J Cell Sci, 2001 Sep, 114(Pt 18), 3345 - 57 DNA double-strand breaks induce formation of RP-A/Ku foci on in vitro reconstituted Xenopus sperm nuclei; Grandi P et al.; Replication protein A (RP-A) is involved in DNA replication, repair and recombination . It has been demonstrated that RP-A clusters in foci prior to DNA replication and redistributes over chromatin during S-phase . Here, we show that RP-A foci also form in response to DNA double-strand (ds) breaks produced on Xenopus laevis sperm nuclei by restriction enzymes and then reconstituted with Xenopus egg high-speed extracts . Ku86 co-localizes with RP-A in the same foci . An unscheduled RP-A-dependent DNA synthesis takes place overlapping with RP-A and Ku86 foci . Immunoelectron-microscopy analysis reveals that these foci correspond to spherical bodies up to 300 nm in diameter, which contain RP-A, Ku86 and DNA . In an independent in vitro assay, we incubated linear dsDNA bound to magnetic beads with Xenopus egg extracts . Here, also RP-A and Ku cluster in foci as seen through immunofluorescence . Both proteins appear to enrich themselves in sequences near the ends of the DNA molecules and influence ligation efficiency of ds linear DNA to these ends . Thus, the Xenopus in vitro system allows for the generation of specific DNA ds breaks, RP-A and Ku can be used as markers for these lesions and the repair of this type of DNA damage can be studied under conditions of a normal nuclear environment. J Cell Sci, 2001 Sep, 114(Pt 18), 3297 - 307 Nuclear membrane protein LAP2beta mediates transcriptional repression alone and together with its binding partner GCL (germ-cell-less); Nili E et al.; LAP2beta is an integral membrane protein of the nuclear envelope involved in chromatin and nuclear architecture . Using the yeast two-hybrid system, we have cloned a novel LAP2beta-binding protein, mGCL, which contains a BTB/POZ domain and is the mouse homologue of the Drosophila germ-cell-less (GCL) protein . In Drosophila embryos, GCL was shown to be essential for germ cell formation and was localized to the nuclear envelope . Here, we show that, in mammalian cells, GCL is co-localized with LAP2beta to the nuclear envelope . Nuclear fractionation studies reveal that mGCL acts as a nuclear matrix component and not as an integral protein of the nuclear envelope . Recently, mGCL was found to interact with the DP3alpha component of the E2F transcription factor . This interaction reduced the transcriptional activity of the E2F-DP heterodimer, probably by anchoring the complex to the nuclear envelope . We demonstrate here that LAP2beta is also capable of reducing the transcriptional activity of the E2F-DP complex and that it is more potent than mGCL in doing so . Co-expression of both LAP2beta and mGCL with the E2F-DP complex resulted in a reduced transcriptional activity equal to that exerted by the pRb protein. Gene, 2001 Oct 3, 276(1-2), 161 - 73 Contribution of sponge genes to unravel the genome of the hypothetical ancestor of Metazoa (Urmetazoa); Muller WE et al.; Recently the term Urmetazoa, as the hypothetical metazoan ancestor, was introduced to highlight the finding that all metazoan phyla including the Porifera (sponges) are derived from one common ancestor . Sponges as the evolutionarily oldest, still extant phylum, are provided with a complex network of structural and functional molecules . Analyses of sponge genomes from Demospongiae (Suberites domuncula and Geodia cydonium), Calcarea (Sycon raphanus) and Hexactinellida (Aphrocallistes vastus) have contributed also to the reconstruction of the evolutionary position of Metazoa with respect to Fungi . Furthermore, these analyses have provided evidence that the characteristic evolutionary novelties of Metazoa, such as the extracellular matrix molecules, the cell surface receptors, the nervous signal transduction molecules as well as the immune molecule existing in Porifera, share high sequence and in some aspects also functional similarities to related polypeptides found in other metazoan phyla . During the transition to Metazoa new domains occurred; as one example, the formation of the death domain from the ankyrin is outlined . In parallel, domanial proteins have been formed, such as the receptor tyrosine kinases . The metazoan essentials have been defined by analyzing and comparing the sponge sequences with the related sequences from the metazoans Homo sapiens, Caenorhabditis elegans and Drosophila melanogaster, the fungus Saccharomyces cerevisiae and the plant Arabidopsis thaliana . The data revealed that those sponge molecules grouped to cell adhesion cell recognition proteins are predominantly found in Protostomia and Deuterostomia while they are missing in Fungi and Viridiplantae . Moreover, evidence is presented allowing the conclusion that the sponge molecules are more closely related to the corresponding molecules from H . sapiens than to those of C . elegans or D . melanogaster . Especially surprising was the finding that the Demospongiae are provided with elements of adaptive immunity. Structure (Camb), 2001 Oct, 9(10), 987 - 97 Crystal structure of imidazole glycerol phosphate synthase: a tunnel through a (beta/alpha)8 barrel joins two active sites; Chaudhuri BN et al.; BACKGROUND: Imidazole glycerol phosphate synthase catalyzes a two-step reaction of histidine biosynthesis at the bifurcation point with the purine de novo pathway . The enzyme is a new example of intermediate channeling by glutamine amidotransferases in which ammonia generated by hydrolysis of glutamine is channeled to a second active site where it acts as a nucleophile . In this case, ammonia reacts in a cyclase domain to produce imidazole glycerol phosphate and an intermediate of purine biosynthesis . The enzyme is also a potential target for drug and herbicide development since the histidine pathway does not occur in mammals . RESULTS: The 2.1 A crystal structure of imidazole glycerol phosphate synthase from yeast reveals extensive interaction of the glutaminase and cyclase catalytic domains . At the domain interface, the glutaminase active site points into the bottom of the (beta/alpha)(8) barrel of the cyclase domain . An ammonia tunnel through the (beta/alpha)(8) barrel connects the glutaminase docking site at the bottom to the cyclase active site at the top . A conserved "gate" of four charged residues controls access to the tunnel . CONCLUSIONS: This is the first structure in which all the components of the ubiquitous (beta/alpha)(8) barrel fold, top, bottom, and interior, take part in enzymatic function . Intimate contacts between the barrel domain and the glutaminase active site appear to be poised for crosstalk between catalytic centers in response to substrate binding at the cyclase active site . The structure provides a number of potential sites for inhibitor development in the active sites and in a conserved interdomain cavity. Structure (Camb), 2001 Oct, 9(10), 881 - 4 A Ku bridge over broken DNA; Jones JM et al.; The Ku heterodimer is essential for the nonhomologous end-joining pathway of DNA double-strand break repair; it both protects the broken ends and recruits some of the many proteins required to complete repair . The recently determined structure of Ku provides insights into how it can both bind to the DNA ends and allow access by the other proteins required to rejoin them. Mol Cell Neurosci, 2001 Sep, 18(3), 307 - 19 Doublecortin interacts with mu subunits of clathrin adaptor complexes in the developing nervous system; Friocourt G et al.; Doublecortin is a microtubule-associated protein required for normal corticogenesis in the developing brain . We carried out a yeast two-hybrid screen to identify interacting proteins . One of the isolated clones encodes the mu1 subunit of the adaptor complex AP-1 involved in clathrin-dependent protein sorting . We found that Doublecortin also interacts in yeast with mu2 from the AP-2 complex . Mutagenesis and pull-down experiments showed that these interactions were mediated through a tyrosine-based sorting signal (YLPL) in the C-terminal part of Doublecortin . The functional relevance of these interactions was suggested by the coimmunoprecipitation of Doublecortin with AP-1 and AP-2 from mouse brain extracts . This interaction was further supported by RNA in situ hybridization and immunofluorescence studies . Taken together these data indicate that a certain proportion of Doublecortin interacts with AP-1 and/or AP-2 in vivo and are consistent with a potential involvement of Doublecortin in protein sorting or vesicular trafficking . Biotechnol Bioeng, 2001 Nov 5, 75(3), 345 - 54 On-line stoichiometry and identification of metabolic state under dynamic process conditions; Herwig C et al.; A method for the on-line calculation of conversion rates and yield coefficients under dynamic process conditions was developed . The method is based on cumulated mass balances using a moving average method . Elemental balances were used to test the measured cumulated quantities for gross errors and inappropriate stoichiometry definition followed by data reconciliation and estimation of non-measured conversion rates, using a bioprocess set-up including multiple on-line analysis techniques . The quantitative potential of the proposed method is demonstrated by executing transient experiments in aerobic cultures of Saccharomyces cerevisiae on glucose . Rates and yield coefficients could be consistently quantified in shift-up, shift-down, and accelerostat experiments . The method shows the capability to describe quantitatively transient changes in metabolism including uncoupling of catabolism and anabolism, also for the case when multiple components of metabolism are not measured . The validity of the experiment can be evaluated on-line . Additionally, the method detects with high sensitivity inappropriate stoichiometry definition, such as a change in state of metabolism . It was shown that concentration values can be misleading for the identification of the metabolic state . In contrast, the proposed method provides a clear picture of the metabolic state and new physiological regulations could be revealed . Hence, the novelty of the proposed method is the on-line availability of consistent stoichiometric coefficients allowing a significant speed up in strain characterization and bioprocess development using minimal knowledge of the metabolism . Additionally, it opens up the use of transient experiments for physiological studies . Mol Vis, 2001 Oct 05, 7, 234 - 9 Identification, genomic structure, and screening of the vacuolar proton-ATPase membrane sector-associated protein M8-9 gene within the COD1 critical region (Xp11.4); Demirci FY et al.; PURPOSE: Our goal is to identify the gene responsible for X-linked cone-rod dystrophy (COD1) that has been localized to a limited region of Xp11.4 . METHODS: A complete physical contig of the COD1 region was partially sequenced and subjected to BLAST searches to identify homologies with GenBank ESTs . ESTs were analyzed for overlapping or related cDNA sequences and retinal expression by PCR screening of multiple human retina cDNA libraries . RACE was performed to complete the missing 5' end of the transcripts . Transcripts were compared with genomic sequences to specify intron-exon boundaries . Genomic DNAs from COD1-affected males from 3 families were screened for mutations using direct PCR sequencing of the exons . RESULTS: The vacuolar proton-ATPase membrane sector-associated protein M8-9 (APT6M8-9) gene was identified within our critical region . We confirmed its retinal expression and its genomic location in our physical contig . Eight exons (with flanking intronic sequences) were characterized from partial cDNA sequence and genomic sequence data . An additional 5' end exon was identified using RACE . No mutations were found in the COD1-affected males . CONCLUSIONS: The combination of disease mapping and information from the Human Genome project has enabled us to identify candidate genes within the COD1 region, including APT6M8-9 gene . We found no evidence that this gene is responsible for COD1 in our families, but it may be an important candidate for other diseases that have been mapped to this region of the X chromosome. J Cell Sci, 2001 Sep, 114(Pt 17), 3093 - 102 A specific targeting signal directs Runx2/Cbfa1 to subnuclear domains and contributes to transactivation of the osteocalcin gene; Zaidi SK et al.; Key components of DNA replication and the basal transcriptional machinery as well as several tissue-specific transcription factors are compartmentalized in specialized nuclear domains . In the present study, we show that determinants of subnuclear targeting of the bone-related Runx2/Cbfa1 protein reside in the C-terminus . With a panel of C-terminal mutations, we further demonstrate that targeting of Runx2 to discrete subnuclear foci is mediated by a 38 amino acid sequence (aa 397-434) . This nuclear matrix-targeting signal (NMTS) directs the heterologous Gal4 protein to nuclear-matrix-associated Runx2 foci and enhances transactivation of a luciferase gene controlled by Gal4 binding sites . Importantly, we show that targeting of Runx2 to the NM-associated foci contributes to transactivation of the osteoblast-specific osteocalcin gene in osseous cells . Taken together, these findings identify a critical component of the mechanisms mediating Runx2 targeting to subnuclear foci and provide functional linkage between subnuclear organization of Runx2 and bone-specific transcriptional control. J Biol Chem, 2001 Dec 14, 276(50), 47684 - 9 Epub 2001 Oct 04. Pex18p is constitutively degraded during peroxisome biogenesis; Purdue PE et al.; Pex18p and Pex21p are structurally related yeast peroxins (proteins required for peroxisome biogenesis) that are partially redundant in function . One or the other is essential for the import into peroxisomes of proteins with type 2 peroxisomal targeting sequences (PTS2) . These sequences bind to the soluble PTS2 receptor, Pex7p, which in turn binds to Pex18p (or Pex21p or possibly both) . Here we show that Pex18p is constitutively degraded with a half-time of less than 10 min in wild-type Saccharomyces cerevisiae . This degradation probably occurs in proteasomes, because it requires the related ubiquitin-conjugating enzymes Ubc4p and Ubc5p and occurs normally in a mutant lacking the Pep4p vacuolar protease . The turnover of Pex18p stops, and Pex18p accumulates to a much higher than normal abundance in pex mutants in which the import of all peroxisomal matrix proteins is blocked . This includes mutants that lack peroxins involved in receptor docking at the membrane (Deltapex13 or Deltapex14), a mutant that lacks the peroxisomal member of the E2 family of ubiquitin-conjugating enzymes (Deltapex4), and others (Deltapex1) . This stabilization in a variety of pex mutants indicates that Pex18p turnover is associated with its normal function . A Pex18p-Pex7p complex is detected by immunoprecipitation in wild type cells, and its abundance increases considerably in the Deltapex14 peroxisome biogenesis mutant . Cells that lack Pex7p fail to stabilize and accumulate Pex18p, indicating an important role for complex formation in the stabilization . Mono- and diubiquitinated forms of Pex18p are detected in wild-type cells, and there is no Pex18p turnover in a yeast doa4 mutant in which ubiquitin homeostasis is defective . These data represent, to the best of our knowledge, the first instance of an organelle biogenesis factor that is degraded constitutively and rapidly. Bioinformatics, 2001 Sep, 17(9), 763 - 74 Principal component analysis for clustering gene expression data; Yeung KY et al.; MOTIVATION: There is a great need to develop analytical methodology to analyze and to exploit the information contained in gene expression data . Because of the large number of genes and the complexity of biological networks, clustering is a useful exploratory technique for analysis of gene expression data . Other classical techniques, such as principal component analysis (PCA), have also been applied to analyze gene expression data . Using different data analysis techniques and different clustering algorithms to analyze the same data set can lead to very different conclusions . Our goal is to study the effectiveness of principal components (PCs) in capturing cluster structure . Specifically, using both real and synthetic gene expression data sets, we compared the quality of clusters obtained from the original data to the quality of clusters obtained after projecting onto subsets of the principal component axes . RESULTS: Our empirical study showed that clustering with the PCs instead of the original variables does not necessarily improve, and often degrades, cluster quality . In particular, the first few PCs (which contain most of the variation in the data) do not necessarily capture most of the cluster structure . We also showed that clustering with PCs has different impact on different algorithms and different similarity metrics . Overall, we would not recommend PCA before clustering except in special circumstances. Trends Plant Sci, 2001 Oct, 6(10), 463 - 70 Ubiquitylation in plants: a post-genomic look at a post-translational modification; Bachmair A et al.; In this article, we summarize Arabidopsis genes encoding ubiquitin, ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzymes (E2s) and an additional selected set of proteins related to ubiquitylation . We emphasize comparisons to components from Saccharomyces cerevisiae, with occasional reference to animals . Among the E1 and E2s, Arabidopsis usually has two to four probable orthologs to one yeast gene . Also, Arabidopsis has genes with no likely ortholog in yeast, although they often have potential orthologs in animals . The large number of components with known function in ubiquitylation indicates that this process plays a complex role in cellular physiology. Chem Biol, 2001 Oct, 8(10), 941 - 50 Selective chemical inactivation of AAA proteins reveals distinct functions of proteasomal ATPases; Russell SJ et al.; BACKGROUND: The 26S proteasome contains six highly related ATPases of the AAA family . We have developed a strategy that allows selective inhibition of individual proteasomal ATPases in the intact proteasome . Mutation of a threonine in the active site of Sug1/Rpt6 or Sug2/Rpt4 to a cysteine sensitizes these proteins to inactivation through alkylation by the sulfhydryl modifying agent NEM . Using this technique the individual contributions of Sug1 and Sug2 to proteasome function can be assessed . RESULTS: We show that both Sug1 and Sug2 can be selectively alkylated by NEM in the context of the intact 26S complex and as predicted by structural modeling, this inactivates the ATPase function . Using this technique we demonstrate that both Sug 1 and 2 are required for full peptidase activity of the proteasome and that their functions are not redundant . Kinetic analysis suggests that Sug2 may have an important role in maintaining the interaction between the 19S regulatory complex and the 20S proteasome . In contrast, inhibition of Sug1 apparently decreases peptidase activity of the 26S proteasome by another mechanism . CONCLUSIONS: These results describe a useful technique for the selective inactivation of AAA proteins . In addition, they also demonstrate that the functions of two related proteasomal AAA proteins are not redundant, suggesting differential roles of proteasomal AAA proteins in protein degradation. Eur J Paediatr Neurol, 2001, 5 Suppl A, 95 - 8 Searching for interacting partners of CLN1, CLN2 and Btn1p with the two-hybrid system; Cottone CD et al.; The neuronal ceroid lipofuscinoses (NCLs) are the most common neurodegenerative disorders of childhood . The CLN1, CLN2 and CLN3 genes are associated to the infantile, late infantile and juvenile forms of NCL, respectively . We have subcloned the cDNAs encoding CLN1, CLN2 and BTN1, the yeast homologue of human CLN3, into plasmid vectors to evaluate whether these proteins interact with other proteins co-expressed from either a cDNA library derived from human cerebellum or from yeast, respectively, using the two-hybrid system . We concluded that CLN1 most likely does not interact with any other proteins in vivo . Furthermore, it is unlikely that CLN2 interacts with other proteins in vivo, although this study utilized a cDNA encoding the CLN2 precursor and it is possible that interacting partners may be excluded by the nature of this protein structure . Finally, we conclude that proteins that interact with Btn1p and therefore CLN3 cannot be identified using the whole proteins in a two-hybrid system, due to the hydrophobic nature of this protein . By understanding the topology of CLN3, specific regions of CLN3 need to be tested by two-hybrid to identify any interacting partners. Stroke, 2001 Oct, 32(10), 2388 - 93 Neuronal expression of the DNA repair protein Ku 70 after ischemic preconditioning corresponds to tolerance to global cerebral ischemia; Sugawara T et al.; BACKGROUND AND PURPOSE: Oxidative stress after ischemia/reperfusion has been shown to induce DNA damage and subsequent DNA repair activity . Ku 70/86, multifunctional DNA repair proteins, bind to broken DNA ends and trigger a DNA repair pathway . We investigated the involvement of these proteins in the development of neuronal tolerance to global cerebral ischemia after ischemic preconditioning . METHODS: Adult male Sprague-Dawley rats were subjected to either 5 minutes of lethal global ischemia with or without 3 minutes of sublethal ischemic preconditioning or 3 minutes of ischemia only . Neuronal injury was histologically assessed, and DNA damage was visualized by in situ labeling of DNA fragmentation and DNA gel electrophoresis . Ku expression was also examined by immunohistochemistry and Western blot analysis . RESULTS: Hippocampal CA1 neurons underwent DNA-fragmented cell death 3 days after 5 minutes of ischemia . However, these neurons showed a strong tolerance to 5 minutes of ischemia 1 to 3 days after ischemic preconditioning . Immunohistochemistry showed virtually no constitutive expression of Ku proteins in CA1 neurons; however, ischemic preconditioning induced neuronal Ku 70 expression 1 to 3 days later . Western blot confirmed an increase in Ku 70 in this region at the same time . CONCLUSIONS: The temporal and spatial expression of Ku 70 corresponded to tolerance of the hippocampal CA1 neurons to subsequent ischemia, suggesting the involvement of Ku proteins in the development of neuronal tolerance after ischemic preconditioning. Biochem Biophys Res Commun, 2001 Oct 12, 287(5), 1083 - 7 The PUB domain: a putative protein-protein interaction domain implicated in the ubiquitin-proteasome pathway; Suzuki T et al.; Cytoplasmic peptide:N-glycanase (PNGase) is a de-N-glycosylating enzyme which may be involved in the proteasome-dependent pathway for degradation of misfolded glycoproteins formed in the endoplasmic reticulum (ER) that are exported into the cytoplasm . A cytoplasmic PNGase found in Saccharomyces cerevisiae, Png1p, is widely distributed in higher eukaryotes as well as in yeast (Suzuki, T., et al . J . Cell Biol . 149, 1039-1051, 2000) . The recently uncovered complete genome sequence of Arabidopsis thaliana prompted us to search for the protein homologue of Png1p in this organism . Interestingly, when the mouse Png1p homologue sequence was used as a query, not only a Png1p homologue containing a transglutaminase-like domain that is believed to contain a catalytic triad for PNGase activity, but also four proteins which had a domain of 46 amino acids in length that exhibited significant similarity to the N-terminus of mouse Png1p were identified . Moreover, three of these homologous proteins were also found to possess a UBA or UBX domain, which are found in various proteins involved in the ubiquitin-related pathway . We name this newly found homologous region the PUB (Peptide:N-glycanase/UBA or UBX-containing proteins) domain and propose that this domain may mediate protein-protein interactions . Pharm Res, 2001 Aug, 18(8), 1102 - 9 Characterizing the expression of CYP3A4 and efflux transporters (P-gp, MRP1, and MRP2) in CYP3A4-transfected Caco-2 cells after induction with sodium butyrate and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate; Cummins CL et al.; PURPOSE: To examine the changes in expression levels of CYP3A4 and efflux transporters in CYP3A4-transfected Caco-2 (colon carcinoma) cells in the presence of the inducers sodium butyrate (NaB) and 12-O-tetradecanoylphorbol-13-acetate (TPA) . To characterize the transport of {3H}-digoxin and the metabolism of midazolam in the cells under different inducing conditions . METHODS: CYP3A4-Caco-2 cells were seeded onto cell culture inserts and were grown for 13-14 days . Transport and metabolism studies were performed on cells induced with NaB and/or TPA for 24 h . The expression and localization of P-gp, MRP1, MRP2, and CYP3A4 were examined by Western blot and confocal microscopy . RESULTS: In the presence of both inducers, CYP3A4 protein levels were increased 40-fold over uninduced cells, MRP2 expression was decreased by 90%, and P-gp and MRP1 expression were unchanged . Midazolam 1-OH formation exhibited a rank order correlation with increased CYP3A4 protein, whereas {3H}-digoxin transport (a measure of P-gp activity) was unchanged with induction . P-gp and MRP2 were found on the apical membrane, whereas MRP1 was found perinuclear within the cell . CYP3A4 displayed a punctate pattern of expression consistent with endoplasmic reticulum localization and exhibited preferential polarization towards the apical side of the cell . CONCLUSIONS: The present study characterized CYP3A4-Caco-2 cell monolayers when induced for 24 h in the presence of both NaB and TPA . These conditions provide intact cells with significant CYP3A4 and P-gp expression suitable for the concurrent study of transport and metabolism. Nature, 2001 Oct 4, 413(6855), 538 - 42 Quality control of mRNA 3'-end processing is linked to the nuclear exosome; Hilleren P et al.; An emerging theme in messenger RNA metabolism is the coupling of nuclear pre-mRNA processing events, which contributes to mRNA quality control . Most eukaryotic mRNAs acquire a poly(A) tail during 3'-end processing within the nucleus, and this is coupled to efficient export of mRNAs to the cytoplasm . In the yeast Saccharomyces cerevisiae, a common consequence of defective nuclear export of mRNA is the hyperadenylation of nascent transcripts, which are sequestered at or near their sites of transcription . This implies that polyadenylation and nuclear export are coupled in a step that involves the release of mRNA from transcription site foci . Here we demonstrate that transcripts which fail to acquire a poly(A) tail are also retained at or near transcription sites . Surprisingly, this retention mechanism requires the protein Rrp6p and the nuclear exosome, a large complex of exonucleolytic enzymes . In exosome mutants, hypo- as well as hyperadenylated mRNAs are released and translated . These observations suggest that the exosome contributes to a checkpoint that monitors proper 3'-end formation of mRNA. Nat Genet, 2001 Oct, 29(2), 105 - 6 The promoter connection; Werner T; The availability of the complete genomic sequence of yeast now enables elucidation of molecular mechanisms governing gene expression patterns . New results from the yeast genome and recent advances in predicting and finding human promoters support the use of similar combinatorial approaches to study genome-wide transcriptional regulation in humans. Mol Cell Biol, 2001 Nov, 21(21), 7366 - 79 Absence of Dbp2p alters both nonsense-mediated mRNA decay and rRNA processing; Bond AT et al.; Dbp2p, a member of the large family of DEAD-box proteins and a yeast homolog of human p68, was shown to interact with Upf1p, an essential component of the nonsense-mediated mRNA decay pathway . Dbp2p:Upf1p interaction occurs within a large conserved region in the middle of Upf1p that is largely distinct from its Nmd2p and Sup35/45p interaction domains . Deletion of DBP2, or point mutations within its highly conserved DEAD-box motifs, increased the abundance of nonsense-containing transcripts, leading us to conclude that Dbp2p also functions in the nonsense-mediated mRNA decay pathway . Dbp2p, like Upf1p, acts before or at decapping, is predominantly cytoplasmic, and associates with polyribosomes . Interestingly, Dbp2p also plays an important role in rRNA processing . In dbp2Delta cells, polyribosome profiles are deficient in free 60S subunits and the mature 25S rRNA is greatly reduced . The ribosome biogenesis phenotype, but not the mRNA decay function, of dbp2Delta cells can be complemented by the human p68 gene . We propose a unifying model in which Dbp2p affects both nonsense-mediated mRNA decay and rRNA processing by altering rRNA structure, allowing specific processing events in one instance and facilitating dissociation of the translation termination complex in the other. Mol Cell Biol, 2001 Nov, 21(21), 7331 - 44 Analysis of TAF90 mutants displaying allele-specific and broad defects in transcription; Durso RJ et al.; Yeast TAF90p is a component of at least two transcription regulatory complexes, the general transcription factor TFIID and the Spt-Ada-Gcn5 histone acetyltransferase complex (SAGA) . Broad transcription defects have been observed in mutants of other TAF(II)s shared by TFIID and SAGA but not in the only two TAF90 mutants isolated to date . Given that the numbers of mutants analyzed thus far are small, we isolated and characterized 11 temperature-sensitive mutants of TAF90 and analyzed their effects on transcription and integrity of the TFIID and SAGA complexes . We found that the mutants displayed a variety of allele-specific defects in their ability to support transcription and maintain the structure of the TFIID and SAGA complexes . Sequencing of the alleles revealed that all have mutations corresponding to the C terminus of the protein, with most clustering within the conserved WD40 repeats; thus, the C terminus of TAF90p is required for its incorporation into TFIID and function in SAGA . Significantly, inactivation of one allele, taf90-20, caused the dramatic reduction in the levels of total mRNA and most specific transcripts analyzed . Analysis of the structure and/or activity of both TAF90p-containing complexes revealed that this allele is the most disruptive of all . Our analysis defines the requirement for the WD40 repeats in preserving TFIID and SAGA function, demonstrates that the defects associated with distinct mutations in TAF90 vary considerably, and indicates that TAF90 can be classified as a gene required for the transcription of a large number of genes. Mol Cell Biol, 2001 Nov, 21(21), 7191 - 8 Repair of DNA strand breaks by the overlapping functions of lesion-specific and non-lesion-specific DNA 3' phosphatases; Vance JR et al.; In Saccharomyces cerevisiae, the apurinic/apyrimidinic (AP) endonucleases Apn1 and Apn2 act as alternative pathways for the removal of various 3'-terminal blocking lesions from DNA strand breaks and in the repair of abasic sites, which both result from oxidative DNA damage . Here we demonstrate that Tpp1, a homologue of the 3' phosphatase domain of polynucleotide kinase, is a third member of this group of redundant 3' processing enzymes . Unlike Apn1 and Apn2, Tpp1 is specific for the removal of 3' phosphates at strand breaks and does not possess more general 3' phosphodiesterase, exonuclease, or AP endonuclease activities . Deletion of TPP1 in an apn1 apn2 mutant background dramatically increased the sensitivity of the double mutant to DNA damage caused by H2O2 and bleomycin but not to damage caused by methyl methanesulfonate . The triple mutant was also deficient in the repair of 3' phosphate lesions left by Tdp1-mediated cleavage of camptothecin-stabilized Top1-DNA covalent complexes . Finally, the tpp1 apn1 apn2 triple mutation displayed synthetic lethality in combination with rad52, possibly implicating postreplication repair in the removal of unrepaired 3'-terminal lesions resulting from endogenous damage . Taken together, these results demonstrate a clear role for the lesion-specific enzyme, Tpp1, in the repair of a subset of DNA strand breaks. Mol Cell Biol, 2001 Nov, 21(21), 7150 - 62 Topoisomerase III acts upstream of Rad53p in the S-phase DNA damage checkpoint; Chakraverty RK et al.; Deletion of the Saccharomyces cerevisiae TOP3 gene, encoding Top3p, leads to a slow-growth phenotype characterized by an accumulation of cells with a late S/G2 content of DNA (S . Gangloff, J . P . McDonald, C . Bendixen, L . Arthur, and R . Rothstein, Mol . Cell . Biol . 14:8391-8398, 1994) . We have investigated the function of TOP3 during cell cycle progression and the molecular basis for the cell cycle delay seen in top3Delta strains . We show that top3Delta mutants exhibit a RAD24-dependent delay in the G2 phase, suggesting a possible role for Top3p in the resolution of abnormal DNA structures or DNA damage arising during S phase . Consistent with this notion, top3Delta strains are sensitive to killing by a variety of DNA-damaging agents, including UV light and the alkylating agent methyl methanesulfonate, and are partially defective in the intra-S-phase checkpoint that slows the rate of S-phase progression following exposure to DNA-damaging agents . This S-phase checkpoint defect is associated with a defect in phosphorylation of Rad53p, indicating that, in the absence of Top3p, the efficiency of sensing the existence of DNA damage or signaling to the Rad53 kinase is impaired . Consistent with a role for Top3p specifically during S phase, top3Delta mutants are sensitive to the replication inhibitor hydroxyurea, expression of the TOP3 mRNA is activated in late G1 phase, and DNA damage checkpoints operating outside of S phase are unaffected by deletion of TOP3 . All of these phenotypic consequences of loss of Top3p function are at least partially suppressed by deletion of SGS1, the yeast homologue of the human Bloom's and Werner's syndrome genes . These data implicate Top3p and, by inference, Sgs1p in an S-phase-specific role in the cellular response to DNA damage . A model proposing a role for these proteins in S phase is presented. J Biol Chem, 2001 Dec 14, 276(50), 47496 - 507 Epub 2001 Oct 03. Mechanism for nucleocytoplasmic shuttling of histone deacetylase 7; Kao HY et al.; Here we show that HDAC7, a member of the class II histone deacetylases, specifically targets several members of myocyte enhancer factors, MEF2A, -2C, and -2D, and inhibits their transcriptional activity . Furthermore, we demonstrate that DNA-bound MEF2C is capable of recruiting HDAC7, demonstrating that the HDAC7-dependent repression of transcription is not due to the inhibition of the MEF2 DNA binding activity . The data also suggest that the promoter bound MEF2 is potentially capable of remodeling adjacent nucleosomes via the recruitment of HDAC7 . We have also observed a nucleocytoplasmic shuttling of HDAC7 and dissected the mechanism involved . In NIH3T3 cells, HDAC7 was primarily localized in the cytoplasm, essentially due to an active CRM1-dependent export of the protein from the nucleus . Interestingly, in HeLa cells, HDAC7 was predominantly nuclear . In these cells we could restore the cytoplasmic localization of HDAC7 by expressing CaMK I . This CaMK I-induced nuclear export of HDAC7 was abolished when three critical serines, Ser-178, Ser-344, and Ser-479, of HDAC7 were mutated . We show that these serines are involved in the direct interaction of HDAC7 with 14-3-3 . Mutations of these serine residues weakened the association with 14-3-3 and dramatically enhanced the repression activity of HDAC7 in NIH3T3 cells, but not in HeLa cells . Data presented in this work clearly show that the signal dependent subcellular localization of HDAC7 is essential in controlling its activities . The data also show that the cellular concentration of factors such as 14-3-3, CaMK I, and other yet unknown molecules may determine the subcellular localization of an individual HDAC member in a cell type and HDAC-specific manner. Nat Rev Mol Cell Biol, 2001 Oct, 2(10), 749 - 59 A decade of CDK5; Dhavan R et al.; Since it was identified a decade ago, cyclin-dependent kinase 5 (CDK5) has emerged as a crucial regulator of neuronal migration in the developing central nervous system . CDK5 phosphorylates a diverse list of substrates, implicating it in the regulation of a range of cellular processes - from adhesion and motility, to synaptic plasticity and drug addiction . Recent evidence indicates that deregulation of this kinase is involved in the pathology of neurodegenerative diseases. Nat Rev Mol Cell Biol, 2001 Oct, 2(10), 721 - 30 The endocytic pathway: a mosaic of domains; Gruenberg J; Organelles in the endocytic pathway are composed of a mosaic of structural and functional regions . These regions consist, at least in part, of specialized protein-lipid domains within the plane of the membrane, or of protein complexes associated with specific membrane lipids . Whereas some of these molecular assemblies can be found in more than one compartment, a given combination seems to be unique to each compartment, indicating that membrane organization might be modular. Nat Rev Genet, 2001 Oct, 2(10), 743 - 55 Conditional control of gene expression in the mouse; Lewandoski M; One of the most powerful tools that the molecular biology revolution has given us is the ability to turn genes on and off at our discretion . In the mouse, this has been accomplished by using binary systems in which gene expression is dependent on the interaction of two components, resulting in either transcriptional transactivation or DNA recombination . During recent years, these systems have been used to analyse complex and multi-staged biological processes, such as embryogenesis and cancer, with unprecedented precision . Here, I review these systems and discuss certain studies that exemplify the advantages and limitations of each system. Nat Cell Biol, 2001 Oct, 3(10), 939 - 43 Proteins containing the UBA domain are able to bind to multi-ubiquitin chains; Wilkinson CR et al.; The UBA domain is a motif found in a variety of proteins, some of which are associated with the ubiquitin-proteasome system . We describe the isolation of a fission-yeast gene, mud1+, which encodes a UBA domain containing protein that is able to bind multi-ubiquitin chains . We show that the UBA domain is responsible for this activity . Two other proteins containing this motif, the fission-yeast homologues of Rad23 and Dsk2, are also shown to bind multi-ubiquitin chains via their UBA domains . These two proteins are implicated, along with the fission-yeast Pus1(S5a/Rpn10) subunit of the 26 S proteasome, in the recognition and turnover of substrates by this proteolytic complex. Nat Cell Biol, 2001 Oct, 3(10), 874 - 82 A flavoprotein oxidase defines a new endoplasmic reticulum pathway for biosynthetic disulphide bond formation; Sevier CS et al.; Ero1 and Pdi1 are essential elements of the pathway for the formation of disulphide bonds within the endoplasmic reticulum (ER) . By screening for alternative oxidation pathways in Saccharomyces cerevisiae, we identified ERV2 as a gene that when overexpressed can restore viability and disulphide bond formation to an ero1-1 mutant strain . ERV2 encodes a luminal ER protein of relative molecular mass 22,000 . Purified recombinant Erv2p is a flavoenzyme that can catalyse O2-dependent formation of disulphide bonds . Erv2p transfers oxidizing equivalents to Pdi1p by a dithiol-disulphide exchange reaction, indicating that the Erv2p-dependent pathway for disulphide bond formation closely parallels that of the previously identified Ero1p-dependent pathway. J Natl Cancer Inst, 2001 Oct 3, 93(19), 1473 - 8 In vitro evidence for homologous recombinational repair in resistance to melphalan; Wang ZM et al.; BACKGROUND: The generation of DNA interstrand cross-links is thought to be important in the cytotoxicity of nitrogen mustard alkylating agents, such as melphalan, which have antitumor activity . Cell lines with mutations in recombinational repair pathways are hypersensitive to nitrogen mustards . Thus, resistance to melphalan may require accelerated DNA repair by either recombinational repair mechanisms involving Rad51-related proteins (including x-ray repair cross-complementing proteins Xrcc2, Xrcc3, and Rad52) or by nonhomologous endjoining involving DNA-dependent protein kinase (DNA-PK) and Ku proteins . We investigated the role of DNA repair in melphalan resistance in epithelial tumor cell lines . METHODS: Melphalan cytotoxicity was determined in 14 epithelial tumor cell lines by use of the sulforhodamine assay . Homologous recombinational repair involving Rad51-related proteins was investigated by determining the levels of Rad51, Rad52, and Xrcc3 proteins and the density of nuclear melphalan-induced Rad51 foci, which represent sites of homologous recombinational repair . Nonhomologous endjoining was investigated by determining the levels of Ku70 and Ku86 proteins and DNA-PK activity . Linear regression analysis was used to analyze correlations between the various protein levels, DNA-PK activity, or Rad51 foci formation and melphalan cytotoxicity . All statistical tests were two-sided . RESULTS: Melphalan resistance was correlated with Xrcc3 levels (r =.587; P =.027) and the density of melphalan-induced Rad51 foci (r =.848; P =.008) . We found no correlation between melphalan resistance and Rad51, Rad52, or Ku protein levels or DNA-PK activity . CONCLUSION: Correlations of melphalan resistance in epithelial tumor cell lines with Xrcc3 protein levels and melphalan-induced Rad51 foci density suggest that homologous recombinational repair is involved in resistance to this nitrogen mustard. Biochim Biophys Acta, 2001 Sep 26, 1540(3), 221 - 32 Characteristics and distribution of endogenous RFamide-related peptide-1; Fukusumi S et al.; We have recently identified RFamide-related peptide (RFRP) gene that would encode three peptides (i.e., RFRP-1, -2, and -3) in human and bovine, and demonstrated that synthetic RFRP-1 and -3 act as specific agonists for a G protein-coupled receptor OT7T022 . However, molecular characteristics and tissue distribution of endogenous RFRPs have not been determined yet . In this study, we prepared a monoclonal antibody for the C-terminal portion of rat RFRP-1 . As this antibody could recognize a consensus sequence among the C-terminal portions of rat, human, and bovine RFRP-1, we purified endogenous RFRP-1 from bovine hypothalamus on the basis of immunoreactivity to the antibody . The purified bovine endogenous RFRP-1 was found to have 35-amino-acid length that corresponds to 37-amino-acid length in human and rat . We subsequently constructed a sandwich enzyme immunoassay using the monoclonal antibody and a polyclonal antibody for the N-terminal portion of rat RFRP-1, and analyzed the tissue distribution of endogenous RFRP-1 in rats . Significant levels of RFRP-1 were detected only in the central nervous system, and the highest concentration of RFRP-1 was detected in the hypothalamus . RFRP-1-positive nerve cells were detected in the rat hypothalamus by immunohistochemical analyses using the monoclonal antibody . In culture, RFRP-1 lowered cAMP production in Chinese hamster ovary cells expressing OT7T022 and it was abolished by pre-treatment with pertussis toxin, suggesting that OT7T022 couples G(i)/G(o) in the signal transduction pathway. Mol Cell, 2001 Sep, 8(3), 713 - 8 Involvement of PIAS1 in the sumoylation of tumor suppressor p53; Kahyo T et al.; Sumoylation of p53 by the ubiquitin-like protein, SUMO-1/sentrin/PIC1, has been shown to stimulate its transcriptional activation activity . The SUMO E3 ligase, a key enzyme in the recognition of substrates to be sumoylated, has not yet been identified . We isolated PIAS1 (protein inhibitor of activated STAT1) as a SUMO-1 binding protein by yeast two-hybrid screening . In addition, PIAS1 bound p53 and Ubc9, the E2 for SUMO . PIAS1 that was mutated in the RING finger-like domain bound p53 and SUMO-1, but not Ubc9 . PIAS1 catalyzed the sumoylation of p53 both in U2OS cells and in vitro in a domain-dependent manner . These data suggest that PIAS1 functions as a SUMO ligase, or possibly as a tightly bound regulator of it, toward p53. Mol Cell, 2001 Sep, 8(3), 671 - 82 The TGF beta receptor activation process: an inhibitor- to substrate-binding switch; Huse M et al.; The type I TGF beta receptor (T beta R-I) is activated by phosphorylation of the GS region, a conserved juxtamembrane segment located just N-terminal to the kinase domain . We have studied the molecular mechanism of receptor activation using a homogeneously tetraphosphorylated form of T beta R-I, prepared using protein semisynthesis . Phosphorylation of the GS region dramatically enhances the specificity of T beta R-I for the critical C-terminal serines of Smad2 . In addition, tetraphosphorylated T beta R-I is bound specifically by Smad2 in a phosphorylation-dependent manner and is no longer recognized by the inhibitory protein FKBP12 . Thus, phosphorylation activates T beta R-I by switching the GS region from a binding site for an inhibitor into a binding surface for substrate . Our observations suggest that phosphoserine/phosphothreonine-dependent localization is a key feature of the T beta R-I/Smad activation process. Mol Cell, 2001 Sep, 8(3), 557 - 69 Linking the Rb and polycomb pathways; Dahiya A et al.; Polycomb group (PcG) proteins associate to form complexes that repress Hox genes, thereby imposing the patterning of Hox expression required for development . However, these proteins have a second Hox-independent role in regulating cell proliferation . Our results suggest that association between Rb and PcG proteins forms a repressor complex that blocks entry of cells into mitosis . Also, we provide evidence that Rb colocalizes with nuclear PcG complexes and is important for association of PcG complexes with nuclear targets . The Rb-PcG complex may provide a means to link cell cycle arrest to differentiation events leading to embryonic pattern formation. Mol Cell, 2001 Sep, 8(3), 517 - 29 Identification of a 60S preribosomal particle that is closely linked to nuclear export; Bassler J et al.; A nuclear GTPase, Nug1p, was identified in a genetic screen for components linked to 60S ribosomal subunit export . Nug1p cosedimented with nuclear 60S preribosomes and was required for subunit export to the cytoplasm . Tagged Nug1p coprecipitated with proteins of the 60S subunit, late precursors to the 25S and 5.8S rRNAs, and at least 21 nonribosomal proteins . These included a homologous nuclear GTPase, Nug2p, the Noc2p/Noc3p heterodimer, Rix1p, and Rlp7p, each of which was implicated in 60S subunit export . Other known ribosome synthesis factors and proteins of previously unknown function, including the 559 kDa protein Ylr106p, also copurified . Eight of these proteins were copurified with nuclear pore complexes, suggesting that this complex represents the transport intermediate for 60S subunit export. Mol Cell, 2001 Sep, 8(3), 489 - 98 Common themes in mechanisms of gene silencing; Moazed D; The assembly of DNA into regions of inaccessible chromatin, called silent chromatin, is involved in the regulation of gene expression and maintenance of chromosome stability in eukaryotes . Recent studies on Sir2-containing silencing complexes in budding yeast and HP1- and Swi6-containing silencing complexes in metazoans and fission yeast suggest a common mechanism for the assembly of these domains, which involves the physical coupling of histone modifying enzymes to histone binding proteins. Mol Cell, 2001 Sep, 8(3), 484 - 6 Protein transport: two translocons are better than one; Robb A et al.; The translocon is the gateway to the endoplasmic reticulum (ER) . In yeast this is the Sec61p complex . However, new evidence suggests that a second translocon containing the Sec61p homolog Ssh1p provides important flexibility to the ER translocation machinery. Biochemistry, 2001 Aug 14, 40(32), 9685 - 94 Mutational analysis of the role of N-glycosylation in alpha-factor receptor function; Mentesana PE et al.; The alpha-factor mating pheromone receptor (encoded by STE2) activates a G protein signaling pathway that stimulates the conjugation of Saccharomyces cerevisiae yeast cells . The alpha-factor receptor is known to undergo several forms of post-translational modification, including phosphorylation, mono-ubiquitination, and N-linked glycosylation . Since phosphorylation and mono-ubiquitination have been shown previously to play key roles in regulating the signaling activity and membrane trafficking of the alpha-factor receptors, the role of N-linked glycosylation was investigated in this study . The Asn residues in the five consensus sites for N-linked glycosylation present in the extracellular regions of the receptor protein were mutated to prevent carbohydrate attachment at these sites . Mutation of two sites near the receptor N-terminus (N25Q and N32Q) diminished the degree of receptor glycosylation, and the corresponding double mutant was not detectably N-glycosylated . The nonglycosylated receptors displayed normal function and subcellular localization, indicating that glycosylation is not important for wild-type receptor activity . However, mutation of the glycosylation sites resulted in improved plasma membrane localization for the Ste2-3 mutant receptors that are normally retained intracellularly at elevated temperatures . These results suggest that N-glycosylation may be involved in the sorting process for misfolded Ste2 proteins, and may similarly affect certain mutant receptors whose altered trafficking is implicated in human diseases. Biochemistry, 2001 Aug 14, 40(32), 9638 - 46 DNA length-dependent cooperative interactions in the binding of Ku to DNA; Ma Y et al.; Despite its central role in the nonhomologous DNA end joining process, we still have an incomplete picture of the interaction between Ku and DNA . Here we describe both kinetic (surface plasmon resonance or SPR) and equilibrium (electrophoretic mobility shift assay or EMSA) studies of Ku binding to linear double-stranded DNA . Ku interaction with 1-site DNA is noncooperative, as expected . Electrophoretic mobility shift assays indicate cooperativity in the binding of Ku molecules to DNA long enough for two Ku molecules to bind (2-site DNA) . For the kinetic studies, we use surface plasmon resonance in which one end of the DNA molecules is linked to a surface while the other end is free to interact with Ku . We find that one Ku molecule dissociates from 1-site DNA with simple Langmuir (i.e., independent) kinetics . However, two Ku molecules associate and dissociate from 2-site DNA with a time course that cannot be described as a simple Langmuir interaction . On 3- and 4-site DNA, EMSA and SPR studies do not reveal any cooperativity, suggesting that the middle Ku does not exhibit cooperative interaction with the two Ku molecules bound at the DNA ends . These results indicate that Ku molecules can demonstrate cooperative interaction, and this is influenced by their positions along the DNA. J Virol, 2001 Nov, 75(21), 10334 - 47 Identification of acidic and aromatic residues in the Zta activation domain essential for Epstein-Barr virus reactivation; Deng Z et al.; Epstein-Barr virus (EBV) lytic cycle transcription and DNA replication require the transcriptional activation function of the viral immediate-early protein Zta . We describe a series of alanine substitution mutations in the Zta activation domain that reveal two functional motifs based on amino acid composition . Alanine substitution of single or paired hydrophobic aromatic amino acid residues resulted in modest transcription activation defects, while combining four substitutions of aromatic residues (F22/F26/W74/F75) led to more severe transcription defects . Substitution of acidic amino acid residue E27, D35, or E54 caused severe transcription defects on most viral promoters . Promoter- and cell-specific defects were observed for some substitution mutants . Aromatic residues were required for Zta interaction with TFIIA-TFIID and the CREB-binding protein (CBP) and for stimulation of CBP histone acetyltransferase activity in vitro . In contrast, acidic amino acid substitution mutants interacted with TFIIA-TFIID and CBP indistinguishably from the wild type . The nuclear domain 10 (ND10) protein SP100 was dispersed by most Zta mutants, but acidic residue mutations led to reduced, while aromatic substitution mutants led to increased SP100 nuclear staining . Acidic residue substitution mutants had more pronounced defects in transcription activation of endogenous viral genes in latently infected cells and for viral replication, as measured by the production of infectious virus . One mutant, K12/F13, was incapable of stimulating EBV lytic replication but had only modest transcription defects . These results indicate that Zta stimulates viral reactivation through two nonredundant structural motifs, one of which interacts with general transcription factors and coactivators, and the other has an essential but as yet not understood function in lytic transcription. J Virol, 2001 Nov, 75(21), 10272 - 80 mRNA decay during herpesvirus infections: interaction between a putative viral nuclease and a cellular translation factor; Feng P et al.; During lytic infections, the virion host shutoff (Vhs) protein (UL41) of herpes simplex virus destabilizes both host and viral mRNAs . By accelerating mRNA decay, it helps determine the levels and kinetics of viral and cellular gene expression . In vivo, Vhs shows a strong preference for mRNAs, as opposed to non-mRNAs, and degrades the 5' end of mRNAs prior to the 3' end . In contrast, partially purified Vhs is not restricted to mRNAs and causes cleavage of target RNAs at various sites throughout the molecule . To explain this discrepancy, we searched for cellular proteins that interact with Vhs using the Saccharomyces cerevisiae two-hybrid system . Vhs was found to interact with the human translation initiation factor, eIF4H . This interaction was verified by glutathione S-transferase pull-down experiments and by coimmunoprecipitation of Vhs and epitope-tagged eIF4H from extracts of mammalian cells . The interaction was abolished by several point mutations in Vhs that abrogate its ability to degrade mRNAs in vivo . The results suggest that Vhs is a viral mRNA degradation factor that is targeted to mRNAs, and to regions of translation initiation, through an interaction with eIF4H. J Struct Biol, 2001 Aug, 135(2), 219 - 29 Review: postchaperonin tubulin folding cofactors and their role in microtubule dynamics; Lopez-Fanarraga M et al.; The microtubule cytoskeleton consists of a highly organized network of microtubule polymers bound to their accessory proteins: microtubule-associated proteins, molecular motors, and microtubule-organizing proteins . The microtubule subunits are heterodimers composed of one alpha-tubulin polypeptide and one beta-tubulin polypeptide that should undergo a complex folding processing before they achieve a quaternary structure that will allow their incorporation into the polymer . Due to the extremely high protein concentration that exists at the cell cytoplasm, there are alpha- and beta-tubulin interacting proteins that prevent the unwanted interaction of these polypeptides with the surrounding protein pool during folding, thus allowing microtubule dynamics . Several years ago, the development of a nondenaturing electrophoretic technique made it possible to identify different tubulin intermediate complexes during tubulin biogenesis in vitro . By these means, the cytosolic chaperonin containing TCP-1 (CCT or TriC) and prefoldin have been demonstrated to intervene through tubulin and actin folding . Various other cofactors also identified along the alpha- and beta-tubulin postchaperonin folding route are now known to have additional roles in tubulin biogenesis such as participating in the synthesis, transport, and storage of alpha- and beta-tubulin . The future characterization of the tubulin-binding sites to these proteins, and perhaps other still unknown proteins, will help in the development of chemicals that could interfere with tubulin folding and thus modulating microtubule dynamics . In this paper, current knowledge of the above postchaperonin folding cofactors, which are in fact chaperones involved in tubulin heterodimer quaternary structure achievement, will be reviewed . Mol Endocrinol, 2001 Oct, 15(10), 1693 - 705 Cloning and characterization of gonadotropin-inducible ovarian transcription factors (GIOT1 and -2) that are novel members of the (Cys)(2)-(His)(2)-type zinc finger protein family; Mizutani T et al.; Gonadotropins are essential for ovarian follicular development and differentiation . To identify genes that are rapidly induced by gonadotropin in the immature rat ovary, ovarian genes were screened by a subtraction cloning procedure . cDNA clones encoding novel members of the (Cys)(2)-(His)(2)-type zinc finger protein family GIOT1 and -2 (gonadotropin-inducible transcription factor 1 and 2), were identified . Two isoforms of GIOT2 (GIOT2 alpha and 2 beta), which are probably produced by alternative splicing, also exist . Nucleotide sequence analysis revealed that GIOT1, but not GIOT2, contains the kruppel-associated box-A domain at the NH(2) terminus . RNA analyses revealed that these mRNAs were rapidly and temporarily induced by gonadotropins in the rat testis as well as in the ovary . In situ hybridization study revealed that expression of GIOT1 was induced in theca interna cells in the ovary and Leydig cells in the testis . Interestingly, the gene expression of GIOT1 is restricted to the pituitary, adrenal, testis, and ovary, while GIOT2 gene is expressed ubiquitously . A functional analysis of GIOT1 and -2 by a GAL4-based mammalian one-hybrid system revealed that GIOT1, but not GIOT2, is a transcriptional repressor and that the kruppel-associated box-A domain of GIOT1 is responsible for the transcriptional repressor activity . A GAL4-based yeast two-hybrid system was also used to identify proteins that interact with the rat GIOT1 . We cloned genes encoding rat homologs of human I-mfa domain containing protein and transcriptional intermediary factor 1 beta, both of which are transcription-regulatory proteins . Interaction of these proteins with GIOT1 was directly demonstrated by GST pull-down assay . Our data strongly suggest that GIOT1 may function as a novel transcriptional repressor by working with rat homologs of human I-mfa domain containing protein and transcriptional intermediary factor 1 beta proteins and may play a significant role at the transcription level in the folliculogenesis. J Biol Chem, 2001 Dec 7, 276(49), 46408 - 13 Epub 2001 Sep 27. Deletion of the RNA polymerase subunit RPB4 acts as a global, not stress-specific, shut-off switch for RNA polymerase II transcription at high temperatures; Miyao T et al.; We used whole genome expression analysis to investigate the changes in the mRNA profile in cells lacking the Saccharomyces cerevisiae RNA polymerase II subunit RPB4 (Delta RPB4) . Our results indicated that an essentially complete shutdown of transcription occurs upon temperature shift of this conditionally lethal mutant; 98% of mRNA transcript levels decrease at least 2-fold, 96% at least 4-fold . This data was supported by in vivo experiments that revealed a rapid and greater than 5-fold decline in steady state poly(A) RNA levels after the temperature shift . Expression of several individual genes, measured by Northern analysis, was also consistent with the whole genome expression profile . Finally we demonstrated that the loss of RNA polymerase II activity causes secondary effects on RNA polymerase I, but not RNA polymerase III, transcription . The transcription phenotype of the Delta RPB4 mutant closely mirrors that of the temperature-sensitive rpb1-1 mutant frequently implemented as a tool to inactivate the RNA polymerase II in vivo . Therefore, the Delta RPB4 mutant can be used to easily design strains that enable the study of distinct post-transcriptional cellular processes in the absence of RNA polymerase II transcription. J Biol Chem, 2001 Nov 30, 276(48), 44502 - 3 Epub 2001 Sep 27. Acetylation of MyoD by p300 requires more than its histone acetyltransferase domain; Polesskaya A et al.; MyoD, an essential transcription factor involved in muscle cell terminal differentiation, is regulated by acetylation, as are a number of other transcription factors, but the histone acetyltransferase enzyme responsible for this acetylation is a matter of controversy . In particular, contradictory findings have been reported concerning the ability of CBP/p300 to acetylate MyoD in vitro . Here we provide an explanation for this discrepancy: although full-length p300 does indeed acetylate MyoD, a fragment of p300 corresponding to its histone acetyltransferase domain does not . In addition to clearly demonstrating that p300 acetylates MyoD in vitro, these results underscore the necessity of using full-length histone acetyltransferase enzymes to draw valid conclusions from acetylation experiments. J Biol Chem, 2001 Nov 30, 276(48), 44413 - 8 Epub 2001 Sep 27. Genetic analysis of the DNA-dependent protein kinase reveals an inhibitory role of Ku in late S-G2 phase DNA double-strand break repair; Fukushima T et al.; Two major complementary double-strand break (DSB) repair pathways exist in vertebrates, homologous recombination (HR), which involves Rad54, and non-homologous end-joining, which requires the DNA-dependent protein kinase (DNA-PK) . DNA-PK comprises a catalytic subunit (DNA-PKcs) and a DNA-binding Ku70 and Ku80 heterodimer . To define the activities of individual DNA-PK components in DSB repair, we targeted the DNA-PKcs gene in chicken DT40 cells . DNA-PKcs deficiency caused a DSB repair defect that was, unexpectedly, suppressed by KU70 disruption . We have shown previously that genetic ablation of Ku70 confers RAD54-dependent radioresistance on S-G(2) phase cells, when sister chromatids are available for HR repair . To test whether direct interference by Ku70 with HR might explain the Ku70(-/-)/DNA-PKcs(-/-/-) radioresistance, we monitored HR activities directly in Ku- and DNA-PKcs-deficient cells . The frequency of intrachromosomal HR induced by the I-SceI restriction enzyme was increased in the absence of Ku but not of DNA-PKcs . Significantly, abrogation of HR activity by targeting RAD54 in Ku70(-/-) or DNA-PKcs(-/-/-) cells caused extreme radiosensitivity, suggesting that the relative radioresistance seen with loss of Ku70 was because of HR-dependent repair pathways . Our findings suggest that Ku can interfere with HR-mediated DSB repair, perhaps competing with HR for DSB recognition. Am J Kidney Dis, 2001 Oct, 38(4 Suppl 1), S34 - 7 Vascular calcification and inorganic phosphate; Giachelli CM et al.; Vascular calcification is highly correlated with elevated serum phosphate levels in uremic patients . To shed light on this process, we examined the ability of extracellular inorganic phosphate (Pi) levels to regulate human aortic smooth muscle cell (HSMC) culture mineralization in vitro . When cultured in media containing normal physiological levels of Pi (1.4 mmol/L Pi), HSMC grew in monolayers and did not mineralize . In contrast, HSMC cultured in media containing Pi levels comparable to those seen in hyperphosphatemic individuals (>1.4 mmol/L), showed dose-dependent increases in cell culture calcium deposition . Mechanistic studies showed that elevated Pi treatment of HSMC also enhanced the expression of the osteogenic markers, osteocalcin and Cbfa-1 . The effects of elevated Pi on HSMC were mediated by a sodium-dependent phosphate cotransporter (NPC), as indicated by the ability of the specific NPC inhibitor, phosphonoformic acid (PFA), to dose-dependently inhibit Pi-induced calcium deposition as well as osteocalcin and Cbfa-1 gene expression . Using polymerase chain reaction and Northern blot analyses, the NPC in HSMC was identified as Pit-1 (Glvr-1), a member of the type III NPCs . Interestingly, platelet-derived growth factor-BB (PDGF-BB), a potent atherogenic stimulus, increased the maximum velocity (Vmax) but not the affinity (Km) of phosphate uptake, enhanced the expression of Pit-1 mRNA, and induced HSMC culture calcification in a time- and dose-dependent manner . Importantly, in the presence of PDGF, HSMC culture calcification occurred under normophosphatemic conditions . These data suggest that elevated Pi may directly stimulate HSMC to undergo phenotypic changes that predispose to calcification and may help explain both the phenomena of human metastatic calcification under hyperphosphatemic conditions as well as increased calcification in PDGF-rich atherosclerotic lesions. FEBS Lett, 2001 Sep 21, 505(3), 393 - 8 Molecular characterization of two ammonium transporters from the ectomycorrhizal fungus Hebeloma cylindrosporum; Javelle A et al.; Heterologous expression of the yeast triple Mep mutant has enabled the first molecular characterization of AMT/MEP family members in an ectomycorrhizal fungus . External hyphae, which play a key role in nitrogen nutrition of trees, are considered as the absorbing structure of the ectomycorrhizal symbiosis and therefore molecular studies on ammonium transport in hyphae are urgently needed . The kinetic properties of AMT2 and AMT3 from Hebeloma cylindrosporum were studied in Saccharomyces cerevisiae . Expression of HcAmts in the yeast triple Mep mutant restored ammonium retention within cells . The HcAmts did not complement the ammonium sensing defect phenotype of Mep2Delta cells during pseudohyphal differentiation . Northern blot analysis in H . cylindrosporum showed that the HcAMTs were up-regulated upon nitrogen deprivation and down-regulated by ammonium. Traffic, 2001 Oct, 2(10), 698 - 704 Regulation of organelle membrane fusion by Pkc1p; Lin A et al.; Membrane fusion relies on complex protein machineries, which act in sequence to catalyze the fusion of bilayers . The fusion of endoplasmic reticulum membranes requires the t-SNARE Ufe1p, and the AAA ATPase p97/Cdc48p . While the mechanisms of membrane fusion events have begun to emerge, little is known about how this fusion process is regulated . We provide first evidence that endoplasmic reticulum membrane fusion in yeast is regulated by the action of protein kinase C . Specifically, Pkc1p kinase activity is needed to protect the fusion machinery from ubiquitin-mediated degradation. Plant J, 2001 Sep, 27(6), 539 - 49 Hydrophobic residues within the predicted N-terminal amphiphilic alpha-helix of a plant mitochondrial targeting presequence play a major role in in vivo import; Duby G et al.; A deletion and mutagenesis study was performed on the mitochondrial presequence of the beta-subunit of the F(1)-ATP synthase from Nicotiana plumbaginifolia linked to the green fluorescent protein (GFP) . The various constructs were tested in vivo by transient expression in tobacco protoplasts . GFP distribution in transformed cells was analysed in situ by confocal microscopy, and in vitro in subcellular fractions by Western blotting . Despite its being highly conserved in different species, deletion of the C-terminal region (residues 48-54) of the presequence did not affect mitochondrial import . Deletion of the conserved residues 40-47 and the less conserved intermediate region (residues 18-39) resulted in 60% reduction in GFP import, whereas mutation of conserved residues within these regions had little effect . Further shortening of the presequence progressively reduced import, with the construct retaining the predicted N-terminal amphiphilic alpha-helix (residues 1-12) being unable to mediate mitochondrial import . However, point mutation showed that this last region plays an important role through its basic residues and amphiphilicity, but also through its hydrophobic residues . Replacing Arg4 and Arg5 by alanine residues and shifting the Arg5 and Leu6 (in order to disturb amphiphilicity) resulted in reduction of the presequence import efficiency . The most dramatic effects were seen with single or double mutations of the four Leu residues (positions 5, 6, 10 and 11), which resulted in marked reduction or abolition of GFP import, respectively . We conclude that the N-terminal helical structure of the presequence is necessary but not sufficient for efficient mitochondrial import, and that its hydrophobic residues play an essential role in in vivo mitochondrial targeting. Prog Mol Subcell Biol, 2001, 26, 155 - 84 Regulation of translation initiation by amino acids in eukaryotic cells; Kimball SR; The translation of mRNA in eukaryotic cells is regulated by amino acids through multiple mechanisms . One such mechanism involves activation of mTOR (Fig . 1) . mTOR controls a myriad of downstream effectors, including RNA polymerase I, S6K1, 4E-BP1, and eEF2 kinase . In yeast, and probably in higher eukaryotes, mTOR signals through Tap42p/alpha 4 to regulate protein phosphatases . Through phosphorylation of Tap42p/alpha 4, mTOR abrogates dephosphorylation of the downstream effectors by PP2 A and/or PP6, resulting in their increased phosphorylation . Although at this time still speculative, in vitro results using mTOR immunoprecipitates suggest that mTOR, or an associated kinase, may also be directly involved in phosphorylating some effectors . Enhanced RNA polymerase I activity results in increased transcription of rDNA genes, whereas increased S6K1 activity promotes preferential translation of TOP mRNAs, such as those encoding ribosomal proteins . Together, stimulated RNA polymerase I and S6K1 activities enhance ribosome biogenesis, increasing the translational capacity of the cell . Phosphorylation of 4E-BP1 prohibits its association with eIF4E, allowing eIF4E to bind to eIF4G and form the active eIF4F complex . Increased eIF4F formation preferentially stimulates translation of mRNAs containing long, highly-structured 5' UTRs . Finally, amino acids cause inhibition of the eEF2 kinase, resulting in an increase in the proportion of eEF2 in the active, dephosphorylated form . By inhibiting eEF2 phosphorylation, amino acids may not only stimulate translation elongation, but may also prevent activation of GCN2 by enhancing the rate of removal of deacylated tRNA from the P-site on the ribosome; a potential activator of GCN2 . GCN2 may also be regulated directly by the accumulation of deacylated-tRNA caused by treatment with inhibitors of tRNA synthetases or in cells incubated in the absence of essential amino acids . However, because the Km of the tRNA synthetases for amino acids is well above the amino acid concentrations found in plasma of fasted animals, such a mechanism may not be operative in mammals in vivo . Activation of GCN2 results in increased phosphorylation of the alpha-subunit of eIF2, which in turn causes inhibition of eIF2B . Thus, by preventing activation of GCN2, amino acids preserve eIF2B activity, which promotes translation of all mRNAs, i.e., global protein synthesis is enhanced. Prog Mol Subcell Biol, 2001, 27, 57 - 89 Initiation factor eIF2 alpha phosphorylation in stress responses and apoptosis; Clemens MJ; The alpha subunit of polypeptide chain initiation factor eIF2 can be phosphorylated by a number of related protein kinases which are activated in response to cellular stresses . Physiological conditions which result in eIF2 alpha phosphorylation include virus infection, heat shock, iron deficiency, nutrient deprivation, changes in intracellular calcium, accumulation of unfolded or denatured proteins and the induction of apoptosis . Phosphorylated eIF2 acts as a dominant inhibitor of the guanine nucleotide exchange factor eIF2B and prevents the recycling of eIF2 between successive rounds of protein synthesis . Extensive phosphorylation of eIF2 alpha and strong inhibition of eIF2B activity can result in the downregulation of the overall rate of protein synthesis; less marked changes may lead to alterations in the selective translation of alternative open reading frames in polycistronic mRNAs, as demonstrated in yeast . These mechanisms can provide a signal transduction pathway linking eukaryotic cellular stress responses to alterations in the control of gene expression at the translational level. Nucleic Acids Res, 2001 Oct 1, 29(19), 3988 - 96 PROSPECT improves cis-acting regulatory element prediction by integrating expression profile data with consensus pattern searches; Fujibuchi W et al.; Consensus pattern and matrix-based searches designed to predict cis-acting transcriptional regulatory sequences have historically been subject to large numbers of false positives . We sought to decrease false positives by incorporating expression profile data into a consensus pattern-based search method . We have systematically analyzed the expression phenotypes of over 6000 yeast genes, across 121 expression profile experiments, and correlated them with the distribution of 14 known regulatory elements over sequences upstream of the genes . Our method is based on a metric we term probabilistic element assessment (PEA), which is a ranking of potential sites based on sequence similarity in the upstream regions of genes with similar expression phenotypes . For eight of the 14 known elements that we examined, our method had a much higher selectivity than a naive consensus pattern search . Based on our analysis, we have developed a web-based tool called PROSPECT, which allows consensus pattern-based searching of gene clusters obtained from microarray data. EMBO J, 2001 Oct 1, 20(19), 5480 - 90 Box C/D small nucleolar RNA trafficking involves small nucleolar RNP proteins, nucleolar factors and a novel nuclear domain; Verheggen C et al.; Nucleolar localization of box C/D small nucleolar (sno) RNAs requires the box C/D motif and, in vertebrates, involves transit through Cajal bodies (CB) . We report that in yeast, overexpression of a box C/D reporter leads to a block in the localization pathway with snoRNA accumulation in a specific sub-nucleolar structure, the nucleolar body (NB) . The human survival of motor neuron protein (SMN), a marker of gems/CB, specifically localizes to the NB when expressed in yeast, supporting similarities between these structures . Box C/D snoRNA accumulation in the NB was decreased by mutation of Srp40 and increased by mutation of Nsr1p, two related nucleolar proteins that are homologous to human Nopp140 and nucleolin, respectively . Box C/D snoRNAs also failed to accumulate in the NB, and became delocalized to the nucleoplasm, upon depletion of any of the core snoRNP proteins, Nop1p/fibrillarin, Snu13p, Nop56p and Nop5p/Nop58p . We conclude that snoRNP assembly occurs either in the nucleoplasm, or during transit of snoRNAs through the NB, followed by routing of the complete snoRNP to functional sites of ribosome synthesis. Gene, 2001 Sep 5, 275(1), 19 - 29 Molecular biology of the chromo domain: an ancient chromatin module comes of age; Eissenberg JC; The chromo domain motif is found in proteins from fungi, protists, plants, fish, insects, amphibians, birds, and mammals . The chromo domain peptide fold may have its origins as a chromosomal protein in a common ancestor of archea and eukaryota, making it a particularly ancient protein structural module . Chromo domains have been found in single or multiple copies in proteins with diverse structures and activities, most or all of which are connected with chromosome structure/function . In this review, our current knowledge of chromo domain properties is summarized and a variety of contexts in which chromo domains participate in aspects of chromatin metabolism are discussed. Genome Biol . 2001;2(9):RESEARCH0039 . Epub 2001 Aug 24. Abundant protein domains occur in proportion to proteome size; Malek JA; BACKGROUND: Conserved domains in proteins have crucial roles in protein interactions, DNA binding, enzyme activity and other important cellular processes . It will be of interest to determine the proportions of genes containing such domains in the proteomes of different eukaryotes . RESULTS: The average proportion of conserved domains in each of five eukaryote genomes was calculated . In pairwise genome comparisons, the ratio of genes containing a given conserved domain in the two genomes on average reflected the ratio of the predicted total gene numbers of the two genomes . These ratios have been verified using a repository of databases and one of its subdivisions of conserved domains . CONCLUSIONS: Many conserved domains occur as a constant proportion of proteome size across the five sequenced eukaryotic genomes . This raises the possibility that this proportion is maintained because of functional constraints on interacting domains . The universality of the ratio in the five eukaryotic genomes attests to its potential importance. Proc Natl Acad Sci U S A, 2001 Sep 25, 98(20), 11806 - 11 LEAFY COTYLEDON2 encodes a B3 domain transcription factor that induces embryo development; Stone SL et al.; The Arabidopsis LEAFY COTYLEDON2 (LEC2) gene is a central embryonic regulator that serves critical roles both early and late during embryo development . LEC2 is required for the maintenance of suspensor morphology, specification of cotyledon identity, progression through the maturation phase, and suppression of premature germination . We cloned the LEC2 gene on the basis of its chromosomal position and showed that the predicted polypeptide contains a B3 domain, a DNA-binding motif unique to plants that is characteristic of several transcription factors . We showed that LEC2 RNA accumulates primarily during seed development, consistent with our finding that LEC2 shares greatest similarity with the B3 domain transcription factors that act primarily in developing seeds, VIVIPAROUS1/ABA INSENSITIVE3 and FUSCA3 . Ectopic, postembryonic expression of LEC2 in transgenic plants induces the formation of somatic embryos and other organ-like structures and often confers embryonic characteristics to seedlings . Together, these results suggest that LEC2 is a transcriptional regulator that establishes a cellular environment sufficient to initiate embryo development. Proc Natl Acad Sci U S A, 2001 Sep 25, 98(20), 11211 - 7 Binding of cyclin-dependent kinases to ORC and Cdc6p regulates the chromosome replication cycle; Weinreich M et al.; Cdc6p and the origin recognition complex (ORC) are essential for assembly of a pre-replicative complex (preRC) at origins of replication, before the initiation of DNA synthesis . In the absence of Cdc6p, cells fail to initiate DNA replication and undergo a "reductional" mitosis, in which the unreplicated chromosomes are randomly segregated to the spindle poles . We show here that the cells harboring a mutation in the essential Cdc6p Walker A-box arrest in late mitosis, probably at anaphase . This cell cycle block requires either the three Cdc28p phosphorylation sites within the N terminus of Cdc6p or a short region (aa 8-17) that contains a Cy (Cyclin) interaction sequence . These same two Cdc6p mutants that allow a reductional mitosis are defective in binding Cdc28p kinase . In addition to Cdc6p, ORC also binds to cyclin-dependent kinases (CDKs) . Interestingly, Sic1p, a CDK inhibitor protein, blocked the S phase-specific Cdc28p-Clb5p kinase from interacting with ORC, but did not prevent the G(1)-specific Cdc28p-Cln2p kinase-ORC interaction . We suggest that ORC, Cdc6p, and Sic1p bind to different CDKs in a cell cycle-dependent manner to temporally regulate events that (i) allow preRC formation after mitosis, (ii) prevent mitosis before DNA replication can occur, and (iii) promote initiation of DNA replication. Proc Natl Acad Sci U S A, 2001 Sep 25, 98(20), 11193 - 8 A computational analysis of sequence features involved in recognition of short introns; Lim LP et al.; Splicing of short introns by the nuclear pre-mRNA splicing machinery is thought to proceed via an "intron definition" mechanism, in which the 5' and 3' splice sites (5'ss, 3'ss, respectively) are initially recognized and paired across the intron . Here, we describe a computational analysis of sequence features involved in recognition of short introns by using available transcript data from five eukaryotes with complete or nearly complete genomic sequences . The information content of five different transcript features was measured by using methods from information theory, and Monte Carlo simulations were used to determine the amount of information required for accurate recognition of short introns in each organism . We conclude: (i) that short introns in Drosophila melanogaster and Caenorhabditis elegans contain essentially all of the information for their recognition by the splicing machinery, and computer programs that simulate splicing specificity can predict the exact boundaries of approximately 95% of short introns in both organisms; (ii) that in yeast, the 5'ss, branch signal, and 3'ss can accurately identify intron locations but do not precisely determine the location of 3' cleavage in every intron; and (iii) that the 5'ss, branch signal, and 3'ss are not sufficient to accurately identify short introns in plant and human transcripts, but that specific subsets of candidate intronic enhancer motifs can be identified in both human and Arabidopsis that contribute dramatically to the accuracy of splicing simulators. J Biol Chem, 2001 Nov 30, 276(48), 44677 - 87 Epub 2001 Sep 25. Coordinate action of the helicase and 3' to 5' exonuclease of Werner syndrome protein; Opresko PL et al.; Werner syndrome is a human disorder characterized by premature aging, genomic instability, and abnormal telomere metabolism . The Werner syndrome protein (WRN) is the only known member of the RecQ DNA helicase family that contains a 3' --> 5'-exonuclease . However, it is not known whether both activities coordinate in a biological pathway . Here, we describe DNA structures, forked duplexes containing telomeric repeats, that are substrates for the simultaneous action of both WRN activities . We used these substrates to study the interactions between the WRN helicase and exonuclease on a single DNA molecule . WRN helicase unwinds at the forked end of the substrate, whereas the WRN exonuclease acts at the blunt end . Progression of the WRN exonuclease is inhibited by the action of WRN helicase converting duplex DNA to single strand DNA on forks of various duplex lengths . The WRN helicase and exonuclease act in concert to remove a DNA strand from a long forked duplex that is not completely unwound by the helicase . We analyzed the simultaneous action of WRN activities on the long forked duplex in the presence of the WRN protein partners, replication protein A (RPA), and the Ku70/80 heterodimer . RPA stimulated the WRN helicase, whereas Ku stimulated the WRN exonuclease . In the presence of both RPA and Ku, the WRN helicase activity dominated the exonuclease activity. Cell, 2001 Sep 21, 106(6), 759 - 69 Hedgehog signaling in germ cell migration; Deshpande G et al.; The primitive gonad of the Drosophila embryo is formed from two cell types, the somatic gonad precursor cells (SGPs) and the germ cells, which originate at distant sites . To reach the SGPs the germ cells must undergo a complex series of cell movements . While there is evidence that attractive and repulsive signals guide germ cell migration through the embryo, the molecular identity of these instructive molecules has remained elusive . Here, we present evidence suggesting that hedgehog (hh) may serve as such an attractive guidance cue . Misexpression of hh in the soma induces germ cells to migrate to inappropriate locations . Conversely, cell-autonomous components of the hh pathway appear to be required in the germline for proper germ cell migration. Cell, 2001 Sep 21, 106(6), 723 - 33 A plant viral "reinitiation" factor interacts with the host translational machinery; Park HS et al.; The cauliflower mosaic virus transactivator, TAV, controls translation reinitiation of major open reading frames on polycistronic RNA . We show here that TAV function depends on its association with polysomes and eukaryotic initiation factor eIF3 in vitro and in vivo . TAV physically interacts with eIF3 and the 60S ribosomal subunit . Two proteins mediating these interactions were identified: eIF3g and 60S ribosomal protein L24 . Transient expression of eIF3g and L24 in plant protoplasts strongly affects TAV-mediated reinitiation activity . We demonstrate that TAV/eIF3/40S and eIF3/TAV/60S ternary complexes form in vitro, and propose that TAV mediates efficient recruitment of eIF3 to polysomes, allowing translation of polycistronic mRNAs by reinitiation, overcoming the normal cell barriers to this process. Cell, 2001 Sep 21, 106(6), 661 - 73 Switching and signaling at the telomere; Blackburn EH; This review describes the structure of telomeres, the protective DNA-protein complexes at eukaryotic chromosomal ends, and several molecular mechanisms involved in telomere functions . Also discussed are cellular responses to compromising the functions of telomeres and of telomerase, which synthesizes telomeric DNA. J Am Chem Soc, 2001 Oct 3, 123(39), 9665 - 72 Theoretical study of the mechanisms of substrate recognition by catalase; Kalko SG et al.; A variety of theoretical methods including classical molecular interaction potentials, classical molecular dynamics, and activated molecular dynamics have been used to analyze the substrate recognition mechanisms of peroxisomal catalase from Saccharomyces cerevisiae . Special attention is paid to the existence of channels connecting the heme group with the exterior of the protein . On the basis of these calculations a rationale is given for the unique catalytic properties of this enzyme, as well as for the change in enzyme efficiency related to key mutations . According to our calculations the water is expected to be a competitive inhibitor of the enzyme, blocking the access of hydrogen peroxide to the active site . The main channel is the preferred route for substrate access to the enzyme and shows a cooperative binding to hydrogen peroxide . However, the overall affinity of the main channel for H(2)O(2) is only slightly larger than that for H(2)O . Alternative channels connecting the heme group with the monomer interface and the NADP(H) binding site are detected . These secondary channels might be important for product release. Glia, 2001 Oct, 36(1), 1 - 10 KUB3 amplification and overexpression in human gliomas; Fischer U et al.; Gene amplification is known to occur frequently in human glioma . Recently we reported cloning of a novel gene termed glioma-amplified sequence 16 (GAS16) by microdissection-mediated cDNA capture . In this article, we demonstrate that GAS16 results from an alternative splicing process of the Ku70 binding protein 3 (KUB3) that is essential for DNA double-strand break repair . The alternative splice product was found in glioblastoma and in normal fetal brain . We determined the amplification frequency of KUB3 in glioma with different grading . We analyzed a total of 102 glioma primary tumors and found KUB3 to be amplified in 12/82 (14%) glioblastomas, 4/13 anaplastic astrocytomas (30%), and 2/4 astrocytomas, but in none of three pilocytic astrocytomas . Northern blot analysis of glioblastoma shows a strong correlation between KUB3 amplification and overexpression . Amplification of KUB3 appears to be independent of other genetic changes frequently associated with the development of gliomas, including EGFR amplification, LOH of TP53, and LOH of chromosome 10 . The KUB3 amplification and overexpression may interfere with the function of KUB3 in the DNA-PK complex involved in the maintenance of genome stability and reduction of mutation frequency . Cell Growth Differ, 2001 Sep, 12(9), 457 - 63 The Smad transcriptional corepressor TGIF recruits mSin3; Wotton D et al.; The homeodomain protein TG-interacting factor (TGIF) represses transcription by histone deacetylase-dependent and -independent means . Heterozygous mutations in human TGIF result in holoprosencephaly, a severe genetic disorder affecting craniofacial development, suggesting that TGIF is critical for normal development . After transforming growth factorbeta (TGFbeta) stimulation, Smad proteins enter the nucleus and form transcriptional activation complexes or interact with TGIF, which functions as a corepressor . The relative levels of Smad corepressors and coactivators present within the cell may determine the outcome of a TGFbeta response . We show that TGIF interacts directly with the paired amphipathic alpha-helix 2 domain of the mSin3 corepressor, and TGIF recruits mSin3 to a TGFbeta-activated Smad complex . The mSin3 interaction domain of TGIF has been shown to be essential for repression of a TGFbeta transcriptional response . Thus, TGIF represents a targeting component of the mSin3 corepressor complex. Biochemistry, 2001 Oct 2, 40(39), 11785 - 93 Use of a combined enzymatic digestion/ESI mass spectrometry assay to study the effect of TATA-binding protein on photoproduct formation in a TATA box; Wang Y et al.; Recently, it was reported that TATA-binding protein (TBP) enhances (6-4) photoproduct formation in a TATA box under UVC irradiation {Aboussekhra and Thoma (1999) EMBO J . 18, 433-443} . The conclusions of that study were based on an indirect enzymatic assay that was not specific for (6-4) photoproducts . Herein we report the use of a recently developed coupled enzymatic digestion/mass spectrometry assay {Wang et al . (1999) Chem . Res . Toxicol . 12, 1077-1082} to identify unambiguously and quantify the photoproducts formed in a TATA box-containing dodecamer duplex sequence in the presence or absence of TBP binding . Exposure of the adenovirus major late promoter TATA box to a high dose of UVC irradiation in the absence of the C-terminal domain of yeast TBP leads to predominant formation of the cis-syn dimer within the T(4) tract, whereas exposure in the presence of TBP leads to almost exclusive formation of the (6-4) photoproduct . In contrast, the (6-4) product is not detected at high doses of UVB irradiation in the absence of TBP but is detected in the presence of TBP, although the cis-syn product predominates . When the products of UVB irradiation were subsequently exposed to a high dose of UVC irradiation in the presence of TBP, the (6-4) photoproduct again becomes nearly the exclusive photoproduct, indicating that the cis-syn dimer is being reversed to TT by UVC light . Both cis-syn and (6-4) photoproducts are formed in approximately equal amounts upon irradiation with small doses of UVC in the presence of TBP, but the fraction of (6-4) photoproduct increases with dose . Through the use of a TATA box containing a site-specifically deuterated thymine, it was found that (6-4) photoproducts formed most selectively at the second and third positions of the T(4) tract upon either UVB or UVC irradiation in the presence of TBP . By using the same substrate, it was found that UVC-induced TA formation was inhibited by TBP binding and that TA formation was greatest at the 5' end of the TATA sequence. Curr Genet, 2001 Aug, 40(1), 27 - 39 Transposon mutagenesis reveals novel loci affecting tolerance to salt stress and growth at low temperature; de Jesus Ferreira MC et al.; Using transposon mutagenesis in the haploid Saccharomyces cerevisiae strain W303-1A we have identified genes required for growth in high salt medium, survival of a hypo-osmotic shock and growth at 15 degrees C . Screening 25,000 transposon insertions revealed a total of 61 insertions that caused salt-sensitivity; and those insertions affected 31 genes . Only 12 of those genes were previously known to be required for salt-tolerance . Among the 61 insertions, three caused general osmo-sensitivity . We identified one single insertion mutant in the already-known gene, FPS1, required for survival of hypo-osmotic shock . A total of 31 insertions caused failure to grow at low temperature . Those identified ten different genes, three of which had previously been reported to affect cold-tolerance . Four genes were identified in both the salt and the cold-sensitivity screen . We found several unusual insertion mutations: (1) insertions in or close to essential genes, (2) insertion in an intergenic region and (3) insertions causing stress-sensitivity in W303-1A, while the deletion mutant in BY4741 did not show such a phenotype . Surprisingly, our mutant set and that reported in the large-scale transposon insertion project (TRIPLES, only marginally overlap . We discuss some of the features of transposon mutagenesis in light of the availability of the complete set of yeast deletion mutants and we discuss the possible roles of the genes we identified. Curr Genet, 2001 Aug, 40(1), 13 - 26 far4, far5, and far6 define three genes required for efficient activation of MAPKs Fus3 and Kss1 and accumulation of glycogen; Cherkasova V et al.; In Saccharomyces cerevisiae, mating pheromones induce G1 arrest through the activation of two MAP kinases, Fus3 and Kss1 . Here we report the isolation of three mutants, far4, far5, and far6, that have the novel phenotype of regulating both the activity of Fus3 and Kss1 and the accumulation of glycogen . A far4 mutation constitutively activates Fus3 and Kss1, reduces glycogen, and blocks G1 arrest in the presence of alpha factor . In contrast, far5 and far6 mutations increase glycogen and reduce activation of Fus3 and Kss1 by pheromone . far4, far5, and far6 are recessive and not allelic to FAR1, FAR3, or 14 genes known to regulate the pheromone response . Non-allelic noncomplementation occurs between far6 and both far4 and far5, suggesting that FAR6 functionally interacts with FAR4 and FAR5 . Additional observations suggest that FAR4 has functional overlap with FAR3, which we also find to regulate glycogen accumulation . Our results suggest that the activation of the mating MAPK cascade and subsequent G1 arrest is influenced by a signal transduction pathway that regulates glycogen . In support of this possibility, we find that Fus3 is activated to a greater extent in a "wimp" strain with defective protein kinase A . Finally, BIM1 and BIK1 have been identified as CEN suppressors of far5, suggesting that the microtubule apparatus may regulate the ability of the pheromone response pathway to promote G1 arrest. Dev Psychobiol, 2001 Sep, 39(2), 107 - 23 Cardiovascular indices of physiological arousal in boys with fragile X syndrome; Roberts JE et al.; In this study, the relationship between physiological arousal, as indexed by heart rate variability, was examined in boys with fragile X syndrome (FXS) and typically developing boys matched on chronological age . In addition, the relationship of heart activity to clinical and molecular factors in the group of boys with FXS was examined . Results suggest that boys with FXS have higher levels of heart activity during the passive phases, as reflected in shorter heart periods . This high level of heart activity appears to be due to increased sympathetic activity and reduced parasympathetic activity . Boys with FXS did not display the expected patterns of heart activity in response to phases of increasing challenge, and sympathetic and parasympathetic systems did not appear coordinated in these boys with FXS . Clinical factors may be related to neural regulation of heart activity while molecular factors do not appear to be . Anal Biochem, 2001 Oct 1, 297(1), 25 - 31 Optimization of the isotope-coded affinity tag-labeling procedure for quantitative proteome analysis; Smolka MB et al.; The combination of isotope coded affinity tag (ICAT) reagents and tandem mass spectrometry constitutes a new method for quantitative proteomics . It involves the site-specific, covalent labeling of proteins with isotopically normal or heavy ICAT reagents, proteolysis of the combined, labeled protein mixture, followed by the isolation and mass spectrometric analysis of the labeled peptides . The method critically depends on labeling protocols that are specific, quantitative, general, robust, and reproducible . Here we describe the systematic evaluation of important parameters of the labeling protocol and describe optimized labeling conditions . The tested factors include the ICAT reagent concentration, the influence of the protein, SDS, and urea concentrations on the labeling reaction, and the reaction time . We demonstrate that using the optimized conditions specific and quantitative labeling was achieved on standard proteins as well as in complex protein mixtures such as a yeast cell lysate . J Bacteriol, 2001 Oct, 183(20), 5942 - 55 Aut5/Cvt17p, a putative lipase essential for disintegration of autophagic bodies inside the vacuole; Epple UD et al.; Selective disintegration of membrane-enclosed autophagic bodies is a feature of eukaryotic cells not studied in detail . Using a Saccharomyces cerevisiae mutant defective in autophagic-body breakdown, we identified and characterized Aut5p, a glycosylated integral membrane protein . Site-directed mutagenesis demonstrated the relevance of its putative lipase active-site motif for autophagic-body breakdown . aut5Delta cells show reduced protein turnover during starvation and are defective in maturation of proaminopeptidase I . Most recently, by means of the latter phenotype, Aut5p was independently identified as Cvt17p . In this study we additionally checked for effects on vacuolar acidification and detected mature vacuolar proteases, both of which are prerequisites for autophagic-body lysis . Furthermore, biologically active hemagglutinin-tagged Aut5p (Aut5-Ha) localizes to the endoplasmic reticulum (nuclear envelope) and is targeted to the vacuolar lumen independent of autophagy . In pep4Delta cells immunogold electron microscopy located Aut5-Ha at approximately 50-nm-diameter intravacuolar vesicles . Characteristic missorting in vps class E and fab1Delta cells, which affects the multivesicular body (MVB) pathway, suggests vacuolar targeting of Aut5-Ha similar to that of the MVB pathway . In agreement with localization of Aut5-Ha at intravacuolar vesicles in pep4Delta cells and the lack of vacuolar Aut5-Ha in wild-type cells, our pulse-chase experiments clearly indicated that Aut5-Ha degradation with 50 to 70 min of half-life is dependent on vacuolar proteinase A. EMBO J, 2001 Sep 17, 20(18), 5269 - 79 Distinct requirements for C.elegans TAF(II)s in early embryonic transcription; Walker AK et al.; TAF(II)s are conserved components of the TFIID, TFTC and SAGA-related mRNA transcription complexes . In yeast (y), yTAF(II)17 is required broadly for transcription, but various other TAF(II)s appear to have more specialized functions . It is important to determine how TAF(II)s contribute to transcription in metazoans, which have larger and more diverse genomes . We have examined TAF(II) functions in early Caenorhabditis elegans embryos, which can survive without transcription for several cell generations . We show that taf-10 (yTAF(II)17) and taf-11 (yTAF(II)25) are required for a significant fraction of transcription, but apparently are not needed for expression of multiple developmental and other metazoan-specific genes . In contrast, taf-5 (yTAF(II)48; human TAF(II)130) seems to be required for essentially all early embryonic mRNA transcription . We conclude that TAF-10 and TAF-11 have modular functions in metazoans, and can be bypassed at many metazoan-specific genes . The broad involvement of TAF-5 in mRNA transcription in vivo suggests a requirement for either TFIID or a TFTC-like complex. EMBO J, 2001 Sep 17, 20(18), 5219 - 31 Antagonistic remodelling by Swi-Snf and Tup1-Ssn6 of an extensive chromatin region forms the background for FLO1 gene regulation; Fleming AB et al.; Novel yeast histone mutations that confer Swi-Snf independence (Sin(-)) were used to investigate the mechanisms by which transcription coactivator complexes relieve chromatin repression in vivo . Derepression of the flocculation gene FLO1, which is normally repressed by the Tup1-Ssn6 corepressor, leads to its identification as a constitutive Swi-Snf-dependent gene . We demonstrate that Tup1-Ssn6 is a chromatin remodelling complex that rearranges and also orders nucleosomal arrays on the promoter and over 5 kb of upstream intergenic region . Our results confirm that the Swi-Snf complex disrupts nucleosome positioning on promoters, but reveal that it can also rearrange nucleosomes several kilobases upstream from the transcription start site . The antagonistic chromatin remodelling activities of Swi-Snf and Tup1-Ssn6 detected in an array of 32 nucleosomes upstream of FLO1 extend far beyond the scale of promoter-based models of chromatin-mediated gene regulation . The Swi-Snf coactivator and Tup1-Ssn6 corepressor control an extensive chromatin domain in which regulation of the FLO1 gene takes place. EMBO J, 2001 Sep 17, 20(18), 5187 - 96 A novel active site-directed probe specific for deubiquitylating enzymes reveals proteasome association of USP14; Borodovsky A et al.; A C-terminally modified ubiquitin (Ub) derivative, ubiquitin vinyl sulfone (UbVS), was synthesized as an active site-directed probe that irreversibly modifies a subset of Ub C-terminal hydrolases (UCHs) and Ub-specific processing proteases (UBPs) . Specificity of UbVS for deubiquitylating enzymes (DUBs) is demonstrated not only by inhibition of {(125)I}UbVS labeling with N-ethylmaleimide and Ub aldehyde, but also by genetic analysis . {(125)I}UbVS modifies six of the 17 known and putative yeast deubiquitylating enzymes (Yuh1p, Ubp1p, Ubp2p, Ubp6p, Ubp12p and Ubp15p), as revealed by analysis of corresponding mutant strains . In mammalian cells, greater numbers of polypeptides are labeled, most of which are likely to be DUBs . Using {(125)I}UbVS as a probe, we report the association of an additional DUB with the mammalian 26S proteasome . In addition to the 37 kDa enzyme reported to be part of the 19S cap, we identified USP14, a mammalian homolog of yeast Ubp6p, as being bound to the proteasome . Remarkably, labeling of 26S-associated USP14 with {(125)I}UbVS is increased when proteasome function is impaired, suggesting functional coupling between the activities of USP14 and the proteasome. EMBO J, 2001 Sep 17, 20(18), 5176 - 86 Sorting of proteins into multivesicular bodies: ubiquitin-dependent and -independent targeting; Reggiori F et al.; Yeast endosomes, like those in animal cells, invaginate their membranes to form internal vesicles . The resulting multivesicular bodies fuse with the vacuole, the lysosome equivalent, delivering the internal vesicles for degradation . We have partially purified internal vesicles and analysed their content . Besides the known component carboxypeptidase S (Cps1p), we identified a polyphosphatase (Phm5p), a presumptive haem oxygenase (Ylr205p/Hmx1p) and a protein of unknown function (Yjl151p/Sna3p) . All are membrane proteins, and appear to be cargo molecules rather than part of the vesicle-forming machinery . We show that both Phm5p and Cps1p are ubiquitylated, and that in a doa4 mutant, which has reduced levels of free ubiquitin, Cps1p, Phm5p and Hmx1p are mis-sorted to the vacuolar membrane . Mutation of Lys 6 in the cytoplasmic tail of Phm5p disrupts its sorting, but sorting is restored, even in doa4 cells, by the biosynthetic addition of a single ubiquitin chain . In contrast, Sna3p enters internal vesicles in a ubiquitin-independent manner . Thus, ubiquitin acts as a signal for the partitioning of some, but not all, membrane proteins into invaginating endosomal vesicles. Biochim Biophys Acta, 2001 Sep 28, 1537(2), 158 - 66 Nuclear aggregation of huntingtin is not prevented by deletion of chaperone Hsp104; Cao F et al.; Polyglutamine expansion causes the disease proteins to aggregate, resulting in stable insoluble aggregates in the nucleus . The in vitro aggregation and cellular toxicity of polyglutamine proteins are reduced by chaperone heat shock proteins (Hsp) . In polyglutamine disease animal models, however, polyglutamine inclusions remain in the nucleus despite the suppression of neurodegeneration by Hsp . Studies using yeast genetic approach revealed that the balance of Hsp is important for regulating protein aggregation in the cytoplasm of yeast cells . Here we report that N-terminal fragments of huntingtin with an expanded polyglutamine tract form aggregates only in the cytoplasm of yeast cells and, when tagged with nuclear localization sequences (NLS), are able to aggregate in the nucleus . Deletion of the Hsp104 gene prevents the aggregation of huntingtin in the cytoplasm but is unable to eliminate the aggregation of NLS-tagged huntingtin in the nucleus . The inhibitory effect of Hsp104 deletion on the cytoplasmic aggregation of huntingtin only occurs in viable yeast cells, as aggregates can be formed in Hsp104 deletion cells that have been frozen for 72 h . Fresh cytosolic extracts of the Hsp104 deletion strain inhibit the aggregation of huntingtin in vitro, suggesting that the deletion of Hsp104 may alter the activities of other cytoplasmic factors to inhibit polyglutamine aggregation in the cytoplasm . We propose that the regulatory effects of chaperones may mainly be restricted to the cytoplasm and have much less influence on polyglutamine-containing aggregates in the nucleus. Curr Biol, 2001 Sep 18, 11(18), 1427 - 31 Origin recognition complex binding to a metazoan replication origin; Bielinsky AK et al.; The initiation of DNA replication in eukaryotic cells at the onset of S phase requires the origin recognition complex (ORC) {1} . This six-subunit complex, first isolated in Saccharomyces cerevisiae {2}, is evolutionarily conserved {1} . ORC participates in the formation of the prereplicative complex {3}, which is necessary to establish replication competence . The ORC-DNA interaction is well established for autonomously replicating sequence (ARS) elements in yeast in which the ARS consensus sequence {4} (ACS) constitutes part of the ORC binding site {2, 5} . Little is known about the ORC-DNA interaction in metazoa . For the Drosophila chorion locus, it has been suggested that ORC binding is dispersed {6} . We have analyzed the amplification origin (ori) II/9A of the fly, Sciara coprophila . We identified a distinct 80-base pair (bp) ORC binding site and mapped the replication start site located adjacent to it . The binding of ORC to this 80-bp core region is ATP dependent and is necessary to establish further interaction with an additional 65-bp of DNA . This is the first time that both the ORC binding site and the replication start site have been identified in a metazoan amplification origin . Thus, our findings extend the paradigm from yeast ARS1 to multicellular eukaryotes, implicating ORC as a determinant of the position of replication initiation. Arch Biochem Biophys, 2001 Oct 1, 394(1), 67 - 75 Chimers of two fused ADP/ATP carrier monomers indicate a single channel for ADP/ATP transport; Huang SG et al.; The mitochondrial ADP/ATP carrier (AAC) is generally believed to function as a homodimer (Wt . Wt) . It remains unknown whether the two monomers possess two independent but fully anticooperative channels or they form a single central channel for nucleotide transport . Here we generated fusion proteins consisting of two tandem covalent-linked AAC monomers and studied the kinetics of ADP/ATP transport in reconstituted proteoliposomes . Functional 64-kDa fusion proteins Wt-Wt and Wt-R294A (wild-type AAC linked to a mutant having low ATP transport activity) were expressed in mitochondria of yeast transformants . Compared to homodimer Wt . Wt, the fusion protein Wt-Wt retained the transport activity and selectivity of ADP versus ATP . The strongly divergent selectivities of Wt and R294A were partially propagated in the Wt-R294A fusion protein, suggesting a limited cooperativity during solute translocation . The rates of ADP or ATP transport were significantly higher than those predicted by the two-channel model . Fusion proteins for Wt-R204L (Wt linked to an inactive mutant) and R204L-Wt were not expressed in aerobically grown yeast cells, which contained plasmid rearrangements that regenerated the fully active 32-kDa homodimer Wt . Wt, suggesting that these fusion proteins are inactive in ADP/ATP transport . These results favor a single binding center gated pore model {Klingenberg, M . (1991) in A Study of Enzymes, Vol . 2: pp . 367-388} in which two AAC subunits cooperate for a coordinated ADP/ATP exchange through a single channel . RNA, 2001 Sep, 7(9), 1317 - 34 Dbp9p, a putative ATP-dependent RNA helicase involved in 60S-ribosomal-subunit biogenesis, functionally interacts with Dbp6p; Daugeron MC et al.; Ribosome synthesis is a highly complex process and constitutes a major cellular activity . The biogenesis of this ribonucleoprotein assembly requires a multitude of protein trans-acting factors including several putative ATP-dependent RNA helicases of the DEAD-box and related protein families . Here we show that the previously uncharacterized Saccharomyces cerevisiae open reading frame YLR276C, hereafter named DBP9 (DEAD-box protein 9), encodes an essential nucleolar protein involved in 60S-ribosomal-subunit biogenesis . Genetic depletion of Dbp9p results in a deficit in 60S ribosomal subunits and the appearance of half-mer polysomes . This terminal phenotype is likely due to the instability of early pre-ribosomal particles, as evidenced by the low steady-state levels and the decreased synthesis of the 27S precursors to mature 25S and 5.8S rRNAs . In agreement with a role of Dbp9p in 60S subunit synthesis, we find that increased Dbp9p dosage efficiently suppresses certain dbp6 alleles and that dbp6/dbp9 double mutants show synthetic lethality . Furthermore, Dbp6p and Dbp9p weakly interact in a yeast two-hybrid assay . Altogether, our findings indicate an intimate functional interaction between Dbp6p and Dbp9p during the process of 60S-ribosomal-subunit assembly. RNA, 2001 Sep, 7(9), 1284 - 97 Evidence for a role of Sky1p-mediated phosphorylation in 3' splice site recognition involving both Prp8 and Prp17/Slu4; Dagher SF et al.; The SRPK family of kinases is specific for RS domain-containing splicing factors and known to play a critical role in protein-protein interaction and intracellular distribution of their substrates in both yeast and mammalian cells . However, the function of these kinases in pre-mRNA splicing remains unclear . Here we report that SKY1, a SRPK family member in Saccharomyces cerevisiae, genetically interacts with PRP8 and PRP17/SLU4, both of which are involved in splice site selection during pre-mRNA splicing . Prp8 is essential for splicing and is known to interact with both 5' and 3' splice sites in the spliceosomal catalytic center, whereas Prp17/Slu4 is nonessential and is required only for efficient recognition of the 3' splice site . Interestingly, deletion of SKY1 was synthetically lethal with all prp17 mutants tested, but only with specific prp8 alleles in a domain implicated in governing fidelity of 3'AG recognition . Indeed, deletion of SKY1 specifically suppressed 3'AG mutations in ACT1-CUP1 splicing reporters . These results suggest for the first time that 3' AG recognition may be subject to phosphorylation regulation by Sky1p during pre-mRNA splicing. Mol Cell Biol, 2001 Oct, 21(20), 7097 - 104 Mitochondrial import driving forces: enhanced trapping by matrix Hsp70 stimulates translocation and reduces the membrane potential dependence of loosely folded preproteins; Geissler A et al.; The mitochondrial heat shock protein Hsp70 (mtHsp70) is essential for driving translocation of preproteins into the matrix . Two models, trapping and pulling by mtHsp70, are discussed, but positive evidence for either model has not been found so far . We have analyzed a mutant mtHsp70, Ssc1-2, that shows a reduced interaction with the membrane anchor Tim44, but an enhanced trapping of preproteins . Unexpectedly, at a low inner membrane potential, ssc1-2 mitochondria imported loosely folded preproteins more efficiently than wild-type mitochondria . The import of a tightly folded preprotein, however, was not increased in ssc1-2 mitochondria . Thus, enhanced trapping by mtHsp70 stimulates the import of loosely folded preproteins and reduces the dependence on the import-driving activity of the membrane potential, directly demonstrating that trapping is one of the molecular mechanisms of mtHsp70 action. Mol Cell Biol, 2001 Oct, 21(20), 7089 - 96 Mitogen-regulated RSK2-CBP interaction controls their kinase and acetylase activities; Merienne K et al.; The protein kinase ribosomal S6 kinase 2 (RSK2) has been implicated in phosphorylation of transcription factor CREB and histone H3 in response to mitogenic stimulation by epidermal growth factor . Binding of phospho-CREB to the coactivator CBP allows gene activation through recruitment of the basal transcriptional machinery . Acetylation of H3 by histone acetyltransferase (HAT) activities, such as the one carried by CBP, has been functionally coupled to H3 phosphorylation . While various lines of evidence indicate that coupled histone acetylation and phosphorylation may act in concert to induce chromatin remodeling events facilitating gene activation, little is known about the coupling of the two processes at the signaling level . Here we show that CBP and RSK2 are associated in a complex in quiescent cells and that they dissociate within a few minutes upon mitogenic stimulus . CBP preferentially interacts with unphosphorylated RSK2 in a complex where both RSK2 kinase activity and CBP acetylase activity are inhibited . Dissociation is dependent on phosphorylation of RSK2 on Ser227 and results in stimulation of both kinase and HAT activities . We propose a model in which dynamic formation and dissociation of the CBP-RSK2 complex in response to mitogenic stimulation allow regulated phosphorylation and acetylation of specific substrates, leading to coordinated modulation of gene expression. Mol Cell Biol, 2001 Oct, 21(20), 7020 - 4 Cse1l is essential for early embryonic growth and development; Bera TK et al.; The CSE1L gene, the human homologue of the yeast chromosome segregation gene CSE1, is a nuclear transport factor that plays a role in proliferation as well as in apoptosis . CSE1 and CSE1L are essential genes in Saccharomyces cerevisiae and mammalian cells, as shown by conditional yeast mutants and mammalian cell culture experime