Protein Pept Lett, 2004 Feb, 11(1), 23 - 8 NMR studies of the prionogenic peptide derived from Sup35 protein; Chae YK et al.; The NMR studies of the prionogenic peptide derived from Sup35 are presented . The peptide molecules were dissolved in the half-aqueous solution to prevent severe aggregation, and were found to be in an extended conformation from the chemical shift and the coupling constant data . They could form higher order multimers by making intermolecular hydrogen bonds, judging from the observation that the NMR sample became a gel-like state at lower temperatures . This work reports the first structural information in the solution state about the prionogenic peptide mimicking the state of amyloid fibrils, and provides a solid foundation for further structural analysis of peptide molecules forming insoluble aggregates. Curr Drug Metab, 2004 Feb, 5(1), 109 - 24 Intestinal drug transporters: in vivo function and clinical importance; Kunta JR et al.; The oral route of drug administration remains the most popular and convenient route of administration, despite its many shortcomings and challenges . Although the advantages associated with this route far outweigh any limitations, a prominent limitation relates to the interactions of drugs with intestinal membrane transporters . The complexities of these interactions and their impact on drug absorption and absorption variability are only now becoming recognized . The rapidly growing awareness of transporter-mediated secretion, saturable absorption, and even the concerted actions of transporters in intestinal drug absorption and secretion has attracted the attention of pharmaceutical scientists in academia, the pharmaceutical industry and the regulatory agencies . This is evidenced by the recent rapid accumulation of data in the literature, the routine conducting of transport studies in the discovery and development of drugs, and finally by the recognition of the importance of transporter (e.g . P-glycoprotein and OATP) mediated secretion of drugs by regulatory authorities such as the U.S . Food and Drug Administration . In this mini-review, we focus on the handful of absorptive and secretory transporters that have been relatively well studied and illustrate the impact of these intestinal transporters on oral drug absorption using published reports from preclinical and clinical studies. Cell Cycle, 2004 Apr, 3(4), 439 - 42 Epub 2004 Apr 01. A field guide to the Mps1 family of protein kinases; Fisk HA et al.; Cell cycle events must be faithfully executed and properly integrated to ensure genetic stability . The Mps1 family of protein kinases has recently emerged as a critical regulator of genetic stability, because they regulate several processes central to mitotic fidelity . The spindle checkpoint monitors alignment of mitotic chromosomes, and centrosomes control cell cycle entry, mitotic spindle assembly, and cytokinesis . Several studies have shown that vertebrate orthologues of budding yeast Mps1p regulate the spindle checkpoint . More recently it has been demonstrated that human Mps1 is also required for centrosome duplication, normal mitotic progression, and cytokinesis. Science, 2004 Feb 13, 303(5660), 1014 - 6 Structural basis of transcription: separation of RNA from DNA by RNA polymerase II; Westover KD et al.; The structure of an RNA polymerase II-transcribing complex has been determined in the posttranslocation state, with a vacancy at the growing end of the RNA-DNA hybrid helix . At the opposite end of the hybrid helix, the RNA separates from the template DNA . This separation of nucleic acid strands is brought about by interaction with a set of proteins loops in a strand/loop network . Formation of the network must occur in the transition from abortive initiation to promoter escape. Science, 2004 Feb 13, 303(5660), 983 - 8 Structural basis of transcription: an RNA polymerase II-TFIIB cocrystal at 4.5 Angstroms; Bushnell DA et al.; The structure of the general transcription factor IIB (TFIIB) in a complex with RNA polymerase II reveals three features crucial for transcription initiation: an N-terminal zinc ribbon domain of TFIIB that contacts the "dock" domain of the polymerase, near the path of RNA exit from a transcribing enzyme; a "finger" domain of TFIIB that is inserted into the polymerase active center; and a C-terminal domain, whose interaction with both the polymerase and with a TATA box-binding protein (TBP)-promoter DNA complex orients the DNA for unwinding and transcription . TFIIB stabilizes an early initiation complex, containing an incomplete RNA-DNA hybrid region . It may interact with the template strand, which sets the location of the transcription start site, and may interfere with RNA exit, which leads to abortive initiation or promoter escape . The trajectory of promoter DNA determined by the C-terminal domain of TFIIB traverses sites of interaction with TFIIE, TFIIF, and TFIIH, serving to define their roles in the transcription initiation process. Genomics, 2004 Mar, 83(3), 473 - 81 Functional assessment of the carboxy-terminus of the Wilson disease copper-transporting ATPase, ATP7B; Hsi G et al.; The carboxy-terminus of ATP7B, the protein defective in the copper-transport disorder Wilson disease, was investigated with respect to its role in copper delivery to the ferroxidase ceruloplasmin . We use yeast as a model system to assess the functional capabilities of ATP7B variants . The yeast ferroxidase, Fet3p, acquires copper from Ccc2p and cannot function if Ccc2p is impaired; expression of wild-type ATP7B in ccc2 yeast complements the iron-deficient phenotype . Our results demonstrate that the C-terminus of ATP7B is necessary for protein stability, as removal of the nonmembranous terminus leads to reduced protein levels and cessation of growth in iron-limited medium . Growth is partially restored when an additional three amino acids are present and is near wild-type levels when only one-third of the C-terminus is present . Measurement of ferroxidase activity is a more sensitive indicator of copper transport function and allowed identification of impaired variants not detected with the growth assay. Nature, 2004 Mar 4, 428(6978), 93 - 7 Epub 2004 Feb 11. Tension between two kinetochores suffices for their bi-orientation on the mitotic spindle; Dewar H et al.; The movement of sister chromatids to opposite spindle poles during anaphase depends on the prior capture of sister kinetochores by microtubules with opposing orientations (amphitelic attachment or bi-orientation) . In addition to proteins necessary for the kinetochore-microtubule attachment, bi-orientation requires the Ipl1 (Aurora B in animal cells) protein kinase and tethering of sister chromatids by cohesin . Syntelic attachments, in which sister kinetochores attach to microtubules with the same orientation, must be either 'avoided' or 'corrected' . Avoidance might be facilitated by the juxtaposition of sister kinetochores such that they face in opposite directions; kinetochore geometry is therefore deemed important . Error correction, by contrast, is thought to stem from the stabilization of kinetochore-spindle pole connections by tension in microtubules, kinetochores, or the surrounding chromatin arising from amphitelic but not syntelic attachment . The tension model predicts that any type of connection between two kinetochores suffices for efficient bi-orientation . Here we show that the two kinetochores of engineered, unreplicated dicentric chromosomes in Saccharomyces cerevisiae bi-orient efficiently, implying that sister kinetochore geometry is dispensable for bi-orientation . We also show that Ipl1 facilitates bi-orientation by promoting the turnover of kinetochore-spindle pole connections in a tension-dependent manner. Bioinformatics, 2004 Feb 12, 20(3), 381 - 8 Epub 2004 Jan 22. A graph-theoretic modeling on GO space for biological interpretation of gene clusters; Lee SG et al.; MOTIVATION: With the advent of DNA microarray technologies, the parallel quantification of genome-wide transcriptions has been a great opportunity to systematically understand the complicated biological phenomena . Amidst the enthusiastic investigations into the intricate gene expression data, clustering methods have been the useful tools to uncover the meaningful patterns hidden in those data . The mathematical techniques, however, entirely based on the numerical expression data, do not show biologically relevant information on the clustering results . RESULTS: We present a novel methodology for biological interpretation of gene clusters . Our graph theoretic algorithm extracts common biological attributes of the genes within a cluster or a group of interest through the modified structure of gene ontology (GO) called GO tree . After genes are annotated with GO terms, the hierarchical nature of GO terms is used to find the representative biological meanings of the gene clusters . In addition, the biological significance of gene clusters can be assessed quantitatively by defining a distance function on the GO tree . Our approach has a complementary meaning to many statistical clustering techniques; we can see clustering problems from a different viewpoint by use of biological ontology . We applied this algorithm to the well-known data set and successfully obtained the biological features of the gene clusters with the quantitative biological assessment of clustering quality through GO Biological Process. Bioinformatics, 2004 Feb 12, 20(3), 340 - 8 Functional topology in a network of protein interactions; Przulj N et al.; MOTIVATION: The building blocks of biological networks are individual protein-protein interactions (PPIs) . The cumulative PPI data set in Saccharomyces cerevisiae now exceeds 78 000 . Studying the network of these interactions will provide valuable insight into the inner workings of cells . RESULTS: We performed a systematic graph theory-based analysis of this PPI network to construct computational models for describing and predicting the properties of lethal mutations and proteins participating in genetic interactions, functional groups, protein complexes and signaling pathways . Our analysis suggests that lethal mutations are not only highly connected within the network, but they also satisfy an additional property: their removal causes a disruption in network structure . We also provide evidence for the existence of alternate paths that bypass viable proteins in PPI networks, while such paths do not exist for lethal mutations . In addition, we show that distinct functional classes of proteins have differing network properties . We also demonstrate a way to extract and iteratively predict protein complexes and signaling pathways . We evaluate the power of predictions by comparing them with a random model, and assess accuracy of predictions by analyzing their overlap with MIPS database . CONCLUSIONS: Our models provide a means for understanding the complex wiring underlying cellular function, and enable us to predict essentiality, genetic interaction, function, protein complexes and cellular pathways . This analysis uncovers structure-function relationships observable in a large PPI network. Eukaryot Cell, 2004 Feb, 3(1), 180 - 9 An ste20 homologue in Ustilago maydis plays a role in mating and pathogenicity; Smith DG et al.; The mitogen-activated protein kinase (MAPK) pathways are conserved from fungi to humans and have been shown to play important roles in mating and filamentous growth for both Saccharomyces cerevisiae and dimorphic fungi and in infectivity for pathogenic fungi . STE20 encodes a protein kinase of the p21-activated protein kinase family that regulates more than one of these cascades in yeasts . We hypothesized that an Ste20p homologue would play a similar role in the dimorphic plant pathogen Ustilago maydis . The full-length copy of the U . maydis gene was obtained from a genomic library; it lacked introns and was predicted to encode a protein of 826 amino acids, whose sequence confirmed its identity as the first Ste20p homologue to be isolated from a plant pathogen . The predicted protein contained both an N-terminal regulatory Cdc42-Rac interactive binding domain and a C-terminal catalytic kinase domain . Disruption of the gene smu1 resulted in a delayed mating response in a mating-type-specific manner and also in a severe reduction in disease production on maize . Unlike the Ustilago bypass of cyclase (ubc) mutations previously identified in genes in the pheromone-responsive MAPK cascade, mutation of smu1 does not by itself act as an extragenic suppressor of the filamentous phenotype of a uac1 mutant . Thus, the direct connection of Smu1p to MAPK cascade function has yet to be established . Even so, Smu1, though not absolutely required for mating, is necessary for wild-type mating and pathogenicity. Eukaryot Cell, 2004 Feb, 3(1), 144 - 56 Chromatin rearrangements in the prnD-prnB bidirectional promoter: dependence on transcription factors; Garcia I et al.; The prnD-prnB intergenic region regulates the divergent transcription of the genes encoding proline oxidase and the major proline transporter . Eight nucleosomes are positioned in this region . Upon induction, the positioning of these nucleosomes is lost . This process depends on the specific transcriptional activator PrnA but not on the general GATA factor AreA . Induction of prnB but not prnD can be elicited by amino acid starvation . A specific nucleosomal pattern in the prnB proximal region is associated with this process . Under conditions of induction by proline, metabolite repression depends on the presence of both repressing carbon (glucose) and nitrogen (ammonium) sources . Under these repressing conditions, partial nucleosomal positioning is observed . This depends on the CreA repressor's binding to two specific cis-acting sites . Three conditions (induction by the defective PrnA80 protein, induction by amino acid starvation, and induction in the presence of an activated CreA) result in similar low transcriptional activation . Each results in a different nucleosome pattern, which argues strongly for a specific effect of each signal on nucleosome positioning . Experiments with trichostatin A suggest that both default nucleosome positioning and partial positioning under induced-repressed conditions depend on deacetylated histones. Eukaryot Cell, 2004 Feb, 3(1), 108 - 20 The Ras/protein kinase A pathway acts in parallel with the Mob2/Cbk1 pathway to effect cell cycle progression and proper bud site selection; Schneper L et al.; In Saccharomyces cerevisiae, Ras proteins connect nutrient availability to cell growth through regulation of protein kinase A (PKA) activity . Ras proteins also have PKA-independent functions in mitosis and actin repolarization . We have found that mutations in MOB2 or CBK1 confer a slow-growth phenotype in a ras2Delta background . The slow-growth phenotype of mob2Delta ras2Delta cells results from a G1 delay that is accompanied by an increase in size, suggesting a G1/S role for Ras not previously described . In addition, mob2Delta strains have imprecise bud site selection, a defect exacerbated by deletion of RAS2 . Mob2 and Cbk1 act to properly localize Ace2, a transcription factor that directs daughter cell-specific transcription of several genes . The growth and budding phenotypes of the double-deletion strains are Ace2 independent but are suppressed by overexpression of the PKA catalytic subunit, Tpk1 . From these observations, we conclude that the PKA pathway and Mob2/Cbk1 act in parallel to determine bud site selection and promote cell cycle progression. Eukaryot Cell, 2004 Feb, 3(1), 52 - 60 C-terminal truncation of alpha-COP affects functioning of secretory organelles and calcium homeostasis in Hansenula polymorpha; Chechenova MB et al.; In eukaryotic cells, COPI vesicles retrieve resident proteins to the endoplasmic reticulum and mediate intra-Golgi transport . Here, we studied the Hansenula polymorpha homologue of the Saccharomyces cerevisiae RET1 gene, encoding alpha-COP, a subunit of the COPI protein complex . H . polymorpha ret1 mutants, which expressed truncated alpha-COP lacking more than 300 C-terminal amino acids, manifested an enhanced ability to secrete human urokinase-type plasminogen activator (uPA) and an inability to grow with a shortage of Ca2+ ions, whereas a lack of alpha-COP expression was lethal . The alpha-COP defect also caused alteration of intracellular transport of the glycosylphosphatidylinositol-anchored protein Gas1p, secretion of abnormal uPA forms, and reductions in the levels of Pmr1p, a Golgi Ca2+-ATPase . Overexpression of Pmr1p suppressed some ret1 mutant phenotypes, namely, Ca2+ dependence and enhanced uPA secretion . The role of COPI-dependent vesicular transport in cellular Ca2+ homeostasis is discussed. Genes Dev, 2004 Feb 1, 18(3), 333 - 43 In vivo target of a transcriptional activator revealed by fluorescence resonance energy transfer; Bhaumik SR et al.; Our understanding of eukaryotic transcriptional activation mechanisms has been hampered by an inability to identify the direct in vivo targets of activator proteins, primarily because of lack of appropriate experimental methods . To circumvent this problem, we have developed a fluorescence resonance energy transfer (FRET) assay to monitor interactions with transcriptional activation domains in living cells . We use this method to show that the Tra1 subunit of the SAGA (Spt/Ada/Gcn5/acetyltransferase) complex is the direct in vivo target of the yeast activator Gal4 . Chromatin-immunoprecipitation experiments demonstrate that the Gal4-Tra1 interaction is required for recruitment of SAGA to the upstream activating sequence (UAS), and SAGA, in turn, recruits the Mediator complex to the UAS . The UAS-bound Mediator is required for recruitment of the general transcription factors to the core promoter . Thus, our results identify the in vivo target of an activator and show how the activator-target interaction leads to transcriptional stimulation . The FRET assay we describe is a general method that can be used to identify the in vivo targets of other activators. Biochim Biophys Acta, 2004 Feb 15, 1608(2-3), 190 - 9 Roles of histidine residues in plant vacuolar H(+)-pyrophosphatase; Hsiao YY et al.; Vacuolar proton pumping pyrophosphatase (H(+)-PPase; EC 3.6.1.1) plays a pivotal role in electrogenic translocation of protons from cytosol to the vacuolar lumen at the expense of PP(i) hydrolysis . Alignment analysis on amino acid sequence demonstrates that vacuolar H(+)-PPase of mung bean contains six highly conserved histidine residues . Previous evidence indicated possible involvement of histidine residue(s) in enzymatic activity and H(+)-translocation of vacuolar H(+)-PPase as determined by using histidine specific modifier, diethylpyrocarbonate {J . Protein Chem . 21 (2002) 51} . In this study, we further attempted to identify the roles of histidine residues in mung bean vacuolar H(+)-PPase by site-directed mutagenesis . A line of mutants with histidine residues singly replaced by alanine was constructed, over-expressed in Saccharomyces cerevisiae, and then used to determine their enzymatic activities and proton translocations . Among the mutants scrutinized, only the mutation of H716 significantly decreased the enzymatic activity, the proton transport, and the coupling ratio of vacuolar H(+)-PPase . The enzymatic activity of H716A is relatively resistant to inhibition by diethylpyrocarbonate as compared to wild-type and other mutants, indicating that H716 is probably the target residue for the attack by this modifier . The mutation at H716 of V-PPase shifted the optimum pH value but not the T(1/2) (pretreatment temperature at which half enzymatic activity is observed) for PP(i) hydrolytic activity . Mutation of histidine residues obviously induced conformational changes of vacuolar H(+)-PPase as determined by immunoblotting analysis after limited trypsin digestion . Furthermore, mutation of these histidine residues modified the inhibitory effects of F(-) and Na(+), but not that of Ca(2+) . Single substitution of H704, H716 and H758 by alanine partially released the effect of K(+) stimulation, indicating possible location of K(+) binding in the vicinity of domains surrounding these residues. Methods Mol Biol, 2004, 257, 17 - 28 Identifying phosphoCTD-associating proteins; Phatnani HP et al.; The C-terminal repeat domain (CTD) of the largest subunit of RNA polymerase II is hyperphosphorylated during transcription elongation . The phosphoCTD is known to bind to a subset of RNA processing factors and to several other nuclear proteins, thereby positioning them to efficiently carry out their elongation-linked functions . The authors propose that additional phosphoCTD-associating proteins (PCAPs) exist and describe a systematic biochemical approach for identifying such proteins . A binding probe is generated by using yeast CTD kinase I to exhaustively phosphorylate a CTD fusion protein . This phosphoCTD is used to probe fractionated yeast or mammalian extracts in a Far Western protein interaction assay . Putative PCAPs are further purified and identified by mass spectrometry. Methods Mol Biol, 2004, 262, 223 - 37 Chromatin immunoprecipitation to investigate protein-DNA interactions during genetic recombination; Goldfarb T et al.; Chromatin immunoprecipitation is a technique that allows one to examine the in vivo localization of proteins to DNA . This technique is well suited for studying genetic recombination since it can provide both a temporal and spatial assessment of the dynamic association of proteins with DNA in both wild-type and mutant backgrounds . To perform this procedure, cells undergoing a synchronous recombination event are treated with a crosslinking agent . Following cell lysis and shearing of the DNA, immunoprecipitation is used to isolate the protein of interest, along with any DNA that is crosslinked to the protein . Polymerase chain reaction (PCR) is then used to determine the relative amounts of DNA associated with the protein of interest throughout the recombination event . This in vivo chemical crosslinking technique can be used to localize proteins to both double-strand breaks and recombination intermediates. Methods Mol Biol, 2004, 262, 209 - 19 Enhancement of in vivo targeted nucleotide exchange by nonspecific carrier DNA; Maguire KK et al.; Targeted nucleotide exchange (TNE) is a process in which an oligonucleotide bearing sequence complementarity aligns with the sequence of a target gene and directs the alteration of a single base . This technique can be used to repair a point mutation or mediate site-specific mutagenesis . A critical factor in the development of this approach centers around the elevation and stabilization of the frequencies with which these events occur . Here we describe a protocol for increasing the frequency of TNE in the true yeast, Saccharomyces cerevisiae, through the use of nonspecific, carrier oligonucleotides . These molecules, when added to the reaction, increase the TNE frequency up to 25-fold in some cases, perhaps by providing a molecular trap to bind factors, which may inactivate the specific targeting oligos. Methods Mol Biol, 2004, 262, 35 - 52 Analysis of recombinational repair of DNA double-strand breaks in mammalian cells with I-SceI nuclease; Nickoloff JA et al.; Eukaryotes repair DNA double-strand breaks (DSBs) by homologous recombination (HR) or by nonhomologous end-joining (NHEJ) . DSBs are a natural consequence of DNA metabolism, occurring, for example, during DNA replication and meiosis . DSBs are also induced by chemicals and radiation . I-SceI endonuclease recognizes an 18-bp sequence with little degeneracy; therefore I-SceI is highly specific, and its recognition sequence is predicted to occur by chance less than once in even the largest known genomes . As such, I-SceI can be used to introduce a DSB into a defined (engineered) site in a mammalian chromosome, and this facilitates detailed studies of DSB repair . DSBs induced in repeated regions can be repaired by several different HR processes, including gene conversion with or without associated crossovers, or single-strand annealing . The specific types of HR events that can be scored depend on the configuration of the repeated regions and whether selection for recombinants is imposed . Nonselective assays detect both HR and NHEJ events . This chapter focuses on the systems for delivering I-SceI nuclease to mammalian cells and the strategies for detecting various outcomes of DSB repair. Proc Natl Acad Sci U S A, 2004 Feb 17, 101(7), 1858 - 62 Epub 2004 Feb 09. Cotranscriptional recruitment of the serine-arginine-rich (SR)-like proteins Gbp2 and Hrb1 to nascent mRNA via the TREX complex; Hurt E et al.; The TREX (transcription/export) complex couples transcription elongation to the nuclear export of mRNAs . In this article, we show that the poly(A)(+) RNA-binding proteins Gbp2 and Hrb1, which resemble the serine-arginine-rich (SR) family of splicing factors found in higher eukaryotes, are specifically associated with the yeast TREX complex . We also show that Gbp2 and Hrb1 interact with Ctk1, a kinase that phosphorylates the C-terminal domain of RNA polymerase II during transcription elongation . Consistent with these findings, Gbp2 and Hrb1 associate with actively transcribed genes throughout their entire lengths . By using an RNA immunoprecipitation assay, we show that Gbp2 and Hrb1 also are bound to transcripts that are derived from these genes . We conclude that recruitment of the SR-like proteins Gbp2 and Hrb1 to mRNA occurs cotranscriptionally by means of association with the TREX complex and/or Ctk1. Mol Biol Cell, 2004 Apr, 15(4), 2038 - 47 Epub 2004 Feb 06. Phosphatidylinositol 4-kinasebeta is critical for functional association of rab11 with the Golgi complex; de Graaf P et al.; Phosphatidylinositol 4-kinasebeta (PI4Kbeta) plays an essential role in maintaining the structural integrity of the Golgi complex . In a search for PI4Kbeta-interacting proteins, we found that PI4Kbeta specifically interacts with the GTP-bound form of the small GTPase rab11 . The PI4Kbeta-rab11 interaction is of functional significance because inhibition of rab11 binding to PI4Kbeta abolished the localization of rab11 to the Golgi complex and significantly inhibited transport of vesicular stomatitis virus G protein from the Golgi complex to the plasma membrane . We propose that a novel function of PI4Kbeta is to act as a docking protein for rab11 in the Golgi complex, which is important for biosynthetic membrane transport from the Golgi complex to the plasma membrane. Endocrinology, 2004 May, 145(5), 2307 - 18 Epub 2004 Feb 05. Cloning and characterization of granulosa cell high-mobility group (HMG)-box protein-1, a novel HMG-box transcriptional regulator strongly expressed in rat ovarian granulosa cells; Kajitani T et al.; Specific events in the ovary are dependent on gene expression in the tissue . By screening a rat ovarian granulosa cell cDNA library, a cDNA clone encoding a novel transcription factor-like protein containing a high-mobility group-box, referred to as granulosa cell high-mobility group-box protein-1 (GCX-1), was identified . The expression of GCX-1 is restricted to the hypothalamus, pituitary, testis, uterus, and ovary but was not detected in the adrenal gland . An in situ hybridization study revealed that the expression of GCX-1 was restricted to granulosa cell layers in early-stage follicles, and the expression was very low in large antral follicles and the corpus luteum, but localized expression in the testis or pituitary was not clear . Endogenous GCX-1 protein in the granulosa cells was identified by a Western blot analysis, and an analysis using the green fluorescence protein-GCX-1 fusion protein revealed that the GCX-1 protein was localized in the cell nucleus . GAL4 fusion protein-based assays demonstrated that GCX-1 is a potent transcriptional activator, and its putative transactivation domain was mapped to the region between amino acid residues 25 and 63 from the N terminus . These data strongly suggest that GCX-1 is likely a novel transcriptional activator that is exclusively expressed in reproductive tissues involving the hypothalamo-pituitary-gonadal axis, and functions as a specific regulator of follicular development, and may also participate in other specific events related to reproduction, particularly in the female. Bioinformatics, 2004 May 1, 20(7), 1119 - 28 Epub 2004 Feb 05. Predicting rules on organization of cis-regulatory elements, taking the order of elements into account; Terai G et al.; MOTIVATION: In eukaryotes, rules regarding organization of cis-regulatory elements are complex . They sometimes govern multiple kinds of elements and positional restrictions on elements . RESULTS: We propose a method for detecting rules, by which the order of elements is restricted . The order restriction is expressed as element patterns . We extract all the element patterns that occur in promoter regions of at least the specified number of genes . Then, we find significant patterns based on the expression similarity of genes with promoter regions containing each of the extracted patterns . When we applied our method to Saccharomyces cerevisiae, we detected significant patterns overlooked by previous methods, thus demonstrating the utility of our method for analyses of eukaryotic gene regulation . We also suggest that several types of element organization exist: (i) those in which only the order of elements is important, (ii) order and distance both are important and (iii) only the combination of elements is important . AVAILABILITY: The program for extracting element patterns is available upon request. Mol Microbiol, 2004 Feb, 51(4), 1129 - 42 Overexpression of Upf1p compensates for mitochondrial splicing deficiency independently of its role in mRNA surveillance; de Pinto B et al.; In yeast the UPF1, UPF2 and UPF3 genes encode three interacting factors involved in translation termination and nonsense-mediated mRNA decay (NMD) . UPF1 plays a central role in both processes . In addition, UPF1 was originally isolated as a multicopy suppressor of mitochondrial splicing deficiency, and its deletion leads to an impairment in respiratory growth . Here, we provide evidence that inactivation of UPF2 or UPF3, like that of UPF1, leads to an impairment in respiratory competence, suggesting that their products, Upf1p, Upf2p and Upf3p, are equivalently involved in mitochondrial biogenesis . In addition, however, we show that only Upf1p acts as a multicopy suppressor of mitochondrial splicing deficiency, and its activity does not require either Upf2p or Upf3p . Mutations in the conserved cysteine- and histidine-rich regions and ATPase and helicase motifs of Upf1p separate the ability of Upf1p to complement the respiratory impairment of a Deltaupf1 strain from its ability to act as a multicopy suppressor of mitochondrial splicing deficiency, indicating that distinct pathways express these phenotypes . In addition, we show that, when overexpressed, Upf1p is not detected within mitochondria, suggesting that its role as multicopy suppressor of mitochondrial splicing deficiency is indirect . Furthermore, we provide evidence that cells overexpressing certain upf1 alleles accumulate a phosphorylated isoform of Upf1p . Altogether, these results indicate that overexpression of Upf1p compensates for mitochondrial splicing deficiency independently of its role in mRNA surveillance, which relies on Upf1p-Upf2p-Upf3p functional interplay. Nucleic Acids Res, 2004 Feb 03, 32(2), 784 - 90 Print 2004. A programmed -1 ribosomal frameshift signal can function as a cis-acting mRNA destabilizing element; Plant EP et al.; Nonsense-mediated mRNA decay (NMD) directs rapid degradation of premature termination codon (PTC)-containing mRNAs, e.g . those containing frameshift mutations . Many viral mRNAs encode polycistronic messages where programmed -1 ribosomal frameshift (-1 PRF) signals direct ribosomes to synthesize polyproteins . A previous study, which identified consensus -1 PRF signals in the yeast genome, found that, in contrast to viruses, the majority of predicted -1 PRF events would direct translating ribosomes to PTCs . Here we tested the hypothesis that a -1 PRF signal can function as a cis-acting mRNA destabilizing element by inserting an L-A viral -1 PRF signal into a PGK1 reporter construct in the 'genomic' orientation . The results show that even low levels of -1 PRF are sufficient to target the reporter mRNA for degradation via the NMD pathway, with half-lives similar to messages containing in-frame PTCs . The demonstration of an inverse correlation between frameshift efficiency and mRNA half-lives suggests that modulation of -1 PRF frequencies can be used to post-transcriptionally regulate gene expression . Analysis of the mRNA decay profiles of the frameshift-signal- containing reporter mRNAs also supports the notion that NMD remains active on mRNAs beyond the 'pioneer round' of translation in yeast. Mol Genet Genomics, 2004 Mar, 271(2), 121 - 9 Epub 2004 Jan 31. An insertional mutation in the rice PAIR2 gene, the ortholog of Arabidopsis ASY1, results in a defect in homologous chromosome pairing during meiosis; Nonomura KI et al.; To elucidate the genetic system that establishes homologous chromosome pairing in monocot plants, we have isolated an asynaptic mutant of rice, designated pair2 (homologous pairing aberration in rice meiosis 2), in which 24 completely unpaired univalents are observed at pachytene and diakinesis . The mutation was caused by an insertion of the retrotransposon Tos17, as demonstrated by complementation of the mutation by transformation with the corresponding wild-type gene . The gene in which the element was inserted is orthologous to the ASY1 gene of Arabidopsis thaliana and the HOP1 gene of Saccharomyces cerevisiae . Mature PAIR2 mRNA and several splicing variants were found to be highly expressed in wild-type reproductive tissues, and lower expression was also detected in vegetative tissues . In situ hybridization and BrdU incorporation experiments revealed that PAIR2 expression is specifically enhanced in male and female meiocytes, but not in those at pre-meiotic S phase or in the pollen maturation stages . The results obtained in this study suggest that the PAIR2 gene is essential for homologous chromosome pairing in meiosis, as in the case of the genes ASY1 and HOP1 . The study also suggested the possibility that a highly homologous copy of the PAIR2 gene located on a different chromosome is in fact a pseudogene. Nat Struct Mol Biol, 2004 Mar, 11(3), 249 - 56 Epub 2004 Feb 01. Crystal structure of Dcp1p and its functional implications in mRNA decapping; She M et al.; A major pathway of eukaryotic mRNA turnover begins with deadenylation, followed by decapping and 5'-->3' exonucleolytic degradation . A critical step in this pathway is decapping, which is carried out by an enzyme composed of Dcp1p and Dcp2p . The crystal structure of Dcp1p shows that it markedly resembles the EVH1 family of protein domains . Comparison of the proline-rich sequence (PRS)-binding sites in this family of proteins with Dcp1p indicates that it belongs to a novel class of EVH1 domains . Mapping of the sequence conservation on the molecular surface of Dcp1p reveals two prominent sites . One of these is required for the function of the Dcp1p-Dcp2p complex, and the other, corresponding to the PRS-binding site of EVH1 domains, is probably a binding site for decapping regulatory proteins . Moreover, a conserved hydrophobic patch is shown to be critical for decapping. J Med Genet, 2004 Feb, 41(2), 120 - 4 Respiratory chain complex V deficiency due to a mutation in the assembly gene ATP12; De Meirleir L et al.; In patients with mitochondrial encephalomyopathies an increasing number of causative gene defects have been detected . The number of identified pathogenic mitochondrial DNA mutations has largely increased over the past 15 years . Recently, much attention has turned to the investigation of nuclear oxidative phosphorylation (OXPHOS) gene defects . Within the OXPHOS defects, complex V deficiency is rarely found and, so far, these defects have only been attributed to mutations in the mitochondrial MTATP6 gene . Mutation analysis of the complete coding regions at the cDNA level of the nuclear ATP11, ATP12, ATPalpha, ATPbeta and ATPgamma genes and the mitochondrial MTATP6 and MTAT8 genes was undertaken in two unrelated patients . Blue Native polyacrylamide gel electrophoresis followed by catalytic staining had already documented their complex V decreased activity . Extensive molecular analysis of five nuclear and two mitochondrial genes revealed a mutation in the ATP12 assembly gene in one patient . This mutation is believed to be the cause of the impaired complex V activity . To our knowledge, this is the first report of a pathogenic mutation in a human nuclear encoded ATPase assembly gene. J Mol Biol, 2004 Feb 13, 336(2), 489 - 96 Cytochrome c and SDS: a molten globule protein with altered axial ligation; Bertini I et al.; Saccharomices cerevisiae (yeast iso-1) cytochrome c has been investigated in the presence of 100 mM SDS in order to simulate the interaction of cytochrome c with membrane . Under these circumstances, a high spin species with detached methionine axial ligand is observed through NMR, in analogy to findings on the horse heart protein . However, at variance with the latter system, for the yeast protein also a low spin species is detected, which appears to be present with a concentration of about 40% with respect to that of the high spin species . The R(1), R(2), {1H}-15N NOE of backbone amides which are not affected by paramagnetism are homogeneous and allow a simultaneous analysis of the data for the two species . The result is that the rotational correlation time is larger than in water and larger than expected on the basis of viscosity of the SDS-containing solution . This finding suggests interactions of cytochrome c with SDS . Furthermore, it appears that there is subnanosecond backbone mobility, which also accounts for the decreased intensity of NOE cross-peaks and may be associated with equilibria between helical and random coil structure . The dynamic behavior appears to be a common feature of the high spin and low spin species and is consistent with the presence of a molten globule state . The molten globule nature of the protein could account for the presence of the different axial coordination of the heme iron . Such findings are meaningful with respect to the physiology of cytochrome c as electron transfer protein and as promoter of apoptosis. Biochemistry, 2004 Feb 10, 43(5), 1204 - 12 Transition state structure for ADP-ribosylation of eukaryotic elongation factor 2 catalyzed by diphtheria toxin; Parikh SL et al.; Bacterial protein toxins are the most powerful human poisons known, exhibiting an LD(50) of 0.1-1 ng kg(-)(1) . A major subset of such toxins is the NAD(+)-dependent ADP-ribosylating exotoxins, which include pertussis, cholera, and diphtheria toxin . Diphtheria toxin catalyzes the ADP ribosylation of the diphthamide residue of eukaryotic elongation factor 2 (eEF-2) . The transition state of ADP ribosylation catalyzed by diphtheria toxin has been characterized by measuring a family of kinetic isotope effects using (3)H-, (14)C-, and (15)N-labeled NAD(+) with purified yeast eEF-2 . Isotope trapping experiments yield a commitment to catalysis of 0.24 at saturating eEF-2 concentrations, resulting in suppression of the intrinsic isotope effects . Following correction for the commitment factor, intrinsic primary kinetic isotope effects of 1.055 +/- 0.003 and 1.022 +/- 0.004 were observed for {1(N)'-(14)C}- and {1(N)-(15)N}NAD(+), respectively; the double primary isotope effect was 1.066 +/- 0.004 for {1(N)'-(14)C, 1(N)-(15)N}NAD(+) . Secondary kinetic isotope effects of 1.194 +/- 0.002, 1.101 +/- 0.003, 1.013 +/- 0.005, and 0.988 +/- 0.002 were determined for {1(N)'-(3)H}-, {2(N)'-(3)H}-, {4(N)'-(3)H}-, and {5(N)'-(3)H}NAD(+), respectively . The transition state structure was modeled using density functional theory (B1LYP/6-31+G) as implemented in Gaussian 98, and theoretical kinetic isotope effects were subsequently calculated using Isoeff 98 . Constraints were varied in a systematic manner until the calculated kinetic isotope effects matched the intrinsic isotope effects . The transition state model most consistent with the intrinsic isotope effects is characterized by the substantial loss in bond order of the nicotinamide leaving group (bond order = 0.18, 1.99 A) and weak participation of the attacking imidazole nucleophile (bond order = 0.03, 2.58 A) . The transition state structure imparts strong oxacarbenium ion character to the ribose ring even though significant bond order remains to the nicotinamide leaving group . The transition state model presented here is asymmetric and consistent with a dissociative S(N)1 type mechanism in which attack of the diphthamide nucleophile lags behind departure of the nicotinamide. Plant Mol Biol, 2003 Sep, 53(1-2), 227 - 36 Cyclin D-knockout uncouples developmental progression from sugar availability; Lorenz S et al.; Multicellular organisms need to modulate proliferation and differentiation in response to external conditions . An important role in these processes plays the mitogen-stimulated induction of cyclin D (cycD) gene expression . D-type cyclins have been identified as the crucial intracellular sensors for cell-cycle regulation in all eukaryotes . However, cycD deletions have been found to cause specific phenotypic alterations in animals but not yet in plants . An insertional mutation of a so far uncharacterized Arabidopsis cycD gene did not alter the plant phenotype . To gain new insights into CycD function of land plants, we generated targeted cycD gene knockouts in the moss Physcomitrella patens and observed a surprisingly limited disruption phenotype . While wild-type plants reacted to exogenous glucose sources with prolonged growth of juvenile stages and retarded differentiation, cycD knockouts exhibited developmental progression independent of sugar supply . On the other hand, growth rate, cell sizes or plant size were not affected . Thus, we conclude that Physcomitrella CycD might not be essential for cell-cycle regulation but is important for coupling the developmental progression to nutrient availability. Plant Mol Biol, 2003 Sep, 53(1-2), 201 - 12 Arabidopsis transportin1 is the nuclear import receptor for the circadian clock-regulated RNA-binding protein AtGRP7; Ziemienowicz A et al.; We characterized the Arabidopsis orthologue of the human nuclear import receptor transportin1 (TRN1) . Like the human receptor, Arabidopsis TRN1 recognizes nuclear import signals on proteins that are different from the classical basic nuclear localization signals . The M9 domain of human heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is the prototype of such signals . We show that AtTRN1 binds to similar domains in hnRNP-like proteins from plants . AtTRN1 also interacts with human hnRNP A1 and with yeast Nab2p, two classical import cargo proteins of transportin in these organisms . Like all nuclear transport receptors of the importin-beta family, AtTRN1 binds to the regulatory GTPase Ran from Arabidopsis . We demonstrated that the amino terminus of AtTRN1 is necessary for this interaction . Recombinant AtTRN1 conferred nuclear import of fluorescently labelled BSA-M9 peptide conjugates in permeabilized HeLa cells, functionally replacing human TRN1 in these in vitro nuclear import assays . We identified three plant substrate proteins that interact with AtTRN1 and contain M9-like domains: a novel Arabidopsis hnRNP that shows high similarity to human hnRNP A1 and two small RNA-binding proteins from Arabidopsis, AtGRP7 and AtGRP8 . Nuclear import activity of the M9-like domains of these plant proteins was demonstrated in vivo by their ability to confer partial nuclear re-localisation of a GFP fusion protein containing a nuclear export signal . In addition, fluorescently labelled AtGRP7 was specifically imported into nuclei of permeabilized HeLa cells by Arabidopsis AtTRN1 and human TRN1 . These results suggest that the transportin-mediated nuclear import pathway is highly conserved between man, yeast and plants. Biochem J, 2004 May 15, 380(Pt 1), 131 - 7 Mutation of leucine-92 selectively reduces the apparent affinity of inosine, guanosine, NBMPR {S6-(4-nitrobenzyl)-mercaptopurine riboside} and dilazep for the human equilibrative nucleoside transporter, hENT1; Endres CJ et al.; We developed a yeast-based assay for selection of hENT1 (human equilibrative nucleoside transporter 1) mutants that have altered affinity for hENT1 inhibitors and substrates . In this assay, expression of hENT1 in a yeast strain deficient in adenine biosynthesis (ade2) permits yeast growth on a plate lacking adenine but containing adenosine, a hENT1 substrate . This growth was prevented when inhibitors of hENT1 {e.g . NBMPR {S6-(4-nitrobenzyl)-mercaptopurine riboside}, dilazep or dipyridamole} were included in the media . To identify hENT1 mutants resistant to inhibition by these compounds, hENT1 was randomly mutagenized and introduced into this strain . Mutation(s) that allowed growth of yeast cells in the presence of these inhibitors were then identified and characterized . Mutants harbouring amino acid changes at Leu92 exhibited resistance to NBMPR and dilazep but not dipyridamole . The IC50 values of NBMPR and dilazep for {3H}adenosine transport by one of these mutants L92Q (Leu92-->Gln) were approx . 200- and 4-fold greater when compared with the value for the wild-type hENT1, whereas that for dipyridamole remained unchanged . Additionally, when compared with the wild-type transporter, {3H}adenosine transport by L92Q transporter was significantly resistant to inhibition by inosine and guanosine but not by adenosine or pyrimidines . The Km value for inosine transport was approx . 4-fold greater for the L92Q mutant (260+/-16 mM) when compared with the wild-type transporter (65+/-7.8 mM) . We have identified for the first time an amino acid residue (Leu92) of hENT1 that, when mutated, selectively alters the affinity of hENT1 to transport the nucleosides inosine and guanosine and its sensitivity to the inhibitors NBMPR and dilazep. J Cell Biochem, 2004 Feb 15, 91(3), 633 - 45 A LIM protein, Hic-5, functions as a potential coactivator for Sp1; Shibanuma M et al.; Hic-5 is a LIM protein with striking similarity to paxillin, and shuttles between focal adhesions and the nucleus . Our previous study suggested that Hic-5 participates in the transcriptional control of several genes such as the c-fos and p21 genes . In the present study, we examined the function of Hic-5 in the nucleus using the transcriptional promoter region of the p21 gene . When localized to the nucleus, Hic-5 was found to transactivate the p21 promoter through two of five Sp1 sites in the region proximal to the TATA box . The Hic-5 effect was mediated by a transactivation domain of Sp1 and functional interaction with p300 through the LIM4 domain . Hic-5 was also shown to interact functionally and physically with Smad3 through the LIM domains and to potentiate p21 promoter activity together with Smad3 and Sp1 . These properties were confirmed in an artificial system using GAL4-fusion protein . Thus, Hic-5 was suggested to have a potential function as a cofactor in the transcriptional complex that contains Sp1, playing a role in gene transcription in the nucleus as well as in integrin signaling at focal adhesion sites . Oncogene, 2004 Mar 11, 23(10), 1801 - 8 An RNF11: Smurf2 complex mediates ubiquitination of the AMSH protein; Li H et al.; RING-finger proteins play crucial roles in ubiquitination events involved in diverse cellular processes including signal transduction, differentiation and apoptosis . Most of the RING-finger proteins have E3-ubiquitin ligase activity . RNF11 is a small RING-finger protein and harbors a RING-H2 domain and a PY motif that could facilitate protein:protein interaction(s) involved in oncogenesis . To isolate RNF11 protein partners and determine its role in normal and cancer cells, we performed yeast two-hybrid screening . Among 18 in-frame positive clones, three were found to be ZBRK1, Eps15 and AMSH (associated molecule with the SH3 domain of STAM) . ZBRK1 is a KRAB domain containing Zinc-finger protein and is known to repress target gene transcription in a BRCA1-dependent manner . Eps15 is monoubiquitinated and is part of an essential complex involved in the endocytosis of plasma membrane receptors via the clathrin-mediated internalization pathway . Recent studies have shown that AMSH protein is involved in BMP/TGF-beta signaling pathway by binding to Smad6 and Smad7 . The association of RNF11 with these binding partners suggests that it would be involved in biological processes such as gene transcription, BMP/TGF-beta signaling and ubiquitination-associated events . Previously, we have shown that RNF11 interacts with the HECT-type E3 ligases AIP4 and Smurf2 . Here, we show that RNF11 binds to AMSH in mammalian cells and that this interaction is independent of the RNF11 RING-finger domain and the PY motif . Our results also demonstrate that AMSH is ubiquitinated by Smurf2 E3 ligase in the presence of RNF11 and that a consequent reduction in its steady-state level requires both RNF11 and Smurf2 . RNF11 therefore recruits AMSH to Smurf2 for ubiquitination, leading to its degradation by the 26S proteasome . The potential functions of RNF11-mediated degradation of AMSH in breast cancer are discussed. J Exp Bot, 2004 Mar, 55(397), 585 - 94 Epub 2004 Jan 30. Effect of salt and osmotic stresses on the expression of genes for the vacuolar H+-pyrophosphatase, H+-ATPase subunit A, and Na+/H+ antiporter from barley; Fukuda A et al.; Two cDNA clones encoding vacuolar H+-inorganic pyrophosphatase (HVP1 and HVP10), one clone encoding the catalytic subunit (68 kDa) of vacuolar H+-ATPase (HvVHA-A), and one clone encoding vacuolar Na+/H+ antiporter (HvNHX1) were isolated from barley (Hordeum vulgare), a salt-tolerant crop . Salt stress increased the transcript levels of HVP1, HVP10, HvVHA-A, and HvNHX1, and osmotic stress also increased the transcript levels of HVP1 and HvNHX1 in barley roots . The transcription of HVP1 in response to salt stress was regulated differently from that of HVP10 . In addition, the HVP1 expression changed in a pattern similar to that of HvNHX1 expression . These results indicate that the expression of HVP1 is co-ordinated with that of HvNHX1 in barley roots in response to salt and osmotic stresses. J Cell Mol Med, 2003 Oct-Dec, 7(4), 388 - 400 Peroxisome biogenesis and the role of protein import; Brown LA et al.; Peroxisomes are metabolic organelles with enzymatic content that are found in virtually all cells and are involved in beta-oxidation of fatty acids, hydrogen peroxide-based respiration and defence against oxidative stress . The steps of their biogenesis involves "peroxins", proteins encoded by PEX genes . Peroxins are involved in three key stages of peroxisome development: (1) . import of peroxisomal membrane proteins; (2) . import of peroxisomal matrix proteins and (3) . peroxisome proliferation . Of these three areas, peroxisomal matrix-protein import is by far the best understood and accounts for most of the available published data on peroxisome biogenesis . Defects in peroxisome biogenesis result in peroxisome biogenesis disorders (PBDs), which although rare, have no known cure to-date . This review explores current understanding of each key area in peroxisome biogenesis, paying particular attention to the role of protein import. J Chromatogr A, 2004 Jan 23, 1024(1-2), 71 - 8 Effect of the detergent Tween-20 on the DNA affinity chromatography of Gal4, C/EBPalpha, and lac repressor with observations on column regeneration; Robinson FD et al.; C/EBPalpha, Gal4, and lac repressor, representing three different transcription factor homology families, were expressed as fusion proteins and used to characterize the effects of column aging, Mg2+, the nonionic detergent Tween-20, column loading, and bovine serum albumin on DNA-affinity chromatography . When lac-repressor-beta-galactosidase fusion protein is loaded onto a new DNA-Sepharose column, less elutes from a new column than one that has been used two or more times . Higher amounts of lac repressor, the Green Fluorescent Protein fusions with CAAT enhancer binding protein (C/EBPalpha) and Gal4, elute from the columns when 0.1% Tween-20 is added to the mobile phase . The amount of improvement found depends upon the transcription factor studied and the amount of the protein loaded on the column; lac repressor and Gal4 are eluted in higher amounts over a large range of protein loads while C/EBP shows the greatest effect at low protein loads . This detergent effect is seen when either Sepharose or silica is used for the stationary phase . Including bovine serum albumin in the mobile phase gives a similar though lesser improvement to that observed with Tween-20 . Mg2+ or EDTA in the mobile phase gave similar chromatography for C/EBP; since EDTA protects columns from DNases, its inclusion in the mobile phase is preferred . After extended use, the DNA affinity columns no longer bind transcription factors and this is not due to losses of DNA from the columns . Two simple methods (sodium dodecylsulfate and KSCN) were developed to regenerate such worn out columns. Cell Motil Cytoskeleton, 2004 Apr, 57(4), 233 - 45 The Roadblock light chains are ubiquitous components of cytoplasmic dynein that form homo- and heterodimers; Nikulina K et al.; The Roadblock/LC7 class of light chains associate with the intermediate chains at the base of the soluble dynein particle . In mammals, there are two Roadblock isoforms (Robl1 and Robl2), one of which (Robl2) is differentially expressed in a tissue-dependent manner and is especially prominent in testis . Here we define the alpha helical content of Robl and demonstrate using both the yeast two-hybrid system and in vitro biochemistry that Robl1 and Robl2 are capable of forming homo- and heterodimers . This is the first report of heterodimer formation by any cytoplasmic dynein component, and it further enlarges the number of potential cytoplasmic dynein isoforms available for binding specific cellular cargoes . In addition, we have generated an antibody that specifically recognizes Robl light chains and shows a 5-10 fold preference for Robl2 over Robl1 . Using this antibody, we show that Robl is a ubiquitous cytoplasmic dynein component, being found in samples purified from brain, liver, kidney, and testis . Immunofluorescence analysis reveals that Robl is present in punctate organelles in rat neuroblastoma cells . In testis, Robl is found in Leydig cells, spermatocytes, and sperm flagella . Cell Cycle, 2004 Apr, 3(4), 401 - 3 Epub 2004 Apr 01. Cdk1: unsung hero of S phase? Pacek M, Prokhorova TA, Walter JC. Cdk2 has been viewed as a key cell cycle regulator that is essential for S phase progression . The recent discovery that Cdk2 is not required for cell proliferation in mice now shows that other factors must be able to replace Cdk2 in stimulating DNA replication . Experiments performed in Xenopus egg extracts identify the mitotic protein kinases Cdk1/Cyclin B and Cdk1/Cyclin A as likely candidates . These observations raise the intriguing possibility that Cdk1 normally participates in genome duplication in wild type cells. Science, 2004 Feb 27, 303(5662), 1367 - 70 Epub 2004 Jan 29. A genome-wide screen identifies genes required for centromeric cohesion; Marston AL et al.; During meiosis, two chromosome segregation phases follow a single round of DNA replication . We identified factors required to establish this specialized cell cycle by examining meiotic chromosome segregation in a collection of yeast strains lacking all nonessential genes . This analysis revealed Sgo1, Chl4, and Iml3 to be important for retaining centromeric cohesin until the onset of anaphase II . Consistent with this role, Sgo1 localizes to centromeric regions but dissociates at the onset of anaphase II . The screen described here provides a comprehensive analysis of the genes required for the meiotic cell cycle and identifies three factors important for the stepwise loss of sister chromatid cohesion. Genes Dev, 2004 Jan 15, 18(2), 184 - 95 Rad6 plays a role in transcriptional activation through ubiquitylation of histone H2B; Kao CF et al.; Covalent modifications of the histone N tails play important roles in eukaryotic gene expression . Histone acetylation, in particular, is required for the activation of a subset of eukaryotic genes through the targeted recruitment of histone acetyltransferases . We have reported that a histone C tail modification, ubiquitylation of H2B, is required for optimal expression of several inducible yeast genes, consistent with a role in transcriptional activation . H2B was shown to be ubiquitylated and then deubiquitylated at the GAL1 core promoter following galactose induction . We now show that the Rad6 protein, which catalyzes monoubiquitylation of H2B, is transiently associated with the GAL1 promoter upon gene activation, and that the period of its association temporally overlaps with the period of H2B ubiquitylation . Rad6 promoter association depends on the Gal4 activator and the Rad6-associated E3 ligase, Bre1, but is independent of the histone acetyltransferase, Gcn5 . The SAGA complex, which contains a ubiquitin protease that targets H2B for deubiquitylation, is recruited to the GAL1 promoter in the absence of H2B ubiquitylation . The data suggest that Rad6 and SAGA function independently during galactose induction, and that the staged recruitment of these two factors to the GAL1 promoter regulates the ubiquitylation and deubiquitylation of H2B . We additionally show that both Rad6 and ubiquitylated H2B are absent from two regions of transcriptionally silent chromatin but present at genes that are actively transcribed . Thus, like histone H3 lysine 4 and lysine 79 methylation, two modifications that it regulates, Rad6-directed H2B ubiquitylation defines regions of active chromatin. Bioinformatics, 2004 Mar 22, 20(5), 750 - 7 Epub 2004 Jan 29. Regression trees for regulatory element identification; Phuong TM et al.; MOTIVATION: The transcription of a gene is largely determined by short sequence motifs that serve as binding sites for transcription factors . Recent findings suggest direct relationships between the motifs and gene expression levels . In this work, we present a method for identifying regulatory motifs . Our method makes use of tree-based techniques for recovering the relationships between motifs and gene expression levels . RESULTS: We treat regulatory motifs and gene expression levels as predictor variables and responses, respectively, and use a regression tree model to identify the structural relationships between them . The regression tree methodology is extended to handle responses from multiple experiments by modifying the split function . The significance of regulatory elements is determined by analyzing tree structures and using a variable importance measure . When applied to two data sets of the yeast Saccharomyces cerevisiae, the method successfully identifies most of the regulatory motifs that are known to control gene transcription under the given experimental conditions, and suggests several new putative motifs . Analysis of the tree structures also reconfirms several pairs of motifs that are known to regulate gene transcription in combination . AVAILABILITY: http://if.kaist.ac.kr/~phuong/RegTree Bioinformatics, 2004 Apr 12, 20(6), 895 - 902 Epub 2004 Jan 29. Mapping Gene Ontology to proteins based on protein-protein interaction data; Deng M et al.; MOTIVATION: Gene Ontology (GO) consortium provides structural description of protein function that is used as a common language for gene annotation in many organisms . Large-scale techniques have generated many valuable protein-protein interaction datasets that are useful for the study of protein function . Combining both GO and protein-protein interaction data allows the prediction of function for unknown proteins . RESULT: We apply a Markov random field method to the prediction of yeast protein function based on multiple protein-protein interaction datasets . We assign function to unknown proteins with a probability representing the confidence of this prediction . The functions are based on three general categories of cellular component, molecular function and biological process defined in GO . The yeast proteins are defined in the Saccharomyces Genome Database (SGD) . The protein-protein interaction datasets are obtained from the Munich Information Center for Protein Sequences (MIPS), including physical interactions and genetic interactions . The efficiency of our prediction is measured by applying the leave-one-out validation procedure to a functional path matching scheme, which compares the prediction with the GO description of a protein's function from the abstract level to the detailed level along the GO structure . For biological process, the leave-one-out validation procedure shows 52% precision and recall of our method, much better than that of the simple guilty-by-association methods. Toxicology, 2004 Feb 15, 195(2-3), 243 - 54 Antiandrogenic activity of extracts of diesel exhaust particles emitted from diesel-engine truck under different engine loads and speeds; Okamura K et al.; To clarify the alteration of androgenic and antiandrogenic activities by diesel engine conditions, we collected diesel exhaust particles (DEP) samples emitted from a diesel-engine truck under different conditions of engine loads and vehicle speeds, and DEP extract (DEPE) samples were prepared from each . The androgenic and antiandrogenic activities of the DEPE samples were examined using a prostate specific antigen (PSA) promoter-luciferase reporter gene assay in PC3/AR human prostate cancer cells . While all DEPE samples did not exhibit androgenic effects, the antiandrogenic effects were enhanced by higher engine load but not by higher vehicle speed . In this study, significant correlations between antiandrogenic and aryl hydrocarbon receptor (AhR) agonistic activities were demonstrated in PC3/AR cells by 16 polycyclic aromatic compounds and beta-naphthoflavone . Yeast two-hybrid assay and cytochrome P450 (CYP) 1A1 promoter-luciferase reporter gene assay showed that the antiandrogenic constituents acting as androgen receptor (AR) antagonists and AhR agonists were increased by only the higher engine load . In conclusion, the antiandrogenic effects of DEPE samples were enhanced by a higher engine load which resulted in DEPC samples with elevated AhR agonistic and AR antagonistic activities. Biochem J, 2004 Apr 1, 379(Pt 1), 23 - 9 Repression of Smad4 transcriptional activity by SUMO modification; Long J et al.; Smad4 plays a key role in TGF-beta (transforming growth factor beta)/Smad-mediated transcriptional responses . We show that Smad4 is sumoylated both in vivo and in vitro . Recent studies showed that sumoylation of Smad4 regulated its stability, but the effect of sumoylation on the intrinsic transcriptional activity of Smad4 was not defined . We show that overexpression of SUMO (small ubiquitin-related modifier)-1 and Ubc9 can inhibit a TGF-beta-responsive reporter gene, whereas co-transfection with SUMO-1 protease-1 (SuPr-1) can increase the TGF-beta response . We show further that mutation of the Smad4 sumoylation sites or co-transfection with SuPr-1 greatly increases Smad4 transcriptional activity . Moreover, direct fusion of SUMO-1 to the sumoylation mutant Smad4 potently inhibits its transcriptional activity . Thus, as it is being rapidly discovered that sumoylation inhibits the activities of many transcription factors, sumoylation also represses Smad4 transcriptional activity . The net effect of sumoylation of Smad4 can therefore be either stimulatory or inhibitory, depending on the target promoter that is analysed. Anal Chem, 2004 Feb 1, 76(3), 720 - 7 Whole protein dissociation in a quadrupole ion trap: identification of an a priori unknown modified protein; Amunugama R et al.; A protein mixture derived from a whole cell lysate fraction of Saccharomyces cerevisiae, which contains roughly 19 proteins, has been analyzed to identify an a priori unknown modified protein using a quadrupole ion trap tandem mass spectrometer . Collection of the experimental data was facilitated by collision-induced dissociation and ion/ion proton-transfer reactions in multistage mass spectrometry procedures . Ion/ion reactions were used to manipulate charge states of both parent ions and product ions for the purpose of concentrating charge into the parent ion of interest and to reduce the product ion charge states for determination of product ion mass and abundance . The identification of the protein was achieved by matching the uninterpreted product ion spectrum against protein sequence databases with varying degrees of annotation, coupled with a scoring scheme weighted for the relative abundances of the experimentally observed product ions and the frequency of fragmentations occurring at preferential sites . The protein was identified to be an acetylated yeast heat shock protein, HS12_Yeast (11.6 kDa), with the initiating methionine residue removed . This constitutes the first example of the identification of an a priori unknown protein that is not present in an annotated protein database using a "top-down" approach with a quadrupole ion trap . This example illustrates the utility of relatively low cost instrumentation with modest mass analysis characteristics for the identification of modified proteins without recourse to enzymatic digestion . It also illustrates how experimental data can be used interactively with protein databases when the modified protein of interest is not initially present in the database. J Org Chem, 2004 Feb 6, 69(3), 613 - 8 The first direct evaluation of the two-active site mechanism for chitin synthase; Yeager AR et al.; Chitin synthase polymerizes UDP-GlcNAc to form chitin (poly-beta(1,4)-GlcNAc) and is essential for fungal cell wall biosynthesis . The alternating orientation of the GlcNAc residues within the chitin chain has led to the proposal that chitin synthase possesses two active sites . We report the results of the first direct test of this possibility . Two simple uridine-derived dimeric inhibitors are shown to exhibit 10-fold greater inhibition than a monomeric control, consistent with the presence of two active sites . This observation has important implications for the development of antifungal agents, as well as the understanding of polymerizing glycosyltransferases. Plant Mol Biol, 2003 Oct, 53(3), 383 - 97 Molecular characterization of Brassica napus NAC domain transcriptional activators induced in response to biotic and abiotic stress; Hegedus D et al.; Subtractive expressed sequence tag analysis and screening of cDNA libraries derived from Brassica napus leaves subjected to mechanical wounding, flea beetle feeding or cold temperatures revealed eight genes encoding NAC-domain transcription factors . The genes were found to be differentially regulated in response to biotic and abiotic stresses including wounding, insect feeding, Sclerotinia sclerotiorum infection, cold shock and dehydration . Five BnNAC proteins were orthologous to Arabidopsis thaliana ATAF1 or ATAF2 and gave rise to developmental abnormalities similar to the A . thaliana nam and cuc mutants when expressed ectopically in A . thaliana . Transgenic lines expressing BnNAC14, exhibited large leaves, thickened stems and hyper-developed lateral root systems similar to that observed with A . thaliana NAC1, but also were delayed in bolting and lacked an apical dominant tap root . Several of the BnNAC proteins were capable of activating gene expression in yeast and recognized an element within the CaMV35S promoter . A yeast two-hybrid screen revealed that BnNAC14 interacted with other select BnNAC proteins in vitro and identified an additional BnNAC gene, BnNAC485 . The protein interaction and transcriptional activation domains were mapped by deletion analysis. Plant Mol Biol, 2003 Oct, 53(3), 273 - 9 Identification of rice (Oryza sativa) proteins linked to the cyclin-mediated regulation of the cell cycle; Cooper B et al.; Yeast two-hybrid assays were used to identify rice proteins interacting with two rice cyclins and other proteins potentially involved in cell cycling . The DNA sequences encoding 119 protein fragments identified were then compared by BLAST against proteins in GenBank . The proteins found include myosin-like proteins, transcription factors, kinesins, centromere proteins and undefined proteins . Based on interactions with cyclins and other elements required for cycling, we believe the undefined proteins may be involved in associated cycling processes . The identification of proteins involved in cell cycle regulation in rice may allow for the control of agronomic traits involving plant growth or development. EMBO J, 2004 Feb 11, 23(3), 616 - 26 Epub 2004 Jan 29. Human Fip1 is a subunit of CPSF that binds to U-rich RNA elements and stimulates poly(A) polymerase; Kaufmann I et al.; In mammals, polyadenylation of mRNA precursors (pre-mRNAs) by poly(A) polymerase (PAP) depends on cleavage and polyadenylation specificity factor (CPSF) . CPSF is a multisubunit complex that binds to the canonical AAUAAA hexamer and to U-rich upstream sequence elements on the pre-mRNA, thereby stimulating the otherwise weakly active and nonspecific polymerase to elongate efficiently RNAs containing a poly(A) signal . Based on sequence similarity to the Saccharomyces cerevisiae polyadenylation factor Fip1p, we have identified human Fip1 (hFip1) and found that the protein is an integral subunit of CPSF . hFip1 interacts with PAP and has an arginine-rich RNA-binding motif that preferentially binds to U-rich sequence elements on the pre-mRNA . Recombinant hFip1 is sufficient to stimulate the in vitro polyadenylation activity of PAP in a U-rich element-dependent manner . hFip1, CPSF160 and PAP form a ternary complex in vitro, suggesting that hFip1 and CPSF160 act together in poly(A) site recognition and in cooperative recruitment of PAP to the RNA . These results show that hFip1 significantly contributes to CPSF-mediated stimulation of PAP activity. Mol Cell Biol, 2004 Feb, 24(4), 1758 - 68 TRAM2 protein interacts with endoplasmic reticulum Ca2+ pump Serca2b and is necessary for collagen type I synthesis; Stefanovic B et al.; Cotranslational insertion of type I collagen chains into the lumen of the endoplasmic reticulum (ER) and their subsequent folding into a heterotrimeric helix is a complex process which requires coordinated action of the translation machinery, components of translocons, molecular chaperones, and modifying enzymes . Here we describe a role for the protein TRAM2 in collagen type I expression in hepatic stellate cells (HSCs) and fibroblasts . Activated HSCs are collagen-producing cells in the fibrotic liver . Quiescent HSCs produce trace amounts of type I collagen, while upon activation collagen synthesis increases 50- to 70-fold . Likewise, expression of TRAM2 dramatically increases in activated HSCs . TRAM2 shares 53% amino acid identity with the protein TRAM, which is a component of the translocon . However, TRAM2 has a C terminus with only a 15% identity . The C-terminal part of TRAM2 interacts with the Ca(2+) pump of the ER, SERCA2b, as demonstrated in a Saccharomyces cerevisiae two-hybrid screen and by immunoprecipitations in human cells . TRAM2 also coprecipitates with anticollagen antibody, suggesting that these two proteins interact . Deletion of the C-terminal part of TRAM2 inhibits type I collagen synthesis during activation of HSCs . The pharmacological inhibitor of SERCA2b, thapsigargin, has a similar effect . Depletion of ER Ca(2+) with thapsigargin results in inhibition of triple helical collagen folding and increased intracellular degradation . We propose that TRAM2, as a part of the translocon, is required for the biosynthesis of type I collagen by coupling the activity of SERCA2b with the activity of the translocon . This coupling may increase the local Ca(2+) concentration at the site of collagen synthesis, and a high Ca(2+) concentration may be necessary for the function of molecular chaperones involved in collagen folding. Mol Cell Biol, 2004 Feb, 24(4), 1721 - 35 Two cyclin-dependent kinases promote RNA polymerase II transcription and formation of the scaffold complex; Liu Y et al.; Three cyclin-dependent kinases, CDK7, -8, and -9, are specifically involved in transcription by RNA polymerase II (Pol II) and target the Pol II C-terminal domain (CTD) . The role of CDK7 and CDK8 kinase activity in transcription has been unclear, with CDK7 shown to have variable effects on transcription and CDK8 suggested to repress transcription and/or to target other gene-specific factors . Using a chemical genetics approach, the Saccharomyces cerevisiae homologs of these kinases, Kin28 and Srb10, were engineered to respond to a specific inhibitor and the inhibitor was used to test the role of these kinases in transcription in vivo and in vitro . In vitro, these kinases can both promote transcription, with up to 70% of transcription abolished when both kinases are inhibited together . Similarly, in vivo inhibition of both kinases together gives the strongest decrease in transcription, as measured by chromatin immunoprecipitation of Pol II . Kin28 and Srb10 also have overlapping roles in promoting ATP-dependent dissociation of the preinitiation complex (PIC) into the Scaffold complex . Using the engineered kinases and an ATP analog, specific kinase substrates within the PIC were identified . In addition to the previously known substrate, the Pol II CTD, it was found that Kin28 phosphorylates two subunits of Mediator and Srb10 targets two subunits of TFIID for phosphorylation. Mol Cell Biol, 2004 Feb, 24(4), 1709 - 20 RNA polymerase II (Pol II)-TFIIF and Pol II-mediator complexes: the major stable Pol II complexes and their activity in transcription initiation and reinitiation; Rani PG et al.; Protein purification and depletion studies were used to determine the major stable forms of RNA polymerase II (Pol II) complexes found in Saccharomyces cerevisiae nuclear extracts . About 50% of Pol II is found associated with the general transcription factor TFIIF (Pol II-TFIIF), and about 20% of Pol II is associated with Mediator (Pol-Med) . No Pol II-Med-TFIIF complex was observed . The activity of Pol II and the purified Pol II complexes in transcription initiation and reinitiation was investigated by supplementing extracts depleted of either total Pol II or total TFIIF with purified Pol II or the Pol II complexes . We found that all three forms of Pol II can complement Pol II-depleted extracts for transcription initiation, but Pol II-TFIIF has the highest specific activity . Similarly, Pol II-TFIIF has a much higher specific activity than TFIIF for complementation of TFIIF transcription activity . Although the Pol II-TFIIF and Pol II-Med complexes were stable when purified, we found these complexes were dynamic in extracts under transcription conditions, with a single polymerase capable of exchanging bound Mediator and TFIIF . Using a purified system to examine transcription reinitiation, we found that Pol II-TFIIF was active in promoting multiple rounds of transcription while Pol II-Med was nearly inactive . These results suggest that both the Pol II-Med and Pol II-TFIIF complexes can be recruited for transcription initiation but that only the Pol II-TFIIF complex is competent for transcription reinitiation. Org Lett, 2004 Feb 5, 6(3), 321 - 4 Water-soluble camptothecin derivatives that are intrinsic topoisomerase I poisons; Rahier NJ et al.; {structure: see text} In an effort to improve the water solubility of camptothecin, four 20-O-phosphate and phosphonate analogues have been prepared . These analogues are freely water soluble, stable at physiological pH, and stabilize the human topoisomerase I-DNA covalent binary complex with the same sequence selectivity as camptothecin itself . All four compounds inhibited the growth of yeast expressing human topoisomerase I in an enzyme-dependent fashion. Acta Crystallogr D Biol Crystallogr, 2004 Feb, 60(Pt 2), 317 - 9 Epub 2004 Jan 23. Crystallization and preliminary crystallographic analysis of rat monoamine oxidase A complexed with clorgyline; Ma J et al.; Monoamine oxidase (MAO) is an FAD-containing mitochondrial outer-membrane protein which catalyzes the degradation of several neurotransmitters in the central nervous system . The two subtypes of MAO, MAOA and MAOB, have similar primary sequences but different substrate and inhibitor specificities . The structure of human MAOB has recently been determined, but the structure of MAOA remains unknown . To clarify the mechanisms underlying their unique substrate and inhibitor recognition and thereby facilitate the development of new specific inhibitors to treat MAO-related neurological disorders, rat MAOA was crystallized in a complex with the specific inhibitor clorgyline . Diffraction data were collected to 3.2 A resolution . The crystal belongs to the space group P4(3)2(1)2, with unit-cell parameters a = b = 158.2, c = 258.4 A. Trends Genet, 2004 Feb, 20(2), 72 - 6 Conservation of protein-protein interactions - lessons from ascomycota; Pagel P et al.; Interacting proteins from Saccharomyces cerevisiae are evolutionarily conserved and their likelihood of having an ortholog in other ascomycota species correlates with the number of interaction partners . Moreover, interacting proteins show a clear preference to be conserved as a pair, indicating that nature maintains selection pressure on the interaction links between proteins . The conservation of interacting protein pairs between different organisms does not exhibit any bias with respect to protein functional roles. Cell, 2004 Jan 23, 116(2), 153 - 66 The mechanisms of vesicle budding and fusion; Bonifacino JS et al.; Genetic and biochemical analyses of the secretory pathway have produced a detailed picture of the molecular mechanisms involved in selective cargo transport between organelles . This transport occurs by means of vesicular intermediates that bud from a donor compartment and fuse with an acceptor compartment . Vesicle budding and cargo selection are mediated by protein coats, while vesicle targeting and fusion depend on a machinery that includes the SNARE proteins . Precise regulation of these two aspects of vesicular transport ensures efficient cargo transfer while preserving organelle identity. Nat Cell Biol, 2004 Feb, 6(2), 138 - 45 Epub 2004 Jan 25. The DNA damage checkpoint and PKA pathways converge on APC substrates and Cdc20 to regulate mitotic progression; Searle JS et al.; The conserved checkpoint kinases Chk1 and Rad53-Dun1 block the metaphase to anaphase transition by the phosphorylation and stabilization of securin, and block the mitotic exit network regulated by the Bfa1-Bub2 complex . However, both chk1 and rad53 mutants are able to exit from mitosis and initiate a new cell cycle, suggesting that both pathways have supporting functions in restraining anaphase and in blocking the inactivation of mitotic cyclin-Cdk1 complexes . Here we find that the cyclic-AMP-dependent protein kinase (PKA) pathway supports Chk1 in the regulation of mitosis by targeting the mitotic inducer Cdc20 . Cdc20 is phosphorylated on PKA consensus sites after DNA damage, and this phosphorylation requires the Atr orthologue Mec1 and the PKA catalytic subunits Tpk1 and Tpk2 . We show that the inactivation of PKA or expression of phosphorylation-defective Cdc20 proteins accelerates securin and Clb2 destruction in chk1 mutants and is sufficient to remove most of the DNA damage-induced delay . Mutation of the Cdc20 phosphorylation sites permitted the interaction of Cdc20 with Clb2 under conditions that should halt cell cycle progression . These data show that PKA pathways regulate mitotic progression through Cdc20 and support the DNA damage checkpoint pathways in regulating the destruction of Clb2 and securin. Mol Biol Cell, 2004 Apr, 15(4), 1785 - 92 Epub 2004 Jan 23. Sti1 and Cdc37 can stabilize Hsp90 in chaperone complexes with a protein kinase; Lee P et al.; Hsp90 functions in association with several cochaperones for folding of protein kinases and transcription factors, although the relative contribution of each to the overall reaction is unknown . We assayed the role of nine different cochaperones in the activation of Ste11, a Saccharomyces cerevisiae mitogen-activated protein kinase kinase kinase . Studies on signaling via this protein kinase pathway was measured by alpha-factor-stimulated induction of FIG1 or lacZ, and repression of HHF1 . Several cochaperone mutants tested had reduced FIG1 induction or HHF1 repression, although to differing extents . The greatest defects were in cpr7Delta, sse1Delta, and ydj1Delta mutants . Assays of Ste11 kinase activity revealed a pattern of defects in the cochaperone mutant strains that were similar to the gene expression studies . Overexpression of CDC37, a chaperone required for protein kinase folding, suppressed defects the sti1Delta mutant back to wild-type levels . CDC37 overexpression also restored stable Hsp90 binding to the Ste11 protein kinase domain in the sti1Delta mutant strain . These data suggest that Cdc37 and Sti1 have functional overlap in stabilizing Hsp90:client complexes . Finally, we show that Cns1 functions in MAP kinase signaling in association with Cpr7. Mol Biol Cell, 2004 Apr, 15(4), 1736 - 45 Epub 2004 Jan 23. Identification of protein complexes required for efficient sister chromatid cohesion; Mayer ML et al.; Ctf8p is a component of Ctf18-RFC, an alternative replication factor C-like complex required for efficient sister chromatid cohesion in Saccharomyces cerevisiae . We performed synthetic genetic array (SGA) analysis with a ctf8 deletion strain as a primary screen to identify other nonessential genes required for efficient sister chromatid cohesion . We then assessed proficiency of cohesion at three chromosomal loci in strains containing deletions of the genes identified in the ctf8 SGA screen . Deletion of seven genes (CHL1, CSM3, BIM1, KAR3, TOF1, CTF4, and VIK1) resulted in defective sister chromatid cohesion . Mass spectrometric analysis of immunoprecipitated complexes identified a physical association between Kar3p and Vik1p and an interaction between Csm3p and Tof1p that we confirmed by coimmunoprecipitation from cell extracts . These data indicate that synthetic genetic array analysis coupled with specific secondary screens can effectively identify protein complexes functionally related to a reference gene . Furthermore, we find that genes involved in mitotic spindle integrity and positioning have a previously unrecognized role in sister chromatid cohesion. Mol Biol Cell, 2004 Apr, 15(4), 1623 - 34 Epub 2004 Jan 23. Mutant telomere sequences lead to impaired chromosome separation and a unique checkpoint response; Lin J et al.; Mutation of the template region in the RNA component of telomerase can cause incorporation of mutant DNA sequences at telomeres . We made all 63 mutant sequence combinations at template positions 474-476 of the yeast telomerase RNA, TLC1 . Mutants contained faithfully incorporated template mutations, as well as misincorporated sequences in telomeres, a phenotype not previously reported for Saccharomyces cerevisiae telomerase template mutants . Although growth rates and telomere profiles varied widely among the tlc1 mutants, chromosome separation and segregation were always aberrant . The mutants showed defects in sister chromatid separation at centromeres as well as telomeres, suggesting activation of a cell cycle checkpoint . Deletion of the DNA damage response genes DDC1, MEC3, or DDC2/SML1 failed to restore chromosome separation in the tlc1 template mutants . These results suggest that mutant telomere sequences elicit a checkpoint that is genetically distinct from those activated by deletion of telomerase or DNA damage. Mol Biol Cell, 2004 Apr, 15(4), 1802 - 15 Epub 2004 Jan 23. Positive and negative regulation of a SNARE protein by control of intracellular localization; Nakanishi H et al.; In Saccharomyces cerevisiae, the developmentally regulated Soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) protein Spo20p mediates the fusion of vesicles with the prospore membrane, which is required for the formation of spores . Spo20p is subject to both positive and negative regulation by separate sequences in its aminoterminal domain . We report that the positive activity is conferred by a short, amphipathic helix that is sufficient to confer plasma membrane or prospore membrane localization to green fluorescent protein . In vitro, this helix binds to acidic phospholipids, and mutations that reduce or eliminate phospholipid binding in vitro inactivate Spo20p in vivo . Genetic manipulation of phospholipid pools indicates that the likely in vivo ligand of this domain is phosphatidic acid . The inhibitory activity is a nuclear targeting signal, which confers nuclear localization in vegetative cells and in cells entering meiosis . However, as cells initiate spore formation, fusions containing the inhibitory domain exit the nucleus and localize to the nascent prospore membrane . Thus, the SNARE Spo20p is both positively and negatively regulated by control of its intracellular localization. Infect Immun, 2004 Feb, 72(2), 949 - 57 CpG oligodeoxynucleotide and Montanide ISA 51 adjuvant combination enhanced the protective efficacy of a subunit malaria vaccine; Kumar S et al.; Unmethylated CpG dinucleotide motifs present in bacterial genomes or synthetic oligodeoxynucleotides (ODNs) serve as strong immunostimulatory agents in mice, monkeys and humans . We determined the adjuvant effect of murine CpG ODN 1826 on the immunogenicity and protective efficacy of the Saccharomyces cerevisiae-expressed 19-kDa C-terminal region of merozoite surface protein 1 (yMSP1(19)) of the murine malaria parasite Plasmodium yoelii . We found that in C57BL/6 mice, following sporozoite challenge, the degree of protective immunity against malaria induced by yMSP1(19) in a formulation of Montanide ISA 51 (ISA) plus CpG ODN 1826 was similar or superior to that conferred by yMSP1(19) emulsified in complete Freund's adjuvant (CFA/incomplete Freund's adjuvant) . In total, among mice immunized with yMSP1(19), 22 of 32 (68.7%) with ISA plus CpG 1826, 0 of 4 (0%) with CFA/incomplete Freund's adjuvant, 0 of 4 (0%) with CpG 1826 mixed with ISA (no yMSP1(19)), and 0 of 11 (0%) with CpG 1826 alone were completely protected against development of erythrocytic stage infection after sporozoite challenge . The adjuvant effect of CpG ODN 1826 was manifested as both significantly improved complete protection from malaria (defined as the absence of detectable erythrocytic form parasites) (P = 0.007, chi square) and reduced parasite burden in infected mice . In vivo depletions of interleukin-12 and gamma interferon cytokines and CD4+ and CD8+ T cells in vaccinated mice had no significant effect on immunity . On the other hand, immunoglobulin G (IgG) isotype levels appeared to correlate with protection . Inclusion of CpG ODN 1826 in the yMSP1(19) plus ISA vaccine contributed towards the induction of higher levels of IgG2a and IgG2b (Th1 type) antibodies, suggesting that CpG ODN 1826 caused a shift towards a Th1 type of immune response that could be responsible for the higher degree of protective immunity . Our results indicate that this potent adjuvant formulation should be further evaluated for use in clinical trials of recombinant malarial vaccine candidates. Biochim Biophys Acta, 2004 Feb 2, 1644(1), 47 - 59 Identification of major proteins in the lipid droplet-enriched fraction isolated from the human hepatocyte cell line HuH7; Fujimoto Y et al.; Recent studies have revealed the presence of intracellular lipid droplets in wide variety of species . In mammalian cells, there exist proteins specifically localize in lipid droplets . However, the protein profile in the droplet remains yet to be clarified . In this study, a fraction enriched with lipid droplets was isolated from a human hepatocyte cell line HuH7 using sucrose density gradient centrifugation, and 17 major proteins in the fraction were identified using nano LC-MS/MS techniques . Adipose differentiation-related protein (ADRP) was the most abundant protein in the fraction . The secondary abundant proteins were identified to be acyl-CoA synthetase 3 (ACS3) and 17beta-hydroxysteroid dehydrogenase 11 (17betaHSD11) . Included in the identified proteins were five lipid-metabolizing enzymes as well as two lipid droplet-specific proteins . When HuH7 cell lysate was fractionated by a density gradient, most of 17betaHSD11 was found in the droplet-enriched fraction . In immunocytochemical analysis, 17betaHSD11 showed ring-shaped images which overlapped with those for ADRP . These results suggest that a specific set of proteins is enriched in the lipid droplet-enriched fraction and that 17betaHSD11 localizes specifically in the fraction. FEBS Lett, 2004 Jan 16, 557(1-3), 185 - 92 Impas 1 possesses endoproteolytic activity against multipass membrane protein substrate cleaving the presenilin 1 holoprotein; Moliaka YK et al.; Presenilins (PS1 and PS2) are supposed to be unusual aspartic proteases and components of the gamma-secretase complex regulating cleavage of type I proteins . Multiple mutations in PS1 are a major cause of familial early-onset Alzheimer's disease (AD) . We and others recently identified PS-related families of proteins (IMPAS/PSH/signal peptide peptidases (SPP)) . The functions of these proteins are yet to be determined . We found that intramembrane protease-associated or intramembrane protease aspartic protein Impas 1 (IMP1)/SPP induces intramembranous cleavage of PS1 holoprotein in cultured cells coexpressing these proteins . Mutations in evolutionary invariant sites in hIMP1 or specific gamma-secretase inhibitors abolish the hIMP1-mediated endoproteolysis of PS1 . In contrast, neither AD-like mutations in hIMP1 nor in PS1 substrate abridge the PS1 cleavage . The data suggest that IMP1 is a bi-aspartic polytopic protease capable of cleaving transmembrane precursor proteins . These data, to our knowledge, are a first observation that a multipass transmembrane protein or the integral protease per se may be a primary substrate for an intramembranous proteolysis. EMBO J, 2004 Jan 28, 23(2), 333 - 42 Epub 2004 Jan 22. Nhp6 facilitates Aft1 binding and Ssn6 recruitment, both essential for FRE2 transcriptional activation; Fragiadakis GS et al.; We found Nhp6a/b yeast HMG-box chromatin-associated architectural factors and Ssn6 (Cyc8) corepressor to be crucial transcriptional coactivators of FRE2 gene . FRE2 encoding a plasma membrane ferric reductase is induced by the iron-responsive, DNA-binding, transcriptional activator Aft1 . We have shown that Nhp6 interacts directly with the Aft1 N-half, including the DNA-binding region, to facilitate Aft1 binding at FRE2 UAS . Ssn6 also interacts directly with the Aft1 N-half and is recruited on FRE2 promoter only in the presence of both Aft1 and Nhp6 . This Nhp6/Ssn6 role in Aft1-mediated transcription is FRE2 promoter context specific, and both regulators are required for activation-dependent chromatin remodeling . Our results provide the first in vivo biochemical evidence for nonsequence-specific HMG-box protein-facilitated recruitment of a yeast gene-specific transactivator to its DNA target site and for Nhp6-mediated Ssn6 promoter recruitment . Ssn6 has an explicitly coactivating role on FRE2 promoter only upon induction . Therefore, transcriptional activation in response to iron availability involves multiple protein interactions between the Aft1 iron-responsive DNA-binding factor and global regulators such as Nhp6 and Ssn6. J Biomol NMR, 2004 Jan, 28(1), 59 - 67 New applications of 2D filtered/edited NOESY for assignment and structure elucidation of RNA and RNA-protein complexes; Peterson RD et al.; NMR spectra of large RNAs are difficult to assign because of extensive spectral overlap and unfavorable relaxation properties . Here we present a new approach to facilitate assignment of RNA spectra using a suite of four 2D-filtered/edited NOESY experiments in combination with base-type-specific isotopically labeled RNA . The filtering method was developed for use in 3D filtered NOESY experiments (Zwahlen et al., 1997), but the 2D versions are both more sensitive and easier to interpret for larger RNAs than their 3D counterparts . These experiments are also useful for identifying intermolecular NOEs in RNA-protein complexes . Applications to NOE assignment of larger RNAs and an RNA-protein complex are presented. Nucleic Acids Res, 2004 Jan 22, 32(2), 447 - 55 Print 2004. Statistical resynchronization and Bayesian detection of periodically expressed genes; Lu X et al.; We propose a periodic-normal mixture (PNM) model to fit transcription profiles of periodically expressed (PE) genes in cell cycle microarray experiments . The model leads to a principled statistical estimation procedure that produces more accurate estimates of the mean cell cycle length and the gene expression periodicity than existing heuristic approaches . A central component of the proposed procedure is the resynchronization of the observed transcription profile of each PE gene according to the PNM with estimated periodicity parameters . By using a two-component mixture-Beta model to approximate the PNM fitting residuals, we employ an empirical Bayes method to detect PE genes . We estimate that about one-third of the genes in the genome of Saccharomyces cerevisiae are likely to be transcribed periodically, and identify 822 genes whose posterior probabilities of being PE are greater than 0.95 . Among these 822 genes, 540 are also in the list of 800 genes detected by Spellman . Gene ontology annotation analysis shows that many of the 822 genes were involved in important cell cycle-related processes, functions and components . When matching the 822 resynchronized expression profiles of three independent experiments, little phase shifts were observed, indicating that the three synchronization methods might have brought cells to the same phase at the time of release. Curr Biol, 2004 Jan 20, 14(2), R70 - 2 Cell division: AAAtacking the mitotic spindle; Cheeseman IM et al.; Cell division requires the assembly of a microtubule-based spindle which captures and segregates sister chromatids . But how is this spindle broken down once chromosome segregation is complete? New evidence implicates a highly conserved AAA-ATPase in spindle disassembly at the end of mitosis. Curr Biol, 2004 Jan 20, 14(2), 125 - 30 Condensin regulates rDNA silencing by modulating nucleolar Sir2p; Machin F et al.; The tandem array of ribosomal DNA (rDNA) in Saccharomyces cerevisiae is subjected to transcriptional silencing of RNA polymerase II-transcribed genes . This form of silencing depends on SIR2 and has been tightly linked to the suppression of rDNA recombination and the control of cellular lifespan . Paradoxically, rDNA silencing takes place in the context of an extremely high rate of RNA polymerase I transcription . Because rDNA silencing requires different factors than HMR and telomere silencing, the chromatin structure and the mechanisms of silencing must be fundamentally different . Here we show that yeast condensin organizes the specialized topology of rDNA chromatin . We then demonstrate that this function is necessary for maintaining the correct balance of telomeric and nucleolar Sir2p . Condensin mutants relocalize telomeric Sir2p to rDNA and show histone hyperacetylation at telomeres . Our data reveal the implication of yeast condensin in the arrangement of rDNA repeats into a heterochromatic-like structure that is important for the correct delineation of silencing domains in the nucleus. Nature, 2004 Jan 22, 427(6972), 370 - 4 The MAPK Hog1 recruits Rpd3 histone deacetylase to activate osmoresponsive genes; De Nadal E et al.; Regulation of gene expression by mitogen-activated protein kinases (MAPKs) is essential for proper cell adaptation to extracellular stimuli . Exposure of yeast cells to high osmolarity results in rapid activation of the MAPK Hog1, which coordinates the transcriptional programme required for cell survival on osmostress . The mechanisms by which Hog1 and MAPKs in general regulate gene expression are not completely understood, although Hog1 can modify some transcription factors . Here we propose that Hog1 induces gene expression by a mechanism that involves recruiting a specific histone deacetylase complex to the promoters of genes regulated by osmostress . Cells lacking the Rpd3-Sin3 histone deacetylase complex are sensitive to high osmolarity and show compromised expression of osmostress genes . Hog1 interacts physically with Rpd3 in vivo and in vitro and, on stress, targets the deacetylase to specific osmostress-responsive genes . Binding of the Rpd3-Sin3 complex to specific promoters leads to histone deacetylation, entry of RNA polymerase II and induction of gene expression . Together, our data indicate that targeting of the Rpd3 histone deacetylase to osmoresponsive promoters by the MAPK Hog1 is required to induce gene expression on stress. Nucleic Acids Res, 2004 Jan 21, 32(2), 415 - 21 Print 2004. The major 5' determinant in stop codon read-through involves two adjacent adenines; Tork S et al.; The aim of this approach was to identify the major determinants, located at the 5' end of the stop codon, that modulate translational read-through in Saccharomyces cerevisiae . We developed a library of oligonucleotides degenerate at the six positions immediately upstream of the termination codon, cloned in the ADE2 reporter gene . Variations at these positions modulated translational read-through efficiency approximately 16-fold . The major effect was imposed by the two nucleotides immediately upstream of the stop codon . We showed that this effect was neither mediated by the last amino acid residues present in the polypeptide chain nor by the tRNA present in the ribosomal P site . We propose that the mRNA structure, depending on the nucleotides in the P site, is the main 5' determinant of read-through efficiency. Biol Reprod, 2004 May, 70(5), 1527 - 33 Epub 2004 Jan 21. Estrogenic activities of nitrophenols in diesel exhaust particles; Furuta C et al.; We recently isolated 3-methyl-4-nitrophenol (4-nitro-m-cresol; PNMC) and 4-nitro-3-phenylphenol (PNMPP) from diesel exhaust particles (DEP) and identified them as vasodilators . Because these compounds are alkylphenolic derivatives that might mimic hormones, we evaluated their estrogenic activity by using recombinant yeast screens, myometrial contractility assays, and in vivo uterotrophic assays . Recombinant yeast screen assays showed that both PNMC and PNMPP possess estrogenic activity . Furthermore, ovariectomized 25-day-old immature female rats injected with PNMC and PNMPP subcutaneously for 2 days showed significant increases in uterine weight among those receiving 100 mg/kg PNMC and 0.1 and 1.0 mg/kg PNMPP . To clarify further the estrogenic activity of PNMC and PNMPP, rat uterine horns were monitored in organ bath chambers for myometrial contractility in response to oxytocin (OT) . Significant differences occurred in the initial and maximum contractilities to OT at 0.25 and 25 mIU/ml in uterine horns obtained from animals treated with 100 mg/kg PNMC and in the maximum contractilities to OT at 0.025, 0.25, and 25 mIU/ml in those from rats treated with 0.1 mg/kg PNMPP . These results clearly demonstrated that PNMC and PNMPP in DEP have estrogenic activity both in vitro and in vivo and might therefore be considered as endocrine-disrupting chemicals. Proc Natl Acad Sci U S A, 2004 Feb 3, 101(5), 1200 - 5 Epub 2004 Jan 20. A genomewide oscillation in transcription gates DNA replication and cell cycle; Klevecz RR et al.; Microarray analysis from a yeast continuous synchrony culture system shows a genomewide oscillation in transcription . Maximums in transcript levels occur at three nearly equally spaced intervals in this approximately 40-min cycle of respiration and reduction . Two temporal clusters (4,679 of 5,329) are maximally expressed during the reductive phase of the cycle, whereas a third cluster (650) is maximally expressed during the respiratory phase . Transcription is organized functionally into redox-state superclusters with genes known to be important in respiration or reduction being synthesized in opposite phases of the cycle . The transcriptional cycle gates synchronous bursts in DNA replication in a constant fraction of the population at 40-min intervals . Restriction of DNA synthesis to the reductive phase of the cycle may be an evolutionarily important mechanism for reducing oxidative damage to DNA during replication. J Biol Chem, 2004 Apr 2, 279(14), 13616 - 23 Epub 2004 Jan 20. Systematic mutagenesis of the leucine-rich repeat (LRR) domain of CCR4 reveals specific sites for binding to CAF1 and a separate critical role for the LRR in CCR4 deadenylase activity; Clark LB et al.; CCR4, a poly(A) deadenylase of the exonuclease III family, is a component of the multiprotein CCR4-NOT complex of Saccharomyces cerevisiae that is involved in mRNA degradation . CCR4, unlike all other exonuclease III family members, contains a leucine-rich repeat (LRR) motif through which it makes contact to CAF1 and other factors . The LRR residues important in contacting CAF1 were identified by constructing 29 CCR4 mutations encompassing a majority (47 of 81) of residues interstitial to the conserved structural residues . Two-hybrid and immunoprecipitation data revealed that physical contact between CAF1 and the LRR is blocked by mutation of just two alpha-helix/beta-helix strand loop residues linking the first and second repeats . In contrast, CAF16, a potential ligand of CCR4, was abrogated in its binding to the LRR by mutations in the N terminus of the second beta-strand . The LRR domain was also found to contact the deadenylase domain of CCR4, and deletion of the LRR region completely inhibited CCR4 enzymatic activity . Mutations throughout the beta-sheet surface of the LRR, including those that did not specifically interfere with contacts to CAF1 or CAF16, significantly reduced CCR4 deadenylase activity . These results indicate that the CCR4-LRR, in addition to binding to CAF1, plays an essential role in the CCR4 deadenylation of mRNA. J Cell Biol, 2004 Jan 19, 164(2), 219 - 31 Novel regulation of mitotic exit by the Cdc42 effectors Gic1 and Gic2; Hofken T et al.; The guanine nucleotide exchange factor Cdc24, the GTPase Cdc42, and the Cdc42 effectors Cla4 and Ste20, two p21-activated kinases, form a signal transduction cascade that promotes mitotic exit in yeast . We performed a genetic screen to identify components of this pathway . Two related bud cortex-associated Cdc42 effectors, Gic1 and Gic2, were obtained as factors that promoted mitotic exit independently of Ste20 . The mitotic exit function of Gic1 was dependent on its activation by Cdc42 and on the release of Gic1 from the bud cortex . Gic proteins became essential for mitotic exit when activation of the mitotic exit network through Cdc5 polo kinase and the bud cortex protein Lte1 was impaired . The mitotic exit defect of cdc5-10 Deltalte1 Deltagic1 Deltagic2 cells was rescued by inactivation of the inhibiting Bfa1-Bub2 GTPase-activating protein . Moreover, Gic1 bound directly to Bub2 and prevented binding of the GTPase Tem1 to Bub2 . We propose that in anaphase the Cdc42-regulated Gic proteins trigger mitotic exit by interfering with Bfa1-Bub2 GTPase-activating protein function. J Cell Biol, 2004 Jan 19, 164(2), 207 - 18 Regulation of polarized growth initiation and termination cycles by the polarisome and Cdc42 regulators; Bidlingmaier S et al.; The dynamic regulation of polarized cell growth allows cells to form structures of defined size and shape . We have studied the regulation of polarized growth using mating yeast as a model . Haploid yeast cells treated with high concentration of pheromone form successive mating projections that initiate and terminate growth with regular periodicity . The mechanisms that control the frequency of growth initiation and termination under these conditions are not well understood . We found that the polarisome components Spa2, Pea2, and Bni1 and the Cdc42 regulators Cdc24 and Bem3 control the timing and frequency of projection formation . Loss of polarisome components and mutation of Cdc24 decrease the frequency of projection formation, while loss of Bem3 increases the frequency of projection formation . We found that polarisome components and the cell fusion proteins Fus1 and Fus2 are important for the termination of projection growth . Our results define the first molecular regulators that control the timing of growth initiation and termination during eukaryotic cell differentiation. FEMS Yeast Res, 2004 Jan, 4(4-5), 459 - 65 ATG23, a novel gene required for maturation of proaminopeptidase I, but not for autophagy; Meiling-Wesse K et al.; In rich media proaminopeptidase I is targeted to the vacuole via the Cvt pathway and during starvation via autophagy . We here identify Atg23 (Ylr431c), a protein of so far unknown function, as a novel component essential for proaminopeptidase I maturation under non-starvation conditions . Maturation of proaminopeptidase I takes place in starved atg23Delta cells . Selective vacuolar targeting of the autophagosomal marker GFP-Aut7 and the accumulation of autophagic bodies during starvation in the presence of phenylmethylsulfonyl fluoride suggest that autophagy occurs in atg23Delta cells but at a reduced rate . In atg23Delta cells mature vacuolar carboxypeptidase Y is present and accumulation of quinacrine suggests no significant defect in vacuolar acidification . Furthermore, growth of atg23Delta cells on nitrocellulose detects no significant secretion of carboxypeptidase Y. Biochem Biophys Res Commun, 2004 Feb 6, 314(2), 586 - 96 Sorting pathway and molecular targeting signals for the Arabidopsis peroxin 3; Hunt JE et al.; Peroxin 3 (Pex3p) has been identified and characterized as a peroxisomal membrane protein in yeasts and mammals . We identified two putative homologs in Arabidopsis (AtPex3p, forms 1 and 2), both with an identical cluster of positively charged amino acid residues (RKHRRK) immediately preceding one of the two predicted transmembrane domains (TMD1) . In transiently transformed Arabidopsis and tobacco BY-2 suspension-cultured cells, epitope-tagged AtPex3p (form 2) sorted post-translationally from the cytosol directly to peroxisomes, the first sorting pathway described for any peroxin in plants . TMD1 and RKHRRK were necessary for targeting form 2 to peroxisomes and sufficient for directing chloramphenicol acetyltransferase to peroxisomes in both cell types . The N and C termini of AtPex3p (form 2) extend into the peroxisomal matrix, different from mammal and yeast Pex3 proteins . Thus, two authentic peroxisomal membrane-bound Pex3p homologs possessing a membrane peroxisomal targeting signal, the first one defined for a plant peroxin and for any Pex3p homolog, exist in plant cells. Neurosci Lett, 2004 Jan 30, 355(3), 217 - 20 Expression of the mammalian homologue of vacuolar protein sorting 16 (Vps16p) in the mouse and rat brain; Kim BY et al.; Synaptic vesicle fusion events are essential for synaptic transmission . Membrane docking and fusion events are highly regulated processes requiring the participation of a large number of soluble N-ethylmaleimide-sensitive factors (SNAREs) and SNARE-interacting proteins . We report the neuronal expression of mammalian homologue of vacuolar protein sorting 16 (mVps16p) which exhibits a high homology to the yeast Vps16p, a component of Class C Vps . Western blot and immunohistochemical analyzes revealed that mVps16p is highly expressed in the various brain areas and developmental stages tested . The immunoreactivities of mVps16p colocalized with microtubule associated protein 2, but not glial fibrillary acidic protein . In the primary culture of cortical neurons, mVps16p immunoreactivities were observed in the cell body and the neuronal processes, and highly enriched in the axonal outgrowths. Fungal Genet Biol, 2004 Feb, 41(2), 148 - 56 A putative high affinity hexose transporter, hxtA, of Aspergillus nidulans is induced in vegetative hyphae upon starvation and in ascogenous hyphae during cleistothecium formation; Wei H et al.; Fungi employ different carbohydrate uptake systems to adapt to certain environmental conditions and to different carbon source concentrations . The hydrolysis of polymeric carbohydrates and the subsequent uptake of monomeric forms may also play a role in development . Aspergillus nidulans accumulates cell wall components during vegetative growth and degrades them during sexual development . We have identified the hxtA (high affinity hexose transporter) gene in a differential library, which was enriched for sexual-specific genes . The hxtA gene is disrupted by 6 introns and predicted to encode a 531 amino acid protein with high similarity to major facilitator superfamily members including the high affinity hexose transporter Gtt1 from Trichoderma harzianum . A . nidulans HxtA contains the 12 predicted transmembrane domains characteristic for this family . Deletion of hxtA did not impair growth of A . nidulans on a variety of carbon sources nor did it inhibit sexual development suggesting redundant sugar uptake systems . We found at least 17 putative hexose transporters in the genome of A . nidulans . Despite the high similarity of HxtA to fungal high affinity glucose transporters, the hxtA gene did not restore growth on glucose of a Saccharomyces cerevisiae mutant, in which all hexose transporters were deleted . Northern blot analysis revealed that the A . nidulans hxtA gene was repressed under high glucose conditions and expressed in vegetative hyphae upon carbon starvation and during sexual development . We found hxtA(p)::sgfp expression in developing cleistothecia specifically in ascogenous hyphae and propose that HxtA is a high affinity glucose transporter involved in sugar metabolism during sexual development. Trends Cell Biol, 1995 Jul, 5(7), 278 - 82 Nuclear migration advances in fungi; Morris NR et al.; Nuclear migration encompasses three areas: separation of daughter nuclei during mitosis, congress of parental nuclei before they fuse during fertilization, and positioning of nuclei in interphase cells . This review deals primarily with interphase nuclear migration, which is crucial for events as disparate as vertebrate embryonic development and growth of fungal mycelia . Mutants of Aspergillus nidulans, Neurospora crassa and Saccharomyces cerevisiae have been particularly informative, and a detailed molecular analysis of this process is now well under way. Trends Cell Biol, 1995 Aug, 5(8), 322 - 7 Ste5: a meeting place for MAP kinases and their associates; Elion EA; Growth and differentiation of the budding yeast Saccharomyces cerevisiae is regulated by six functionally distinct but structurally similar MAP kinase cascades . Three of the protein kinases in the cascade that regulates G1-phase arrest and mating have recently been shown to form a multikinase complex with a LIM-domain-containing protein called Ste5 . These studies implicate Ste5 as a tethering protein that physically links protein kinases operating sequentially in a cascade . The significance of this complex for the regulation and specificity of signal transduction is explored in this review. Trends Cell Biol, 1994 May, 4(5), 160 - 6 The MCM2-3-5 proteins: are they replication licensing factors? Tye BK. DNA replication occurs only once in each normal mitotic cell cycle . To explain this strict control, a 'licensing factor' was proposed to enter the nucleus periodically as the nuclear envelope disintegrates and reassembles at the end of mitosis . Inactivation of licensing factor immediately following initiation of DNA synthesis would prevent reinitiation until after the next mitosis . The MCM2-3-5 proteins of Saccharomyces cerevisiae may be yeast's equivalent of licensing factor: they are present in the nucleus only between M and S phase, bind to chromatin and are important for the initiation of DNA replication. Mol Cell, 2004 Jan 16, 13(1), 67 - 76 Phosphorylation of serine 2 within the RNA polymerase II C-terminal domain couples transcription and 3' end processing; Ahn SH et al.; The largest subunit of RNA polymerase II contains a unique C-terminal domain important for coupling of transcription and mRNA processing . This domain consists of a repeated heptameric sequence (YSPTSPS) phosphorylated at serines 2 and 5 . Serine 5 is phosphorylated during initiation and recruits capping enzyme . Serine 2 is phosphorylated during elongation by the Ctk1 kinase, a protein similar to mammalian Cdk9/P-TEFb . Chromatin immunoprecipitation was used to map positions of transcription elongation and mRNA processing factors in strains lacking Ctk1 . Ctk1 is not required for association of elongation factors with transcribing polymerase . However, in ctk1Delta strains, the recruitment of polyadenylation factors to 3' regions of genes is disrupted and changes in 3' ends are seen . Therefore, Serine 2 phosphorylation by Ctk1 recruits factors for cotranscriptional 3' end processing in vivo. Mol Cell, 2004 Jan 16, 13(1), 7 - 18 Indecent exposure: when telomeres become uncapped; Ferreira MG et al.; The protective "cap" that assembles at chromosome ends recruits and controls an intricate network of biochemical activities, each one critical for telomere structure and the maintenance of genomic stability . Recent studies have uncovered the components of telomere caps and have started to define the pathways that lead from telomere dysfunction to chromosomal catastrophe. Biochemistry, 2004 Jan 27, 43(3), 718 - 27 Monomeric and dimeric bZIP transcription factor GCN4 bind at the same rate to their target DNA site; Cranz S et al.; Basic leucine zipper (bZIP) transcription factors are dimeric proteins that recognize dyadic and mostly palindromic DNA sites . Dimerization of