S. cerevisiae

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 determine the role of MSH3 in mammalian mismatch repair, we employed MSH3-deficient Chinese hamster ovary (CHO) cell lines . No significant changes in mutation rate were detected in the MSH3-deficient strain and there were no differences in sensitivity to DNA-damaging agents . Further analysis of hprt mutants did not show a MSH3-dependent shift in the mutant spectrum . Interestingly, thorough examination of four dinucleotide microsatellite regions revealed instability at only one locus in one of the MSH3-deficient cell lines . These data support the idea of a high degree of redundancy in the function of the MutS homologs MSH3 and MSH6, at least with respect to the control of microsatellite instability.

Dev Biol, 1999 Mar 15, 207(2), 322 - 33
Histone ubiquitination and chromatin remodeling in mouse spermatogenesis; Baarends WM et al.; Male infertility in HR6B knockout mice is associated with impairment of spermatogenesis . The HR6B gene is a mammalian, autosomal homolog of the Saccharomyces cerevisiae gene Rad6 encoding a ubiquitin-conjugating enzyme . In addition, X-chromosomal HR6A has been identified, in human and mouse . RAD6 in yeast is required for a variety of cellular functions, including sporulation, DNA repair, and mutagenesis . Since RAD6 and its mammalian homologs can ubiquitinate histones in vitro, we have investigated the pattern of histone ubiquitination in mouse testis . By immunoblot and immunohistochemical analysis of wild-type mouse testis, a high amount of ubiquitinated H2A (uH2A) was detected in pachytene spermatocytes . This signal became undetectable in round spermatids, but then increased again during a relatively short developmental period, in elongating spermatids . No other ubiquitinated histones were observed . In the HR6B knockout mice, we failed to detect an overt defect in the overall pattern of histone ubiquitination . For somatic cell types, it has been shown that histone ubiquitination is associated with destabilization of nucleosomes, in relation to active gene transcription . Unexpectedly, the most intense uH2A signal in pachytene spermatocytes was detected in the sex body, an inactive nuclear structure that contains the heterochromatic X and Y chromosomes . The postmeiotic uH2A immunoexpression in elongating spermatids indicates that nucleosome destabilization induced by histone ubiquitination may play a facilitating role during histone-to-protamine replacement .

Mol Immunol, 1998 Nov, 35(16), 1057 - 67
The mouse genome contains two expressed intronless retroposed pseudogenes for the sentrin/sumo-1/PIC1 conjugating enzyme Ubc9; Tsytsykova AV et al.; The ubiquitin conjugating (ubc) E2 enzyme ubc-9 conjugates the ubiquitin-like peptide sentrin/SUMO-1/PIC1 to target proteins which include the Fas antigen . We show that the mouse genome contains four copies of the ubc-9 gene . These include a structural ubc-9 gene consisting of seven exons which encode a protein identical to human ubc-9, and three intronless processed pseudogenes . The open reading frames (ORF) of two of the pseudogenes, ubc9-psi1 and ubc9-psi2, correspond to the cDNA of ubc-9 and encode for proteins which differ from ubc9 by three and one amino acid substitutions respectively . The third pseudogene, ubc9-psi3, contains many mutations and stop codons . ubc9-psi1 and ubc9-psi2 are flanked by 5'- and 3'-untranslated (UT) regions homologous to those of the structural ubc-9 gene . Both genes contain a polyA tail and direct repeats at both ends suggesting that they arose by mRNA retroposition . Both ubc9-psi1 and ubc9-psi2 are transcribed into mRNA in murine cells . In contrast to ubc9, the protein products of ubc9-psil and ubc9-psi2 fail to bind Fas and to complement an yeast conditional ubc9 mutant . These results suggest that ubc9-psi1 and ubc9-psi2 encode for proteins that may interact with targets that differ from those recognized by ubc-9.

Microbiol Mol Biol Rev, 1999 Mar, 63(1), 54 - 105
Cdc42: An essential Rho-type GTPase controlling eukaryotic cell polarity; Johnson DI; Cdc42p is an essential GTPase that belongs to the Rho/Rac subfamily of Ras-like GTPases . These proteins act as molecular switches by responding to exogenous and/or endogenous signals and relaying those signals to activate downstream components of a biological pathway . The 11 current members of the Cdc42p family display between 75 and 100% amino acid identity and are functional as well as structural homologs . Cdc42p transduces signals to the actin cytoskeleton to initiate and maintain polarized gorwth and to mitogen-activated protein morphogenesis . In the budding yeast Saccharomyces cerevisiae, Cdc42p plays an important role in multiple actin-dependent morphogenetic events such as bud emergence, mating-projection formation, and pseudohyphal growth . In mammalian cells, Cdc42p regulates a variety of actin-dependent events and induces the JNK/SAPK protein kinase cascade, which leads to the activation of transcription factors within the nucleus . Cdc42p mediates these processes through interactions with a myriad of downstream effectors, whose number and regulation we are just starting to understand . In addition, Cdc42p has been implicated in a number of human diseases through interactions with its regulators and downstream effectors . While much is known about Cdc42p structure and functional interactions, little is known about the mechanism(s) by which it transduces signals within the cell . Future research should focus on this question as well as on the detailed analysis of the interactions of Cdc42p with its regulators and downstream effectors.

J Biol Chem, 1999 Mar 12, 274(11), 7576 - 82
Identification of determinants in E2 ubiquitin-conjugating enzymes required for hect E3 ubiquitin-protein ligase interaction; Nuber U et al.; Members of the hect domain protein family are characterized by sequence similarity of their C-terminal regions to the C terminus of E6-AP, an E3 ubiquitin-protein ligase . An essential intermediate step in E6-AP-dependent ubiquitination is the formation of a thioester complex between E6-AP and ubiquitin in the presence of distinct E2 ubiquitin-conjugating enzymes including human UbcH5, a member of the UBC4/UBC5 subfamily of E2s . Similarly, several hect domain proteins, including Saccharomyces cerevisiae RSP5, form ubiquitin thioester complexes, indicating that hect domain proteins in general have E3 activity . We show here, by the use of chimeric E2s generated between UbcH5 and other E2s, that a region of UbcH5 encompassing the catalytic site cysteine residue is critical for its ability to interact with E6-AP and RSP5 . Of particular importance is a phenylalanine residue at position 62 of UbcH5 that is conserved among the members of the UBC4/UBC5 subfamily but is not present in any of the other known E2s, whereas the N-terminal 60 amino acids do not contribute significantly to the specificity of these interactions . The conservation of this phenylalanine residue throughout evolution underlines the importance of the ability to interact with hect domain proteins for the cellular function of UBC4/UBC5 subfamily members.

J Biol Chem, 1999 Mar 12, 274(11), 7405 - 11
Molecular cloning and biochemical characterization of a novel anthocyanin 5-O-glucosyltransferase by mRNA differential display for plant forms regarding anthocyanin; Yamazaki M et al.; UDP-glucose: anthocyanin 5-O-glucosyltransferase (5-GT) is responsible for the modification of anthocyanins to more stable molecules in complexes for co-pigmentation, supposedly resulting in a purple hue . The cDNA encoding 5-GT was isolated by a differential display applied to two different forms of anthocyanin production in Perilla frutescens var . crispa . Differential display was carried out for mRNA from the leaves of reddish-purple and green forms of P . frutescens, resulting in the isolation of five cDNA clones predominantly expressed in the red form . The cDNA encoded a polypeptide of 460 amino acids, exhibiting a low homology with the sequences of several glucosyltransferases including UDP-glucose: anthocyanidin 3-O-glucosyltransferase . By using this cDNA as the probe, we also isolated a homologous cDNA clone from a petal cDNA library of Verbena hybrida . To identify the biochemical function of the encoded proteins, these cDNAs were expressed in Saccharomyces cerevisiae cells . The recombinant proteins in the yeast extracts catalyzed the conversion of anthocyanidin 3-O-glucosides into the corresponding anthocyanidin 3,5-di-O-glucosides using UDP-glucose as a cofactor, indicating the identity of the cDNAs encoding 5-GT . Several biochemical properties (optimum pH, Km values, and sensitivity to inhibitors) were similar to those reported previously for 5-GTs . Southern blot analysis indicated the presence of two copies of 5-GT genes in the genome of both red and green forms of P . frutescens . The mRNA accumulation of the 5-GT gene was detected in the leaves of the red form but not in those of the green form and was induced by illumination of light, as observed for other structural genes for anthocyanin biosynthesis in P . frutescens.

J Biol Chem, 1999 Mar 12, 274(11), 7334 - 40
Cell cycle-dependent expression and centrosome localization of a third human aurora/Ipl1-related protein kinase, AIK3; Kimura M et al.; We earlier isolated cDNAs encoding novel human protein kinases AIK and AIK2 sharing high amino acid sequence identities with Drosophila Aurora and Saccharomyces cerevisiae Ipl1 kinases whose mutations cause abnormal chromosome segregation . In the present study, a third human cDNA (AIK3) highly homologous to aurora/IPL1 was isolated, and the nucleotide sequence was determined . This cDNA encodes 309 amino acids with a predicted molecular mass of 35.9 kDa . C-terminal kinase domain of AIK3 protein shares high amino acid sequence identities with those of Aurora/Ipl1 family protein kinases including human AIK, human AIK2, Xenopus pEg2, Drosophila Aurora, and yeast Ipl1, whereas the N-terminal domain of AIK3 protein shares little homology with any other Aurora/Ipl1 family members . AIK3 gene was assigned to human chromosome 19q13.43, which is a frequently deleted or rearranged region in several tumor tissues, by fluorescence in situ hybridization, somatic cell hybrid panel, and radiation hybrid cell panel . Northern blot analyses revealed that AIK3 expression was limited to testis . The expression levels of AIK3 in several cancer cell lines were elevated severalfold compared with normal fibroblasts . In HeLa cells, the endogenous AIK3 protein level is low in G1/S, accumulates during G2/M, and reduces after mitosis . Immunofluorescence studies using a specific antibody have shown that AIK3 is localized to centrosome during mitosis from anaphase to cytokinesis . These results suggest that AIK3 may play a role(s) in centrosome function at later stages of mitosis.

J Biol Chem, 1999 Mar 12, 274(11), 7302 - 8
The topoisomerase-related function gene TRF4 affects cellular sensitivity to the antitumor agent camptothecin; Walowsky C et al.; Camptothecin is an antitumor agent that kills cells by converting DNA topoisomerase I into a DNA-damaging poison . Although camptothecin derivatives are now being used to treat tumors in a variety of clinical protocols, the cellular factors that influence sensitivity to the drug are only beginning to be understood . We report here that two genes required for sister chromatid cohesion, TRF4 and MCD1/SCC1, are also required to repair camptothecin-mediated damage to DNA . The hypersensitivity to camptothecin in the trf4 mutant does not result from elevated expression of DNA topoisomerase I . We show that Trf4 is a nuclear protein whose expression is cell cycle-regulated at a post-transcriptional level . Suppression of camptothecin hypersensitivity in the trf4 mutant by gene overexpression resulted in the isolation of three genes: another member of the TRF4 gene family, TRF5, and two genes that may influence higher order chromosome structure, ZDS1 and ZDS2 . We have isolated and sequenced two human TRF4 family members, hTRF4-1 and hTRF4-2 . The hTRF4-1 gene maps to chromosome 5p15, a region of frequent copy number alteration in several tumor types . The evolutionary conservation of TRF4 suggests that it may also influence mammalian cell sensitivity to camptothecin.

Curr Opin Plant Biol, 1998 Dec, 1(6), 470 - 4
Regulated nuclear targeting; Nagatani A; Extracellular signals are transduced to the nucleus through respective signal transduction pathways . Evidence in animals and yeast indicates the importance of regulated nuclear targeting in these processes . Although little is known about plants in this regard, some plant signaling factors have recently been shown to translocate to the nucleus upon receipt of a signal.

Curr Opin Plant Biol, 1998 Dec, 1(6), 458 - 62
Meiosis: vive la difference!
Shaw P, Moore G.
The application of molecular and modern cell biological techniques is beginning to show that the classical picture of the meiotic process is an oversimplification . The comparison of different species and analysis of mutants have recently demonstrated that many of the features previously thought to be an integral part of meiosis can be altered in their timing or even entirely dispensed with . In plants, differences in the meiotic pathway have been observed, by using methods for 3-D optical imaging, between the polyploids maize and wheat . The fact that two such closely related species differ may be the result of different mechanisms for dealing with polyploidy or may be species differences, but shows that a detailed understanding of the process in the particular species of interest is necessary.

Curr Opin Plant Biol, 1998 Aug, 1(4), 299 - 304
Genetic dissection of R gene signal transduction pathways; Innes RW; Mutant screens have identified several genes in tomato, barley and Arabidopsis that are required for the function of specific plant disease resistance (R) genes . Two of these genes, NDR1 and EDS1, have recently been cloned from Arabidopsis . Most Arabidopsis R genes require NDR1 or EDS1, but not both . In a complementary approach, yeast two-hybrid screens have identified several proteins in tomato that interact with the Pto R gene protein, including a kinase and three putative transcription factors . The present data indicate that R gene proteins directly activate multiple signal transduction pathways, and that common defense responses can be activated via independent pathways.

Curr Opin Plant Biol, 1998 Jun, 1(3), 267 - 74
Plant stress adaptations--making metabolism move; Bohnert HJ et al.; Persistently sub-optimal environmental conditions constitute stress . Perception and signaling lead to protein expression changes, the activation of new biochemical pathways, and repression of others which are characteristic of the unstressed state . Protective metabolic adaptations alter physiological reactions of the whole plant . Paramount among the mechanisms are oxygen radical scavenging, maintenance of ion uptake and water balance, and reactions altering carbon and nitrogen allocation, such that reducing power is defused . Elements of the stress signaling pathways and proteins that lead to stress protection have recently become known.

Curr Opin Microbiol, 1998 Aug, 1(4), 406 - 10
Green fluorescent protein as a reporter of transcription and protein localization in fungi; Cormack B; Green fluorescent protein (GFP) is a versatile and powerful tool for analysis of diverse biological processes . The recent development of GFP variants with altered spectral properties and altered codon composition has allowed efficient expression of GFP in a number of fungal species . GFP has been successfully used to analyze transcription regulation as well as protein and organelle localization, and promises to give an unprecedented view into the dynamic subcellular processes that shape the fungal cell.

Biochem Biophys Res Commun, 1999 Mar 5, 256(1), 45 - 51
Recruitment of TBP or TFIIB to a promoter proximal position leads to stimulation of RNA polymerase II transcription without activator proteins both in vivo and in vitro; Huh JR et al.; Eukaryotic transcriptional activators may function, at least in part, to facilitate the assembly of the RNA polymerase II (pol II) preinitiation complex at the core promoter region through their interaction with a subset of components of the basal transcription machinery . Previous studies have shown that artificial tethering of TATA-binding protein (TBP) to the promoter region is sufficient to stimulate pol II transcription in yeast . To test whether this phenomenon is a general one in eukaryotic pol II transcription, the DNA-binding domain of yeast GAL4 was fused to either Xenopus laevis TBP or TFIIB in order to enable these factors to be efficiently positioned near the transcription start site in a GAL4-binding site-dependent manner . We found that GAL4-xTBP as well as GAL4-xTFIIB directed an increased level of transcription without involvement of the transcriptional activator, suggesting that incorporation of these basal factors into a preinitiation complex (PIC) is a major rate-limiting step accelerated by activator proteins in metazoans . These results show that transcription activation by artificial recruitment of basal transcription machinery can be observed in general among eukaryotic transcription both in vivo and in vitro . Furthermore, failure of recovery of transcription by adding GAL4-xTFIIB after depletion of endogenous TBP with TATA oligo competitor suggests that recruitment of TBP cannot be bypassed for Pol II transcription .

Exp Cell Res, 1999 Mar 15, 247(2), 554 - 62
Association of human SCF(SKP2) subunit p19(SKP1) with interphase centrosomes and mitotic spindle poles; Gstaiger M et al.; In Saccharomyces cerevisiae, the initiation of DNA replication and mitotic progression requires SKP1p function . SKP1p is an essential subunit of a newly identified class of E3 ubiquitin protein ligases, the SCF complexes, that catalyze ubiquitin-mediated proteolysis of key cell-cycle-regulatory proteins at distinct times in the cell cycle . SKP1p is also required for proper kinetochore assembly . Little is known about the corresponding human homolog, p19(SKP1), except that it is expressed throughout the cell cycle and that it too is a component of an S-phase-regulating SCF-E3 ligase complex . Here we show by immunofluorescence microscopy that p19(SKP1) localizes to the centrosomes . Centrosome association occurs throughout the mammalian cell cycle, including all stages of mitosis . These findings suggest that p19(SKP1) is a novel component of the centrosome and the mitotic spindle, which, in turn, implies a physiological role of this protein in the regulation of one or more aspects of the centrosome cycle .

Science, 1999 Mar 5, 283(5407), 1493 - 7
The machinery of mitochondrial inheritance and behavior; Yaffe MP; The distribution of mitochondria to daughter cells during cell division is an essential feature of cell proliferation . Until recently, it was commonly believed that inheritance of mitochondria and other organelles was a passive process, a consequence of their random diffusion throughout the cytoplasm . A growing recognition of the reticular morphology of mitochondria in many living cells, the association of mitochondria with the cytoskeleton, and the coordinated movements of mitochondria during cellular division and differentiation has illuminated the necessity for a cellular machinery that mediates mitochondrial behavior . Characterization of the underlying molecular components of this machinery is providing insight into mechanisms regulating mitochondrial morphology and distribution.

EMBO J, 1999 Mar 1, 18(5), 1397 - 406
Replication-mediated DNA damage by camptothecin induces phosphorylation of RPA by DNA-dependent protein kinase and dissociates RPA:DNA-PK complexes; Shao RG et al.; Replication protein A (RPA) is a DNA single-strand binding protein essential for DNA replication, recombination and repair . In human cells treated with the topoisomerase inhibitors camptothecin or etoposide (VP-16), we find that RPA2, the middle-sized subunit of RPA, becomes rapidly phosphorylated . This response appears to be due to DNA-dependent protein kinase (DNA-PK) and to be independent of p53 or the ataxia telangiectasia mutated (ATM) protein . RPA2 phosphorylation in response to camptothecin required ongoing DNA replication . Camptothecin itself partially inhibited DNA synthesis, and this inhibition followed the same kinetics as DNA-PK activation and RPA2 phosphorylation . DNA-PK activation and RPA2 phosphorylation were prevented by the cell-cycle checkpoint abrogator 7-hydroxystaurosporine (UCN-01), which markedly potentiates camptothecin cytotoxicity . The DNA-PK catalytic subunit (DNA-PKcs) was found to bind RPA which was replaced by the Ku autoantigen upon camptothecin treatment . DNA-PKcs interacted directly with RPA1 in vitro . We propose that the encounter of a replication fork with a topoisomerase-DNA cleavage complex could lead to a juxtaposition of replication fork-associated RPA and DNA double-strand end-associated DNA-PK, leading to RPA2 phosphorylation which may signal the presence of DNA damage to an S-phase checkpoint mechanism . Keywords: camptothecin/DNA damage/DNA-dependent protein kinase/RPA2 phosphorylation

EMBO J, 1999 Mar 1, 18(5), 1137 - 45
Physical interactions among circadian clock proteins KaiA, KaiB and KaiC in cyanobacteria; Iwasaki H et al.; The kai gene cluster, which is composed of three genes, kaiA, kaiB and kaiC, is essential for the generation of circadian rhythms in the unicellular cyanobacterium Synechococcus sp . strain PCC 7942 . Here we demonstrate the direct association of KaiA, KaiB and KaiC in yeast cells using the two-hybrid system, in vitro and in cyanobacterial cells . KaiC enhanced KaiA-KaiB interaction in vitro and in yeast cells, suggesting that the three Kai proteins were able to form a heteromultimeric complex . We also found that a long period mutation kaiA1 dramatically enhanced KaiA-KaiB interaction in vitro . Thus, direct protein-protein association among the Kai proteins may be a critical process in the generation of circadian rhythms in cyanobacteria.

Cell, 1999 Feb 19, 96(4), 575 - 85
Replication-dependent marking of DNA by PCNA facilitates CAF-1-coupled inheritance of chromatin; Shibahara K et al.; Chromatin assembly factor 1 (CAF-1) is required for inheritance of epigenetically determined chromosomal states in vivo and promotes assembly of chromatin during DNA replication in vitro . Herein, we demonstrate that after DNA replication, replicated, but not unreplicated, DNA is also competent for CAF-1-dependent chromatin assembly . The proliferating cell nuclear antigen (PCNA), a DNA polymerase clamp, is a component of the replication-dependent marking of DNA for chromatin assembly . The clamp loader, replication factor C (RFC), can reverse this mark by unloading PCNA from the replicated DNA . PCNA binds directly to p150, the largest subunit of CAF-1, and the two proteins colocalize at sites of DNA replication in cells . We suggest that PCNA and CAF-1 connect DNA replication to chromatin assembly and the inheritance of epigenetic chromosome states.

Proc Natl Acad Sci U S A, 1999 Mar 2, 96(5), 2116 - 21
Human PEX19: cDNA cloning by functional complementation, mutation analysis in a patient with Zellweger syndrome, and potential role in peroxisomal membrane assembly; Matsuzono Y et al.; At least 11 complementation groups (CGs) have been identified for the peroxisome biogenesis disorders (PBDs) such as Zellweger syndrome, for which seven pathogenic genes have been elucidated . We have isolated a human PEX19 cDNA (HsPEX19) by functional complementation of peroxisome deficiency of a mutant Chinese hamster ovary cell line, ZP119, defective in import of both matrix and membrane proteins . This cDNA encodes a hydrophilic protein (Pex19p) comprising 299 amino acids, with a prenylation motif, CAAX box, at the C terminus . Farnesylated Pex19p is partly, if not all, anchored in the peroxisomal membrane, exposing its N-terminal part to the cytosol . A stable transformant of ZP119 with HsPEX19 was morphologically and biochemically restored for peroxisome biogenesis . HsPEX19 expression also restored peroxisomal protein import in fibroblasts from a patient (PBDJ-01) with Zellweger syndrome of CG-J . This patient (PBDJ-01) possessed a homozygous, inactivating mutation: a 1-base insertion, A764, in a codon for Met255, resulted in a frameshift, inducing a 24-aa sequence entirely distinct from normal Pex19p . These results demonstrate that PEX19 is the causative gene for CG-J PBD and suggest that the C-terminal part, including the CAAX homology box, is required for the biological function of Pex19p . Moreover, Pex19p is apparently involved at the initial stage in peroxisome membrane assembly, before the import of matrix protein.

Proc Natl Acad Sci U S A, 1999 Mar 2, 96(5), 2104 - 9
Immunophilins, Refsum disease, and lupus nephritis: the peroxisomal enzyme phytanoyl-COA alpha-hydroxylase is a new FKBP-associated protein; Chambraud B et al.; FKBP52 (FKBP59, FKBP4) is a "macro" immunophilin that, although sharing high structural and functional homologies in its amino-terminal domain with FKBP12 (FKBP1), does not have immunosuppressant activity when complexed with FK506, unlike FKBP12 . To investigate the physiological function of FKBP52, we used the yeast two-hybrid system as an approach to find its potential protein partners and, from that, its cellular role . This methodology, which already has allowed us to find the FK506-binding protein (FKBP)-associated protein FAP48, also led to the detection of another FKBP-associated protein . Determination of the sequence of this protein permitted its identification as phytanoyl-CoA alpha-hydroxylase (PAHX), a peroxisomal enzyme that so far was unknown as an FKBP-associated protein . Inactivation of this enzyme is responsible for Refsum disease in humans . The protein also corresponds to the mouse protein LN1, which could be involved in the progress of lupus nephritis . We show here that PAHX has the physical capacity to interact with the FKBP12-like domain of FKBP52, but not with FKBP12, suggesting that it is a particular and specific target of FKBP52 . Whereas the binding of calcineurin to FKBP12 is potentiated by FK506, the specific association of PAHX and FKBP52 is maintained in the presence of FK506 . This observation suggests that PAHX is a serious candidate for studying the cellular signaling pathway(s) involving FKBP52 in the presence of immunosuppressant drugs.

Proc Natl Acad Sci U S A, 1999 Mar 2, 96(5), 1881 - 5
Interaction of 5-lipoxygenase with cellular proteins; Provost P et al.; 5-Lipoxygenase (5LO) plays a pivotal role in cellular leukotriene synthesis . To identify proteins interacting with human 5LO, we used a two-hybrid approach to screen a human lung cDNA library . From a total of 1.5 x 10(7) yeast transformants, nine independent clones representing three different proteins were isolated and found to specifically interact with 5LO . Four 1.7- to 1.8-kb clones represented a 16-kDa protein named coactosin-like protein for its significant homology with coactosin, a protein found to be associated with actin in Dictyostelium discoideum . Coactosin-like protein thus may provide a link between 5LO and the cytoskeleton . Two other yeast clones of 1.5 kb encoded transforming growth factor (TGF) type beta receptor-I-associated protein 1 partial cDNA . TGF type beta receptor-I-associated protein 1 recently has been reported to associate with the activated form of the TGF beta receptor I and may be involved in the TGF beta-induced up-regulation of 5LO expression and activity observed in HL-60 and Mono Mac 6 cells . Finally, three identical 2.1-kb clones contained the partial cDNA of a human protein with high homology to a hypothetical helicase K12H4 . 8 from Caenorhabditis elegans and consequently was named DeltaK12H4 . 8 homologue . Analysis of the predicted amino acid sequence revealed the presence of a RNase III motif and a double-stranded RNA binding domain, indicative of a protein of nuclear origin . The identification of these 5LO-interacting proteins provides additional approaches to studies of the cellular functions of 5LO.

Proc Natl Acad Sci U S A, 1999 Mar 2, 96(5), 1869 - 74
Studies on the interactions between human replication factor C and human proliferating cell nuclear antigen; Zhang G et al.; Proliferating cell nuclear antigen (PCNA) is a processivity factor required for DNA polymerase delta (or epsilon)-catalyzed DNA synthesis . When loaded onto primed DNA templates by replication factor C (RFC), PCNA acts to tether the polymerase to DNA, resulting in processive DNA chain elongation . In this report, we describe the identification of two separate peptide regions of human PCNA spanning amino acids 36-55 and 196-215 that bind RFC by using the surface plasmon resonance technique . Site-directed mutagenesis of residues within these regions in human PCNA identified two specific sites that affected the biological activity of PCNA . Replacement of the aspartate 41 residue by an alanine, serine, or asparagine significantly impaired the ability of PCNA to (i) support the RFC/PCNA-dependent polymerase delta-catalyzed elongation of a singly primed DNA template; (ii) stimulate RFC-catalyzed DNA-dependent hydrolysis of ATP; (iii) be loaded onto DNA by RFC; and (iv) activate RFC-independent polymerase delta-catalyzed synthesis of poly dT . Introduction of an alanine at position 210 in place of an arginine also reduced the efficiency of PCNA in supporting RFC-dependent polymerase delta-catalyzed elongation of a singly primed DNA template . However, this mutation did not significantly alter the ability of PCNA to stimulate DNA polymerase delta in the absence of RFC but substantially lowered the efficiency of RFC-catalyzed reactions . These results are in keeping with a model in which surface exposed regions of PCNA interact with RFC and the subsequent loading of PCNA onto DNA orients the elongation complex in a manner essential for processive DNA synthesis.

Mamm Genome, 1999 Mar, 10(3), 266 - 70
Molecular cloning and chromosomal assignment of the porcine 54 and 56 kDa vacuolar H(+)-ATPase subunit gene (V-ATPase); Hui D et al.; Vacuolar proton-translocating ATPases (V-ATPase) are multisubunit enzyme complexes located in the membranes of eukaryotic cells regulating cytoplasmic pH . So far, nothing is known about the genomic organization and chromosomal location of the various subunit genes in higher eukaryotes . Here we describe the isolation and analysis of a cDNA coding for the 54- and 56-kDa porcine V-ATPase subunit alpha and beta isoforms . We have determined the genomic structure of the V-ATPase subunit gene spanning at least 62 kb on Chromosome (Chr) 4q14-q16 . It consists of 14 exons with sizes ranging from 54 bp to 346 bp, with a non-coding first exon and an alternatively spliced seventh exon leading to two isoforms . The 5' end of the V-ATPase cDNA was isolated by RACE-PCR . The V-ATPase alpha isoform mRNA, lacking the seventh exon, has an open reading frame of 1395 nucleotides encoding a hydrophilic protein of 465 amino acids with a calculated molecular mass of 54.2 kDa and a pI of 7.8, whereas the beta isoform has a length of 1449 nucleotides encoding a protein of 483 amino acids with a calculated molecular mass of 55.8 kDa . Amino acid and DNA sequence comparison revealed that the porcine V-ATPase subunit exhibits a significant homology to the VMA13 subunit of Saccharomyces cerevisiae V-ATPase complex and V-ATPase subunit of Caenorhabditis elegans.

Appl Environ Microbiol, 1999 Mar, 65(3), 1168 - 74
Thaumatin production in Aspergillus awamori by use of expression cassettes with strong fungal promoters and high gene dosage; Moralejo FJ et al.; Four expression cassettes containing strong fungal promoters, a signal sequence for protein translocation, a KEX protease cleavage site, and a synthetic gene (tha) encoding the sweet protein thaumatin II were used to overexpress this protein in Aspergillus awamori lpr66, a PepA protease-deficient strain . The best expression results were obtained with the gdhA promoter of A . awamori or with the gpdA promoter of Aspergillus nidulans . There was good correlation of tha gene dosage, transcript levels, and thaumatin secretion . The thaumatin gene was expressed as a transcript of the expected size in each construction (1.9 or 1.4 kb), and the transcript levels and thaumatin production rate decayed at the end of the growth phase, except in the double transformant TB2b1-44-GD5, in which secretion of thaumatin continued until 96 h . The recombinant thaumatin secreted by a high-production transformant was purified to homogeneity, giving one major component and two minor components . In all cases, cleavage of the fused protein occurred at the KEX recognition sequence . This work provides new expression systems in A . awamori that result in very high levels of thaumatin production.

Arch Biochem Biophys, 1999 Mar 1, 363(1), 116 - 20
The significance of amino acid residue Asp446 for enzymatic stability of rat UDP-glucuronosyltransferase UGT1A6; Iwano H et al.; Asp446 in rat UDP-glucuronosyltransferase (UGT), UGT1A6, is an essential amino acid residue for its enzymatic activity (H . Iwano et al . Biochem . J . 325, 587-591, 1997) . The role of Asp446 in UGT1A6 was investigated by comparing some properties of UGT mutant proteins that have a single mutation (D446K, D446E, D446N, D446Q, D446A, and D446T) . These mutants, except D446K, had catalytic activities toward 1-naphthol and 4-methylumbelliferone . The UGT activities of D446E and D446N were about half of that of the wild type, and the activities of the other mutants were only about 1/5-1/10 of that of the wild type . The Km values for 1-naphthol of these mutants were similar to that of the wild type, while the values for UDP-glucuronic acid were slightly higher . The mutants were unstable in a low-pH buffer solution and were dramatically inactivated by heat treatments . Interestingly, after 30 min of treatment at 37 degrees C in the presence of UDP-glucuronic acid, the UGT activities of all functional mutants were elevated . These results suggest that Asp446 is an indispensable residue for folding a functional conformation of rat UGT1A6 by cooperation with UDP-glucuronic acid .

Nat Struct Biol, 1999 Feb, 6(2), 117 - 23
Folding intermediates of SNARE complex assembly; Fiebig KM et al.; SNARE (soluble NSF attachment protein receptor) proteins assemble into a stable complex essential for vesicle-membrane fusion . To further understand SNARE function we have used solution nuclear magnetic resonance (NMR) spectroscopy to characterize three assembly states of a yeast SNARE complex: first, the 'closed' conformation of Sso1; second, the binary complex of Sso1 and Sec9; and third, the ternary complex of Sso1, Sec9 and Snc1 . Sec9 and Snc1 are unstructured in isolation . Sso1 likely consists of a four helix bundle formed by part of the C-terminal Hcore domain and the N-terminal H(A)H(B)H(C) domain, and this bundle is flanked on both sides by large flexible regions . Sso1 switches to an 'open' state when its Hcore domain binds Sec9 . Conformational switching of the Hcore domain, via H(A)H(B)H(C), may provide a key regulatory mechanism in SNARE assembly . Formation of binary and ternary complexes induces additional alpha-helical structure in previously unstructured regions . Our data suggest a directed assembly process beginning distal to the membrane surfaces and proceeding toward them, bringing membranes into close proximity and possibly leading to membrane fusion.

Toxicol Lett, 1998 Nov 23, 100-101, 293 - 300
Baseline K+ channels as targets of general anesthetics: studies of the action of volatile anesthetics on TOK1; Yost CS et al.; A large body of evidence has accumulated in recent years pointing towards the GABA(A) receptor as a primary determinant of volatile anesthetic action (Franks and Lieb, 1994) . Nevertheless, our understanding of the function of the central nervous system (CNS) remains sufficiently incomplete that other mechanisms of CNS depression remain to be examined . We have studied a new family of potassium (K+) channels which function as regulators of the baseline excitability of neuronal tissue . As such they must be considered potential targets for volatile anesthetic action and as a possible mechanism by which volatile anesthetics act to allow patients to undergo noxious surgical stimulation.

J Antibiot (Tokyo), 1998 Dec, 51(12), 1069 - 74
Novel selective inhibitors for human topoisomerase I, BM2419-1 and -2 derived from saintopin; Ishiyama D et al.; Compounds BM2419-1 and -2 were isolated from a culture broth of a fungus Paecilomyces sp . BM2419 . It was shown that these novel compounds were artifacts derived from saintopin, a dual inhibitor of topoisomerase I and II by independent processes . In the human topoisomerase I inhibition assay using the recombinant Saccharomyces cerevisiae, BM2419-1 and -2 inhibited selectively the yeast growth dependent on human topoisomerase I induction with IC50 values of 0.3 ng/ml and 6.0 ng/ml, respectively.

Hum Gene Ther, 1999 Feb 10, 10(3), 341 - 53
Keratinocyte growth factor stimulates transduction of the respiratory epithelium by retroviral vectors; Zsengeller ZK et al.; Cell proliferation is required for transduction by standard retrovirus vectors derived from viruses in the murine leukemia virus (MuLV) group . Since proliferation rates are low in the mature pulmonary epithelium, we tested the hypothesis that the efficiency of retrovirus-mediated transduction of respiratory epithelial cells can be enhanced by stimulation of cell proliferation with recombinant human keratinocyte growth factor (rhKGF) . A marked increase in proliferation of bronchiolar and alveolar epithelial cells was observed after intratracheal administration of rhKGF (30 mg/kg) to adult FVB/N mice . Two days after rhKGF or saline treatment, 10(7) AP+ FFU of LAPSN, a recombinant amphotropic retrovirus that expresses human placental alkaline phosphatase (AP), was instilled intratracheally into the mice . Transduction efficiency, measured 2 days after infection, was increased approximately 70-fold by rhKGF pretreatment . However, even after KGF treatment the total numbers of AP-expressing cells were few . Transduction efficiency was similar using either LAPSN packaged by amphotropic host range packaging cells or LAPSN pseudotyped with 10A1 MuLV envelope protein (0.091 +/- 0.006 versus 0.094 +/- 0.028 transduction events/mm2, respectively) . Amphotropic vectors use Pit-2 for cell entry, while 10A1 MuLV vectors can use Pit-1 or Pit-2 for cell entry . By in situ hybridization the retroviral receptor Pit-2 (Ram-1) mRNA was expressed only in the pulmonary vasculature, and Pit-1 (Glvr-1) mRNA was expressed at low levels throughout the lung . In vitro studies demonstrated that retrovirus was inactivated by pulmonary surfactant . Stimulating proliferation of the respiratory epithelium increased retroviral transduction in vivo, but the paucity of retroviral receptors and inactivation by surfactant are additional barriers to high-level retroviral gene transfer in the lung.

Nucleosides Nucleotides, 1999 Jan, 18(1), 125 - 36
Preparation of N2, N2,7-trimethylguanosine affinity columns; Espuny R et al.; 2,2,7-trimethylguanosine (TMG) binding proteins from human cells were purified through TMG-affinity columns . TMG synthesis was improved and the TMG obtained was shown to be similar to the TMG in the 5' cap of the UsnRNAs . The eluates obtained with TMG-affinity chromatographies were very different from those isolated with m7G-affinity columns, thus suggesting that specific TMG-binding proteins were obtained . The fraction may be enriched with factors associated with import and/or hypermethylation of UsnRNPs.

Curr Opin Cell Biol, 1999 Feb, 11(1), 68 - 80
Cytokinesis in eukaryotes: a mechanistic comparison; Field C et al.; Cytokinesis is a crucial but poorly understood process of cell proliferation . Recently, molecular genetic analyses of fungal cytokinesis have led to an appreciation of contractile mechanisms in simple eukaryotes, and studies in animal and plant cells have led to new insights into the role of microtubules in the cleavage process . These findings suggest that fundamental mechanisms of cytokinesis may be highly conserved among eukaryotic organisms.

Curr Opin Cell Biol, 1999 Feb, 11(1), 129 - 33
Action at the ends of microtubules; Saunders WS; Microtubule-based motility in the cell is directly associated with changes in microtubule numbers through nucleation and growth and shrinkage of the polymer from the ends . Recent analysis of spindle pole bodies and kinetochores in yeast reveal how the cell builds specialized structures for association with the ends of microtubules.

Curr Opin Cell Biol, 1999 Feb, 11(1), 45 - 53
Cytoplasmic dynein and dynactin in cell division and intracellular transport; Karki S et al.; Since the initial discovery of cytoplasmic dynein, it has become apparent that this microtubule-based motor is involved in several cellular functions including cell division and intracellular transport . Another multisubunit complex, dynactin, may be required for most, if not all, cytoplasmic dynein-driven activities and may provide clues to dynein's functional diversity . Recent genetic and biochemical findings have illuminated the cellular roles of dynein and dynactin and provided insight into the functional mechanism of this complex motor.

J Mol Biol, 1999 Mar 5, 286(4), 983 - 7
A novel feature of DNA recognition: a mutant Gcn4p bZip peptide with dual DNA binding specificities dependent of half-site spacing; Suckow M et al.; Homodimeric DNA-binding proteins with relaxed half-site spacing requirements for their DNA targets have been described . As an example, the yeast transcriptional activator Gcn4p binds in vitro equally well to the AP1 site (5'A4T3G2A1C0T1'C2'A3'T4'3') and the ATF/CREB site (5'A4T3G2A1C0G0'T1'C2'A3'T4'3'), which have identical but differently spaced half-site blocks . We describe a novel feature for the bZip class of DNA-binding proteins . The N-14 mutant of a Gcn4p-derived bZip peptide shows a diametrically opposed base-pair recognition specificity depending on the half-site spacing of its DNA target: on pseudo-palindromic, AP1 site-like binding sites, guanine is required in position 2 for proper binding; in contrast, on palindromic, ATF/CREB site-like targets, position 2 must be cytosine to prevent a loss of binding . Modeling studies suggest that the different base-pair requirements on differently spaced DNA targets are due to minimal alterations of the distances between the relevant atoms of the N-14 side-chain and the corresponding target groups on the DNA . Although the N-14 peptide does not have a natural counterpart, its behavior hints at the possibility that dual binding modi dependent on half-site spacing may occur also for natural homodimeric DNA-binding proteins .

J Biol Chem, 1999 Mar 5, 274(10), 6754 - 62
Molecular enzymology of mammalian Delta1-pyrroline-5-carboxylate synthase . Alternative splice donor utilization generates isoforms with different sensitivity to ornithine inhibition; Hu CA et al.; Delta1-Pyrroline-5-carboxylate synthase (P5CS; EC not assigned), a mitochondrial inner membrane, ATP- and NADPH-dependent, bifunctional enzyme, catalyzes the reduction of glutamate to Delta1-pyrroline-5-carboxylate, a critical step in the de novo biosynthesis of proline and ornithine . We utilized published plant P5CS sequence to search the expressed sequence tag data base and cloned two full-length human P5CS cDNAs differing in length by 6 base pairs (bp) in the open reading frame . The short cDNA has a 2379-bp open reading frame encoding a protein of 793 residues; the long cDNA, generated by "exon sliding," a form of alternative splicing, contains an additional 6-bp insert following bp +711 of the short form resulting in inclusion of two additional amino acids in the region predicted to be the gamma-glutamyl kinase active site of P5CS . The long form predominates in all tissues examined except gut . We also isolated the corresponding long and short murine P5CS transcripts . To confirm the identity of the putative P5CS cDNAs, we expressed both human forms in gamma-glutamyl kinase- and gamma-glutamyl phosphate reductase-deficient strains of Saccharomyces cerevisiae and showed that they conferred the proline prototrophy . Additionally, we found expression of the murine putative P5CS cDNAs conferred proline prototrophy to P5CS-deficient Chinese hamster ovary cells (CHO-K1) . We utilized stable CHO-K1 cell transformants to compare the biochemical characteristics of the long and short murine P5CS isoforms . We found that both confer P5CS activity and that the short isoform is inhibited by L-ornithine with a Ki of approximately 0.25 mM . Surprisingly, the long isoform is insensitive to ornithine inhibition . Thus, the two amino acid insert in the long isoform abolishes feedback inhibition of P5CS activity by L-ornithine.

Science, 1999 Feb 26, 283(5406), 1339 - 43
Prion domain initiation of amyloid formation in vitro from native Ure2p; Taylor KL et al.; The {URE3} non-Mendelian genetic element of Saccharomyces cerevisiae is an infectious protein (prion) form of Ure2p, a regulator of nitrogen catabolism . Here, synthetic Ure2p1-65 were shown to polymerize to form filaments 40 to 45 angstroms in diameter with more than 60 percent beta sheet . Ure2p1-65 specifically induced full-length native Ure2p to copolymerize under conditions where native Ure2p alone did not polymerize . Like Ure2p in extracts of {URE3} strains, these 180- to 220-angstrom-diameter filaments were protease resistant . The Ure2p1-65-Ure2p cofilaments could seed polymerization of native Ure2p to form thicker, less regular filaments . All filaments stained with Congo Red to produce the green birefringence typical of amyloid . This self-propagating amyloid formation can explain the properties of {URE3}.

FEBS Lett, 1999 Feb 5, 444(1), 15 - 21
Isocitrate lyase of Ashbya gossypii--transcriptional regulation and peroxisomal localization; Maeting I et al.; The isocitrate lyase-encoding gene AgICL1 from the filamentous hemiascomycete Ashbya gossypii was isolated by heterologous complementation of a Saccharomyces cerevisiae icl1d mutant . The open reading frame of 1680 bp encoded a protein of 560 amino acids with a calculated molecular weight of 62584 . Disruption of the AgICL1 gene led to complete loss of AgIcl1p activity and inability to grow on oleic acid as sole carbon source . Compartmentation of AgIcl1p in peroxisomes was demonstrated both by Percoll density gradient centrifugation and by immunogold labeling of ultrathin sections using specific antibodies . This fitted with the peroxisomal targeting signal AKL predicted from the C-terminal DNA sequence . Northern blot analysis with mycelium grown on different carbon sources as well as AgICL1 promoter replacement with the constitutive AgTEF promoter revealed a regulation at the transcriptional level . AgICL1 was subject to glucose repression, derepressed by glycerol, partially induced by the C2 compounds ethanol and acetate, and fully induced by soybean oil.

Genet Res, 1998 Dec, 72(3), 199 - 204
Loss of heterozygosity at the dilute-short ear (Myo5a-Bmp5) region of the mouse: mitotic recombination or double non-disjunction?
Favor J, Neuhauser-Klaus A.
The occurrence of homozygous-viable dilute-short ear (Myo5a-Bmp5) double mutants in mouse specific locus mutation experiments has generally been assumed to be the result of double non-disjunction such that the mutant inherits two copies of chromosome 9 carrying the recessive alleles from the test-stock . A homozygous viable Myo5a-Bmp5 double mutant was recovered recently in our laboratory . We were able to genetically analyse both the Myo5a-Bmp5 region and proximal and distal markers in the original mutant as well as in offspring of the original mutant . Our results indicate the mutational event to be due to mitotic recombination and not double non-disjunction.

Plant J, 1998 Dec, 16(5), 613 - 9
A rice homolog of Cdk7/MO15 phosphorylates both cyclin-dependent protein kinases and the carboxy-terminal domain of RNA polymerase II; Yamaguchi M et al.; The activation of cyclin-dependent protein kinases (CDKs) requires phosphorylation of a threonine residue within the T-loop by a CDK-activating kinase (CAK) . The R2 protein of rice is very similar to CAKs of animals and fission yeast at the amino acid level but phosphorylation by R2 has not yet been demonstrated . When R2 was overexpressed in a CAK-deficient mutant of budding yeast, it suppressed the temperature sensitivity of the mutation . Immunoprecipitates of rice proteins with the anti-R2 antibody phosphorylated human CDK2, one of the rice CDKs (Cdc2Os1), and the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II of Arabidopsis . Mutational analysis indicated that R2 phosphorylated the threonine residue within the T-loop of CDK2 and Cdc2Os1 . R2 was found mainly in two protein complexes which had molecular masses of 190 kDa and 70 kDa, respectively, whilst the CDK- and CTD-kinase activities associated with R2 were identified in a complex of 105 kDa . These results indicate that R2 is closely related to CAKs of animals and fission yeast in terms of its phosphorylation activity and, moreover, that this CAK of rice is distinct from a CAK of the dicotyledonous plant Arabidopsis.

Genomics, 1999 Feb 15, 56(1), 59 - 69
A gene upregulated in the acoustically damaged chick basilar papilla encodes a novel WD40 repeat protein; Adler HJ et al.; The chick WDR1 gene is expressed at higher levels in the chick basilar papilla after acoustic overstimulation . The 3.3-kb WDR1 cDNA encodes a novel 67-kDa protein containing nine WD40 repeats, motifs that mediate protein-protein interactions . The predicted WDR1 protein has high sequence identity to WD40-repeat proteins in budding yeast (Saccharomyces cerevisiae), two slime molds (Dictyostelium discoideum and Physarum polycephalum), and the roundworm (Caenorhabditis elegans) . The yeast and P . polycephalum proteins bind actin, suggesting that the novel chick protein may be an actin-binding protein . Sequence database comparisons identified mouse and human cDNAs with high sequence identity to the chick WDR1 cDNA . The mouse Wdr1 and human WDR1 proteins showed 95% sequence identity to each other and 86% identity to the chick WDR1 protein . Northern blot analysis of total RNA from the chick basilar papilla after noise trauma revealed increased levels of a 3.1-kb transcript in the lesioned area . The WDR1 gene was mapped to human chromosome 4, between 22 and 24 cM from the telomere of 4p .

Yeast, 1999 Jan 30, 15(2), 133 - 7
A PDR5-independent pathway of multi-drug resistance regulated by the SIN4 gene product; Fleckenstein A et al.; The SIN4 locus encodes a global transcriptional regulator of various yeast genes . In this report, we demonstrate that loss of function mutations in SIN4 create a multi-drug hypersensitive phenotype that is independent of PDR5 mediated resistance . Thus, double sin4, pdr5 mutants are more sensitive than single mutants . Furthermore, SIN4 does not regulate the PDR5 locus . These observations establish that yeast cells have two genetically distinct pathways conferring resistance towards similar substrates.

Biochemistry, 1999 Feb 23, 38(8), 2601 - 9
Viscosity dependence of the folding kinetics of a dimeric and monomeric coiled coil; Bhattacharyya RP et al.; We measured whether solvent viscosity, and hence chain diffusion, plays a role in the rate-limiting step of the folding reactions of GCN4-p2', a simple alpha-helical coiled coil derived from the leucine zipper region of bZIP transcriptional activator GCN4 . To deconvolute the dual effects of viscosogenic solvents on both viscosity, eta, and stability, earlier attempts assumed that the cosolvent and denaturant interact to the same degree in the transition state . Applying this analysis to GCN4-p2' yielded a nearly 1/eta dependence between folding rates and viscosity for both the dimeric and the cross-linked, monomeric versions of the coiled coil, but it revealed no such coherent relationship for cytochrome c . We also developed a method to determine the relative viscosity dependence of the dimeric and monomeric forms of the coiled coil independent of the assumption concerning the transition state's relative interaction with cosolvents and denaturants . Application of this method indicated that the effect of viscosity on both the folding and the unfolding rates was the same for the dimeric and monomeric versions, further supporting the view that the folding of the dimeric version is folding-limited rather than encounter-limited . The finding that GCN4-p2' folding appears to exhibit a 1/eta viscosity dependence implies that the rate-limiting step in folding is opposed predominantly by solvent-derived rather than internal frictional forces . These results are interpreted in relation to various models for protein folding.

Biochemistry, 1999 Feb 23, 38(8), 2514 - 22
The core histone N-terminal domains are required for multiple rounds of catalytic chromatin remodeling by the SWI/SNF and RSC complexes; Logie C et al.; SWI/SNF and RSC are large, distinct multi-subunit complexes that use the energy of ATP hydrolysis to disrupt nucleosome structure, facilitating the binding of transcription factors or restriction enzymes to nucleosomes {Cote, J., Quinn, J., Workman, J . L., and Peterson, C . L . (1994) Science 265, 53-60 (1); Lorch, Y., Cairns, B . R., Zhang, M., and Kornberg, R . D . (1998) Cell 94, 29-34 (2)} . Here we have used a quantitative assay to measure the activities of these ATP-dependent chromatin remodeling complexes using nucleosomal arrays reconstituted with hypoacetylated, hyperacetylated, or partially trypsinized histones . This assay is based on measuring the kinetics of restriction enzyme digestion of a site located within the central nucleosome of a positioned 11-mer array {Logie, C., and Peterson, C . L . (1997) EMBO J . 16, 6772-6782 (3)} . We find that the DNA-stimulated ATPase activities of SWI/SNF and RSC are not altered by the absence of the histone N-termini . Furthermore, ATP-dependent nucleosome remodeling is also equivalent on all three substrate arrays under reaction conditions where the concentrations of nucleosomal array and either SWI/SNF or RSC are equivalent . However, SWI/SNF and RSC cannot catalytically remodel multiple nucleosomal arrays in the absence of the histone termini, and this catalytic activity of SWI/SNF is decreased by histone hyperacetylation . These results indicate that the histone termini are important for SWI/SNF and RSC function; and, furthermore, our data defines a step in the remodeling cycle where the core histone termini exert their influence . This step appears to be after remodeling, but prior to intermolecular transfer of the remodelers to new arrays.

Cancer Res, 1999 Feb 15, 59(4), 816 - 22
hMSH5: a human MutS homologue that forms a novel heterodimer with hMSH4 and is expressed during spermatogenesis; Bocker T et al.; MutS homologues have been identified in nearly all organisms examined to date . They play essential roles in maintaining mitotic genetic fidelity and meiotic segregation fidelity . MutS homologues appear to function as a molecular switch that signals genomic manipulation events . Here we describe the identification of the human homologue of the Saccharomyces cerevisiae MSH5, which is known to participate in meiotic segregation fidelity and crossing-over . The human MSH5 (hMSH5) was localized to chromosome 6p22-21 and appears to play a role in meiosis because expression is induced during spermatogenesis between the late primary spermatocytes and the elongated spermatid phase . hMSH5 interacts specifically with hMSH4, confirming the generality of functional heterodimeric interactions in the eukaryotic MutS homologue, which also includes hMSH2-hMSH3 and hMSH2-hMSH6.

Biochemistry, 1999 Feb 16, 38(7), 2167 - 78
A structure-based mechanism for copper-zinc superoxide dismutase; Hart PJ et al.; A reaction cycle is proposed for the mechanism of copper-zinc superoxide dismutase (CuZnSOD) that involves inner sphere electron transfer from superoxide to Cu(II) in one portion of the cycle and outer sphere electron transfer from Cu(I) to superoxide in the other portion of the cycle . This mechanism is based on three yeast CuZnSOD structures determined by X-ray crystallography together with many other observations . The new structures reported here are (1) wild type under 15 atm of oxygen pressure, (2) wild type in the presence of azide, and (3) the His48Cys mutant . Final R-values for the three structures are respectively 20.0%, 17.3%, and 20.9% . Comparison of these three new structures to the wild-type yeast Cu(I)ZnSOD model, which has a broken imidazolate bridge, reveals the following: (i) The protein backbones (the "SOD rack") remain essentially unchanged . (ii) A pressure of 15 atm of oxygen causes a displacement of the copper ion 0.37 A from its Cu(I) position in the trigonal plane formed by His46, His48, and His120 . The displacement is perpendicular to this plane and toward the NE2 atom of His63 and is accompanied by elongated copper electron density in the direction of the displacement suggestive of two copper positions in the crystal . The copper geometry remains three coordinate, but the His48-Cu bond distance increases by 0.18 A . (iii) Azide binding also causes a displacement of the copper toward His63 such that it moves 1.28 A from the wild-type Cu(I) position, but unlike the effect of 15 atm of oxygen, there is no two-state character . The geometry becomes five-coordinate square pyramidal, and the His63 imidazolate bridge re-forms . The His48-Cu distance increases by 0.70 A, suggesting that His48 becomes an axial ligand . (iv) The His63 imidazole ring tilts upon 15 atm of oxygen treatment and azide binding . Its NE2 atom moves toward the trigonal plane by 0.28 and 0.66 A, respectively, in these structures . (v) The replacement of His48 by Cys, which does not bind copper, results in a five-coordinate square pyramidal, bridge-intact copper geometry with a novel chloride ligand . Combining results from these and other CuZnSOD crystal structures, we offer the outlines of a structure-based cyclic mechanism.

Biochemistry, 1999 Feb 16, 38(7), 2152 - 9
The solution structure of the DNA double-stranded break repair protein Ku and its complex with DNA: a neutron contrast variation study; Zhao J et al.; Small-angle X-ray and neutron scattering with contrast variation has been used to study the structure of the DNA targeting component (Ku) of the DNA-dependent protein kinase and its complex with DNA . The Ku protein in solution has the approximate shape of a prolate ellipsoid with semi-axes of 24, 43, and 89 A . In the presence of a minimal-length DNA binding sequence (a 24-base-pair duplex DNA), a 1:1 Ku/DNA complex forms . This 1:1 stoichiometry is observed when either the Ku or the DNA is in excess . Analysis of the contrast variation data on Ku complexed with either the 24-mer duplex DNA or a slightly longer 30-mer duplex DNA shows that both the DNA and Ku structures have the same overall conformations within the 1:1 complex as the uncomplexed components . The separation of the centers-of-mass for the Ku/24-mer DNA complex is 46 A, while that for the Ku/30-mer DNA is 56 A . The DNA binds within what appears to be a preformed channel that penetrates deeply into the Ku protein such that the entire length of the 24-mer DNA spans the protein . The slightly longer 30-mer binds in a similar fashion, but with its extra length protruding from the protein envelop . The scattering data are consistent with the idea that the Ku "threads" onto the duplex DNA via a channel that can completely bury approximately 24 base pairs.

Biochemistry, 1999 Feb 9, 38(6), 1819 - 28
DNA-dependent protein kinase phosphorylation sites in Ku 70/80 heterodimer; Chan DW et al.; Ku antigen is composed of 70 and 82 kDa subunits (Ku70 and Ku80, respectively) that together bind with high affinity to ends of double-stranded DNA and other DNA structures in vitro . When bound to DNA, the Ku 70/80 heterodimer enhances the kinase activity of the catalytic subunit of the DNA-dependent protein kinase, DNA-PKcs . Ku and DNA-PKcs are required for V(D)J recombination and DNA double-strand break repair in vivo and may also play a role in regulation of transcription . Ku is phosphorylated by DNA-PKcs in vitro, and cells that lack DNA-PKcs are deficient in Ku phosphorylation in vitro, suggesting that Ku may be a physiological target for DNA-PK . Here we have identified the sites of DNA-PK phosphorylation in human Ku protein . We find that Ku70 is phosphorylated at a single serine residue, serine 6, located in the putative transcriptional activation domain, and Ku80 is phosphorylated at serines 577 and 580 and at threonine 715 . Interestingly, none of the phosphorylation sites identified in Ku correspond to the serine-glutamine consensus for DNA-PK phosphorylation, consistent with previous reports that DNA-PK can recognize additional phosphorylation motifs.

J Biol Chem, 1999 Feb 26, 274(9), 5895 - 900
Expanded lysine acetylation specificity of Gcn5 in native complexes; Grant PA et al.; The coactivator/adaptor protein Gcn5 is a conserved histone acetyltransferase, which functions as the catalytic subunit in multiple yeast transcriptional regulatory complexes . The ability of Gcn5 to acetylate nucleosomal histones is significantly reduced relative to its activity on free histones, where it predominantly modifies histone H3 at lysine 14 . However, the association of Gcn5 in multisubunit complexes potentiates its nucleosomal histone acetyltransferase activity . Here, we show that the association of Gcn5 with other proteins in two native yeast complexes, Ada and SAGA (Spt-Ada-Gcn5-acetyltransferase), directly confers upon Gcn5 the ability to acetylate an expanded set of lysines on H3 . Furthermore Ada and SAGA have overlapping, yet distinct, patterns of acetylation, suggesting that the association of specific subunits determines site specificity.

J Biol Chem, 1999 Feb 26, 274(9), 5810 - 22
The L1 major capsid protein of human papillomavirus type 11 recombinant virus-like particles interacts with heparin and cell-surface glycosaminoglycans on human keratinocytes; Joyce JG et al.; The L1 major capsid protein of human papillomavirus (HPV) type 11, a 55-kDa polypeptide, forms particulate structures resembling native virus with an average particle diameter of 50-60 nm when expressed in the yeast Saccharomyces cerevisiae . We show in this report that these virus-like particles (VLPs) interact with heparin and with cell-surface glycosaminoglycans (GAGs) resembling heparin on keratinocytes and Chinese hamster ovary cells . The binding of VLPs to heparin is shown to exhibit an affinity comparable to that of other identified heparin-binding proteins . Immobilized heparin chromatography and surface plasmon resonance were used to show that this interaction can be specifically inhibited by free heparin and dextran sulfate and that the effectiveness of the inhibitor is related to its molecular weight and charge density . Sequence comparison of nine human L1 types revealed a conserved region of the carboxyl terminus containing clustered basic amino acids that bear resemblance to proposed heparin-binding motifs in unrelated proteins . Specific enzymatic cleavage of this region eliminated binding to both immobilized heparin and human keratinocyte (HaCaT) cells . Removal of heparan sulfate GAGs on keratinocytes by treatment with heparinase or heparitinase resulted in an 80-90% reduction of VLP binding, whereas treatment of cells with laminin, a substrate for alpha6 integrin receptors, provided minimal inhibition . Cells treated with chlorate or substituted beta-D-xylosides, resulting in undersulfation or secretion of GAG chains, also showed a reduced affinity for VLPs . Similarly, binding of VLPs to a Chinese hamster ovary cell mutant deficient in GAG synthesis was shown to be only 10% that observed for wild type cells . This report establishes for the first time that the carboxyl-terminal portion of HPV L1 interacts with heparin, and that this region appears to be crucial for interaction with the cell surface.

J Biol Chem, 1999 Feb 26, 274(9), 5762 - 8
Cell adhesion regulates the interaction between the docking protein p130(Cas) and the 14-3-3 proteins; Garcia-Guzman M et al.; Integrin ligand binding induces a signaling complex formation via the direct association of the docking protein p130(Cas) (Cas) with diverse molecules . We report here that the 14-3-3zeta protein interacts with Cas in the yeast two-hybrid assay . We also found that the two proteins associate in mammalian cells and that this interaction takes place in a phosphoserine-dependent manner, because treatment of Cas with a serine phosphatase greatly reduced its ability to bind 14-3-3zeta . Furthermore, the Cas-14-3-3zeta interaction was found to be regulated by integrin-mediated cell adhesion . Thus, when cells are detached from the extracellular matrix, the binding of Cas to 14-3-3zeta is greatly diminished, whereas replating the cells onto fibronectin rapidly induces the association . Consistent with these results, we found that the subcellular localization of Cas and 14-3-3 is also regulated by integrin ligand binding and that the two proteins display a significant co-localization during cell attachment to the extracellular matrix . In conclusion, our results demonstrate that 14-3-3 proteins participate in integrin-activated signaling pathways through their interaction with Cas, which, in turn, may contribute to important biological responses regulated by cell adhesion to the extracellular matrix.

FEBS Lett, 1999 Jan 29, 443(3), 241 - 5
Re-entering the translocon from the lumenal side of the endoplasmic reticulum . Studies on mutated carboxypeptidase yscY species; Plemper RK et al.; Misfolded or unassembled secretory proteins are retained in the endoplasmic reticulum (ER) and subsequently degraded by the cytosolic ubiquitin-proteasome system . This requires their retrograde transport from the ER lumen into the cytosol, which is mediated by the Sec61 translocon . It had remained a mystery whether ER-localised soluble proteins are at all capable of re-entering the Sec61 channel de novo or whether a permanent contact of the imported protein with the translocon is a prerequisite for retrograde transport . In this study we analysed two new variants of the mutated yeast carboxypeptidase yscY, CPY*: a carboxy-terminal fusion protein of CPY* and pig liver esterase and a CPY* species carrying an additional glycosylation site at its carboxy-terminus . With these constructs it can be demonstrated that the newly synthesised CPY* chain is not retained in the translocation channel but reaches its ER lumenal side completely . Our data indicate that the Sec61 channel provides the essential pore for protein transport through the ER membrane in either direction; persistent contact with the translocon after import seems not to be required for retrograde transport.

Neurology, 1999 Feb, 52(3), 614 - 6
Adrenoleukodystrophy protein enhances association of very long-chain acyl-coenzyme A synthetase with the peroxisome; Yamada T et al.; OBJECTIVE: To clarify the function of adrenoleukodystrophy protein (ALDP) using our ALDP-deficient mice established by gene targeting . BACKGROUND: X-linked adrenoleukodystrophy (ALD) is characterized biochemically by the accumulation of very long-chain fatty acids (VLCFA) in tissues and body fluids, and is caused by impairment of peroxisomal beta-oxidation . In ALD, very long-chain acyl-coenzyme A synthetase (VLACS), which is necessary for peroxisomal beta-oxidation, does not function . METHODS: The ALDP-deficient mice and C57BL/6J mice were used . VLACS or ALDP were transiently expressed by lipofection in murine fibroblasts, and VLCFA beta-oxidation was assayed . Liver peroxisomes were purified by sequential centrifugations and a Nycodenz gradient centrifugation . The peroxisomal localization of VLACS was compared between the mutant and control mice using a Western blot analysis . RESULTS: Impairment of VLCFA beta-oxidation in ALDP-deficient fibroblasts was not corrected by the additional expression of VLACS alone but was by the coexpression of VLACS and ALDP . Although the tissue-specific expression of VLACS was similar in ALDP-deficient and normal mice, peroxisomal VLACS was clearly lower in ALDP-deficient than in normal mice . CONCLUSIONS: ALDP plays a role in the peroxisomal localization of VLACS, and VLACS does not function unless localized in the peroxisome.

Cell, 1999 Feb 5, 96(3), 405 - 13
Regulation of histone acetyltransferases p300 and PCAF by the bHLH protein twist and adenoviral oncoprotein E1A; Hamamori Y et al.; Histone acetyltransferases (HAT) play a critical role in transcriptional control by relieving repressive effects of chromatin, and yet how HATs themselves are regulated remains largely unknown . Here, it is shown that Twist directly binds two independent HAT domains of acetyltransferases, p300 and p300/CBP-associated factor (PCAF), and directly regulates their HAT activities . The N terminus of Twist is a primary domain interacting with both acetyltransferases, and the same domain is required for inhibition of p300-dependent transcription by Twist . Adenovirus E1A protein mimics the effects of Twist by inhibiting the HAT activities of p300 and PCAF . These findings establish a cogent argument for considering the HAT domains as a direct target for acetyltransferase regulation by both a cellular transcription factor and a viral oncoprotein.

Cell, 1999 Feb 5, 96(3), 393 - 403
A viral mechanism for inhibition of p300 and PCAF acetyltransferase activity; Chakravarti D et al.; Nucleosomal histone modification is believed to be a critical step in the activation of RNA polymerase II-dependent transcription . p300/CBP and PCAF histone acetyltransferases (HATs) are coactivators for several transcription factors, including nuclear hormone receptors, p53, and Stat1alpha, and participate in transcription by forming an activation complex and by promoting histone acetylation . The adenoviral E1A oncoprotein represses transcriptional signaling by binding to p300/CBP and displacing PCAF and p/CIP proteins from the complex . Here, we show that E1A directly represses the HAT activity of both p300/CBP and PCAF in vitro and p300-dependent transcription in vivo . Additionally, E1A inhibits nucleosomal histone modifications by the PCAF complex and blocks p53 acetylation . These results demonstrate the modulation of HAT activity as a novel mechanism of transcriptional regulation.

Cell, 1999 Feb 5, 96(3), 389 - 92
Histone octamer transfer by a chromatin-remodeling complex; Lorch Y et al.; RSC, an abundant, essential chromatin-remodeling complex related to SWI/SNF complex, catalyzes the transfer of a histone octamer from a nucleosome core particle to naked DNA . The newly formed octamer-DNA complex is identical with a nucleosome in all respects . The reaction requires ATP and involves an activated RSC-nucleosome intermediate . The mechanism may entail formation of a duplex displacement loop on the nucleosome, facilitating the entry of exogeneous DNA and the release of the endogenous molecule.

Mol Cell, 1999 Jan, 3(1), 109 - 18
Elongator, a multisubunit component of a novel RNA polymerase II holoenzyme for transcriptional elongation; Otero G et al.; The form of RNA polymerase II (RNAPII) engaged in transcriptional elongation was isolated . Elongating RNAPII was associated with a novel multisubunit complex, termed elongator, whose stable interaction was dependent on a hyperphosphorylated state of the RNAPII carboxy-terminal domain (CTD) . A free form of elongator was also isolated, demonstrating the discrete nature of the complex, and free elongator could bind directly to RNAPII . The gene encoding the largest subunit of elongator, ELP1, was cloned . Phenotypes of yeast elp1 delta cells demonstrated an involvement of elongator in transcriptional elongation as well as activation in vivo . Our data indicate that the transition from transcriptional initiation to elongation involves an exchange of the multiprotein mediator complex for elongator in a reaction coupled to CTD hyperphosphorylation.

Mol Cell, 1999 Jan, 3(1), 97 - 108
A novel human SRB/MED-containing cofactor complex, SMCC, involved in transcription regulation; Gu W et al.; A novel human complex that can either repress activator-dependent transcription mediated by PC4, or, at limiting TFIIH, act synergistically with PC4 to enhance activator-dependent transcription has been purified . This complex contains homologs of a subset of yeast mediator/holoenzyme components (including SRB7, SRB10, SRB11, MED6, and RGR1), homologs of other yeast transcriptional regulatory factors (SOH1 and NUT2), and, significantly, some components (TRAP220, TRAP170/hRGR1, and TRAP100) of a human thyroid hormone receptor-associated coactivator complex . The complex shows direct activator interactions but, unlike yeast mediator, can act independently of the RNA polymerase II CTD . These findings demonstrate both positive and negative functional capabilities for the human complex, emphasize novel (CTD-independent) regulatory mechanisms, and link the complex to other human coactivator complexes.

Mol Cell, 1999 Jan, 3(1), 65 - 75
Splicing factor Prp8 governs U4/U6 RNA unwinding during activation of the spliceosome; Kuhn AN et al.; The pre-mRNA 5' splice site is recognized by the ACAGA box of U6 spliceosomal RNA prior to catalysis of splicing . We previously identified a mutant U4 spliceosomal RNA, U4-cs1, that masks the ACAGA box in the U4/U6 complex, thus conferring a cold-sensitive splicing phenotype in vivo . Here, we show that U4-cs1 blocks in vitro splicing in a temperature-dependent, reversible manner . Analysis of splicing complexes that accumulate at low temperature shows that U4-cs1 prevents U4/U6 unwinding, an essential step in spliceosome activation . A novel mutation in the evolutionarily conserved U5 snRNP protein Prp8 suppresses the U4-cs1 growth defect . We propose that wild-type Prp8 triggers unwinding of U4 and U6 RNAs only after structurally correct recognition of the 5' splice site by the U6 ACAGA box and that the mutation (prp8-201) relaxes control of unwinding.

Mol Cell, 1999 Jan, 3(1), 55 - 64
An RNA switch at the 5' splice site requires ATP and the DEAD box protein Prp28p; Staley JP et al.; Pre-mRNA splicing requires dramatic RNA rearrangements hypothesized to be catalyzed by ATP-dependent RNA unwindases of the DExD/H box family . In a rearrangement critical for the fidelity of 5' splice site recognition, a base-pairing interaction between the 5' splice site and U1 snRNA must be switched for a mutually exclusive interaction between the 5' splice site and U6 snRNA . By lengthening the U1:5' splice site duplex, we impeded this switch in a temperature-dependent manner and prevented formation of the spliceosome's catalytic core . Using genetics, we identified the DExD/H box protein Prp28p as a potential mediator of the switch . In vitro, the switch requires both Prp28p and ATP . We propose that Prp28p directs isomerization of RNA at the 5' splice site and promotes fidelity in splicing.

Biochem J, 1999 Mar 1, 338 ( Pt 2), 499 - 505
Cloning and characterization of a maize cytochrome-b5 reductase with Fe3+-chelate reduction capability; Bagnaresi P et al.; We previously purified an NADH-dependent Fe3+-chelate reductase (NFR) from maize roots with biochemical features of a cytochrome-b5 reductase (b5R) {Sparla, Bagnaresi, Scagliarini and Trost (1997) FEBS Lett . 414, 571-575} . We have now cloned a maize root cDNA that, on the basis of sequence information, calculated parameters and functional assay, codes for NFR . Maize NFR has 66% and 65% similarity to mammal and yeast b5R respectively . It has a deduced molecular mass of 31.17 kDa and a pI of 8.53 . An uncharged region is observed at its N-terminus but no myristoylation consensus site is present . Taken together, these results, coupled with previous biochemical evidence, prove that NFR belongs to the b5R class and document b5R from a plant at the molecular level for the first time . We have also identified a putative Arabidopsis thaliana NFR gene . Its organization (nine exons) closely resembles mammalian b5Rs . Several NFR isoforms are expected to exist in maize . They are probably not produced by alternative translational mechanisms as occur in mammals, because of specific constraints observed in the maize NFR cDNA sequence . In contrast with yeast and mammals, tissue-specific and various subcellular localizations of maize b5R isoforms could result from differential expression of the various members of a multigene family . The first molecular characterization of a plant b5R indicates an overall remarkable evolutionary conservation for these versatile reductase systems . In addition, the well-characterized Fe3+-chelate reduction capabilities of NFR, in addition to known Fe3+-haemoglobin reduction roles for mammal b5R isoforms, suggest further and more generalized roles for the b5R class in endocellular iron reduction.

Biochem J, 1999 Mar 1, 338 ( Pt 2), 375 - 86
Comparison of the fibrin-binding activities in the N- and C-termini of fibronectin; Rostagno AA et al.; Fibronectin (Fn) binds to fibrin in clots by covalent and non-covalent interactions . The N- and C-termini of Fn each contain one non-covalent fibrin-binding site, which are composed of type 1 (F1) structural repeats . We have previously localized the N-terminal site to the fourth and fifth F1 repeats (4F1.5F1) . In the current studies, using proteolytic and recombinant proteins representing both the N- and C-terminal fibrin-binding regions, we localized and characterized the C-terminal fibrin-binding site, compared the relative fibrin-binding activities of both sites and determined the contribution of each site to the fibrin-binding activity of intact Fn . By fibrin-affinity chromatography, a protein composed of the 10F1 repeat through to the C-terminus of Fn (10F1-COOH), expressed in COS-1 cells, and 10F1-12F1, produced in Saccharomyces cerevisiae, displayed fibrin-binding activity . However, since 10F1 and 10F1.11F1 were not active, the presence of 12F1 is required for fibrin binding . A proteolytic fragment of 14.4 kDa, beginning 14 residues N-terminal to 10F1, was isolated from the fibrin-affinity matrix . Radio-iodinated 14.4 kDa fibrin-binding peptide/protein (FBP) demonstrated a dose-dependent and saturable binding to fibrin-coated wells that was both competitively inhibited and reversed by unlabelled 14.4 kDa FBP . Comparison of the fibrin-binding affinities of proteolytic FBPs from the N-terminus (25.9 kDa FBP), the C-terminus (14.4 kDa) and intact Fn by ELISA yielded estimated Kd values of 216, 18 and 2.1 nM, respectively . The higher fibrin-binding affinity of the N-terminus was substantiated by the ability of both a recombinant 4F1.5F1 and a monoclonal antibody (mAb) to this site to maximally inhibit biotinylated Fn binding to fibrin by 80%, and by blocking the 90% inhibitory activity of a polyclonal anti-Fn, by absorption with the 25.9 kDa FBP . We propose that whereas the N-terminal site appears to contribute to most of the binding activity of native Fn to fibrin, the specific binding of the C-terminal site may strengthen this interaction.

Protein Expr Purif, 1999 Feb, 15(1), 127 - 45
A kinetic locking-on strategy for bioaffinity purification: further studies with alcohol dehydrogenase; O'flaherty M et al.; The kinetic locking-on strategy improves the selectivity of protein purification procedures based on immobilized cofactor derivatives through use of enzyme-specific substrate analogues in irrigants to promote biospecific adsorption . This paper describes the development and application of this strategy to the one-chromatographic step affinity purification of NAD(P)+-dependent alcohol dehydrogenases using 8'-azo-linked immobilized NAD(P)+, S6-linked and N6-linked immobilized NAD+, and N6-linked immobilized NADP+ derivatives . These studies were carried out using alcohol dehydrogenases from Saccharomyces cerevisiae (YADH, EC 1.1.1.1), equine liver (HLADH, EC 1.1.1.1), and Thermoanaerobium brockii (TBADH, EC 1.1.1.2) . The results reveal that the factors which require careful consideration before development of a truly biospecific system based on the locking-on strategy include: (i) the stability of the immobilized cofactor derivative; (ii) the spacer-arm composition of the affinity derivative; (iii) the accessible immobilized cofactor concentration; (iv) the soluble locking-on ligand concentration; (v) the dissociation constant of locking-on ligand, and (vi) the identification and elimination of nonbiospecific interference . The S6-linked immobilized NAD+ derivative (synthesized with a hydrophilic spacer arm) proved to be the most suitable of the affinity adsorbents investigated in the present study for use with the locking-on strategy . This conclusion was based primarily on the observations that this affinity adsorbent was stable, retained cofactor activity with the "test" enzymes under study, and was not prone to nonbiospecific interactions . Using this immobilized derivative in conjunction with the locking-on strategy, alcohol dehydrogenase from Saccharomyces cerevisiae was purified to electrophoretic homogeneity in a single affinity chromatographic step .

RNA, 1999 Feb, 5(2), 272 - 80
Arginine methylation and binding of Hrp1p to the efficiency element for mRNA 3'-end formation; Valentini SR et al.; Hrp1p is a heterogeneous ribonucleoprotein (hnRNP) from the yeast Saccharomyces cerevisiae that is involved in the cleavage and polyadenylation of the 3'-end of mRNAs and mRNA export . In addition, Hrplp is one of several RNA-binding proteins that are posttranslationally modified by methylation at arginine residues . By using functional recombinant Hrp1p, we have identified RNA sequences with specific high affinity binding sites . These sites correspond to the efficiency element for mRNA 3'-end formation, UAUAUA . To examine the effect of methylation on specific RNA binding, purified recombinant arginine methyltransferase (Hmt1p) was used to methylate Hrp1p . Methylated Hrp1p binds with the same affinity to UAUAUA-containing RNAs as unmethylated Hrpl p indicating that methylation does not affect specific RNA binding . However, RNA itself inhibits the methylation of Hrp1p and this inhibition is enhanced by RNAs that specifically bind Hrpl p . Taken together, these data support a model in which protein methylation occurs prior to protein-RNA binding in the nucleus.

Scand J Immunol, 1999 Jan, 49(1), 73 - 7
Peripheral blood neutrophils of germ-free rats modified by in vivo granulocyte-colony-stimulating factor and exposure to natural environment; Ohkubo T et al.; Peripheral blood neutrophils from germ-free (GF), specific-pathogen-free (SPF) and conventional (CV) rats were compared . Besides neutropenia and impaired superoxide anion generation as previously reported, it was found that GF rats had lower phagocytic function (70%) and generated less nitric oxide than the other rats . GF and SPF rats were injected with recombinant human granulocyte-colony-stimulating factor (rhG-CSF), or were transferred to natural environment . It was found that total number, phagocytosis, intracellular killing, ratio of phagocytosis versus killing (killing efficiency) and nitric oxide production induced by recombinant rat interferon-gamma (rrIFN-gamma) were normalized upon injection of rhG-CSF . These results indicate that rhG-CSF may stimulate neutrophil production and induce the expression of neutrophil receptors for phagocytosis and nitric oxide production in GF rats . Although lower than in CV rats, the level of superoxide produced was sufficient for normal neutrophil-killing efficiency in SPF and GF rats . In SPF rats, this could be amended by exposure to natural environment . However, neither rhG-CSF injection nor transfer to natural environment could increase the generation of superoxide anion in GF rats.

Biochim Biophys Acta, 1999 Feb 16, 1444(2), 276 - 82
Characterization and expression of the cDNA encoding a new kind of phospholipid transfer protein, the phosphatidylglycerol/phosphatidylinositol transfer protein from Aspergillus oryzae: evidence of a putative membrane targeted phospholipid transfer protein in fungi; Record E et al.; The full-length cDNA of a phospholipid transfer protein (PLTP) was isolated from Aspergillus oryzae by a RACE-PCR procedure using degenerated primer pool selected from the N-terminal sequence of the purified phosphatidylinositol/phosphatidylglycerol transfer protein (PG/PI-TP) . The cDNA encodes a 173 amino acid protein of 18823 Da . The deduced amino acid sequence from position 38 to 67 is 100% identical to the N-terminal sequence (first 30 amino acids) of the purified PG/PI-TP . This amino acid sequence is preceded by a leader peptide of 37 amino acids which is predicted to be composed of a signal peptide of 21 amino acids followed by an extra-sequence of 16 amino acids, or a membrane anchor protein signal (amino acid 5-29) . This strongly suggests that the PG/PI-TP is a targeted protein . The deduced mature protein is 138 amino acids long with a predicted molecular mass of 14933 Da . Comparison of the deduced PG/PI-TP sequence with other polypeptide sequences available in databases revealed a homology with a protein deduced from an open reading frame coding for an unknown protein in Saccharomyces cerevisiae (36% identity and 57% similarity) . Apart from this homology, the PG/PI-TP is unique and specific to the filamentous fungi on the basis of comparison of PLTP protein sequences . Northern blot analysis of RNA isolated from A . oryzae cultures grown on glucose or glucose supplemented with phospholipids suggests that the PG/PI-TP is transcribed by only one RNA species and allows us to show that expression of the protein is regulated at the messenger RNA level.

Gene, 1999 Feb 18, 227(2), 149 - 55
Identification and complete cDNA sequence of the missing Drosophila MCMs: DmMCM3, DmMCM6 and DmMCM7; Feger G; The minichromosome maintenance (MCM) gene family consists of six members (MCM2, 3, 4, 5, 6 and 7) in Saccharomyces cerevisiae as well as in humans . Each family member plays an essential role in the replication of DNA . In Drosophila melanogaster only three members, DmMCM2, DmMCM4/dpa and DmMCM5/DmCDC46, have been studied . In addition, two other partial sequences were recently reported . Using degenerate primers and low stringency PCR conditions six different DNA sequences were identified with highest sequence similarity to MCM2, 3, 4, 5, 6 and 7 . Sequence analysis of full length cDNA clones corresponding to the MCM3, 6 and 7 fragment proves the existence of six MCM genes in Drosophila melanogaster . Strong homology to the human counterparts, mRNA expression analysis and physico-chemical properties suggest a conserved function in DNA replication for DmMCM3, 6 and 7.

J Cell Biochem, 1999 Mar 1, 72(3), 356 - 67
180-kD bullous pemphigoid antigen/type XVII collagen: tissue-specific expression and molecular interactions with keratin 18; Aho S et al.; The 180-kD bullous pemphigoid antigen (BPAG2) is a hemidesmosomal transmembrane protein, also known as type XVII collagen . In this study, potential interactions of BPAG2 with other proteins expressed in epidermal keratinocytes were explored by yeast two-hybrid system using the amino-terminal intracellular domain of BPAG2 as a bait . Several independent interacting clones encoding keratin 18 (K18) were identified when the keratinocyte cDNA library, cloned into the yeast two-hybrid activation domain vector, was screened . The peptide sequence responsible for the interaction of BPAG2 was restricted to amino acids 15-25, and substitution of a valine residue in the middle of this sequence by a proline (V23P) by site-directed mutagenesis abolished the interaction . Further examination of the K18 sequences by restricted cDNA constructs in yeast two-hybrid system identified a carboxyl-terminal segment corresponding to helix 2B domain as critical for BPAG2 binding . The interaction of BPAG2/K18 was confirmed by an in vitro protein-protein interaction assay, which also confirmed that normal human keratinocytes express K18 in culture . The tissue specific expression of BPAG2 was first examined using a multi-tissue RNA blot . Human multiple tissue cDNA panels representing a variety of adult and fetal tissues as well as tumor cells were used as PCR-templates to study the expression patterns of both BPAG2 and K18 . The results demonstrated significant level of expression of BPAG2, besides in epidermal keratinocytes, also in a variety of tissues with predominant epithelial component, such as mammary, salivary and thyroid glands, colon, prostate, testis, placenta, and adult and fetal thymus, as well as in colon, pancreatic and prostatic adenocarcinoma cell lines, and an ovarian carcinoma . As expected, K18 transcript is present in liver, pancreas, colon, placenta, and in fetal kidney . Collectively, the results suggest that BPAG2 has a relatively broad tissue distribution including specialized and simple epithelia, and that within the tissues such as colon and placenta, BPAG2 may have direct interactions with K18, a keratin characteristically expressed in a simple epithelia.

Mol Cell Biol, 1999 Mar, 19(3), 2189 - 97
The Caenorhabditis elegans sex determination gene mog-1 encodes a member of the DEAH-Box protein family; Puoti A et al.; In the Caenorhabditis elegans hermaphrodite germ line, the sex-determining gene fem-3 is repressed posttranscriptionally to arrest spermatogenesis and permit oogenesis . This repression requires a cis-acting regulatory element in the fem-3 3' untranslated region; the FBF protein, which binds to this element; and at least six mog genes . In this paper, we report the molecular characterization of mog-1 as well as additional phenotypic characterization of this gene . The mog-1 gene encodes a member of the DEAH-box family . Three mog-1 alleles possess premature stop codons and are likely to be null alleles, and one is a missense mutation and is likely to retain residual activity . mog-1 mRNA is expressed in both germ line and somatic tissues and appears to be ubiquitous . The MOG-1 DEAH-box protein is most closely related to proteins essential for splicing in the yeast Saccharomyces cerevisiae, but splicing appears to occur normally in a mog-1-null mutant . In addition to its involvement in the sperm-oocyte switch and control of fem-3, zygotic mog-1 is required for robust germ line proliferation and for normal growth during development . We suggest that mog-1 plays a broader role in RNA regulation than previously considered.

Mol Cell Biol, 1999 Mar, 19(3), 1810 - 20
The histone acetylase PCAF is a phorbol-ester-inducible coactivator of the IRF family that confers enhanced interferon responsiveness; Masumi A et al.; Transcription factors of the interferon regulatory factor (IRF) family bind to the type I interferon (IFN)-responsive element (ISRE) and activate transcription from IFN-inducible genes . To identify cofactors that associate with IRF proteins, DNA affinity binding assays were performed with nuclear extracts prepared from tissue culture cells . The results demonstrated that the endogenous IRFs bound to the ISRE are complexed with the histone acetylases, PCAF, GCN5, and p300/CREB binding protein and that histone acetylase activities are accumulated on the IRF-ISRE complexes . By testing recombinant proteins, we show that PCAF directly binds to some but not all members of the IRF family through distinct domains of the two proteins . This interaction was functionally significant, since transfection of PCAF strongly enhanced IRF-1- and IRF-2-dependent promoter activities . Further studies showed that expression of PCAF and other histone acetylases was markedly induced in U937 cells upon phorbol ester treatment, which led to increased recruitment of PCAF to the IRF-ISRE complexes . Coinciding with the induction of histone acetylases, phorbol ester markedly enhanced IFN-alpha-stimulated gene expression in U937 cells . Supporting the role for PCAF in conferring IFN responsiveness, transfection of PCAF into U937 cells led to a large increase in IFN-alpha-inducible promoter activity . These results demonstrate that PCAF is a phorbol ester-inducible coactivator of the IRF proteins which contributes to the establishment of type I IFN responsiveness.

EMBO J, 1999 Feb 15, 18(4), 1071 - 80
The exocyst is an effector for Sec4p, targeting secretory vesicles to sites of exocytosis; Guo W et al.; Polarized secretion requires proper targeting of secretory vesicles to specific sites on the plasma membrane . Here we report that the exocyst complex plays a key role in vesicle targeting . Sec15p, an exocyst component, can associate with secretory vesicles and interact specifically with the rab GTPase, Sec4p, in its GTP-bound form . A chain of protein-protein interactions leads from Sec4p and Sec15p on the vesicle, through various subunits of the exocyst, to Sec3p, which marks the sites of exocytosis on the plasma membrane . Sec4p may control the assembly of the exocyst . The exocyst may therefore function as a rab effector system for targeted secretion.

Oncogene, 1999 Feb 11, 18(6), 1333 - 9
B-MYB transactivates its own promoter through SP1-binding sites; Sala A et al.; B-MYB is an ubiquitous protein required for mammalian cell growth . In this report we show that B-MYB transactivates its own promoter through a 120 bp segment proximal to the transcription start site . The B-MYB-responsive element does not contain myb-binding sites and gel-shift analysis shows that SP1, but not B-MYB, protein contained in SAOS2 cell extracts binds to the 120 bp B-myb promoter fragment . B-MYB-dependent transactivation is cooperatively increased in the presence of SP1, but not SP3 overexpression . When the SP1 elements of the B-myb promoter are transferred in front of a heterologous promoter, an increased response to B-MYB results . In contrast, c-MYB, the prototype member of the Myb family, is not able to activate the luciferase construct containing the SP1 elements . With the use of an SP1-GAL4 fusion protein, we have determined that the cooperative activation occurs through the domain A of SP1 . These observations suggest that B-MYB functions as a coactivator of SP1, and that diverse combinations of myb and SP1 sites may dictate the responsiveness of myb-target genes to the various members of the myb family.

Curr Opin Chem Biol, 1999 Feb, 3(1), 64 - 70
Progress and variations in two-hybrid and three-hybrid technologies; Drees BL; The original yeast two-hybrid system and its variants have proven to be effective tools for identification and analysis of protein-protein, protein-DNA and protein-RNA interactions . The two-hybrid assay is being applied to the entire complement of proteins of the yeast Saccharomyces cerevisiae to characterize the network of protein-protein interactions in the eukaryotic cell . The development of nontranscriptional cytosolic and membrane-associated two-hybrid methods has made it possible to detect and examine a number of protein-protein interactions in their normal cellular locations . Small-molecule hybrid systems have been developed which can be used to study protein-ligand interactions and to activate cellular processes by forcing protein associations.

Curr Biol, 1999 Feb 11, 9(3), 159 - 62
Vac1p coordinates Rab and phosphatidylinositol 3-kinase signaling in Vps45p-dependent vesicle docking/fusion at the endosome; Peterson MR et al.; The vacuolar protein sorting (VPS) pathway of Saccharomyces cerevisiae mediates transport of vacuolar protein precursors from the late Golgi to the lysosome-like vacuole . Sorting of some vacuolar proteins occurs via a prevacuolar endosomal compartment and mutations in a subset of VPS genes (the class D VPS genes) interfere with the Golgi-to-endosome transport step . Several of the encoded proteins, including Pep12p/Vps6p (an endosomal target (t) SNARE) and Vps45p (a Sec1p homologue), bind each other directly {1} . Another of these proteins, Vac1p/Pep7p/Vps19p, associates with Pep12p and binds phosphatidylinositol 3-phosphate (PI(3)P), the product of the Vps34 phosphatidylinositol 3-kinase (PI 3-kinase) {1} {2} . Here, we demonstrate that Vac1p genetically and physically interacts with the activated, GTP-bound form of Vps21p, a Rab GTPase that functions in Golgi-to-endosome transport, and with Vps45p . These results implicate Vac1p as an effector of Vps21p and as a novel Sec1p-family-binding protein . We suggest that Vac1p functions as a multivalent adaptor protein that ensures the high fidelity of vesicle docking and fusion by integrating both phosphoinositide (Vps34p) and GTPase (Vps21p) signals, which are essential for Pep12p- and Vps45p-dependent targeting of Golgi-derived vesicles to the prevacuolar endosome.

Curr Biol, 1999 Jan 28, 9(2), 101 - 4
A Drosophila TNF-receptor-associated factor (TRAF) binds the ste20 kinase Misshapen and activates Jun kinase; Liu H et al.; Two families of protein kinases that are closely related to Ste20 in their kinase domain have been identified - the p21-activated protein kinase (Pak) and SPS1 families {1-3} . In contrast to Pak family members, SPS1 family members do not bind and are not activated by GTP-bound p21Rac and Cdc42 . We recently placed a member of the SPS1 family, called Misshapen (Msn), genetically upstream of the c-Jun amino-terminal (JNK) mitogen-activated protein (MAP) kinase module in Drosophila {4} . The failure to activate JNK in Drosophila leads to embryonic lethality due to the failure of these embryos to stimulate dorsal closure {5-8} . Msn probably functions as a MAP kinase kinase kinase kinase in Drosophila, activating the JNK pathway via an, as yet, undefined MAP kinase kinase kinase . We have identified a Drosophila TNF-receptor-associated factor, DTRAF1, by screening for Msn-interacting proteins using the yeast two-hybrid system . In contrast to the mammalian TRAFs that have been shown to activate JNK, DTRAF1 lacks an amino-terminal 'Ring-finger' domain, and overexpression of a truncated DTRAF1, consisting of only its TRAF domain, activates JNK . We also identified another DTRAF, DTRAF2, that contains an amino-terminal Ring-finger domain . Msn specifically binds the TRAF domain of DTRAF1 but not that of DTRAF2 . In Drosophila, DTRAF1 is thus a good candidate for an upstream molecule that regulates the JNK pathway by interacting with, and activating, Msn . Consistent with this idea, expression of a dominant-negative Msn mutant protein blocks the activation of JNK by DTRAF1 . Furthermore, coexpression of Msn with DTRAF1 leads to the synergistic activation of JNK . We have extended some of these observations to the mammalian homolog of Msn, Nck-interacting kinase (NIK), suggesting that TRAFs also play a critical role in regulating Ste20 kinases in mammals.

Development, 1999 Mar, 126(6), 1129 - 38
Apontic binds the translational repressor Bruno and is implicated in regulation of oskar mRNA translation; Lie YS et al.; The product of the oskar gene directs posterior patterning in the Drosophila oocyte, where it must be deployed specifically at the posterior pole . Proper expression relies on the coordinated localization and translational control of the oskar mRNA . Translational repression prior to localization of the transcript is mediated, in part, by the Bruno protein, which binds to discrete sites in the 3' untranslated region of the oskar mRNA . To begin to understand how Bruno acts in translational repression, we performed a yeast two-hybrid screen to identify Bruno-interacting proteins . One interactor, described here, is the product of the apontic gene . Coimmunoprecipitation experiments lend biochemical support to the idea that Bruno and Apontic proteins physically interact in Drosophila . Genetic experiments using mutants defective in apontic and bruno reveal a functional interaction between these genes . Given this interaction, Apontic is likely to act together with Bruno in translational repression of oskar mRNA . Interestingly, Apontic, like Bruno, is an RNA-binding protein and specifically binds certain regions of the oskar mRNA 3' untranslated region.

Genes Dev, 1999 Feb 1, 13(3), 307 - 19
Ctf7p is essential for sister chromatid cohesion and links mitotic chromosome structure to the DNA replication machinery; Skibbens RV et al.; CTF7 (chromosome transmission fidelity) gene in budding yeast encodes an essential protein that is required for high-fidelity chromosome transmission and contains regions of identity conserved from yeast to man . ctf7 mutant cells arrested prior to anaphase onset contain separated sister chromatids . Thus, Ctf7p is essential for cohesion . Cohesion is established during S phase and then maintained until mitosis . However, Ctf7p activity is required only during S phase, suggesting that Ctf7p functions in the establishment of cohesion . In addition, ctf7 genetically interacts with DNA metabolism mutations pol30 (PCNA) and ctf18 (an RF-C like protein) and ctf7 temperature sensitivity and chromosome loss are rescued by high levels of POL30 . These findings provide the first evidence that links the establishment of sister chromatid cohesion to the DNA replication machinery and suggest that the assembly of cohesion (and possibly condensation) complexes are coupled to PCNA-dependent DNA replication . The analysis of Ctf7p also reveals an important connection between sister chromatid cohesion, spindle integrity and the spindle assembly checkpoint.

J Biochem (Tokyo), 1999 Feb, 125(2), 319 - 27
Complete inhibition of mouse macrophage-derived foam cell formation by triacsin C; Namatame I et al.; Primary mouse peritoneal macrophages effectively take up and metabolize phosphatidylcholine/cholesterol liposomes containing a small amount of phosphatidylserine, which results in the massive accumulation in the cytoplasm of oil red O positive lipid droplets consisting of cholesteryl ester (CE) and triacylglycerol (TG) {Nishikawa et al . (1990) J . Biol . Chem . 265, 5226-5231} . A number of inhibitors of CE synthesis have been reported, but their effects on the lipid droplet formation have not been fully examined . Furthermore, the contribution of TG synthesis to lipid droplet formation has been poorly investigated . We have investigated the relationship between CE and TG syntheses and cytosolic lipid droplet formation in macrophages cultured in the presence of inhibitors with different modes of action . When macrophages were cultured with liposomes and {14C}oleic acid in the presence of triacsin C, a potent inhibitor of long chain acyl-CoA synthetase, both {14C}CE and {14C}TG syntheses were inhibited to similar extents with IC50 values of 0.19 and 0.10 microM, respectively . On the other hand, pregnenolone, a well-known inhibitor of cellular cholesterol transport, and CL-283,546, a potent inhibitor of acyl-CoA:cholesterol acyltransferase, inhibited {14C}CE synthesis specifically with IC50 values of 5.0 and 0.038 microM, respectively . Microscopic observation revealed that the inhibitors of cholesterol metabolism produce only a partial decrease in cytosolic lipid droplets even at the highest doses which cause almost complete inhibition of {14C}CE synthesis . However, the triacsin C-dose dependent inhibition of lipid droplet formation was almost complete at 0.59 microM, a concentration that inhibits {14C}CE and {14C}TG syntheses by about 90% . These results show that inhibiton of acyl-CoA synthetase by triacsin C causes a decrease in the cellular levels of acyl-CoA, the common substrate for CE and TG syntheses, leading to an inhibiton of neutral lipid synthesis and eventually to the complete disappearance of cytosolic lipid droplets from macrophages . These findings suggest that TG synthesis, as well as CE synthesis, is responsible for macrophage-derived foam cell formation, and is therefore a potential target for new antiatherosclerotic agents.

Proc Natl Acad Sci U S A, 1999 Feb 16, 96(4), 1498 - 503
The {URE3} prion is an aggregated form of Ure2p that can be cured by overexpression of Ure2p fragments; Edskes HK et al.; The {URE3} nonchromosomal genetic element is a prion of Ure2p, a regulator of nitrogen catabolism in Saccharomyces cerevisiae . Ure2p1-65 is the prion domain of Ure2p, sufficient to propagate {URE3} in vivo . We show that full length Ure2p-green fluorescent protein (GFP) or a Ure2p1-65-GFP fusion protein is aggregated in cells carrying {URE3} but is evenly distributed in cells lacking the {URE3} prion . This indicates that {URE3} involves a self-propagating aggregation of Ure2p . Overexpression of Ure2p1-65 induces the de novo appearance of {URE3} by 1,000-fold in a strain initially {ure-o}, but cures {URE3} from a strain initially carrying the {URE3} prion . Overexpression of several other fragments of Ure2p or Ure2-GFP fusion proteins also efficiently cures the prion . We suggest that incorporation of fragments or fusion proteins into a putative {URE3} "crystal" of Ure2p poisons its propagation.

Proc Natl Acad Sci U S A, 1999 Feb 16, 96(4), 1439 - 44
The molecular chaperone Hsp90 can negatively regulate the activity of a glucocorticosteroid-dependent promoter; Kang KI et al.; Hsp90, a molecular chaperone required for the functioning of glucocorticosteroid receptor (GR), ensures, by direct interaction, the conformational competence of the steroid-binding pocket . In addition to having this positive function, Hsp90 maintains steroid receptors in an inactive form in the absence of hormone . However, neither the participation of Hsp90 once the pathway has been activated by the ligand nor the importance of increased Hsp90 levels in determining the amplitude of the response has ever been assessed directly . Here, by increasing the Hsp90/GR ratio in the nuclear compartment, we found an attenuation of the response to glucocorticosteroids that was not due to a nonspecific or toxic effect of the Hsp90 modified by nuclear targeting . Since this negative effect was more pronounced at high levels of hormone, when receptor and Hsp90 are maximally dissociated, the possibility of an interaction between Hsp90 and GR, already activated to a DNA-binding form, was directly investigated . Indeed GR, after in vivo activation by ligand, was still able to reassociate with Hsp90, suggesting that this interaction plays a role in vivo, possibly in receptor recycling . Moreover, the GR binding to its DNA response element was inhibited by an excess of Hsp90, pointing to a function of Hsp90 in the nuclear compartment . It is thus proposed that an increased Hsp90/GR ratio influences the responsiveness to ligand at a step that is after receptor activation . This increased ratio may be of pathophysiological relevance in the different circumstances that lead to an elevated level of nuclear Hsp90.

Proc Natl Acad Sci U S A, 1999 Feb 16, 96(4), 1403 - 8
Catalytic subunit of DNA-dependent protein kinase: impact on lymphocyte development and tumorigenesis; Kurimasa A et al.; The DNA-dependent protein kinase (DNA-PK) consists of a heterodimer DNA-binding complex, Ku70 and Ku80, and a large catalytic subunit, DNA-PKcs . To examine the role of DNA-PKcs in lymphocyte development, radiation sensitivity, and tumorigenesis, we disrupted the mouse DNA-PKcs by homologous recombination . DNA-PKcs-null mice exhibit neither growth retardation nor a high frequency of T cell lymphoma development, but show severe immunodeficiency and radiation hypersensitivity . In contrast to the Ku70-/- and Ku80-/- phenotype, DNA-PKcs-null mice are blocked for V(D)J coding but not for signal-end joint formation . Furthermore, inactivation of DNA-PKcs leads to hyperplasia and dysplasia of the intestinal mucosa and production of aberrant crypt foci, suggesting a novel role of DNA-PKcs in tumor suppression.

Proc Natl Acad Sci U S A, 1999 Feb 16, 96(4), 1385 - 90
A human cell-surface receptor for xenotropic and polytropic murine leukemia viruses: possible role in G protein-coupled signal transduction; Battini JL et al.; Although present in many copies in the mouse genome, xenotropic murine leukemia viruses cannot infect cells from laboratory mice because of the lack of a functional cell surface receptor required for virus entry . In contrast, cells from many nonmurine species, including human cells, are fully permissive . Using an expression library approach, we isolated a cDNA from HeLa cell RNA that conferred susceptibility to xenotropic envelope protein binding and virus infection when expressed in nonpermissive cells . The deduced product is a 696-aa multiple-membrane spanning molecule, is widely expressed in human tissues, and shares homology with nematode, fly, and plant proteins of unknown function as well as with the yeast SYG1 protein, which has been shown to interact with a G protein . This molecule also acts as a receptor for polytropic murine leukemia viruses, consistent with observed interference between xenotropic and polytropic viruses in some cell types . This xenotropic and polytropic retrovirus receptor (XPR1) is the fourth identified molecule having multiple membrane spanning domains among mammalian type C oncoretrovirus receptors and may play a role in G protein-coupled signal transduction, as do the chemokine receptors required for HIV entry.

Proc Natl Acad Sci U S A, 1999 Feb 16, 96(4), 1321 - 6
Mammalian subtilisin/kexin isozyme SKI-1: A widely expressed proprotein convertase with a unique cleavage specificity and cellular localization; Seidah NG et al.; Using reverse transcriptase-PCR and degenerate oligonucleotides derived from the active-site residues of subtilisin/kexin-like serine proteinases, we have identified a highly conserved and phylogenetically ancestral human, rat, and mouse type I membrane-bound proteinase called subtilisin/kexin-isozyme-1 (SKI-1) . Computer databank searches reveal that human SKI-1 was cloned previously but with no identified function . In situ hybridization demonstrates that SKI-1 mRNA is present in most tissues and cells . Cleavage specificity studies show that SKI-1 generates a 28-kDa product from the 32-kDa brain-derived neurotrophic factor precursor, cleaving at an RGLT downward arrowSL bond . In the endoplasmic reticulum of either LoVo or HK293 cells, proSKI-1 is processed into two membrane-bound forms of SKI-1 (120 and 106 kDa) differing by the nature of their N-glycosylation . Late along the secretory pathway some of the membrane-bound enzyme is shed into the medium as a 98-kDa form . Immunocytochemical analysis of stably transfected HK293 cells shows that SKI-1 is present in the Golgi apparatus and within small punctate structures reminiscent of endosomes . In vitro studies suggest that SKI-1 is a Ca2+-dependent serine proteinase exhibiting a wide pH optimum for cleavage of pro-brain-derived neurotrophic factor.

Proc Natl Acad Sci U S A, 1999 Feb 16, 96(4), 1315 - 20
The retinitis pigmentosa GTPase regulator, RPGR, interacts with the delta subunit of rod cyclic GMP phosphodiesterase; Linari M et al.; Recently, the retinitis pigmentosa 3 (RP3) gene has been cloned and named retinitis pigmentosa GTPase regulator (RPGR) . The amino-terminal half of RPGR is homologous to regulator of chromosome condensation (RCC1), the nucleotide exchange factor for the small GTP-binding protein Ran . In a yeast two-hybrid screen we identified the delta subunit of rod cyclic GMP phosphodiesterase (PDEdelta) as interacting with the RCC1-like domain (RLD) of RPGR (RPGR392) . The interaction of RPGR with PDEdelta was confirmed by pull-down assays and plasmon surface resonance . The binding affinity was determined to be 90 nM . Six missense mutations at evolutionary conserved residues within the RLD, which were found in RP3 patients, were analyzed by using the two-hybrid system . All missense mutations showed reduced interaction with PDEdelta . A non-RP3-associated missense substitution outside the RLD, V36F, did not abolish the interaction with PDEdelta . PDEdelta is widely expressed and highly conserved across evolution and is proposed to regulate the membrane insertion or solubilization of prenylated proteins, including the catalytic subunits of the PDE holoenzyme involved in phototransduction and small GTP-binding proteins of the Rab family . These results suggest that RPGR mutations give rise to retinal degeneration by dysregulation of intracellular processes that determine protein localization and protein transport.

Proc Natl Acad Sci U S A, 1999 Feb 16, 96(4), 1303 - 8
A role for FEN-1 in nonhomologous DNA end joining: the order of strand annealing and nucleolytic processing events; Wu X et al.; Eukaryotic repair of double-strand DNA breaks can occur either by homologous recombination or by nonhomologous DNA end joining (NHEJ) . NHEJ relies on Ku70/86, XRCC4, DNA ligase IV, and DNA-dependent protein kinase . NHEJ involves a synapsis step in which the two ends are maintained in proximity, processing steps in which nucleases and polymerases act on the ends, an alignment step in which a few nucleotides of terminal homology guide the ends into preferred alignments, and a ligation step . Some of the steps, such as ligation, rely on a single enzymatic component . However, the processing steps begin and end with a wide array of alternative substrates and products, respectively, and likely involve multiple nucleases and polymerases . Given the alternative pathways that can be catalyzed by the remaining nucleases and polymerases, no one of these processing enzymes is likely to be essential . The only requirement for the processing enzymes, as a collective, is to generate a ligatable configuration, namely a ligatable nick on each strand . Here, we have tested the two major known 5'-specific nucleases of Saccharomyces cerevisiae for involvement in NHEJ . Whereas EXO1 does not appear to be involved to any detectable level, deleting RAD27 (FEN-1 of yeast) leads to a 4.4-fold reduction specifically of those NHEJ events predicted to proceed by means of 5' flap intermediates . Because Rad27/FEN-1 acts specifically at 5' flap structures, these results suggest that the NHEJ alignment step precedes nucleolytic processing steps in a significant fraction of NHEJ events.

Proc Natl Acad Sci U S A, 1999 Feb 16, 96(4), 1297 - 302
The charged region of Hsp90 modulates the function of the N-terminal domain; Scheibel T et al.; Hsp90, an abundant heat shock protein that is highly expressed even under physiological conditions, is involved in the folding of key molecules of the cellular signal transduction system such as kinases and steroid receptors . It seems to contain two chaperone sites differing in substrate specificity . Binding of ATP or the antitumor drug geldanamycin alters the substrate affinity of the N-terminal chaperone site, whereas both substances show no influence on the C-terminal one . In wild-type Hsp90 the fragments containing the chaperone sites are connected by a highly charged linker of various lengths in different organisms . As this linker region represents the most striking difference between bacterial and eukaryotic Hsp90s, it may be involved in a gain of function of eukaryotic Hsp90s . Here, we have analyzed a fragment of yeast Hsp90 consisting of the N-terminal domain and the charged region (N272) in comparison with the isolated N-terminal domain (N210) . We show that the charged region causes an increase in the affinity of the N-terminal domain for nonnative protein and establishes a crosstalk between peptide and ATP binding . Thus, the binding of peptide to N272 decreases its affinity for ATP and geldanamycin, whereas the ATP-binding properties of the monomeric N-terminal domain N210 are not influenced by peptide binding . We propose that the charged region connecting the two chaperone domains plays an important role in regulating chaperone function of Hsp90.

Nat Genet, 1999 Feb, 21(2), 204 - 8
Interaction between Set1p and checkpoint protein Mec3p in DNA repair and telomere functions; Corda Y et al.; The yeast protein Set1p, inactivation of which alleviates telomeric position effect (TPE), contains a conserved SET domain present in chromosomal proteins involved in epigenetic control of transcription . Mec3p is required for efficient DNA-damage-dependent checkpoints at G1/S, intra-S and G2/M (refs 3-7) . We show here that the SET domain of Set1p interacts with Mec3p . Deletion of SET1 increases the viability of mec3delta mutants after DNA damage (in a process that is mostly independent of Rad53p kinase, which has a central role in checkpoint control) but does not significantly affect cell-cycle progression . Deletion of MEC3 enhances TPE and attenuates the Set1delta-induced silencing defect . Furthermore, restoration of TPE in a Set1delta mutant by overexpression of the isolated SET domain requires Mec3p . Finally, deletion of MEC3 results in telomere elongation, whereas cells with deletions of both SET1 and MEC3 do not have elongated telomeres . Our findings indicate that interactions between SET1 and MEC3 have a role in DNA repair and telomere function.

J Biol Chem, 1999 Feb 19, 274(8), 5227 - 35
In vitro evolution of preferred topoisomerase II DNA cleavage sites; Burden DA et al.; Topoisomerase II is an essential enzyme that is the target for several clinically important anticancer drugs . Although this enzyme must create transient double-stranded breaks in the genetic material in order to carry out its indispensable DNA strand passage reaction, the factors that underlie its nucleotide cleavage specificity remain an enigma . Therefore, to address the critical issue of enzyme specificity, a modified systematic evolution of ligands by exponential enrichment (SELEX) protocol was employed to select/evolve DNA sequences that were preferentially cleaved by Drosophila melanogaster topoisomerase II . Levels of DNA scission rose substantially (from 3 to 20%) over 20 rounds of SELEX . In vitro selection/evolution converged on an alternating purine/pyrmidine sequence that was highly AT-rich (TATATATACATATATATA) . The preference for this sequence was more pronounced for Drosophila topoisomerase II over other species and was increased in the presence of DNA cleavage-enhancing anticancer drugs . Enhanced cleavage appeared to be based on higher rates of DNA scission rather than increased binding affinity or decreased religation rates . The preferred sequence for topoisomerase II-mediated DNA cleavage is dramatically overrepresented ( approximately 10,000-fold) in the euchromatic genome of D . melanogaster, implying that it may be a site for the physiological action of this enzyme.

Mol Microbiol, 1998 Dec, 30(5), 1041 - 50
The E protein of satellite phage P4 acts as an anti-repressor by binding to the C protein of helper phage P2; Liu T et al.; Temperate phage P2 has the capacity to function as a helper for the defective, unrelated, satellite phage P4 . In the absence of a helper, P4 can either lysogenize its host or establish itself as a plasmid . For lytic growth, P4 requires the structural genes, packaging and lysis functions of the helper . P4 can get access to the late genes of prophage P2 by derepression, which is mediated by the P4 E protein . E has been hypothesized to function as an anti-repressor . To locate possible epitopes interacting with E, an epitope display library was screened against E, and the most frequent sequence found had some identities to a region within P2 C . Using the yeast two-hybrid system, a clear activation of a reporter gene was found, strongly supporting an interaction between E and C . The P2 C repressor is believed to act as a dimer, which is confirmed in this work using in vivo dimerization studies . The E protein was also found to form dimers in vivo . The E protein only affects dimerization of C marginally, but the presence of E enhances multimeric forms of C . Furthermore, binding of the C protein to its operator is inhibited by E in vitro, indicating that the anti-repressor function of E is mediated by the formation of multimeric complexes of E and C that interfere with the binding of C to its operator.

Kidney Int, 1999 Feb, 55(2), 476 - 85
Activation of mesangial cell signaling cascades in response to mechanical strain; Ingram AJ et al.; BACKGROUND: Mesangial cells (MCs) are constantly exposed to pulsatile stretch and relaxation in their role as architectural support for the glomerulus . There is no cell proliferation in normal glomeruli . In contrast, animal models of increased glomerular capillary pressure are characterized by resident glomerular cell proliferation and elaboration of extracellular matrix (ECM) protein, resulting in glomerulosclerosis . This process can be ameliorated by maneuvers, such as angiotensin converting enzyme inhibition, that reduce glomerular capillary pressure . MCs grown on ECM-coated plates and exposed to cyclic stretch/relaxation proliferate and produce ECM protein, suggesting that this may be a useful in vitro model for MC behavior in response to increased physical forces . Previous work has shown induction of c-fos in response to application of mechanical strain to MCs, which may induce increases in AP-1 transcription factor activity, which, in turn, may augment ECM protein and transforming growth factor beta transcription and cell proliferation . Stimuli that lead to c-fos induction pass through mitogen-activated protein kinase (MAPK) pathways . Three MAPK cascades have been characterized in mammalian cells--p44/42 (classic MAPK), the stress-activated protein kinase/Jun terminal kinase (SAPK/JNK) pathway, and p38/HOG--and mechanical strain activates p44/42 and SAPK/JNK in cardiac fibroblasts . However, in contrast to MCs, these cells do not proliferate in response to physical force . Accordingly, we studied activation of the MAPK pathways in MCs exposed to mechanical strain . METHODS: MCs (passages 5 to 10) cultured on type 1 collagen-coated, flexible-bottom plates were exposed to 30, 60, or 120 minutes of cyclic strain (60 cycles/min) by computer-driven generation of vacuums of -14 and -28 kPa, inducing 20% and 29% elongations in the diameter of the surfaces, respectively . Control MCs were grown on coated rigid bottom plates . Proliferation was assessed at 24 hours by 3H-thymidine incorporation . Protein levels (by Western blot) and activity assays for all three kinase cascades were performed at 30, 60, and 120 minutes . RESULTS: Cyclic strain/relaxation lead to an approximate doubling of 3H-thymidine incorporation at 24 hours (N = 3, P < 0.05) only in cultures stretched 29%, but not in cultures stretched 20% . At -29% elongation, the increase in 3H-thymidine incorporation was preceded by early activation of MAPK signaling pathways . p44/42 activity increased to a maximum of eightfold greater than control at 60 minutes . p38/HOG activity was not measurable at baseline but was increased markedly at 30 minutes, which was sustained through to 120 minutes . SAPK/JNK activity was present at a very low level in MCs and was not changed by stretch . However, it was markedly increased by sorbitol . In MCs stretched to 20% elongation, lesser increases in p44/42 were seen with a similar time course, whereas no increases in p38/HOG or SAPK could be detected at the time points studied . No increase in any kinase pathway activity was seen at any time in static cultures . CONCLUSIONS: High-pressure cyclic stretch leads to MC proliferation, preceded by marked activation of p44/42 and p38/HOG MAPKs . Cell proliferation is not seen with low-pressure stretch, and there is only modest p44/42 MAPK activation, suggesting that glomerular capillary hypertension may lead to cell proliferation and injury partly through differential activation of kinase cascades.

J Magn Reson, 1999 Feb, 136(2), 137 - 42
Dielectric resonator-based flow and stopped-flow EPR with rapid field scanning: A methodology for increasing kinetic information; Sienkiewicz A et al.; We report methodology which combines recently developed dielectric resonator-based, rapid-mix, stopped-flow EPR (appropriate for small, aqueous, lossy samples) with rapid scanning of the external (Zeeman) magnetic field where the scanning is preprogrammed to occur at selected times after the start of flow . This methodology gave spectroscopic information complementary to that obtained by stopped-flow EPR at single fields, and with low reactant usage, it yielded more graphic insight into the time evolution of radical and spin-labeled species . We first used the ascorbyl radical as a test system where rapid scans triggered after flow was stopped provided "snapshots" of simultaneously evolving and interacting radical species . We monitored ascorbyl radical populations either as brought on by biologically damaging peroxynitrite oxidant or as chemically and kinetically interacting with a spectroscopically overlapping nitroxide radical . In a different biophysical application, where a spin-label lineshape reflected rapidly changing molecular dynamics of folding spin-labeled protein, rapid scan spectra were taken during flow with different flow rates and correspondingly different times after the mixing-induced inception of protein folding . This flow/rapid scan method is a means for monitoring early immobilization of the spin probe in the course of the folding process .

Science, 1999 Feb 12, 283(5404), 996 - 8
A copper cofactor for the ethylene receptor ETR1 from Arabidopsis; Rodriguez FI et al.; The ETR1 receptor from Arabidopsis binds the gaseous hormone ethylene . A copper ion associated with the ethylene-binding domain is required for high-affinity ethylene-binding activity . A missense mutation in the domain that renders the plant insensitive to ethylene eliminates both ethylene binding and the interaction of copper with the receptor . A sequence from the genome of the cyanobacterium Synechocystis sp . strain 6803 that shows homology to the ethylene-binding domain of ETR1 encodes a functional ethylene-binding protein . On the basis of sequence conservation between the Arabidopsis and the cyanobacterial ethylene-binding domains and on in vitro mutagenesis of ETR1, a structural model for this copper-based ethylene sensor domain is presented.

Nucleic Acids Res, 1999 Mar 1, 27(5), 1350 - 8
Binding of the glucose-dependent Mig1p repressor to the GAL1 and GAL4 promoters in vivo: regulationby glucose and chromatin structure; Frolova E et al.; Binding of the MIG1 repressor to the glucose-repressible GAL1 and GAL4 promoters was analyzed in vivo by cyclobutane dimer footprinting in two yeast strains that show different glucose repression responses . Mig1p binding to the two promoters in both strains was glucose-induced . In cells subject to rapid and stringent glucose repression (S288c), long-term Mig1p binding in glucose-grown cells was inhibited by the formation of a competing chromatin structure . Under conditions where glucose repression was only partially effective (gal80 - or low glucose), the chromatin structure did not form and long-term Mig1p binding was observed The same long-term binding of Mig1p was seen in cells of a different strain (W303A) that shows only partial glucose repression of the GAL1 promoter . We conclude from these experiments that Mig1p binding to glucose-repressed promoters is glucose-dependent but transient . We suggest that Mig1p functions at an early step in repression, but is not required to maintain the repressed state.

Nucleic Acids Res, 1999 Mar 1, 27(5), 1308 - 15
Transfer RNA modification enzymes from Pyrococcus furiosus: detection of the enzymatic activities in vitro; Constantinesco F et al.; The modification patterns of in vitro transcripts of two yeast Saccharomyces cerevisiae tRNAs (tRNAPheand tRNAAsp) and one archaeal Haloferax volcanii tRNA (tRNAIle) were investigated in the cell-free extract of Pyrococcus furiosus supplemented with S -adenosyl-l-methionine (AdoMet) . The results indicate that the enzymatic formation of 11 distinct modified nucleotides corresponding to 12 enzymatic activities can be detected in vitro . They correspond to the formation of pseudouridines (Psi) at positions 39 and 55, 2' -O- ribose methylations at positions 6 (Am) and 56 (Cm), base methylations at positions 10 (m2G), 26 (m22G), 37 (m1G), 49 (m5C), 54 (m5U) and 58 (m1A) and both the deamination and methylation of adenosine into m1I at position 57 . Most of the detected modified nucleotides are common modifications found in other phylogenetic groups, while Am6, Cm56and m1I57are specific modifications found exclusively in Archaea . It is also shown that the enzymatic formation of m5C49, m5U54, Psi55and m1I57does not depend on the three-dimensional architecture of the tRNA substrate, since these modi-fications also occur in fragmented tRNAs as substrate.

J Immunol, 1999 Feb 1, 162(3), 1376 - 83
Monocytes stimulate expression of the Bcl-2 family member, A1, in endothelial cells and confer protection against apoptosis; Noble KE et al.; We have investigated the molecular mechanisms underlying the ability of peripheral blood monocytes to block apoptosis induction in endothelial cells . Monocytes stimulated the expression of the bcl-2 homologue A1 in serum-starved endothelial cells after 6 h of coincubation, with elevated A1 levels persisting for up to 21 h . IL-1 and TNF also stimulated A1 expression at 6 h, but A1 transcript levels fell by 21 h . Direct cellular contact with monocytes was required for stimulation of A1 mRNA in endothelial cells . Stimulation of endothelial cell A1 mRNA by monocytes was not inhibited by anti-beta2 integrin Abs, but anti-platelet endothelial cell adhesion molecule-1 (PECAM-1) mAb reduced A1 transcript levels at 21 h . Studies employing either TNF on its own, or anti-TNF in endothelium/monocyte cocultures showed that TNF plays a role in the early (6-h) stimulation of A1, but is less important for the sustained elevation of A1 levels at 21 h . Serum-starved endothelial cells demonstrated increased survival and decreased apoptosis after coculture with monocytes . IL-10 reduced A1 mRNA expression in, as well as survival of, endothelial cells that were cocultured with monocytes . In comparison with A1, Bcl-2 was expressed at low levels and was up-regulated by monocytes only at 21 h, while neither Bax nor Bcl-xL levels were altered by monocytes . The interaction of monocytes with endothelium during the course of an inflammatory reaction may provide survival signals to endothelial cells.

Mol Endocrinol, 1999 Feb, 13(2), 286 - 96
Estrogen receptor domains E and F: role in dimerization and interaction with coactivator RIP-140; Peters GA et al.; We have used the yeast two-hybrid system to localize the ligand-dependent dimerization domain of the estrogen receptor-alpha (ER) to region E in vivo . In this system, the cDNAs corresponding to the A-D, E, E/F, A-E (deltaF), and full-length (wtER) domains of the human ER were each cloned into the yeast two-hybrid vectors GAL4 DB and GAL4 TA and expressed in different combinations in yeast harboring a GAL1-lacZ reporter . The reporter was used as a relative measure of the interaction between the ER domains, through reconstitution of GAL4 activity . We found that the interaction of E or E/F domains of the ER with full-length ER is estradiol dependent and estrogen responsive element independent, as measured by the reconstitution of GAL4 activity from GAL4-E domain-containing fusion protein interactions . In the presence of F domain, this activity is reduced 10-fold . The results suggest that sequences in the F domain are inhibitory to the dimerization signal that is present in the E region . We propose that the full-length ER contains intrinsic dimerization restraints contributed by regions outside domain E that are released upon binding hormone agonist . In addition, we have demonstrated that coactivator RIP140 is able to interact with the ER in vivo at the E domain of the receptor in the presence of estrogen . Yeast two-hybrid analysis shows that RIP140 does not homodimerize in the presence or absence of estrogens . We present evidence showing that the ER has the inherent ability to interact with RIP140 in the presence of antiestrogens, but sequences inherent in the ER itself that are present outside of the E domain compromise this ability.

J Electron Microsc (Tokyo), 1998, 47(6), 671 - 4
Molecular structure of human topoisomerase II alpha revealed by atomic force microscopy; Nettikadan SR et al.; The entire human topoisomerase II alpha (hTopoII alpha) dimer was expressed in the yeast Saccaromyces cerevisiae, purified to homogeneity, and subjected to atomic force microscopy (AFM) under a tapping mode . Molecular images obtained exhibited a 'heart or donut-like' structure with a large axial hole . The main benefit of the application of AFM to study the hTopoII alpha is that clear images of the internal 'pore' have been achieved without crystallization, staining, or fixation of the sample . These images are consistent with the model in which topoisomerase II has a large internal gate for DNA strand passage.

Biosci Biotechnol Biochem, 1998 Dec, 62(12), 2364 - 70
Isolation of the creA gene from the cellulolytic fungus Humicola grisea and analysis of CreA binding sites upstream from the cellulase genes; Takashima S et al.; A carbon catabolite repressor gene, creA, was isolated from the cellulolytic fungus Humicola grisea by using a portion of the Trichoderma reesei cre1 gene as a probe . The deduced amino acid sequence predicts a zinc finger protein of 419 amino acids in length, and its zinc finger regions show high similarity with those of Aspergillus CreAs, T . reesei Cre1, and Saccharomyces cerevisiae MIG1 . Northern blot analysis showed that the H . grisea creA gene was highly transcribed when the mycelia were grown on glucose-containing media, but the transcription of the H . grisea endoglucanase 1 gene (egl1) and the exoglucanase 1 gene (exo1) were repressed under these conditions . Results of binding assays with the maltose-binding protein::CreA(1-166) fusion protein and the egl1 and the exo1 upstream regions showed that some 6-bp sites having an identical or similar sequence to the consensus sequence for CreA binding were protected from DNase I digestion.

J Cell Biol, 1999 Feb 8, 144(3), 435 - 46
The intermediate filament protein peripherin is the specific interaction partner of mouse BPAG1-n (dystonin) in neurons; Leung CL et al.; The dystonia musculorum (dt) mouse suffers from severe degeneration of primary sensory neurons . The mutated gene product is named dystonin and is identical to the neuronal isoform of bullous pemphigoid antigen 1 (BPAG1-n) . BPAG1-n contains an actin-binding domain at its NH2 terminus and a putative intermediate filament-binding domain at its COOH terminus . Because the degenerating sensory neurons of dt mice display abnormal accumulations of intermediate filaments in the axons, BPAG1-n has been postulated to organize the neuronal cytoskeleton by interacting with both the neurofilament triplet proteins (NFTPs) and microfilaments . In this paper we show by a variety of methods that the COOH-terminal tail domain of mouse BPAG1 interacts specifically with peripherin, but in contrast to a previous study (Yang, Y., J . Dowling, Q.C . Yu, P . Kouklis, D.W . Cleveland, and E . Fuchs . 1996 . Cell . 86:655-665), mouse BPAG1 fails to associate with full-length NFTPs . The tail domains interfered with the association of the NFTPs with BPAG1 . In dt mice, peripherin is present in axonal swellings of degenerating sensory neurons in the dorsal root ganglia and is downregulated even in other neural regions, which have no obvious signs of pathology . Since peripherin and BPAG1-n also display similar expression patterns in the nervous system, we suggest that peripherin is the specific interaction partner of BPAG1-n in vivo.

J Cell Biol, 1999 Feb 8, 144(3), 403 - 11
Mammalian homologue of the Caenorhabditis elegans UNC-76 protein involved in axonal outgrowth is a protein kinase C zeta-interacting protein; Kuroda S et al.; By the yeast two-hybrid screening of a rat brain cDNA library with the regulatory domain of protein kinase C zeta (PKCzeta) as a bait, we have cloned a gene coding for a novel PKCzeta-interacting protein homologous to the Caenorhabditis elegans UNC-76 protein involved in axonal outgrowth and fasciculation . The protein designated FEZ1 (fasciculation and elongation protein zeta-1) consisting of 393 amino acid residues shows a high Asp/Glu content and contains several regions predicted to form amphipathic helices . Northern blot analysis has revealed that FEZ1 mRNA is abundantly expressed in adult rat brain and throughout the developmental stages of mouse embryo . By the yeast two-hybrid assay with various deletion mutants of PKC, FEZ1 was shown to interact with the NH2-terminal variable region (V1) of PKCzeta and weakly with that of PKCepsilon . In the COS-7 cells coexpressing FEZ1 and PKCzeta, FEZ1 was present mainly in the plasma membrane, associating with PKCzeta and being phosphorylated . These results indicate that FEZ1 is a novel substrate of PKCzeta . When the constitutively active mutant of PKCzeta was used, FEZ1 was found in the cytoplasm of COS-7 cells . Upon treatment of the cells with a PKC inhibitor, staurosporin, FEZ1 was translocated from the cytoplasm to the plasma membrane, suggesting that the cytoplasmic translocation of FEZ1 is directly regulated by the PKCzeta activity . Although expression of FEZ1 alone had no effect on PC12 cells, coexpression of FEZ1 and constitutively active PKCzeta stimulated the neuronal differentiation of PC12 cells . Combined with the recent finding that a human FEZ1 protein is able to complement the function of UNC-76 necessary for normal axonal bundling and elongation within axon bundles in the nematode, these results suggest that FEZ1 plays a crucial role in the axon guidance machinery in mammals by interacting with PKCzeta.

J Cell Biol, 1999 Feb 8, 144(3), 389 - 401
A novel in vivo assay reveals inhibition of ribosomal nuclear export in ran-cycle and nucleoporin mutants; Hurt E et al.; To identify components involved in the nuclear export of ribosomes in yeast, we developed an in vivo assay exploiting a green fluorescent protein (GFP)-tagged version of ribosomal protein L25 . After its import into the nucleolus, L25-GFP assembles with 60S ribosomal subunits that are subsequently exported into the cytoplasm . In wild-type cells, GFP-labeled ribosomes are only detected by fluorescence in the cytoplasm . However, thermosensitive rna1-1 (Ran-GAP), prp20-1 (Ran-GEF), and nucleoporin nup49 and nsp1 mutants are impaired in ribosomal export as revealed by nuclear accumulation of L25-GFP . Furthermore, overexpression of dominant-negative RanGTP (Gsp1-G21V) and the tRNA exportin Los1p inhibits ribosomal export . The pattern of subnuclear accumulation of L25-GFP observed in different mutants is not identical, suggesting that transport can be blocked at different steps . Thus, nuclear export of ribosomes requires the nuclear/cytoplasmic Ran-cycle and distinct nucleoporins . This assay can be used to identify soluble transport factors required for nuclear exit of ribosomes.

Plant Physiol, 1999 Feb, 119(2), 543 - 52
Apyrase functions in plant phosphate nutrition and mobilizes phosphate from extracellular ATP; Thomas C et al.; ATP, which is present in the extracellular matrix of multicellular organisms and in the extracellular fluid of unicellular organisms, has been shown to function as a signaling molecule in animals . The concentration of extracellular ATP (xATP) is known to be functionally modulated in part by ectoapyrases, membrane-associated proteins that cleave the gamma- and beta-phosphates on xATP . We present data showing a previously unreported (to our knowledge) linkage between apyrase and phosphate transport . An apyrase from pea (Pisum sativum) complements a yeast (Saccharomyces cerevisiae) phosphate-transport mutant and significantly increases the amount of phosphate taken up by transgenic plants overexpressing the gene . The transgenic plants show enhanced growth and augmented phosphate transport when the additional phosphate is supplied as inorganic phosphate or as ATP . When scavenging phosphate from xATP, apyrase mobilizes the gamma-phosphate without promoting the transport of the purine or the ribose.

Plant Physiol, 1999 Feb, 119(2), 507 - 10
Arabidopsis ent-kaurene oxidase catalyzes three steps of gibberellin biosynthesis; Helliwell CA et al.; The Arabidopsis GA3 cDNA was expressed in yeast (Saccharomyces cerevisiae) and the ability of the transformed yeast cells to metabolize ent-kaurene was tested . We show by full-scan gas chromatography-mass spectrometry that the transformed cells produce ent-kaurenoic acid, and demonstrate that the single enzyme GA3 (ent-kaurene oxidase) catalyzes the three steps of gibberellin biosynthesis from ent-kaurene to ent-kaurenoic acid.

Exp Gerontol, 1998 Nov-Dec, 33(7-8), 773 - 83
Genetics of longevity; Jazwinski SM; Recent studies on the genetics of aging in the yeast Saccharomyces cerevisiae, the roundworm Caenorhabditis elegans, and the fruit fly Drosophila melanogaster have converged revealing the central role of metabolic capacity and resistance to stress in determining life span . Signal transduction has emerged from these studies as an important molecular mechanism underlying longevity . In their broad features, the results obtained in these genetic models are applicable to the dietary restriction paradigm in mammals, suggesting a general significance . It will be of interest to determine whether many of the molecular details will also pertain . The examination of centenarian populations for the frequency of certain alleles of pertinent genes may provide insights into the relevance of the conclusions of studies in invertebrates to human aging . These population genetic studies can be augmented by mechanistic studies in transgenic mice.

Nature, 1999 Jan 28, 397(6717), 363 - 8
TPL-2 kinase regulates the proteolysis of the NF-kappaB-inhibitory protein NF-kappaB1 p105; Belich MP et al.; The transcription factor NF-kappaB is composed of homodimeric and heterodimeric complexes of Rel/NF-kappaB-family polypeptides, which include Rel-A, c-Rel, Rel-B, NF-kappaB/p50 and NF-kappaB2/p52 . The NF-kappaB1 gene encodes a larger precursor protein, p105, from which p50 is produced constitutively by proteasome-mediated removal of the p105 carboxy terminus . The p105 precursor also acts as an NFkappaB-inhibitory protein, retaining associated p50, c-Rel and Rel-A proteins in the cytoplasm through its carboxy terminus . Following cell stimulation by agonists, p105 is proteolysed more rapidly and released Rel subunits translocate into the nucleus . Here we show that TPL-2 , which is homologous to MAP-kinase-kinase kinases in its catalytic domain, forms a complex with the carboxy terminus of p105 . TPL-2 was originally identified, in a carboxy-terminal-deleted form, as an oncoprotein in rats and is more than 90% identical to the human oncoprotein COT . Expression of TPL-2 results in phosphorylation and increased degradation of p105 while maintaining p50 production . This releases associated Rel subunits or p50-Rel heterodimers to generate active nuclear NF-kappaB . Furthermore, kinase-inactive TPL-2 blocks the degradation of p105 induced by tumour-necrosis factor-alpha . TPL-2 is therefore a component of a new signalling pathway that controls proteolysis of NF-kappaB1 p105.

Nature, 1999 Jan 28, 397(6717), 355 - 9
Secondary V(D)J recombination in B-1 cells; Qin XF et al.; B-1 B cells are a self-renewing population of B cells that differ from conventional B cells (B-2 cells) in that they are particularly predisposed to auto-antibody production . Although much is known about the signalling pathways that control B-1-cell growth and development, less is known about why these cells are prone to produce autoreactive antibodies . Here we show that B-1 cells, like germinal-centre B cells, can express recombinase-activating genes 1 and 2 (RAG1 and RAG2) and undergo secondary V(D)J recombination of immunoglobulin genes . In addition, B cells from autoimmune-prone NZB mice show high levels of RAG messenger RNA and recombination . We propose that secondary immunoglobulin-gene rearrangements outside organized lymphoid organs may contribute to the development of autoreactive antibodies.

Mol Biol Cell, 1999 Feb, 10(2), 435 - 53
Morphological and functional association of Sec22b/ERS-24 with the pre-Golgi intermediate compartment; Zhang T et al.; Yeast Sec22p participates in both anterograde and retrograde vesicular transport between the endoplasmic reticulum (ER) and the Golgi apparatus by functioning as a v-SNARE (soluble N-ethylmaleimide-sensitive factor {NSF} attachment protein receptor) of transport vesicles . Three mammalian proteins homologous to Sec22p have been identified and are referred to as Sec22a, Sec22b/ERS-24, and Sec22c, respectively . The existence of three homologous proteins in mammalian cells calls for detailed cell biological and functional examinations of each individual protein . The epitope-tagged forms of all three proteins have been shown to be primarily associated with the ER, although functional examination has not been carefully performed for any one of them . In this study, using antibodies specific for Sec22b/ERS-24, it is revealed that endogenous Sec22b/ERS-24 is associated with vesicular structures in both the perinuclear Golgi and peripheral regions . Colabeling experiments for Sec22b/ERS-24 with Golgi mannosidase II, the KDEL receptor, and the envelope glycoprotein G (VSVG) of vesicular stomatitis virus (VSV) en route from the ER to the Golgi under normal, brefeldin A, or nocodazole-treated cells suggest that Sec22b/ERS-24 is enriched in the pre-Golgi intermediate compartment (IC) . In a well-established semi-intact cell system that reconstitutes transport from the ER to the Golgi, transport of VSVG is inhibited by antibodies against Sec22b/ERS-24 . EGTA is known to inhibit ER-Golgi transport at a stage after vesicle/transport intermediate docking but before the actual fusion event . Antibodies against Sec22b/ERS-24 inhibit ER-Golgi transport only when they are added before the EGTA-sensitive stage . Transport of VSVG accumulated in pre-Golgi IC by incubation at 15 degreesC is also inhibited by Sec22b/ERS-24 antibodies . Morphologically, VSVG is transported from the ER to the Golgi apparatus via vesicular intermediates that scatter in the peripheral as well as the Golgi regions . In the presence of antibodies against Sec22b/ERS-24, VSVG is seen to accumulate in these intermediates, suggesting that Sec22b/ERS-24 functions at the level of the IC in ER-Golgi transport.

Mol Biol Cell, 1999 Feb, 10(2), 329 - 44
Detection of transient in vivo interactions between substrate and transporter during protein translocation into the endoplasmic reticulum; Dunnwald M et al.; The split-ubiquitin technique was used to detect transient protein interactions in living cells . Nub, the N-terminal half of ubiquitin (Ub), was fused to Sec62p, a component of the protein translocation machinery in the endoplasmic reticulum of Saccharomyces cerevisiae . Cub, the C-terminal half of Ub, was fused to the C terminus of a signal sequence . The reconstitution of a quasi-native Ub structure from the two halves of Ub, and the resulting cleavage by Ub-specific proteases at the C terminus of Cub, serve as a gauge of proximity between the two test proteins linked to Nub and Cub . Using this assay, we show that Sec62p is spatially close to the signal sequence of the prepro-alpha-factor in vivo . This proximity is confined to the nascent polypeptide chain immediately following the signal sequence . In addition, the extent of proximity depends on the nature of the signal sequence . Cub fusions that bore the signal sequence of invertase resulted in a much lower Ub reconstitution with Nub-Sec62p than otherwise identical test proteins bearing the signal sequence of prepro-alpha-factor . An inactive derivative of Sec62p failed to interact with signal sequences in this assay . These in vivo findings are consistent with Sec62p being part of a signal sequence-binding complex.

Bioorg Med Chem Lett, 1998 Dec 15, 8(24), 3627 - 30
Synthesis of 6(alpha, beta)-aminocholestanols as ergosterol biosynthesis inhibitors; Beuchet P et al.; delta 7-5-Desaturase catalyses one of the last steps in ergosterol biosynthesis in fungi . Moreover delta 5-unsaturation is necessary for the sparking function . Synthesis of three pairs of C-6 epimeric cholestanol derivatives are described as potential growth inhibitors . Preliminary results suggest that 6 beta-aminocholestanol is a potent antifungal agent.

Can J Microbiol, 1998 Oct, 44(10), 945 - 53
Localization of a phosphatidylglycerol/phosphatidylinositol transfer protein in Aspergillus oryzae; Record E et al.; The subcellular localization of the phosphatidylglycerol/phosphatidylinositol transfer protein (PG/PI-TP) of Aspergillus oryzae was investigated using Western blot analysis of the cell protein extracts, a cellular membrane fractionation technique, and transmission electron microscopy . The PG/PI-TP, as detected by Western blot analysis with a specific immune serum, was found to be mainly cytoplasmic and partly associated with intracellular membranes . A fractionation experiment was conducted after homogenization of the filamentous fungus mycelium . The endoplasmic reticulum, Golgi-like vesicles, and the plasma membrane were separated by isopycnic ultracentrifugation on a sucrose gradient, and our data revealed that the immunodetected PG/PI-TP was only associated with the Golgi-like apparatus . All these results were documented by electron microscopy and indicate here for the first time that there exists a specific phospholipid transfer protein in a filamentous fungus that is localized in the cytoplasm and associated with Golgi-like vesicles.

J Biol Chem, 1999 Feb 12, 274(7), 4389 - 99
Cargo can modulate COPII vesicle formation from the endoplasmic reticulum; Aridor M et al.; The COPII coat complex found on endoplasmic reticulum (ER)-derived vesicles plays a critical role in cargo selection . We now address the potential role of biosynthetic cargo in modulating COPII coat assembly and vesicle budding . The ER accumulation of vesicular stomatitis glycoprotein (VSV-G), a transmembrane protein, or the soluble PiZ variant of alpha1-antitrypsin, reduced levels of general COPII vesicle formation in vivo . Consistent with this result, conditions that prevent the export of VSV-G from the ER led to a significant inhibition of general COPII vesicle budding from ER microsomes and the export of an endogenous recycling protein p58 in vitro . In contrast, synchronized export of VSV-G stimulated COPII vesicle budding both in vivo and in vitro . Under conditions where VSV-G is retained in the ER, we find that it can to be recovered in pre-budding complexes containing COPII components . These results suggest that the export of biosynthetic cargo is integrated with ER functions involved in protein folding and oligomerization . The ability of biosynthetic cargo to prevent or enhance ER export suggests that interactions of cargo with the COPII machinery contribute to the formation of vesicles budding from the ER.

J Biol Chem, 1999 Feb 12, 274(7), 4347 - 53
Sustained activation of the mitogen-activated protein kinase pathway . A mechanism underlying receptor tyrosine kinase specificity for matrix metalloproteinase-9 induction and cell migration; McCawley LJ et al.; Activation of the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) pathway is required for ligand-dependent regulation of numerous cellular functions by receptor tyrosine kinases . We have shown previously that although many receptor tyrosine kinase ligands are mitogens for keratinocytes, cell migration and induction of the 92-kilodalton gelatinase/matrix metalloproteinase (MMP)-9 are selectively regulated by the epidermal growth factor and scatter factor/hepatocyte growth factor receptors . In this report we present evidence of an underlying mechanism to account for these observed differences in receptor tyrosine kinase-mediated response . Ligands that are mitogenic, but do not induce MMP-9 or colony dispersion, transiently activate the p42/p44 ERK/MAP kinases . In contrast, ligands that stimulate MMP-9 induction and colony dispersion induced sustained activation of these kinases . The functional significance of sustained MAPK activation was demonstrated by inhibition of the MAP kinase kinase MEK1 . Disruption of the prolonged signal by addition of the MEK1 inhibitor PD 98059 up to 4 h after growth factor stimulation substantially impaired ligand-dependent colony dispersion and MMP-9 induction . These findings support the conclusion that duration of MAPK activation is an important determinant for certain growth factor-mediated functions in keratinocytes.

J Biol Chem, 1999 Feb 12, 274(7), 4147 - 54
A short conserved motif is required for repressor domain function in the myeloid-specific transcription factor CCAAT/enhancer-binding protein epsilon; Angerer ND et al.; CCAAT/enhancer-binding protein epsilon (C/EBPepsilon) is expressed almost exclusively in the myeloid lineage of the hematopoietic system and functions during terminal differentiation of neutrophils and macrophages, and in the regulation of cytokine gene expression in macrophages and T cells . We have undertaken a series of structure/function studies on the murine C/EBPepsilon polypeptide to investigate the mechanism by which C/EBPepsilon activates transcription . Studies with deletion mutants and fusion proteins consisting of C/EBPepsilon sequences joined to the Gal4 DNA-binding protein identified two transcriptional activation domains in C/EBPepsilon . Removal of sequences between the two activation domains or sequences between the second activation domain and the C-terminal DNA binding domain significantly increased the activity of C/EBPepsilon, suggesting the presence of two separate regulatory domains (designated RD-1epsilon and RD-2epsilon) . RD-1epsilon behaved as a classic active repressor domain being capable of inhibiting adjacent activation domains irrespective of their origin and when linked to a heterologous DNA binding domain . Mutagenesis studies revealed a short motif in RD-1epsilon that appears to be a target site for protein-protein interactions and is conserved in repressor domains from C/EBPbeta, Sp3, c-Fos, and FosB . The juxtaposition of activation and repressor domains may permit C/EBPepsilon to function as a transcriptional activator or repressor at different stages of myeloid differentiation or as an inducible transcriptional activator of cytokine genes.

J Biol Chem, 1999 Feb 12, 274(7), 4027 - 35
Regulation of exocytosis by cyclin-dependent kinase 5 via phosphorylation of Munc18; Fletcher AI et al.; Munc18a, a mammalian neuronal homologue of Saccharomyces cerevisiae Sec1p protein, is essential for secretion, likely as a result of its high affinity interaction with the target SNARE protein syntaxin 1a (where SNARE is derived from SNAP receptor (the soluble N-ethylmaleimide-sensitive fusion protein)) . However, this interaction inhibits vesicle SNARE interactions with syntaxin that are required for secretory vesicles to achieve competency for membrane fusion . As such, regulation of the interaction between Munc18a and syntaxin 1a may provide an important mechanism controlling secretory responsiveness . Cyclin-dependent kinase 5 (Cdk5), a member of the Cdc2 family of cell division kinases, co-purifies with Munc18a from rat brain, interacts directly with Munc18a in vitro, and utilizes Munc18a as a substrate for phosphorylation . We have now demonstrated that Cdk5 is capable of phosphorylating Munc18a in vitro within a preformed Munc18a.syntaxin 1a heterodimer complex and that this results in the disassembly of the complex . Using site-directed mutagenesis, the Cdk5 phosphorylation site on Munc18a was identified as Thr574 . Stimulation of secretion from neuroendocrine cells produced a corresponding rapid translocation of cytosolic Cdk5 to a particulate fraction and an increase of Cdk5 kinase activity . Inhibition of Cdk5 with olomoucine decreased evoked norepinephrine secretion from chromaffin cells, an effect not observed with the inactive analogue iso-olomoucine . The effects of olomoucine were independent of calcium influx as evidenced by secretory inhibition in permeabilized chromaffin cells and in cells under whole-cell voltage clamp . Furthermore, transfection and expression in chromaffin cells of a neural specific Cdk5 activator, p25, led to a strong increase in nicotinic agonist-induced secretory responses . Our data suggest a model whereby Cdk5 acts to regulate Munc18a interaction with syntaxin 1a and thereby modulates the level of vesicle SNARE interaction with syntaxin 1a and secretory responsiveness.

J Biol Chem, 1999 Feb 12, 274(7), 3941 - 5
Mitochondrial citrate synthase is immobilized in vivo; Haggie PM et al.; The enzymes of the tricarboxylic acid cycle in the mitochondrial matrix are proposed to form a multienzyme complex, in which there is channeling of substrates between enzyme active sites . However no direct evidence has been obtained in vivo for the involvement of these enzymes in such a complex . We have labeled the tricarboxylic acid cycle enzyme, citrate synthase 1, in the yeast Saccharomyces cerevisiae, by biosynthetic incorporation of 5-fluorotryptophan . Comparison of the 19F NMR resonance intensities from the labeled enzyme in the intact cell and in cell-free lysates indicated that the enzyme is motionally restricted in vivo, consistent with its participation in a multienzyme complex.

Curr Genet, 1999 Jan, 34(6), 419 - 29
GRC5 and NMD3 function in translational control of gene expression and interact genetically; Karl T et al.; The yeast gene, GRC5 (growth control), is a member of the highly conserved QM gene family, the human member of which has been associated with the suppression of Wilms' tumor . GRC5 encodes ribosomal protein L10, which is thought to play a regulatory role in the translational control of gene expression . A revertant screen identified four spontaneous revertants of the mutant grc5-1ts allele . Genetic and phenotypic analysis showed that these represent one gene, NMD3, and that the interaction of NMD3 and GRC5 is gene-specific . NMD3 was previously identified as a component of the nonsense-mediated mRNA decay pathway . The point mutations within NMD3 reported here may define a domain important for the functional interaction of Grc5p and Nmd3p.

J Pediatr Gastroenterol Nutr, 1999 Feb, 28(2), 137 - 42
Sacrosidase therapy for congenital sucrase-isomaltase deficiency; Treem WR et al.; BACKGROUND: The purpose of this study was to determine if sacrosidase, a liquid produced from Saccharomyces cerevisiae containing 6000 IU of sucrase activity per mg protein, prevented symptoms of diarrhea, abdominal cramps, gas, and bloating in patients with congenital sucrase-isomaltase deficiency (CSID) consuming a normal sucrose and carbohydrate-containing diet . METHODS: Twenty-eight children (aged 5 months to 11 years) underwent a randomized, double-blind trial consisting of two phases: 1) three sucrose breath H2 tests with three single-dose treatments (placebo, sacrosidase, and sacrosidase plus milk), and 2) a dose-response phase consisting of four multidose treatments, each for 10 days of full-strength sacrosidase, 1:10 dilution, 1:100 dilution, and 1:1000 dilution . Patients who weighed less than or equal to 15 kg received a dose of sacrosidase and those who weighed more than 15 kg received 2 ml . For the dose-response phase each patient consumed a normal diet . The number of stools and severity of symptoms were recorded daily for each concentration of sacrosidase administered and compared to a baseline period during which the patient took no sacrosidase and consumed a sucrose/starch-free diet . Data were analyzed using an ANOVA model and the nonparameter Wilcoxon signed-rank test . RESULTS: Breath H2 excretion decreased significantly when patients received sacrosidase or sacrosidase plus milk compared to placebo during sucrose breath tests . During the dose-response phase significant treatment differences were observed between the two higher concentrations and the two lower concentrations of sacrosidase for both total stools (p < 0.001) and total symptom score (p = 0.003) . Higher concentrations of sacrosidase were associated with fewer stools and a greater number of formed or hard stools compared to lower concentrations and compared to the baseline period . Higher concentrations were also associated with fewer symptoms of gas, abdominal cramps, or bloating, but no differences in vomiting . The only significant adverse event was wheezing in one child with a history of asthma . CONCLUSIONS: Sacrosidase is a safe, effective, well-accepted treatment to prevent gastrointestinal symptoms in patients with CSID consuming a normal diet.

J Bioenerg Biomembr, 1998 Oct, 30(5), 465 - 75
Genetic evidence for coenzyme Q requirement in plasma membrane electron transport; Santos-Ocana C et al.; Plasma membranes isolated from wild-type Saccharomyces cerevisiae crude membrane fractions catalyzed NADH oxidation using a variety of electron acceptors, such as ferricyanide, cytochrome c, and ascorbate free radical . Plasma membranes from the deletion mutant strain coq3delta, defective in coenzyme Q (ubiquinone) biosynthesis, were completely devoid of coenzyme Q6 and contained greatly diminished levels of NADH-ascorbate free radical reductase activity (about 10% of wild-type yeasts) . In contrast, the lack of coenzyme Q6 in these membranes resulted in only a partial inhibition of either the ferricyanide or cytochrome-c reductase . Coenzyme Q dependence of ferricyanide and cytochrome-c reductases was based mainly on superoxide generation by one-electron reduction of quinones to semiquinones . Ascorbate free radical reductase was unique because it was highly dependent on coenzyme Q and did not involve superoxide since it was not affected by superoxide dismutase (SOD) . Both coenzyme Q6 and NADH-ascorbate free radical reductase were rescued in plasma membranes derived from a strain obtained by transformation of the coq3delta strain with a single-copy plasmid bearing the wild type COQ3 gene and in plasma membranes isolated form the coq3delta strain grown in the presence of coenzyme Q6 . The enzyme activity was inhibited by the quinone antagonists chloroquine and dicumarol, and after membrane solubilization with the nondenaturing detergent Zwittergent 3-14 . The various inhibitors used did not affect residual ascorbate free radical reductase of the coq3delta strain . Ascorbate free radical reductase was not altered significantly in mutants atp2delta and cor1delta which are also respiration-deficient but not defective in ubiquinone biosynthesis, demonstrating that the lack of ascorbate free radical reductase in coq3delta mutants is related solely to the inability to synthesize ubiquinone and not to the respiratory-defective phenotype . For the first time, our results provide genetic evidence for the participation of ubiquinone in NADH-ascorbate free radical reductase, as a source of electrons for transmembrane ascorbate stabilization.

Toxicol Appl Pharmacol, 1999 Feb 1, 154(3), 228 - 35
Molecular genotoxicity profiles of apoptosis-inducing vanadocene complexes; Aubrecht J et al.; Metallocene complexes containing vanadium induce apoptosis in human cancer cells by an as yet unknown mechanism and may therefore be useful as a new class of cytotoxic anticancer drugs . Ultrastructural studies showing the formation of metallocene-DNA complexes prompted the hypothesis that their mechanism of action may resemble the DNA damage induced by cisplatin . Molecular genotoxicity testing provides insights into the mechanisms of action of new chemotherapeutic agents . Therefore, we determined the effects of three cytotoxic vanadocene complexes, vanadocene dichloride, vanadocene dithiocyanate, and vanadocene dioxycyanate, on genomic stability using the yeast DEL recombination assay and transcriptional activation of genotoxic stress-specific promoters in human HepG2 cells using the CAT-Tox(L) assay . Cisplatin caused an 11-fold increase of recombination frequency in yeast and induced transcriptional activation of the DNA damage-associated promoters such as the minimum promoter containing p53 response elements and the GADD45 promoter in addition to activating the promoters for c-fos, heat shock protein 70, metallothionine IIa, and the minimum promoter containing nuclear factor kappa(kappa)B response elements . In contrast to cisplatin, vanadocene complexes did not increase the DEL recombination frequency in yeast nor did they activate any of the DNA damage-associated promoters in HepG2 cells . Vanadocene complexes triggered activation of the c-fos promoter without affecting the minimum promoter containing p53 response elements or the GADD45 promoter . These results indicate that the apoptotic signal of vanadocene complexes is not triggered by primary DNA damage and it does not require p53 induction, thereby disproving the hypothesis that it mechanistically resembles the cytotoxic action of cisplatin .

J Mol Biol, 1999 Feb 12, 286(1), 1 - 13
DNA-sequence asymmetry directs the alignment of recombination sites in the FLP synaptic complex; Huffman KE et al.; The FLP recombinase promotes site-specific recombination in the 2 micrometer circle of Saccharomyces cerevisiae . FLP recognizes a 48 bp target site (FLP recombination target, or FRT) consisting of three 13 bp protein binding sites, or symmetry elements, flanking an 8 bp spacer region . Efficient recombination also occurs with DNA substrates that have minimal FRT sites, consisting only of the spacer and two surrounding 13 bp symmetry elements arranged in inverse orientation; thus, the wild-type spacer sequence is the main asymmetric feature of the minimal recombination site . FLP carries out recombination with many minimal target sites bearing symmetric or asymmetric mutant spacer sequences; however, the overall directionality of recombination defined in terms of inversion or excision of a DNA domain is determined by spacer-sequence asymmetry . In order to evaluate the potential influence of spacer-sequence asymmetry on structures formed during early steps in recombination, we used electron microscopy to investigate the structure of the FLP synaptic complex, which is the intermediate protein-DNA complex involved in site pairing and strand exchange . Using linear substrate DNAs that have minimal FRTs with wild-type spacer sequences, we find that 85 to 90% of the FLP synaptic complexes examined contain the two FRTs aligned in parallel . This strong preference for parallel site alignment stands in contrast with prevailing models for lambda integrase-class recombination systems, which postulate antiparallel site alignment, and results from biophysical studies on synthetic, immobile four-way DNA junctions . Our results show that the strong preference for parallel alignment can be attributed to conformational preferences of Holliday junctions present in the synaptosome .

Biochemistry, 1999 Jan 26, 38(4), 1365 - 70
Nonspecific weak actomyosin interactions: relocation of charged residues in subdomain 1 of actin does not alter actomyosin function; Wong WW et al.; Yeast actin mutants with relocated charged residues within subdomain 1 were constructed so we could investigate the functional importance of individual clusters of acidic residues in mediating actomyosin weak-binding states in the cross-bridge cycle . Past studies have established a functional role for three distinct pairs of charged residues within this region of yeast actin (D2/E4, D24/D25, and E99/E100); the loss of any one of these pairs resulted in the same impairment in weak actomyosin interaction and in its function . However, the specificity of myosin interaction with these sites has not yet been addressed . To investigate this, we made and analyzed two new actin mutants, 4Ac/D24A/D25A and 4Ac/E99A/E100A . In these mutants, the acidic residues of the D24/D25 or E99/E100 sites were replaced with uncharged residues (alanines) and a pair of acidic residues was inserted at the N-terminus, maintaining the overall charge density of subdomain 1 . Using the in vitro motility assays, we found that the sliding and force generation properties of these mutant actins were identical to those of wild-type actin . Similarly, actin-activated ATPase activities of the mutant and wild-type actins were also indistinguishable . Additionally, the binding of S1 to these mutant actins in the presence of ATP was similar to that of wild-type actin . These results show that relocation of charged residues in subdomain 1 of actin does not affect the weak actomyosin interactions and actomyosin function.

Protein Eng, 1998 Dec, 11(12), 1121 - 8
Crystallographic and mutational analyses of an extremely acidophilic and acid-stable xylanase: biased distribution of acidic residues and importance of Asp37 for catalysis at low pH; Fushinobu S et al.; Xylanase C from Aspergillus kawachii has an optimum pH of 2.0 and is stable at pH 1.0 . The crystal structure of xylanase C was determined at 2.0 A resolution (R-factor = 19.4%) . The overall structure was similar to those of other family 11 xylanases . Asp37 and an acid-base catalyst, Glu170, are located at a hydrogen-bonding distance (2.8 A), as in other xylanases with low pH optima . Asp37 of xylanase C was replaced with asparagine and other residues by site-directed mutagenesis . Analyses of the wild-type and mutant enzymes showed that Asp37 is important for high enzyme activity at low pH . In the case of the asparagine mutant, the optimum pH shifted to 5.0 and the maximum specific activity decreased to about 15% of that of the wild-type enzyme . On structural comparison with xylanases with higher pH optima, another striking feature of the xylanase C structure was found; the enzyme has numerous acidic residues concentrated on the surface (so-called 'Ser/Thr surface' in most family 11 xylanases) . The relationship of the stability against extreme pH conditions and high salt concentrations with the spatially biased distribution of charged residues on the proteins is discussed.

Eur J Cell Biol, 1998 Dec, 77(4), 263 - 8
The Sec1p homologue Vps45p binds to the syntaxin Tlg2p; Nichols BJ et al.; SNAREs are compartmentally specific membrane proteins required for intracellular membrane fusion . Homologues of the Saccharomyces cerevisiae protein Sec1p interact with, and are likely to be involved in regulation of, the syntaxin family of SNAREs . In yeast there are 7 functionally distinct syntaxins but only four clearly identifiable homologues of Sec1p . One of these, Vps45p, is required for transport from Golgi to late endosomes, and has been implicated in the function of the late endosomal syntaxin Pep12p . However, there is evidence that not all the functions of Pep12p are equally dependent on Vps45p, and conversely that the phenotypes of vps45 mutants cannot be explained entirely by loss of Pep12p activity . We have recently characterised two yeast syntaxins which function in trans-Golgi or endosomal compartments, Tlg1p and Tlg2p . We show here that the principal binding site for Vps45p on intracellular membranes is provided by Tlg2p rather than Pep12p, and that Vps45p is required for stable expression of Tlg2p . Vps45p is also associated with Tlg1p as part of a triple complex containing both Tlg1p and Tlg2p . Since a deltavps45 deltatlg2 double mutant has a more severe vacuolar protein sorting defect than a deltatlg2 mutant, Vps45p cannot only interact with Tlg2p . It appears that the role of Vps45p in protein traffic is more complex than has previously been assumed.

Horm Metab Res, 1998 Dec, 30(12), 705 - 10
Insulin increases liver protein phosphatase-1 and protein phosphatase-2C activities in lean, young adult rhesus monkeys; Ortmeyer HK; Liver glycogen synthase activity is increased, and glycogen phosphorylase activity and glucose 6-phosphate content reduced by in vivo insulin during a euglycemic hyperinsulinemic clamp in lean young adult rhesus monkeys . To examine the mechanism of dephosphorylation of liver glycogen synthase and glycogen phosphorylase, the enzyme activities of protein phosphatase-1, protein phosphatase-2C, cAMP-dependent protein kinase, glycogen synthase kinase-3, protein kinase C and protein tyrosine kinase were determined before and after three hours of in vivo insulin in these same monkeys . The bioactivity of an inositol phosphoglycan insulin mediator (pH 2.0) and cAMP concentrations were also measured in the liver before and after insulin administration . Insulin caused significant increases in protein phosphatase-1 (p = 0.005) and in protein phosphatase-2C activities (p = 0.001) . Insulin-stimulated minus basal bioactivity of the pH 2.0 insulin mediator was strongly inversely related to the insulin-stimulated minus basal glucose 6-phosphate content (r = -0.93, p < 0.0001) . These findings suggest that protein phosphatase-1 and protein phosphatase-2C may be involved in the mechanism of in vivo insulin activation of liver glycogen synthase and inactivation of liver glycogen phosphorylase.

Drug Metab Dispos, 1999 Feb, 27(2), 188 - 92
Developmental changes in the catalytic activity and expression of CYP2D isoforms in the rat liver; Chow T et al.; Developmental changes in bufuralol 1'-hydroxylation activity, which is known as a typical activity of cytochrome P-450 (CYP)2D isoforms, in the liver of rats were investigated . The catalytic activities of hepatic microsomes increased with development especially from 3 to 7 weeks . Eadie-Hofstee plots of bufuralol 1'-hydroxylation were obtained for monophasic kinetics (Km: 0.037 microM) at 1 week and for biphasic kinetics (Km: 0.051 and 6.4 microM) at 7 weeks of age . Quinine completely inhibited bufuralol 1'-hydroxylation activity of hepatic microsomes of 1- and 7-week-old rats . These results indicated that at least two kinds of CYP2D isoforms, which differ markedly in their affinity for bufuralol, were present at 7 weeks of age and that the CYP2D isoform that had low affinity for bufuralol was expressed with development . To assess the affinity of CYP2D isoforms for bufuralol, the kinetic properties of CYP2D1, 2D2, 2D3, and 2D4 expressed in yeast cells were investigated . The Km value of CYP2D2, 0.044 microM, was extremely small compared with that of the other rat CYP2D isoforms . We further investigated developmental changes of CYP2D isoform mRNA by reverse transcription-polymerase chain reaction . CYP2D3 mRNA increased with development although CYP2D1 and 2D2 mRNA were not changed . The CYP2D4 mRNA was not detected . These findings indicated that CYP2D2, which had high affinity for bufuralol, was expressed in immature and mature rats, but CYP2D3, which had low affinity for bufuralol, was expressed only in mature rats.

FEBS Lett, 1999 Jan 22, 443(1), 41 - 7
C-terminal domains of human translation termination factors eRF1 and eRF3 mediate their in vivo interaction; Merkulova TI et al.; At the termination step of protein synthesis, hydrolysis of the peptidyl-tRNA is jointly catalysed at the ribosome by the termination codon and the polypeptide release factor (eRF1 in eukaryotes) . eRF1 forms in vivo and in vitro a stable complex with release factor eRF3, an eRF1-dependent and ribosome-dependent GTPase . The role of the eRF1-eRF3 complex in translation remains unclear . We have undertaken a systematic analysis of the interactions between the human eRF1 and eRF3 employing a yeast two-hybrid assay . We show that the N-terminal parts of eRF1 (positions 1-280) and of eRF3 (positions 1477) are either not involved or non-essential for binding . Two regions in each factor are critical for mutual binding: positions 478-530 and 628-637 of eRF3 and positions 281-305 and 411-415 of eRF1 . The GTP binding domain of eRF3 is not involved in complex formation with eRF1 . The GILRY pentamer (positions 411-415) conserved in eukaryotes and archaebacteria is critical for eRF1's ability to stimulate eRF3 GTPase . The human eRF1 lacking 22 C-terminal amino acids remains active as a release factor and promotes an eRF3 GTPase activity whereas C-terminally truncated eRF3 is inactive as a GTPase.

Toxicol Sci, 1998 Nov, 46(1), 45 - 60
An ongoing validation of a Tier I screening battery for detecting endocrine-active compounds (EACs); O'Connor JC et al.; After previously examining an estrogen receptor agonist (17beta-estradiol), several additional compounds have been evaluated in a Tier I screening battery for detecting endocrine-active compounds (EACs): an estrogen receptor antagonist (ICI-182,780, ICI), an androgen receptor antagonist (flutamide, FLUT), a testosterone biosynthesis inhibitor (ketoconazole, KETO), a 5alpha-reductase inhibitor (finasteride, FIN), and an aromatase inhibitor (anastrozole, ANA) . The Tier I battery incorporates two short-term in vivo tests (a 5-day ovariectomized female battery and a 15-day intact male battery) and an in vitro yeast transactivation system (YTS) . The Tier I battery is designed to identify compounds that have the potential to act as agonists or antagonists to the estrogen, androgen, progesterone, or dopamine receptors, steroid biosynthesis inhibitors (aromatase, 5alpha-reductase, and testosterone biosynthesis), or compounds that alter thyroid function . ICI administration decreased uterine estrogen and progesterone receptor number in the female battery, increased serum follicle-stimulating hormone (FSH) levels and caused spermatid retention in the male battery, and activated gene transcription in the YTS containing the estrogen receptor . FLUT administration increased uterine stromal cell proliferation in the female battery and decreased weights for all androgen-dependent tissues, induced Leydig cell hyperplasia, and caused hormonal alterations (increased testosterone (T), estradiol (E2), dihydrotestosterone (DHT), luteinizing hormone (LH), and FSH) in the male battery, and competed for binding to the androgen receptor in the YTS competition assay . In the male battery KETO decreased weights for all androgen-dependent tissues, caused hormonal alterations (decreased T and DHT and increased LH and FSH), and induced spermatid retention . FIN decreased seminal vesicle and accessory sex gland (ASG) unit weight and caused hormonal alterations (decreased DHT and increased LH, and PRL) in the male battery . KETO was judged not to affect any of the endpoints in the female battery . ANA decreased ASG unit weight and serum E2 levels in the male battery . Using the responses obtained for all the endpoints in the Tier I battery, a distinct "fingerprint" was produced for each type of endocrine activity against which compounds with unknown activity can be compared . These data demonstrate that the described Tier I battery is useful for identifying EACs.

Annu Rev Genet, 1998, 32, 619 - 97
The leptotene-zygotene transition of meiosis; Zickler D et al.; The leptotene/zygotene transition of meiosis, as defined by classical cytological studies, is the period when homologous chromosomes, already being discernible individualized entities, begin to be close together or touching over portions of their lengths . This period also includes the bouquet stage: Chromosome ends, which have already become integral components of the inner nuclear membrane, move into a polarized configuration, along with other nuclear envelope components . Chromosome movements, active or passive, also occur . The detailed nature of interhomologue interactions during this period, with special emphasis on the involvement of chromosome ends, and the overall role for meiosis and recombination of chromosome movement and, especially, the bouquet stage are discussed.

Annu Rev Genet, 1998, 32, 307 - 37
Kinetochores and the checkpoint mechanism that monitors for defects in the chromosome segregation machinery; Skibbens RV et al.; Whether we consider the division of the simplest unicellular organisms into two daughter cells or the generation of haploid gametes by the most complex eukaryotes, no two processes secure the continuance of life more than the proper replication and segregation of the genetic material . The cell cycle, marked in part by the periodic rise and fall of cyclin-dependent kinase (CDK) activities, is the means by which these two processes are separated . DNA damage and mistakes in chromosome segregation are costly, so nature has further devised elaborate checkpoint mechanisms that halt cell cycle progression, allowing time for repairs or corrections . In this article, we review the mitotic checkpoint mechanism that responds to defects in the chromosome segregation machinery and arrests cells in mitosis prior to anaphase onset . At opposite ends of this pathway are the kinetochore, where many checkpoint proteins reside, and the anaphase-promoting complex (APC), the metaphase-to-interphase transition regulator . Throughout this review we focus on budding yeast but reference parallel processes found in other organisms.

Nucleic Acids Res, 1999 Feb 15, 27(4), 993 - 1000
A new human topoisomerase III that interacts with SGS1 protein; Ng SW et al.; Eukaryotic DNA topoisomerase III was first identified by studying the hyper-recombination and slow growth phenotypes of yeast mutants . Topoisomerase III interacts with DNA helicase SGS1 and the two proteins are involved in DNA recombination, cellular aging and maintenance of genome stability . A human homolog of topoisomerase III has previously been identified . Here we report the identification of cDNAs and the determination of gene structure for a second human topoisomerase III gene . This novel gene expresses three alternatively spliced transcripts, which encode gene products different in the putative DNA-binding C-termini . The largest gene product of the novel topoisomerase III was expressed and shown to interact with SGS1 protein and partially rescue the slow growth defect of a yeast topoisomerase III mutant . The presence of more than one human topoisomerase III is reminiscent of mammalian topoisomerase II, which has two genetically distinct isoforms with different expression patterns and probably different functions in mammalian cells.

Bioinformatics, 1998, 14(10), 830 - 8
Empirical estimation of the reliability of ribosomal RNA alignments; O'Brien EA et al.; MOTIVATION: The automatic alignment of rRNA sequences can reproduce manual expert alignments with high, but not perfect, fidelity . We examine the use of empirical methods for the identification of regions of an alignment of a new sequence with an existing large alignment which can confidently be predicted to be correctly aligned . RESULTS: We show how to use a simple jack-knife procedure to derive an estimate of the reliability that is to be expected at each position of a large alignment of eukaryotic rRNA sequences . These reliabilities are then improved using measures that are specific to the input sequence . Regions where the sequence-specific reliability method performs particularly well are identified and seen to correspond with elements in the structure of the rRNA molecules that vary between species in the alignment . We also compare these reliability measures to an algorithmic alignment stability measure . AVAILABILITY: The software is available free of charge by sending an e-mail message to emmet@chah.ucc.ie . CONTACT: emmet@chah.ucc.ie

Proc Natl Acad Sci U S A, 1999 Feb 2, 96(3), 950 - 5
FOG-2: A novel GATA-family cofactor related to multitype zinc-finger proteins Friend of GATA-1 and U-shaped; Tevosian SG et al.; GATA factors are transcriptional regulatory proteins that play critical roles in the differentiation of multiple cell types in both vertebrates and invertebrates . Recent evidence suggests that the biological activities of both mammalian and Drosophila GATA factors are controlled in part by physical interaction with multitype zinc-finger proteins, Friend of GATA-1 (FOG) and U-shaped (Ush), respectively . Here we describe a new FOG-related polypeptide, designated FOG-2, that is likely to participate in differentiation mediated by GATA factors in several tissues . Expression of FOG-2 mRNA differs from that of FOG and is largely restricted to heart, neurons, and gonads in the adult . Somewhat broader expression is evident during mouse embryonic development . Similar to FOG and Ush, FOG-2 protein interacts specifically with the amino finger of GATA factors in the yeast two-hybrid system and in mammalian cells . Remarkably, though FOG-2 is quite divergent from FOG in its primary sequence, forced expression of FOG-2 rescues terminal erythroid maturation of FOG-/- hematopoietic cells . Thus, members of the FOG family of cofactors share highly specific association with GATA factors and are substantially interchangeable with respect to some aspects of function in vivo . The interaction of GATA and FOG family members constitutes an evolutionarily conserved paradigm for transcriptional control in differentiation and organogenesis.

Proc Natl Acad Sci U S A, 1999 Feb 2, 96(3), 927 - 32
Cloning and characterization of a cell surface receptor for xenotropic and polytropic murine leukemia viruses; Tailor CS et al.; Xenotropic and polytropic murine leukemia viruses (X-MLVs and P-MLVs) cross-interfere to various extents in non-mouse species and in wild Asian mice, suggesting that they might use a common receptor for infection . Consistent with this hypothesis, the susceptibility of some wild mice to X-MLVs has been mapped to the P-MLV receptor locus at the distal end of mouse chromosome 1 . In this study, we report the isolation and characterization of a cDNA for the human X-MLV cell surface receptor (X-receptor) by using a human T lymphocyte cDNA library in a retroviral vector . The predicted X-receptor contains 696 amino acids with multiple hydrophobic potential membrane-spanning sequences and with weak homologies to the yeast proteins SYG1, of unknown function, and PHO81, which has been implicated in a system that regulates transport of inorganic phosphate . Expression of the X-receptor in Chinese hamster ovary cells, which are substantially resistant to P-MLVs and to X-MLVs, made them susceptible to both of these virus groups . The mouse homologue of the X-receptor was mapped by hybridization to the distal end of chromosome 1 at the same position as the P-MLV receptor gene Rmc1 . These results strongly support the hypothesis that a common gene encodes the receptors for X-MLVs and P-MLVs, with the human X-receptor preferentially mediating X-MLV infections and the homologous protein of inbred mice mediating only P-MLV infections . We propose that X-MLVs and P-MLVs comprise a single family of retroviruses that have coevolved in response to diversification in X-receptor genes of the host.

Proc Natl Acad Sci U S A, 1999 Feb 2, 96(3), 915 - 20
Expression cloning of LDLB, a gene essential for normal Golgi function and assembly of the ldlCp complex; Chatterton JE et al.; The Chinese hamster ovary (CHO) cell mutants ldlC and ldlB, which exhibit almost identical phenotypes, define two genes required for multiple steps in the normal medial and trans Golgi-associated processing of glycoconjugates . The LDLC gene encodes ldlCp, an approximately 80-kDa protein, which in wild-type, but not ldlB, cells associates reversibly with the cytoplasmic surface of the Golgi apparatus . Here, we have used a retrovirus-based expression cloning system to clone a murine cDNA, LDLB, that corrects the pleiotropic mutant phenotypes of ldlB cells . The corresponding mRNA was not detected in ldlB mutants . LDLB encodes an approximately 110-kDa protein, ldlBp, which lacks homology to known proteins and contains no common structural motifs . Database searches identified short segments of homology to sequences from Drosophila melanogaster, Arabidopsis thaliana, and Caenorhabditis elegans, and the essentially full-length homologous human sequence (82% identity); however, as was the case for ldlCp, no homologue was identified in Saccharomyces cerevisiae . We have found that in wild-type cell cytosols, ldlCp is a component of an approximately 950-kDa "ldlCp complex," which is smaller, approximately 700 kDa, in ldlB cytosols . Normal assembly of this complex is ldlBp-dependent and may be required for Golgi association of ldlCp and for the normal activities of multiple luminal Golgi processes.

Proc Natl Acad Sci U S A, 1999 Feb 2, 96(3), 881 - 6
Similarity in the catalysis of DNA breakage and rejoining by type IA and IIA DNA topoisomerases; Liu Q et al.; Studies of yeast DNA topoisomerase II with various alanine-substitution mutations provide strong biochemical support of a recent hypothesis that the type IA and IIA DNA topoisomerases act similarly in their cleavage and rejoining of DNA . DNA breakage and rejoining by either a type IA or a type IIA enzyme are shown to involve cooperation between a DNA-binding domain containing the active-site tyrosine and a Rossmann fold containing several highly conserved acidic residues . For a homodimeric type IIA enzyme, cooperation occurs in trans: the active-site tyrosine in the DNA-binding domain of one protomer cooperates with several residues in the Rossmann fold as well as other regions of the other protomer.

Virology, 1999 Feb 1, 254(1), 160 - 8
Complementarity between 3' terminal nucleotides of tRNA and primer binding site is a major determinant for selection of the tRNA primer used for initiation of HIV-1 reverse transcription; Yu Q et al.; The initiation of reverse transcription of human immunodeficiency virus type 1 (HIV-1) exclusively utilizes tRNALys,3 as a primer . Previous studies have shown that HIV-1 could use alternative tRNAs, such as tRNAIle or tRNAHis, to initiate reverse transcription only if the primer binding site (PBS) was made complementary to the 3' terminal 18 nucleotides of the cognate tRNA . However, upon in vitro culture, the viruses with a PBS complementary to the alternative tRNAs rapidly reverted to generate a PBS complementary to tRNALys,3 . To investigate the process of reversion, we have constructed defective proviral genomes that contain a PBS complementary to tRNAIle or tRNAHis . The genomes contain the gene for xanthine-guanosine phosphoribosyl transferase (gpt) in place of env . Cotransfection of these proviral genomes with a plasmid-encoding vesicular stomatitis virus G protein (VSV-G) results in viruses that undergo a single round of HIV-1 infection; successful infections are scored as cells resistant to the drug mycophenolic acid . Using this single-round infection system, we demonstrated that HIV-1 with a PBS complementary to tRNAIle or tRNAHis is three- to fivefold less efficient in replication as measured by production of drug-resistant cell colonies compared to the wild-type virus . These viruses predominantly used the cognate tRNA as primer in their initial round of replication, although we did obtain a single cell colony in which the PBS was complementary to tRNALys,3 . Using an HIV-1 provirus with a PBS complementary to yeast tRNAPhe, we established a single-round infection system in which the infectivity of this mutant HIV-1 relies on transfected yeast tRNAPhe . The results of our studies suggest that the mechanism for selection of the tRNA primer for initiation of reverse transcription relies primarily on the complementarity between the tRNA primerthe PBS .

Cytogenet Cell Genet, 1998, 83(1-2), 18 - 20
Alternative splicing, genomic structure, and fine chromosome localization of REV3L; Morelli C et al.; We have localized a human homolog, REV3L, of the Saccharomyces cerevisiae REV3 gene on chromosome region 6q21 . The full-length cDNA consists of 10,919 nucleotides, with a putative open reading frame of 9,159 bp for a predicted protein of 3,053 amino acids . The gene contains 33 exons in about 200 kb of genomic DNA . In contrast to the previously reported sequence, an additional exon and an alternative splicing site are demonstrated.

J Mol Biol, 1999 Feb 5, 285(5), 2133 - 46
Temperature dependence of intramolecular dynamics of the basic leucine zipper of GCN4: implications for the entropy of association with DNA; Bracken C et al.; The basic leucine zipper domain of the yeast transcription factor GCN4 consists of a C-terminal leucine zipper and an N-terminal basic DNA-binding region that achieves a stable structure only after association with DNA . Backbone dynamics of a peptide encompassing the basic and leucine zipper bZip domain (residues 226-281) are described using NMR spectroscopy . The 15N longitudinal relaxation rates, 15N transverse relaxation rates, and inverted question mark1H inverted question mark-15N nuclear Overhauser effects were measured for the backbone amide nitrogen atoms at 290 K, 300 K, and 310 K . The relaxation data were interpreted using reduced spectral density mapping to determine values of the spectral density function, J(omega), at the frequencies 0, omegaN, and 0.87omegaH to characterize overall and intramolecular motions on picosecond-nanosecond timescales . To account for the temperature dependence of overall rotational diffusion, the J(0) values were normalized using Stoke's Law . At 310 K, the 13Calpha and 13CO chemical shifts in conjunction with the spectral density values indicate that the leucine zipper sequence forms a highly ordered alpha-helix, while the basic region populates an ensemble of highly dynamic transient structures with substantial helical character . The normalized values of J(0) and the values of J(0.87omegaH) for residues in the leucine zipper dimerization domain are independent of temperature . In contrast, residues in the basic region exhibit pronounced increases in the normalized J(0) and decreases in J(0.87omegaH) as temperature is decreased . A strong correlation exists between the temperature dependence of 13CO chemical shifts and of J(0.87omegaH) . These results suggest that, for the basic region, lowering the temperature increases the population of transient helical conformations, and concomitantly reduces the amplitude or timescale of conformational fluctuations on picosecond-nanosecond timescales . Changes in the conformational dynamics of the peptide backbone of the basic region that accompany DNA binding contribute to the overall thermodynamics of complex formation . The change in backbone conformational entropy derived from NMR spin-relaxation data agrees well with the result calculated from calorimetric measurements . Restriction of the conformational space accessible to the basic region may significantly reduce the entropic cost associated with formation of the basic region helices consequent to DNA binding .

EMBO J, 1999 Feb 1, 18(3), 754 - 62
Regulation of Hsp90 ATPase activity by tetratricopeptide repeat (TPR)-domain co-chaperones; Prodromou C et al.; The in vivo function of the heat shock protein 90 (Hsp90) molecular chaperone is dependent on the binding and hydrolysis of ATP, and on interactions with a variety of co-chaperones containing tetratricopeptide repeat (TPR) domains . We have now analysed the interaction of the yeast TPR-domain co-chaperones Sti1 and Cpr6 with yeast Hsp90 by isothermal titration calorimetry, circular dichroism spectroscopy and analytical ultracentrifugation, and determined the effect of their binding on the inherent ATPase activity of Hsp90 . Sti1 and Cpr6 both bind with sub-micromolar affinity, with Sti1 binding accompanied by a large conformational change . Two co-chaperone molecules bind per Hsp90 dimer, and Sti1 itself is found to be a dimer in free solution . The inherent ATPase activity of Hsp90 is completely inhibited by binding of Sti1, but is not affected by Cpr6, although Cpr6 can reactivate the ATPase activity by displacing Sti1 from Hsp90 . Bound Sti1 makes direct contact with, and blocks access to the ATP-binding site in the N-terminal domain of Hsp90 . These results reveal an important role for TPR-domain co-chaperones as regulators of the ATPase activity of Hsp90, showing that the ATP-dependent step in Hsp90-mediated protein folding occurs after the binding of the folding client protein, and suggesting that ATP hydrolysis triggers client-protein release.

EMBO J, 1999 Feb 1, 18(3), 717 - 26
Destruction of Myc by ubiquitin-mediated proteolysis: cancer-associated and transforming mutations stabilize Myc; Salghetti SE et al.; The human proto-oncogene c-myc encodes a highly unstable transcription factor that promotes cell proliferation . Although the extreme instability of Myc plays an important role in preventing its accumulation in normal cells, little is known about how Myc is targeted for rapid destruction . Here, we have investigated mechanisms regulating the stability of Myc . We show that Myc is destroyed by ubiquitin-mediated proteolysis, and define two elements in Myc that oppositely regulate its stability: a transcriptional activation domain that promotes Myc destruction, and a region required for association with the POZ domain protein Miz-1 that stabilizes Myc . We also show that Myc is stabilized by cancer-associated and transforming mutations within its transcriptional activation domain . Our data reveal a complex network of interactions regulating Myc destruction, and imply that enhanced protein stability contributes to oncogenic transformation by mutant Myc proteins.

EMBO J, 1999 Feb 1, 18(3), 632 - 43
Bcl-xL regulates apoptosis by heterodimerization-dependent and -independent mechanisms; Minn AJ et al.; A hydrophobic cleft formed by the BH1, BH2 and BH3 domains of Bcl-xL is responsible for interactions between Bcl-xL and BH3-containing death agonists . Mutants were constructed which did not bind to Bax but retained anti-apoptotic activity . Since Bcl-xL can form an ion channel in synthetic lipid membranes, the possibility that this property has a role in heterodimerization-independent cell survival was tested by replacing amino acids within the predicted channel-forming domain with the corresponding amino acids from Bax . The resulting chimera showed a reduced ability to adopt an open conductance state over a wide range of membrane potentials . Although this construct retained the ability to heterodimerize with Bax and to inhibit apoptosis, when a mutation was introduced that rendered the chimera incapable of heterodimerization, the resulting protein failed to prevent both apoptosis in mammalian cells and Bax-mediated growth defect in yeast . Similar to mammalian cells undergoing apoptosis, yeast cells expressing Bax exhibited changes in mitochondrial properties that were inhibited by Bcl-xL through heterodimerization-dependent and -independent mechanisms . These data suggest that Bcl-xL regulates cell survival by at least two distinct mechanisms; one is associated with heterodimerization and the other with the ability to form a sustained ion channel.

J Physiol, 1999 Feb 15, 515 ( Pt 1), 19 - 30
ATP inhibition of KATP channels: control of nucleotide sensitivity by the N-terminal domain of the Kir6.2 subunit; Koster JC et al.; 1 . To gain insight into the role of the cytoplasmic regions of the Kir6.2 subunit in regulating channel activity, we have expressed the sulphonylurea receptor SUR1 with Kir6.2 subunits containing systematic truncations of the N- and C-termini . Up to 30 amino acids could be truncated from the N-terminus, and up to 36 amino acids from the C-terminus without loss of functional channels in co-expression with SUR1 . Furthermore, Kir6.2DeltaC25 and Kir6 . 2DeltaC36 subunits expressed functional channels in the absence of SUR1 . 2 . In co-expression with SUR1, N-terminal truncations increased Ki,ATP ({ATP} causing half-maximal inhibition of channel activity) by as much as 10-fold, accompanied by an increase in the ATP-insensitive open probability, whereas the C-terminal truncations did not affect the ATP sensitivity of co-expressed channels . 3 . A mutation in the near C-terminal region, K185Q, reduced ATP sensitivity of co-expressed channels by approximately 30-fold, and on the Kir6.2DeltaN2-30 background, this mutation decreased ATP sensitivity of co-expressed channels by approximately 400-fold . 4 . Each of these mutations also reduced the sensitivity to inhibition by ADP, AMP and adenosine tetraphosphate . 5 . The results can be quantitatively explained by assuming that the N-terminal deletions stabilize the ATP-independent open state, whereas the Kir6.2K185Q mutation may alter the stability of ATP binding . These two effects are energetically additive, causing the large reduction of ATP sensitivity in the double mutant channels.

Genes Dev, 1999 Jan 15, 13(2), 146 - 51
Telomeric chromatin modulates replication timing near chromosome ends; Stevenson JB et al.; Saccharomyces cerevisiae telomeric DNA replicates late in S phase, and telomeric genes are transcriptionally silent . Transcriptional repression of telomere-proximal genes results from silent chromatin initiating at the chromosome end, but the relationship between telomeric chromatin and DNA replication is unknown . Mutations in SIR3, a silent chromatin component, cause telomeric DNA on chromosome V to replicate much earlier because of earlier initiation of a nearby replication origin, the Y' ARS . A second telomere-proximal ARS, from an X element, does not act as an origin in a wild-type strain, whereas in a sir3 cell it does . We conclude that telomeric chromatin has a Sir3-dependent inhibitory effect on DNA replication.

Biochimie, 1998 Dec, 80(12), 977 - 85
A pseudoknotted tRNA variant is a substrate for tRNA (cytosine-5)-methyltransferase from Xenopus laevis; Brule H et al.; tRNA post-transcriptional modification enzymes of Xenopus laevis were proposed previously to belong to two major groups according to their sensitivity to structural perturbations in their substrates . To further investigate the structural variations tolerated by these enzymes, the tRNA-like domain of turnip yellow mosaic virus RNA (88 nucleotides in length) has been microinjected into the oocytes of Xenopus laevis . This RNA possesses 12 potential target nucleotides for modification within a structure including a pseudoknotted folding, an extended anticodon stem, and unusual D-loop/T-loop interactions . Results indicate that only cytosine-42, a position equivalent to C-49 in canonical tRNAs, was quantitatively modified into m5C in the microinjected RNA . Modification was detected to high levels, indicating that at least one enzyme tolerates non-canonical structural features.

FEBS Lett, 1999 Jan 8, 442(1), 83 - 8
Identification and cloning of a novel cellular protein Naf1, Nef-associated factor 1, that increases cell surface CD4 expression; Fukushi M et al.; The nef gene of human and simian immunodeficiency virus is a key factor in acquired immunodeficiency syndrome pathogenesis and virus replication . Several Nef-induced phenomena, including the down-regulation of CD4 molecule, have been previously reported . In this study, we have identified and cloned a novel cellular protein Naf1 (Nef-associated factor 1), which associated with Nef in the yeast two-hybrid system and pull-down assay . The Naf1 gene generates two isoforms (Naf1alpha and beta) containing four coiled-coil structures . The Naf1 mRNA is ubiquitously expressed in human tissues with strong expression in peripheral blood lymphocytes and spleen . Naf1 overexpression increased cell surface CD4 expression . Nef suppressed this Naf1-induced augmentation of CD4 expression, providing a novel mode of Nef action in CD4 down-regulation.

Biochemistry, 1998 Dec 22, 37(51), 18018 - 25
Synthesis of (2R,3R)-erythro- and (2R,3S)-threo-fluoromalate using malic dehydrogenase; stereoselectivity of malic dehydrogenase; Urbauer JL et al.; 3-Fluorooxalacetate is a substrate for malic dehydrogenase . When enzymatic reduction is slower than the rate of epimerization of the two enantiomers, only (2R,3R)-erythro-fluoromalate is formed . Conversely, when a high enzyme level and excess of NADH lead to reduction that is fast relative to the epimerization rate, equal amounts of (2R,3R)-erythro- and (2R,3S)-threo-fluoromalate are formed . These data suggest that the V/K value for reduction of the R enantiomer to give the erythro isomer is approximately 100 times greater than for reduction of the S enantiomer to give the threo isomer . The equilibrium constant for the oxidation of fluoromalate is an order of magnitude less favorable than for oxidation of malate, while the equilibrium deuterium isotope effect from deuteration at C-2 of the substrate is 1.09 for fluoromalate versus 1.18 for malate . These effects reflect the inductive effect of fluorine at the 3-position.

Biochemistry, 1998 Dec 22, 37(51), 17673 - 9
Dual function C-terminal domain of dynamin-1: modulation of self-assembly by interaction of the assembly site with SH3 domains; Scaife R et al.; Impairment of endocytosis by mutational targeting of dynamin-1 GTPases can result in paralysis and embryonic lethality . Dynamin-1 assembles at coated pits where it functions to cleave vesicles from donor membranes . Receptor endocytosis is modulated by SH3 (src homology 3) domain proteins, which directly bind to dynamin C-terminal proline motif sequences, affecting both the dynamin GTPase activity and its recruitment to coated pits . We have determined that dynamin-dynamin interactions, which are required for dynamin helix formation, involve these same SH3 domain-binding C-terminal proline motif sequences . Consequently, SH3 domain proteins induce the in vitro disassembly of dynamin helices . Our results therefore suggest the the dual function of the dynamin C-terminus (involving amino acids 800-840) permits direct regulation of dynamin assembly and function through interaction with SH3 domain proteins . Additionally, the N-terminal GTPase domain plays an important role in assembly . Finally, we show that the central PH (pleckstrin homology) domain exerts a strong inhibitory effect on the capacity for dynamin-1 self-assembly.

Biochemistry, 1998 Dec 22, 37(51), 17651 - 8
How fumarase recycles after the malate --> fumarate reaction . Insights into the reaction mechanism; Rose IA; Recycling of yeast fumarase to permit repetition of its reaction chemistry requires two proton transfers and two conformational changes, in pathways that are different in detail but thematically similar in the two directions . In the malate --> fumarate direction, simple anions such as acetate accelerate the fumarate-off step producing E(H(f)), a fumarate-specific isoform that retains the C3R-proton of malate . Fumarate specificity is shown with S-2,3-dicarboxyaziridine, which is competitive vs fumarate and noncompetitive with malate as substrate . The steady-state level of E(H(f)), based on Kii (S-2,3-dicarboxyaziridine), is increased by D2O and decreased by imidazole acting as a general acid for conversion of E(H(f)) to E(H(f))H . E(H(f))H is fumarate-specific as shown by the inhibition pattern with ClO4- . The pKa of this step is approximately 7.25 based on the pH dependence of Kii (ClO4-) . A conformational change occurs next as shown by high sensitivity of k(cat) but not k(cat)/Km, to the microviscosogen, glycerol, and change to a nonspecific isoform, E(H(mf))H, probably the same species formed in the fumarate --> malate direction from malate-specific intermediates by a different conformational change . Malate enters the cycle by reaction with E(H(mf))H and returns to E(m)H x malate after a second conformational change . When fumarate-off is slow, as in low anion medium, malate itself becomes an activator of malate --> fumarate . This effect occurs with changes in inhibition patterns suggestive of the bypass of the slow E(f) --> E(mf) conversion in favor of direct formation of E(mf) when free fumarate is formed . 3-Nitro-2-hydroxypropionate, a strong inhibitor of fumarase {Porter, D . J . T., and Bright, H . J . (1980) J . Biol . Chem . 255, 4772-4780} in its carbanion form, is competitive with both malate and fumarate . Therefore, 3-nitro-2-hydroxypropionic acid interacts with E(H(mf))H and not with E(m) or E(f) isoforms . Occurrence of two different conformational changes in the recycling process suggests that the reaction chemistry employs a two-step mechanism . The specificity of inhibition for E(H(mf))H is consistent with the expected intermediate of a carbanion mechanism, E(H)H x carbanion- . The proton transfers and conformational changes of recycling occur in the same sequence that is expected for this reaction chemistry . Several examples of ligand-activated conformational changes are reported.

Biochemistry, 1998 Dec 22, 37(51), 17637 - 41
Structure and function of the core histone N-termini: more than meets the eye; Hansen JC et al.; For two decades, the core histone N-termini generally have been thought of as unstructured domains whose function is to bind to DNA and screen negative charge . New data indicates that both the molecular mechanisms of action and biological functions of the core histone N-termini in chromatin are considerably more complex . At the level of the chromatin fiber, multiple distinct functions of the N-termini are required to achieve higher order chromatin condensation, two of which apparently involve protein-protein rather than protein-DNA interactions . In addition, the N-termini have been documented to participate in specific interactions with many chromatin-associated regulatory proteins . Here, we discuss evidence supporting the new concepts that when functioning in their natural chromatin context, (1) the N-termini are engaged primarily in protein-protein interactions, (2) as a consequence of these interactions the N-termini adopt specific secondary structure, (3) posttranslational modifications such as acetylation disrupt the ability of the N-termini to form secondary structure, and (4) because the N-termini perform essential roles in both chromatin condensation and also bind specific chromatin-associated proteins, the global structure and function of any given region of the genome will be determined predominantly by the core histone N-termini and their specific interaction partners.

J Biol Chem, 1999 Feb 5, 274(6), 3453 - 60
The requirement for molecular chaperones during endoplasmic reticulum-associated protein degradation demonstrates that protein export and import are mechanistically distinct; Brodsky JL et al.; Polypeptide import into the yeast endoplasmic reticulum (ER) requires two hsp70s, Ssa1p in the cytosol and BiP (Kar2p) in the ER lumen . After import, aberrant polypeptides may be exported to the cytoplasm for degradation by the proteasome, and defects in the ER chaperone calnexin (Cne1p) compromise their degradation . Both import and export require BiP and the Sec61p translocation complex, suggesting that import and export may be mechanistically related . We now show that the cne1Delta and two kar2 mutant alleles exhibit a synthetic interaction and that the export and degradation of pro-alpha factor is defective in kar2 mutant microsomes . Pulse-chase analysis indicates that A1PiZ, another substrate for degradation, is stabilized in the kar2 strains at the restrictive temperature . Because two of the kar2 mutants examined are proficient for polypeptide import, the roles of BiP during ER protein export and import differ, indicating that these processes must be mechanistically distinct . To examine whether Ssa1p drives polypeptides from the ER and is also required for degradation, we assembled reactions using strains either containing a mutation in SSA1 or in which the level of Ssa1p could be regulated . We found that pro-alpha factor and A1PiZ were degraded normally, indicating further that import and export are distinct and that other cytosolic factors may pull polypeptides from the ER.

J Biol Chem, 1999 Feb 5, 274(6), 3402 - 6
Dependence of peroxisomal beta-oxidation on cytosolic sources of NADPH; Minard KI et al.; Growth of Saccharomyces cerevisiae with a fatty acid as carbon source was shown previously to require function of either glucose-6-phosphate dehydrogenase (ZWF1) or cytosolic NADP+-specific isocitrate dehydrogenase (IDP2), suggesting dependence of beta-oxidation on a cytosolic source of NADPH . In this study, we find that DeltaIDP2DeltaZWF1 strains containing disruptions in genes encoding both enzymes exhibit a rapid loss of viability when transferred to medium containing oleate as the carbon source . This loss of viability is not observed following transfer of a DeltaIDP3 strain lacking peroxisomal isocitrate dehydrogenase to medium with docosahexaenoate, a nonpermissive carbon source that requires function of IDP3 for beta-oxidation . This suggests that the fatty acid- phenotype of DeltaIDP2DeltaZWF1 strains is not a simple defect in utilization . Instead, we propose that the common function shared by IDP2 and ZWF1 is maintenance of significant levels of NADPH for enzymatic removal of the hydrogen peroxide generated in the first step of peroxisomal beta-oxidation in yeast and that inadequate levels of the reduced form of the cofactor can produce lethality . This proposal is supported by the finding that the sensitivity to exogenous hydrogen peroxide previously reported for DeltaZWF1 mutant strains is less pronounced when analyses are conducted with a nonfermentable carbon source, a condition associated with elevated expression of IDP2 . Under those conditions, similar slow growth phenotypes are observed for DeltaZWF1 and DeltaIDP2 strains, and co-disruption of both genes dramatically exacerbates the H2O2s phenotype . Collectively, these results suggest that IDP2, when expressed, and ZWF1 have critical overlapping functions in provision of reducing equivalents for defense against endogenous or exogenous sources of H2O2.

Biochem Biophys Res Commun, 1999 Jan 27, 254(3), 693 - 8
In vitro SUMO-1 modification requires two enzymatic steps, E1 and E2; Okuma T et al.; The SUMO-1 has been identified as a protein that is highly similar to ubiquitin and shown to conjugate to RanGAP1, PML, Sp200 and I kappa B alpha . The conjugation steps are thought to be similar to those of ubiquitination; and human Ubc9, which is homologous to the E2 enzyme for the ubiquitin conjugation step, was identified and shown to be necessary for the conjugation of SUMO-1 to its target protein . Other essential enzymes involved in this modification, however, remain to be clarified . Here we cloned human Sua1 (SUMO-1 activating enzyme) and hUba2, which are human homologs of yeast Saccharomyces cerevisiae Aos1 and Uba2, respectively . The recombinant proteins, Sua1p and hUba2p, formed a complex . In this complex, hUba2 bound SUMO-1 and this complex had the activity of the SUMO-1 activating enzyme . Furthermore, in an in vitro system, RanGAP1 was modified by SUMO-1 in the presence of Sua1p/Uba2p and hUbc9p, showing that the modification of SUMO-1 could be catalyzed by two enzyme steps, although ubiquitination usually requires three enzyme steps .

J Neurosci, 1999 Feb 1, 19(3), 1018 - 26
A novel pineal night-specific ATPase encoded by the Wilson disease gene; Borjigin J et al.; We have identified a pineal night-specific ATPase (PINA), a novel splice variant of the ATP7B gene disrupted in Wilson disease (WD) . PINA expression exhibits a dramatic diurnal rhythm in both pineal gland and retina with 100-fold greater expression at night than at day . PINA is expressed in pinealocytes and a subset of photoreceptors in adult rats and is transiently expressed in the retinal pigment epithelium and the ciliary body during retinal development . Nocturnal pineal expression of PINA is under the control of a suprachiasmatic nucleus clock mediated by superior cervical ganglion innervation of the pineal . In vitro, PINA expression in pineal cells can be stimulated by agents activating the cAMP signal transduction pathway . PINA is able to restore copper transport activity in Saccharomyces cerevisiae deficient in the homologous copper-transporting ATPase CCC2, suggesting that this protein may function as a copper transporter in rat pinealocytes . These studies suggest a potential role of rhythmic copper metabolism in pineal and/or retina circadian function.

Mutat Res, 1998 Nov 9, 422(1), 77 - 83
Differences in the mutational specificities of sunlight and UVB radiation suggest a role for transversion-inducing DNA damage in solar photocarcinogenesis; Kunz BA et al.; Mutations induced by UVB radiation and natural sunlight in a plasmid-borne yeast (Saccharomyces cerevisiae) tRNA gene (SUP4-o) were characterised by DNA sequencing . For both agents, the majority (> 90%) of the total mutations analysed were single base-pair substitutions, but tandem substitutions and single base-pair deletions also were detected . Each agent induced all six types of base-pair change but the tandem substitutions involved exclusively G.C-->A.T transitions . However, the fractions of single and tandem G.C-->A.T transitions were reduced by about 50%, and the fraction of transversions at G.C pairs was increased by 11-fold for sunlight relative to UVB . Comparisons of the site and strand specificities of the substitutions suggested that dipyrimidine adducts were responsible for the transitions, and that other lesions induced by sunlight may have given rise to the transversions . The relevance of these findings to skin cancer is discussed.

Hepatology, 1999 Feb, 29(2), 483 - 93
Hepatic sequestration and modulation of the canalicular transport of the organic cation, daunorubicin, in the Rat; Hayes JH et al.; In contrast to organic anions, substrates for the canalicular mdr1a and b are usually organic cations and are often sequestered in high concentrations in intracellular acidic compartments . Because many of these compounds are therapeutic agents, we investigated if their sequestration could be regulated . We used isolated perfused rat liver (IPRL), isolated rat hepatocyte couplets (IRHC), and WIF-B cells to study the cellular localization and biliary excretion of the fluorescent cation, daunorubicin (DNR) . Despite rapid (within 15 minutes) and efficient (>90%) cellular uptake in the IPRL, only approximately 10% of the dose administered (0.2-20 micromol) was excreted in bile after 85 minutes . Confocal microscopy revealed fluorescence predominantly in vesicles in the pericanalicular region in IPRL, IRHC, and WIF-B cells . Treatment of these cells with chloroquine and bafilomycin A, agents that disrupt the pH gradient across the vesicular membrane, resulted in a loss of vesicular fluorescence, reversible in the case of bafilomycin A . Taurocholate (TC) and dibutyryl cAMP (DBcAMP), stimulators of transcytotic vesicular transport, increased the biliary recovery of DNR significantly above controls, by 70% and 35%, respectively . The microtubule destabilizer, nocodazole, decreased biliary excretion of DNR . No effect on secretion was noted in TR- mutant rats deficient in mrp2 . Coadministration of verapamil, an inhibitor of mdr1, also decreased DNR excretion . While TC and DBcAMP did not affect the fluorescent intensity or pattern of distribution in IRHC, nocodazole resulted in redistribution of DNR to peripheral punctuate structures . These findings suggest that the organic cation, DNR, is largely sequestered in cells such as hepatocytes, yet its excretion can still be modulated.

Melanoma Res, 1998 Dec, 8(6), 471 - 81
Inhibition of Ku autoantigen binding activity to the E2F motif after ultraviolet B irradiation of melanocytic cells; Pedley J et al.; In human melanocytes and a human melanoma cell line (MM96L), the level of Ku sequence-specific binding to a 37-mer oligonucleotide containing a single E2F-1 binding site of the c-myc promoter (E2cM) significantly decreased 12 24h after cytostatic exposure to 300 J/m2 ultraviolet B radiation (UVB) . No UVB-induced loss was found in fibroblasts, while HeLa cells showed an earlier (4 h) but less significant decrease than melanocytic cells . Equitoxic doses of gamma radiation, cisplatin or UVC had little effect on E2cM-specific binding . The loss of Ku binding in MM96L cells was not the result of translocation of Ku or a decrease in Ku protein or DNA-dependent protein kinase activity . The level of E2cM-specific binding in MM96L cells was increased by tunicamycin (2 microg/ml), an inhibitor of N-linked glycosylation, and decreased by the glucosidase inhibitor castanospermine (50 microg/ml) . These results, which parallel the reported loss in melanocytes of the cell cycle regulator pRB after UVB, suggest that the DNA binding activity of Ku is affected by post-translational modification and may play a role in regulating the cell cycle response to UVB.

J Mol Biol, 1999 Jan 29, 285(4), 1429 - 40
The RNA polymerase III-recruiting factor TFIIIB induces a DNA bend between the TATA box and the transcriptional start site; Grove A et al.; TFIIIB, the RNA polymerase III-recruiting factor of Saccharomyces cerevisiae, may be assembled upstream of the transcriptional start site, either through the interaction of its constituent TATA-binding protein (TBP) with a strong TATA-box, or by means of the multisubunit assembly factor, TFIIIC . Missing nucleoside interference analysis of TFIIIC-dependent TFIIIB-DNA complex formation revealed enhanced complex formation at 0 degreesC when the DNA is missing nucleosides in two broad 7-10 bp regions centered around base-pairs -17 and -3 relative to the transcriptional start site; no effect of missing nucleosides was evident at 20 degreesC . The implication of these results for required DNA flexure in TFIIIC-mediated TFIIIB-DNA complex formation was pursued in a TFIIIC-independent context, using DNA with a suboptimal 6 bp TATA box (TATAAA) . A unique missing nucleoside at the downstream end of the TATA box, corresponding to the position of one of two TBP-mediated DNA kinks, significantly enhances TBP-DNA complex formation . In contrast, TFIIIB displays a broad preference for missing nucleosides within an approximately 15 bp region immediately downstream of the TATA box . Consecutive mismatches (4-nt loops), either at the sites of TBP-mediated DNA kinking at both ends of the TATA box or within the identified region where missing nucleosides promote TFIIIB-DNA complex formation, also result in enhanced and specific TFIIIB assembly; 4-nt loops further downstream do not lead to preferential placement of TFIIIB . We conclude that TFIIIB induces an additional DNA deformation between the TATA box and the start site of transcription that is likely to be more extended than the sharp kinks generated by TBP .

Arch Biochem Biophys, 1999 Feb 1, 362(1), 175 - 82
Isolation and characterization of a Delta 5-fatty acid desaturase from Caenorhabditis elegans; Watts JL et al.; Arachidonic acid and eicosapentaenoic acid are important precursors for the production of prostaglandins and other hormone-like eicosanoid molecules . These fatty acids are synthesized by animals by elongating and desaturating precursor fatty acids such as linoleic acid (18:2Delta9,12) and alpha-linolenic acid (18:3Delta9, 12,15) . We have identified a Delta5 fatty acid desaturase gene (fat-4) from the nematode Caenorhabditis elegans . We have expressed this gene product in Saccharomyces cerevisiae and demonstrate that it readily converts di-homo-gamma-linolenic acid (20:3Delta8,11,14) to arachidonic acid (20:4Delta5,8,11,14) . The FAT-4 Delta5-desaturase also acts on a number of other substrates, including fatty acids that do not contain a double bond at the Delta8 position .

Nat Genet, 1999 Jan, 21(1), 123 - 7
Mammalian MutS homologue 5 is required for chromosome pairing in meiosis; Edelmann W et al.; MSH5 (MutS homologue 5) is a member of a family of proteins known to be involved in DNA mismatch repair . Germline mutations in MSH2, MLH1 and GTBP (also known as MSH6) cause hereditary non-polyposis colon cancer (HNPCC) or Lynch syndrome . Inactivation of Msh2, Mlh1, Gtmbp (also known as Msh6) or Pms2 in mice leads to hereditary predisposition to intestinal and other cancers . Early studies in yeast revealed a role for some of these proteins, including Msh5, in meiosis . Gene targeting studies in mice confirmed roles for Mlh1 and Pms2 in mammalian meiosis . To assess the role of Msh5 in mammals, we generated and characterized mice with a null mutation in Msh5 . Msh5-/- mice are viable but sterile . Meiosis in these mice is affected due to the disruption of chromosome pairing in prophase I . We found that this meiotic failure leads to a diminution in testicular size and a complete loss of ovarian structures . Our results show that normal Msh5 function is essential for meiotic progression and, in females, gonadal maintenance.

J Immunol, 1999 Jan 15, 162(2), 635 - 8
Cutting edge: protective effects of notch-1 on TCR-induced apoptosis; Jehn BM et al.; The Notch receptor protein was originally identified in Drosophila and is known to mediate cell to cell communication and influence cell fate decisions . Members of this family have been isolated from invertebrates as well as vertebrates . We isolated mouse Notch-1 in a yeast two-hybrid screen with Nur77, which is a protein that has been shown previously to be required for apoptosis in T cell lines . The data presented below indicate that Notch-1 expression provides significant protection to T cell lines from TCR-mediated apoptosis . These data demonstrate a new antiapoptotic role for Notch-1, providing evidence that, in addition to regulating cell fate decisions, Notch-1 can play a critical role in controlling levels of cell death in T cells.

J Immunol, 1999 Jan 15, 162(2), 911 - 9
A role for RAD51 in the generation of immunoglobulin gene diversity in rabbits; Barrington RA et al.; Ig VDJ genes in rabbit somatically diversify by both hyperpointmutation and gene conversion . To elucidate the mechanism of gene conversion of IgH genes, we cloned a rabbit homologue of RAD51, a gene involved in gene conversion in Saccharomyces cerevisiae (yeast), and tested whether it could complement a yeast rad51 mutant deficient in recombination repair . We found that rabbit RAD51 partially complemented the defect in switching mating types by gene conversion as well as in DNA double-strand break repair after gamma-irradiation . Further, by Western blot analysis, we found that levels of Rad51 were higher in appendix-derived B lymphocytes of 6-wk-old rabbits, a time at which IgH genes diversify by somatic gene conversion . We suggest that Rad51 is involved in somatic gene conversion of rabbit Ig genes.

Mycoses, 1998 Nov, 41(9-10), 355 - 62
Ribosomal genes of Histoplasma capsulatum var . duboisii and var . farciminosum; Okeke CN et al.; A total of 1704 basepairs of the 18S rDNA of Histoplasma capsulatum var . duboisii (HCD, strain CBS175.57) and H . capsulatum var . farciminosum (HCF, strain CBS478.64) were sequenced (EMBL accession no . Z75306 and no . Z75307) . The 18S rDNA of HCD was 100% identical to a published sequence of H . capsulatum var . capsulatum (HCC) . The 18S rDNA of HCF showed one transversional point mutation at the nucleotide position 114 (ref . Saccharomyces cerevisiae) . Hybridization confirmed that, in the 18S rDNA of two out of five strains of HCF, guanine was substituted for cytosine at the nucleotide position 114 . Furthermore, identical group 1C1 introns (403 bp) were found to be inserted after position 1165 in four out of five strains of HCF, including the two strains with point mutations in the 18S rDNA, and a slightly different group 1C1 intron (408 bp) was detected in one strain of HCC without this point mutation . Intraspecific sequence variability in the highly conserved 18S rDNA because of occurrence of introns and mutations as a possible source of error in molecular diagnostics is discussed . In addition, internal transcribed spacer regions between the 18S rDNA and the 5.8S rDNA (ITS1) of three strains of HCF, and one strain each of HCC and HCD showed significant sequence variability between varieties and strains of H . capsulatum.

Nat Genet, 1999 Jan, 21(1 Suppl), 25 - 32
Options available--from start to finish--for obtaining expression data by microarray; Bowtell DD; The excitement surrounding microarray technology has been tempered by the limited ability of the general biomedical research community to gain access to it . Given the hardware required for exploitation of the technology is becoming increasingly available, it is an appropriate moment to review options, be they commercially or publically available . Here, we provide a snapshot of the rapidly changing field of microarray-based RNA expression analysis and consider the components and procedures for putting together a complete system.

Eur J Biochem, 1999 Jan, 259(1-2), 385 - 95
Synergism between a half-site and an imperfect estrogen-responsive element, and cooperation with COUP-TFI are required for estrogen receptor (ER) to achieve a maximal estrogen-stimulation of rainbow trout ER gene; Petit FG et al.; In all oviparous, liver represents one of the main E2-target tissues where estrogen receptor (ER) constitutes the key mediator of estrogen action . The rainbow trout estrogen receptor (rtER) gene expression is markedly up-regulated by estrogens and the sequences responsible for this autoregulation have been located in a 0.2 kb upstream transcription start site within - 40/- 248 enhancer region . Absence of interference with steroid hormone receptors and tissue-specific factors and a conserved basal transcriptional machinery between yeast and higher eukaryotes, make yeast a simple assay system that will enable determination of important cis-acting regulatory sequences within rtER gene promoter and identification of transcription factors implicated in the regulation of this gene . Deletion analysis allowed to show a synergistic effect between an imperfect estrogen-responsive element (ERE) and a consensus half-ERE to achieve a high hormone-dependent transcriptional activation of the rtER gene promoter in the presence of stably expressed rtER . As in mammalian cells, here we observed a positive regulation of the rtER gene promoter by the chicken ovalbumin upstream promoter-transcription factor I (COUP-TFI) through enhancing autoregulation . Using a point mutation COUP-TFI mutant unable to bind DNA demonstrates that enhancement of rtER gene autoregulation requires the interaction of COUP-TFI to the DNA . Moreover, this enhancement of transcriptional activation by COUP-TFI requires specifically the AF-1 transactivation function of ER and can be observed in the presence of E2 or 4-hydroxytamoxifen but not ICI 164384 . Thus, this paper describes the reconstitution of a hormone-responsive transcription unit in yeast in which the regulation of rtER gene promoter could be enhanced by the participation of cis-elements and/or trans-acting factors, such as ER itself or COUP-TF.

Chromosoma, 1998 Dec, 107(6-7), 471 - 8
Regulation of tubulin polypeptides and microtubule function: Luv1p {correction of Rki1p} interacts with the beta-tubulin binding protein Rbl2p; Smith AM et al.; Genetic analysis of microtubule functions in the yeast Saccharomyces cerevisiae suggests that cells manage the levels and activities of the tubulin polypeptides . These reactions may be involved in protein folding, formation of the heterodimer, and maintenance of the appropriate balance between alpha- and beta-tubulin . One protein involved in these functions is Rbl2p, which forms a complex with beta-tubulin . Here we describe the identification of a novel yeast gene, LUV1 {corrected}, that interacts genetically with RBL2 . Deletion of rki1 causes conditional defects in microtubule assembly and cell growth . Luv1p {corrected} can be isolated in a complex containing Rbl2p . The results support the existence of cellular mechanisms for regulating microtubule function through the tubulin polypeptides.

Curr Opin Genet Dev, 1998 Dec, 8(6), 694 - 700
Eukaryote genome duplication - where's the evidence?
Skrabanek L, Wolfe KH.
Several eukaryotes, including maize, yeast and Xenopus, are degenerate polyploids formed by relatively recent whole-genome duplications . Ohno's conjecture that more ancient genome duplications occurred in an ancestor of vertebrates is probably at least partly true but the present shortage of gene sequence and map information from vertebrates makes it difficult to either prove or disprove this hypothesis . Candidate paralogous segments in mammalian genomes have been identified but the lack of statistical rigour means that many of the proposals in the literature are probably artefacts.

J Cell Sci, 1999 Feb, 112 ( Pt 4), 503 - 13
The Ku70 autoantigen interacts with p40phox in B lymphocytes; Grandvaux N et al.; Ku70, a regulatory component of the DNA-dependent protein kinase, was identified by a yeast two-hybrid screen of a B lymphocyte cDNA library as a partner of p40phox, a regulatory component of the O2--producing NADPH oxidase . Truncated constructs of p40phox and Ku70 were used to map the interacting sites . The 186 C-terminal amino acids (aa) of Ku70 were found to interact with two distinct regions of p40phox, the central core region (aa 50-260) and the C-terminal extremity (aa 260-339) . In complementary experiments, it was observed that Ku70 binds to immobilized recombinant p40phox fusion protein and that p40phox and Ku70 from a B lymphocyte cell extract comigrate in successive chromatographies on Q Separose, Superose 12 and hydroxylapatite columns . Moreover, we report that Ku70 and p40phox colocalize in B lymphocytes and in transfected Cos-7 cells . We also show that the two NADPH oxidase activating factors, p47phox and p67phox are substrates for DNA-PK in vitro and that they are present together with p40phox in the nucleus of B cells . These results may help solve the paradox that the phox protein triad, p40phox, p47phox and p67phox, is expressed equally in B lymphocytes and neutrophils, whereas the redox component of the NADPH oxidase, a flavocytochrome b, which is well expressed in neutrophils, is barely detectable in B lymphocytes.

Biochemistry, 1999 Jan 19, 38(3), 1119 - 27
Histidine77, glutamic acid81, glutamic acid123, threonine126, asparagine194, and tryptophan197 of the human emopamil binding protein are required for in vivo sterol delta 8-delta 7 isomerization; Moebius FF et al.; The human emopamil binding protein (hEBP) exhibits sterol Delta8-Delta7 isomerase activity (EC 5.3.3.5) upon heterologous expression in a sterol Delta8-Delta7 isomerization-deficient erg2-3 yeast strain . Ala scanning mutagenesis was used to identify residues in the four putative transmembrane alpha-helices of hEBP that are required for catalytic activity . Isomerization was assayed in vivo by spectrophotometric quantification of Delta5,7-sterols . Out of 64 Ala mutants of hEBP only H77A-, E81A-, E123A-, T126A-, N194A-, and W197A-expressing yeast strains contained 10% or less of wild-type (wt) Delta5,7-sterols . All substitutions of these six residues with functionally or structurally similar amino acid residues failed to fully restore catalytic activity . Mutants E81D, T126S, N194Q, and W197F, but not H77N and E123D, still bound the enzyme inhibitor 3H-ifenprodil . Changed equilibrium and kinetic binding properties of the mutant enzymes confirmed our previous suggestion that residues required for catalytic activity are also involved in inhibitor binding {Moebius et al . (1996) Biochemistry 35, 16871-16878} . His77, Glu81, Glu123, Thr126, Asn194, and Trp197 are localized in the cytoplasmic halves of the transmembrane segments 2-4 and are proposed to line the catalytic cleft . Ala mutants of Trp102, Tyr105, Asp109, Arg111, and Tyr112 in a conserved cytoplasmic domain (WKEYXKGDSRY) between transmembrane segments 2 and 3 contained less than 10% of wt Delta5,7-sterols, implying that this region also could be functionally important . The in vivo complementation of enzyme-deficient yeast strains with mutated cDNAs is a simple and sensitive method to rapidly analyze the functional consequences of mutations in sterol modifying enzymes.

Biochemistry, 1999 Jan 19, 38(3), 923 - 8
Hourglass pore-forming domains restrict aquaporin-1 tetramer assembly; Mathai JC et al.; The AQP1 water channel protein is a homotetramer with 28 kDa subunits containing six transmembrane domains . The sequence-related loops B (cytoplasmic) and E (extracellular) were predicted to overlap within the membrane, forming an aqueous pore ("the hourglass") flanked by the corresponding B and E residues 73 and 189 . Cryoelectron microscopy of AQP1 previously revealed the central hourglass structure surrounded by six transmembrane helices which provide contact points between subunits . Several mutants in loop B and E residues were nonfunctional when expressed in X . laevis oocytes, but their ability to form tetramers is unknown . To explore the possible functional dependence of hourglass domains in adjacent subunits, we prepared a series of tandem dimers as single 55 kDa polypeptides containing different combinations of wild-type (AQP1) or mutant subunits (A73M or C189M) . In oocytes, AQP1-AQP1 exhibited high osmotic water permeability, and AQP1-C189M exhibited half activity . Dimer polypeptides with A73M were nonfunctional or not expressed . In yeast secretory vesicles, AQP1-AQP1 exhibited high water permeability, AQP1-C189M exhibited half activity, and both were inhibited by pCMBS . Although expressed, the dimer polypeptides with A73M were all nonfunctional . Tetramer formation was investigated by detergent solubilization and velocity sedimentation through sucrose gradients . Dimer polypeptides containing one A73M subunit or two C189M subunits migrated with slower velocity (s < 3.5 S) . In contrast, dimer polypeptides with one C189M subunit migrated with velocity similar to native AQP1 tetramers (s approximately 6 S) . Thus, although hourglass pore-forming domains are not points of subunit-subunit contact, the structure of loop B is important to normal tetramer assembly.

J Cell Biochem Suppl, 1998, 30-31, 8 - 17
Initiation of DNA replication in eukaryotic chromosomes; DePamphilis ML; Our understanding of the process by which eukaryotes regulate initiation of DNA replication has made remarkable advances in the past few years, thanks in large part to the explosion of genetic and biochemical information on the budding yeast, Saccharomyces cerevisiae . At least three major concepts have emerged: 1) The sequence of molecular events that determines when replication begins and how frequently each replication site is used are conserved among most, if not all, eukaryotes; 2) specific replication origins are used in most, if not all, eukaryotes that consist of a flexible modular anatomy; and 3) epigenetic factors such as chromatin structure and nuclear organization determine which of many potential replication origins are used at different stages in animal development . Thus, the current state of our knowledge suggests a simple unifying concept--all eukaryotes utilize the same basic proteins and DNA sequences to initiate replication, but the metazoa can change both the number and locations of replication origins in response to the demands of animal development.

Mamm Genome, 1999 Jan, 10(1), 30 - 4
Rat Chromosome 2: assignment of the genes encoding cyclin B1, interleukin 6 signal transducer, and proprotein convertase 1 to the Mcs1-containing region and identification of new microsatellite markers; Szpirer C et al.; The rat Chromosome (Chr) 2 harbors several genes controlling tumor growth or development, blood pressure, and non-insulin-dependent diabetes mellitus . We report that the region (2q1) containing the mammary susceptibility cancer gene Mcs1 also harbors the genes encoding cyclin B1, interleukin 6 signal transducer (gp130), and proprotein convertase 1 . We also generated 13 new anonymous microsatellite markers from Chr 2-sorted DNA . These markers, as well as a microsatellite marker in the cyclin B1 gene, were genetically mapped in combination with known markers . A cyclin B1-related gene was also cytogenetically assigned to rat Chr 11q22-q23.

Proc Natl Acad Sci U S A, 1999 Jan 19, 96(2), 453 - 8
Trapping of megabase-sized DNA molecules during agarose gel electrophoresis; Gurrieri S et al.; Megabase DNA molecules become trapped in agarose gels during electrophoresis if the electric field exceeds a few volts per cm . Fluorescence microscopy reveals that these molecules invariably arrest in U-shaped conformations . The field-vs.-size dependence for trapping indicates that a critical molecular tension is required for trapping . The size of unligated lambda-ladders, sheared during gel electrophoresis at a given field, coincides with the size of molecules trapped at that field, suggesting that both processes occur through nick melting near the vertex of the U-shape . Consistently, molecules nicked by exposure to UV radiation trap more readily than unexposed ones . The critical trapping tension at the vertex is estimated to be 15 pN, a force sufficient to melt nicks bent around gel fibers, and, according to our model, trap a molecule . Strategies to reduce molecular tension and avoid trapping are discussed.

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