S. cerevisiae

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-specific phospholipase C and resistance to mild alkaline hydrolysis . Unlike the Saccharomyces cerevisiae IPC synthase, the T . cruzi enzyme is stimulated by Triton X-100 but not by divalent cations, CHAPS or fatty acid-free BSA, and it is not inhibited by rustmicin, aureobasidin A, or both in combination . Further studies showed that aureobasidin A had effects on macrophages independent of the infecting T . cruzi cells . These results suggest that T . cruzi synthesize its own IPC, but by a mechanism that is not affected by rustmicin and aureobasidin A.

Biochemistry, 2004 Dec 7, 43(48), 15267 - 75
In vitro biosynthesis of glycosylphosphatidylinositol in Aspergillus fumigatus; Fontaine T et al.; Glycosylphosphatidylinositol (GPI) represents a mechanism for the attachment of proteins to the plasma membrane found in all eukaryotic cells . GPI biosynthesis has been mainly studied in parasites, yeast, and mammalian cells . Aspergillus fumigatus, a filamentous fungus, produces GPI-anchored molecules, some of them being essential in the construction of the cell wall . An in vitro assay was used to study the GPI biosynthesis in the mycelium form of this organism . In the presence of UDP-GlcNAc and coenzyme A, the cell-free system produces the initial intermediates of the GPI biosynthesis: GlcNAc-PI, GlcN-PI, and GlcN-(acyl)PI . Using GDP-Man, two types of mannosylation are observed . First, one or two mannose residues are added to GlcN-PI . This mannosylation, never described in fungi, does not require dolichol phosphomannoside (Dol-P-Man) as the monosaccharide donor . Second, one to five mannose residues are added to GlcN-(acyl)PI using Dol-P-Man as the mannose donor . The addition of ethanolamine phosphate groups to the first, second, and third mannose residue is also observed . This latter series of GPI intermediates identified in the A . fumigatus cell-free system indicates that GPI biosynthesis in this filamentous fungus is similar to the mammalian or yeast systems . Thus, these biochemical data are in agreement with a comparative genome analysis that shows that all but 3 of the 21 genes described in the Saccharomyces cerevisiae GPI pathways are found in A . fumigatus.

Science, 2004 Nov 26, 306(5701), 1511 - 2
When the stress of your environment makes you go HOG wild; Westfall PJ et al.; When exposed to increased dissolved solute in their environment (hyperosmotic stress), all eukaryotic cells respond by rapidly activating a conserved mitogen-activated protein kinase cascade, known in budding yeast Saccharomyces cerevisiae as the high osmolarity glycerol (HOG) pathway . Intensive genetic and biochemical analysis in this organism has revealed the presumptive osmosensors, downstream signaling components, and metabolic and transcriptional changes that allow cells to cope with this stressful condition . These findings have had direct application to understanding stress sensing and control of transcription by stress-activated mitogen-activated protein kinases in mammalian cells.

Planta . 2004 Nov 24; {Epub ahead of print}
AtSTP11, a pollen tube-specific monosaccharide transporter in Arabidopsis; Schneidereit A et al.; Pollen development, as well as pollen germination and pollen tube growth, requires a highly regulated supply of sugars . In this paper we describe the molecular, kinetic, and physiological characterization of AtSTP11, a new member of the H(+)/monosaccharide transporter family in Arabidopsis thaliana (L.) Heynh . Heterologous expression in yeast ( Saccharomyces cerevisiae) showed that AtSTP11 is a high-affinity ( K(m)=25 muM), broad-spectrum, and uncoupler-sensitive monosaccharide transporter of the plasma membrane . In reverse transcription-polymerase chain reaction analyses we found that AtSTP11 expression is restricted to flowers . Furthermore, AtSTP11-promoter::GFP plants revealed that AtSTP11 expression is only found in pollen tubes . Using a specific antibody we could also detect the AtSTP11 protein exclusively in pollen tubes but not in other flower tissues or in pollen grains of any developmental stage . These results suggest that the newly identified AtSTP11 transporter plays a role in the supply of monosaccharides to growing pollen tubes.

Nucleic Acids Res . 2004 Nov;32(20):e160.
Systematic analysis of bicistronic reporter assay data; Jacobs JL et al.; Bicistronic reporter assay systems have become a mainstay of molecular biology . While the assays themselves encompass a broad range of diverse and unrelated experimental protocols, the numerical data garnered from these experiments often have similar statistical properties . In general, a primary dataset measures the paired expression of two internally controlled reporter genes . The expression ratio of these two genes is then normalized to an external control reporter . The end result is a 'ratio of ratios' that is inherently sensitive to propagation of the error contributed by each of the respective numerical components . The statistical analysis of this data therefore requires careful handling in order to control for the propagation of error and its potentially misleading effects . A careful survey of the literature found no consistent method for the statistical analysis of data generated from these important and informative assay systems . In this report, we present a detailed statistical framework for the systematic analysis of data obtained from bicistronic reporter assay systems . Specifically, a dual luciferase reporter assay was employed to measure the efficiency of four programmed -1 frameshift signals . These frameshift signals originate from the L-A virus, the SARS-associated Coronavirus and computationally identified frameshift signals from two Saccharomyces cerevisiae genes . Furthermore, these statistical methods were applied to prove that the effects of anisomycin on programmed -1 frameshifting are statistically significant . A set of Microsoft Excel spreadsheets, which can be used as templates for data generated by dual reporter assay systems, and an online tutorial are available at our website . These spreadsheets could be easily adapted to any bicistronic reporter assay system.

Biochem J . 2004 Nov 24; {Epub ahead of print}
Peptide mimotopes of Mycobacterium tuberculosis carbohydrate immunodeterminants; Gevorkian G et al.; Cell-surface saccharides of Mycobacterium tuberculosis appear as crucial factors in tuberculosis pathogenicity and could be useful antigens in tuberculosis immunodiagnosis . Here we report the successful antigenic and immunogenic mimicry of mannose-containing cell-wall compounds of Mycobacterium tuberculosis by dodecamer peptides identified by phage-display technology . Using a rabbit antiserum raised against Mycobacterium tuberculosis cell-surface saccharides as a target for biopanning, peptides with three different consensus sequences were identified . Phage-displayed and chemically synthesised peptides bound to the anti-carbohydrate antiserum . Rabbit antibodies elicited against the peptide QEPLMGTVPIRAGGGS recognise mannosylated Mycobacterium tuberculosis cell-wall antigens arabinomannan, lipoarabinomannan and the glycosylated recombinant protein Apa . Furthermore, antibodies were also able to react with mannan from Saccharomyces cerevisiae, but not with phosphatidyl-inositol-di-mannosides or arabinogalactan from mycobacteria . These results suggest that the immunogenic peptide mimics oligomannosidic epitopes . Interestingly, this report provides evidence that, in contrast to previously known carbohydrate mimotopes, no aromatic residues are necessary in a peptide sequence for mimicking unusual glycoconjugates synthesised by mycobacteria . The possible usefulness of the identified peptide mimotopes as surrogate reagents for immunodiagnosis and for the study of functional roles of the native non-peptide epitopes is discussed.

Cell Mol Life Sci, 2004 Nov, 61(22), 2799 - 811
Mitochondrial DNA mutators; Foury F et al.; In this article we review our current knowledge of the mechanisms by which point mutations arise in the mitochondrial DNA (mtDNA) of Saccharomyces cerevisiae and discuss to what extent these mechanisms operate in human mtDNA mutagenesis . The 3'-5' exonuclease proofreading activity of Pol gamma ensures accuracy of mtDNA replication in both yeast and humans, while the role of base excision repair in mtDNA error avoidance remains debated . The mitochondrial mismatch repair Msh1 protein, which removes transitions in yeast, is absent in humans, a particularity that might cause accumulation of transitions, while the most frequent substitution in yeast mtDNA is A:T to T:A transversion . Proofreading-deficient mutator human cell lines and knockin mice have been created . They will be useful for studying the mechanisms by which mtDNA mutations accumulate in diseases, ageing, malignancy and drug therapy.

J Cell Biol, 2004 Nov 22, 167(4), 605 - 11
Mlp-dependent anchorage and stabilization of a desumoylating enzyme is required to prevent clonal lethality; Zhao X et al.; Myosin-like proteins 1 and 2 (Mlp1 and Mlp2) form filaments attached to the nucleoplasmic side of the nuclear pore complexes via interaction with the nucleoporin Nup60 . Here, we show that Mlps and Nup60, but not several other nucleoporins, are required to localize and stabilize a desumoylating enzyme Ulp1 . Moreover, like Mlps, Ulp1 exhibits a unique asymmetric distribution on the nuclear envelope . Consistent with a role in regulating Ulp1, removal of either or both MLPs affects the SUMO conjugate pattern . We also show that deleting MLPs or the localization domains of Ulp1 results in DNA damage sensitivity and clonal lethality, the latter of which is caused by increased levels of 2-micron circle DNA . Epistatic and dosage suppression analyses further demonstrate that Mlps function upstream of Ulp1 in 2-micron circle maintenance and the damage response . Together, our results reveal that Mlps play important roles in regulating Ulp1 and subsequently affect sumoylation stasis, growth, and DNA repair.

J Cell Biol, 2004 Nov 22, 167(4), 583 - 90
The FG-repeat asymmetry of the nuclear pore complex is dispensable for bulk nucleocytoplasmic transport in vivo; Zeitler B et al.; Nucleocytoplasmic transport occurs through gigantic proteinaceous channels called nuclear pore complexes (NPCs) . Translocation through the NPC is exquisitely selective and is mediated by interactions between soluble transport carriers and insoluble NPC proteins that contain phenylalanine-glycine (FG) repeats . Although most FG nucleoporins (Nups) are organized symmetrically about the planar axis of the nuclear envelope, very few localize exclusively to one side of the NPC . We constructed Saccharomyces cerevisiae mutants with asymmetric FG repeats either deleted or swapped to generate NPCs with inverted FG asymmetry . The mutant Nups localize properly within the NPC and exhibit exchanged binding specificity for the export factor Xpo1 . Surprisingly, we were unable to detect any defects in the Kap95, Kap121, Xpo1, or mRNA transport pathways in cells expressing the mutant FG Nups . These findings suggest that the biased distribution of FG repeats is not required for major nucleocytoplasmic trafficking events across the NPC.

FEBS Lett, 2004 Nov 19, 577(3), 422 - 6
A genome-wide screen identifies Yos9p as essential for ER-associated degradation of glycoproteins; Buschhorn BA et al.; We undertook a growth-based screen exploiting the degradation of CTL*, a chimeric membrane-bound ERAD substrate derived from soluble lumenal CPY* . We screened the Saccharomyces cerevisiae genomic deletion library containing approximately 5000 viable strains for mutants defective in endoplasmic reticulum (ER) protein quality control and degradation (ERAD) . Among the new gene products we identified Yos9p, an ER-localized protein previously involved in the processing of GPI anchored proteins . We show that deficiency in Yos9p affects the degradation only of glycosylated ERAD substrates . Degradation of non-glycosylated substrates is not affected in cells lacking Yos9p . We propose that Yos9p is a lectin or lectin-like protein involved in the quality control of N-glycosylated proteins . It may act sequentially or in concert with the ERAD lectin Htm1p/Mnl1p (EDEM) to prevent secretion of malfolded glycosylated proteins and deliver them to the cytosolic ubiquitin-proteasome machinery for elimination.

Biochem Biophys Res Commun, 2004 Dec 24, 325(4), 1495 - 502
Drosophila melanogaster CYP6A8, an insect P450 that catalyzes lauric acid (omega-1)-hydroxylation; Helvig C et al.; Only a handful of P450 genes have been functionally characterized from the approximately 90 recently identified in the genome of Drosophila melanogaster . Cyp6a8 encodes a 506-amino acid protein with 53.6% amino acid identity with CYP6A2 . CYP6A2 has been shown to catalyze the metabolism of several insecticides including aldrin and heptachlor . CYP6A8 is expressed at many developmental stages as well as in adult life . CYP6A8 was produced in Saccharomyces cerevisiae and enzymatically characterized after catalytic activity was reconstituted with D . melanogaster P450 reductase and NADPH . Although several saturated or non-saturated fatty acids were not metabolized by CYP6A8, lauric acid (C12:0), a short-chain unsaturated fatty acid, was oxidized by CYP6A8 to produce 11-hydroxylauric acid with an apparent V(max) of 25 nmol/min/nmol P450 . This is the first report showing that a member of the CYP6 family catalyzes the hydroxylation of lauric acid . Our data open new prospects for the CYP6 P450 enzymes, which could be involved in important physiological functions through fatty acid metabolism.

Am J Gastroenterol, 2004 Nov, 99(11), 2186 - 94
A prospective comparative study of ASCA and pANCA in Chinese and Caucasian IBD patients; Lawrance IC et al.; BACKGROUND: Inflammatory bowel disease manifests throughout all ethnic groups . Antisaccharomyces cerevisiae (ASCA) and antineutrophil cytoplasmic antibodies (pANCA) can aid in the differentiation between Crohn's disease (CD) and ulcerative colitis (UC), but their sensitivity may vary between races . OBJECTIVES: This study compared the sensitivity, specificity, and positive and negative predictive values (PPV, NPV) of pANCA and ASCA between Chinese and Caucasian IBD populations and identified disease subtype associations . RESULTS: Three hundred patients were prospectively recruited from Caucasian and Chinese populations (CD, n = 50, UC, n = 50, controls, n = 50 each) . pANCA detection was greater in Caucasian than Chinese UC patients (p= 0.046) . ASCA IgG detection was similar, but IgA was lower in Chinese CD patients (p < 0.001) . Differentiation between UC and CD (+ve pANCA/-ve ASCA) demonstrated a PPV of 92% in isolated colonic disease . Logistic regression in CD identified positive pANCA had a lower association with ileal (OR = 6.8, p= 0.0067) and complicated disease (OR = 5.5, p= 0.015) . Caucasian CD patients with positive ASCA IgA/IgG had a greater association with ileal (OR = 6.7, p= 0.022) or complicated disease (OR = 9.4, p= 0.0073) and in Chinese CD patients positive ASCA IgA/IgG was associated with isolated ileal disease (OR = 16.8, p= 0.032) . Linear regression demonstrated that higher ASCA titers predicted complicated CD and isolated ileal disease . CONCLUSIONS: This study identified that pANCA is more sensitive in Caucasian than Chinese UC and that ASCA IgA has a low yield in Chinese CD . pANCA and ASCA are useful for differentiating between UC and CD in both populations, and ASCA IgG and IgA titers have potential use in determining the risk of developing complicated CD.

Mol Microbiol, 2004 Dec, 54(5), 1173 - 85
Mitochondrial beta-oxidation in Aspergillus nidulans; Maggio-Hall LA et al.; Beta-oxidation (beta-ox) occurs exclusively in the peroxisomes of Saccharomyces cerevisiae and other yeasts, leading to the supposition that fungi lack mitochondrial beta-ox . Here we present unequivocal evidence that the filamentous fungus Aspergillus nidulans houses both peroxisomal and mitochondrial beta-ox . While growth of a peroxisomal beta-ox disruption mutant (DeltafoxA) was eliminated on a very long-chain fatty acid (C(22:1)), growth was only partially impeded on a long-chain fatty acid (C(18:1)) and was not affected at all on short chain (C4-C6) fatty acids . In contrast, growth of a putative enoyl-CoA hydratase mutant (DeltaechA) was abolished on short-chain and severely restricted on long- and very long-chain fatty acids . Furthermore fatty acids inhibited growth of the DeltaechA mutant but not the DeltafoxA mutant in the presence of an alternate carbon source (lactose) . Disruption of echA led to a 28-fold reduction in 2-butenoyl-CoA hydratase activity in a preparation of organelles . EchA was also required for growth on isoleucine and valine . The subcellular localization of the FoxA and EchA proteins was confirmed through the use of red and green fluorescent protein fusions.

Mar Biotechnol (NY) . 2004 Nov 4; {Epub ahead of print}
Molecular Cloning and Functional Characterization of Fatty Acyl Desaturase and Elongase cDNAs Involved in the Production of Eicosapentaenoic and Docosahexaenoic Acids from ALPHA-Linolenic Acid in Atlantic Salmon ( Salmo salar); Hastings N et al.; Fish are the only major dietary source for humans of omega-3 highly unsaturated fatty acids (HUFAs) and with declining fisheries farmed fish such as Atlantic salmon ( Salmo salar) constitute an increasing proportion of the fish in the human diet . However, the current high use of fish oils, derived from wild capture marine fisheries, in aquaculture feeds is not sustainable in the longer term and will constrain continuing growth of aquaculture activities . Greater understanding of how fish metabolize and biosynthesize HUFA may lead to more sustainable aquaculture diets . The study described here contributes to an effort to determine the molecular genetics of the HUFA biosynthetic pathway in salmon, with the overall aim being to determine mechanisms for optimizing the use of vegetable oils in Atlantic salmon culture . In this paper we describe the cloning and functional characterization of 2 genes from salmon involved in the biosynthesis of HUFA . A salmon desaturase complementary DNA, SalDes, was isolated that include an open reading frame of 1362 bp specifying a protein of 454 amino acids . The protein sequence includes all the characteristics of microsomal fatty acid desaturases, including 3 histidine boxes, 2 transmembrane regions, and an N-terminal cytochrome b(5) domain containing a heme-binding motif similar to that of other fatty acid desaturases . Functional expression in the yeast Saccharomyces cerevisiae showed SalDes is predominantly an omega-3 delta5 desaturase, a key enzyme in the synthesis of eicosapentaenoic acid (20:5n-3) from alpha-linolenic acid (18:3n-3) . The desaturase showed only low levels of delta6 activity toward C(18) polyunsaturated fatty acids . In addition, a fatty acid elongase cDNA, SalElo, was isolated that included an open reading frame of 888 bp, specifying a protein of 295 amino acids . The protein sequence of SalElo included characteristics of microsomal fatty acid elongases, including a histidine box and a transmembrane region . Upon expression in yeast SalElo showed broad substrate specificity for polyunsaturated fatty acids with a range of chain lengths, with the rank order being C(18) > C(20) > C(22) . Thus this one polypeptide product displays all fatty acid elongase activities required for the biosynthesis of docosahexaenoic acid (22:6n-3) from 18:3n-3.

EMBO J, 2004 Dec 8, 23(24), 4868 - 75 Epub 2004 Dec 8.
The CDK regulates repair of double-strand breaks by homologous recombination during the cell cycle; Aylon Y et al.; DNA double-strand breaks (DSBs) are dangerous lesions that can lead to genomic instability and cell death . Eukaryotic cells repair DSBs either by nonhomologous end-joining (NHEJ) or by homologous recombination . We investigated the ability of yeast cells (Saccharomyces cerevisiae) to repair a single, chromosomal DSB by recombination at different stages of the cell cycle . We show that cells arrested at the G(1) phase of the cell cycle restrict homologous recombination, but are able to repair the DSB by NHEJ . Furthermore, we demonstrate that recombination ability does not require duplicated chromatids or passage through S phase, and is controlled at the resection step by Clb-CDK activity.

Plant Cell, 2004 Dec, 16(12), 3216 - 29 Epub 2004 Dec.
Heterodimerization and endocytosis of Arabidopsis brassinosteroid receptors BRI1 and AtSERK3 (BAK1); Russinova E et al.; In Arabidopsis thaliana brassinosteroid (BR), perception is mediated by two Leu-rich repeat receptor-like kinases, BRASSINOSTEROID INSENSITIVE1 (BRI1) and BRI1-ASSOCIATED RECEPTOR KINASE1 (BAK1) (Arabidopsis SOMATIC EMBRYOGENESIS RECEPTOR-like KINASE3 {AtSERK3}) . Genetic, biochemical, and yeast (Saccharomyces cerevisiae) interaction studies suggested that the BRI1-BAK1 receptor complex initiates BR signaling, but the role of the BAK1 receptor is still not clear . Using transient expression in protoplasts of BRI1 and AtSERK3 fused to cyan and yellow fluorescent green fluorescent protein variants allowed us to localize each receptor independently in vivo . We show that BRI1, but not AtSERK3, homodimerizes in the plasma membrane, whereas BRI1 and AtSERK3 preferentially heterodimerize in the endosomes . Coexpression of BRI1 and AtSERK3 results in a change of the steady state distribution of both receptors because of accelerated endocytosis . Endocytic vesicles contain either BRI1 or AtSERK3 alone or both . We propose that the AtSERK3 protein is involved in changing the equilibrium between plasma membrane-located BRI1 homodimers and endocytosed BRI1-AtSERK3 heterodimers.

Proc Natl Acad Sci U S A, 2004 Nov 30, 101(48), 16843 - 8 Epub 2004 Nov 30.
Changes in genomewide occupancy of core transcriptional regulators during heat stress; Zanton SJ et al.; Organisms respond to heat stress by reprogramming gene expression . In Saccharomyces cerevisiae, heat-induced genes tend to be regulated by factors that belong to the Spt-Ada-Gcn5 acetyltransferase (SAGA) transcription regulatory pathway, whereas heat-repressed genes tend to be regulated by a parallel pathway involving transcription factor IID (TFIID) . Here, we examine whether heat stress affects the occupancy of representative factors of each pathway at promoter regions throughout the yeast genome . Representatives of the SAGA pathway include the TATA binding protein, Spt3, and Mot1 . Representatives of the TFIID pathway include the TATA binding protein, TAF1, and Bdf1 . We find that heat stress causes disassembly of the TFIID pathway at genes that are inhibited by stress . In contrast, heat induces assembly of the SAGA pathway at stress-induced genes, although many also assemble along the TFIID pathway . Other genes were found to assemble almost exclusively along the TFIID pathway . Strikingly, these genes are lowly transcribed and are generally not induced . Thus, heat stress leads to factor assembly along each pathway but with distinct transcriptional outcomes . Further investigation of these pathways reveals that Bdf1 and Mot1 negatively regulate the SAGA pathway in different ways . The findings suggest that Bdf1 blocks assembly, whereas Mot1 promotes disassembly of the transcription machinery.

Biochem Pharmacol, 2004 Dec 15, 68(12), 2417 - 25
Human CYP4F12 genetic polymorphism: identification and functional characterization of seven variant allozymes; Cauffiez C et al.; The human cytochrome CYP4F12 has been shown to be metabolically active toward inflammatory mediators and exogenous compounds such as antihistaminic drugs . We recently identified a genetic polymorphism within the promoter region, associated with a decreased level of enzyme expression . In the present study, we report the further identification of single nucleotide polymorphisms in the coding sequence of the CYP4F12 gene . A polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) analysis of DNA samples from 53 unrelated French Caucasians, allowed the identification of ten mutations, comprising seven missense mutations, 31C>T (Leu11Phe), 38C>T (Pro13Leu), 47C>T (Met16Thr), 4759G>A (Asp76Asn), 4801G>A (Val90Leu), 8896C>T (Arg188Cys) and 23545G>A (Gly522Ser) . Their functional impact toward ebastine hydroxylation was evaluated using heterologous expression in Saccharomyces cerevisiae cells of site-directed mutated cDNA variants . Five out seven variants did not exhibit any significant difference in CYP4F12 catalytic activity, whereas two variants, Val90Ile and Arg188Cys, displayed significant changes in their Michaelis-Menten (Km, Vm) parameters . These data on CYP4F12 genetic polymorphism provide tools for further studies of association with pathological processes involving an inflammatory component and with variations in anti-histaminic drug response.

J Cell Biochem, 2005 Feb 1, 94(2), 247 - 56
Origins of bidirectional replication of Epstein-Barr virus: Models for understanding mammalian origins of DNA synthesis; Wang J et al.; Epstein-Barr virus (EBV), provides unique advantages to understand origins of replication in higher eukaryotes . EBV establishes itself efficiently in infected B lymphocytes, where it exists as a 165 kb, circular chromosome which is duplicated once per cell cycle (Adams {1987} J Virol 61:1743-1746) . Five to twenty copies of the EBV chromosome are usually present in each cell, increasing the signal/noise ratio for mapping and analyzing its replication origins . Remarkably only one viral protein is required for the synthesis and partitioning of the viral chromosomes: EBV nuclear antigen-1, or EBNA1 . EBV uses distinct origins related to the ARS1 origin of Saccharomyces cerevisiae and to that of the dihydrofolate reductase (DHFR) locus in Chinese hamster ovary (CHO) cells {Bogan et al., 2000} . We shall review the properties and the regulation of these two kinds of origins in EBV and relate them to their cellular cousins . (c) 2004 Wiley-Liss, Inc.

Proc Natl Acad Sci U S A, 2004 Nov 23, 101(47), 16577 - 82 Epub 2004 Nov 23.
Integrative analysis of genome-scale data by using pseudoinverse projection predicts novel correlation between DNA replication and RNA transcription; Alter O et al.; We describe an integrative data-driven mathematical framework that formulates any number of genome-scale molecular biological data sets in terms of one chosen set of data samples, or of profiles extracted mathematically from data samples, designated the "basis" set . By using pseudoinverse projection, the molecular biological profiles of the data samples are least-squares-approximated as superpositions of the basis profiles . Reconstruction of the data in the basis simulates experimental observation of only the cellular states manifest in the data that correspond to those of the basis . Classification of the data samples according to their reconstruction in the basis, rather than their overall measured profiles, maps the cellular states of the data onto those of the basis and gives a global picture of the correlations and possibly also causal coordination of these two sets of states . We illustrate this framework with an integration of yeast genome-scale proteins' DNA-binding data with cell cycle mRNA expression time course data . Novel correlation between DNA replication initiation and RNA transcription during the yeast cell cycle, which might be due to a previously unknown mechanism of regulation, is predicted.

J Mol Biol, 2004 Dec 3, 344(4), 885 - 92
Crystal structure of the spindle assembly checkpoint protein Bub3; Larsen NA et al.; Bub3 is one of at least six proteins that transmit the spindle assembly checkpoint signal . These proteins delay cell cycle progression from metaphase to anaphase in response to attachment defects between kinetochores and spindle microtubules and to tension defects between sister chromatids . To explore the molecular interactions mediated by Bub3, we have determined the crystal structure of the Saccharomyces cerevisiae protein Bub3p at 2.35 A resolution . Bub3p is a seven-blade beta-propeller, although its sequence diverges from that of other WD40 family members . Several loops are substantially elongated, but extra domains or insertions are not present at the termini . In particular, two extended loops project from the top face of the propeller, forming a cleft . Amino acid residues across the top face and one aspect of the lateral surface (spanning blades 5-6) are highly conserved among Bub3 proteins . We propose that these conserved surfaces are the loci for key interactions with conserved motifs in spindle checkpoint proteins Bub1 and Mad3/BubR1 . Comparison of the Bub3 sequence to the WD40 protein, Rae1, shows high sequence conservation along the same surfaces . Rae1 interaction with Bub1 is, therefore, likely to involve a similar mode of binding.

Nat Struct Mol Biol, 2004 Dec, 11(12), 1237 - 42 Epub 2004 Dec.
U2-U6 RNA folding reveals a group II intron-like domain and a four-helix junction; Sashital DG et al.; Intron removal in nuclear precursor mRNA is catalyzed through two transesterification reactions by a multi-megaDalton ribonucleoprotein machine called the spliceosome . A complex between U2 and U6 small nuclear RNAs is a core component of the spliceosome . Here we present an NMR structural analysis of a protein-free U2-U6 complex from Saccharomyces cerevisiae . The observed folding of the U2-U6 complex is a four-helix junction, in which the catalytically important AGC triad base-pairs only within U6 and not with U2 . The base-pairing of the AGC triad extends the U6 intramolecular stem-loop (U6 ISL), and the NMR structure of this extended U6 ISL reveals structural similarities with domain 5 of group II self-splicing introns . The observed conformation of the four-helix junction could be relevant to the first, but not the second, step of splicing and may help to position the U6 ISL adjacent to the 5' splice site.

Mol Cell Biol, 2004 Dec, 24(23), 10448 - 55
Deficiency in SNM1 abolishes an early mitotic checkpoint induced by spindle stress; Akhter S et al.; Spindle poisons represent an important class of anticancer drugs that act by interfering with microtubule polymerization and dynamics and thereby induce mitotic checkpoints and apoptosis . Here we show that mammalian SNM1 functions in an early mitotic stress checkpoint that is distinct from the well-characterized spindle checkpoint that regulates the metaphase-to-anaphase transition . Specifically, we found that compared to wild-type cells, Snm1-deficient mouse embryonic fibroblasts exposed to spindle poisons exhibited elevated levels of micronucleus formation, decreased mitotic delay, a failure to arrest in mitosis prior to chromosome condensation, supernumerary centrosomes, and decreased viability . In addition, we show that both Snm1 and 53BP1, previously shown to interact, coimmunoprecipitate with components of the anaphase-promoting complex (APC)/cyclosome . These findings suggest that Snm1 is a component of a mitotic stress checkpoint that negatively targets the APC prior to chromosome condensation.

Mol Cell Biol, 2004 Dec, 24(23), 10193 - 207
Transcription factor binding and induced transcription alter chromosomal c-myc replicator activity; Ghosh M et al.; The observation that transcriptionally active genes generally replicate early in S phase and observations of the interaction between transcription factors and replication proteins support the thesis that promoter elements may have a role in DNA replication . To test the relationship between transcription and replication we constructed HeLa cell lines in which inducible green fluorescent protein (GFP)-encoding genes replaced the proximal approximately 820-bp promoter region of the c-myc gene . Without the presence of an inducer, basal expression occurred from the GFP gene in either orientation and origin activity was restored to the mutant c-myc replicator . In contrast, replication initiation was repressed upon induction of transcription . When basal or induced transcription complexes were slowed by the presence of alpha-amanitin, origin activity depended on the orientation of the transcription unit . To test mechanistically whether basal transcription or transcription factor binding was sufficient for replication rescue by the uninduced GFP genes, a GAL4p binding cassette was used to replace all regulatory sequences within approximately 1,400 bp 5' to the c-myc gene . In these cells, expression of a CREB-GAL4 fusion protein restored replication origin activity . These results suggest that transcription factor binding can enhance replication origin activity and that high levels of expression or the persistence of transcription complexes can repress it.

Mol Cell Biol, 2004 Dec, 24(23), 10126 - 44
A Tel1/MRX-dependent checkpoint inhibits the metaphase-to-anaphase transition after UV irradiation in the absence of Mec1; Clerici M et al.; In Saccharomyces cerevisiae, Mec1/ATR plays a primary role in sensing and transducing checkpoint signals in response to different types of DNA lesions, while the role of the Tel1/ATM kinase in DNA damage checkpoints is not as well defined . We found that UV irradiation in G(1) in the absence of Mec1 activates a Tel1/MRX-dependent checkpoint, which specifically inhibits the metaphase-to-anaphase transition . Activation of this checkpoint leads to phosphorylation of the downstream checkpoint kinases Rad53 and Chk1, which are required for Tel1-dependent cell cycle arrest, and their adaptor Rad9 . The spindle assembly checkpoint protein Mad2 also partially contributes to the G(2)/M arrest of UV-irradiated mec1Delta cells independently of Rad53 phosphorylation and activation . The inability of UV-irradiated mec1Delta cells to undergo anaphase can be relieved by eliminating the anaphase inhibitor Pds1, whose phosphorylation and stabilization in these cells depend on Tel1, suggesting that Pds1 persistence may be responsible for the inability to undergo anaphase . Moreover, while UV irradiation can trigger Mec1-dependent Rad53 phosphorylation and activation in G(1)- and G(2)-arrested cells, Tel1-dependent checkpoint activation requires entry into S phase independently of the cell cycle phase at which cells are UV irradiated, and it is decreased when single-stranded DNA signaling is affected by the rfa1-t11 allele . This indicates that UV-damaged DNA molecules need to undergo structural changes in order to activate the Tel1-dependent checkpoint . Active Clb-cyclin-dependent kinase 1 (CDK1) complexes also participate in triggering this checkpoint and are required to maintain both Mec1- and Tel1-dependent Rad53 phosphorylation, suggesting that they may provide critical phosphorylation events in the DNA damage checkpoint cascade.

Mol Cell Biol, 2004 Dec, 24(23), 10111 - 7
Evidence for eviction and rapid deposition of histones upon transcriptional elongation by RNA polymerase II; Schwabish MA et al.; Biochemical experiments indicate that transcriptional elongation by RNA polymerase II (Pol II) is inhibited by nucleosomes and hence requires chromatin-modifying activities . Here, we examine the fate of histones upon passage of elongating Pol II in vivo . Histone density throughout the entire Saccharomyces cerevisiae GAL10 coding region is inversely correlated with Pol II association and transcriptional activity, suggesting that the elongating Pol II machinery efficiently evicts core histones from the DNA . Furthermore, new histones appear to be deposited onto DNA less than 1 min after passage of Pol II . Transcription-dependent deposition of histones requires the FACT complex that travels with elongating Pol II . Our results suggest that Pol II transcription generates a highly dynamic equilibrium of histone eviction and histone deposition and that there is significant histone exchange throughout most of the yeast genome within a single cell cycle.

Mol Cell Biol, 2004 Dec, 24(23), 10101 - 10
Interaction between a G-patch protein and a spliceosomal DEXD/H-box ATPase that is critical for splicing; Silverman EJ et al.; Prp2 is an RNA-dependent ATPase that activates the spliceosome before the first transesterification reaction of pre-mRNA splicing . Prp2 has extensive homology throughout the helicase domain characteristic of DEXD/H-box helicases and a conserved carboxyl-terminal domain also found in the spliceosomal helicases Prp16, Prp22, and Prp43 . Despite the extensive homology shared by these helicases, each has a distinct, sequential role in splicing; thus, uncovering the determinants of specificity becomes crucial to the understanding of Prp2 and the other DEAH-splicing helicases . Mutations in an 11-mer near the C-terminal end of Prp2 eliminate its spliceosome binding and splicing activity . Here we show that a helicase-associated protein interacts with this domain and that this interaction contributes to the splicing process . First, a genome-wide yeast two-hybrid screen using Prp2 as bait identified Spp2, which contained a motif with glycine residues found in a number of RNA binding proteins . SPP2 was originally isolated as a genetic suppressor of a prp2 mutant . In a reciprocal screen, Spp2 specifically pulled out the C-terminal half of Prp2 . Mutations in the Prp2 C-terminal 11-mer that disrupted function or spliceosome binding also disrupted Spp2 interaction . A screen of randomly mutagenized SPP2 clones identified an Spp2 protein with a mutation in the G patch that could restore interaction with Prp2 and enhanced splicing in a prp2 mutant strain . The study identifies a potential mechanism for Prp2 specificity mediated through a unique interaction with Spp2 and elucidates a role for a helicase-associated protein in the binding of a DEXD/H-box protein to the spliceosome.

Arch Biochem Biophys, 2004 Dec 15, 432(2), 244 - 51
The iSH2 domain of PI 3-kinase is a rigid tether for p110 and not a conformational switch; Fu Z et al.; Class IA PI 3-kinases are heterodimeric proteins with distinct catalytic (p110) and regulatory (p85) subunits . The minimal fragment of p85 capable of regulating p110 activity (p85ni) is the N-terminal SH2 domain linked to the iSH2 coiled-coil domain . We used cysteine mutagenesis and (14)C-NEM-labeling to show that the p110-binding site in the iSH2 domain includes two regions: residues 482-484 and 532-541 . These regions are adjacent to each other in the three-dimensional structural model of the iSH2 domain, and define a coherent binding site . We then used spin labeling and EPR spectroscopy to demonstrate that the conformation of the iSH2 domain is unaffected by binding to the N-terminal fragment of p110 (residues 1-108), and/or by phosphopeptide binding to p85ni/p110(1-108) heterodimers . Finally, we show that the cSH2 domain cannot substitute for the nSH2 domain with regard to inhibition of p110 . These data support a model in which the iSH2 domain is a rigid tether for p110, and regulation of p85/p110 is mediated by nSH2-p110 contacts.

Biochem Biophys Res Commun, 2004 Dec 17, 325(3), 892 - 8
Differential regulation of gene expression by RNA polymerase II in response to DNA damage; Heo JH et al.; Cells change their gene expression profile dynamically in various conditions . By taking the advantage of ChIP, we examined the transcription profile of Saccharomyces cerevisiae genes in response to DNA damaging agents such as MMS or 4NQO . Gene expression profiles of different groups of genes roughly correlated with that revealed by Northern blot assay or microarray method . Damage-inducible genes showed increased cross-linking signals of RNA polymerase II, TFIIH, and TFIIF, meanwhile damage repressible genes decreased them, which means that gene expression is mainly regulated at the level of transcription . Interestingly, the characteristic occupancy pattern of TFIIH and polymerase with phosphorylated carboxy-terminal domain (CTD) in promoter or in coding regions was not changed by the presence of DNA damaging agents in both non-inducible and inducible genes . ChIP data showed that the extent of phosphorylation of CTD per elongating polymerase complex was still maintained . These findings suggest that overall increase in CTD phosphorylation in response to DNA damage is attributed to the global shift of gene expression profile rather than modification of specific polymerase function.

Proc Natl Acad Sci U S A, 2004 Nov 23, 101(47), 16594 - 9 Epub 2004 Nov 23.
Finding new components of the target of rapamycin (TOR) signaling network through chemical genetics and proteome chips; Huang J et al.; The TOR (target of rapamycin) proteins play important roles in nutrient signaling in eukaryotic cells . Rapamycin treatment induces a state reminiscent of the nutrient starvation response, often resulting in growth inhibition . Using a chemical genetic modifier screen, we identified two classes of small molecules, small-molecule inhibitors of rapamycin (SMIRs) and small-molecule enhancers of rapamycin (SMERs), that suppress and augment, respectively, rapamycin's effect in the yeast Saccharomyces cerevisiae . Probing proteome chips with biotinylated SMIRs revealed putative intracellular target proteins, including Tep1p, a homolog of the mammalian PTEN (phosphatase and tensin homologue deleted on chromosome 10) tumor suppressor, and Ybr077cp (Nir1p), a protein of previously unknown function that we show to be a component of the TOR signaling network . Both SMIR target proteins are associated with PI(3,4)P2, suggesting a mechanism of regulation of the TOR pathway involving phosphatidylinositides . Our results illustrate the combined use of chemical genetics and proteomics in biological discovery and map a path for creating useful therapeutics for treating human diseases involving the TOR pathway, such as diabetes and cancer.

EMBO J, 2004 Dec 8, 23(24), 4857 - 67 Epub 2004 Dec 8.
Polyamines regulate their synthesis by inducing expression and blocking degradation of ODC antizyme; Palanimurugan R et al.; Polyamines are essential organic cations with multiple cellular functions . Their synthesis is controlled by a feedback regulation whose main target is ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis . In mammals, ODC has been shown to be inhibited and targeted for ubiquitin-independent degradation by ODC antizyme (AZ) . The synthesis of mammalian AZ was reported to involve a polyamine-induced ribosomal frameshifting mechanism . High levels of polyamine therefore inhibit new synthesis of polyamines by inducing ODC degradation . We identified a previously unrecognized sequence in the genome of Saccharomyces cerevisiae encoding an orthologue of mammalian AZ . We show that synthesis of yeast AZ (Oaz1) involves polyamine-regulated frameshifting as well . Degradation of yeast ODC by the proteasome depends on Oaz1 . Using this novel model system for polyamine regulation, we discovered another level of its control . Oaz1 itself is subject to ubiquitin-mediated proteolysis by the proteasome . Degradation of Oaz1, however, is inhibited by polyamines . We propose a model, in which polyamines inhibit their ODC-mediated biosynthesis by two mechanisms, the control of Oaz1 synthesis and inhibition of its degradation.

Cell, 2004 Nov 12, 119(4), 503 - 16
Mammalian Pins is a conformational switch that links NuMA to heterotrimeric G proteins; Du Q et al.; During asymmetric cell divisions, mitotic spindles align along the axis of polarization . In invertebrates, spindle positioning requires Pins or related proteins and a G protein alpha subunit . A mammalian Pins, called LGN, binds Galphai and also interacts through an N-terminal domain with the microtubule binding protein NuMA . During mitosis, LGN recruits NuMA to the cell cortex, while cortical association of LGN itself requires the C-terminal Galpha binding domain . Using a FRET biosensor, we find that LGN behaves as a conformational switch: in its closed state, the N and C termini interact, but NuMA or Galphai can disrupt this association, allowing LGN to interact simultaneously with both proteins, resulting in their cortical localization . Overexpression of Galphai or YFP-LGN causes a pronounced oscillation of metaphase spindles, and NuMA binding to LGN is required for these spindle movements . We propose that a related switch mechanism might operate in asymmetric cell divisions in the fly and nematode.

Cell, 2004 Nov 12, 119(4), 491 - 502
She2p is a novel RNA binding protein with a basic helical hairpin motif; Niessing D et al.; Selective transport of mRNAs in ribonucleoprotein particles (mRNP) ensures asymmetric distribution of information within and among eukaryotic cells . Actin-dependent transport of ASH1 mRNA in yeast represents one of the best-characterized examples of mRNP translocation . Formation of the ASH1 mRNP requires recognition of zip code elements by the RNA binding protein She2p . We determined the X-ray structure of She2p at 1.95 A resolution . She2p is a member of a previously unknown class of nucleic acid binding proteins, composed of a single globular domain with a five alpha helix bundle that forms a symmetric homodimer . After demonstrating potent, dimer-dependent RNA binding in vitro, we mapped the RNA binding surface of She2p to a basic helical hairpin in vitro and in vivo and present a mechanism for mRNA-dependent initiation of ASH1 mRNP complex assembly.

J Biol Chem, 2005 Jan 21, 280(3), 1921 - 30 Epub 2004 Nov 09.
Ubiquitination of the Peroxisomal Targeting Signal Type 1 Receptor, Pex5p, Suggests the Presence of a Quality Control Mechanism during Peroxisomal Matrix Protein Import; Kiel JA et al.; PEX genes encode proteins (peroxins) that are required for the biogenesis of peroxisomes . One of these peroxins, Pex5p, is the receptor for matrix proteins with a type 1 peroxisomal targeting signal (PTS1), which shuttles newly synthesized proteins from the cytosol into the peroxisome matrix . We observed that in various Saccharomyces cerevisiae pex mutants disturbed in the early stages of PTS1 import, the steady-state levels of Pex5p are enhanced relative to wild type controls . Furthermore, we identified ubiquitinated forms of Pex5p in deletion mutants of those PEX genes that have been implicated in recycling of Pex5p from the peroxisomal membrane into the cytosol . Pex5p ubiquitination required the presence of the ubiquitin-conjugating enzyme Ubc4p and the peroxins that are required during early stages of PTS1 protein import . Finally, we provide evidence that the proteasome is involved in the turnover of Pex5p in wild type yeast cells, a process that requires Ubc4p and occurs at the peroxisomal membrane . Our data suggest that during receptor recycling a portion of Pex5p becomes ubiquitinated and degraded by the proteasome . We propose that this process represents a conserved quality control mechanism in peroxisome biogenesis.

Antivir Chem Chemother, 2004 Sep, 15(5), 261 - 8
Treatment of mannan-enhanced influenza B virus infections in mice with oseltamivir, ribavirin and viramidine; Smee DF et al.; Mannan, a polysaccharide preparation from Saccharomyces cerevisiae, has previously been shown to enhance influenza virus replication in mice by inhibiting host defense collectins . The use of mannan in infections may serve to broaden the types of influenza viruses that can be studied in rodent infection models . When mannan was co-administered with influenza B/Sichuan/379/99 virus to mice, the animals died from the infection, whereas mice infected with only virus survived . Three types of influenza A (H1N1) and another influenza B (Hong Kong/330/01) virus infection were also enhanced by mannan, but not four types of influenza A (H3N2) viruses . Mannan was used at 0.16 or 0.5 mg/mouse for optimal disease-enhancing activity using influenza B/Sichuan/379/99 virus . Using this model, influenza B/Sichuan/379/99 infections were treated with oseltamivir, ribavirin or viramidine (the carboxamidine derivative of ribavirin) . When oral gavage treatments started 4 h before virus and mannan challenge, oseltamivir was effective at 2.5, 5 and 10 mg/kg/day . Ribavirin was active at 20, 40 and 80 mg/kg/day . Viramidine was effective at 80 and 160 mg/kg/day but not at 40 mg/kg/day . Active drug doses improved lung consolidation scores and lung weights, with decreases in lung virus titres also noted . Arterial oxygen saturation values in treated groups were significantly better than those of the placebo group on days 7-11 of the infection . Oseltamivir (5 mg/kg/day) and ribavirin (40 mg/kg/day) were used alone and in combination to determine how late after infection they could be beneficially administered . Ribavirin alone was very effective (90-100% survival of mice) when treatments started as late as 3 days after infection . Forty percent survival was evident even when treatments started 4 days post-infection . Oseltamivir was active starting treatments 1 day after virus exposure, but lost considerable efficacy when treatments began after that time . The combination of ribavirin and oseltamivir appeared to be no better than ribavirin alone, due to the stronger beneficial effect of ribavirin in this model . The overall results demonstrate that mannan can be used to enhance certain non-lethal influenza virus infections sufficiently to allow antiviral studies.

Proc Natl Acad Sci U S A, 2004 Nov 16, 101(46), 16234 - 9 Epub 2004 Nov 16.
Interacting models of cooperative gene regulation; Das D et al.; Cooperativity between transcription factors is critical to gene regulation . Current computational methods do not take adequate account of this salient aspect . To address this issue, we present a computational method based on multivariate adaptive regression splines to correlate the occurrences of transcription factor binding motifs in the promoter DNA and their interactions to the logarithm of the ratio of gene expression levels . This allows us to discover both the individual motifs and synergistic pairs of motifs that are most likely to be functional, and enumerate their relative contributions at any arbitrary time point for which mRNA expression data are available . We present results of simulations and focus specifically on the yeast cell-cycle data . Inclusion of synergistic interactions can increase the prediction accuracy over linear regression to as much as 1.5- to 3.5-fold . Significant motifs and combinations of motifs are appropriately predicted at each stage of the cell cycle . We believe our multivariate adaptive regression splines-based approach will become more significant when applied to higher eukaryotes, especially mammals, where cooperative control of gene regulation is absolutely essential.

Proc Natl Acad Sci U S A, 2004 Nov 23, 101(47), 16437 - 41 Epub 2004 Nov 23.
Remote hot spots mediate protein substrate recognition for the Cdc25 phosphatase; Sohn J et al.; Cdc25B is a phosphatase that catalyzes the dephosphorylation and activation of the cyclin-dependent kinases, thus driving cell cycle progression . We have identified two residues, R488 and Y497, located >20 A from the active site, that mediate protein substrate recognition without affecting activity toward small-molecule substrates . Injection of Cdc25B wild-type but not the R488L or Y497A variants induces germinal vesicle breakdown and cyclin-dependent kinase activation in Xenopus oocytes . The conditional knockout of the cdc25 homolog (mih1) in Saccharomyces cerevisiae can be complemented by the wild type but not by the hot spot variants, indicating that protein substrate recognition by the Cdc25 phosphatases is an essential and evolutionarily conserved feature.

Plant Physiol, 2004 Dec, 136(4), 4198 - 204 Epub 2004 Nov 05.
Root-to-shoot transport of sulfate in Arabidopsis . Evidence for the role of SULTR3;5 as a component of low-affinity sulfate transport system in the root vasculature; Kataoka T et al.; Xylem transport of sulfate regulates distribution of sulfur in vascular plants . Here, we describe SULTR3;5 as an essential component of the sulfate transport system that facilitates the root-to-shoot transport of sulfate in the vasculature . In Arabidopsis (Arabidopsis thaliana), SULTR3;5 was colocalized with the SULTR2;1 low-affinity sulfate transporter in xylem parenchyma and pericycle cells in roots . In a yeast (Saccharomyces cerevisiae) expression system, sulfate uptake was hardly detectable with SULTR3;5 expression alone; however, cells coexpressing both SULTR3;5 and SULTR2;1 showed substantial uptake activity that was considerably higher than with SULTR2;1 expression alone . The V(max) value of sulfate uptake activity with SULTR3;5-SULTR2;1 coexpression was approximately 3 times higher than with SULTR2;1 alone . In Arabidopsis, the root-to-shoot transport of sulfate was restricted in the sultr3;5 mutants, under conditions of high SULTR2;1 expression in the roots after sulfur limitation . These results suggested that SULTR3;5 is constitutively expressed in the root vasculature, but its function to reinforce the capacity of the SULTR2;1 low-affinity transporter is only essential when SULTR2;1 mRNA is induced by sulfur limitation . Consequently, coexpression of SULTR3;5 and SULTR2;1 provides maximum capacity of sulfate transport activity, which facilitates retrieval of apoplastic sulfate to the xylem parenchyma cells in the vasculature of Arabidopsis roots and may contribute to the root-to-shoot transport of sulfate.

Biomacromolecules, 2004 Nov-Dec, 5(6), 2176 - 85
Comprehensive conformational study of key interactions involved in zearalenone complexation with beta-D-glucans; Yiannikouris A et al.; The beta-D-glucans from the cell wall of Saccharomyces cerevisiae have shown in vitro affinity for zearalenone . For this reason, their utilization as dietary adsorbent, to reduce the bioavailability of zearalenone, is of practical interest . Our study used powerful devices to elucidate the spatial conformation and molecular sites of interaction between ZEN and beta-D-glucans . In this respect, 1H NMR spectroscopy implicated the hydroxyl groups of the phenol moiety of zearalenone in the complexation by laminarin, a pure beta-(1,3)-D-glucan . X-ray diffraction determined that laminarin displays the conformation of a single-helix with six beta-D-glucopyranose residues per turn . At this stage, molecular modeling was useful to locate the interaction sites and to propose highly probable complexes of zearalenone with laminarin fragment . Interestingly, the beta-(1,3)-D-glucan chain favors a very stable intra-helical association with zearalenone, nicely stabilized by beta-(1,6)-D-glucans side chains . Both hydrogen bonds and van der Waals interactions were precisely identified in the complex and could thus be proposed as driving interactions to monitor the association between the two molecules.

Gene, 2004 Nov 10, 342(1), 125 - 36
WD dipeptide motifs and LXXLL motif of chicken HIRA are essential for interactions with the p48 subunit of chromatin assembly factor-1 and histone deacetylase-2 in vitro and in vivo; Ahmad A et al.; We cloned cDNA encoding chicken HIRA, a homolog of Saccharomyces cerevisiae transcriptional corepressors Hir1p and Hir2p, possessing seven WD dipeptide motifs and a LXXLL motif in its N-terminal and C-terminal regions, respectively . It binds to CAF-1p48, HDAC-1 and 2, but not to CAF-1p60, p46 polypeptide and HDAC-3 . The immunoprecipitation experiment involving truncated and missense mutants of HIRA and CAF-1p48 revealed not only that even one of seven WD dipeptide motifs in the N-terminal half of HIRA are necessary for the interaction with CAF-1p48, but also that those of CAF-1p48 are necessary for the interaction with HIRA . These findings indicate that the proper propeller structures of both HIRA and CAF-1p48 are necessary for their in vitro interaction . The immunoprecipitation experiment involving truncated and missense mutants of HIRA and HDAC-2 revealed that the LXXLL motif in the C-terminal half of HIRA and a C-terminal region of HDAC-2 are necessary for their in vitro interaction . Moreover, the WD dipeptide motifs and LXXLL motif of HIRA are essential for the interaction with CAF-1p48 and HDAC-2 in vivo . Taken together, these results indicate that HIRA should participate differentially in a number of DNA-utilizing processes through interactions of its distinct regions with these proteins.

Curr Gastroenterol Rep, 2004 Dec, 6(6), 482 - 7
Relevance of serologic studies in inflammatory bowel disease; Vernier G et al.; The serologic panel for inflammatory bowel disease (IBD) is rapidly expanding . Antineutrophil cytoplasmic antibodies (ANCA) and anti-Saccharomyces cerevisiae mannan antibodies (ASCA) have remained the most widely studied markers, but immune reactivity against a new group of bacterial antigens such as I2, OmpC (outer membrane porin C), and flagellin, has been described in Crohn's disease . Several clinical avenues have been explored, such as the usefulness of serologic markers as screening tools for IBD and in accelerating a diagnosis in patients with indeterminate colitis . Another area of interest is disease stratification . Emerging data suggest there is a diversity of qualitative and quantitative responses to environmental antigens that differs among groups of IBD patients and may be associated with different clinical behaviors . As a result, it may be possible to tailor therapy on the basis of serologic responses . Prospective studies are needed before translating this concept into clinical practice . Clustering of IBD patients into more homogeneous subgroups based on antibody responses may help to unravel the pathophysiology of subsets of IBD.

Curr Genet . 2004 Nov 4; {Epub ahead of print}
Characterization of the Arxula adeninivorans AHOG1 gene and the encoded mitogen-activated protein kinase; Boer E et al.; Arxula adeninivorans is an osmo-resistant yeast species that can tolerate high levels of osmolytes like NaCl, PEG400 and ethylene glycol . As in other yeast species, this tolerance is elicited by components of the high osmolarity glycerol (HOG) response pathway . In the present study, we isolated and characterized as a key component of this pathway the A . adeninivorans AHOG1 gene encoding the mitogen-activated protein (MAP) kinase Ahog1p, an enzyme of 45.9 kDa . The gene includes a coding sequence of 1,203 bp disrupted by a 57-bp intron . The identity of the gene was confirmed by complementation of a hog1 mutation in a Saccharomyces cerevisiae mutant strain and the high degree of homology of the derived amino acid sequence with that of MAP kinases from other yeasts and fungi . Under stress-free conditions, the inactive Ahog1p is present in low levels . When exposed to osmotic stress, Ahog1p is rendered active by phosphorylation . In addition, AHOG1 expression is increased . Assessment of the AHOG1 promoter activity with a lacZ reporter gene confirmed its inducibility by osmolytes, a characteristic not observed in homologous HOG1 genes of other yeast species . This specific property could account for the fast adaptation and high osmo-resistance encountered in this species.

Nature, 2004 Nov 4, 432(7013), 112 - 8
A faux 3'-UTR promotes aberrant termination and triggers nonsense-mediated mRNA decay; Amrani N et al.; Nonsense-mediated messenger RNA decay (NMD) is triggered by premature translation termination, but the features distinguishing premature from normal termination are unknown . One model for NMD suggests that decay-inducing factors bound to mRNAs during early processing events are routinely removed by elongating ribosomes but remain associated with mRNAs when termination is premature, triggering rapid turnover . Recent experiments challenge this notion and suggest a model that posits that mRNA decay is activated by the intrinsically aberrant nature of premature termination . Here we use a primer extension inhibition (toeprinting) assay to delineate ribosome positioning and find that premature translation termination in yeast extracts is indeed aberrant . Ribosomes encountering premature UAA or UGA codons in the CAN1 mRNA fail to release and, instead, migrate to upstream AUGs . This anomaly depends on prior nonsense codon recognition and is eliminated in extracts derived from cells lacking the principal NMD factor, Upf1p, or by flanking the nonsense codon with a normal 3'-untranslated region (UTR) . Tethered poly(A)-binding protein (Pab1p), used as a mimic of a normal 3'-UTR, recruits the termination factor Sup35p (eRF3) and stabilizes nonsense-containing mRNAs . These findings indicate that efficient termination and mRNA stability are dependent on a properly configured 3'-UTR.

Mol Biol Evol . 2004 Nov 8; {Epub ahead of print}
Codon Volatility as an Indicator of Positive Selection: Data from Eukaryotic Genome Comparisons; Friedman R et al.; It has been suggested that codon volatility (the proportion of the point-mutation neighbors of a codon that encode different amino acids) can be used as an index of past positive selection . We compared codon volatility with patterns of synonymous and nonsynonymous nucleotide substitution in genome-wide comparisons of orthologous genes between three pairs of related genomes: (1) the protists Plasmodium falciparum and P . yoelii; (2) the fungi Saccharomyces cerevisiae and S . paradoxus; and the mammals mouse and rat . Codon volatility was not consistently associated with an elevated rate of nonsynonymous substitution, as would be expected under positive selection . Rather, the most consistent and powerful correlate of elevated codon volatility was nucleotide content at the second codon position, as expected given the nature of the genetic code.

Mol Biol Cell, 2005 Jan, 16(1), 141 - 52 Epub 2004 Nov 03.
Analysis of a spindle pole body mutant reveals a defect in biorientation and illuminates spindle forces; Yoder TJ et al.; The spindle pole body (SPB) is the microtubule organizing center in Saccharomyces cerevisiae . An essential task of the SPB is to ensure assembly of the bipolar spindle, which requires a proper balancing of forces on the microtubules and chromosomes . The SPB component Spc110p connects the ends of the spindle microtubules to the core of the SPB . We previously reported the isolation of a mutant allele spc110-226 that causes broken spindles and SPB disintegration 30 min after spindle formation . By live cell imaging of mutant cells with green fluorescent protein (GFP)-Tub1p or Spc97p-GFP, we show that spc110-226 mutant cells have early defects in spindle assembly . Short spindles form but do not advance to the 1.5-mum stage and frequently collapse . Kinetochores are not arranged properly in the mutant cells . In 70% of the cells, no stable biorientation occurs and all kinetochores are associated with only one SPB . Examination of the SPB remnants by electron microscopy tomography and fluorescence microscopy revealed that the Spc110-226p/calmodulin complex is stripped off of the central plaque of the SPB and coalesces to from a nucleating structure in the nucleoplasm . The central plaque components Spc42p and Spc29p remain behind in the nuclear envelope . The delamination is likely due to a perturbed interaction between Spc42p and Spc110-226p as detected by fluorescence resonance energy transfer analysis . We suggest that the force exerted on the SPB by biorientation of the chromosomes pulls the Spc110-226p out of the SPB; removal of force exerted by coherence of the sister chromatids reduced fragmentation fourfold . Removal of the forces exerted by the cytoplasmic microtubules had no effect on fragmentation . Our results provide insights into the relative contributions of the kinetochore and cytoplasmic microtubules to the forces involved in formation of a bipolar spindle.

Mol Biol Cell, 2005 Jan, 16(1), 248 - 59 Epub 2004 Nov 03.
Formation of Membrane-bound Ring Complexes by Prohibitins in Mitochondria; Tatsuta T et al.; Prohibitins comprise a remarkably conserved protein family in eukaryotic cells with proposed functions in cell cycle progression, senescence, apoptosis, and the regulation of mitochondrial activities . Two prohibitin homologues, Phb1 and Phb2, assemble into a high molecular weight complex of approximately 1.2 MDa in the mitochondrial inner membrane, but a nuclear localization of Phb1 and Phb2 also has been reported . Here, we have analyzed the biogenesis and structure of the prohibitin complex in Saccharomyces cerevisiae . Both Phb1 and Phb2 subunits are targeted to mitochondria by unconventional noncleavable targeting sequences at their amino terminal end . Membrane insertion involves binding of newly imported Phb1 to Tim8/13 complexes in the intermembrane space and is mediated by the TIM23-translocase . Assembly occurs via intermediate-sized complexes of approximately 120 kDa containing both Phb1 and Phb2 . Conserved carboxy-terminal coiled-coil regions in both subunits mediate the formation of large assemblies in the inner membrane . Single particle electron microscopy of purified prohibitin complexes identifies diverse ring-shaped structures with outer dimensions of approximately 270 x 200 A . Implications of these findings for proposed cellular activities of prohibitins are discussed.

Mol Cell, 2004 Nov 5, 16(3), 479 - 85
The INO80 protein controls homologous recombination in Arabidopsis thaliana; Fritsch O et al.; Homologous recombination (HR) serves a dual role in providing genetic flexibility and in maintaining genome integrity . Little is known about the regulation of HR and other repair pathways in the context of chromatin . We report on a mutant affected in the expression of the Arabidopsis INO80 ortholog of the SWI/SNF ATPase family, which shows a reduction of the HR frequency to 15% of that in wild-type plants . In contrast, sensitivity to genotoxic agents and efficiency of T-DNA integration remain unaffected, suggesting that INO80 is a positive regulator of HR, while not affecting other repair pathways . So far, INO80 function has only been reported in a lower eukaryote . Profiling studies on three ino80 allelic mutants show that INO80 regulates nearly 100 Arabidopsis genes . However, the transcriptional regulation of repair-related genes is unaffected in the mutant . This suggests a dual role for INO80 in transcription and DNA repair by HR.

Mol Cell, 2004 Nov 5, 16(3), 465 - 77
Rvb1p/Rvb2p recruit Arp5p and assemble a functional Ino80 chromatin remodeling complex; Jonsson ZO et al.; The Rvb1p and Rvb2p (or TIP48 and TIP49) nuclear ATP binding proteins are universally conserved in eukaryotes and essential for viability of yeasts . Rvbp associate with each other as a double hexamer, with YHR034c and with two complexes involved in chromatin remodeling, Ino80.com and Swr1.com . Loss of Rvb1p or Ino80p affects many yeast promoters similarly . Rvbp are not essential for the recruitment of Ino80p to promoters but are essential for the catalytic activity of Ino80.com . Loss of Rvbp leads to loss of the functionally critical Arp5p in Ino80.com . Rvb2p associates with Arp5p in vitro in a reaction dependent on the presence of ATP and Ino80p . Therefore, Rvbp are required for the structural and functional integrity of the Ino80 chromatin remodeling complex.

Nat Struct Mol Biol, 2004 Nov, 11(11), 1049 - 53
SRP meets the ribosome; Wild K et al.; Cotranslational targeting directly couples synthesis of proteins to their translocation across or insertion into membranes . The signal recognition particle (SRP) and its membrane-bound receptor facilitate the targeting of the translation machinery, the ribosome, via recognition of a signal sequence in the nascent peptide chain . By combining structures of free and ribosome-bound SRP we derive a structural model describing the dynamic nature of SRP when it meets the ribosome.

Biochim Biophys Acta, 2004 Nov 8, 1686(1-2), 30 - 6
The ERG28-encoded protein, Erg28p, interacts with both the sterol C-4 demethylation enzyme complex as well as the late biosynthetic protein, the C-24 sterol methyltransferase (Erg6p); Mo C et al.; In Saccharomyces cerevisiae, the C-24 sterol methyltransferase (Erg6p) converts zymosterol to fecosterol, an enzymatic step following C-4 demethylation of 4,4-dimethylzymosterol . Our previous study showed that an endoplasmic reticulum (ER) transmembrane protein, Erg28p, functions as a scaffold to tether the C-4 demethylation enzymatic complex (Erg25p-Erg26p-Erg27p) to the ER . To determine whether Erg28p also interacts with other ergosterol biosynthetic proteins, we compared protein levels of Erg3p, Erg6p, Erg7p, Erg11p and Erg25p in three pairs of erg28 and ERG28 strains . In erg28 strains, the Erg6p level in the ER fraction was decreased by about 50% relative to the wild-type strain, while ER protein levels of the four other ergosterol proteins showed no significant differences . Co-immunoprecipitation experiments, using an erg28 strain transformed with the epitope-tagged plasmid pERG28-HA and proteins detected with anti-HA and anti-Erg6p antibodies, indicated that Erg6p and Erg28p reciprocally co-immunoprecipitate . Further, the split ubiquitin yeast membrane two-hybrid system designed to detect protein interactions between membrane bound proteins also indicated an Erg28p-Erg6p interaction when pERG6-Cub was used as the bait and pERG28-NubG was used as the prey . We conclude that Erg28p may not only anchor the C-4 demethylation enzyme complex to the ER but also acts as a protein bridge to the Erg6p enzyme required for the next ergosterol biosynthetic step.

Adv Genet, 2004, 52, 165 - 207
Metabolic highways of Neurospora crassa revisited; Radford A; This chapter describes the metabolic pathways for Neurospora crassa in the biosynthesis of amino acids, purines, pyrimidines, vitamins, and cofactors, and for glycolysis, the TCA and glyoxylate cycles and the initial stages of the pentose phosphate pathway . For each step in metabolism, the gene or genes within the genome sequence of the species is identified, correlations are made with previously identified genes, and new gene designations are assigned to others . For each gene, details given are the function of the gene product, contig location, comparison of the genetic and physical map location, Saccharomyces cerevisiae homolog, and perhaps others, and the level of similarity.

J Mol Biol, 2004 Nov 19, 344(2), 443 - 54
The N-terminal extension of plant mitochondrial carrier proteins is removed by two-step processing: the first cleavage is by the mitochondrial processing peptidase; Murcha MW et al.; In contrast to yeast, many plants encode mitochondrial inner membrane carrier proteins with an N-terminal extension that is removed upon organelle import . Investigations using yeast and plant mitochondria models and purified general mitochondrial processing peptidase (MPP) indicate that the extension was removed in a two-step process . The first processing was carried out by MPP, while the second processing most probably occurs in the inter-membrane space by an as yet undefined peptidase, putatively a serine protease . Purified MPP from potato processed two carrier proteins to an intermediate size, this processing was sensitive to an MPP inhibitor (1,10-phenanthroline) and further, processing could be inhibited by changing arginine residues to glycine residues at a -3 arginine consensus processing site for MPP . Interestingly, yeast mitochondria only processed plant mitochondrial carrier proteins to the same intermediate size as purified plant MPP, and this intermediary processing did not occur in a temperature sensitive yeast mutant for MPP at the restrictive temperature . Incubation of carrier proteins with intact or lysed plant mitochondria under conditions designed to slow down the rate of import revealed that the MPP processed intermediate could be observed and chased to the mature form . The second processing step is inhibited by Pefabloc, suggesting it is carried out by a serine protease . A model for the processing of the N-terminal extension of plant mitochondrial carrier proteins is presented.

Traffic, 2004 Dec, 5(12), 963 - 78
The function of the endocytic scaffold protein Pan1p depends on multiple domains; Miliaras NB et al.; Pan1p is an essential protein of the yeast Saccharomyces cerevisiae that is required for the internalization step of endocytosis and organization of the actin cytoskeleton . Pan1p, which binds several other endocytic proteins, is composed of multiple protein-protein interaction domains including two Eps15 Homology (EH) domains, a coiled-coil domain, an acidic Arp2/3-activating region, and a proline-rich domain . In this study, we have induced high-level expression of various domains of Pan1p in wild-type cells to assess the dominant consequences on viability, endocytosis, and actin organization . We found that the most severe phenotypes, with blocked endocytosis and aggregated actin, required expression of nearly full length Pan1p, and also required the endocytic regulatory protein kinase Prk1p . The central coiled-coil domain was the smallest fragment whose overexpression caused any dominant effects; these effects were more pronounced by inclusion of the second EH domain . Co-overexpressing nonoverlapping amino- and carboxy-terminal fragments did not mimic the effects of the intact protein, whereas fragments that overlapped within the coiled-coil region could . Yeast two-hybrid and in vivo coimmunoprecipitation analyses suggest that Pan1 may form dimers or higher order oligomers . Collectively, our data support a view of Pan1p as a dimeric/oligomeric scaffold whose functions require both the amino- and carboxy-termini, linked by the central region.

Traffic, 2004 Dec, 5(12), 925 - 35
Functional and physical interactions between autonomously replicating sequence-binding factor 1 and the nuclear transport machinery; Loch CM et al.; Autonomously replicating sequence-binding factor 1 (Abf1p) is a site-specific DNA binding protein in Saccharomyces cerevisiae that functions to regulate multiple nuclear events including DNA replication, transcriptional activation, and gene silencing . Previous work indicates that the multiple functions of Abf1p are conferred by the carboxy-terminus of the protein, which can be further dissected into two important clusters of amino acid residues (CS1 and CS2) . Here we present genetic and cell biological evidence for a critical role of CS1 in proper nuclear localization of Abf1p . Mutations in CS1 cause severe defects in cell growth, nuclear translocation, and Abf1p-mediated gene regulation, which can be rescued by a heterologous nuclear localization sequence (NLS) . In addition, the CS1-domain can mediate the import of a CS1-GFP fusion protein . Importantly, the CS1-mediated nuclear import depends on the Ran guanine nucleotide exchange factor Prp20p . Interestingly, a single amino acid change in CS1 (K625I) also causes the protein to be exported out of the nucleus via the Crm1p-dependent pathway . The temperature-sensitive growth phenotype of this particular mutant can be overcome by overexpression of Kap121p/Pse1p, a well-established nuclear transport receptor . Biochemical studies indicate that Pse1p binds to a region of Abf1p upstream of CS1 in a RanGTP-sensitive manner, suggesting that Abf1p has a second distinct NLS and can be imported into the nucleus by several overlapping pathways . We propose that the link between Abf1p and the nuclear transport machinery may also be important for partitioning multiple Abf1p-mediated nuclear processes.

Nat Rev Mol Cell Biol, 2004 Nov, 5(11), 886 - 96
Targeting Rab GTPases to distinct membrane compartments; Pfeffer S et al.; Rab GTPases are key to membrane-trafficking events in eukaryotic cells, and human cells contain more than 60 Rab proteins that are localized to distinct compartments . The recent determination of the structure of a monoprenylated Rab GTPase bound to GDP-dissociation inhibitor provides new molecular details that are relevant to models of Rab delivery . The further discovery of an integral membrane protein that can dissociate prenylated Rab proteins from GDP-dissociation inhibitor gives new insights into the mechanisms of Rab localization.

EMBO Rep, 2004 Nov, 5(11), 1053 - 7
On the mechanism of protein palmitoylation; Dietrich LE et al.; Protein palmitoylation or, more specifically, S-acylation is a reversible post-translational lipid modification . Despite the identification of several proteins that are altered in this way, our understanding of the enzymology of this process has been hampered by the lack of well-characterized acyltransferases . We now know of three proteins in Saccharomyces cerevisiae that promote palmitoylation: effector of Ras function (Erf2), ankyrin-repeat-containing protein (Akr1) and the SNARE protein Ykt6 . Erf2 and Akr1 are integral membrane proteins that contain a cysteine-rich domain and an Asp-His-His-Cys motif, both of which catalyse acylation at the carboxyl terminus of their target proteins . Recently, we discovered that Ykt6 mediates the amino-terminal acylation of the fusion protein Vac8 . Even though these three proteins differ in sequence, topology, size and substrate specificity, they might function in a similar manner . In this review, we discuss these observations in the context of a potential general mechanism of acylation.

Nucleic Acids Res . 2004 Nov 01;32(19):e151.
Quantitative sequential chromatin immunoprecipitation, a method for analyzing co-occupancy of proteins at genomic regions in vivo; Geisberg JV et al.; Sequential chromatin immunoprecipitation (SeqChIP) is a procedure in which formaldehyde-crosslinked, protein-DNA complexes from living cells are subjected to two sequential immunoprecipitations with antibodies of different specificity . SeqChIP has been used to address, in a qualitative manner, whether two proteins can simultaneously co-occupy a stretch of DNA in vivo . Here, we expand on our earlier work and describe theoretical and practical considerations for performing and interpreting SeqChIP experiments in a quantitative manner . We provide a detailed experimental procedure for designing and performing SeqChIP experiments as well as experimental examples of the three possible outcomes: full co-occupancy, no co-occupancy and partial co-occupancy . In some cases of partial co-occupancy, the order of immunoprecipitations in SeqChIP can strongly influence the outcome . We experimentally confirm a quantitative parameter that provides a measure of co-occupancy of two proteins on a given region of DNA and provide information on how to interpret the results of SeqChIP experiments . Our quantitative treatment of SeqChIP data substantially expands the usefulness of the technique for elucidating molecular mechanisms in vivo.

Genes Dev, 2004 Nov 1, 18(21), 2581 - 95
At the interface between signaling and executing anaphase--Cdc14 and the FEAR network; D'Amours D et al.; Anaphase is the stage of the cell cycle when the duplicated genome is separated to opposite poles of the cell . The irreversible nature of this event confers a unique burden on the cell and it is therefore not surprising that the regulation of this cell cycle stage is complex . In budding yeast, a signaling network known as the Cdc fourteen early anaphase release (FEAR) network and its effector, the protein phosphatase Cdc14, play a key role in the coordination of the multiple events that occur during anaphase, such as partitioning of the DNA, regulation of spindle stability, activation of microtubule forces, and initiation of mitotic exit . These functions of the FEAR network contribute to genomic stability by coordinating the completion of anaphase and the execution of mitotic exit.

Genes Dev, 2004 Nov 15, 18(22), 2764 - 73 Epub 2004 Nov 01.
Idling by DNA polymerase delta maintains a ligatable nick during lagging-strand DNA replication; Garg P et al.; During each yeast cell cycle, approximately 100,000 nicks are generated during lagging-strand DNA replication . Efficient nick processing during Okazaki fragment maturation requires the coordinated action of DNA polymerase delta (Pol delta) and the FLAP endonuclease FEN1 . Misregulation of this process leads to the accumulation of double-stranded breaks and cell lethality . Our studies highlight a remarkably efficient mechanism for Okazaki fragment maturation in which Pol delta by default displaces 2-3 nt of any downstream RNA or DNA it encounters . In the presence of FEN1, efficient nick translation ensues, whereby a mixture of mono- and small oligonucleotides are released . If FEN1 is absent or not optimally functional, the ability of Pol delta to back up via its 3'-5'-exonuclease activity, a process called idling, maintains the polymerase at a position that is ideal either for ligation (in case of a DNA-DNA nick) or for subsequent engagement by FEN1 (in case of a DNA-RNA nick) . Consistent with the hypothesis that DNA polymerase epsilon is the leading-strand enzyme, we observed no idling by this enzyme and no cooperation with FEN1 for creating a ligatable nick.

Genes Dev, 2004 Nov 15, 18(22), 2774 - 84 Epub 2004 Nov 01.
PAF1-complex-mediated histone methylation of FLOWERING LOCUS C chromatin is required for the vernalization-responsive, winter-annual habit in Arabidopsis; He Y et al.; The winter-annual habit (which typically involves a requirement for exposure to the cold of winter to flower in the spring) in Arabidopsis thaliana is mainly due to the repression of flowering by relatively high levels of FLC expression . Exposure to prolonged cold attenuates FLC expression through a process known as vernalization and thus permits flowering to occur in the spring . Here we show that the elevated FLC expression characteristic of nonvernalized winter annuals requires two genes, EARLY FLOWERING 7 (ELF7) and EARLY FLOWERING 8 (ELF8), that are homologs of components of the PAF1 complex of Saccharomyces cerevisiae . Furthermore, ELF7 and ELF8 are also required for the expression of other genes in the FLC clade of flowering repressors such as MAF2 and FLM . FLC, FLM, and MAF2 are involved in multiple flowering pathways that account for the broad effects of elf7 and elf8 mutations on flowering behavior . ELF7 and ELF8 are required for the enhancement of histone 3 trimethylation at Lys 4 in FLC chromatin . This modification of FLC chromatin appears to be required to elevate FLC expression to levels that can delay flowering in plants that have not been vernalized . A model of the role of ELF7, ELF8, and other previously described genes in the modification of the chromatin of flowering repressors is presented.

J Cell Biol, 2004 Nov 8, 167(3), 445 - 56 Epub 2004 Nov 01.
A role for BiP as an adjustor for the endoplasmic reticulum stress-sensing protein Ire1; Kimata Y et al.; In the unfolded protein response, the type I transmembrane protein Ire1 transmits an endoplasmic reticulum (ER) stress signal to the cytoplasm . We previously reported that under nonstressed conditions, the ER chaperone BiP binds and represses Ire1 . It is still unclear how this event contributes to the overall regulation of Ire1 . The present Ire1 mutation study shows that the luminal domain possesses two subregions that seem indispensable for activity . The BiP-binding site was assigned not to these subregions, but to a region neighboring the transmembrane domain . Phenotypic comparison of several Ire1 mutants carrying deletions in the indispensable subregions suggests these subregions are responsible for multiple events that are prerequisites for activation of the overall Ire1 proteins . Unexpectedly, deletion of the BiP-binding site rendered Ire1 unaltered in ER stress inducibility, but hypersensitive to ethanol and high temperature . We conclude that in the ER stress-sensory system BiP is not the principal determinant of Ire1 activity, but an adjustor for sensitivity to various stresses.

Biochemistry, 2004 Nov 9, 43(44), 13955 - 62
Localization of the antimitotic peptide and depsipeptide binding site on beta-tubulin; Mitra A et al.; Several naturally occurring peptides and depsipeptides which include the cryptophycins, dolastatin 10, hemiasterlin, and phomopsin A have been found to be potent antimitotic agents, causing cell death at picomolar or low nanomolar concentrations . These compounds inhibit microtubule growth, modulate the dynamics of microtubules, and induce the self-association of tubulin dimers into single-walled rings and spirals . These peptides exhibit mutual competitive inhibition in binding to beta-tubulin, while noncompetitively inhibiting the binding of vinblastine and vincristine to beta-tubulin . Despite the abundance of biochemical information, the details of their molecular interactions with tubulin are not known . In this study, using a combination of molecular dynamics simulations and molecular docking studies, a common binding site for cryptophycin 1, cryptophycin 52, dolastatin 10, hemiasterlin, and phomopsin A on beta-tubulin has been identified . Application of these same methods to alpha-tubulin indicated no interaction between alpha-tubulin and any of the peptides . On the basis of the docking results, a model for the mechanism of microtubule disruption and formation of aberrant nonmicrotubule structures is proposed . Both the active site and mechanism of microtubule depolymerization predictions are in good agreement with experimental findings.

Oncogene, 2004 Nov 1, 23(51), 8346 - 52
Towards full employment: using RNAi to find roles for the redundant; Fraser A; Cancer is a genetic disease that ultimately results from the failure of cells to respond correctly to diverse signals . Signal transduction and signal integration are highly complex, requiring the combinatorial interaction of multiple genes . Classical genetics in model organisms including Caenorhabditis elegans has been of immense use in identifying nonredundant components of conserved signalling pathways . However, it is likely that there is much functional redundancy in the informational processing machinery of metazoan cells; we therefore need to develop methods for uncovering such redundant functions in model organisms if we are to use them to understand complex gene interactions and oncogene cooperation . RNAi may provide a powerful tool to probe redundancy in informational networks . In this review, I set out some of the progress made so far by classical genetics in understanding redundancy in gene networks, and outline how RNAi may allow us to approach this problem more systematically in C . elegans . In particular, I discuss the use of genome-wide RNAi screens in C . elegans to identify synthetic lethal interactions and compare this with synthetic lethal interaction analysis in Saccharomyces cerevisiae.

Plant Physiol, 2004 Nov, 136(3), 3814 - 23 Epub 2004 Oct 29.
Identification of Thlaspi caerulescens genes that may be involved in heavy metal hyperaccumulation and tolerance . Characterization of a novel heavy metal transporting ATPase; Papoyan A et al.; Thlaspi caerulescens is a heavy metal hyperaccumulator plant species that is able to accumulate extremely high levels of zinc (Zn) and cadmium (Cd) in its shoots (30,000 microg g(-1) Zn and 10,000 microg g(-1) Cd), and has been the subject of intense research as a model plant to gain a better understanding of the mechanisms of heavy metal hyperaccumulation and tolerance and as a source of genes for developing plant species better suited for the phytoremediation of metal-contaminated soils . In this study, we report on the results of a yeast (Saccharomyces cerevisae) complementation screen aimed at identifying candidate heavy metal tolerance genes in T . caerulescens . A number of Thlaspi genes that conferred Cd tolerance to yeast were identified, including possible metal-binding ligands from the metallothionein gene family, and a P-type ATPase that is a member of the P1B subfamily of purported heavy metal-translocating ATPases . A detailed characterization of the Thlaspi heavy metal ATPase, TcHMA4, demonstrated that it mediates yeast metal tolerance via active efflux of a number of different heavy metals (Cd, Zn, lead {Pb}, and copper {Cu}) out of the cell . However, in T . caerulescens, based on differences in tissue-specific and metal-responsive expression of this transporter compared with its homolog in Arabidopsis (Arabidopsis thaliana), we suggest that it may not be involved in metal tolerance . Instead, we hypothesize that it may play a role in xylem loading of metals and thus could be a key player in the hyperaccumulation phenotype expressed in T . caerulescens . Additionally, evidence is presented showing that the C terminus of the TcHMA4 protein, which contains numerous possible heavy metal-binding His and Cys repeats residues, participates in heavy metal binding . When partial peptides from this C-terminal domain were expressed in yeast, they conferred an extremely high level of Cd tolerance and Cd hyperaccumulation . The possibilities for enhancing the metal tolerance and phytoremediation potential of higher plants via expression of these metal-binding peptides are also discussed.

Bioinformatics . 2004 Oct 28; {Epub ahead of print}
Comparison of computational methods for the identification of cell cycle regulated genes; de Lichtenberg U et al.; MOTIVATION: DNA microarrays have been used extensively to study the cell cycle transcription programme in a number of model organisms . The Saccharomyces cerevisiae data in particular have been subjected to a wide range of bioinformatics analysis methods, aimed at identifying the correct and complete set of periodically expressed genes . RESULTS: Here, we provide the first thorough benchmark of such methods, surprisingly revealing that most new and more mathematically advanced methods actually perform worse than the analysis published with the original microarray data sets . We show that this loss of accuracy specifically affects methods that only model the shape of the expression profile without taking into account the magnitude of regulation . We present a simple permutation-based method that performs better than most existing methods . SUPPLEMENTARY INFORMATION: Results and benchmark sets are available at http://www.cbs.dtu.dk/cellcycle.

Bioinformatics . 2004 Oct 28; {Epub ahead of print}
Clustering of gene expression data using a local shape-based similarity measure; Balasubramaniyan R et al.; MOTIVATION: Microarray technology enables the study of gene expression in large scale . The application of methods for data analysis then allows for grouping genes that show a similar expression profile and that are thus likely to be co-regulated . A relationship among genes at the biological level often presents itself by locally similar and potentially time-shifted patterns in their expression profiles . RESULTS: Here, we thus propose a new method (CLARITY; Clustering with Local shApe-based similaRITY) for the analysis of microarray time course experiments that uses a local shape-based similarity measure based on Spearman rank correlation . This measure does not require a normalization of the expression data and is comparably robust towards noise . It is also able to detect similar and even time-shifted sub-profiles . To this end, we implemented an approach motivated by the BLAST algorithm for sequence alignment . We used CLARITY to cluster the times series of gene expression data during the mitotic cell cycle of the yeast Saccharomyces cerevisiae . The obtained clusters were related to the MIPS functional classification to assess their biological significance . We found that several clusters were significantly enriched with genes that share similar or related functions . AVAILABILITY: Upon request from the authors.

Eur J Biochem, 2004 Nov, 271(21), 4213 - 21
Solution structure of Cu6 metallothionein from the fungus Neurospora crassa; Cobine PA et al.; The 3D-solution structure of Neurospora crassa Cu(6)-metallothionein (NcMT) polypeptide backbone was determined using homonuclear, multidimensional (1)H-NMR spectroscopy . It represents a new metallothionein (MT) fold with a protein chain where the N-terminal half is left-handed and the C-terminal half right-handedly folded around a copper(I)-sulfur cluster . As seen with other MTs, the protein lacks definable secondary structural elements; however, the polypeptide fold is unique . The metal coordination and the cysteine spacing defines this unique fold . NcMT is only the second MT in the copper-bound form to be structurally characterized and the first containing the -CxCxxxxxCxC- motif . This motif is found in a variety of mammalian MTs and metalloregulatory proteins . The in vitro formation of the Cu(6)NcMT identical to the native Cu(6)NcMT was dependent upon the prior formation of the Zn(3)NcMT and its titration with Cu(I) . The enhanced sensitivity and resolution of the 800 MHz (1)H-NMR spectral data permitted the 3D structure determination of the polypeptide backbone without the substitution and utilization of the NMR active spin 1/2 metals such as (113)Cd and (109)Ag . These restraints have been necessary to establish specific metal to cysteine restraints in 3D structural studies on this family of proteins when using lower field, less sensitive (1)H-NMR spectral data . The accuracy of the structure calculated without these constraints is, however, supported by the similarities of the 800 MHz structures of the alpha-domain of mouse MT1 compared to the one recalculated without metal-cysteine connectivities.

Eur J Biochem, 2004 Nov, 271(21), 4189 - 94
Donor substrate regulation of transketolase; Esakova OA et al.; The influence of substrates on the interaction of apotransketolase with thiamin diphosphate was investigated in the presence of magnesium ions . It was shown that the donor substrates, but not the acceptor substrates, enhance the affinity of the coenzyme either to only one active center of transketolase or to both active centers, but to different degrees in each, resulting in a negative cooperativity for coenzyme binding . In the absence of donor substrate, negative cooperativity is not observed . The donor substrate did not affect the interaction of the apoenzyme with the inactive coenzyme analogue, N3'-pyridyl-thiamin diphosphate . The influence of the donor substrate on the coenzyme-apotransketolase interaction was predicted as a result of formation of the transketolase reaction intermediate 2-(alpha,beta-dihydroxyethyl)-thiamin diphosphate, which exhibited a higher affinity to the enzyme than thiamin diphosphate . The enhancement of thiamin diphosphate's affinity to apotransketolase in the presence of donor substrate is probably one of the mechanisms underlying the substrate-affected transketolase regulation at low coenzyme concentrations.

Plant Cell Physiol, 2004 Sep, 45(9), 1313 - 9
Functional identification of the glycerol transport activity of Chlamydomonas reinhardtii CrMIP1; Anderca MI et al.; By searching a Chlamydomonas expressed sequence tag database and by comparing the retrieved data with homologous sequences from a DNA database, we identified an expressed Chlamydomonas reinhardtii putative major intrinsic protein (MIP) gene . The nucleotide sequence, consisting of 1,631 bp, contains an open reading frame coding for a 300-amino-acid protein named CrMIP1 . It possesses conserved NPA motifs, but is not highly homologous to known aquaporins . CrMIP1 was expressed in Saccharomyces cerevisiae and assayed for water and glycerol transport activity . By the stopped-flow spectrophotometric assay, CrMIP1 did not enhance the osmotic water permeability of membrane vesicles of the yeast transformant . However, the transformant cells showed glycerol transport activity in the in vivo assay using {14C}glycerol . This is the first report on the isolation and functional identification of a MIP member from algae.

Mol Cell Biol, 2004 Nov, 24(22), 10072 - 82
NC2alpha interacts with BTAF1 and stimulates its ATP-dependent association with TATA-binding protein; Klejman MP et al.; Transcriptional activity of the TATA-binding protein (TBP) is controlled by a variety of proteins . The BTAF1 protein (formerly known as TAF(II)170/TAF-172 and the human ortholog of Saccharomyces cerevisiae Mot1p) and the NC2 complex composed of NC2alpha (DRAP1) and NC2beta (Dr1) are able to bind to TBP directly and regulate RNA polymerase II transcription both positively and negatively . Here, we present evidence that the NC2alpha subunit interacts with BTAF1 . In contrast, the NC2beta subunit is not able to associate with BTAF1 and seems to interfere with the BTAF1-TBP interaction . Addition of NC2alpha or the NC2 complex can stimulate the ability of BTAF1 to interact with TBP . This function is dependent on the presence of ATP in cell extracts but does not involve the ATPase activity of BTAF1 nor phosphorylation of NC2alpha . Together, our results constitute the first evidence of the physical cooperation between BTAF1 and NC2alpha in TBP regulation and provide a framework to understand transcription functions of NC2alpha and NC2beta in vivo.

Mol Cell Biol, 2004 Nov, 24(22), 10047 - 57
Functional role of extranucleosomal DNA and the entry site of the nucleosome in chromatin remodeling by ISW2; Zofall M et al.; A minimal amount of extranucleosomal DNA was required for nucleosome mobilization by ISW2 as shown by using a photochemical histone mapping approach to analyze nucleosome movement on a set of nucleosomes with varied lengths of extranucleosomal DNA . ISW2 was ineffective in repositioning or mobilizing nucleosomes with <or=20 bp of extranucleosomal DNA . In addition, ISW2 was able to slide nucleosomes to within only 10 to 13 bp of the edge of DNA fragments . The nucleosome mobilization was promoted by extranucleosomal single-stranded DNA with modest strand preference . Gaps (10 bp) just inside the nucleosome and in the extranucleosomal DNA showed that the transfer of torsional strain (twist) into the nucleosomal DNA region was not required for mobilizing nucleosomes . However, indications are that the extranucleosomal DNA immediately adjacent to the nucleosome has an important role in the initial stage of nucleosome movement by ISW2.

Mol Cell Biol, 2004 Nov, 24(22), 10036 - 46
Regulation of cell cycle-specific gene expression through cyclin-dependent kinase-mediated phosphorylation of the forkhead transcription factor Fkh2p; Pic-Taylor A et al.; The forkhead transcription factor Fkh2p acts in a DNA-bound complex with Mcm1p and the coactivator Ndd1p to regulate cell cycle-dependent expression of the CLB2 gene cluster in Saccharomyces cerevisiae . Here, we demonstrate that Fkh2p is a target of cyclin-dependent protein kinases and that phosphorylation of Fkh2p promotes interactions between Fkh2p and the coactivator Ndd1p . These phosphorylation-dependent changes in the Fkh2p-Ndd1p complex play an important role in the cell cycle-regulated expression of the CLB2 cluster . Our data therefore identify an important regulatory target for cyclin-dependent kinases in the cell cycle and further our molecular understanding of the key cell cycle regulatory transcription factor Fkh2p.

Mol Cell Biol, 2004 Nov, 24(22), 10016 - 25
Requirement of the Mre11 complex and exonuclease 1 for activation of the Mec1 signaling pathway; Nakada D et al.; The large protein kinases, ataxia-telangiectasia mutated (ATM) and ATM-Rad3-related (ATR), orchestrate DNA damage checkpoint pathways . In budding yeast, ATM and ATR homologs are encoded by TEL1 and MEC1, respectively . The Mre11 complex consists of two highly related proteins, Mre11 and Rad50, and a third protein, Xrs2 in budding yeast or Nbs1 in mammals . The Mre11 complex controls the ATM/Tel1 signaling pathway in response to double-strand break (DSB) induction . We show here that the Mre11 complex functions together with exonuclease 1 (Exo1) in activation of the Mec1 signaling pathway after DNA damage and replication block . Mec1 controls the checkpoint responses following UV irradiation as well as DSB induction . Correspondingly, the Mre11 complex and Exo1 play an overlapping role in activation of DSB- and UV-induced checkpoints . The Mre11 complex and Exo1 collaborate in producing long single-stranded DNA (ssDNA) tails at DSB ends and promote Mec1 association with the DSBs . The Ddc1-Mec3-Rad17 complex associates with sites of DNA damage and modulates the Mec1 signaling pathway . However, Ddc1 association with DSBs does not require the function of the Mre11 complex and Exo1 . Mec1 controls checkpoint responses to stalled DNA replication as well . Accordingly, the Mre11 complex and Exo1 contribute to activation of the replication checkpoint pathway . Our results provide a model in which the Mre11 complex and Exo1 cooperate in generating long ssDNA tracts and thereby facilitate Mec1 association with sites of DNA damage or replication block.

Biochim Biophys Acta, 2004 Nov 5, 1680(3), 171 - 5
Isolation and characterization of Arabidopsis thaliana ISU1 gene; Tone Y et al.; We describe the isolation of a cDNA encoding Arabidopsis thaliana ISU1 (AtISU1), which regulates iron homeostasis in the mitochondria . The AtISU1 gene contained an open reading frame that encoded 167 amino acid residues . Northern blot analysis demonstrated that AtISU1 gene was ubiquitously expressed in plant tissues examined . The yeast seo5-1, which harbors a single base-pair deletion in ScISU1, is a suppressor of oxidative damage in sod1-deficient mutant . Based on comparative expression analyses using yeast ISU1 gene (ScISU1) in seo5-1 mutant, we found that AtISU1 acts as a counterpart of ScISU1.

Proc Natl Acad Sci U S A, 2004 Nov 2, 101(44), 15621 - 6 Epub 2004 Oct 25.
AtTPK4, an Arabidopsis tandem-pore K+ channel, poised to control the pollen membrane voltage in a pH- and Ca2+-dependent manner; Becker D et al.; The Arabidopsis tandem-pore K(+) (TPK) channels displaying four transmembrane domains and two pore regions share structural homologies with their animal counterparts of the KCNK family . In contrast to the Shaker-like Arabidopsis channels (six transmembrane domains/one pore region), the functional properties and the biological role of plant TPK channels have not been elucidated yet . Here, we show that AtTPK4 (KCO4) localizes to the plasma membrane and is predominantly expressed in pollen . AtTPK4 (KCO4) resembles the electrical properties of a voltage-independent K(+) channel after expression in Xenopus oocytes and yeast . Hyperpolarizing as well as depolarizing membrane voltages elicited instantaneous K(+) currents, which were blocked by extracellular calcium and cytoplasmic protons . Functional complementation assays using a K(+) transport-deficient yeast confirmed the biophysical and pharmacological properties of the AtTPK4 channel . The features of AtTPK4 point toward a role in potassium homeostasis and membrane voltage control of the growing pollen tube . Thus, AtTPK4 represents a member of plant tandem-pore-K(+) channels, resembling the characteristics of its animal counterparts as well as plant-specific features with respect to modulation of channel activity by acidosis and calcium.

Proc Natl Acad Sci U S A, 2004 Nov 2, 101(44), 15639 - 44 Epub 2004 Oct 25.
Barrier-limited, microsecond folding of a stable protein measured with hydrogen exchange: Implications for downhill folding; Meisner WK et al.; Folding experiments are conducted to test whether a covalently cross-linked coiled-coil folds so quickly that the process is no longer limited by a free-energy barrier . This protein is very stable and topologically simple, needing merely to "zipper up," while having an extrapolated folding rate of k(f) = 2 x 10(5) s(-1) . These properties make it likely to attain the elusive "downhill folding" limit, at which a series of intermediates can be characterized . To measure the ultra-fast kinetics in the absence of denaturant, we apply NMR and hydrogen-exchange methods . The stability and its denaturant dependence for the hydrogen bonds in the central part of protein equal the values calculated for whole-molecule unfolding . Like-wise, their closing and opening rates indicate that these hydrogen bonds are broken and reformed in a single cooperative event representing the folding transition from the fully unfolded state to the native state . Additionally, closing rates for these hydrogen bonds agree with the extrapolated barrier-limited folding rate observed near the melting transition . Therefore, even in the absence of denaturant, where DeltaG(eq) approximately -6 kcal.mol(-1) (1 cal = 4.18 J) and tau(f) approximately 6 mus, folding remains cooperative and barrier-limited . Given that this prime candidate for downhill folding fails to do so, we propose that protein folding will remain barrier-limited for proteins that fold cooperatively.

J Cell Biol, 2004 Oct 25, 167(2), 281 - 92
The GTPase Arf1p and the ER to Golgi cargo receptor Erv14p cooperate to recruit the golgin Rud3p to the cis-Golgi; Gillingham AK et al.; Rud3p is a coiled-coil protein of the yeast cis-Golgi . We find that Rud3p is localized to the Golgi via a COOH-terminal domain that is distantly related to the GRIP domain that recruits several coiled-coil proteins to the trans-Golgi by binding the small Arf-like GTPase Arl1p . In contrast, Rud3p binds to the GTPase Arf1p via this COOH-terminal "GRIP-related Arf-binding" (GRAB) domain . Deletion of RUD3 is lethal in the absence of the Golgi GTPase Ypt6p, and a screen of other mutants showing a similar genetic interaction revealed that Golgi targeting of Rud3p also requires Erv14p, a cargo receptor that cycles between the endoplasmic reticulum and Golgi . The one human protein with a GRAB domain, GMAP-210 (CEV14/Trip11/Trip230), is known to be on the cis-Golgi, but the COOH-terminal region that contains the GRAB domain has been reported to bind to centrosomes and gamma-tubulin (Rios, R.M, A . Sanchis, A.M . Tassin, C . Fedriani, and M . Bornens . 2004 . Cell . 118:323-335) . In contrast, we find that this region binds to the Golgi in a GRAB domain-dependent manner, suggesting that GMAP-210 may not link the Golgi to gamma-tubulin and centrosomes.

J Biol Chem, 2004 Dec 31, 279(53), 55218 - 23 Epub 2004 Oct 25.
Rpn4 is a physiological substrate of the Ubr2 ubiquitin ligase; Wang L et al.; The homeostatic abundance of the proteasome in Saccharomyces cerevisiae is controlled by a feedback circuit in which transcriptional activator Rpn4 up-regulates the proteasome genes and is destroyed by the assembled, active proteasome . Remarkably, the degradation of Rpn4 can be mediated by two independent pathways . One pathway is independent of ubiquitin, whereas the other involves ubiquitination on internal lysines . In the present study, we investigated the mechanism underlying the ubiquitin-dependent degradation of Rpn4 . We demonstrated, through in vivo and in vitro assays, that Rpn4 is a physiological substrate of the Ubr2 ubiquitin ligase, which was originally identified as a sequence homolog of Ubr1, the E3 component of the N-end rule pathway . The ubiquitin-conjugating enzyme Rad6, which directly interacts with Ubr2, is also required for the ubiquitin-dependent degradation of Rpn4 . Furthermore, we showed that deletion of UBR2 exhibited a strong synthetic growth defect with a mutation in the Rpt1 proteasome subunit when Rpn4 was overexpressed . This study not only identified the ubiquitination apparatus for Rpn4 but also unveiled the first physiological substrate of Ubr2 . The biological significance of Ubr2-mediated degradation of Rpn4 is also discussed.

BMC Bioinformatics . 2004 Oct 26;5(1):161.
Implications for domain fusion protein-protein interactions based on structural information; Chia JM et al.; BACKGROUND: Several in silico methods exist that were developed to predict protein interactions from the copious amount of genomic and proteomic data . One of these methods is Domain Fusion, which has proven to be effective in predicting functional links between proteins . RESULTS: Analyzing the structures of multi-domain single-chain peptides, we found that domain pairs located less than 30 residues apart on a chain are almost certain to share a physical interface . The majority of these interactions are also conserved across separate chains . We make use of this observation to improve domain fusion based protein interaction predictions, and demonstrate this by implementing it on a set of Saccharomyces cerevisiae proteins . CONCLUSION: We show that existing structural data supports the domain fusion hypothesis . Empirical information from structural data also enables us to refine and assess domain fusion based protein interaction predictions . These interactions can then be integrated with downstream biochemical and genetic assays to generate more reliable protein interaction data sets.

Biochemistry, 2004 Nov 2, 43(43), 13816 - 26
Control of Hap1-DNA site recognition through the interplay of multiple distinct intermolecular interactions; Wang LL et al.; Hap1 belongs to the Zn(2)Cys(6) zinc binuclear cluster family of transcription factors that typically bind as dimers to symmetric DNA sites containing two CGG triplets separated by spacer DNA . The cluster domain binds CGG while an adjoining C-terminal linker and dimerization helix specifies the length of spacer DNA recognized . Hap1 is unusual in binding a direct repeat of CGG triplets, in contacting a TA in the spacer DNA, and in making direct dimer contacts between its cluster domains . Binding of Hap1 fragments to different DNA sites was tested to determine how these interactions control Hap1-DNA recognition . The spacer TA contacts were found to facilitate monomer binding of Hap1 to a single CGG . When the spacer-binding residues were deleted, binding was still specific for the direct repeat but was much weaker and appeared to require dimerization . When the dimerization helix and all subsequent C-terminal residues were deleted, the remaining linker, cluster domain, and spacer-binding residues still dimerized on DNA . The energy of this dimerization was comparable to that of the Hap1-spacer TA interaction . Moving the TA from the spacer to a position following the second CGG maintained Hap1 monomer binding but greatly weakened dimerization . This suggested that binding a TA after the second CGG triplet required a geometry that impaired dimerization with a Hap1 molecule on the first CGG . The geometric restraints for optimal TA binding and dimerization thus drive Hap1 selectivity for CGG direct repeat sites that contain an asymmetrically positioned spacer TA following the first CGG triplet.

Biochemistry, 2004 Nov 2, 43(43), 13739 - 47
Dynamics in the U6 RNA intramolecular stem-loop: a base flipping conformational change; Reiter NJ et al.; The U6 RNA intramolecular stem-loop (ISL) structure is an essential component of the spliceosome and binds a metal ion required for pre-messenger RNA splicing . The metal binding internal loop region of the stem contains a partially protonated C67-(+)A79 base pair (pK(a) = 6.5) and an unpaired U80 nucleotide that is stacked within the helix at pH 7.0 . Here, we determine that protonation occurs with an exchange lifetime of approximately 20 micros and report the solution structures of the U6 ISL at pH 5.7 . The differences between pH 5.7 and 7.0 structures reveal that the pH change significantly alters the RNA conformation . At lower pH, U80 is flipped out into the major groove . Base flipping involves a purine stacking interaction of flanking nucleotides, inversion of the sugar pucker 5' to the flipped base, and phosphodiester backbone rearrangement . Analysis of residual dipolar couplings as a function of pH indicates that base flipping is not restricted to a local conformational change . Rather, base flipping alters the alignment of the upper and lower helices . The alternative conformations of the U6 ISL reveal striking structural similarities with both the NMR and crystal structures of domain 5 of self-splicing group II introns . These structures suggest that base flipping at an essential metal binding site is a conserved feature of the splicing machinery for both the spliceosome and group II self-splicing introns.

Phytochemistry, 2004 Nov, 65(21), 2885 - 91
HPLC-based bioactivity profiling of plant extracts: a kinetic assay for the identification of monoamine oxidase-A inhibitors using human recombinant monoamine oxidase-A; Dittmann K et al.; An assay for the HPLC-based search for monoamine oxidase-A (MAO-A) inhibitors in plant extracts was established . It combines human recombinant MAO-A, expressed as GST-fusion protein in yeast, with a kinetic measurement of the conversion of kynuramine to 4-hydroxyquinoline . Substrate selectivity and kinetic parameters of the GST-fusion protein were comparable to the wild-type enzyme . The applicability of the assay to HPLC-based activity profiling was tested with plant extracts spiked with small amounts of known MAO inhibitors.

Mol Biochem Parasitol, 2004 Nov, 138(1), 107 - 22
On the biogenesis of lipid bodies in ancient eukaryotes: synthesis of triacylglycerols by a Toxoplasma DGAT1-related enzyme; Quittnat F et al.; In mammalian cells, the main stored neutral lipids are triacylglycerol and cholesteryl esters, which are produced by two related enzymes, acyl-CoA:diacylglycerol acyltransferase (DGAT) and acyl-CoA:cholesterol acyltransferase (ACAT), respectively . Very little is known about the metabolism, intracellular storage and function of neutral lipids in many pathogenic lower eukaryotes . In this paper, we have characterized the activity of an important triacylglycerol synthetic enzyme in the protozoan Toxoplasma gondii . A full-length cDNA and gene encoding a T . gondii DGAT1-related enzyme were identified and designated TgDGAT1 . The gene is composed of 15 exons and 14 introns, and encodes a protein with a predicted M(r) 63.5kDa, containing signature motifs characteristic of the DGAT1 family . The native protein migrates at 44kDa under reducing conditions . TgDGAT1 is an integral membrane protein localized to the parasite cortical and perinuclear endoplasmic reticulum, with the C-terminus oriented to the lumen of the organelle . When a Saccharomyces cerevisiae mutant strain lacking neutral lipid production is transformed with TgDGAT1 cDNA, a significant DGAT activity is reconstituted, resulting in triacylglycerol synthesis and biogenesis of cytosolic lipid inclusions, resembling lipid bodies in T . gondii . No production of steryl esters is observed upon TgDGAT1 expression in yeast . In contrast to human DGAT1 lacking fatty acid specificity, TgDGAT1 preferentially incorporates palmitate . Our results indicate that parasitic protozoa are also neutral lipid accumulators and illustrate the first example of the existence of a functional DGAT gene in an ancient eukaryote, demonstrating that diacylglycerol esterification is evolutionarily conserved.

Bull Exp Biol Med, 2003 Dec, 136(6), 615 - 7
A method for biological control of a complex phytoadaptogen; Bocharova OA et al.; We propose a method for standardization of complex adaptogen-containing preparations . The method is based on acceleration of baking yeast strain growth on energy-depleted medium in the presence of the test agent . This method allows simple quantitative biological control of phytoadaptogens and comparison of adaptogenic activity of mono- and complex preparations.

Bioessays, 2004 Nov, 26(11), 1243 - 53
The genetics of Drosophila transgenics; Roman G; In Drosophila, the genetic approach is still the method of choice for answering fundamental questions on cell biology, signal transduction, development, physiology and behavior . In this approach, a gene's function is ascertained by altering either the amount or quality of the gene product, and then observing the consequences . The genetic approach is itself polymorphous, encompassing new and more complex techniques that typically employ the growing collections of transgenes . The keystone of these modern Drosophila transgenic techniques has been the Gal4 binary system . Recently, several new techniques have modified this binary system to offer greater control over the timing, tissue specificity and magnitude of gene expression . Additionally, the advances in post-transcriptional gene silencing, or RNAi, have greatly expanded the ability to knockdown almost any gene's function . Regardless of the growing experimental intricacy, the application of these advances to modify gene activity still obeys the fundamental principles of genetic analysis . Several of these transgenic techniques, which offer more precise control over a gene's activity, will be reviewed here with a discussion on how they may be used for determining a gene's function.

J Biol Chem, 2005 Jan 7, 280(1), 94 - 103 Epub 2004 Oct 21.
Phosphorylation of Candida glabrata ATP-binding Cassette Transporter Cdr1p Regulates Drug Efflux Activity and ATPase Stability; Wada S et al.; Fungal ATP-binding cassette transporter regulation was investigated using Candida glabrata Cdr1p and Pdh1p expressed in Saccharomyces cerevisiae . Rephosphorylation of Pdh1p and Cdr1p was protein kinase A inhibitor-sensitive but responded differentially to Tpk isoforms, stressors, and glucose concentration . Cdr1p Ser(307), which borders the nucleotide binding domain 1 ABC signature motif, and Ser(484), near the membrane, were dephosphorylated on glucose depletion and independently rephosphorylated during glucose exposure or under stress . The S484A enzyme retained half the wild type ATPase activity without affecting azole resistance, but the S307A enzyme was unstable to plasma membrane isolation . Studies of pump function suggested conformational interaction between Ser(484) and Ser(307) . An S307A/S484A double mutant, which failed to efflux the Cdr1p substrate rhodamine 6G, had a fluconazole susceptibility 4-fold greater than the Cdr1p expressing strain, twice that of the S307A mutant, but 64-fold less than the control null strain . Stable intragenic suppressors indicative of homodimer nucleotide binding domain 1-nucleotide binding domain 1 interactions partially restored rhodamine 6G pumping and increased fluconazole and rhodamine 6G resistance in the S307A/S484A mutant . Nucleotide binding domain 1 of Cdr1p is a sensor of important physiological stimuli.

Nature, 2004 Oct 21, 431(7011), 1011 - 7
DNA end resection, homologous recombination and DNA damage checkpoint activation require CDK1; Ira G et al.; A single double-strand break (DSB) induced by HO endonuclease triggers both repair by homologous recombination and activation of the Mec1-dependent DNA damage checkpoint in budding yeast . Here we report that DNA damage checkpoint activation by a DSB requires the cyclin-dependent kinase CDK1 (Cdc28) in budding yeast . CDK1 is also required for DSB-induced homologous recombination at any cell cycle stage . Inhibition of homologous recombination by using an analogue-sensitive CDK1 protein results in a compensatory increase in non-homologous end joining . CDK1 is required for efficient 5' to 3' resection of DSB ends and for the recruitment of both the single-stranded DNA-binding complex, RPA, and the Rad51 recombination protein . In contrast, Mre11 protein, part of the MRX complex, accumulates at unresected DSB ends . CDK1 is not required when the DNA damage checkpoint is initiated by lesions that are processed by nucleotide excision repair . Maintenance of the DSB-induced checkpoint requires continuing CDK1 activity that ensures continuing end resection . CDK1 is also important for a later step in homologous recombination, after strand invasion and before the initiation of new DNA synthesis.

Nature, 2004 Oct 28, 431(7012), 1118 - 23 Epub 2004 Oct 20.
Phosphorylation-dependent binding of mitotic cyclins to Cdc6 contributes to DNA replication control; Mimura S et al.; Cyclin-dependent kinases (CDKs) limit the activation of DNA replication origins to once per cell cycle by preventing the assembly of pre-replicative complexes (pre-RCs) during S, G2 and M phases of the cell cycle in the budding yeast Saccharomyces cerevisiae . CDKs inhibit each pre-RC component (ORC, Cdc6, Cdt1/Mcm2-7) by different mechanisms . We show here that the mitotic CDK, Clb2/Cdc28, binds tightly to an amino-terminal domain (NTD) of Cdc6, and that Cdc6 in this complex is unable to assemble pre-RCs . We present evidence indicating that this Clb2-dependent mechanism contributes to preventing re-replication in vivo . CDK interaction with the NTD of Cdc6 is mediated by the cyclin subunit Clb2, and could be reconstituted with recombinant Clb2 protein and synthetic NTD peptides . Tight Clb2 binding occurred only when the NTD was phosphorylated on CDK consensus sites . Human CDKs containing cyclins A, B and E also bound specifically to phospho-NTD peptides . We propose that direct binding of cyclins to phosphopeptide motifs may be a widespread phenomenon contributing to the targeting of CDKs to substrates.

Proc Natl Acad Sci U S A, 2004 Nov 2, 101(44), 15682 - 7 Epub 2004 Oct 20.
Combining biological networks to predict genetic interactions; Wong SL et al.; Genetic interactions define overlapping functions and compensatory pathways . In particular, synthetic sick or lethal (SSL) genetic interactions are important for understanding how an organism tolerates random mutation, i.e., genetic robustness . Comprehensive identification of SSL relationships remains far from complete in any organism, because mapping these networks is highly labor intensive . The ability to predict SSL interactions, however, could efficiently guide further SSL discovery . Toward this end, we predicted pairs of SSL genes in Saccharomyces cerevisiae by using probabilistic decision trees to integrate multiple types of data, including localization, mRNA expression, physical interaction, protein function, and characteristics of network topology . Experimental evidence demonstrated the reliability of this strategy, which, when extended to human SSL interactions, may prove valuable in discovering drug targets for cancer therapy and in identifying genes responsible for multigenic diseases.

J Biol Chem, 2004 Dec 24, 279(52), 54221 - 9 Epub 2004 Oct 19.
Mobilization of intracellular copper stores by the ctr2 vacuolar copper transporter; Rees EM et al.; Copper plays an essential role in processes including signaling to the transcription and protein trafficking machinery, oxidative phosphorylation, iron mobilization, neuropeptide maturation, and normal development . Whereas much is known about intracellular mobilization of ions such as calcium, little information is available on how eukaryotic cells mobilize intracellular copper stores . We describe a mechanism by which the Saccharomyces cerevisiae Ctr2 protein provides bioavailable copper via mobilization of intracellular copper stores . Whereas Ctr2 exhibits structural similarity to the Ctr1 plasma membrane copper importer, microscopic and biochemical fractionation studies localize Ctr2 to the vacuole membrane . We demonstrate that Ctr2 mobilizes vacuolar copper stores in a manner dependent on amino acid residues conserved between the Ctr1 and Ctr2 copper transport family and that ctr2 Delta mutants hyper-accumulate vacuolar copper . Furthermore, a Ctr2 mutant that is mislocalized to the plasma membrane stimulates extracellular copper uptake, supporting a direct role for Ctr2 in copper transport across membranes . These studies identify a novel mechanism for copper mobilization and suggest that organisms cope with copper deprivation via the use of intracellular vesicular stores.

J Cell Sci, 2004 Nov 1, 117(Pt 23), 5687 - 96 Epub 2004 Oct 19.
Localization of GDP-mannose transporter in the Golgi requires retrieval to the endoplasmic reticulum depending on its cytoplasmic tail and coatomer; Abe M et al.; The Saccharomyces cerevisiae GDP-mannose transporter (GMT) encoded by the essential gene VRG4/VIG4 is a member of the nucleotide-sugar transporter family in the Golgi apparatus . We examined GMT in the secretory mutant cells to investigate the mechanism of its localization in the Golgi . At the nonpermissive temperature, most GMT was found in the endoplasmic reticulum of sec23ts cells, which have defective COPII, and in the vacuole of sec21ts cells, which have defective COPI . The C-terminal hydrophilic peptide of GMT that is exposed to the cytosol binds to Ret2p, a subunit of the COPI coat . Mutant peptide derivatives that have lost a cluster of lysine in the vicinity of the transmembrane domain had reduced binding activity to Ret2p and the GMT with this sequence was delivered to the vacuole . Our results indicate that GMT escapes from delivery to the vacuole by recycling to the endoplasmic reticulum and retrieval requires the lysine-rich C-terminal tail that can bind to the COPI coat.

Mol Cell, 2004 Oct 22, 16(2), 173 - 85
Mcm10 regulates the stability and chromatin association of DNA polymerase-alpha; Ricke RM et al.; Mcm10 is a conserved eukaryotic DNA replication factor whose function has remained elusive . We report here that Mcm10 binding to replication origins in budding yeast is cell cycle regulated and dependent on the putative helicase, Mcm2-7 . Mcm10 is also an essential component of the replication fork . A fraction of Mcm10 binds to DNA, as shown by histone association assays that allow for the study of chromatin binding in vivo . However, Mcm10 is also required to maintain steady-state levels of DNA polymerase-alpha (polalpha) . In temperature-sensitive mcm10-td mutants, depletion of Mcm10 during S phase results in degradation of the catalytic subunit of polalpha, without affecting other fork components such as Cdc45 . We propose that Mcm10 stabilizes polalpha and recruits the complex to replication origins . During elongation, Mcm10 is required for the presence of polalpha at replication forks and may coordinate DNA synthesis with DNA unwinding by the Mcm2-7 complex.

Biochem Soc Trans, 2004 Nov, 32(Pt 5), 774 - 6
Moving proteins from the cytosol into mitochondria; Chacinska A et al.; Mitochondria of the yeast Saccharomyces cerevisiae contain at least 750 different proteins, which perform diverse roles . Most of these proteins (approx . 99%) are translated on cytosolic ribosomes, and their import into mitochondria is essential for mitochondrial function . Proteinaceous machineries of great complexity, the so-called translocases, in the mitochondrial membranes mediate the import of these proteins.

Proc Natl Acad Sci U S A, 2004 Nov 2, 101(44), 15561 - 6 Epub 2004 Oct 18.
A system for enhancing genome-wide coexpression dynamics study; Li KC et al.; Statistical similarity analysis has been instrumental in elucidation of the voluminous microarray data . Genes with correlated expression profiles tend to be functionally associated . However, the majority of functionally associated genes turn out to be uncorrelated . One conceivable reason is that the expression of a gene can be sensitively dependent on the often-varying cellular state . The intrinsic state change has to be plastically accommodated by gene-regulatory mechanisms . To capture such dynamic coexpression between genes, a concept termed "liquid association" (LA) has been introduced recently . LA offers a scoring system to guide a genome-wide search for critical cellular players that may interfere with the coexpression of a pair of genes, thereby weakening their overall correlation . Although the LA method works in many cases, a direct extension to more than two genes is hindered by the "curse of dimensionality." Here we introduce a strategy of finding an informative 2D projection to generalize LA for multiple genes . A web site is constructed that performs on-line LA computation for any user-specified group of genes . We apply this scoring system to study yeast protein complexes by using the Saccharomyces cerevisiae protein complexes database of the Munich Information Center for Protein Sequences . Human genes are also investigated by profiling of 60 cancer cell lines of the National Cancer Institute . In particular, our system links the expression of the Alzheimer's disease hallmark gene APP (amyloid-beta precursor protein) to the beta-site-cleaving enzymes BACE and BACE2, the gamma-site-cleaving enzymes presenilin 1 and 2, apolipoprotein E, and other Alzheimer's disease-related genes.

Metab Eng, 2004 Oct, 6(4), 326 - 39
A Boolean algorithm for reconstructing the structure of regulatory networks; Mehra S et al.; Advances in transcriptional analysis offer great opportunities to delineate the structure and hierarchy of regulatory networks in biochemical systems . We present an approach based on Boolean analysis to reconstruct a set of parsimonious networks from gene disruption and over expression data . Our algorithms, Causal Predictor (CP) and Relaxed Causal Predictor (RCP) distinguish the direct and indirect causality relations from the non-causal interactions, thus significantly reducing the number of miss-predicted edges . The algorithms also yield substantially fewer plausible networks . This greatly reduces the number of experiments required to deduce a unique network from the plausible network structures . Computational simulations are presented to substantiate these results . The algorithms are also applied to reconstruct the entire network of galactose utilization pathway in Saccharomyces cerevisiae . These algorithms will greatly facilitate the elucidation of regulatory networks using large scale gene expression profile data.

Metab Eng, 2004 Oct, 6(4), 285 - 93
Integration of gene expression data into genome-scale metabolic models; Akesson M et al.; A framework for integration of transcriptome data into stoichiometric metabolic models to obtain improved flux predictions is presented . The key idea is to exploit the regulatory information in the expression data to give additional constraints on the metabolic fluxes in the model . Measurements of gene expression from chemostat and batch cultures of Saccharomyces cerevisiae were combined with a recently developed genome-scale model, and the computed metabolic flux distributions were compared to experimental values from carbon labeling experiments and metabolic network analysis . The integration of expression data resulted in improved predictions of metabolic behavior in batch cultures, enabling quantitative predictions of exchange fluxes as well as qualitative estimations of changes in intracellular fluxes . A critical discussion of correlation between gene expression and metabolic fluxes is given.

Genome Res, 2004 Oct, 14(10B), 2111 - 20
Concerted assembly and cloning of multiple DNA segments using in vitro site-specific recombination: functional analysis of multi-segment expression clones; Cheo DL et al.; The ability to clone and manipulate DNA segments is central to molecular methods that enable expression, screening, and functional characterization of genes, proteins, and regulatory elements . We previously described the development of a novel technology that utilizes in vitro site-specific recombination to provide a robust and flexible platform for high-throughput cloning and transfer of DNA segments . By using an expanded repertoire of recombination sites with unique specificities, we have extended the technology to enable the high-efficiency in vitro assembly and concerted cloning of multiple DNA segments into a vector backbone in a predefined order, orientation, and reading frame . The efficiency and flexibility of this approach enables collections of functional elements to be generated and mixed in a combinatorial fashion for the parallel assembly of numerous multi-segment constructs . The assembled constructs can be further manipulated by directing exchange of defined segments with alternate DNA segments . In this report, we demonstrate feasibility of the technology and application to the generation of fusion proteins, the linkage of promoters to genes, and the assembly of multiple protein domains . The technology has broad implications for cell and protein engineering, the expression of multidomain proteins, and gene function analysis.

Genes Dev, 2004 Oct 15, 18(20), 2529 - 44
Identification of C . elegans DAF-12-binding sites, response elements, and target genes; Shostak Y et al.; Intracellular receptor DAF-12 regulates dauer formation and developmental age and affects Caenorhabditis elegans lifespan . Genetic analyses place DAF-12 at the convergence of several signal transduction pathways; however, the downstream effectors and the molecular basis for the receptor's multiple physiological outputs are unknown . Beginning with C . elegans genomic DNA, we devised a procedure for multiple rounds of selection and amplification that yielded fragments bearing DAF-12-binding sites . These genomic fragments mediated DAF-12-dependent transcriptional regulation both in Saccharomyces cerevisiae and in C . elegans; that is, they served as functional DAF-12 response elements . We determined that most of the genomic fragments that displayed DAF-12 response element activity in yeast were linked to genes that were regulated by DAF-12 in C . elegans; indeed, the response element-containing fragments typically resided within clusters of DAF-12-regulated genes . DAF-12 target gene regulation was developmental program and stage specific, potentially predicting a fit of these targets into regulatory networks governing aspects of C . elegans reproductive development and dauer formation.

Genes Dev, 2004 Oct 15, 18(20), 2506 - 17
RNA polymerase I transcription and pre-rRNA processing are linked by specific SSU processome components; Gallagher JE et al.; Sequential events in macromolecular biosynthesis are often elegantly coordinated . The small ribosomal subunit (SSU) processome is a large ribonucleoprotein (RNP) required for processing of precursors to the small subunit RNA, the 18S, of the ribosome . We have found that a subcomplex of SSU processome proteins, the t-Utps, is also required for optimal rRNA transcription in vivo in the yeast Saccharomyces cerevisiae . The t-Utps are ribosomal chromatin (r-chromatin)-associated, and they exist in a complex in the absence of the U3 snoRNA . Transcription is required neither for the formation of the subcomplex nor for its r-chromatin association . The t-Utps are associated with the pre-18S rRNAs independent of the presence of the U3 snoRNA . This association may thus represent an early step in the formation of the SSU processome . Our results indicate that rRNA transcription and pre-rRNA processing are coordinated via specific components of the SSU processome.

Genes Dev, 2004 Nov 1, 18(21), 2652 - 62 Epub 2004 Oct 15.
Genome-wide mRNA surveillance is coupled to mRNA export; Hieronymus H et al.; Nuclear export of mRNA is a central step in gene expression that shows extensive coupling to transcription and transcript processing . However, little is known about the fate of mRNA and its export under conditions that damage the DNA template and RNA itself . Here we report the discovery of four new factors required for mRNA export through a screen of all annotated nonessential Saccharomyces cerevisiae genes . Two of these factors, mRNA surveillance factor Rrp6 and DNA repair protein Lrp1, are nuclear exosome components that physically interact with one another . We find that Lrp1 mediates specific mRNA degradation upon DNA-damaging UV irradiation as well as general mRNA degradation . Lrp1 requires Rrp6 for genomic localization to genes encoding its mRNA targets, and Rrp6 genomic localization in turn correlates with transcription . Further, Rrp6 and Lrp1 are both required for repair of UV-induced DNA damage . These results demonstrate coupling of mRNA surveillance to mRNA export and suggest specificity of the RNA surveillance machinery for different transcript populations . Broadly, these findings link DNA and RNA surveillance to mRNA export.

Proc Natl Acad Sci U S A, 2004 Oct 26, 101(43), 15301 - 6 Epub 2004 Oct 15.
Imp3p and Imp4p mediate formation of essential U3-precursor rRNA (pre-rRNA) duplexes, possibly to recruit the small subunit processome to the pre-rRNA; Gerczei T et al.; In eukaryotes, formation of short duplexes between the U3 small nucleolar RNA (snoRNA) and the precursor rRNA (pre-rRNA) at multiple sites is a prerequisite for three endonucleolytic cleavages that initiate small subunit biogenesis by releasing the 18S rRNA precursor from the pre-rRNA . The most likely role of these RNA duplexes is to guide the U3 snoRNA and its associated proteins, designated the small subunit processome, to the target cleavage sites on the pre-rRNA . Studies by others in Saccharomyces cerevisiae have identified the proteins Mpp10p, Imp3p, and Imp4p as candidates to mediate U3-pre-rRNA interactions . We report here that Imp3p and Imp4p appear to stabilize an otherwise unstable duplex between the U3 snoRNA hinge region and complementary bases in the external transcribed spacer of the pre-rRNA . In addition, Imp4p, but not Imp3p, seems to rearrange the U3 box A stem structure to expose the site that base-pairs with the 5' end of the 18S rRNA, thereby mediating duplex formation at a second site . By mediating formation of both essential U3-pre-rRNA duplexes, Imp3p and Imp4p may help the small subunit processome to dock onto the pre-rRNA, an event indispensable for ribosome biogenesis and hence for cell growth.

Science, 2004 Oct 15, 306(5695), 482 - 4
Regulation of gene expression by a metabolic enzyme; Hall DA et al.; Gene expression in eukaryotes is normally believed to be controlled by transcriptional regulators that activate genes encoding structural proteins and enzymes . To identify previously unrecognized DNA binding activities, a yeast proteome microarray was screened with DNA probes; Arg5,6, a well-characterized mitochondrial enzyme involved in arginine biosynthesis, was identified . Chromatin immunoprecipitation experiments revealed that Arg5,6 is associated with specific nuclear and mitochondrial loci in vivo, and Arg5,6 binds to specific fragments in vitro . Deletion of Arg5,6 causes altered transcript levels of both nuclear and mitochondrial target genes . These results indicate that metabolic enzymes can directly regulate eukaryotic gene expression.

Mol Cell Biol, 2004 Nov, 24(21), 9682 - 94
The Mre11 nuclease is not required for 5' to 3' resection at multiple HO-induced double-strand breaks; Llorente B et al.; Current hypotheses suggest the Mre11 nuclease activity could be directly involved in double-strand break (DSB) resection in the presence of a large number of DSBs or limited to processing abnormal DNA ends . To distinguish between these possibilities, we used two methods to create large numbers of DSBs in Saccharomyces cerevisiae chromosomes, without introducing other substrates for the Mre11 nuclease . Multiple DSBs were created either by expressing the HO endonuclease in strains containing several HO cut sites embedded within randomly dispersed Ty1 elements or by phleomycin treatment . Analysis of resection by single-strand DNA formation in these systems showed no difference between strains containing MRE11 or the mre11-D56N nuclease defective allele, suggesting that the Mre11 nuclease is not involved in the extensive 5' to 3' resection of DSBs . We postulate that the ionizing radiation (IR) sensitivity of mre11 nuclease-defective mutants results from the accumulation of IR-induced DNA damage that is normally processed by the Mre11 nuclease . We also report that the processivity of 5' to 3' DSB resection and the yield of repaired products are affected by the number of DSBs in a dose-dependent manner . Finally, we show that the exonuclease Exo1 is involved in the processivity of 5' to 3' resection of an HO-induced DSB at the MAT locus.

Mol Cell Biol, 2004 Nov, 24(21), 9568 - 79
A coordinated temporal interplay of nucleosome reorganization factor, sister chromatin cohesion factor, and DNA polymerase alpha facilitates DNA replication; Zhou Y et al.; DNA replication depends critically upon chromatin structure . Little is known about how the replication complex overcomes the nucleosome packages in chromatin during DNA replication . To address this question, we investigate factors that interact in vivo with the principal initiation DNA polymerase, DNA polymerase alpha (Polalpha) . The catalytic subunit of budding yeast Polalpha (Pol1p) has been shown to associate in vitro with the Spt16p-Pob3p complex, a component of the nucleosome reorganization system required for both replication and transcription, and with a sister chromatid cohesion factor, Ctf4p . Here, we show that an N-terminal region of Polalpha (Pol1p) that is evolutionarily conserved among different species interacts with Spt16p-Pob3p and Ctf4p in vivo . A mutation in a glycine residue in this N-terminal region of POL1 compromises the ability of Pol1p to associate with Spt16p and alters the temporal ordered association of Ctf4p with Pol1p . The compromised association between the chromatin-reorganizing factor Spt16p and the initiating DNA polymerase Pol1p delays the Pol1p assembling onto and disassembling from the late-replicating origins and causes a slowdown of S-phase progression . Our results thus suggest that a coordinated temporal and spatial interplay between the conserved N-terminal region of the Polalpha protein and factors that are involved in reorganization of nucleosomes and promoting establishment of sister chromatin cohesion is required to facilitate S-phase progression.

Mol Cell Biol, 2004 Nov, 24(21), 9542 - 56
Recruitment of the Swi/Snf complex by Ste12-Tec1 promotes Flo8-Mss11-mediated activation of STA1 expression; Kim TS et al.; In the yeast Saccharomyces diastaticus, expression of the STA1 gene, which encodes an extracellular glucoamylase, is activated by the specific DNA-binding activators Flo8, Mss11, Ste12, and Tec1 and the Swi/Snf chromatin-remodeling complex . Here we show that Flo8 interacts physically and functionally with Mss11 . Flo8 and Mss11 bind cooperatively to the inverted repeat sequence TTTGC-n-GCAAA (n = 97) in UAS1-2 of the STA1 promoter . In addition, Flo8 and Mss11 bind indirectly to UAS2-1 of the STA1 promoter by interacting with Ste12 and Tec1, which bind to the filamentation and invasion response element (FRE) in UAS2-1 . Furthermore, our findings indicate that the Ste12, Tec1, Flo8, and Mss11 activators and the Swi/Snf complex bind sequentially to the STA1 promoter, as follows: Ste12 and Tec1 bind first to the FRE, whereby they recruit the Swi/Snf complex to the STA1 promoter . Next, the Swi/Snf complex enhances Flo8 and Mss11 binding to UAS1-2 . In the final step, Flo8 and Mss11 directly promote association of RNA polymerase II with the STA1 promoter to activate STA1 expression . In the absence of glucose, the levels of Flo8 and Tec1 are greatly increased, whereas the abundances of two repressors, Nrg1 and Sfl1, are reduced, suggesting that the balance of transcriptional regulators may be important for determining activation or repression of STA1 expression.

Mol Cell Biol, 2004 Nov, 24(21), 9437 - 55
Interactions of eukaryotic translation initiation factor 3 (eIF3) subunit NIP1/c with eIF1 and eIF5 promote preinitiation complex assembly and regulate start codon selection; Valasek L et al.; The N-terminal domain (NTD) of NIP1/eIF3c interacts directly with eIF1 and eIF5 and indirectly through eIF5 with the eIF2-GTP-Met-tRNA(i)(Met) ternary complex (TC) to form the multifactor complex (MFC) . We investigated the physiological importance of these interactions by mutating 16 segments spanning the NIP1-NTD . Mutations in multiple segments reduced the binding of eIF1 or eIF5 to the NIP1-NTD . Mutating a C-terminal segment of the NIP1-NTD increased utilization of UUG start codons (Sui(-) phenotype) and was lethal in cells expressing eIF5-G31R that is hyperactive in stimulating GTP hydrolysis by the TC at AUG codons . Both effects of this NIP1 mutation were suppressed by eIF1 overexpression, as was the Sui(-) phenotype conferred by eIF5-G31R . Mutations in two N-terminal segments of the NIP1-NTD suppressed the Sui(-) phenotypes produced by the eIF1-D83G and eIF5-G31R mutations . From these and other findings, we propose that the NIP1-NTD coordinates an interaction between eIF1 and eIF5 that inhibits GTP hydrolysis at non-AUG codons . Two NIP1-NTD mutations were found to derepress GCN4 translation in a manner suppressed by overexpressing the TC, indicating that MFC formation stimulates TC recruitment to 40S ribosomes . Thus, the NIP1-NTD is required for efficient assembly of preinitiation complexes and also regulates the selection of AUG start codons in vivo.

Mol Cell Biol, 2004 Nov, 24(21), 9424 - 36
The Yaf9 component of the SWR1 and NuA4 complexes is required for proper gene expression, histone H4 acetylation, and Htz1 replacement near telomeres; Zhang H et al.; Yaf9, Taf14, and Sas5 comprise the YEATS domain family in Saccharomyces cerevisiae, which in humans includes proteins involved in acute leukemias . The YEATS domain family is essential, as a yaf9Delta taf14Delta sas5Delta triple mutant is nonviable . We verify that Yaf9 is a stable component of NuA4, an essential histone H4 acetyltransferase complex . Yaf9 is also associated with the SWR1 complex, which deposits the histone H2A variant Htz1 . However, the functional contribution of Yaf9 to these complexes has not been determined . Strains lacking YAF9 are sensitive to DNA-damaging agents, cold, and caffeine, and the YEATS domain is required for full Yaf9 function . NuA4 lacking Yaf9 retains histone acetyltransferase activity in vitro, and Yaf9 does not markedly reduce bulk H4 acetylation levels, suggesting a role for Yaf9 in the targeting or regulation of NuA4 . Interestingly, yaf9Delta strains display reduced transcription of genes near certain telomeres, and their repression is correlated with reduced H4 acetylation, reduced occupancy by Htz1, and increased occupancy by the silencing protein Sir3 . Additionally, the spectra of phenotypes, genes, and telomeres affected in yaf9Delta and htz1Delta strains are significantly similar, further supporting a role for Yaf9 in Htz1 deposition . Taken together, these data indicate that Yaf9 may function in NuA4 and SWR1 complexes to help antagonize silencing near telomeres.

Biochem Biophys Res Commun, 2004 Nov 19, 324(3), 981 - 5
Ammodytoxin, a neurotoxic secreted phospholipase A(2), can act in the cytosol of the nerve cell; Petrovic U et al.; Recent identification of intracellular proteins that bind ammodytoxin (calmodulin, 14-3-3 proteins, and R25) suggests that this snake venom presynaptically active phospholipase A(2) acts intracellularly . As these ammodytoxin acceptors are cytosolic and mitochondrial proteins, the toxin should be able to enter the cytosol of a target cell and remain stable there to interact with them . Using laser scanning confocal microscopy we show here that Alexa-labelled ammodytoxin entered the cytoplasm of the rat hippocampal neuron and subsequently also its nucleus . The transport of proteins into the nucleus proceeds via the cytosol of a cell, therefore, ammodytoxin passed the cytosol of the neuron on its way to the nucleus . Although it is not yet clear how ammodytoxin is translocated into the cytosol of the neuron, our results demonstrate that its stability in the cytosol is not in question, providing the evidence that the toxin can act in this cellular compartment.

Mol Biol Cell, 2004 Dec, 15(12), 5456 - 69 Epub 2004 Dec.
Dictyostelium PAKc is required for proper chemotaxis; Lee S et al.; We have identified a new Dictyostelium p21-activated protein kinase, PAKc, that we demonstrate to be required for proper chemotaxis . PAKc contains a Rac-GTPase binding (CRIB) and autoinhibitory domain, a PAK-related kinase domain, an N-terminal phosphatidylinositol binding domain, and a C-terminal extension related to the Gbetagamma binding domain of Saccharomyces cerevisiae Ste20, the latter two domains being required for PAKc transient localization to the plasma membrane . In response to chemoattractant stimulation, PAKc kinase activity is rapidly and transiently activated, with activity levels peaking at approximately 10 s . pakc null cells exhibit a loss of polarity and produce multiple lateral pseudopodia when placed in a chemoattractant gradient . PAKc preferentially binds the Dictyostelium Rac protein RacB, and point mutations in the conserved CRIB that abrogate this binding result in misregulated kinase activation and chemotaxis defects . We also demonstrate that a null mutation lacking the PAK family member myosin I heavy chain kinase (MIHCK) shows mild chemotaxis defects, including the formation of lateral pseudopodia . A null strain lacking both PAKc and the PAK family member MIHCK exhibits severe loss of cell movement, suggesting that PAKc and MIHCK may cooperate to regulate a common chemotaxis pathway.

Mol Genet Genomics, 2004 Nov, 272(4), 443 - 51 Epub 2004 Nov.
The ire1 and ptc2 genes involved in the unfolded protein response pathway in the filamentous fungus Trichoderma reesei; Valkonen M et al.; A signal transduction pathway called the unfolded protein response is activated when increased levels of misfolded proteins or incorrectly assembled subunits accumulate in the endoplasmic reticulum (ER) . The expression of several genes for ER-resident foldases and chaperones, as well as genes encoding proteins that are involved in functions associated with the secretory process, are induced by this pathway . This paper describes the cloning and characterisation of genes for two components of the pathway, ire1 and ptc2, from the filamentous fungus Trichoderma reesei (Hypocrea jecorina) . The data presented demonstrates that the T . reesei genes can complement Saccharomyces cerevisiae mutants that are deficient in the corresponding homologues . The T . reesei IREI protein has intrinsic kinase activity, as revealed by an in vitro autophosphorylation assay . Overexpression of ire1 in a T . reesei strain that expresses a foreign protein (laccase 1 from Phlebia radiata), results in up-regulation of the UPR pathway, as indicated by the increased expression levels of the known UPR target genes bip1 and pdi1 . Splicing of the mRNA encoding the transcription factor HAC1 is also observed . Other genes encoding proteins from different parts of the secretory pathway also respond to ire1 overexpression.

J Cell Biol, 2004 Oct 11, 167(1), 65 - 74
COPII-dependent export of cystic fibrosis transmembrane conductance regulator from the ER uses a di-acidic exit code; Wang X et al.; Cystic fibrosis (CF) is a childhood hereditary disease in which the most common mutant form of the CF transmembrane conductance regulator (CFTR) DeltaF508 fails to exit the endoplasmic reticulum (ER) . Export of wild-type CFTR from the ER requires the coat complex II (COPII) machinery, as it is sensitive to Sar1 mutants that disrupt normal coat assembly and disassembly . In contrast, COPII is not used to deliver CFTR to ER-associated degradation . We find that exit of wild-type CFTR from the ER is blocked by mutation of a consensus di-acidic ER exit motif present in the first nucleotide binding domain . Mutation of the code disrupts interaction with the COPII coat selection complex Sec23/Sec24 . We propose that the di-acidic exit code plays a key role in linking CFTR to the COPII coat machinery and is the primary defect responsible for CF in DeltaF508-expressing patients.

Cell, 2004 Oct 15, 119(2), 169 - 80
Mapping the location of TFIIB within the RNA polymerase II transcription preinitiation complex: a model for the structure of the PIC; Chen HT et al.; Biochemical probes positioned on the surface of the general transcription factor TFIIB were used to probe the architecture of the RNA polymerase II (Pol II) transcription preinitiation complex (PIC) . In PICs, the TFIIB linker and core domains are positioned over the central cleft and wall of Pol II . This positioning is not observed in the smaller Pol II-TFIIB complex . These results lead to a new model for the structure of the PIC, which agrees with most previously documented protein-DNA interactions within Pol II and archaea PICs . Specific interaction of the TFIIB core domain with Pol II positions and orients the promoter DNA over the Pol II central cleft, and TBP-DNA bending leads to bending of the promoter around the surface of Pol II . The TFIIF subunit Tfg1 was found in close proximity to the TFIIB B finger, linker, and core domains, suggesting that these two factors closely cooperate during initiation.

Mol Biochem Parasitol, 2004 Aug, 136(2), 265 - 72
A novel purine nucleoside transporter whose expression is up-regulated in the short stumpy form of the Trypanosoma brucei life cycle; Sanchez MA et al.; Purine nucleoside and nucleobase transporters play a vital role in the metabolism and survival of Trypanosoma brucei because this parasitic protozoan is unable to synthesize purines de novo and thus must acquire preformed purines from its hosts . These parasites express a variety of nucleoside and nucleobase permeases with diverse substrate specificities and distinct patterns of expression during the trypanosome life cycle . We report here that expression of the newly characterized T . brucei nucleoside transporter 10 gene (TbNT10) is up-regulated in the short stumpy form of the life cycle, the bloodstream form of the parasite that is pre-adapted for infection of the tsetse fly vector . Functional expression of TbNT10 in Saccharomyces cerevisiae reveals that the TbNT10 gene encodes an adenosine/guanosine/inosine transporter with apparent Km values of approximately 1 microM and hence is a high affinity purine nucleoside transporter . The restricted expression of TbNT10 during the life cycle suggests that the functional properties of this permease may be specialized to support development and growth of the differentiated short stumpy form or to promote the transformation of short stumpy to procyclic forms within the insect vector.

Nucleic Acids Res, 2004 Oct 11, 32(18), 5464 - 70 Print 2004.
Interaction of mitochondrial initiation factor 2 with mitochondrial fMet-tRNA; Spencer AC et al.; The mammalian mitochondrial genome contains a single tRNA(Met) gene that gives rise to the initiator and elongator tRNA(Met) . It is generally believed that mitochondrial protein synthesis begins with formylmethionyl-tRNA, which indicates that the formylation of mitochondrial Met-tRNA specifies its participation in initiation through its interaction with initiation factor 2 (IF-2) . However, recent studies in yeast mitochondria, suggest that formylation is not required for protein synthesis . In addition, bovine IF-2(mt) could replace yeast IF-2(mt) in strains that lack fMet-tRNA which suggests that this paradigm may extend to mammalian mitochondria . Here, the importance of the formylation of mitochondrial Met-tRNA for the interaction with IF-2(mt) was investigated by measuring the ability of bovine IF-2(mt) to bind mitochondrial fMet-tRNA . In direct binding experiments, bovine IF-2(mt) has a 25-fold greater affinity for mitochondrial fMet-tRNA than Met-tRNA, using either the native mitochondrial tRNA(Met) or an in vitro transcript of bovine mitochondrial tRNA(Met) . In addition, IF-2(mt) will not effectively stimulate mitochondrial Met-tRNA binding to mitochondrial ribosomes, exhibiting a 50-fold preference for fMet-tRNA over Met-tRNA in this assay . Finally, the region of IF-2(mt) responsible for the interaction with fMet-tRNA was mapped to the C2 sub-domain of domain VI of this factor.

J Mol Biol, 2004 Oct 29, 343(4), 865 - 78
Thermodynamic signature of GCN4-bZIP binding to DNA indicates the role of water in discriminating between the AP-1 and ATF/CREB sites; Dragan AI et al.; The energetic basis of GCN4-bZIP complexes with the AP-1 and ATF/CREB sites was investigated by optical methods and scanning and isothermal titration microcalorimetry . The dissociation constant of the bZIP dimer was found to be significantly higher than that of its isolated leucine zipper domain: at 20 degrees C it is 1.45microM and increases with temperature . To avoid complications from dissociation of this dimer, DNA binding experiments were carried out using an SS crosslinked version of the bZIP . The thermodynamic characteristics of the bZIP/DNA association measured at different temperatures and salt concentrations were corrected for the contribution of refolding the basic segment upon binding, determined from the scanning calorimetric experiments . Fluorescence anisotropy titration experiments showed that the association constants of the bZIP at 20 degrees C with the AP-1 and ATF/CREB binding sites do not differ much, being 1.5nM and 6.4nM, corresponding to Gibbs energies of -49kJmol(-1) and -46kJmol(-1), respectively . Almost half of the Gibbs energy is attributable to the electrostatic component, resulting from the entropic effect of counterion release upon DNA association with the bZIP and is identical for both sites . In contrast to the Gibbs energies, the enthalpies of association of the fully folded bZIP with the AP-1 and ATF/CREB sites, and correspondingly the entropies of association, are very different . bZIP binding to the AP-1 site is characterized by a substantially larger negative enthalpy and non-electrostatic entropy than to the ATF/CREB site, implying that the AP-1 complex incorporates significantly more water molecules than the ATF/CREB complex.

Plant Physiol, 2004 Nov, 136(3), 3712 - 23 Epub 2004 Oct 08.
Arabidopsis HMA2, a divalent heavy metal-transporting P(IB)-type ATPase, is involved in cytoplasmic Zn2+ homeostasis; Eren E et al.; PIB-type ATPases transport heavy metal ions (Cu+, Cu2+, Zn2+, Cd2+, Co2+, etc.) across biological membranes . Several members of this subfamily are present in plants . Higher plants are the only eukaryotes where putative Zn(2+)-ATPases have been identified . We have cloned HMA2, a PIB-ATPase present in Arabidopsis (Arabidopsis thaliana), and functionally characterized this enzyme after heterologous expression in yeast (Saccharomyces cerevisiae) . HMA2 is a Zn(2+)-dependent ATPase that is also activated by Cd2+ and, to a lesser extent, by other divalent heavy metals (Pb2+, Ni2+, Cu2+, and Co2+) . The enzyme forms an acid-stable phosphorylated intermediate and is inhibited by vanadate . HMA2 interacts with Zn2+ and Cd2+ with high affinity (Zn2+ K(1/2) = 0.11 +/- 0.03 microm and Cd2+ K(1/2) = 0.031 +/- 0.007 microm) . However, its activity is dependent on millimolar concentrations of Cys in the assay media . Zn2+ transport determinations indicate that the enzyme drives the outward transport of metals from the cell cytoplasm . Analysis of HMA2 mRNA suggests that the enzyme is present in all plant organs and transcript levels do not change in plants exposed to various metals . Removal of HMA2 full-length transcript results in Zn2+ accumulation in plant tissues . hma2 mutant plants also accumulate Cd2+ when exposed to this metal . These results suggest that HMA2 is responsible for Zn2+ efflux from the cells and therefore is required for maintaining low cytoplasmic Zn2+ levels and normal Zn2+ homeostasis.

Nucleic Acids Res, 2004 Oct 08, 32(18), 5321 - 38 Print 2004.
Human MutL homolog (MLH1) function in DNA mismatch repair: a prospective screen for missense mutations in the ATPase domain; Ellison AR et al.; Germline mutations in the DNA mismatch repair (MMR) genes MSH2 and MLH1 are responsible for the majority of hereditary non-polyposis colorectal cancer (HNPCC), an autosomal-dominant early-onset cancer syndrome . Genetic testing of both MSH2 and MLH1 from individuals suspected of HNPCC has revealed a considerable number of missense codons, which are difficult to classify as either pathogenic mutations or silent polymorphisms . To identify novel MLH1 missense codons that impair MMR activity, a prospective genetic screen in the yeast Saccharomyces cerevisiae was developed . The screen utilized hybrid human-yeast MLH1 genes that encode proteins having regions of the yeast ATPase domain replaced by homologous regions from the human protein . These hybrid MLH1 proteins are functional in MMR in vivo in yeast . Mutagenized MLH1 fragments of the human coding region were synthesized by error-prone PCR and cloned directly in yeast by in vivo gap repair . The resulting yeast colonies, which constitute a library of hybrid MLH1 gene variants, were initially screened by semi-quantitative in vivo MMR assays . The hybrid MLH1 genes were recovered from yeast clones that exhibited a MMR defect and sequenced to identify alterations in the mutagenized region . This investigation identified 117 missense codons that conferred a 2-fold or greater decreased efficiency of MMR in subsequent quantitative MMR assays . Notably, 10 of the identified missense codons were equivalent to codon changes previously observed in the human population and implicated in HNPCC . To investigate the effect of all possible codon alterations at single residues, a comprehensive mutational analysis of human MLH1 codons 43 (lysine-43) and 44 (serine-44) was performed . Several amino acid replacements at each residue were silent, but the majority of substitutions at lysine-43 (14/19) and serine-44 (18/19) reduced the efficiency of MMR . The assembled data identifies amino acid substitutions that disrupt MLH1 structure and/or function, and should assist the interpretation of MLH1 genetic tests.

Toxicol Lett, 2004 Dec 1, 154(1-2), 81 - 8
The role of glutathione transferases in cadmium stress; Adamis PD et al.; Using Saccharomyces cerevisiae as experimental model, we observed that cells mutated in the GTT1 or GTT2 genes showed twice as much cadmium absorption than the control strain . We proposed that the formation of the cadmium-glutathione complex is dependent on that transferase, since it was previously demonstrated that the cytoplasmic levels of this complex affect cadmium uptake . The addition of glutathione monoethyl ester (GME), a drug that mimics glutathione (GSH), to gtt1Delta cells restored the levels of metal absorption to those of the control strain . However, with respect to gtt2Delta cells, addition of GME did not alter the capacity of removing cadmium from the medium . Taken together, these results suggest that Gtt1 and Gtt2 play different roles in the mechanism of cadmium detoxification . By analyzing the toxic effect of this metal, we verified that gtt2Delta and gsh1Delta cells showed, respectively, higher and lower tolerance to cadmium stress than control cells, suggesting that although GSH plays a relevant role in cell protection, formation of the GSH-Cd conjugate is deleterious to the mechanism of defense.

Trends Genet, 2004 Nov, 20(11), 555 - 62
Mitochondriomics or what makes us breathe; Reichert AS et al.; Mitochondria perform several fundamental cellular processes in higher eukaryotes including oxidative phosphorylation, Fe/S cluster formation and apoptosis . Dysfunction of the organelle is associated with a wide range of human diseases . To gain a better understanding of mitochondrial function, several recent proteomic, genetic, transcriptomic and bioinformatic approaches have set out to determine the complete set of mitochondrially located proteins in yeast, plants and mammals . Here, we review these studies and discuss the advances and limitations of individual strategies . Integration of various approaches proves to be a successful and useful way to identify the mitochondrial proteome with high sensitivity and specificity . The most comprehensive dataset is available for Saccharomyces cerevisiae, giving an estimated number of 700 different proteins located in mitochondria.

Biochem Biophys Res Commun, 2004 Nov 12, 324(2), 768 - 72
PLP2/A4 interacts with CCR1 and stimulates migration of CCR1-expressing HOS cells; Lee SM et al.; Multiple CC chemokines bind to CCR1, which plays important roles in immune and inflammatory responses . To search for proteins involved in the CCR1 signaling pathway, we screened a yeast two-hybrid library using the cytoplasmic tail of CCR1 as the bait . One of the positive clones contained an open reading frame of 456bp, of which the nucleotide sequence was identical to that of proteolipid protein 2 (PLP2), also known as protein A4 . Mammalian two-hybrid and coimmunoprecipitation analyses demonstrated the association of PLP2/A4 with CCR1 . Indirect immunofluorescence analysis revealed that PLP2/A4 was predominantly located in plasma membrane and colocalized with CCR1 in transfected human HEK293 cells . In addition, focal staining of CCR1 appeared on the periphery of the membrane upon short exposure to Leukotactin-1(Lkn-1)/CCL15, a CCR1 agonist, and was costained with PLP2/A4 on the focal regions . PLP2/A4 mRNAs were detected in various cells such as U-937, HL-60, HEK293, and HOS cells . Overexpression of PLP2/A4 stimulated a twofold increase in the agonist-induced migration of HOS/CCR1 cells, implicating a functional role for PLP2/A4 in the chemotactic processes via CCR1.

Gene, 2004 Oct 27, 341, 219 - 25
Translation termination in Arabidopsis thaliana: characterisation of three versions of release factor 1; Chapman B et al.; Translation termination is mediated in all eukaryotes by the two release factors eRF1 and eRF3 . Most organisms have a single eRF1 gene, however, three isogenes of eRF1 are found in Arabidopsis thaliana . They have no introns in the coding region which may indicate that some are pseudogenes . However, each was expressed and able to rescue a temperature sensitive eRF1-mutant of Saccharomyces cerevisiae indicating functional redundancy in A . thaliana . While normally a highly accurate process, translation termination can be directed to fail by sequence elements within an messenger RNA (mRNA) . Interestingly, a well-characterised readthrough element follows the stop codon in one of these three isogenes (designated eRF1-1) . This element was shown to be capable of inducing readthrough in an in vitro assay using a dual luciferase reporter, but surprisingly readthrough could not be detected using the complete gene context . The results highlight the diversity and duplication of genes within plant genomes, but also emphasize the conservation of the translation process across kingdoms.

FEBS Lett, 2004 Oct 8, 576(1-2), 190 - 200
ERR-10: a new repressor in transcriptional signaling activation of estrogen receptor-alpha; Meng Q et al.; Estrogen receptor-alpha (ER-alpha) is a nuclear transcriptional factor that is part of the nuclear receptor superfamily . In this study, we isolated and identified a new LXXLL-containing protein that interacts with the ER-alpha via a yeast two-hybrid assay . We have termed this protein estrogen receptor repressor-10 (ERR-10) . The ERR-10 cDNA is predicted to encode a polypeptide of 94 amino acids, with a molecular mass of about 10 kDa . Although the ERR-10 mRNA transcript is expressed in a wide range of normal human tissues, higher expression levels are found in endocrinal tissues relative to other tissues . We have demonstrated, through immunoprecipitation, Western blot and GST pull-down assays, that ERR-10 associates with ER-alpha . Moreover, ERR-10 decreased 17beta-estrodial-induced activation of ER-alpha transcriptional activity in transient transfection assays of mammalian cells . The ERR-10 N-terminus, which resembles two LXXLL motifs, is essential for ER-alpha binding and repression activity . Estrogen modulation of estrogen-responsive gene expression was markedly blocked by ERR-10 . These results suggest that ERR-10 is a novel mediator in ER transcriptional activation .

FEBS Lett, 2004 Oct 8, 576(1-2), 5 - 8
The Arabidopsis thaliana MEK AtMKK6 activates the MAP kinase AtMPK13; Melikant B et al.; Mitogen-activated protein (MAP) kinases mediate cellular responses to a wide variety of stimuli . Activation of a MAP kinase occurs after phosphorylation by an upstream dual-specificity protein kinase, known as a MAP kinase kinase or MEK . The Arabidopsis thaliana genome encodes 10 MEKs but few of these have been shown directly to activate any of the 20 Arabidopsis MAP kinases . We show here that functional complementation of the cell lysis phenotype of a mutant yeast strain depends on the co-expression of the Arabidopsis MEK AtMKK6 and the MAP kinase AtMPK13 . The kinase activity of AtMPK13 is stimulated in the presence of AtMKK6 in yeast cells . RT-PCR analysis showed the co-expression of these two genes in diverse plant tissues . These data show that AtMKK6 can functionally activate the MAP kinase AtMPK13 .

Yi Chuan Xue Bao, 2004 Jul, 31(7), 740 - 9
{Cloning and heterologous expression of a novel delta6 -desaturase gene from Rhizopus arrhizus NK030037}; Zhang Q et al.; A 593 bp DNA fragment was amplified from Rhizopus arrhizus NK030037 with degenerate oligonucleotide primers designed based on the sequences information for fungi delta6-fatty acid desaturase genes by RT-PCR and sequenced . Gene specific primers derived from this partial sequence were used for the amplification of the 3'- and 5'-ends of this cDNA by RACE method, and this lead to a full-length cDNA sequence of 1 482 bp was amplified . Sequence analysis showed this cDNA sequence had an open reading frame(ORF) of 1 377 bp coding 458 amino acids of 52 kD . The deduced amino-acid sequence of the ORF showed similarity to those of the above delta6-fatty acid desaturases which comprised the characteristics of membrane-bound desaturases, including three conserved histidine-rich boxes and hydropathy profile . A cytochrome b5-like domain was observed at the N-terminus . The full-length cDNA sequence is a putative novel delta6-fatty acid desaturase gene . To elucidate the function of the protein, two specific primers corresponding to the nucleotide sequences of start and stop codons were used to amplify the coding sequence . The amplified cDNA RAD6 was subcloned into the expression vector pYES2.0 to generate a recombinant plasmid pYRAD6, which was subsequently transformed into Saccharomyces cerevisiae strain INVScl for heterologous expression by lithium acetate method . Grown to logarithmic phase at 30 degrees C, the transformed cells were supplemented with 0.5 mmol/L . Linoleic acid and induced by 2% galactose for a further 48 h of cultivation at 20 degrees . Total fatty acids were extracted from the induced cells and subjected to methyl-esterification . The resultant fatty acid methyl esters(FAME) were analyzed by gas chromatography(GC) . A novel peak corresponding to gamma-linolenic acid (GLA) methyl ester standards was detected with the same retention time, which was absent in the cell transformed with empty vector . The percentage of this new fatty acid to total fatty acids was 3.85% . Gas chromatography-mass spectrometry( GC-MS) analysis of this fatty acid methyl derivative demonstrated that the novel peak was GLA methyl ester . These results showed that the transgenic product exhibited delta6-fatty acid desaurase activity, converting LA to GLA specifically.

Eukaryot Cell, 2004 Oct, 3(5), 1227 - 32
Linkage between mitochondrial hypovirulence and viral hypovirulence in the chestnut blight fungus revealed by cDNA microarray analysis; Allen TD et al.; The phenomenon of transmissible hypovirulence (virulence attenuation) associated with biological control of natural populations of the chestnut blight fungus Cryphonectria parasitica can be experimentally reproduced by infection with hypovirus cDNA clones (viral hypovirulence) or by mutation of mitochondrial DNA (mtDNA) in the absence of virus infection (mitochondrial hypovirulence) . We now report the use of an established C . parasitica cDNA microarray to monitor nuclear transcriptional responses to an mtDNA mutation of C . parasitica strain EP155, designated EP155/mit2, which was previously shown to induce elevated alternative oxidase activity and hypovirulence (C . B . Monterio-Vitorello, J . A . Bell, D . W . Fulbright, and H . A . Bertrand, Proc . Natl . Acad . Sci . USA 92:5935-5939, 1995) . Approximately 10% of the 2,200 genes represented on the microarray exhibited altered transcript accumulation as a result of the mit2 mtDNA mutation . While genes involved in mitochondrial function were clearly represented in the EP155/mit2-responsive gene list, direct parallels to the well-characterized Saccharomyces cerevisiae retrograde response to mitochondrial dysfunction were not observed . Remarkably, 47% of the genes that were differentially expressed following the infection of strain EP155 by the prototypic hypovirus CHV1-EP713 had similarly changed transcript accumulation in the virus-free EP155/mit2 mutant . These results establish a linkage between viral and mitochondrial hypovirulence and raise questions regarding the relationship between hypovirus infection and mitochondrial dysfunction . The combined set of transcriptional profile data provides a foundation for future studies on mitochondrion-to-nucleus communications in the context of hypovirus infection and senescence associated with mitochondrial dysfunction in filamentous fungi.

Microbiology, 2004 Oct, 150(Pt 10), 3281 - 8
Mutational analysis of the cytoplasmic domain of the Wsc1 cell wall stress sensor; Vay HA et al.; Wsc1 is a member of a family of highly O-glycosylated cell surface proteins that reside in the plasma membrane of Saccharomyces cerevisiae and function as sensors of cell wall stress . These proteins activate the cell wall integrity signalling pathway by stimulating the small G-protein Rho1, protein kinase C (Pkc1) and a MAP kinase cascade . The cytoplasmic domains of Wsc1 family members interact with the Rom2 guanine nucleotide exchange factor to stimulate GTP-binding of Rho1 . Here, a mutational analysis of the cytoplasmic domain of Wsc1 is presented . The data identify two regions of the Wsc1 cytoplasmic tail that are conserved with other family members as important for Rom2 interaction . These regions are separated by an inhibitory region, which includes a cluster of seryl residues that appear to be phosphorylated . Mutational analysis of these residues supports a model in which Wsc1 interaction with Rom2 is negatively regulated by phosphorylation.

Microbiology, 2004 Oct, 150(Pt 10), 3175 - 87
Role of chitin synthase genes in Fusarium oxysporum; Martin-Udiroz M et al.; Three structural chitin synthase genes, chs1, chs2 and chs3, were identified in the genome of Fusarium oxysporum f . sp . lycopersici, a soilborne pathogen causing vascular wilt disease in tomato plants . Based on amino acid identities with related fungal species, chs1, chs2 and chs3 encode structural chitin synthases (CSs) of class I, class II and class III, respectively . A gene (chs7) encoding a chaperone-like protein was identified by comparison of the deduced protein with Chs7p from Saccharomyces cerevisiae, an endoplasmic reticulum (ER) protein required for the export of ScChs3p (class IV) from the ER . So far no CS gene belonging to class IV has been isolated from F . oxysporum, although it probably contains more than one gene of this class, based on the genome data of the closely related species Fusarium graminearum . F . oxysporum chs1-, chs2- and chs7-deficient mutants were constructed through targeted gene disruption by homologous recombination . No compensatory mechanism seems to exist between the CS genes studied, since chitin content determination and expression analysis of the chs genes showed no differences between the disruption mutants and the wild-type strain . By fluorescence microscopy using Calcofluor white and DAPI staining, the wild-type strain and Deltachs2 and Deltachs7 mutants showed similar septation and even nuclear distribution, with each hyphal compartment containing only one nucleus, whereas the Deltachs1 mutant showed compartments containing up to four nuclei . Pathogenicity assays on tomato plants indicated reduced virulence of Deltachs2 and Deltachs7 null mutants . Stress conditions affected normal development in Deltachs2 but not in Deltachs1 or Deltachs7 disruptants, and the three chs-deficient mutants showed increased hyphal hydrophobicity compared to the wild-type strain when grown in sorbitol-containing medium . The chitin synthase mutants will be useful for elucidating cell wall biogenesis in F . oxysporum and the relationship between fungal cell wall integrity and pathogenicity.

Brain Res Mol Brain Res, 2004 Oct 22, 129(1-2), 117 - 23
Effect of genetic polymorphism on the metabolism of endogenous neuroactive substances, progesterone and p-tyramine, catalyzed by CYP2D6; Niwa T et al.; Metabolic activities toward endogenous substrates in the brain, progesterone and p-tyramine, by cytochrome P450 2D6.2 (CYP2D6.2), CYP2D6.10A, CYP2D6.10C, and P34S, G42R, R296C, and S486T mutants expressed in recombinant Saccharomyces cerevisiae were compared with those by CYP2D6.1 (wild-type) in order to clarify the effects of genetic polymorphism of CYP2D6 on the metabolism of neuroactive steroids and amines in the brain . For the 6beta-hydroxylation of progesterone, the V(max) values for CYP2D6.2, CYP2D6.10A, and the P34S and G42R mutants, were less than half of those for CYP2D6.1, and CYP2D6.10C had a higher K(m) and a lower V(max) than the wild-type . The V(max)/K(m) values for CYP2D6.10A, CYP2D6.10C, and the P34S and G42R mutants were 12-31% of that for CYP2D6 . The 16alpha-hydroxylation and 21-hydroxylation of progesterone by CYP2D6.10A, CYP2D6.10C, and the P34S and G42R mutants were not detected, and the R296C mutant had a higher K(m) for the 16alpha-hydroxylation and a lower V(max) for the 21-hydroxylation than those for CYP2D6.1 . For dopamine formation from p-tyramine, the K(m) values for CYP2D6.2 and the R296C mutant were higher than those for CYP2D6.1, CYP2D6.10A, and CYP2D6.10C had a higher K(m) and a lower V(max) than the wild-type . The V(max)/K(m) values for CYP2D6.2, CYP2D6.10A, CYP2D6.10C and the P34S, G42R and R296C mutants were less than 45% of those for the wild-type . These results suggest the possibility that the polymorphism of CYP2D6, including CYP2D6*2, CYP2D6*10 and CYP2D6*12, might affect an individual behavior and the central nervous system through endogenous compounds, such as neuroactive steroids and tyramine, in the brain.

Dev Cell, 2004 Oct, 7(4), 457 - 8
The structure of an endosomal protein sorter; Slagsvold T et al.; Three "endosomal sorting complexes required for transport," ESCRT-I, -II, and -III, mediate sorting of ubiquitinated membrane proteins into intraluminal endosomal vesicles that are destined for degradation in lysosomes . Two recent reports, one in Nature and one in this issue of Developmental Cell, reveal the crystal structure of the yeast form of ESCRT-II.

Mol Cell, 2004 Oct 8, 16(1), 117 - 25
Hot spots for modulating toxicity identified by genomic phenotyping and localization mapping; Begley TJ et al.; DNA repair and checkpoint pathways protect against carcinogen-induced toxicity . Here, we describe additional, equally protective pathways discovered by interrogating 4,733 yeast proteins for their ability to diminish toxicity induced by four known carcinogens . A computational mapping strategy for global phenotypic data was developed to build a systems toxicology model detailing recovery from carcinogen exposure and identifying protein complexes that modulate toxicity . Global phenotypic data were merged with global subcellular localization and protein interactome data to generate an integrated picture of cellular recovery after carcinogen exposure . Statistically validated results from this systems-wide integration demonstrate that, in addition to the nucleus, subnetworks of toxicity-modulating proteins were overrepresented in the vacuolar membrane, endosome, endoplasmic reticulum, and mitochondrion . In addition, we show that many proteins associated with RNA polymerase II, macromolecular trafficking, and vacuole function can now be counted among the many proteins that modulate carcinogen-induced toxicity.

Oncogene, 2004 Nov 11, 23(53), 8597 - 602
Inactivation of the RRB1-Pescadillo pathway involved in ribosome biogenesis induces chromosomal instability; Killian A et al.; Since chromosomal instability (CIN) is a hallmark of most cancer cells, it is essential to identify genes whose alteration results into this genetic instability . Using a yeast CIN indicator strain, we show that inactivation of the YMR131c/RRB1 gene, which is involved in early ribosome assembly and whose expression is induced when the spindle checkpoint is activated, alters chromosome segregation and blocks mitosis at the metaphase/anaphase transition . We demonstrate that RRB1 interacts with YPH1 (yeast pescadillo homologue 1) and other members of the Yph1 complex, RPL3, ERB1 and ORC6, involved in ribosome biogenesis and DNA replication . Transient depletion of the human homologues GRWD, Pescadillo, Rpl3, Bop1 and Orc6L resulted in an increase of abnormal mitoses with appearance of binucleate or hyperploid cells, of cells with multipolar spindles and of aberrant metaphase plates . If deregulation of proteins involved in ribosome biogenesis, commonly observed in malignant tumors, could contribute to cancer through an aberrant protein synthesis, our study demonstrates that alteration of proteins linking ribosome biogenesis and DNA replication may directly cause CIN.

Oncogene, 2004 Nov 11, 23(53), 8611 - 8
Human SNM1B is required for normal cellular response to both DNA interstrand crosslink-inducing agents and ionizing radiation; Demuth I et al.; DNA interstrand crosslinks (ICLs) are critical lesions for the mammalian cell since they affect both DNA strands and block transcription and replication . The repair of ICLs in the mammalian cell involves components of different repair pathways such as nucleotide-excision repair and the double-strand break/homologous recombination repair pathways . However, the mechanistic details of mammalian ICL repair have not been fully delineated . We describe here the complete coding sequence and the genomic organization of hSNM1B, one of at least three human homologs of the Saccharomyces cerevisiae PSO2 gene . Depletion of hSNM1B by RNA interference rendered cells hypersensitive to ICL-inducing agents . This requirement for hSNM1B in the cellular response to ICL has been hypothesized before but never experimentally verified . In addition, siRNA knockdown of hSNM1B rendered cells sensitive to ionizing radiation, suggesting the possibility of hSNM1B involvement in homologous recombination repair of double-strand breaks arising as intermediates of ICL repair . Monoubiquitination of FANCD2, a key step in the FANC/BRCA pathway, is not affected in hSNM1B-depleted HeLa cells, indicating that hSNM1B is probably not a part of the Fanconi anemia core complex . Nonetheless, similarities in the phenotype of hSNM1B-depleted cells and cultured cells from patients suffering from Fanconi anemia make hSNM1B a candidate for one of the as yet unidentified Fanconi anemia genes not involved in monoubiquitination of FANCD2.

J Biol Chem, 2004 Dec 17, 279(51), 53892 - 8 Epub 2004 Oct 05.
A single PXY motif located within the carboxyl terminus of Spt23p and Mga2p mediates a physical and functional interaction with ubiquitin ligase Rsp5p; Shcherbik N et al.; Proteasome-dependent processing of the endoplasmic reticulum localized transcription factor Spt23p of Saccharomyces cerevisiae generates its transcriptionally competent form and requires the WW domain containing Rsp5p ubiquitin ligase . Although previous studies documented an Rsp5p-Spt23p association in cells, very little is known about the nature of this interaction . We report here the identification of an imperfect type I WW domain-binding site (LPKY) within the carboxyl-terminal region of Spt23p that is required for Rsp5p binding in vitro and in vivo . Deletion of this motif abrogates Rsp5p-induced ubiquitination of Spt23p in vitro and reduces ubiquitination of the Spt23p precursor in yeast . In addition, the Spt23pDeltaLPKY mutant is inefficiently processed and is defective at up-regulating target gene (OLE1) expression in cells . Deletion of the corresponding LPKY site within Mga2p, an Spt23p homologue, also abrogates Rsp5p binding and Rsp5p-dependent ubiquitination in vitro as well as Rsp5p binding and Mga2p polyubiquitination in cells . However, the Mga2pDeltaLPKY mutant undergoes efficient proteasome-dependent processing . These experiments indicate that the LPKY motif of Spt23p is required for Rsp5p binding, Rsp5-induced ubiquitination, proteasome-dependent processing, and its OLE1 inducing function . They also suggest that the LPKY motif of Mga2p is required for Rsp5p binding and ubiquitination, and Rsp5p regulates Mga2p function by a mechanism that is independent of providing the partial degradation signal.

J Biol Chem, 2004 Dec 17, 279(51), 53533 - 43 Epub 2004 Oct 05.
Functional regulation of FEZ1 by the U-box-type ubiquitin ligase E4B contributes to neuritogenesis; Okumura F et al.; E4B (also known as UFD2a) is a mammalian homolog of Saccharomyces cerevisiae Ufd2, which was originally described as a ubiquitin chain assembly factor (E4) . E4B is a U-box-type ubiquitin-protein isopeptide ligase (E3) and likely functions as either an E3 or an E4 . With a yeast two-hybrid screen, we have now identified FEZ1 (fasciculation and elongation protein zeta 1) as a protein that interacts with E4B . FEZ1 is implicated in neuritogenesis when phosphorylated by protein kinase Czeta (PKCzeta) . Interaction between E4B and FEZ1 in mammalian cells was enhanced by coexpression of constitutively active PKCzeta . E4B mediated the polyubiquitylation of FEZ1 but did not affect its intracellular stability, suggesting that such modification of FEZ1 is not a signal for its proteolysis . Polyubiquitylation of FEZ1 by E4B required Lys(27) of ubiquitin . Expression of a dominant-negative mutant of E4B in rat pheochromocytoma PC12 cells resulted in inhibition of neurite extension induced either by nerve growth factor or by coexpression of FEZ1 and constitutively active PKCzeta . These findings indicate that E4B serves as a ubiquitin ligase for FEZ1 and thereby regulates its function but not its degradation.

Appl Environ Microbiol, 2004 Oct, 70(10), 6157 - 65
Discrimination of modes of action of antifungal substances by use of metabolic footprinting; Allen J et al.; Diploid cells of Saccharomyces cerevisiae were grown under controlled conditions with a Bioscreen instrument, which permitted the essentially continuous registration of their growth via optical density measurements . Some cultures were exposed to concentrations of a number of antifungal substances with different targets or modes of action (sterol biosynthesis, respiratory chain, amino acid synthesis, and the uncoupler) . Culture supernatants were taken and analyzed for their "metabolic footprints" by using direct-injection mass spectrometry . Discriminant function analysis and hierarchical cluster analysis allowed these antifungal compounds to be distinguished and classified according to their modes of action . Genetic programming, a rule-evolving machine learning strategy, allowed respiratory inhibitors to be discriminated from others by using just two masses . Metabolic footprinting thus represents a rapid, convenient, and information-rich method for classifying the modes of action of antifungal substances.

Plant Physiol, 2004 Oct, 136(2), 3255 - 65 Epub 2004 Oct 01.
Biochemical characterization of the tobacco 42-kD protein kinase activated by osmotic stress; Kelner A et al.; In tobacco (Nicotiana tabacum), hyperosmotic stress induces rapid activation of a 42-kD protein kinase, referred to as Nicotiana tabacum osmotic stress-activated protein kinase (NtOSAK) . cDNA encoding the kinase was cloned and, based on the predicted amino acid sequence, the enzyme was assigned to the SNF1-related protein kinase type 2 (SnRK2) family . The identity of the enzyme was confirmed by immunoprecipitation of the active kinase from tobacco cells subjected to osmotic stress using antibodies raised against a peptide corresponding to the C-terminal sequence of the kinase predicted from the cloned cDNA . A detailed biochemical characterization of NtOSAK purified from stressed tobacco cells was performed . Our results show that NtOSAK is a calcium-independent Ser/Thr protein kinase . The sequence of putative phosphorylation sites recognized by NtOSAK, predicted by the computer program PREDIKIN, resembled the substrate consensus sequence defined for animal and yeast (Saccharomyces cerevisiae) AMPK/SNF1 kinases . Our experimental data confirmed these results, as various targets for AMPK/SNF1 kinases were also efficiently phosphorylated by NtOSAK . A range of protein kinase inhibitors was tested as potential modulators of NtOSAK, but only staurosporine, a rather nonspecific protein kinase inhibitor, was found to abolish the enzyme activity . In phosphorylation reactions, NtOSAK exhibited a preference for Mg(2+) over Mn(2+) ions and an inability to use GTP instead of ATP as a phosphate donor . The enzyme activity was not modulated by 5'-AMP . To our knowledge, these results represent the first detailed biochemical characterization of a kinase of the SnRK2 family.

Genes Dev, 2004 Oct 15, 18(20), 2491 - 505 Epub 2004 Oct 01.
A dynamic transcriptional network communicates growth potential to ribosome synthesis and critical cell size; Jorgensen P et al.; Cell-size homeostasis entails a fundamental balance between growth and division . The budding yeast Saccharomyces cerevisiae establishes this balance by enforcing growth to a critical cell size prior to cell cycle commitment (Start) in late G1 phase . Nutrients modulate the critical size threshold, such that cells are large in rich medium and small in poor medium . Here, we show that two potent negative regulators of Start, Sfp1 and Sch9, are activators of the ribosomal protein (RP) and ribosome biogenesis (Ribi) regulons, the transcriptional programs that dictate ribosome synthesis rate in accord with environmental and intracellular conditions . Sfp1 and Sch9 are required for carbon-source modulation of cell size and are regulated at the level of nuclear localization and abundance, respectively . Sfp1 nuclear concentration responds rapidly to nutrient and stress conditions and is regulated by the Ras/PKA and TOR signaling pathways . In turn, Sfp1 influences the nuclear localization of Fhl1 and Ifh1, which bind to RP gene promoters . Starvation or the absence of Sfp1 causes Fhl1 and Ifh1 to localize to nucleolar regions, concomitant with reduced RP gene transcription . These findings suggest that nutrient signals set the critical cell-size threshold via Sfp1 and Sch9-mediated control of ribosome biosynthetic rates.

Comp Biochem Physiol B Biochem Mol Biol, 2004 Oct, 139(2), 269 - 79
Characterization and comparison of fatty acyl Delta6 desaturase cDNAs from freshwater and marine teleost fish species; Zheng X et al.; Fish are the most important dietary source of the n-3 highly unsaturated fatty acids (HUFA), eicosapentaenoic (EPA) and docosahexaenoic acid (DHA), that have particularly important roles in human nutrition reflecting their roles in critical physiological processes . The objective of the study described here was to clone, functionally characterize and compare expressed fatty acid desaturase genes involved in the production of EPA and DHA in freshwater and marine teleost fish species . Putative fatty acid desaturase cDNAs were isolated and cloned from common carp (Cyprinus carpio) and turbot (Psetta maximus) . The enzymic activities of the products of these cDNAs, together with those of cDNAs previously cloned from rainbow trout (Oncorhynchus mykiss) and gilthead sea bream (Sparus aurata), were determined by heterologous expression in the yeast Saccharomyces cerevisiae . The carp and turbot desaturase cDNAs included open reading frames (ORFs) of 1335 and 1338 base pairs, respectively, specifying proteins of 444 and 445 amino acids . The protein sequences possessed all the characteristic features of microsomal fatty acid desaturases, including three histidine boxes, two transmembrane regions, and N-terminal cytochrome b(5) domains containing the haem-binding motif, HPGG . Functional expression showed all four fish cDNAs encode basically unifunctional Delta6 fatty acid desaturase enzymes responsible for the first and rate-limiting step in the biosynthesis of HUFA from 18:3n-3 and 18:2n-6 . All the fish desaturases were more active towards the n-3 substrate with 59.5%, 31.5%, 23.1% and 7.0% of 18:3n-3 being converted to 18:4n-3 in the case of turbot, trout, sea bream and carp, respectively . The enzymes also showed very low, probably physiologically insignificant, levels of Delta5 desaturase activity, but none of the products showed Delta4 desaturase activity . The cloning and characterization of desaturases from these fish is an important advance, as they are species in which there is a relative wealth of data on the nutritional regulation of fatty acid desaturation and HUFA synthesis, and between which substantive differences occur.

Biochem Biophys Res Commun, 2004 Nov 5, 324(1), 451 - 7
Metabolism of vitamin D by human microsomal CYP2R1; Shinkyo R et al.; The activation of vitamin D requires 25-hydroxylation in the liver and 1alpha-hydroxylation in the kidney . However, it remains unclear which enzyme is relevant to vitamin D 25-hydroxylation . Recently, human CYP2R1 has been reported to be a potential candidate for a hepatic vitamin D 25-hydroxylase . Thus, vitamin D metabolism by CYP2R1 was compared with human mitochondrial CYP27A1, which used to be considered a physiologically important vitamin D(3) 25-hydroxylase . A clear difference was observed between CYP2R1 and CYP27A1 in the metabolism of vitamin D(2) . CYP2R1 hydroxylated vitamin D(2) at the C-25 position while CYP27A1 hydroxylated it at positions C-24 and C-27 . The K(m) and k(cat) values for the CYP2R1-dependent 25-hydroxylation activity toward vitamin D(3) were 0.45microM and 0.97min(-1), respectively . The k(cat)/K(m) value of CYP2R1 was 26-fold higher than that of CYP27A1 . These results strongly suggest that CYP2R1 plays a physiologically important role in the vitamin D 25-hydroxylation in humans.

Arch Biochem Biophys, 2004 Nov 1, 431(1), 63 - 70
Heterologous expression and biophysical characterization of soluble oligosaccharyl transferase subunits; Dempski RE Jr et al.; Oligosaccharyl transferase (OT) catalyzes the first committed step in N-linked protein glycosylation, a co-translational process that occurs in the lumen of the endoplasmic reticulum . The yeast Saccharomyces cerevisiae enzyme complex comprises nine integral membrane proteins, five of which are essential for catalysis . Due to the challenges with purifying the active enzyme complex for detailed biophysical studies, a systematic study to express, isolate, and characterize the soluble domains of three of the largest subunits in the complex (Nlt1p, Wbp1p, and Swp1p) is reported . The proteins are expressed using the lytic baculovirus expression system and the new constructs are well behaved, monomeric in solution, and glycosylated . Two of the proteins interact with each other as seen by gel filtration and circular dichroism . This study provides a framework to study the roles of these three essential subunits of the eukaryotic OT complex.

Arzneimittelforschung, 2004, 54(8), 483 - 97
Expression, purification, biochemical and pharmacological characterization of a recombinant aprotinin variant; Apeler H et al.; Aprotinin (GAS 9087-70-1) is known as a potent inhibitor of serine proteases such as trypsin, plasmin, tissue and plasma kallikrein . In this study, an aprotinin variant was designed by means of rationale mutagenesis that differs from aprotinin by two amino acids in the active site and by seven amino acids in the backbone . The recombinant protein is expressed in a secretory yeast system enabling large scale production . A purification procedure was developed to yield high amounts of pure and correctly processed aprotinin variant . The changes in the active site of the aprotinin variant increase the potency towards inhibition of plasma kallikrein whereas the inhibition of plasmin is only marginally reduced . The net charge of the molecule is reduced from the basic (IP 10.5) to the neutral range (IP 5.6) . The recombinant aprotinin variant shows a decrease of immunogenicity in several models . No cross-reactivity with human and rabbit antibodies directed against aprotinin was observed both in in vivo and in ex vivo studies . In addition, the variant is more potent in a rat brain edema model of acute subdural hematoma compared to aprotinin.

Science, 2004 Oct 1, 306(5693), 117 - 20
Ubistatins inhibit proteasome-dependent degradation by binding the ubiquitin chain; Verma R et al.; To identify previously unknown small molecules that inhibit cell cycle machinery, we performed a chemical genetic screen in Xenopus extracts . One class of inhibitors, termed ubistatins, blocked cell cycle progression by inhibiting cyclin B proteolysis and inhibited degradation of ubiquitinated Sic1 by purified proteasomes . Ubistatins blocked the binding of ubiquitinated substrates to the proteasome by targeting the ubiquitin-ubiquitin interface of Lys(48)-linked chains . The same interface is recognized by ubiquitin-chain receptors of the proteasome, indicating that ubistatins act by disrupting a critical protein-protein interaction in the ubiquitin-proteasome system.

Nucleic Acids Res, 2004 Sep 30, 32(17), 5192 - 7 Print 2004.
DNA-binding domain of GCN4 induces bending of both the ATF/CREB and AP-1 binding sites of DNA; Dragan AI et al.; The interaction of proteins with DNA results, in some cases, in DNA bending, and this might have functional importance . However, when the protein-induced bending of DNA is small, its measurement presents a problem . It is shown that the fluorescence resonance energy transfer between fluorophores placed on the ends of the specially designed U-shaped DNA, which contains the DNA-binding sites at its central part, can be successfully used for this purpose . The lever effect of the arms of such U-shaped DNA ensures that the distance between the fluorophores is very sensitive to bending of the central part . Using this technique, it was shown that (i) the AP-1 and ATF/CREB binding sites of GCN4 transcription factor are pre-bent to the same extent (approximately 12 degrees toward the major groove) and (ii) binding of the GCN4 DNA-binding domain (GCN4-bZIP) results in additional bending of both these target sites but to a greater extent at the ATF/CREB site . In total, in the complex with GCN4-bZIP, the ATF/CREB site is bent by (25 +/- 2) degrees and the AP-1 site by (20 +/- 2) degrees toward the minor groove.

J Biol Chem, 2004 Dec 3, 279(49), 51022 - 32 Epub 2004 Dec 3.
Cytoplasmic inositol hexakisphosphate production is sufficient for mediating the Gle1-mRNA export pathway; Miller AL et al.; Production of inositol hexakisphosphate (IP6) by Ipk1, the inositol-1,3,4,5,6-pentakisphosphate 2-kinase, is required for Gle1-mediated mRNA export in Saccharomyces cerevisiae cells . To examine the network of interactions that require IP6 production, an analysis of fitness defects was conducted in mutants harboring both an ipk1 null allele and a mutant allele in genes encoding nucleoporins or transport factors . Enhanced lethality was observed with a specific subset of mutants, including nup42, nup116, nup159, dbp5, and gle2, all of which had been previously connected to Gle1 function . Complementation of the nup116Deltaipk1Delta and nup42Deltaipk1Delta double mutants did not require the Phe-Gly repeat domains in the respective nucleoporins, suggesting that IP6 was acting subsequent to heterogeneous nuclear ribonucleoprotein targeting to the nuclear pore complex . With Nup42 and Nup159 localized exclusively to the nuclear pore complex cytoplasmic side, we speculated that IP6 may regulate a cytoplasmic step in mRNA export . To test this prediction, the spatial requirements for the production of IP6 were investigated . Restriction of Ipk1 to the cytoplasm did not block IP6 production . Moreover, coincident sequestering of both Ipk1 and Mss4 (an enzyme required for phosphatidylinositol 4,5-bisphosphate production) to the cytoplasm also did not block IP6 production . Given that the kinase required for inositol 1,3,4,5,6-pentakisphosphate production (Ipk2) is localized in the nucleus, these results indicated that soluble inositides were diffusing between the nucleus and the cytoplasm . Additionally, the cytoplasmic production of IP6 by plasma membrane-anchored Ipk1 rescued a gle1-2 ipk1-4 synthetic lethal mutant . Thus, cytoplasmic IP6 production is sufficient for mediating the Gle1-mRNA export pathway.

Neuropharmacology, 2004 Oct, 47(5), 724 - 33
The complexity of PDZ domain-mediated interactions at glutamatergic synapses: a case study on neuroligin; Meyer G et al.; The postsynaptic specialisation at glutamatergic synapses is composed of a network of proteins located within the membrane and the underlying postsynaptic density . The strong interconnectivity between the protein components is mediated by a limited number of interaction modes . Particularly abundant are PDZ domain-mediated interactions . An obstacle in understanding the fidelity of postsynaptic processes involving PDZ domains is the high degree of overlap with respect to their binding specificities . Focussing on transsynaptic adhesion molecules, we used the yeast two-hybrid system to obtain an overview of the binding specificities of selected C-terminal PDZ binding motifs . Neuroligin, a postsynaptic cell surface protein that spans the synaptic cleft and interacts with beta-neurexin, served as a starting point . Neuroligin binds to the PDZ domain-containing proteins PSD95, SAP102, Chapsyn110, S-SCAM, Magi1 and 3, Shank1 and 3, Pick1, GOPC, SPAR, Semcap3 and PDZ-RGS3 . Next, we examined the relationship between neuroligin and synaptic cell adhesion molecules or glutamate receptor subunits with respect to PDZ-mediated interactions . We found a limited overlap in the PDZ-domain binding specificities of neuroligin with those of Sidekick2 and Ephrin-B2 . In contrast, Syndecan2 and IgSF4 show no overlap with the PDZ-domain specificity of neuroligin, instead, they bind to GRIP and syntenin . The AMPA receptor subunit GluR2 interacts with Semcap3 and PDZ-RGS3, whereas the kainate receptor subunits GluR5 and GluR6 show weak interactions with PSD95 . In summary, we can sketch a complex pattern of overlap in the binding specificities of synaptic cell surface proteins towards PDZ-domain proteins.

Curr Biol, 2004 Oct 5, 14(19), 1703 - 11
Distribution and dynamics of chromatin modification induced by a defined DNA double-strand break; Shroff R et al.; BACKGROUND: In response to DNA double-strand breaks (DSBs), eukaryotic cells rapidly phosphorylate histone H2A isoform H2AX at a C-terminal serine (to form gamma-H2AX) and accumulate repair proteins at or near DSBs . To date, these events have been defined primarily at the resolution of light microscopes, and the relationship between gamma-H2AX formation and repair protein recruitment remains to be defined . RESULTS: We report here the first molecular-level characterization of regional chromatin changes that accompany a DSB formed by the HO endonuclease in Saccharomyces cerevisiae . Break induction provoked rapid gamma-H2AX formation and equally rapid recruitment of the Mre11 repair protein . gamma-H2AX formation was efficiently promoted by both Tel1p and Mec1p, the yeast ATM and ATR homologs; in G1-arrested cells, most gamma-H2AX formation was dependent on Tel1 and Mre11 . gamma-H2AX formed in a large (ca . 50 kb) region surrounding the DSB . Remarkably, very little gamma-H2AX could be detected in chromatin within 1-2 kb of the break . In contrast, this region contains almost all the Mre11p and other repair proteins that bind as a result of the break . CONCLUSIONS: Both Mec1p and Tel1p can respond to a DSB, with distinct roles for these checkpoint kinases at different phases of the cell cycle . Part of this response involves histone phosphorylation over large chromosomal domains; however, the distinct distributions of gamma-H2AX and repair proteins near DSBs indicate that localization of repair proteins to breaks is not likely to be the main function of this histone modification.

Mol Cell Biol, 2004 Oct, 24(20), 8994 - 9005
Regulation and recognition of SCFGrr1 targets in the glucose and amino acid signaling pathways; Spielewoy N et al.; SCFGrr1, one of several members of the SCF family of E3 ubiquitin ligases in budding Saccharomyces cerevisiae, is required for both regulation of the cell cycle and nutritionally controlled transcription . In addition to its role in degradation of Gic2 and the CDK targets Cln1 and Cln2, Grr1 is also required for induction of glucose- and amino acid-regulated genes . Induction of HXT genes by glucose requires the Grr1-dependent degradation of Mth1 . We show that Mth1 is ubiquitinated in vivo and degraded via the proteasome . Furthermore, phosphorylated Mth1, targeted by the casein kinases Yck1/2, binds to Grr1 . That binding depends upon the Grr1 leucine-rich repeat (LRR) domain but not upon the F-box or basic residues within the LRR that are required for recognition of Cln2 and Gic2 . Those observations extend to a large number of Grr1-dependent genes, some targets of the amino acid-regulated SPS signaling system, which are properly regulated in the absence of those basic LRR residues . Finally, we show that regulation of the SPS targets requires the Yck1/2 casein kinases . We propose that casein kinase I plays a similar role in both nutritional signaling pathways by phosphorylating pathway components and targeting them for ubiquitination by SCFGrr1.

Mol Cell Biol, 2004 Oct, 24(20), 8938 - 50
The interaction between Sgt1p and Skp1p is regulated by HSP90 chaperones and is required for proper CBF3 assembly; Lingelbach LB et al.; Sgt1p is a well-conserved protein proposed to be involved in a number of cellular processes . Genetic studies of budding yeast suggest a role for SGT1 in signal transduction, cell cycle advance, and chromosome segregation . Recent evidence has linked Sgt1p to HSP90 chaperones, although the precise relationship between these proteins is unclear . To further explore the role of Sgt1p in these processes, we have characterized the interactions among Sgt1p, the inner kinetochore complex CBF3, and HSP90 chaperones . We show that the amino terminus of Sgt1p interacts with CBF3 subunits Skp1p and Ctf13p . HSP90 interacts with Sgt1p and, in combination with the carboxy terminus of Sgt1p, regulates the interaction between Sgt1p and Skp1p in a nucleotide-dependent manner . While the Sgt1p-Skp1p interaction is required for CBF3 assembly, mutations that stabilize this interaction prevent the turnover of protein complexes important for CBF3 assembly . We propose that HSP90 and Sgt1p act together as a molecular switch, maintaining transient interactions required to balance protein complex assembly with turnover.

Mol Cell Biol, 2004 Oct, 24(20), 8895 - 906
Functional similarity between the peroxisomal PTS2 receptor binding protein Pex18p and the N-terminal half of the PTS1 receptor Pex5p; Schafer A et al.; Within the extended receptor cycle of peroxisomal matrix import, the function of the import receptor Pex5p comprises cargo recognition and transport . While the C-terminal half (Pex5p-C) is responsible for PTS1 binding, the contribution of the N-terminal half of Pex5p (Pex5p-N) to the receptor cycle has been less clear . Here we demonstrate, using different techniques, that in Saccharomyces cerevisiae Pex5p-N alone facilitates the import of the major matrix protein Fox1p . This finding suggests that Pex5p-N is sufficient for receptor docking and cargo transport into peroxisomes . Moreover, we found that Pex5p-N can be functionally replaced by Pex18p, one of two auxiliary proteins of the PTS2 import pathway . A chimeric protein consisting of Pex18p (without its Pex7p binding site) fused to Pex5p-C is able to partially restore PTS1 protein import in a PEX5 deletion strain . On the basis of these results, we propose that the auxiliary proteins of the PTS2 import pathway fulfill roles similar to those of the N-terminal half of Pex5p in the PTS1 import pathway.

J Biol Chem, 2004 Dec 3, 279(49), 50895 - 903 Epub 2004 Dec 3.
The mode of action of centrin . Binding of Ca2+ and a peptide fragment of Kar1p to the C-terminal domain; Hu H et al.; Centrin is an EF-hand calcium-binding protein closely related to the prototypical calcium sensor protein calmodulin . It is found in microtubule-organizing centers of organisms ranging from algae and yeast to man . In vitro, the C-terminal domain of centrin binds to the yeast centrosomal protein Kar1p in a calcium-dependent manner, whereas the N-terminal domain does not show any appreciable affinity for Kar1p . To obtain deeper insights into the structural basis for centrin's function, we have characterized the affinities of the C-terminal domain of Chlamydomonas reinhardtii centrin for calcium and for a peptide fragment of Kar1p using CD, fluorescence, and NMR spectroscopy . Calcium binding site IV in C . reinhardtii centrin was found to bind Ca2+ approximately 100-fold more strongly than site III . In the absence of Ca2+, the protein occupies a mixture of closed conformations . Binding of a single ion in site IV is sufficient to radically alter the conformational equilibrium, promoting occupancy of an open conformation . However, an exchange between closed and open conformations remains even at saturating levels of Ca2+ . The population of the open conformation is substantially stabilized by the presence of the target peptide Kar1p-(239-257) to a point where a single ion bound in site IV is sufficient to completely shift the conformational equilibrium to the open conformation . This is reflected in the enhancement of the Ca2+ affinity in this site by more than an order of magnitude . These data confirm the direct coupling of the Ca2+ binding-induced shift in the equilibrium between the closed and open conformations to the binding of the peptide . Combined with the common localization of the two proteins in the microtubule organizing center, our results suggest that centrin is constitutively bound to Kar1p through its C-terminal domain and that centrin's calcium sensor activities are mediated by the N-terminal domain.

J Mol Biol, 2004 Oct 15, 343(2), 445 - 55
A domain in the N-terminal part of Hsp26 is essential for chaperone function and oligomerization; Haslbeck M et al.; Small heat-shock proteins (Hsps) are ubiquitous molecular chaperones which prevent the unspecific aggregation of non-native proteins . For Hsp26, a cytosolic sHsp from of Saccharomyces cerevisiae, it has been shown that, at elevated temperatures, the 24 subunit complex dissociates into dimers . This dissociation is required for the efficient interaction with non-native proteins . Deletion analysis of the protein showed that the N-terminal half of Hsp26 (amino acid residues 1-95) is required for the assembly of the oligomer . Limited proteolysis in combination with mass spectrometry suggested that this region can be divided in two parts, an N-terminal segment including amino acid residues 1-30 and a second part ranging from residues 31-95 . To analyze the structure and function of the N-terminal part of Hsp26 we created a deletion mutant lacking amino acid residues 1-30 . We show that the oligomeric state and the structure, as determined by size exclusion chromatography and electron microscopy, corresponds to that of the Hsp26 wild-type protein . Furthermore, this truncated version of Hsp26 is active as a chaperone . However, in contrast to full length Hsp26, the truncated version dissociates at lower temperatures and complexes with non-native proteins are less stable than those found with wild-type Hsp26 . Our results suggest that the N-terminal segment of Hsp26 is involved in both, oligomerization and chaperone function and that the second part of the N-terminal region (amino acid residues 31-95) is essential for both functions.

J Mol Biol, 2004 Oct 15, 343(2), 361 - 70
Ubiquitin-dependent degradation of Id1 and Id3 is mediated by the COP9 signalosome; Berse M et al.; Recently, evidence is accumulating pointing to a function of the COP9 signalosome (CSN) in regulation of ubiquitination by specific ubiquitin ligases . Here, we demonstrate by mammalian two-hybrid analysis that the transcriptional regulators and substrates of the ubiquitin system Id1 and Id3, but not Id2 and Id4, bind to the CSN subunit CSN5 . Pull-down experiments revealed that Id3 physically interacts with the CSN complex . Additional far Western and pull-down studies with Id3 support our two-hybrid data and show that the transcription regulator can bind to CSN5 and CSN7 . Recombinant Id3 is not phosphorylated by the CSN-associated kinases CK2 and PKD . However, it inhibits c-Jun and CSN2 phosphorylation by the isolated CSN complex and by the recombinant CK2 . The inhibitors of CSN associated kinases, curcumin and emodin, significantly induce ubiquitination and proteasome-dependent degradation of transiently expressed Id3 in HeLa cells . Proteasome-dependent degradation of endogenous Id1 in HeLa cells is also stimulated by treatment with curcumin or emodin . Ubiquitination of Id3 is shown directly by cotransfection of HeLa cells with Id3 and His-ubiquitin cDNA . Curcumin increased Id3-ubiquitin conjugate formation, as shown by Western blotting and His-pull-downs . In addition, overexpression of CSN2 leads to stabilization of Id3 protein . On the basis of these data, it is speculated that CSN-mediated phosphorylation inhibits ubiquitination of Id1 and Id3.

Trends Biochem Sci, 2004 Oct, 29(10), 556 - 64
The eukaryotic plasma membrane as a nutrient-sensing device; Holsbeeks I et al.; In eukaryotic cells, G-protein-coupled receptors (GPCRs), non-transporting nutrient carrier homologues and active nutrient carriers have been recently shown to function as sensors that directly monitor the level of nutrients in the extracellular environment . The plasma membrane is not only the cellular boundary at which signalling molecules that govern metabolism and proliferation are detected, but also the boundary across which nutrients that sustain the generation of energy and building blocks are transported . Nutrient sensors combine these functions in various ways . Classical receptor proteins detect the presence of nutrients, carriers combine the functions of nutrient transporters and receptors, and carrier homologues have lost their transport capacity and become pure receptors . The activation of signal transduction pathways by nutrients adds a new layer to the regulatory network that controls metabolism and proliferation . Nutrient sensors highlight the importance of both nutrients as signalling molecules and nutrient carriers as receptors for signalling pathways.

Trends Biochem Sci, 2004 Oct, 29(10), 516 - 9
Disulfide relays between and within proteins: the Ero1p structure; Hiniker A et al.; The essential flavoenzyme Ero1p both creates de novo disulfide bonds and transfers these disulfides to the folding catalyst protein disulfide isomerase (PDI) . The recently solved crystal structure of Ero1p, in combination with previous biochemical, genetic and structural data, provides insight into the mechanism by which Ero1p accomplishes these tasks . A comparison of Ero1p with the smaller flavoenzyme Erv2p highlights important structural elements that are shared by these flavin adenine dinucleotide (FAD)-binding sulfhydryl oxidases and suggests some general themes that might be common to proteins that generate disulfide bonds.

Mutat Res, 2004 Oct 4, 554(1-2), 53 - 65
In vitro chemopreventive activity of Camellia sinensis, Ilex paraguariensis and Ardisia compressa tea extracts and selected polyphenols; Ramirez-Mares MV et al.; Several herbal teas contain bioactive compounds that have been associated with a lower risk of chronic diseases including cancer . The aim of this study was to evaluate the chemopreventive activity of tea aqueous extracts and selected constituent pure polyphenols using a battery of in vitro marker systems relevant for the prevention of cancer . The effects of (-) epigallocatechin gallate (EGCG), quercetin (Q), gallic acid (GA), green tea (GT, Camellia sinensis), ardisia tea (AT, Ardisia compressa) and mate tea (MT, Ilex paraguariensis) extracts were tested . Cytotoxicity, TPA-induced ornithine decarboxylase (ODC) and quinone reductase (QR) activities were evaluated in vitro using HepG2 cells . The topoisomerase inhibitory activity was also tested, using the Saccharomyces cerevisiae yeast system . Results suggest that MT, AT and GT are cytotoxic to the HepG2 cells, with MT demonstrating dominant cytotoxicity . EGCG showed greater cytotoxicity than Q and GA against HepG2 cells . The greatest inhibition (82%) of TPA-induced ODC activity was shown by Q, with 25 microM (IC50 = 11.90 microM) . Topoisomerase II, but not topoisomerase I, was the cellular target of MT, AT, EGCG, Q and GA, which acted mainly as true catalytic inhibitors . The cytotoxic activity and the inhibition of topoisomerase II may contribute to the overall chemopreventive activity of AT and MT extracts . Ardisia and mate teas may thus share a public health potential as chemopreventive agents.

FEMS Yeast Res, 2004 Sep, 4(8), 815 - 20
DER7, encoding alpha-glucosidase I is essential for degradation of malfolded glycoproteins of the endoplasmic reticulum; Hitt R et al.; Proteins entering the endoplasmic reticulum (ER) have to acquire an export-competent structure before they are delivered to their final destination . This folding process is monitored by an ER protein quality control system . Folding-incompetent conformers are eliminated via a mechanism called ER-associated protein degradation (ERAD) . Genetic studies in the yeast Saccharomyces cerevisiae have revealed that carbohydrate modification plays a crucial role in these processes . Here we show that a previously isolated der mutant (der7-1) is defective in ERAD . We identify DER7 as the gene encoding N-glycan-processing alpha-glucosidase I (EC 3.2.1.106) of the ER and demonstrate that its inactivity, due to a substitution of the conserved glycine residue at position 725 by arginine (G725R) in the der7-1 mutant, leads to ER-stress.

Yeast, 2004 Sep, 21(12), 1021 - 3
Expression of the expanded YFL007w ORF and assignment of the gene name BLM10; Doherty K et al.; This report assigns a gene name to an extended ORF in Saccharomyces cerevisiae, and provides the first evidence from Northern analyses that the ORF is expressed and the transcript is the predicted size . Copyright (c) 2004 John Wiley & Sons, Ltd.

Nat Cell Biol, 2004 Oct, 6(10), 997 - 1002 Epub 2004 Sep 19.
Hog1 mediates cell-cycle arrest in G1 phase by the dual targeting of Sic1; Escote X et al.; Activation of stress-activated protein kinases (SAPKs) is essential for proper cell adaptation to extracellular stimuli . The exposure of yeast cells to high osmolarity, or mutations that lead to activation of the Hog1 SAPK, result in cell-cycle arrest . The mechanisms by which Hog1 and SAPKs in general regulate cell-cycle progression are not completely understood . Here we show that Hog1 regulates cell cycle progression at the G1 phase by a dual mechanism that involves downregulation of cyclin expression and direct targeting of the CDK-inhibitor protein Sic1 . Hog1 interacts physically with Sic1 in vivo and in vitro, and phosphorylates a single residue at the carboxyl terminus of Sic1, which, in combination with the downregulation of cyclin expression, results in Sic1 stabilization and inhibition of cell-cycle progression . Cells lacking Sic1 or containing a Sic1 allele mutated in the Hog1 phosphorylation site are unable to arrest at G1 phase after Hog1 activation, and become sensitive to osmostress . Together, our data indicate that the Sic1 CDK-inhibitor is the molecular target for the SAPK Hog1 that is required to modulate cell-cycle progression in response to stress.

Science, 2004 Sep 24, 305(5692), 1968 - 71
Nitric oxide represses the Arabidopsis floral transition; He Y et al.; The correct timing of flowering is essential for plants to maximize reproductive success and is controlled by environmental and endogenous signals . We report that nitric oxide (NO) repressed the floral transition in Arabidopsis thaliana . Plants treated with NO, as well as a mutant overproducing NO (nox1), flowered late, whereas a mutant producing less NO (nos1) flowered early . NO suppressed CONSTANS and GIGANTEA gene expression and enhanced FLOWERING LOCUS C expression, which indicated that NO regulates the photoperiod and autonomous pathways . Because NO is induced by environmental stimuli and constitutively produced, it may integrate both external and internal cues into the floral decision.

Nucleic Acids Res, 2004 Sep 24, 32(17), 5029 - 35 Print 2004.
Comparative analysis of complete genomes reveals gene loss, acquisition and acceleration of evolutionary rates in Metazoa, suggests a prevalence of evolution via gene acquisition and indicates that the evolutionary rates in animals tend to be conserved; Babenko VN et al.; In this study we systematically examined the differences between the proteomes of Metazoa and other eukaryotes . Metazoans (Homo sapiens, Ceanorhabditis elegans and Drosophila melanogaster) were compared with a plant (Arabidopsis thaliana), fungi (Saccharomyces cerevisiae and Schizosaccaromyces pombe) and Encephalitozoan cuniculi . We identified 159 gene families that were probably lost in the Metazoan branch and 1263 orthologous families that were specific to Metazoa and were likely to have originated in their last common ancestor (LCA) . We analyzed the evolutionary rates of pan-eukaryotic protein families and identified those with higher rates in animals . The acceleration was shown to occur in: (i) the LCA of Metazoa or (ii) independently in the Metazoan phyla . A high proportion of the accelerated Metazoan protein families was found to participate in translation and ribosome biogenesis, particularly mitochondrial . By functional analysis we show that no metabolic pathway in animals evolved faster than in other organisms . We conclude that evolution in the LCA of Metazoa was extensive and proceeded largely by gene duplication and/or invention rather than by modification of extant proteins . Finally, we show that the rate of evolution of a gene family in animals has a clear, but not absolute, tendency to be conserved.

J Biol Chem, 2004 Dec 3, 279(49), 50840 - 9 Epub 2004 Dec 3.
Dna2p helicase/nuclease is a tracking protein, like FEN1, for flap cleavage during Okazaki fragment maturation; Kao HI et al.; During cellular DNA replication the lagging strand is generated as discontinuous segments called Okazaki fragments . Each contains an initiator RNA primer that is removed prior to joining of the strands . Primer removal in eukaryotes requires displacement of the primer into a flap that is cleaved off by flap endonuclease 1 (FEN1) . FEN1 employs a unique tracking mechanism that requires the recognition of the free 5' terminus and then movement to the base of the flap for cleavage . Abnormally long flaps are coated by replication protein A (RPA), inhibiting FEN1 cleavage . A second nuclease, Dna2p, is needed to cleave an RPA-coated flap producing a short RPA-free flap, favored by FEN1 . Here we show that Dna2p is also a tracking protein . Annealed primers or conjugated biotin-streptavidin complex block Dna2p entry and movement . Single-stranded binding protein-coated flaps inhibit Dna2p cleavage . Like FEN1, Dna2p can track over substrates with a non-Watson Crick base, such as a biotin, or a missing base within a chain . Unlike FEN1, Dna2p shows evidence of a "threading-like" mechanism that does not support tracking over a branched substrate . We propose that the two nucleases both track, Dna2p first and then FEN1, to remove initiator RNA via long flap intermediates.

J Cell Physiol, 2005 Jan, 202(1), 49 - 66
Interactions among IQGAP1, Cdc42, and the cadherin/catenin protein complex regulate Sertoli-germ cell adherens junction dynamics in the testis; Lui WY et al.; The movement of developing germ cells across the seminiferous epithelium during spermatogenesis involves extensive adherens junction (AJ) restructuring between Sertoli cells, as well as between Sertoli and germ cells . In this report, we show that the intricate interactions between Cdc42 (a Rho family protein of Mr approximately 23 kDa originally identified in membranes of human platelets and placenta, and is the homolog of CDC42Sc, which is known to regulate of bud-site assembly in Saccharomyces cerevisiae) and its effector, IQ motif containing GTPase activating protein (IQGAP1, Mr approximately 189 kDa, it is also an actin-binding protein known to interact with Cdc42 and Rac1 GTPases), regulate Sertoli-germ cell, but not Sertoli-Sertoli cell, AJ dynamics . Using testis lysates for immunoprecipitation (IP), IQGAP1 was shown to associate with E-cadherin, N-cadherin, and beta-catenin (but not beta1-integrin and nectin-2), as well as with actin and vimentin (but not alpha-tubulin) . Moreover, IQGAP1 was found to localize to the periphery of both Sertoli and germ cells in the seminiferous epithelium, at sites of cell-cell contacts . Using fluorescent microscopy with dual fluorescent probes, IQGAP1 was found to co-localize, at least in part, with N-cadherin in the seminiferous epithelium consistent with their localization at the basal and apical ES . Using Sertoli-germ cell cocultures, it was demonstrated that AJ assembly associated with a transient induction of Cdc42 and IQGAP1, which was not found when Sertoli cells were cultured alone . Lastly, a shift in the interactions of Cdc42, IQGAP1, beta-catenin, and N-cadherin was detected in Sertoli-germ cell cocultures using an Ca2+-induced AJ disruption model, which was used to examine AJ disassembly and its reassembly . In the presence of Ca2+, IQGAP1 bound preferentially to Cdc42 rather than to beta-catenin . However, when Ca2+ was depleted from cocultures using EGTA, a Ca2+ chelating agent, IQGAP1 lost its affinity for Cdc42 and became tightly associated with beta-catenin, destabilizing cadherin-mediated AJs between Sertoli and germ cells . Yet this shift of protein-protein interaction was not detected in Sertoli cells cultured alone . These results illustrate that the interactions among IQGAP1, Cdc42, and beta-catenin are crucial to the regulation of Sertoli-germ cell, but not Sertoli-Sertoli cell, AJ dynamics in the seminiferous epithelium . 2005 Wiley-Liss, Inc.

RNA, 2004 Nov, 10(11), 1698 - 701 Epub 2004 Sep 23.
PIN domain of Nob1p is required for D-site cleavage in 20S pre-rRNA; Fatica A et al.; Nob1p (Yor056c) is essential for processing of the 20S pre-rRNA to the mature 18S rRNA . It is part of a pre-40S ribosomal particle that is transported to the cytoplasm and subsequently cleaved at the 3' end of mature 18S rRNA (D-site) . Nob1p is also reported to participate in proteasome biogenesis, and it was therefore unclear whether its primary activity is in ribosome synthesis . In this work, we describe a homology model of the PIN domain of Nob1p, which structurally mimics Mg(2+)-dependent exonucleases despite negligible similarity in primary sequence . Insights gained from this model were used to design a point mutation that was predicted to abolish the postulated enzymatic activity . Cells expressing Nob1p with this mutation failed to cleave the 20S pre-rRNA . This supports both the significance of the structural model and the idea that Nob1p is the long-sought D-site endonuclease.

RNA, 2004 Nov, 10(11), 1704 - 12 Epub 2004 Sep 23.
Cbf5p, the putative pseudouridine synthase of H/ACA-type snoRNPs, can form a complex with Gar1p and Nop10p in absence of Nhp2p and box H/ACA snoRNAs; Henras AK et al.; Box C/D and box H/ACA small ribonucleoprotein particles (sRNPs) are found from archaea to humans, and some of these play key roles during the biogenesis of ribosomes or components of the splicing apparatus . The protein composition of the core of both types of particles is well established and the assembly pathway of box C/D sRNPs has been extensively investigated both in archaeal and eukaryotic systems . In contrast, knowledge concerning the mode of assembly and final structure of box H/ACA sRNPs is much more limited . In the present study, we have investigated the protein/protein interactions taking place between the four protein components of yeast box H/ACA small nucleolar RNPs (snoRNPs), Cbf5p, Gar1p, Nhp2p, and Nop10p . We provide evidence that Cbf5p, Gar1p, and Nop10p can form a complex devoid of Nhp2p and small nucleolar RNA (snoRNA) components of the particles and that Cbf5p and Nop10p can directly bind to each other . We also show that the absence of any component necessary for assembly of box H/ACA snoRNPs inhibits accumulation of Cbf5p, Gar1p, or Nop10p, whereas Nhp2p levels are little affected.

Antimicrob Agents Chemother, 2004 Oct, 48(10), 3773 - 81
Azole resistance in Candida glabrata: coordinate upregulation of multidrug transporters and evidence for a Pdr1-like transcription factor; Vermitsky JP et al.; Candida glabrata has emerged as a common cause of fungal infection . This yeast has intrinsically low susceptibility to azole antifungals such as fluconazole, and mutation to frank azole resistance during treatment has been documented . Potential resistance mechanisms include changes in expression or sequence of ERG11 encoding the azole target . Alternatively, resistance could result from upregulated expression of multidrug transporter genes; in C . glabrata these include CDR1 and PDH1 . By RNA hybridization, 10 of 12 azole-resistant clinical isolates showed 6- to 15-fold upregulation of CDR1 compared to susceptible strains . In 4 of these 10 isolates PDH1 was similarly upregulated, and in the remainder it was upregulated three- to fivefold, while ERG11 expression was minimally changed . Laboratory mutants were selected on fluconazole-containing medium with glycerol as carbon source (to eliminate mitochondrial mutants) . Similar to the clinical isolates, six of seven laboratory mutants showed unchanged ERG11 expression but coordinate CDR1-PDH1 upregulation ranging from 2- to 20-fold . Effects of antifungal treatment on gene expression in susceptible C . glabrata strains were also studied: azole exposure induced CDR1-PDH1 expression 4- to 12-fold . These findings suggest that these transporter genes are regulated by a common mechanism . In support of this, a mutation associated with laboratory resistance was identified in the C . glabrata homolog of PDR1 which encodes a regulator of multidrug transporter genes in Saccharomyces cerevisiae . The mutation falls within a putative activation domain and was associated with PDR1 autoupregulation . Additional regulatory factors remain to be identified, as indicated by the lack of PDR1 mutation in a clinical isolate with coordinately upregulated CDR1-PDH1.

Antimicrob Agents Chemother, 2004 Oct, 48(10), 3711 - 4
In vitro activities of 2,4-diaminoquinazoline and 2,4-diaminopteridine derivatives against Plasmodium falciparum; Ommeh S et al.; The activities of 28 6-substituted 2,4-diaminoquinazolines, 2,4-diamino-5,6,7,8-tetrahydroquinazolines, and 2,4-diaminopteridines against Plasmodium falciparum were tested . The 50% inhibitory concentrations (IC(50)s) of six compounds were <50 nM, and the most potent compound was 2,4-diamino-5-chloro-6-{N-(2,5-dimethoxybenzyl)amino}quinazoline (compound 1), with an IC(50) of 9 nM . The activity of compound 1 was potentiated by the dihydropteroate synthase inhibitor dapsone, an indication that these compounds are inhibitors of dihydrofolate reductase . Further studies are warranted to assess the therapeutic potential of this combination in vivo.

J Biol Chem, 2004 Nov 26, 279(48), 50243 - 9 Epub 2004 Nov 26.
Zim17, a novel zinc finger protein essential for protein import into mitochondria; Burri L et al.; Translocation of precursor proteins across the mitochondrial membranes requires the coordinated action of multisubunit translocases in the outer and inner membrane, and the driving force for translocation across the inner membrane is provided by the matrix-located heat shock protein 70 (mtHsp70) . The central components of the protein import machinery are essential . Here we describe Zim17, an essential protein with a zinc finger motif involved in protein import into mitochondria . Comparative genomics suggested a correction to the open reading frame of YNL310c, the gene encoding Zim17 in Saccharomyces cerevisiae . The revised open reading frame codes for a classic mitochondrial targeting signal, which is processed from Zim17 in the mitochondrial matrix . Loss of Zim17 selectively diminishes import of proteins into the matrix of mitochondria, but this loss of Zim17 is partially suppressed by overexpression of the J-protein Pam18/Tim14 . We propose that Zim17 functions as an example of a "fractured" J-protein, where a protein like Zim17 contributes a zinc finger domain to Type III J-proteins, in toto providing for substrate loading onto Hsp70.

Mol Biochem Parasitol, 2004 Oct, 137(2), 253 - 65
TbRAB1 and TbRAB2 mediate trafficking through the early secretory pathway of Trypanosoma brucei; Dhir V et al.; The African trypanosome possesses a total of 16 small GTPases of the Rab family, which are involved in control of various membrane transport events . Recently the roles of these proteins in the endocytosis and recycling of the major surface antigen of the bloodstream form, the variant surface glycoprotein (VSG), have been described but little has been reported on the roles of Rab proteins in exocytic pathways in trypanosomatids . Whilst phylogenetic analysis based on sequence similarity indicates a comparatively well conserved core set of Rab proteins, the evolutionary distance of the trypanosome lineage from crown eukaryote model systems requires direct experimental evidence to support these sequence data . By database searching we identified two further Rab genes, TbRAB1 and TbRAB2, which are the trypanosome sequence orthologues of mammalian Rab1 and Rab2, important mediators of ER to Golgi and intra-Golgi transport processes . A remarkably high level of sequence conservation is retained between the trypanosome and higher eukaryote orthologues . By immunolocalisation we find that both TbRAB1 and TbRAB2 reside on membranes in intimate association with the Golgi complex . By heterologous expression in mammalian cells we also demonstrate conservation of targeting information in the TbRAB1 and TbRAB2 proteins, whilst TbRAB1, but not TbRAB2, can complement a Ypt1(ts) conditional mutant in Saccharomyces cerevisiae . The roles of TbRAB1 and TbRAB2 in exocytosis were examined using RNAi . Suppression of TbRAB1 or TbRAB2 was strongly inhibitory to growth and most importantly both TbRAB1 and TbRAB2 were required for normal progression of VSG through the early secretory pathway . These data indicate conservation of function for these proteins between trypanosomes and crown eukaryotes.

Mol Cell, 2004 Sep 24, 15(6), 951 - 64
Structure and stability of cohesin's Smc1-kleisin interaction; Haering CH et al.; A multisubunit complex called cohesin forms a huge ring structure that mediates sister chromatid cohesion, possibly by entrapping sister DNAs following replication . Cohesin's kleisin subunit Scc1 completes the ring, connecting the ABC-like ATPase heads of a V-shaped Smc1/3 heterodimer . Proteolytic cleavage of Scc1 by separase triggers sister chromatid disjunction, presumably by breaking the Scc1 bridge . One half of the SMC-kleisin bridge is revealed here by a crystal structure of Smc1's ATPase complexed with Scc1's C-terminal domain . The latter forms a winged helix that binds a pair of beta strands in Smc1's ATPase head . Mutation of conserved residues within the contact interface destroys Scc1's interaction with Smc1/3 heterodimers and eliminates cohesin function . Interaction of Scc1's N terminus with Smc3 depends on prior C terminus connection with Smc1 . There is little or no turnover of Smc1-Scc1 interactions within cohesin complexes in vivo because expression of noncleavable Scc1 after DNA replication does not hinder anaphase .

IEEE Trans Nanobioscience, 2004 Mar, 3(1), 32 - 8
Nanocapsules: coating for living cells; Krol S et al.; One of the most promising tools for future applications in science and medicine is the use of nanotechnologies . Especially self-assembly systems, e.g., polyelectrolyte (PE) capsules prepared by means of the layer-by-layer technique with tailored properties, fulfill the requirements for nano-organized systems in a satisfactory manner . The nano-organized shells are suitable as coating for living cells or artificial tissue to prevent immune response . With these shells, material can be delivered to predefined organs . In this paper, some preliminary results are presented, giving a broad overview over the possibilities to use nano-organized capsules . Based on the observations that the cells while duplicating break the capsule a mutant yeast strain (Saccharomyces cerevisiae), which express GFP-tubulin under galactose promotion, was investigated by means of confocal laser scanning microscopy . The measurements reveal an increased surface charge in the region of buds developed prior encapsulation . In order to test the used PE pair for cytotoxicity, germinating conidia of the fungi Neurospora crassa were coated . The investigation with fluorescence microscopy shows a variation in the surface charge for the growing region and the conidium poles . The capsules exhibit interesting properties as valuable tool in science and a promising candidate for application in the field of medicine.

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