth defect phenotype . Hypomorphic bel mutants are male-sterile . Bel is also closely related to the Drosophila DEAD-box protein Vasa (Vas), a germ line-specific translational regulator . We find that Bel and Vas colocalize in nuage and at the oocyte posterior during oogenesis, and that bel function is required for female fertility . However, unlike Vas, Bel is not specifically enriched in embryonic pole cells . We conclude that the DEAD-box protein Bel has evolutionarily conserved roles in fertility and development. J Mol Biol, 2005 Jan 14, 345(2), 211 - 27 Proprotein convertase models based on the crystal structures of furin and kexin: explanation of their specificity; Henrich S et al.; In eukaryotes, many secreted proteins and peptide hormones are excised from larger precursors by calcium-dependent serine proteinases, the proprotein/prohormone convertases (PCs) . These PCs cleave their protein substrates very specifically following multiple basic residues . The seven mammalian PCs and their yeast orthologue kexin are multi-domain proteinases consisting of a subtilisin-related catalytic domain, a conserved P-domain and a variable, often cysteine-rich domain, which in some PCs is followed by an additional C-terminal trans-membrane domain and a short cytoplasmic domain . The recently published crystal structures of the soluble mouse furin and yeast kexin ectodomains have revealed the relative arrangement of catalytic and P domains, the exact domain fold and the detailed architecture of the substrate binding clefts . Based on these experimental structures, we now have modelled the structures of the other human/mouse PCs . According to topology and to structure-based sequence comparisons, these other PCs closely resemble furin, with PC4, PACE4 and PC5/6 being more similar, and PC1/3, PC2 and PC7 being less similar to furin . Except for PC1 and PC2, this order of similarity is valid for the catalytic as well as for the P domains, and is almost reversed using kexin as a reference molecule . A similar order results from the number and clustering of negative charges lining the non-prime subsites, explaining the gradually decreasing requirement for basic residues N-terminal to substrate cleavage sites . The preference of the different PCs for distinct substrates seems to be governed by overall charge compensation and matching of the detailed charge distribution pattern. Front Biosci, 2005 Jan 1, 10, 866 - 72 Print 2005 Jan 1. Histone modifications as key regulators of transcription; Khan AU et al.; Covalent modifications of the amino-termini of the core histones in nucleosomes have been shown to be one of the key regulatory mechanisms in transcription regulation . Recently, new roles for histone modifications have been uncovered for the efficient functioning of RNA Pol II . Besides acetylation, which is the most characterized to date these modifications comprise phosphorylation, methylation, and ubiquitination . This review gives comprehensive view of all the major histone modifications and their effect on transcriptional regulation, in Saccharomyces cerevisiae. Biochem J . 2004 Nov 30; {Epub ahead of print} Characterization of the inositol phosphorylceramide synthase activity from Trypanosoma cruzi; Figueiredo JM et al.; Inositol phosphorylceramide (IPC) synthase is an essential enzyme for fungal viability and is the target of potent antifungal compounds as rustmicin and aureobasidin A . Like fungi and some other lower eukaryotes, the human harmful protozoan parasite Trypanosoma cruzi is able to synthesize free or protein-linked glycoinositolphospholipids containing IPC . As a first step towards understanding the importance and mechanism of IPC synthesis in T . cruzi, we have investigated the effects of rustmicin and aureobasidin A on the proliferation of different life cycle stages of the parasite . The compounds did not interfere with the axenic growth of epimastigotes, but aureobasidin A reduced the release of trypomastigotes from infected murine peritoneal macrophages and the number of intracellular amastigotes in a dose-dependent manner . We have demonstrated for the first time that all forms of T . cruzi express an IPC synthase activity which is able to transfer inositol-phosphate from phosphatidylinositol (PI) to the C-1 hydroxyl group of 6-{N-(7-nitro-2-1,3-benzoxadiazol-4-yl)amino}-hexanoyl ceramide (C 6-NBD-cer) to form inositol phosphophoryl-C 6-NBD-cer (IP-C 6-NBD-cer) . The produced IP-C 6-NBD-cer was purified and characterized by its chromatographic behaviour on TLC and HPLC, sensitivity to PI-specif