Biochemistry, 1999 Mar 23, 38(12), 3457 - 61 Binding of etoposide to topoisomerase II in the absence of DNA: decreased affinity as a mechanism of drug resistance; Kingma PS et al.; Despite the prevalence of topoisomerase II-targeted drugs in cancer chemotherapy and the impact of drug resistance on the efficacy of treatment, interactions between these agents and topoisomerase II are not well understood . Therefore, to further define interactions between anticancer drugs and the type II enzyme, a nitrocellulose filter assay was used to characterize the binding of etoposide to yeast topoisomerase II . Results indicate that etoposide binds to the enzyme in the absence of DNA . The apparent Kd value for the interaction was approximately 5 microM drug . Etoposide also bound to ytop2H1012Y, a mutant yeast type II enzyme that is approximately 3-4-fold resistant to etoposide . However, the apparent Kd value for the drug (approximately 16 microM) was approximately 3 times higher than that determined for wild-type topoisomerase II . Although it has been widely speculated that resistance to topoisomerase II-targeted anticancer agents results from a decreased drug-enzyme binding affinity, these data provide the first direct evidence in support of this hypothesis . Finally, the ability of yeast topoisomerase II to bind etoposide was dependent on the presence of the hydroxyl moiety of Tyr783, suggesting specific interactions between etoposide and the active site residue that is involved in DNA scission. J Rheumatol, 1999 Mar, 26(3), 563 - 7 Clinical and serological associations of anti-Ku antibody; Cooley HM et al.; OBJECTIVE: To ascertain the clinical and serological associations of anti-Ku antibody . METHODS: Twenty-seven patients over a 7 year period (1987-1996) had anti-Ku antibody detected by counterimmunoelectrophoresis (CIEP) . Nineteen patients were available for clinical review . Five patients were assessed by chart review . Serum was taken at review for repeat antibody analysis . Patients were assigned to diagnostic groups based on the American College of Rheumatology criteria . RESULTS: There were 22 women and 5 men . The duration of symptoms ranged from one year to 28 years . Nine patients fulfilled criteria for systemic lupus erythematosus (SLE), 4 scleroderma, 3 rheumatoid arthritis (RA), one discoid lupus, and 7 had an undifferentiated connective tissue disease . There was a low incidence of renal (2/24) and central nervous system involvement (1/24); 19/24 had Raynaud's phenomenon, 15/24 had inflammatory arthritis but only one had erosions on radiograph; 11/24 reported esophageal reflux symptoms . Three of 24 patients had myositis . All patients had anti-nuclear antibody using indirect immunofluorescence of > 640 titer with a speckled and nucleolar pattern . Anti-Ku antibody was detected on CIEP in 15/19 sera available for repeat testing . Three patients had anti-Ro antibody, 2 had anti-U1RNP antibody, one patient had anti-topoisomerase-1 and anti-Ro . CONCLUSION: Anti-Ku antibody is found in a wide variety of connective tissue syndromes . While several patients fulfilled diagnostic criteria for SLE, scleroderma, and RA, their clinical features were usually mild and did not form a distinctive clinical pattern . Common features associated with anti-Ku were Raynaud's phenomenon, arthralgia, skin thickening, and esophageal reflux . Few patients had associated autoantibody specificities found in SLE or scleroderma. Cell, 1999 Mar 5, 96(5), 645 - 53 Noncanonical MMS2-encoded ubiquitin-conjugating enzyme functions in assembly of novel polyubiquitin chains for DNA repair; Hofmann RM et al.; Ubiquitin-conjugating enzyme variant (UEV) proteins resemble ubiquitin-conjugating enzymes (E2s) but lack the defining E2 active-site residue . The MMS2-encoded UEV protein has been genetically implicated in error-free postreplicative DNA repair in Saccharomyces cerevisiae . We show that Mms2p forms a specific heteromeric complex with the UBC13-encoded E2 and is required for the Ubc13p-dependent assembly of polyubiquitin chains linked through lysine 63 . A ubc13 yeast strain is UV sensitive, and single, double, and triple mutants of the UBC13, MMS2, and ubiquitin (ubiK63R) genes display a comparable phenotype . These findings support a model in which an Mms2p/Ubc13p complex assembles novel polyubiquitin chains for signaling in DNA repair, and they suggest that UEV proteins may act to increase diversity and selectivity in ubiquitin conjugation. Cell, 1999 Mar 5, 96(5), 635 - 44 A novel ubiquitination factor, E4, is involved in multiubiquitin chain assembly; Koegl M et al.; Proteins modified by multiubiquitin chains are the preferred substrates of the proteasome . Ubiquitination involves a ubiquitin-activating enzyme, E1, a ubiquitin-conjugating enzyme, E2, and often a substrate-specific ubiquitin-protein ligase, E3 . Here we show that efficient multiubiquitination needed for proteasomal targeting of a model substrate requires an additional conjugation factor, named E4 . This protein, previously known as UFD2 in yeast, binds to the ubiquitin moieties of preformed conjugates and catalyzes ubiquitin chain assembly in conjunction with E1, E2, and E3 . Intriguingly, E4 defines a novel protein family that includes two human members and the regulatory protein NOSA from Dictyostelium required for fruiting body development . In yeast, E4 activity is linked to cell survival under stress conditions, indicating that eukaryotes utilize E4-dependent proteolysis pathways for multiple cellular functions. J Cell Biochem, 1999 Apr 1, 73(1), 56 - 69 Regulatory domain of human heat shock transcription factor-2 is not regulated by hemin or heat shock; Zhu Z et al.; Heat shock transcription factor 2 (HSF-2) activates transcription of heat shock proteins in response to hemin in the human erythroleukemia cell line, K562 . To understand the regulation of HSF-2 activation, a series of deletion mutants of HSF-2 fused to the GAL-4 DNA binding domain were generated . We have found that human HSF-2 has a regulatory domain located in the carboxyl-terminal portion of the protein which represses the activity of its activation domain under normal physiological conditions . The repressive effects of this domain can be eliminated by its deletion in GAL4-HSF-2 fusion constructs . The regulatory domain of HSF-2 can also repress a heterologous chimeric activator that contains a portion of the VP16 activation domain . The activation domain of HSF-2 is a segment of approximately 77 amino acids located proximal to the carboxyl-terminal hydrophobic heptad repeat (leucine zipper 4) of the molecule . Interestingly, the GAL4-HSF-2 fusion protein and the 77 amino acids activation domain are inactive and are not activated by pretreatment of cells with either hemin or elevated temperature . Our data suggest that regulation of HSF-2 differs from HSF-1 in that its regulatory domain is not responsive to hemin or heat directly. J Cell Biochem, 1999 Apr 1, 73(1), 31 - 5 In vivo and in vitro association of 14-3-3 epsilon isoform with calmodulin: implication for signal transduction and cell proliferation; Luk SC et al.; Using a yeast two-hybrid screen, human 14-3-3 epsilon protein was found to interact with human calmodulin . In vitro binding assay between human 14-3-3 epsilon protein/peptide and calmodulin was demonstrated by native gel electrophoresis, and the interaction was shown to be calcium dependent . Our results, along with the association of the 14-3-3 epsilon protein with other signaling proteins, suggest that the 14-3-3 protein could provide a link between signal transduction and cell proliferation. J Cell Biochem, 1999 Apr 1, 73(1), 1 - 10 New insights into the mechanisms of nuclear segmentation in human neutrophils; Sanchez JA et al.; During human neutrophil differentiation, large portions of the genome condense and associate with the nuclear envelope to form filament-like structures . As a result, the nucleus of the mature neutrophil typically consists of a linear array of three or four lobes joined by thin, DNA-containing filaments . Despite the medical significance of neutrophil nuclear morphology, little is known about the events regulating neutrophil nuclear differentiation and its pathological states . This work presents a new model of the mechanisms governing nuclear filament formation in human neutrophils . This model is based on recent chromosome mapping studies in human neutrophils and on studies of genetic and pathological conditions affecting neutrophil nuclear shape . According to this model, filament assembly is initiated by factors that interact with specific regions of the genome in a hierarchical and dose-dependent manner . In this regard, the strategies governing the molecular interactions responsible for filament formation appear to resemble those involved in transcriptional silencing, a phenomenon that also affects the properties of extended chromosomal regions . According to the silencing paradigm, bound filament control Factors must recruit additional Filament Foehn factors which spread along adjacent DNA to mediate filament formation . A better understanding of the factors that shape the neutrophil nucleus may lead to new clinical tools for the diagnosis and manipulation of abnormal neutrophil differentiation. J Cell Biol, 1999 Mar 22, 144(6), 1219 - 33 Differential regulation of the Kar3p kinesin-related protein by two associated proteins, Cik1p and Vik1p; Manning BD et al.; The mechanisms by which kinesin-related proteins interact with other proteins to carry out specific cellular processes is poorly understood . The kinesin-related protein, Kar3p, has been implicated in many microtubule functions in yeast . Some of these functions require interaction with the Cik1 protein (Page, B.D., L.L . Satterwhite, M.D . Rose, and M . Snyder . 1994 . J . Cell Biol . 124:507-519) . We have identified a Saccharomyces cerevisiae gene, named VIK1, encoding a protein with sequence and structural similarity to Cik1p . The Vik1 protein is detected in vegetatively growing cells but not in mating pheromone-treated cells . Vik1p physically associates with Kar3p in a complex separate from that of the Kar3p-Cik1p complex . Vik1p localizes to the spindle-pole body region in a Kar3p-dependent manner . Reciprocally, concentration of Kar3p at the spindle poles during vegetative growth requires the presence of Vik1p, but not Cik1p . Phenotypic analysis suggests that Cik1p and Vik1p are involved in different Kar3p functions . Disruption of VIK1 causes increased resistance to the microtubule depolymerizing drug benomyl and partially suppresses growth defects of cik1Delta mutants . The vik1Delta and kar3Delta mutations, but not cik1Delta, partially suppresses the temperature-sensitive growth defect of strains lacking the function of two other yeast kinesin-related proteins, Cin8p and Kip1p . Our results indicate that Kar3p forms functionally distinct complexes with Cik1p and Vik1p to participate in different microtubule-mediated events within the same cell. J Cell Biol, 1999 Mar 22, 144(6), 1151 - 62 Involvement of Pex13p in Pex14p localization and peroxisomal targeting signal 2-dependent protein import into peroxisomes; Girzalsky W et al.; Pex13p is the putative docking protein for peroxisomal targeting signal 1 (PTS1)-dependent protein import into peroxisomes . Pex14p interacts with both the PTS1- and PTS2-receptor and may represent the point of convergence of the PTS1- and PTS2-dependent protein import pathways . We report the involvement of Pex13p in peroxisomal import of PTS2-containing proteins . Like Pex14p, Pex13p not only interacts with the PTS1-receptor Pex5p, but also with the PTS2-receptor Pex7p; however, this association may be direct or indirect . In support of distinct peroxisomal binding sites for Pex7p, the Pex7p/Pex13p and Pex7p/ Pex14p complexes can form independently . Genetic evidence for the interaction of Pex7p and Pex13p is provided by the observation that overexpression of Pex13p suppresses a loss of function mutant of Pex7p . Accordingly, we conclude that Pex7p and Pex13p functionally interact during PTS2-dependent protein import into peroxisomes . NH2-terminal regions of Pex13p are required for its interaction with the PTS2-receptor while the COOH-terminal SH3 domain alone is sufficient to mediate its interaction with the PTS1-receptor . Reinvestigation of the topology revealed both termini of Pex13p to be oriented towards the cytosol . We also found Pex13p to be required for peroxisomal association of Pex14p, yet the SH3 domain of Pex13p may not provide the only binding site for Pex14p at the peroxisomal membrane. J Cell Biol, 1999 Mar 22, 144(6), 1097 - 112 A conserved biogenesis pathway for nucleoporins: proteolytic processing of a 186-kilodalton precursor generates Nup98 and the novel nucleoporin, Nup96; Fontoura BM et al.; The mammalian nuclear pore complex (NPC) is comprised of approximately 50 unique proteins, collectively known as nucleoporins . Through fractionation of rat liver nuclei, we have isolated >30 potentially novel nucleoporins and have begun a systematic characterization of these proteins . Here, we present the characterization of Nup96, a novel nucleoporin with a predicted molecular mass of 96 kD . Nup96 is generated through an unusual biogenesis pathway that involves synthesis of a 186-kD precursor protein . Proteolytic cleavage of the precursor yields two nucleoporins: Nup98, a previously characterized GLFG-repeat containing nucleoporin, and Nup96 . Mutational and functional analyses demonstrate that both the Nup98-Nup96 precursor and the previously characterized Nup98 (synthesized independently from an alternatively spliced mRNA) are proteolytically cleaved in vivo . This biogenesis pathway for Nup98 and Nup96 is evolutionarily conserved, as the putative Saccharomyces cerevisiae homologues, N-Nup145p and C-Nup145p, are also produced through proteolytic cleavage of a precursor protein . Using immunoelectron microscopy, Nup96 was localized to the nucleoplasmic side of the NPC, at or near the nucleoplasmic basket . The correct targeting of both Nup96 and Nup98 to the nucleoplasmic side of the NPC was found to be dependent on proteolytic cleavage, suggesting that the cleavage process may regulate NPC assembly . Finally, by biochemical fractionation, a complex containing Nup96, Nup107, and at least two Sec13- related proteins was identified, revealing that a major sub-complex of the NPC is conserved between yeast and mammals. J Pharmacol Exp Ther, 1999 Apr, 289(1), 31 - 7 In vitro metabolism of quinidine: the (3S)-3-hydroxylation of quinidine is a specific marker reaction for cytochrome P-4503A4 activity in human liver microsomes; Nielsen TL et al.; The aim of this study was to evaluate the (3S)-3-hydroxylation and the N-oxidation of quinidine as biomarkers for cytochrome P-450 (CYP)3A4 activity in human liver microsome preparations . An HPLC method was developed to assay the metabolites (3S)-3-hydroxyquinidine (3-OH-Q) and quinidine N-oxide (Q-N-OX) formed during incubation with microsomes from human liver and from Saccharomyces cerevisiae strains expressing 10 human CYPs . 3-OH-Q formation complied with Michaelis-Menten kinetics (mean values of Vmax and Km: 74.4 nmol/mg/h and 74.2 microM, respectively) . Q-N-OX formation followed two-site kinetics with mean values of Vmax, Km and Vmax/Km for the low affinity isozyme of 15.9 nmol/mg/h, 76.1 microM and 0.03 ml/mg/h, respectively . 3-OH-Q and Q-N-OX formations were potently inhibited by ketoconazole, itraconazole, and triacetyloleandomycin . Isozyme specific inhibitors of CYP1A2, -2C9, -2C19, -2D6, and -2E1 did not inhibit 3-OH-Q or Q-N-OX formation, with Ki values comparable with previously reported values . Statistically significant correlations were observed between CYP3A4 content and formations of 3-OH-Q and Q-N-OX in 12 human liver microsome preparations . Studies with yeast-expressed isozymes revealed that only CYP3A4 actively catalyzed the (3S)-3-hydroxylation . CYP3A4 was the most active enzyme in Q-N-OX formation, but CYP2C9 and 2E1 also catalyzed minor proportions of the N-oxidation . In conclusion, our studies demonstrate that only CYP3A4 is actively involved in the formation of 3-OH-Q . Hence, the (3S)-3-hydroxylation of quinidine is a specific probe for CYP3A4 activity in human liver microsome preparations, whereas the N-oxidation of quinidine is a somewhat less specific marker reaction for CYP3A4 activity, because the presence of a low affinity enzyme is demonstrated by different approaches. Spectrochim Acta A Mol Biomol Spectrosc, 1999 Feb, 55A(2), 415 - 20 The interaction of the nitrate anion with cytochrome c peroxidase: a 15N-NMR study; Banci L et al.; The interaction of the nitrate anion with cytochrome c peroxidase has been demonstrated by using 15N-NMR spectroscopy . The results indicate that the nitrate anion binds to the protein in a specific binding site and are consistent with the hypothesis of an interaction of this small anion in the active cavity of the enzyme, possibly in the proximity of the distal histidine and the distal arginine. J Cell Biol, 1999 Mar 8, 144(5), 963 - 75 The cortical localization of the microtubule orientation protein, Kar9p, is dependent upon actin and proteins required for polarization; Miller RK et al.; In the yeast Saccharomyces cerevisiae, positioning of the mitotic spindle requires both the cytoplasmic microtubules and actin . Kar9p is a novel cortical protein that is required for the correct position of the mitotic spindle and the orientation of the cytoplasmic microtubules . Green fluorescent protein (GFP)- Kar9p localizes to a single spot at the tip of the growing bud and the mating projection . However, the cortical localization of Kar9p does not require microtubules (Miller, R.K., and M.D . Rose . 1998 . J . Cell Biol . 140: 377), suggesting that Kar9p interacts with other proteins at the cortex . To investigate Kar9p's cortical interactions, we treated cells with the actin-depolymerizing drug, latrunculin-A . In both shmoos and mitotic cells, Kar9p's cortical localization was completely dependent on polymerized actin . Kar9p localization was also altered by mutations in four genes, spa2Delta, pea2Delta, bud6Delta, and bni1Delta, required for normal polarization and actin cytoskeleton functions and, of these, bni1Delta affected Kar9p localization most severely . Like kar9Delta, bni1Delta mutants exhibited nuclear positioning defects during mitosis and in shmoos . Furthermore, like kar9Delta, the bni1Delta mutant exhibited misoriented cytoplasmic microtubules in shmoos . Genetic analysis placed BNI1 in the KAR9 pathway for nuclear migration . However, analysis of kar9Delta bni1Delta double mutants suggested that Kar9p retained some function in bni1Delta mitotic cells . Unlike the polarization mutants, kar9Delta shmoos had a normal morphology and diploids budded in the correct bipolar pattern . Furthermore, Bni1p localized normally in kar9Delta . We conclude that Kar9p's function is specific for cytoplasmic microtubule orientation and that Kar9p's role in nuclear positioning is to coordinate the interactions between the actin and microtubule networks. J Cell Biol, 1999 Mar 8, 144(5), 839 - 55 Proteins connecting the nuclear pore complex with the nuclear interior; Strambio-de-Castillia C et al.; While much has been learned in recent years about the movement of soluble transport factors across the nuclear pore complex (NPC), comparatively little is known about intranuclear trafficking . We isolated the previously identified Saccharomyces protein Mlp1p (myosin-like protein) by an assay designed to find nuclear envelope (NE) associated proteins that are not nucleoporins . We localized both Mlp1p and a closely related protein that we termed Mlp2p to filamentous structures stretching from the nucleoplasmic face of the NE into the nucleoplasm, similar to the homologous vertebrate and Drosophila Tpr proteins . Mlp1p can be imported into the nucleus by virtue of a nuclear localization sequence (NLS) within its COOH-terminal domain . Overexpression experiments indicate that Mlp1p can form large structures within the nucleus which exclude chromatin but appear highly permeable to proteins . Remarkably, cells harboring a double deletion of MLP1 and MLP2 were viable, although they showed a slower net rate of active nuclear import and faster passive efflux of a reporter protein . Our data indicate that the Tpr homologues are not merely NPC-associated proteins but that they can be part of NPC-independent, peripheral intranuclear structures . In addition, we suggest that the Tpr filaments could provide chromatin-free conduits or tracks to guide the efficient translocation of macromolecules between the nucleoplasm and the NPC. J Cell Sci, 1999 Apr, 112 ( Pt 8), 1247 - 56 Polarized targeting of epithelial cell proteins in thyrocytes and MDCK cells; Prabakaran D et al.; Polarized trafficking signals may be interpreted differently in different cell types . In this study, we have compared the polarized trafficking of different proteins expressed endogenously in primary porcine thyroid epithelial cells to similar proteins expressed in MDCK cells . As in MDCK cells, NH4Cl treatment of filter-grown thyrocytes caused mis-sorted soluble proteins to exhibit enhanced secretion to the apical medium . In independent studies, thrombospondin 1 (a thyroid basolaterally secreted protein) was secreted basolaterally from MDCK cells . Likewise, the 5'-deiodinase (a thyroid basolateral membrane protein) encoded by the DIO1 gene was also distributed basolaterally in transfected MDCK cells . Consistent with previous reports, when the secretion of human growth hormone (an unglycosylated regulated secretory protein) was examined from transfected MDCK cells, the release was nonpolarized . However, transfected thyrocytes secreted growth hormone apically in a manner dependent upon zinc addition . Moreover, two additional regulated secretory proteins expressed in thyrocytes, thyroglobulin (the major endogenous glycoprotein) and parathyroid hormone (an unglycosylated protein expressed transiently), were secreted apically even in the absence of zinc . We hypothesize that while cellular mechanisms for interpreting polarity signals are generally similar between thyrocytes and MDCK cells, thyrocytes allow for specialized packaging of regulated secretory proteins for apical delivery, which does not require glycosylation but may involve availability of certain ions as well as appropriate intracellular compartmentation. J Cell Sci, 1999 Apr, 112 ( Pt 8), 1159 - 68 Cloning, sequencing, and nucleolar targeting of the basal-body-binding nucleolar protein BN46/51; Trimbur GM et al.; BN46/51 is an acidic protein found in the granular component of the nucleolus of the amebo-flagellate Naegleria gruberi . When Naegleria amebae differentiate into swimming flagellates, BN46/51 is found associated with the basal body complex at the base of the flagella . In order to determine the factors responsible for targeting BN46/51 to a specific subnucleolar region, cDNAs coding for both subunits were isolated and sequenced . Two clones, JG4.1 and JG12.1 representing the 46 kDa and 51 kDa subunits, respectively, were investigated in detail . JG12.1 encoded a polypeptide of 263 amino acids with a predicted size of 30.1 kDa that co-migrated with the 51 kDa subunit of BN46/51 when expressed in yeast . JG4.1 encoded a polypeptide of 249 amino acids with a predicted size of 28.8 kDa that co-migrated with the 46 kDa subunit of BN46/51 . JG4.1 was identical to JG12.1 except for the addition of an aspartic acid between positions 94 and 95 of the JG12.1 sequence and the absence of 45 amino acids beginning at position 113 . The predicted amino acid sequences were not closely related to any previously reported . However, the sequences did have 26-31% identity to a group of FKPBs (FK506 binding proteins) but lacked the peptidyl-prolyl cis-trans isomerase domain of the FKBPs . Both subunits contained two KKE and three KKX repeats found in other nucleolar proteins and in some microtubule binding proteins . Using 'Far Western' blots of nucleolar proteins, BN46/51 bound to polypeptides of 44 kDa and 74 kDa . The 44 kDa component was identified as the Naegleria homologue of fibrillarin . BN46/51 bound specifically to the nucleoli of fixed mammalian cells, cells which lack a BN46/51 related polypeptide . When the JG4.1 and JG12.1 cDNAs were expressed in yeast, each subunit was independently targeted to the yeast nucleolus . We conclude that BN46/51 represents a unique nucleolar protein that can form specific complexes with fibrillarin and other nucleolar proteins . We suggest that the association of BN46/51 with the MTOC of basal bodies may reflect its role in connecting the nucleolus with the MTOC activity for the mitotic spindle . This would provide a mechanism for nucleolar segregation during the closed mitosis of Naegleria amebae. Biochem J, 1999 Apr 1, 339 ( Pt 1), 1 - 10 The protein disulphide-isomerase family: unravelling a string of folds; Ferrari DM et al.; The mammalian protein disulphide-isomerase (PDI) family encompasses several highly divergent proteins that are involved in the processing and maturation of secretory proteins in the endoplasmic reticulum . These proteins are characterized by the presence of one or more domains of roughly 95-110 amino acids related to the cytoplasmic protein thioredoxin . All but the PDI-D subfamily are composed entirely of repeats of such domains, with at least one domain containing and one domain lacking a redox-active -Cys-Xaa-Xaa-Cys- tetrapeptide . In addition to their known roles as redox catalysts and isomerases, the last few years have revealed additional functions of the PDI proteins, including peptide binding, cell adhesion and perhaps chaperone activities . Attention is now turning to the non-redox-active domains of the PDIs, which may play an important role in all of the known activities of these proteins . Thus the presence of both redox-active and -inactive domains within these proteins portends a complexity of functions differentially accommodated by the various family members. J Biol Chem, 1999 Mar 26, 274(13), 9068 - 75 Transmembrane topology of pmt1p, a member of an evolutionarily conserved family of protein O-mannosyltransferases; Strahl-Bolsinger S et al.; The identification of the evolutionarily conserved family of dolichyl-phosphate-D-mannose:protein O-mannosyltransferases (Pmts) revealed that protein O-mannosylation plays an essential role in a number of physiologically important processes . Strikingly, all members of the Pmt protein family share almost identical hydropathy profiles; a central hydrophilic domain is flanked by amino- and carboxyl-terminal sequences containing several putative transmembrane helices . This pattern is of particular interest because it diverges from structural models of all glycosyltransferases characterized so far . Here, we examine the transmembrane topology of Pmt1p, an integral membrane protein of the endoplasmic reticulum, from Saccharomyces cerevisiae . Structural predictions were directly tested by site-directed mutagenesis of endogenous N-glycosylation sites, by fusing a topology-sensitive monitor protein domain to carboxyl-terminal truncated versions of the Pmt1 protein and, in addition, by N-glycosylation scanning . Based on our results we propose a seven-transmembrane helical model for the yeast Pmt1p mannosyltransferase . The Pmt1p amino terminus faces the cytoplasm, whereas the carboxyl terminus faces the lumen of the endoplasmic reticulum . A large hydrophilic segment that is oriented toward the lumen of the endoplasmic reticulum is flanked by five amino-terminal and two carboxyl-terminal membrane spanning domains . We could demonstrate that this central loop is essential for the function of Pmt1p. J Biol Chem, 1999 Mar 26, 274(13), 8746 - 56 Effects of phosphorylation of threonine 160 on cyclin-dependent kinase 2 structure and activity; Brown NR et al.; We have prepared phosphorylated cyclin-dependent protein kinase 2 (CDK2) for crystallization using the CDK-activating kinase 1 (CAK1) from Saccharomyces cerevisiae and have grown crystals using microseeding techniques . Phosphorylation of monomeric human CDK2 by CAK1 is more efficient than phosphorylation of the binary CDK2-cyclin A complex . Phosphorylated CDK2 exhibits histone H1 kinase activity corresponding to approximately 0.3% of that observed with the fully activated phosphorylated CDK2-cyclin A complex . Fluorescence measurements have shown that Thr160 phosphorylation increases the affinity of CDK2 for both histone substrate and ATP and decreases its affinity for ADP . By contrast, phosphorylation of CDK2 has a negligible effect on the affinity for cyclin A . The crystal structures of the ATP-bound forms of phosphorylated CDK2 and unphosphorylated CDK2 have been solved at 2.1-A resolution . The structures are similar, with the major difference occurring in the activation segment, which is disordered in phosphorylated CDK2 . The greater mobility of the activation segment in phosphorylated CDK2 and the absence of spontaneous crystallization suggest that phosphorylated CDK2 may adopt several different mobile states . The majority of these states are likely to correspond to inactive conformations, but a small fraction of phosphorylated CDK2 may be in an active conformation and hence explain the basal activity observed. J Biol Chem, 1999 Mar 26, 274(13), 8383 - 90 Disruption of the mouse Rce1 gene results in defective Ras processing and mislocalization of Ras within cells; Kim E et al.; Little is known about the enzyme(s) required for the endoproteolytic processing of mammalian Ras proteins . We identified a mouse gene (designated Rce1) that shares sequence homology with a yeast gene (RCE1) implicated in the proteolytic processing of Ras2p . To define the role of Rce1 in mammalian Ras processing, we generated and analyzed Rce1-deficient mice . Rce1 deficiency was lethal late in embryonic development (after embryonic day 15.5) . Multiple lines of evidence revealed that Rce1-deficient embryos and cells lacked the ability to endoproteolytically process Ras proteins . First, Ras proteins from Rce1-deficient cells migrated more slowly on SDS-polyacrylamide gels than Ras proteins from wild-type embryos and fibroblasts . Second, metabolic labeling of Rce1-deficient cells revealed that the Ras proteins were not carboxymethylated . Finally, membranes from Rce1-deficient fibroblasts lacked the capacity to proteolytically process farnesylated Ha-Ras, N-Ras, and Ki-Ras or geranylgeranylated Ki-Ras . The processing of two other prenylated proteins, the farnesylated Ggamma1 subunit of transducin and geranylgeranylated Rap1B, was also blocked . The absence of endoproteolytic processing and carboxymethylation caused Ras proteins to be mislocalized within cells . These studies indicate that Rce1 is responsible for the endoproteolytic processing of the Ras proteins in mammals and suggest a broad role for this gene in processing other prenylated CAAX proteins. J Biol Chem, 1999 Mar 26, 274(13), 8379 - 82 Cloning and characterization of a mammalian prenyl protein-specific protease; Otto JC et al.; Proteins containing C-terminal "CAAX" sequence motifs undergo three sequential post-translational processing steps: modification of the cysteine with either a 15-carbon farnesyl or 20-carbon geranylgeranyl isoprenyl lipid, proteolysis of the C-terminal -AAX tripeptide, and methylation of the carboxyl group of the now C-terminal prenylcysteine . A putative prenyl protein protease in yeast, designated Rce1p, was recently identified . In this study, a portion of a putative human homologue of RCE1 (hRCE1) was identified in a human expressed sequence tag data base, and the corresponding cDNA was cloned . Expression of hRCE1 was detected in all tissues examined . Both yeast and human RCE1 proteins were produced in Sf9 insect cells by infection with a recombinant baculovirus; membrane preparations derived from the infected Sf9 cells exhibited a high level of prenyl protease activity . Recombinant hRCE1 so produced recognized both farnesylated and geranylgeranylated proteins as substrates, including farnesyl-Ki-Ras, farnesyl-N-Ras, farnesyl-Ha-Ras, and the farnesylated heterotrimeric G protein Ggamma1 subunit, as well as geranylgeranyl-Ki-Ras and geranylgeranyl-Rap1b . The protease activity of hRCE1 activity was specific for prenylated proteins, because unprenylated peptides did not compete for enzyme activity . hRCE1 activity was also exquisitely sensitive to a prenyl peptide analogue that had been previously described as a potent inhibitor of the prenyl protease activity in mammalian tissues . These data indicate that both the yeast and the human RCE1 gene products are bona fide prenyl protein proteases and suggest that they play a major role in the processing of CAAX-type prenylated proteins. Biochim Biophys Acta, 1999 Mar 9, 1410(3), 237 - 47 Aromatic amino acids in the Rieske iron-sulfur protein do not form an obligatory conduit for electron transfer from the iron-sulfur cluster to the heme of cytochrome c1 in the cytochrome bc1 complex; Snyder CH et al.; We have changed nine conserved aromatic amino acids by site-directed mutagenesis of the cloned iron-sulfur protein gene to determine if any of these residues form an obligatory conduit for electron transfer within the iron-sulfur protein of the yeast cytochrome bc1 complex . The residues include W111, F117, W152, F173, W176, F177, H184, Y205 and F207 . Greater than 70% of the catalytic activity was retained for all of the mutated iron-sulfur proteins, except for those containing a W152L and a W176L-F177L double mutation, for which the activity was approximately 45% . The crystal structures of the bc1 complex indicate that F177 and H184 are at the surface of the iron-sulfur protein near the surface of cytochrome c1, but not directly in a linear pathway between the iron-sulfur cluster and the c1 heme . The pre-steady-state rates of reduction of cytochromes b and c1 in mutants in which F177 and H184 were changed to non-aromatic residues were approximately 70-85% of the wild-type rates . There was a large decrease in iron-sulfur protein levels in mitochondrial membranes resulting from the W152L mutation and the W176L-F177L double mutation, and a small decrease for the Y205L, W176L and F177L mutations . This indicates that the decreases in activity resulting from these amino acid changes are due to instability of the altered proteins . These results show that these aromatic amino acids are unnecessary for electron transfer, but several are required for structural stability. Biochem Biophys Res Commun, 1999 Feb 5, 255(1), 150 - 6 A model phosphatase 2C --> phosphatase 1 activation cascade via dual control of inhibitor-1 (INH-1) and DARPP-32 dephosphorylation by two inositol glycan putative insulin mediators from beef liver; Huang LC et al.; Two inositol phosphoglycans (IPG) isolated from beef liver and designated as putative insulin mediators were demonstrated to reciprocally enhance the dephosphorylation of inhibitor-1 (INH-1) and DARPP-32, thus directly activating phosphatase 2C and disinhibiting phosphatase 1 in a potential protein phosphatase 2C --> phosphatase 1 cascade mechanism . One IPG termed pH 2.0, containing Dchiro-inositol and galactosamine, stimulated the dephosphorylation of INH-1 and DARPP-32 in a dose-dependent manner in the low micromolar range . A second, termed pH 1.3, containing myo-inositol glucosamine and mannose acted reciprocally to inhibit the cAMP-dependent protein kinase phosphorylation of INH-1 and DARPP-32 in a dose-dependent manner in the low micromolar range . These model experiments are discussed in terms of the observed dephosphorylation of INH-1 with insulin action documented in the literature and the activation of both phosphatase 1 and 2C described in intact cells and in vivo with insulin action . Mol Cell Biol, 1999 Apr, 19(4), 3184 - 97 A genetic screen for ribosomal DNA silencing defects identifies multiple DNA replication and chromatin-modulating factors; Smith JS et al.; Transcriptional silencing in Saccharomyces cerevisiae occurs at several genetic loci, including the ribosomal DNA (rDNA) . Silencing at telomeres (telomere position effect {TPE}) and the cryptic mating-type loci (HML and HMR) depends on the silent information regulator genes, SIR1, SIR2, SIR3, and SIR4 . However, silencing of polymerase II-transcribed reporter genes integrated within the rDNA locus (rDNA silencing) requires only SIR2 . The mechanism of rDNA silencing is therefore distinct from TPE and HM silencing . Few genes other than SIR2 have so far been linked to the rDNA silencing process . To identify additional non-Sir factors that affect rDNA silencing, we performed a genetic screen designed to isolate mutations which alter the expression of reporter genes integrated within the rDNA . We isolated two classes of mutants: those with a loss of rDNA silencing (lrs) phenotype and those with an increased rDNA silencing (irs) phenotype . Using transposon mutagenesis, lrs mutants were found in 11 different genes, and irs mutants were found in 22 different genes . Surprisingly, we did not isolate any genes involved in rRNA transcription . Instead, multiple genes associated with DNA replication and modulation of chromatin structure were isolated . We describe these two gene classes, and two previously uncharacterized genes, LRS4 and IRS4 . Further characterization of the lrs and irs mutants revealed that many had alterations in rDNA chromatin structure . Several lrs mutants, including those in the cdc17 and rfc1 genes, caused lengthened telomeres, consistent with the hypothesis that telomere length modulates rDNA silencing . Mutations in the HDB (RPD3) histone deacetylase complex paradoxically increased rDNA silencing by a SIR2-dependent, SIR3-independent mechanism . Mutations in rpd3 also restored mating competence selectively to sir3Delta MATalpha strains, suggesting restoration of silencing at HMR in a sir3 mutant background. Mol Cell Biol, 1999 Apr, 19(4), 2880 - 6 Functional domains of c-myc promoter binding protein 1 involved in transcriptional repression and cell growth regulation; Ghosh AK et al.; We initially identified c-myc promoter binding protein 1 (MBP-1), which negatively regulates c-myc promoter activity, from a human cervical carcinoma cell expression library . Subsequent studies on the biological role of MBP-1 demonstrated induction of cell death in fibroblasts and loss of anchorage-independent growth, reduced invasive ability, and tumorigenicity of human breast carcinoma cells . To investigate the potential role of MBP-1 as a transcriptional regulator, a chimeric protein containing MBP-1 fused to the DNA binding domain of the yeast transactivator factor GAL4 was constructed . This fusion protein exhibited repressor activity on the herpes simplex virus thymidine kinase promoter via upstream GAL4 DNA binding sites . Structure-function analysis of mutant MBP-1 in the context of the GAL4 DNA binding domain revealed that MBP-1 transcriptional repressor domains are located in the N terminus (amino acids 1 to 47) and C terminus (amino acids 232 to 338), whereas the activation domain lies in the middle (amino acids 140 to 244) . The N-terminal domain exhibited stronger transcriptional repressor activity than the C-terminal region . When the N-terminal repressor domain was transferred to a potent activator, transcription was strongly inhibited . Both of the repressor domains contained hydrophobic regions and had an LXVXL motif in common . Site-directed mutagenesis in the repressor domains indicated that the leucine residues in the LXVXL motif are required for transcriptional repression . Mutation of the leucine residues in the common motif of MBP-1 also abrogated the repressor activity on the c-myc promoter . In addition, the leucine mutant forms of MBP-1 failed to suppress cell growth in fibroblasts like wild-type MBP-1 . Taken together, our results indicate that MBP-1 is a complex cellular factor containing multiple transcriptional regulatory domains that play an important role in cell growth regulation. Mol Cell Biol, 1999 Apr, 19(4), 2835 - 45 MOT1 can activate basal transcription in vitro by regulating the distribution of TATA binding protein between promoter and nonpromoter sites; Muldrow TA et al.; MOT1 is an ATPase which can dissociate TATA binding protein (TBP)-DNA complexes in a reaction requiring ATP hydrolysis . Consistent with this observation, MOT1 can repress basal transcription in vitro . Paradoxically, however, some genes, such as HIS4, appear to require MOT1 as an activator of transcription in vivo . To further investigate the function of MOT1 in basal transcription, we performed in vitro transcription reactions using yeast nuclear extracts depleted of MOT1 . Quantitation of MOT1 revealed that it is an abundant protein, with nuclear extracts from wild-type cells containing a molar excess of MOT1 over TBP . Surprisingly, MOT1 can weakly activate basal transcription in vitro . This activation by MOT1 is detectable with amounts of MOT1 that are approximately stoichiometric to TBP . With amounts of MOT1 similar to those present in wild-type nuclear extracts, MOT1 behaves as a weak repressor of basal transcription . These results suggest that MOT1 might activate transcription via an indirect mechanism in which limiting TBP can be liberated from nonpromoter sites for use at promoters . In support of this idea, excess nonpromoter DNA sequesters TBP and represses transcription, but this effect can be reversed by addition of MOT1 . These results help to reconcile previous in vitro and in vivo results and expand the repertoire of transcriptional control strategies to include factor-assisted redistribution of TBP between promoter and nonpromoter sites. Mol Cell Biol, 1999 Apr, 19(4), 2817 - 27 An in vitro system recapitulates chromatin remodeling at the PHO5 promoter; Haswell ES et al.; The Saccharomyces cerevisiae gene PHO5 is an excellent system with which to study regulated changes in chromatin structure . The PHO5 promoter is packaged into four positioned nucleosomes under repressing conditions; upon induction, the structure of these nucleosomes is altered such that the promoter DNA becomes accessible to nucleases . We report here the development and characterization of an in vitro system in which partially purified PHO5 minichromosomes undergo promoter chromatin remodeling . Several hallmarks of the PHO5 chromatin transition in vivo were reproduced in this system . Chromatin remodeling of PHO5 minichromosomes required the transcription factors Pho4 and Pho2, was localized to the promoter region of PHO5, and was independent of the chromatin-remodeling complex Swi-Snf . In vitro chromatin remodeling also required the addition of fractionated nuclear extract and hydrolyzable ATP . This in vitro system should serve as a useful tool for identifying the components required for this reaction and for elucidating the mechanism by which the PHO5 promoter chromatin structure is changed. Mol Cell Biol, 1999 Apr, 19(4), 2672 - 80 Rpb7 can interact with RNA polymerase II and support transcription during some stresses independently of Rpb4; Sheffer A et al.; Rpb4 and Rpb7 are two yeast RNA polymerase II (Pol II) subunits whose mechanistic roles have recently started to be deciphered . Although previous data suggest that Rpb7 can stably interact with Pol II only as a heterodimer with Rpb4, RPB7 is essential for viability, whereas RPB4 is essential only during some stress conditions . To resolve this discrepancy and to gain a better understanding of the mode of action of Rpb4, we took advantage of the inability of cells lacking RPB4 (rpb4Delta, containing Pol IIDelta4) to grow above 30 degrees C and screened for genes whose overexpression could suppress this defect . We thus discovered that overexpression of RPB7 could suppress the inability of rpb4Delta cells to grow at 34 degrees C (a relatively mild temperature stress) but not at higher temperatures . Overexpression of RPB7 could also partially suppress the cold sensitivity of rpb4Delta strains and fully suppress their inability to survive a long starvation period (stationary phase) . Notably, however, overexpression of RPB4 could not override the requirement for RPB7 . Consistent with the growth phenotype, overexpression of RPB7 could suppress the transcriptional defect characteristic of rpb4Delta cells during the mild, but not during a more severe, heat shock . We also demonstrated, through two reciprocal coimmunoprecipitation experiments, a stable interaction of the overproduced Rpb7 with Pol IIDelta4 . Nevertheless, fewer Rpb7 molecules interacted with Pol IIDelta4 than with wild-type Pol II . Thus, a major role of Rpb4 is to augment the interaction of Rpb7 with Pol II . We suggest that Pol IIDelta4 contains a small amount of Rpb7 that is sufficient to support transcription only under nonstress conditions . When RPB7 is overexpressed, more Rpb7 assembles with Pol IIDelta4, enough to permit appropriate transcription also under some stress conditions. Mol Cell Biol, 1999 Apr, 19(4), 2650 - 6 Differential protein S-thiolation of glyceraldehyde-3-phosphate dehydrogenase isoenzymes influences sensitivity to oxidative stress; Grant CM et al.; The irreversible oxidation of cysteine residues can be prevented by protein S-thiolation, in which protein -SH groups form mixed disulfides with low-molecular-weight thiols such as glutathione . We report here the identification of glyceraldehyde-3-phosphate dehydrogenase as the major target of protein S-thiolation following treatment with hydrogen peroxide in the yeast Saccharomyces cerevisiae . Our studies reveal that this process is tightly regulated, since, surprisingly, despite a high degree of sequence homology (98% similarity and 96% identity), the Tdh3 but not the Tdh2 isoenzyme was S-thiolated . The glyceraldehyde-3-phosphate dehydrogenase enzyme activity of both the Tdh2 and Tdh3 isoenzymes was decreased following exposure to H2O2, but only Tdh3 activity was restored within a 2-h recovery period . This indicates that the inhibition of the S-thiolated Tdh3 polypeptide was readily reversible . Moreover, mutants lacking TDH3 were sensitive to a challenge with a lethal dose of H2O2, indicating that the S-thiolated Tdh3 polypeptide is required for survival during conditions of oxidative stress . In contrast, a requirement for the nonthiolated Tdh2 polypeptide was found during exposure to continuous low levels of oxidants, conditions where the Tdh3 polypeptide would be S-thiolated and hence inactivated . We propose a model in which both enzymes are required during conditions of oxidative stress but play complementary roles depending on their ability to undergo S-thiolation. Mol Cell Biol, 1999 Apr, 19(4), 2585 - 93 Ku-dependent nonhomologous DNA end joining in Xenopus egg extracts; Labhart P; An extract from activated Xenopus eggs joins both matching and nonmatching ends of exogenous linear DNA substrates with high efficiency and fidelity (P . Pfeiffer and W . Vielmetter, Nucleic Acids Res . 16:907-924, 1988) . In mammalian cells, such nonhomologous end joining (NHEJ) is known to require the Ku heterodimer, a component of DNA-dependent protein kinase . Here I investigated whether Ku is also required for the in vitro reaction in the egg extract . Immunological assays indicate that Ku is very abundant in the extract . I found that all NHEJ was inhibited by autoantibodies against Ku and that NHEJ between certain combinations of DNA ends was also decreased after immunodepletion of Ku from the extract . The formation of a joint between a DNA end with a 5'-protruding single strand (PSS) and an end with a 3'-PSS, between two ends with 3'-PSS, and between two blunt ends was most Ku dependent . On the other hand, NHEJ between two DNA ends bearing 5'-PSS was Ku independent . These results show that the Xenopus cell-free system will be useful to biochemically dissect the role of Ku in eukaryotic NHEJ. Mol Cell Biol, 1999 Apr, 19(4), 2515 - 26 Esa1p is an essential histone acetyltransferase required for cell cycle progression; Clarke AS et al.; Histones are dynamically modified during chromatin assembly, as specific transcriptional patterns are established, and during mitosis and development . Modifications include acetylation, phosphorylation, ubiquitination, methylation, and ADP-ribosylation, but the biological significance of each of these is not well understood . For example, distinct acetylation patterns correlate with nucleosome formation and with transcriptionally activated or silenced chromatin, yet mutations in genes encoding several yeast histone acetyltransferase (HAT) activities result in either no cellular phenotype or only modest growth defects . Here we report characterization of ESA1, an essential gene that is a member of the MYST family that includes two yeast silencing genes, human genes associated with leukemia and with the human immunodeficiency virus type 1 Tat protein, and Drosophila mof, a gene essential for male dosage compensation . Esa1p acetylates histones in a pattern distinct from those of other yeast enzymes, and temperature-sensitive mutant alleles abolish enzymatic activity in vitro and result in partial loss of an acetylated isoform of histone H4 in vivo . Strains carrying these mutations are also blocked in the cell cycle such that at restrictive temperatures, esa1 mutants succeed in replicating their DNA but fail to proceed normally through mitosis and cytokinesis . Recent studies show that Esa1p enhances transcription in vitro and thus may modulate expression of genes important for cell cycle control . These observations therefore link an essential HAT activity to cell cycle progression, potentially through discrete transcriptional regulatory events. Mol Cell Biol, 1999 Apr, 19(4), 2465 - 74 Structural and functional analysis of interferon regulatory factor 3: localization of the transactivation and autoinhibitory domains; Lin R et al.; The interferon regulatory factor 3 (IRF-3) gene encodes a 55-kDa protein which is expressed constitutively in all tissues . In unstimulated cells, IRF-3 is present in an inactive cytoplasmic form; following Sendai virus infection, IRF-3 is posttranslationally modified by protein phosphorylation at multiple serine and threonine residues located in the carboxy terminus . Virus-induced phosphorylation of IRF-3 leads to cytoplasmic to nuclear translocation of phosphorylated IRF-3, association with the transcriptional coactivator CBP/p300, and stimulation of DNA binding and transcriptional activities of virus-inducible genes . Using yeast and mammalian one-hybrid analysis, we now demonstrate that an extended, atypical transactivation domain is located in the C terminus of IRF-3 between amino acids (aa) 134 and 394 . We also show that the C-terminal domain of IRF-3 located between aa 380 and 427 participates in the autoinhibition of IRF-3 activity via an intramolecular association with the N-terminal region between aa 98 and 240 . After Sendai virus infection, an intermolecular association between IRF-3 proteins is detected, demonstrating a virus-dependent formation of IRF-3 homodimers; this interaction is also observed in the absence of virus infection with a constitutively activated form of IRF-3 . Substitution of the C-terminal Ser-Thr phosphorylation sites with the phosphomimetic Asp in the region ISNSHPLSLTSDQ between amino acids 395 and 407 {IRF-3(5D)}, but not the adjacent S385 and S386 residues, generates a constitutively activated DNA binding form of IRF-3 . In contrast, substitution of S385 and S386 with either Ala or Asp inhibits both DNA binding and transactivation activities of the IRF-3(5D) protein . These studies thus define the transactivation domain of IRF-3, two domains that participate in the autoinhibition of IRF-3 activity, and the regulatory phosphorylation sites controlling IRF-3 dimer formation, DNA binding activity, and association with the CBP/p300 coactivator. Virus Res, 1999 Feb, 59(2), 211 - 7 Functional analysis of a transrepressor domain in the hepatitis C virus core protein; Ray RB et al.; Hepatitis C virus (HCV) is one of the major causative agents of chronic liver disease with the potential for development of hepatocellular carcinoma . The putative core protein of the virus has many intriguing properties, including transcriptional regulation of cellular and unrelated viral promoters . To further characterize the transregulatory function, a number of chimeric constructs were made by fusion of the core gene to the DNA binding domain of the yeast transactivator factor GAL4 . The fusion protein exhibited a repressor activity on the herpes simplex virus thymidine kinase promoter via the upstream GAL4 DNA binding sites . A structure /function analysis of HCV core mutants in the context of the GAL4 DNA binding domain revealed that the transcriptional repressor activity was located near the N-terminus (amino acids 26 85) . Transcription was strongly inhibited upon transfer of this repressor domain to a heterologous activation domain, (3CGln) of Epstein Barr virus transcription factor EBNA3C . Results from this study suggest that the HCV core protein contains an overall repressor activity, and that the repressor domain is located near the N-terminus. Protein Sci, 1998 Aug, 7(8), 1789 - 95 The magnitude of changes in guanidine-HCl unfolding m-values in the protein, iso-1-cytochrome c, depends upon the substructure containing the mutation; Hammack B et al.; Hydrophilic to hydrophobic mutations have been made at 11 solvent exposed sites on the surface of iso-1-cytochrome c . Most of these mutations involve the replacement of lysine with methionine, which is nearly isosteric with lysine . Minimal perturbation to the native structure is expected, and this expectation is confirmed by infrared amide I spectroscopy . Guanidine hydrochloride denaturation studies demonstrate that these variants affect the magnitude of the m-value, the rate of change of free energy with respect to denaturant concentration, to different degrees . Changes in m-values are indicative of changes in the equilibrium folding mechanism of a protein . Decreases in m-values are normally thought to result either from an increased population of intermediates during unfolding or from a more compact denatured state . When cytochrome c is considered in terms of its thermodynamic substructures, the changes in the m-value for a given variant appear to depend upon the substructure in which the mutation is made . These data indicate that the relative stabilities and physical properties of substructures of cytochrome c play an important determining role in the equilibrium folding mechanism of this protein. Jpn J Pharmacol, 1999 Jan, 79(1), 65 - 73 The novel analgesic compound OT-7100 (5-n-butyl-7-(3,4,5-trimethoxybenzoylamino)pyrazolo{1,5-a}pyrimid ine) attenuates mechanical nociceptive responses in animal models of acute and peripheral neuropathic hyperalgesia; Yasuda T et al.; We investigated the effects of OT-7100, a novel analgesic compound (5-n-butyl-7-(3,4,5-trimethoxybenzoylamino)pyrazolo{1,5-a}pyrimidi ne), on prostaglandin E2 biosynthesis in vitro, acute hyperalgesia induced by yeast and substance P in rats and hyperalgesia in rats with a chronic constriction injury to the sciatic nerve (Bennett model), which is a model for peripheral neuropathic pain . OT-7100 did not inhibit prostaglandin E2 biosynthesis at 10(-8)-10(-4) M . Single oral doses of 3 and 10 mg/kg OT-7100 were effective on the hyperalgesia induced by yeast . Single oral doses of 0.1, 0.3, 1 and 3 mg/kg OT-7100 were effective on the hyperalgesia induced by substance P in which indomethacin had no effect . Repeated oral administration of OT-7100 (10 and 30 mg/kg) was effective in normalizing the mechanical nociceptive threshold in the injured paw without affecting the nociceptive threshold in the uninjured paw in the Bennett model . Indomethacin had no effect in this model . While amitriptyline (10 and 30 mg/kg) and clonazepam (3 and 10 mg/kg) significantly normalized the nociceptive threshold in the injured paw, they also increased the nociceptive threshold in the uninjured paw . These results suggest that OT-7100 is a new type of analgesic with the effect of normalizing the nociceptive threshold in peripheral neuropathic hyperalgesia. Jpn J Cancer Res, 1998 Dec, 89(12), 1284 - 91 Mutational analyses of multiple target genes in histologically heterogeneous gastric cancer with microsatellite instability; Wang Y et al.; It has been recognized that gastric cancer often shows histological heterogeneity in a single tumor . Although microsatellite instability (MSI) has been reported in gastric cancer, the significance of genomic instability in gastric cancers with histological heterogeneity within a single tumor has never been addressed . We investigated MSI at 8 microsatellite loci in 40 normal/tumor DNA pairs from 20 gastric cancers with histological heterogeneity . Six of 20 patients (10 DNAs of 40 tumor DNAs) had severe MSI in more than 3 loci . Four of the MSI-positive cases had frameshift mutations in the poly(A)10 tract of the TGF beta RII gene . This mutation was found only in the MSI-positive component in the 2 cases (cases 4 and 5) in which only 1 component exhibited MSI . The other 4 cases demonstrated homozygous or heteroclonal mutations (1 and 2 base deletions) in the poly(A)8 tract of the hMSH3 gene; no mutation was detected in the poly(C)8 tract of the hMSH6 gene in any of the MSI-positive cases . The profile of alterations in multiple targets was different between the 2 components in most of the cases (5/6) . These findings suggest that mismatch repair deficiency in MSI-positive tumors causes multiple gene inactivations through frameshift mutations in short repetitive sequences in a heterogeneous way within a histologically heterogeneous tumor. Nat Genet, 1999 Mar, 21(3), 318 - 22 Prox1 function is crucial for mouse lens-fibre elongation; Wigle JT et al.; Although insights have emerged regarding genes controlling the early stages of eye formation, little is known about lens-fibre differentiation and elongation . The expression pattern of the Prox1 homeobox gene suggests it has a role in a variety of embryonic tissues, including lens . To analyse the requirement for Prox1 during mammalian development, we inactivated the locus in mice . Homozygous Prox1-null mice die at mid-gestation from multiple developmental defects; here we describe the specific effect on lens development . Prox1 inactivation causes abnormal cellular proliferation, downregulated expression of the cell-cycle inhibitors Cdkn1b (also known as p27KIP1) and Cdkn1c (also known as p57KIP2), misexpression of E-cadherin and inappropriate apoptosis . Consequently, mutant lens cells fail to polarize and elongate properly, resulting in a hollow lens . Our data provide evidence that the progression of terminal fibre differentiation and elongation is dependent on Prox1 activity during lens development. Nat Genet, 1999 Mar, 21(3), 278 - 83 Genomic profiling of drug sensitivities via induced haploinsufficiency; Giaever G et al.; Lowering the dosage of a single gene from two copies to one copy in diploid yeast results in a heterozygote that is sensitized to any drug that acts on the product of this gene . This haploinsufficient phenotype thereby identifies the gene product of the heterozygous locus as the likely drug target . We exploited this finding in a genomic approach to drug-target identification . Genome sequence information was used to generate molecularly tagged heterozygous yeast strains that were pooled, grown competitively in drug and analysed for drug sensitivity using high-density oligonucleotide arrays . Individual heterozygous strain analysis verified six known drug targets . Parallel analysis identified the known target and two hypersensitive loci in a mixed culture of 233 strains in the presence of the drug tunicamycin . Our discovery that both drug target and hypersensitive loci exhibit drug-induced haploinsufficiency may have important consequences in pharmacogenomics and variable drug toxicity observed in human populations. Pathol Oncol Res, 1999, 5(1), 41 - 5 p53 and p16INK4A mutations during the progression of glomus tumor; Guran S et al.; Glomus tumors are significantly rare tumors of carotid body . The great majority of these tumors are benign in character . Here we present two brothers with hereditary glomus jugulare tumor who had consanguineous parents . Radiotherapy was applied approximately 8 and 10 years ago for treatment in both cases . Eight years later, one of these cases came to our notice due to relapse . The mutation pattern of p53, p57KIP2, p16INK4A and p15NK4B genes which have roles in the cell cycle, was analyzed in tumor samples obtained from the two affected cases in the initial phase and from one of these cases at relapse . The DNA sample obtained from the case in initial diagnosis phase revealed no p53, p57KIP2, p16INK4A or p15INK4B mutation . He is still in remission phase . Despite the lack of p53, p57KIP2, p16INK4A and p15INK4B mutation at initial diagnosis the tumor DNA of the other case in relapse revealed p53 codon 243 (ATG-->ATC; met-->ile) and p16 codon 97 (GAC-->AAC; asp-->asn) missense point mutations . No loss of heterozygosity in p53 and p16INK4A was observed by microsatellite analysis of tumoral tissues in these cases . P53 and p16INK4A mutations observed in relapse phase were in conserved regions of both genes . No previous reports have been published with these mutations in glomus tumor during progression . The mutation observed in this case may due to radiotherapy . In spite of this possibility, the missense point mutations in conserved region of p53 and p16INK4A genes may indicate the role of p53 and p16INK4A in tumor progression of glomus tumors. Curr Genet, 1999 Mar, 35(2), 118 - 26 Cloning, characterisation and regulation of the ornithine transaminase (otaA) gene of Aspergillus nidulans; Dzikowska A et al.; The ornithine transaminase (otaA) gene of Aspergillus nidulans has been cloned by transformation of the A . nidulans pro-ota- mutant strain with a cosmid gene library . The otaA gene contains two introns and potentially codes for a 453-aa-long protein . The deduced amino-acid sequence is homologous to known ornithine transaminases from Saccharomyces cerevisiae, Plasmodium falciparum, Vigna aconitifolia, rat, mouse and man, particularly in the pyridoxal phosphate-binding domain . The expression of the otaA gene is specifically induced by arginine, and is also under the control of nitrogen-metabolite and carbon-catabolite repression . Regulation of the gene occurs at both transcriptional and post-transcriptional levels . The promoter region of otaA contains putative AREA and CREA binding-sites . Fusion proteins containing AREA or CREA DNA-binding domains bind some of these sites . CREA binding-sites correspond very well to the CREA-binding consensus sequence which is SYGGRG . AREA binding-sites are composed of GATT sequences which are not typical binding sites for the GATA - binding family of transcription factors. Development, 1999 Apr, 126(8), 1689 - 702 Notch signaling imposes two distinct blocks in the differentiation of C2C12 myoblasts; Nofziger D et al.; Notch signal transduction regulates expression of downstream genes through the activation of the DNA-binding protein Su(H)/CBF1 . In Drosophila most of Notch signaling requires Su(H); however, some Notch-dependent processes occur in the absence of Su(H) suggesting that Notch signaling does not always involve activation of this factor . Using constitutively active forms of Notch lacking CBF1-interacting sequences we identified a Notch signaling pathway that inhibits myogenic differentiation of C2C12 myoblasts in the absence of CBF1 activation . Here we show that ligand-induced Notch signaling suppresses myogenesis in C2C12 myoblasts that express a dominant negative form of CBF1, providing additional evidence for CBF1-independent Notch signal transduction . Surprisingly mutant forms of Notch deficient in CBF1 activation are unable to antagonize MyoD activity, despite the fact that they inhibit myogenesis . Moreover, Notch-induced antagonism of MyoD requires CBF1 suggesting that the CBF1-dependent pathway mediates a cell-type-specific block in the myogenic program . However, Notch signaling in the absence of CBF1 activation blocks both myogenesis and osteogenesis, indicative of a general block in cellular differentiation . Taken together our data provide evidence for two distinct Notch signaling pathways that function to block differentiation at separate steps during the process of myogenesis in C2C12 myoblasts. Biochem Biophys Res Commun, 1999 Mar 16, 256(2), 299 - 306 Epithelin/granulin growth factors: extracellular cofactors for HIV-1 and HIV-2 Tat proteins; Trinh DP et al.; Epithelin/granulin growth factor is synthesized as a 593 amino acid precursor protein that contains 7.5 imperfectly conserved repeats of approximately 57 amino acids . Processed epithelin/granulin peptides have been isolated from vertebrate/invertebrate species and are growth factors implicated in epithelial and haemic cell function . Here they are identified as Human Immunodeficiency Virus (HIV) Tat binding proteins using the yeast two-hybrid assay . Intracellularly in yeast, mutation of selected cysteines in an epithelin/granulin dimeric repeat caused loss of binding to Tat exon 1 . In vitro binding of HIV-1 and HIV-2 Tat to epithelin/granulin dimeric and monomeric repeats was also observed by GST-glutathione bead "pulldown" assays . Because Tat is actively secreted from HIV-infected cells and has been shown to serve as a mitogenic factor for angiogenesis and for Kaposi-like cells, our observations suggest that epithelin/granulin growth factors may function as biologically important extracellular Tat co-factors . Biochem Biophys Res Commun, 1999 Mar 16, 256(2), 278 - 83 Appropriately spaced nuclear localizing signals are necessary for efficient nuclear import of nonnuclear proteins; Borgeld HJ et al.; To deliver nonnuclear proteins into the nucleus, we have examined the locations and number of nuclear localizing signals by use of simian virus 40 large T-antigen (SV40Ta) and yeast enhanced green fluorescent protein (yEGFP) in Saccharomyces cerevisiae as a model system . When only one SV40Ta was added to either the N- or C-terminus of yEGFP, the fluorescence of yEGFP was detected in both the nucleus and the cytoplasm . When two SV40Ta signals were added, one to the N-terminus and one to the C-terminus of yEGFP (SV40Ta-yEGFP-SV40Ta), the fluorescence of yEGFP was localized in only the nucleus . When the presequence of cytochrome oxidase subunit IV (pCOXIV) was inserted between the SV40Ta and the N-terminus of yEGFP (SV40Ta-pCOXIV-yEGFP-SV40Ta) in this construct, the fluorescence was located in both the nucleus and the cytoplasm, suggesting that the increased distance between the two SV40Ta signals decreased the efficiency of transport into the nucleus . When an additional SV40Ta signal was inserted between pCOXIV and yEGFP (SV40Ta-pCOXIV-SV40Ta-yEGFP), the fluorescence was localized only in the nucleus, indicating that two SV40Ta signals spaced by pCOXIV of 28 amino acid residues forming an alpha-helix are potent in transporting yEGFP into the nucleus . These results indicate that two SV40Ta signals spaced appropriately are essential for the efficient transport of the nonnuclear protein into the nucleus . Proc Natl Acad Sci U S A, 1999 Mar 16, 96(6), 2937 - 42 Detecting patterns of protein distribution and gene expression in silico; Geraghty MT et al.; Most biological information is contained within gene and genome sequences . However, current methods for analyzing these data are limited primarily to the prediction of coding regions and identification of sequence similarities . We have developed a computer algorithm, CoSMoS (for context sensitive motif searches), which adds context sensitivity to sequence motif searches . CoSMoS was challenged to identify genes encoding peroxisome-associated and oleate-induced genes in the yeast Saccharomyces cerevisiae . Specifically, we searched for genes capable of encoding proteins with a type 1 or type 2 peroxisomal targeting signal and for genes containing the oleate-response element, a cis-acting element common to fatty acid-regulated genes . CoSMoS successfully identified 7 of 8 known PTS-containing peroxisomal proteins and 13 of 14 known oleate-regulated genes . More importantly, CoSMoS identified an additional 18 candidate peroxisomal proteins and 300 candidate oleate-regulated genes . Preliminary localization studies suggest that these include at least 10 previously unknown peroxisomal proteins . Phenotypic studies of selected gene disruption mutants suggests that several of these new peroxisomal proteins play roles in growth on fatty acids, one is involved in peroxisome biogenesis and at least two are required for synthesis of lysine, a heretofore unrecognized role for peroxisomes . These results expand our understanding of peroxisome content and function, demonstrate the utility of CoSMoS for context-sensitive motif scanning, and point to the benefits of improved in silico genome analysis. Yeast, 1999 Feb, 15(3), 181 - 9 Characterization of the Hansenula polymorpha CPY gene encoding carboxypeptidase Y; Bellu AR et al.; We have isolated the Hansenula polymorpha CPY gene encoding carboxypeptidase Y (Hp-CPY) . The deduced amino acid sequence revealed that Hp-CPY consists of 541 amino acids and has a calculated Mr of 60,793 . The protein is highly similar to Saccharomyces cerevisiae CPY (61.8% identity) . At the N-terminus of Hp-CPY signals for the entry into the secretory pathway and subsequent sorting to the vacuole were identified . Immunocytochemically, using monospecific antibodies raised against Hp-CPY, the protein was localized to the vacuole . On Western blots, a diffuse protein band was observed in extracts of H . polymorpha cells, suggesting that the protein is glycosylated . This was confirmed by endoglycosidase H treatment, which resulted in a strong reduction of the apparent Mr of the protein . We have investigated the effect of CPY deletion on the degradation of peroxisomes, an autophagous process that occurs when the organelles become redundant for growth . In deltacpy cells peroxisomal proteins were degraded in the vacuole as efficiently as in wild-type H . polymorpha cells, indicating that CPY is not a major proteinase in this pathway. Mol Cell, 1999 Feb, 3(2), 219 - 28 Use of altered specificity mutants to probe a specific protein-protein interaction in differentiation: the GATA-1:FOG complex; Crispino JD et al.; GATA-1 and FOG (Friend of GATA-1) are each essential for erythroid and megakaryocyte development . FOG, a zinc finger protein, interacts with the amino (N) finger of GATA-1 and cooperates with GATA-1 to promote differentiation . To determine whether this interaction is critical for GATA-1 action, we selected GATA-1 mutants in yeast that fail to interact with FOG but retain normal DNA binding, as well a compensatory FOG mutant that restores interaction . These novel GATA-1 mutants do not promote erythroid differentiation of GATA-1- erythroid cells . Differentiation is rescued by the second-site FOG mutant . Thus, interaction of FOG with GATA-1 is essential for the function of GATA-1 in erythroid differentiation . These findings provide a paradigm for dissecting protein-protein associations involved in mammalian development. Proc Natl Acad Sci U S A, 1999 Mar 16, 96(6), 2907 - 12 Interpreting patterns of gene expression with self-organizing maps: methods and application to hematopoietic differentiation; Tamayo P et al.; Array technologies have made it straightforward to monitor simultaneously the expression pattern of thousands of genes . The challenge now is to interpret such massive data sets . The first step is to extract the fundamental patterns of gene expression inherent in the data . This paper describes the application of self-organizing maps, a type of mathematical cluster analysis that is particularly well suited for recognizing and classifying features in complex, multidimensional data . The method has been implemented in a publicly available computer package, GENECLUSTER, that performs the analytical calculations and provides easy data visualization . To illustrate the value of such analysis, the approach is applied to hematopoietic differentiation in four well studied models (HL-60, U937, Jurkat, and NB4 cells) . Expression patterns of some 6,000 human genes were assayed, and an online database was created . GENECLUSTER was used to organize the genes into biologically relevant clusters that suggest novel hypotheses about hematopoietic differentiation-for example, highlighting certain genes and pathways involved in "differentiation therapy" used in the treatment of acute promyelocytic leukemia. Proc Natl Acad Sci U S A, 1999 Mar 16, 96(6), 2800 - 4 DNA replication in vertebrates requires a homolog of the Cdc7 protein kinase; Roberts BT et al.; CDC7 is an essential gene required for DNA replication in Saccharomyces cerevisiae . Cdc7p homologs have recently been identified in vertebrates, but their role in DNA replication has not yet been addressed . Here we show that antibodies to the Xenopus laevis homolog, xCdc7, interfere with DNA replication in vivo in developing embryos and in vitro in cycling egg extracts . We also demonstrate cell cycle-dependent association of xCdc7 with the Mcm complex, which binds to replication origins and also is required for DNA synthesis . Taken together, these data indicate that the function of xCdc7 is conserved from fungi to vertebrates . xCdc7 protein accumulates after stimulation of resting oocytes with progesterone, suggesting a molecular explanation for previous observations that the development of the capacity for DNA replication requires protein synthesis late in meiosis I. Proc Natl Acad Sci U S A, 1999 Mar 16, 96(6), 2764 - 9 An activation-specific role for transcription factor TFIIB in vivo; Wu WH et al.; A yeast mutant was isolated encoding a single amino acid substitution {serine-53 --> proline (S53P)} in transcription factor TFIIB that impairs activation of the PHO5 gene in response to phosphate starvation . This effect is activation-specific because S53P did not affect the uninduced level of PHO5 expression, yet is not specific to PHO5 because Adr1-mediated activation of the ADH2 gene also was impaired by S53P . Pho4, the principal activator of PHO5, directly interacted with TFIIB in vitro, and this interaction was impaired by the S53P replacement . Furthermore, Pho4 induced a conformational change in TFIIB, detected by enhanced sensitivity to V8 protease . The S53P replacement also impaired activation of a lexA(op)-lacZ reporter by a LexA fusion protein to the activation domain of Adr1, thereby indicating that the transcriptional effect on ADH2 expression is specific to the activation function of Adr1 . These results define an activation-specific role for TFIIB in vivo and suggest that certain activators induce a conformational change in TFIIB as part of their mechanism of transcriptional stimulation. Chem Res Toxicol, 1999 Mar, 12(3), 286 - 96 Opposite behaviors of reactive metabolites of tienilic acid and its isomer toward liver proteins: use of specific anti-tienilic acid-protein adduct antibodies and the possible relationship with different hepatotoxic effects of the two compounds; Bonierbale E et al.; Tienilic acid (TA) is responsible for an immune-mediated drug-induced hepatitis in humans, while its isomer (TAI) triggers a direct hepatitis in rats . In this study, we describe an immunological approach developed for studying the specificity of the covalent binding of these two compounds . For this purpose, two different coupling strategies were used to obtain TA-carrier protein conjugates . In the first strategy, the drug was linked through its carboxylic acid function to amine residues of carrier proteins (BSA-N-TA and casein-N-TA), while in the second strategy, the thiophene ring of TA was attached to proteins through a short 3-thiopropanoyl linker, the corresponding conjugates (BSA-S-5-TA and betaLG-S-5-TA) thus preferentially presenting the 2, 3-dichlorophenoxyacetic moiety of the drug for antibody recognition . The BSA-S-5-TA conjugate proved to be 30 times more immunogenic than BSA-N-TA . Anti-TA-protein adduct antibodies were obtained after immunization of rabbits with BSA-S-5-TA (1/35000 titer against betaLG-S-5-TA in ELISA) . These antibodies strongly recognized the 2, 3-dichlorophenoxyacetic moiety of TA but poorly the part of the drug engaged in the covalent binding with the proteins . This powerful tool was used in immunoblots to compare TA or TAI adduct formation in human liver microsomes as well as on microsomes from yeast expressing human liver cytochrome P450 2C9 . TA displayed a highly specific covalent binding focused on P450 2C9 which is the main cytochrome P450 responsible for its hepatic activation in humans . On the contrary, TAI showed a nonspecific alkylation pattern, targeting many proteins upon metabolic activation . Nevertheless, this nonspecific covalent binding could be completely shifted to a thiol trapping agent like GSH . The difference in alkylation patterns for these two compounds is discussed with regard to their distinct toxicities . A relationship between the specific covalent binding of P450 2C9 by TA and the appearance of the highly specific anti-LKM2 autoantibodies (known to specifically recognize P450 2C9) in patients affected with TA-induced hepatitis is strongly suggested. Nucleic Acids Res, 1999 Apr 1, 27(7), 1656 - 63 The repressor which binds the -75 GATA motif of the GPB promoter contains Ku70 as the DNA binding subunit; Camara-Clayette V et al.; Glycophorin B (GPB) is an abundant cell surface glycoprotein which is only expressed in human erythroid cells . Previous functional analysis demonstrated that the repression of the GPB promoter is determined by the binding of a ubiquitous factor which recognizes a GATA motif centered at position -75 . In erythroid cells this ubiquitous factor is displaced by the binding of the erythroid-specific factor hGATA1 . Here, we have identified the Ku70 protein as a candidate GPB repressor DNA binding subunit through the screening of a human HeLa expression library using the -75 GATA sequence as bait (one-hybrid method) . Electrophoretic mobility shift assays demonstrated that the ubiquitous factor that binds the -75 GATA sequence was the Ku70-Ku80 (Ku) heterodimer . Co-transfection experiments demonstrated that overexpression of Ku70 in the K562 erythroleukeamic cell line resulted in transcriptional repression of the chloramphenicol acetyltransferase reporter gene when placed under the control of the wild-type GPB promoter . Conversely, no repression was observed when a mutation that abolished the binding of Ku was introduced in the GPB promoter construct . Altogether, these results indicate that Ku binds in vivo to the -75 WGATAR motif and is involved in negative regulation of the GPB promoter . These findings suggest that, besides its role in many functions, Ku is also involved in transcriptional regulation of erythroid genes. EMBO J, 1999 Mar 15, 18(6), 1506 - 15 Sac1p plays a crucial role in microsomal ATP transport, which is distinct from its function in Golgi phospholipid metabolism; Kochendorfer KU et al.; Analysis of microsomal ATP transport in yeast resulted in the identification of Sac1p as an important factor in efficient ATP uptake into the endoplasmic reticulum (ER) lumen . Yet it remained unclear whether Sac1p is the authentic transporter in this reaction . Sac1p shows no homology to other known solute transporters but displays similarity to the N-terminal non-catalytic domain of a subset of inositol 5'-phosphatases . Furthermore, Sac1p was demonstrated to be involved in inositol phospholipid metabolism, an activity whose absence contributes to the bypass Sec14p phenotype in sac1 mutants . We now show that purified recombinant Sac1p can complement ATP transport defects when reconstituted together with sac1Delta microsomal extracts, but is unable to catalyze ATP transport itself . In addition, we demonstrate that sac1Delta strains are defective in ER protein translocation and folding, which is a direct consequence of impaired ATP transport function and not related to the role of Sac1p in Golgi inositol phospholipid metabolism . These data suggest that Sac1p is an important regulator of microsomal ATP transport providing a possible link between inositol phospholipid signaling and ATP-dependent processes in the yeast ER. Methods, 1999 Feb, 17(2), 151 - 60 Modeling transcriptional regulation using microinjection into Xenopus oocytes; Robinett CC et al.; Transcriptional regulation is a complex process that requires cooperation between specific DNA sequence elements, the DNA-binding proteins that bind to these sequences, the general transcriptional machinery, and chromatin . Oocyte microinjection offers a technique to study the integrated transcription process while still providing the opportunity to experimentally perturb this process . We describe here techniques for manipulating DNA templates and the protein complement of the oocyte to study multiple facets of transcriptional regulation . We present sample results showing that the GAL4-VP16 fusion activator is sensitive to distance in constructs containing only a minimal promoter, but can activate transcription at greater distances when proximal promoter elements are present . Methods, 1999 Feb, 17(2), 95 - 103 Targeted linearization of DNA in vivo; Liang CP et al.; In the past decade, site-specific chromosomal DNA cleavage mediated by DNA endonucleases has been used to examine diverse aspects of chromosome structure and function in eukaryotes, such as DNA topology, replication, transcription, recombination, and repair . Here we describe a method with which chromosomes can be linearized at any predefined position in vivo . Yeast homothallic switching endonuclease (HO endo), a sequence-specific double-strand nuclease involved in mating-type switching, is employed for targeting DNA cleavage . HO endo contains discrete functional domains: a N-terminal nuclease and a C-terminal DNA-binding domain, thereby allowing construction of a chimeric nuclease with the cutting site distinct from the original HO recognition sequence . The expression of the nuclease is engineered to be controlled by a tightly regulated, inducible promoter . The cut sites recognized by HO endo or its derivatives are introduced specifically at desired positions in the yeast genome by homologous recombination . Here we present experimental procedures and review some applications based on this approach in yeast and other biological systems . Methods, 1999 Jan, 17(1), 46 - 51 Monitoring mRNA decapping activity; Zhang S et al.; mRNA decapping is a common step shared between two important mRNA decay pathways in yeast, Saccharomyces cerevisiae . To investigate how mRNAs are decapped, we have developed an assay that can be easily used to measure the decapping activity . This assay has been used to isolate yeast strains with altered decapping activities . The results demonstrated that decreased decapping activity in vitro corresponds well with the decapping-deficient phenotype in vivo . This assay has been applied to the purified yeast decapping enzyme Dcp1 protein as well as crude yeast extracts and Xenopus oocyte extracts . J Biol Chem, 1999 Mar 19, 274(12), 8085 - 92 Structure and function of the human transcription elongation factor DSIF; Yamaguchi Y et al.; 5,6-Dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) is a classic inhibitor of transcription elongation by RNA polymerase II (pol II) . We have previously identified and purified a novel transcription elongation factor, termed DSIF (for DRB sensitivity-inducing factor), that makes transcription sensitive to DRB . DSIF is composed of 160- and 14-kDa subunits, which are homologs of the Saccharomyces cerevisiae transcription factors Spt5 and Spt4 . DSIF may either repress or stimulate transcription in vitro, depending on conditions, but its physiological function remains elusive . Here we characterize the structure and function of DSIF p160 . p160 is shown to be a ubiquitous nuclear protein that forms a stable complex with p14 and interacts directly with the pol II largest subunit . Mutation analysis of p160 is used to identify structural features essential for its in vitro activity and to map the domains required for its interaction with p14 and pol II . Finally, a p160 mutant that represses DSIF activity in a dominant-negative manner is identified and used to demonstrate that DSIF represses transcription from various promoters in vivo. J Biol Chem, 1999 Mar 19, 274(12), 8022 - 8 Multisite autophosphorylation of p21-activated protein kinase gamma-PAK as a function of activation; Gatti A et al.; p21-activated protein kinase (PAK) is a family of serine/threonine kinases whose activity is stimulated by binding to small G-proteins such as Cdc42 and subsequent autophosphorylation . Focusing on the ubiquitous gamma-isoform of PAK in this study, baculovirus-infected insect cells were used to obtain recombinant gamma-PAK, while native gamma-PAK was isolated from rabbit reticulocytes . Two-dimensional gel electrophoresis of gamma-PAK followed by immunoblot analysis revealed a similar profile for native and recombinant gamma-PAK, both consisting of multiple protein spots . Following Cdc42-stimulated autophosphorylation, the two-dimensional profiles of native and recombinant gamma-PAK were characterized by a similar acidic shift, suggesting a common response to Cdc42 . To understand the effect of differential phosphorylation on its activation status, gamma-PAK autophosphorylation was conducted in the presence or absence of activators such as Cdc42 and histone II-AS, followed by tryptic digestion and comparative two-dimensional phosphopeptide mapping . The major phosphopeptides were subjected to a combination of manual and automated amino acid sequencing . Overall, eight autophosphorylation sites were identified in Cdc42-activated gamma-PAK, six of which are in common with those previously reported in alpha-PAK, while Ser-19 and Ser-165 appear to be uniquely phosphorylated in the gamma-form . Further, the phosphorylation of Ser-141, Ser-165, and Thr-402 was found to correlate with gamma-PAK activation. J Biol Chem, 1999 Mar 19, 274(12), 7982 - 6 KSR-1 binds to G-protein betagamma subunits and inhibits beta gamma-induced mitogen-activated protein kinase activation; Bell B et al.; The protein kinase KSR-1 is a recently identified participant in the Ras signaling pathway . The subcellular localization of KSR-1 is variable . In serum-deprived cultured cells, KSR-1 is primarily found in the cytoplasm; in serum-stimulated cells, a significant portion of KSR-1 is found at the plasma membrane . To identify the mechanism that mediates KSR-1 translocation, we performed a yeast two-hybrid screen . Three clones that interacted with KSR-1 were found to encode the full-length gamma10 subunit of heterotrimeric G-proteins . KSR-1 also interacted with gamma2 and gamma3 in a two-hybrid assay . Deletion analysis demonstrated that the isolated CA3 domain of KSR-1, which contains a cysteine-rich zinc finger-like domain, interacted with gamma subunits . Coimmunoprecipitation experiments demonstrated that KSR-1 bound to beta1 gamma3 subunits when all three were transfected into cultured cells . Lysophosphatidic acid treatment of cells induced KSR-1 translocation to the plasma membrane from the cytoplasm that was blocked by administration of pertussis toxin but not by dominant-negative Ras . Finally, transfection of wild-type KSR-1 inhibited beta1 gamma3-induced mitogen-activated protein kinase activation in cultured cells . These results demonstrate that KSR-1 translocation to the plasma membrane is mediated, at least in part, by an interaction with beta gamma and that this interaction may modulate mitogen-activated protein kinase signaling. J Biol Chem, 1999 Mar 19, 274(12), 7848 - 56 The DNA-dependent protein kinase catalytic activity regulates DNA end processing by means of Ku entry into DNA; Calsou P et al.; The DNA-dependent protein kinase (DNA-PK) is required for double-strand break repair in mammalian cells . DNA-PK contains the heterodimer Ku and a 460-kDa serine/threonine kinase catalytic subunit (p460) . Ku binds in vitro to DNA termini or other discontinuities in the DNA helix and is able to enter the DNA molecule by an ATP-independent process . It is clear from in vitro experiments that Ku stimulates the recruitment to DNA of p460 and activates the kinase activity toward DNA-binding protein substrates in the vicinity . Here, we have examined in human nuclear cell extracts the influence of the kinase catalytic activity on Ku binding to DNA . We demonstrate that, although Ku can enter DNA from free ends in the absence of p460 subunit, the kinase activity is required for Ku translocation along the DNA helix when the whole Ku/p460 assembles on DNA termini . When the kinase activity is impaired, DNA-PK including Ku and p460 is blocked at DNA ends and prevents their processing by either DNA polymerization, degradation, or ligation . The control of Ku entry into DNA by DNA-PK catalytic activity potentially represents an important regulation of DNA transactions at DNA termini. J Biol Chem, 1999 Mar 19, 274(12), 7833 - 40 Sec24 proteins and sorting at the endoplasmic reticulum; Pagano A et al.; COPII proteins are necessary to generate secretory vesicles at the endoplasmic reticulum . In yeast, the Sec24p protein is the only COPII component in which two close orthologues have been identified . By using gene knock-out in yeast, we found that the absence of one of these Sec24 orthologues resulted in a selective secretion defect for a subset of proteins released into the medium . Data base searches revealed the existence of an entire family of Sec24-related proteins in humans, worms, flies, and plants . We identified and cloned two new human cDNAs encoding proteins homologous to yeast Sec24p, in addition to two human cDNAs already present within the data bases . The entire Sec24 family identified to date is characterized by clusters of highly conserved residues within the 2/3 carboxyl-terminal domain of all the proteins and a divergent amino terminus domain . Human (h) Sec24 orthologues co-immunoprecipitate with hSec23Ap and migrate as a complex by size exclusion chromatography . Immunofluorescence microscopy confirmed that these proteins co-localize with hSec23p and hSec13p . Together, our data suggest that in addition to its role in the shaping up of the vesicle, the Sec23-24p complex may be implicated in cargo selection and concentration. Plant J, 1999 Jan, 17(2), 119 - 30 KCS1 encodes a fatty acid elongase 3-ketoacyl-CoA synthase affecting wax biosynthesis in Arabidopsis thaliana; Todd J et al.; An Arabidopsis fatty acid elongase gene, KCS1, with a high degree of sequence identity to FAE1, encodes a 3-ketoacyl-CoA synthase which is involved in very long chain fatty acid synthesis in vegetative tissues, and which also plays a role in wax biosynthesis . Sequence analysis of KCS1 predicted that this synthase was anchored to a membrane by two adjacent N-terminal, membrane-spanning domains . Analysis of a T-DNA tagged kcs1-1 mutant demonstrated the involvement of the KCS1 in wax biosynthesis . Phenotypic changes in the kcs1-1 mutant included thinner stems and less resistance to low humidity stress at a young age . Complete loss of KCS1 expression resulted in decreases of up to 80% in the levels of C26 to C30 wax alcohols and aldehydes, but much smaller effects were observed on the major wax components, i.e . the C29 alkanes and C29 ketones on leaves, stems and siliques . In no case did the loss of KCS1 expression result in complete loss of any individual wax component or significantly decrease the total wax load . This indicated that there was redundancy in the elongase KCS activities involved in wax synthesis . Furthermore, since alcohol, aldehyde, alkane and ketone levels were affected to varying degrees, involvement of the KCS1 synthase in both the decarbonylation and acyl-reduction wax synthesis pathways was demonstrated. J Clin Microbiol, 1999 Apr, 37(4), 1200 - 2 Contaminations occurring in fungal PCR assays; Loeffler J et al.; Successful in vitro amplification of fungal DNA in clinical specimens has been reported recently . In a collaboration among five European centers, the frequency and risk of contamination due to airborne spore inoculation or carryover contamination in fungal PCR were analyzed . The identities of all contaminants were specified by cycle sequencing and GenBank analysis . Twelve of 150 PCR assays that together included over 2,800 samples were found to be contaminated (3.3% of the negative controls were contaminated during the DNA extraction, and 4.7% of the PCR mixtures were contaminated during the amplification process) . Contaminants were specified as Aspergillus fumigatus, Saccharomyces cerevisiae, and Acremonium spp . Further analysis showed that commercially available products like zymolyase powder or 10x PCR buffer may contain fungal DNA . In conclusion, the risk of contamination is not higher in fungal PCR assays than in other diagnostic PCR-based assays if general precautions are taken. Curr Biol, 1999 Mar 11, 9(5), 227 - 36 Inhibitory phosphorylation of the APC regulator Hct1 is controlled by the kinase Cdc28 and the phosphatase Cdc14; Jaspersen SL et al.; BACKGROUND: Exit from mitosis requires inactivation of mitotic cyclin-dependent kinases (CDKs) . A key mechanism of CDK inactivation is ubiquitin-mediated cyclin proteolysis, which is triggered by the late mitotic activation of a ubiquitin ligase known as the anaphase-promoting complex (APC) . Activation of the APC requires its association with substoichiometric activating subunits termed Cdc20 and Hct1 (also known as Cdh1) . Here, we explore the molecular function and regulation of the APC regulatory subunit Hct1 in Saccharomyces cerevisiae . RESULTS: Recombinant Hct1 activated the cyclin-ubiquitin ligase activity of APC isolated from multiple cell cycle stages . APC isolated from cells arrested in G1, or in late mitosis due to the cdc14-1 mutation, was more responsive to Hct1 than APC isolated from other stages . We found that Hct1 was phosphorylated in vivo at multiple CDK consensus sites during cell cycle stages when activity of the cyclin-dependent kinase Cdc28 is high and APC activity is low . Purified Hct1 was phosphorylated in vitro at these sites by purified Cdc28-cyclin complexes, and phosphorylation abolished the ability of Hct1 to activate the APC in vitro . The phosphatase Cdc14, which is known to be required for APC activation in vivo, was able to reverse the effects of Cdc28 by catalyzing Hct1 dephosphorylation and activation . CONCLUSIONS: We conclude that Hct1 phosphorylation is a key regulatory mechanism in the control of cyclin destruction . Phosphorylation of Hct1 provides a mechanism by which Cdc28 blocks its own inactivation during S phase and early mitosis . Following anaphase, dephosphorylation of Hct1 by Cdc14 may help initiate cyclin destruction. Curr Biol, 1999 Feb 25, 9(4), 211 - 4 The RLF-B component of the replication licensing system is distinct from Cdc6 and functions after Cdc6 binds to chromatin; Tada S et al.; Replication licensing factor (RLF) is an essential initiation factor that can prevent re-replication of DNA in a single cell cycle {1} {2} . It is required for the initiation of DNA replication, binds to chromatin early in the cell cycle, is removed from chromatin as DNA replicates and is unable to re-bind replicated chromatin until the following mitosis . Chromatography of RLF from Xenopus extracts has shown that it consists of two components termed RLF-B and RLF-M {3} . The RLF-M component consists of complexes of all six Xenopus minichromosome maintenance (MCM/P1) proteins (XMcm2-7), which bind to chromatin in late mitosis and are removed as replication occurs {3} {4} {5} {6} {7} . The identity of RLF-B is currently unknown . At least two factors must be present on chromatin before licensing can occur: the Xenopus origin recognition complex (XORC) {8} {9} and Xenopus Cdc6 (XCdc6) {10} . XORC saturates Xenopus sperm chromatin at approximately one copy per replication origin whereas XCdc6 binds to chromatin only if XORC is bound first {9} {10} {11} . Although XORC has been shown to be a distinct activity from RLF-B {9}, the relationship between XCdc6 and RLF-B is currently unclear . Here, we show that active XCdc6 is loaded onto chromatin in extracts with defective RLF, and that both RLF-M and RLF-B are still required for the licensing of XCdc6-containing chromatin . Furthermore, RLF-B can be separated from XCdc6 by immunoprecipitation and standard chromatography . These experiments demonstrate that RLF-B is both functionally and physically distinct from XCdc6, and that XCdc6 is loaded onto chromatin before RLF-B function is executed. J Mol Biol, 1999 Mar 19, 287(1), 1 - 7 The bromodomain of Gcn5p interacts in vitro with specific residues in the N terminus of histone H4; Ornaghi P et al.; Whereas the histone acetyltransferase activity of yeast Gcn5p has been widely studied, its structural interactions with the histones and the role of the carboxy-terminal bromodomain are still unclear . Using a glutathione S-transferase pull down assay we show that Gcn5p binds the amino-terminal tails of histones H3 and H4, but not H2A and H2B . The deletion of bromodomain abolishes this interaction and bromodomain alone is able to interact with the H3 and H4 N termini . The amino acid residues of the H4 N terminus involved in the binding with Gcn5p have been studied by site-directed mutagenesis . The substitution of amino acid residues R19 or R23 of the H4 N terminus with a glutamine (Q) abolishes the interaction with Gcn5p and the bromodomain . These residues differ from those known to be acetylated or to be involved in binding the SIR proteins . This evidence and the known dispensability of the bromodomain for Gcn5p acetyltransferase activity suggest a new structural role for the highly evolutionary conserved bromodomain . Biochemistry, 1999 Mar 9, 38(10), 3157 - 67 Unexpected metal ion requirements specific for catalysis of the branching reaction in a group II intron; Deme E et al.; The splicing process catalyzed by group II intron ribozymes follows the same two-step pathway as nuclear pre-mRNA splicing . In vivo, the first splicing step of wild-type introns is a transesterification reaction giving rise to a branched lariat intron-3'-exon intermediate characteristic of this splicing mode . In the wild-type introns, the ribozyme core and the substrate intron-exon junctions are carried by the same precursor molecule, making it difficult to distinguish between RNA folding and catalysis under normal splicing reactions . To characterize the catalytic step of the first transesterification reaction, we studied the reversal of this reaction, reverse branching . In this reverse reaction, the excised lariat intron and the substrate 5'-exon can be preincubated and folded separately, allowing the measure of the catalytic rate of the reaction . To measure the catalytic rate of the second splicing step, purified lariat intron-3'-exon intermediate molecules were preincubated and folded prior to the addition of 5'-exon . Conditions could be found where chemistry appeared rate limiting for both catalytic steps . Study of the metal ion requirements under these conditions resulted in the unexpected finding that, for the intron studied, substitution of magnesium ions by manganese ions enhanced the rate of the first transesterification reaction by two orders of magnitude but had virtually no effect on the second transesterification reaction or the 5' splice site cleavage by hydrolysis . Finally, the catalytic rates measured under optimal conditions for both splicing steps were faster by three orders of magnitude in the branching pathway than in the hydrolytic pathway. J Virol, 1999 Apr, 73(4), 2622 - 32 A brome mosaic virus intergenic RNA3 replication signal functions with viral replication protein 1a to dramatically stabilize RNA in vivo; Sullivan ML et al.; Brome mosaic virus (BMV), a positive-strand RNA virus in the alphavirus-like superfamily, encodes two RNA replication proteins . The 1a protein has putative helicase and RNA-capping domains, whereas 2a contains a polymerase-like domain . Saccharomyces cerevisiae expressing 1a and 2a is capable of replicating a BMV RNA3 template produced by in vivo transcription of a DNA copy of RNA3 . Although insufficient for RNA3 replication, the expression of 1a protein alone results in a dramatic and specific stabilization of the RNA3 template in yeast . As one step toward understanding 1a-induced stabilization of RNA3, the interactions involved, and its possible relation to RNA replication, we have identified the cis-acting sequences required for this effect . We find that 1a-induced stabilization is mediated by a 150- to 190-base segment of the RNA3 intergenic region corresponding to a previously identified enhancer of RNA3 replication . Moreover, this segment is sufficient to confer 1a-induced stability on a heterologous beta-globin RNA . Within this intergenic segment, partial deletions that inhibited 1a-induced stabilization in yeast expressing 1a alone resulted in parallel decreases in the levels of negative- and positive-strand RNA3 replication products in yeast expressing 1a and 2a . In particular, a small deletion encompassing a motif corresponding to the box B element of RNA polymerase III promoters dramatically reduced the ability of RNAs to respond to 1a or 1a and 2a . These and other findings suggest that 1a-induced stabilization likely reflects an early template selection step in BMV RNA replication. J Bacteriol, 1999 Mar, 181(6), 1963 - 7 LB-AUT7, a novel symbiosis-regulated gene from an ectomycorrhizal fungus, Laccaria bicolor, is functionally related to vesicular transport and autophagocytosis; Kim SJ et al.; We have identified LB-AUT7, a gene differentially expressed 6 h after ectomycorrhizal interaction between Laccaria bicolor and Pinus resinosa . LB-Aut7p can functionally complement its Saccharomyces cerevisiae homolog, which is involved in the attachment of autophagosomes to microtubules . Our findings suggest the induction of an autophagocytosis-like vesicular transport process during ectomycorrhizal interaction. Mech Dev, 1999 Feb, 80(2), 153 - 8 Use of the Gal4-UAS technique for targeted gene expression in the zebrafish; Scheer N et al.; The most common way to analyze the function of cloned genes in zebrafish is to misexpress the gene product or an altered variant of it by mRNA injection . However, mRNA injection has several disadvantages . The GAL4-UAS system for targeted gene expression allows one to overcome some of these disadvantages . To test the GAL4-UAS system in zebrafish, we generated two different kinds of stable transgenic lines, carrying activator and effector constructs, respectively . In the activator lines the gene for the yeast transcriptional activator GAL4 is under the control of a given promoter, while in the effectors the gene of interest is fused to the sequence of the DNA-binding motif of GAL4 (UAS) . Crosses of animals from the activator and effector lines show that effector genes are transcribed with the spatial pattern of the activators . This work smoothes the way for a novel method of misexpression of gene products in zebrafish in order to analyze the function of genes in developmental processes . Genes Dev, 1999 Mar 1, 13(5), 569 - 80 Interaction of the U1 snRNP with nonconserved intronic sequences affects 5' splice site selection; Puig O et al.; Intron definition and splice site selection occur at an early stage during assembly of the spliceosome, the complex mediating pre-mRNA splicing . Association of U1 snRNP with the pre-mRNA is required for these early steps . We report here that the yeast U1 snRNP-specific protein Nam8p is a component of the commitment complexes, the first stable complexes assembled on pre-mRNA . In vitro and in vivo, Nam8p becomes indispensable for efficient 5' splice site recognition when this process is impaired as a result of the presence of noncanonical 5' splice sites or the absence of a cap structure . Nam8p stabilizes commitment complexes in the latter conditions . Consistent with this, Nam8p interacts with the pre-mRNA downstream of the 5' splice site, in a region of nonconserved sequence . Substitutions in this region affect splicing efficiency and alternative splice site choice in a Nam8p-dependent manner . Therefore, Nam8p is involved in a novel mechanism by which a snRNP component can affect splice site choice and regulate intron removal through its interaction with a nonconserved sequence . This supports a model where early 5' splice recognition results from a network of interactions established by the splicing machinery with various regions of the pre-mRNA. Curr Opin Genet Dev, 1999 Feb, 9(1), 97 - 103 Telomere maintenance mechanisms and cellular immortalization; Colgin LM et al.; Immortal cell populations are able to proliferate indefinitely . Immortalization is associated with activation of processes that compensate for the telomeric shortening that accompanies cell division in normal somatic cells . In many immortal cell lines, telomere maintenance is provided by the action of the ribonucleoprotein enzyme complex, telomerase . Some immortal cell lines have undetectable or very low levels of telomerase activity and there is evidence that these cells maintain their telomeres by an alternative mechanism. Curr Opin Genet Dev, 1999 Feb, 9(1), 49 - 54 Target of rapamycin (TOR): balancing the opposing forces of protein synthesis and degradation; Dennis PB et al.; Mitogenic and nutritional signals must be integrated for a cell to grow . The target of rapamycin (TOR) is emerging as an effector for signals which indicate to the cell whether the external environment is conducive for growth . Use of the immunosuppressant rapamycin, a bacterial macrolide, has been instructive in identifying potential signaling components downstream of TOR, leading to the observation that both protein synthesis and turnover are under TOR control . The central issues concerning TOR are the identification of the proliferative and anti-proliferative signals which mediate its function and the mechanisms by which these signals are transduced to downstream molecules. Curr Opin Genet Dev, 1999 Feb, 9(1), 40 - 8 Histone acetylases and deacetylases in cell proliferation; Kouzarides T; There are several enzymes, acetylases and deacetylases, that can regulate transcription by modifying the acetylation state of histones or other promoter-bound transcription factors . Some of these enzymes are present in multisubunit complexes . Recent efforts to understand the biological role of these enzymes reveals their involvement in cell-cycle regulation and differentiation . Furthermore, accumulating evidence suggests that deregulation of acetylase and deacetylase activity plays a causative role in the generation of cancer. Int J Radiat Biol, 1999 Feb, 75(2), 253 - 8 Heat sensitivity of double-stranded DNA-dependent protein kinase (DNA-PK) activity; Ihara M et al.; PURPOSE: The heat sensitivity of DNA-PK activity in hybrid cells and the possible restoration of this activity with extracts from scid cells (defective in DNA-PKcs), sxi-3 cells (defective in Ku80) and V794 (sxi-3 parental wild-type cells) was analysed . MATERIALS AND METHODS: Heat treatment of cells was performed in a water bath at 44 degrees C . The cell extract from scid cells or sxi-3 cells was added to heat-treated hybrid cell extracts, and the DNA-PK activity was assayed . RESULTS: When hybrid cells were heated at 44 degrees C for 15 min, DNA-PK activity was reduced to undetectable levels . The decreased DNA-PK activity could be restored in a concentration-dependent manner with the addition of scid cell extract . The sxi-3 cell extract could not restore heat-inactivated DNA-PK activity . CONCLUSIONS: DNA-PK was inactivated by heat treatment at 44 degrees C . Ku70/Ku80, but not Ku70 alone, could restore heat-inactivated DNA-PK. Am J Physiol, 1999 Mar, 276(3 Pt 1), G606 - 12 Epidermal growth factor regulates fatty acid uptake and metabolism in Caco-2 cells; Darimont C et al.; Epidermal growth factor (EGF) has been reported to stimulate carbohydrate, amino acid, and electrolyte transport in the small intestine, but its effects on lipid transport are poorly documented . This study aimed to investigate EGF effects on fatty acid uptake and esterification in a human enterocyte cell line (Caco-2) . EGF inhibited cell uptake of {14C}palmitate and markedly reduced its incorporation into triglycerides . In contrast, the incorporation in phospholipids was enhanced . To elucidate the mechanisms involved, key steps of lipid synthesis were investigated . The amount of intestinal fatty acid-binding protein (I-FABP), which is thought to be important for fatty acid absorption, and the activity of diacylglycerol acyltransferase (DGAT), an enzyme at the branch point of diacylglycerol utilization, were reduced . EGF effects on DGAT and on palmitate esterification occurred at 2-10 ng/ml, whereas effects on I-FABP and palmitate uptake occurred only at 10 ng/ml . This suggests that EGF inhibited palmitate uptake by reducing the I-FABP level and shifted its utilization from triglycerides to phospholipids by inhibiting DGAT . This increase in phospholipid synthesis might play a role in the restoration of enterocyte absorption function after intestinal mucosa injury. Mol Biol Cell, 1999 Mar, 10(3), 665 - 76 The DNA helicase activity of BLM is necessary for the correction of the genomic instability of bloom syndrome cells; Neff NF et al.; Bloom syndrome (BS) is a rare autosomal recessive disorder characterized by growth deficiency, immunodeficiency, genomic instability, and the early development of cancers of many types . BLM, the protein encoded by BLM, the gene mutated in BS, is localized in nuclear foci and absent from BS cells . BLM encodes a DNA helicase, and proteins from three missense alleles lack displacement activity . BLM transfected into BS cells reduces the frequency of sister chromatid exchanges and restores BLM in the nucleus . Missense alleles fail to reduce the sister chromatid exchanges in transfected BS cells or restore the normal nuclear pattern . BLM complements a phenotype of a Saccharomyces cerevisiae sgs1 top3 strain, and the missense alleles do not . This work demonstrates the importance of the enzymatic activity of BLM for its function and nuclear localization pattern. Mol Biol Cell, 1999 Mar, 10(3), 567 - 80 OBA/Ku86: DNA binding specificity and involvement in mammalian DNA replication; Ruiz MT et al.; Ors-binding activity (OBA) was previously semipurified from HeLa cells through its ability to interact specifically with the 186-basepair (bp) minimal replication origin of ors8 and support ors8 replication in vitro . Here, through competition band-shift analyses, using as competitors various subfragments of the 186-bp minimal ori, we identified an internal region of 59 bp that competed for OBA binding as efficiently as the full 186-bp fragment . The 59-bp fragment has homology to a 36-bp sequence (A3/4) generated by comparing various mammalian replication origins, including the ors . A3/4 is, by itself, capable of competing most efficiently for OBA binding to the 186-bp fragment . Band-shift elution of the A3/4-OBA complex, followed by Southwestern analysis using the A3/4 sequence as probe, revealed a major band of approximately 92 kDa involved in the DNA binding activity of OBA . Microsequencing analysis revealed that the 92-kDa polypeptide is identical to the 86-kDa subunit of human Ku antigen . The affinity-purified OBA fraction obtained using an A3/4 affinity column also contained the 70-kDa subunit of Ku and the DNA-dependent protein kinase catalytic subunit . In vitro DNA replication experiments in the presence of A3/4 oligonucleotide or anti-Ku70 and anti-Ku86 antibodies implicate Ku in mammalian DNA replication. Food Chem Toxicol, 1999 Jan, 37(1), 13 - 22 Safety evaluation of phytosterol esters . Part 1 . Assessment of oestrogenicity using a combination of in vivo and in vitro assays; Baker VA et al.; Phytosterols are natural constituents of the human diet, and as part of an extensive programme of safety evaluation studies investigating their use as a novel food ingredient, the possible oestrogenic effects of phytosterols have been investigated using a combination of in vitro and in vivo assays . Competitive binding with the immature rat uterine oestrogen receptor (ER) has been used to measure the ability of phytosterols to bind to ERs while the transcriptional activation of oestrogen-responsive genes has been examined in an oestrogen-inducible yeast screen . Phytosterols did not display any activity in these in vitro assays . Uterotrophic assays have been conducted to investigate the potential for phytosterols to elicit an oestrogenic response when administered orally to immature female rats (n = 10) at doses of 0, 5, 50 or 500 mg/kg/day for 3 consecutive days . Phytosterols (a well characterized mixture of beta-sitosterol, campesterol and stigmasterol) and phytosterol esters (the previous phytosterol mixture esterified with fatty acids from sunflower oil) did not exhibit oestrogenic activity in the immature female rat using uterine wet weight as the endpoint . Beta-oestradiol (0.4 mg/kg/day) consistently produced a significant increase in uterus weights . Coumestrol (a known phytoestrogen) was also tested as a weak positive control and produced a dose response at doses of 20, 40 and 80 mg/kg/day in the uterotrophic assay . In conclusion, we have shown that phytosterols do not bind to the ER and do not stimulate transcriptional activity of the human ER in a recombinant yeast strain . In addition, there was no indication of oestrogenicity from the uterotrophic assay when the material was administered by oral gavage to immature female rats. Carcinogenesis, 1999 Feb, 20(2), 215 - 20 MSH3 deficiency is not sufficient for a mutator phenotype in Chinese hamster ovary cells; Hinz JM et al.; In the yeast Saccharomyces cerevisiae, the mutS homolog protein products MSH3 and MSH6, each in cooperation with MSH2, play well-defined and specific roles in the repair of DNA mismatches and nucleotide loops . The discrete functions of the human homologs hMSH3 and hMSH6 are less clear and current evidence suggests that the substrate specificity of these proteins may be less strict . To de