FEBS Lett, 1996 Apr 8, 384(1), 48 - 52 Cloning of a novel human RNA polymerase II subunit downregulated by doxorubicin: new potential mechanisms of drug related toxicity; Fanciulli M et al.; Using the differential display PCR method, we have isolated an mRNA downregulated in doxorubicin resistant human cell lines . The full length cDNA clone was identified as the human homologue of yeast RPB11 subunit of RNA polymerase II . Northern blot analysis of normal tissues detected a particularly high expression of RPB11 mRNA in heart and skeletal muscle . Reduction of this mRNA expression was observed in all the cell lines tested after drug treatment and was paralleled by a similar decrease of the protein levels . These findings suggest that doxorubicin may exert in vivo specific inhibitory effects on a major component of the transcription machinery. Science, 1996 Apr 5, 272(5258), 120 - 2 Role of the nuclear transport factor p10 in nuclear import; Nehrbass U et al.; The nuclear import factor p10 was cloned from Saccharomyces cerevisiae and found to be essential . The protein p10 can bind directly to several peptide repeat-containing nucleoporins . It also binds to the guanosine triphosphatase (GTPase) Ran in its guanosine diphosphate (GDP)-bound form and to karyopherin beta . Assembly of the karyopherin heterodimer on immobilized nucleoporin yielded cooperative binding of p10 and Ran-GDP . Addition of GTP to this pentameric complex led to dissociation of karyopherin (chi, presumably via in situ formation of Ran-GTP from Ran-GDP . Thus, p10 appears to coordinate the Ran-dependent association and dissociation reactions underlying nuclear import. Oncogene, 1996 Apr 4, 12(7), 1577 - 81 SH3 domain-dependent interaction of the proto-oncogene product Vav with the focal contact protein zyxin; Hobert O et al.; Scr homology 3 (SH3) domain-mediated protein-protein interactions have been implicated in the localization of proteins to specific sites within the cell . We present evidence that the product of the vav proto-oncogene, p95vav, interacts specifically with the focal adhesion protein zyxin both in vitro and in yeast two hybrid system . Solution binding and two-hybrid system experiments demonstrate that association of Vav with the LIM domain protein zyxin is mediated by the C-terminal SH3 domain of the Vav and involves the proline-rich N-terminus of zyxin . The interaction appears to be selective, since no binding of the proline-rich N-terminus of zyxin with other SH3 domain-containing proteins such as GRB-2, phospholipase C gamma, GTPase-activating protein, or p85 was detected. Oncogene, 1996 Apr 4, 12(7), 1537 - 44 Use of the two hybrid system to detect the association of the protein-tyrosine-phosphatase, SHPTP2, with another SH2-containing protein, Grb7; Keegan K et al.; SHPTP2 is a ubiquitously-expressed SH2-containing tyrosine phosphatase that is tyrosine phosphorylated in response to activation of various receptor and nonreceptor tyrosine kinases . SHPTP2 associates with the platelet-derived growth factor (PDGF) receptor after ligand stimulation, and binding of SHPTP2 to this receptor promotes tyrosine phosphorylation of SHPTP2 . The yeast two-hybrid system was modified to identify partners of tyrosine-phosphorylated proteins . Using SHPTP2 as bait and supplying an exogenous tyrosine kinase gene to the yeast cells, we have found that SHPTP2 interacts with another signaling protein, Grb7 . We have localized the region of interaction to tyrosine 580 in the carboxyl end of SHPTP2 and to the SH2 domain in the carboxy-terminus of Grb7 . We demonstrate that Grb7 binds to SHPTP2 in vitro under conditions where the latter is tyrosine-phosphorylated . These experiments show that this modified two hybrid technique may be useful for the identification of proteins involved in tyrosine kinase signal transduction cascades. Biochemistry, 1996 Apr 2, 35(13), 3970 - 9 Contribution to activity of histidine-aromatic, amide-aromatic, and aromatic-aromatic interactions in the extended catalytic site of cysteine proteinases; Bromme D et al.; Within the papain family of cysteine proteinases few other residues in addition to the catalytic triad, Cys25-His159-Asn175 (papain numbering) are completely conserved {Berti & Storer (1995) J . Mol . Biol . 246, 273-283} . One such residue is tryptophan 177 which participates in a Trp-His-type interaction with the catalytic His159 . In all enzymes of this class for which a three-dimensional structure has been reported, an additional highly conserved tryptophan, Trp181, also interacts with Trp177 via an aromatic-aromatic interaction in which the planes of the indole rings are essentially perpendicular . Also, both indole rings participate as pseudo-hydrogen bond acceptors in interactions with the two side chain amide protons of Asn175 . Clearly, the proximity of Trp177 and Trp181 to the catalytic triad residues His159 and Asn175 and their network of interactions points to potential contributions of these aromatic residues to catalysis . In this paper, using cathepsin S, a naturally occurring variant that has a phenylalanine residue at position 181, we report the kinetic characterization of mutants of residues 175, 177, and 181 . The results are interpreted in terms of the side chain contributions to catalytic activity and thiolate-imidazolium ion-pair stability . For example, the side chain of Asn175 has a major influence on the ion-pair stability presumably through its hydrogen bond to His159 . The magnitude of this effect is modulated by Trp177, which shields the His159-Asn175 hydrogen bond from solvent . The His159-Trp177 interaction also contributes significantly to ion-pair stability; however, Trp181 and its interactions with Asn175 and Trp177 do not influence ion-pair stability to a significant degree . The observation that certain mutations at positions 177 and 181 result in a reduction of kcat/Km but do not appear to influence ion-pair stability probably reflects the contributions of these residues to substrate binding. Proc Natl Acad Sci U S A, 1996 Apr 2, 93(7), 3011 - 5 Isolation of Drosophila cyclin D, a protein expressed in the morphogenetic furrow before entry into S phase; Finley RL Jr et al.; During Drosophila development, nuclear and cell divisions are coordinated in response to developmental signals . In yeast and mammalian cells, signals that control cell division regulate the activity of cyclin-dependent kinases (Cdks) through proteins such as cyclins that interact with the Cdks . Here we describe two Drosophila cyclins identified from a set of Cdk-interacting proteins . One, cyclin J, is of a distinctive sequence type; its exclusive maternal expression pattern suggests that it may regulate oogenesis or the early nuclear divisions of embryogenesis . The other belongs to the D class of cyclins, previously identified in mammalian cells . We show that Drosophila cyclin D is expressed in early embryos and in imaginal disc cells in a pattern that anticipates cell divisions . Expression in the developing eye disc at the anterior edge of the morphogenetic furrow suggests that cyclin D acts early, prior to cyclin E, in inducing G1-arrested cells to enter S phase . Our results also suggest that, although cyclin D may be necessary, its expression alone is not sufficient to initiate the events leading to S phase. Proc Natl Acad Sci U S A, 1996 Apr 2, 93(7), 2975 - 80 Intradimerically tethered DNA topoisomerase II is catalytically active in DNA transport; Lindsley JE; A covalently cross-linked dimer of yeast DNA topoisomerase II was created by fusing the enzyme with the GCN4 leucine zipper followed by two glycines and a cysteine . Upon oxidation of the chimeric protein, a disulfide bond forms between the two carboxyl termini, covalently and intradimerically cross-linking the two protomers . In addition, all nine of the cysteines naturally occurring in topoisomerase II have been changed to alanines in this construct . This cross-linked, cysteine-less topoisomerase II is catalytically active in DNA duplex passage as indicated by ATP-dependent DNA supercoil relaxation and kinetoplast DNA decatenation assays . However, these experiments do not directly distinguish between a "one-gate" and a "two-gate" mechanism for the enzyme. Proc Natl Acad Sci U S A, 1996 Apr 2, 93(7), 2719 - 23 Regulation of mitochondrial pyruvate dehydrogenase activity by tau protein kinase I/glycogen synthase kinase 3beta in brain; Hoshi M et al.; According to the amyloid hypothesis for the pathogenesis of Alzheimer disease, beta-amyloid peptide (betaA) directly affects neurons, leading to neurodegeneration and tau phosphorylation . In rat hippocampal culture, betaA exposure activates tau protein kinase I/glycogen synthase kinase 3beta (TPKI/GSK-3beta), which phosphorylates tau protein into Alzheimer disease-like forms, resulting in neuronal death . To elucidate the mechanism of betaA-induced neuronal death, we searched for substrates of TPKI/GSK-3beta in a two-hybrid system and identified pyruvate dehydrogenase (PDH), which converts pyruvate to acetyl-CoA in mitochondria . PDH was phosphorylated and inactivated by TPKI/GSK-3beta in vitro and also in betaA-treated hippocampal cultures, resulting in mitochondrial dysfunction, which would contribute to neuronal death . In cholinergic neurons, betaA impaired acetylcholine synthesis without affecting choline acetyltransferase activity, which suggests that PDH is inactivated by betaA-induced TPKI/GSK-3beta . Thus, TPKI/GSK-3beta regulates PDH and participates in energy metabolism and acetylcholine synthesis . These results suggest that TPKI/GSK-3beta plays a key role in the pathogenesis of Alzheimer disease. J Bioenerg Biomembr, 1996 Apr, 28(2), 101 - 8 Characterization and function of the mitochondrial outer membrane peptide-sensitive channel; Henry JP et al.; The PSC (peptide-sensitive Channel), a cationic channel of large conductance, has been characterized in yeast and mammalian mitochondria by three different methods, "tip-dip," patch clamp of giant liposomes, and planar bilayers . The yeast and mammalian PSC share the common property to be blocked by basic peptides such as pCyt OX IV (1-12)Y which contains the first 12 residues of the presequence of cytochrome C oxidase subunit IV . The electrophysiological data are consistent with a translocation of the peptide through the pore . Analysis of the frequency of observation of the PSC in different fractions indicates that the channel is located in the outer mitochondrial membrane . Uptake measurements of iodinated peptides by intact mitochondria from a porin-less mutant show that the peptides are translocated through the outer membrane, presumably at the level of PSC . Among the peptides active on PSC, several, such as pCyt OX IV (1-22) and the reduced form of the mast cell degranulating peptide, induce an alteration of the voltage dependence or of the inactivation rate subsisting after washing and which is eliminated only by proteolysis of the interacting peptide . These irreversible effects may account for the variability of the properties of the PSC which would interact with cytosolic or intermembrane cations, peptides, or proteins, thus modulating the channel permeability . Finally, several lines of evidence suggest the participation of the PSC in protein translocation and some interaction with the general insertion pore of the outer membrane translocation machinery. Boll Chim Farm, 1996 Apr, 135(4), 248 - 58 {Biological activity of a carrier molecule-iron complex: probable mechanisms of intestinal absorption}; Curri SB; The physical-chemical properties of a new Iron-Protein complex, the so called "Iron Yeast" biotecnologically produced by Saccharomyces cerevisiae cell cultures, are reported . Using atomic absorption methods the amount of free iron in the complex has been evaluated, ascertaining that 99% of Iron is structurally bound to the protein matrix . Iron Yeast is a further, original contribution to the treatment of iron-deficiency anemias, showing a satisfactory clinical activity and a lower amount of undesired side effects in comparison with other Iron-Protein complexes. Plant Physiol, 1996 Apr, 110(4), 1395 - 404 Physicochemical and serological characterization of rice alpha-amylase isoforms and identification of their corresponding genes; Mitsui T et al.; We have identified, purified, and characterized 10 alpha-amylase isoforms from suspension-cultured rice (Oryza sativa L.) cells having different isoelectric point values . They had distinguishable optimum temperatures for enzymatic activity and molecular sizes . The results of immunoblotting indicated that polyclonal anti-A + B antibodies bound well to isoforms A, B, Y, and Z but weakly or not at all to E, F, G, H, I, and J . However, the anti-A + B antibodies inhibited the enzyme activities of only isoforms A and B . Polyclonal anti-H antibodies strongly bound to isoforms F, G, H, I, and J, whereas polyclonal anti-E antibodies preferentially recognized isoform E . A monoclonal antibody against isoform H (H-G49) inhibited the activities of isoforms E, G, H, I, and J, whereas it did not inhibit those of isoforms A, B, Y, and Z . Judging from their physicochemical and serological properties, we classified the rice alpha-amylase isoforms into two major classes, class I (A, B, Y, and Z) and class II (E, F, G, H, I, and J), and into four subgroups, group 1 (A and B), group 2 (Y and Z), group 3 (E), and group 4 (F, G, H, I, and J) . Partial amino acid sequences for isoforms A, E, G, and H were also determined . In addition, the recombinant alpha-amylases expressed by plasmid pEno/103 containing the rice alpha-amylase gene RAmy1A in yeast were identified as both isoforms A and B . These analyses indicated that isoforms A and B were encoded by the gene RAmy1A, isoforms G and H were encoded by the gene RAmy3D, and isoform E was encoded by RAmy3E . The results strongly suggest that some isoforms within subgroups are formed by posttranslational modifications. Plant Physiol, 1996 Apr, 110(4), 1337 - 47 Tomato Rab1A homologs as molecular tools for studying Rab geranylgeranyl transferase in plant cells; Loraine AE et al.; Rab proteins attach to membranes along the secretory pathway where they contribute to distinct steps in vesicle-mediated transport . To bind membranes, Rab proteins in fungal and animal cells must be isoprenylated by the enzyme Rab geranylgeranyl transferase (Rab GGTase) . We have isolated three tomato (Lycopersicon esculentum, M.) cDNAs (LeRab 1A, B, and C) encoding Rab-like proteins and show here that all three are substrates for a Rab GGTase-like activity in plant cells . The plant enzyme is similar to mammalian Rab GGTase in that the plant activity (a) is enhanced by detergent and (b) is inhibited by mutant Rab lacking a prenylation consensus sequence . LeRab1B contains a rare prenylation target motif and was the best substrate for the plant, but not the yeast, Rab GGTase . LeRab1A, B, and C are functional homologs of the Saccharomyces cerevisiae Rab protein encoded by YPT1 and are differentially expressed in tomato . LeRab1A mRNA, but not that of LeRab1B or C, is induced by ethylene in tomato seedlings and is also upregulated in ripening fruit . The increase in LeRab1A mRNA expression in ripe fruit may be linked to increased synthesis and export of enzymes like polygalacturonase, pectin esterase, and other enzymes important in fruit softening. FEBS Lett, 1996 Apr 1, 383(3), 273 - 6 SAP kinase-3, a new member of the family of mammalian stress-activated protein kinases; Mertens S et al.; Stress-activated protein kinases are MAP kinase homologues that are activated by cellular stresses, bacterial endotoxin and inflammatory cytokines . They are activated by a dual threonine/tyrosine phosphorylation within a TPY sequence in the case of stress-activated protein kinase-1 and its isoforms (also called JNKs) or a TGY sequence in the case of stress-activated protein kinase-2 and its isoforms (also called p38, p40, RK, CSBPs, XMpk2 and Mxi2) . Here we report the cloning and sequencing of a new protein kinase from rat with a TGY sequence in the activation domain . This stress-activated protein kinase-3 is 60% identical to mouse stress-activated protein kinase-2 and 45% identical to HOG1 from Saccharomyces cerevisiae . Transcripts encoding stress-activated protein kinase-3 are widely expressed, with high levels in skeletal muscle. FEBS Lett, 1996 Apr 1, 383(3), 213 - 8 A 45-bp proximal region containing AACA and GCN4 motif is sufficient to confer endosperm-specific expression of the rice storage protein glutelin gene, GluA-3; Yoshihara T et al.; A 45-bp proximal region of the rice glutelin promoter (-104/-60) containing two putative cis-elements, the AACA motif and GCN4 motifs, was fused to a truncated CaMV 35S promoter (-90/+9; -90 delta 35S)/GUS . The 45-bp fragment specifically enhanced the promoter activity in endosperm tissue of transformed tobacco . A substitution mutation of the GCN4 motif reduced the promoter activity, whereas mutation of the AACA motif increased the activity in the embryo as well as in the endosperm . These results suggest that the GCN4 motif generally enhances the promoter activity but that the combination of the two motifs confers the endosperm specificity . Furthermore, the function of the two motifs was dependent on the orientation and/or distance from a G-box element in -90 delta 35S, suggesting that synergistic interaction between the factors that recognize those motifs and the G-box element is important for transcriptional regulation. FEBS Lett, 1996 Apr 1, 383(3), 185 - 90 Influence of substrate structure on cleavage by hammerhead ribozyme; Scarabino D et al.; We compared the cleavage by a hammerhead ribozyme of a wild-type precursor tRNA (pre-tRNA leu(3)) and a structurally altered mutant form . We also analyzed the cleavage reactions of these tRNAs catalyzed by a ribozyme variant that was designed to complement the mutant precursor tRNA . Kinetic analyses reveal that the kcat values are nearly the same for the wild-type and the mutant substrate RNAs . However, the Km values differ considerably, being higher for the wild-type substrate . Thus, the formation of the ribozyme-substrate complex, but not the chemical cleavage step, is affected by these changes . Time course studies were performed, at different temperatures, to estimate the efficiency of the cleavage reactions and the effect of temperature . The cleavage of mutant precursor tRNA is generally faster than the wild-type at all temperatures analyzed . These results suggest that substrate structures can limit ribozyme efficiency, presumably by hindering the hybridization step. Biol Pharm Bull, 1996 Apr, 19(4), 506 - 11 Characterization of cyclophilin 40: highly conserved protein that directly associates with Hsp90; Yokoi H et al.; Cyclophilin 40 (CyP4O) is a recently identified member of the cyclophilin family that may be a component of unactivated steroid receptor complexes . It consists of an N-half portion that is highly homologous to cyclophilin A and has peptidyl prolyl isomerase (PPIase) activity, and a C-half portion that resembles the C-terminal portion of FKBP52 (FK506 binding protein 52), another component of unactivated steroid receptor complexes . To better understand the structure and functional characteristics of this new class of cyclophilin, we have raised monoclonal antibodies against the C-half portion of human CyP4O . Immunostaining with the antibodies showed its preferential localization in cytoplasm . One antibody cross-reacted with a 45 kDa protein in yeast, suggesting high conservation throughout evolution . A CyP4O-associated protein was isolated from rabbit reticulocyte lysate by means of an affinity resin, and was identified as hsp90 . The C-half portion of CyP4O was necessary and sufficient for the interaction. Genes Dev, 1996 Apr 1, 10(7), 862 - 72 Functional interactions between the pelle kinase, Toll receptor, and tube suggest a mechanism for activation of dorsal; Norris JL et al.; A complex signal transduction pathway functions in the early Drosophila embryo to establish dorsal-ventral polarity . Activation of this pathway results in the nuclear transport of the protein dorsal (dl), a member of the rel/NF-kappaB family of transcription factors . Genetic studies have identified three intracellular components whose activity is required for activation of dl: Toll, a transmembrane receptor; pelle (pll), a serine/threonine protein kinase; and tube, a protein of unknown function . Here we examine the activities of these proteins when coexpressed in Drosophila Schneider cells . Coexpression of pll with dl enhanced dl nuclear localization and resulted in a modest increase in transcriptional activity . However, when pll was coexpressed with a specific mutant derivative of Toll (TlNaeI), although not with wild-type Toll, a striking synergistic activation of dl was detected . Unexpectedly, coexpression of pll plus TlNaeI, in the absence of dl, resulted in a similar synergistic activation of a GAL4-tube fusion protein . Based on these and other results, we propose a model in which pll receives a signal from activated Toll and phosphorylates tube, which then participates directly in dl activation. Protein Sci, 1996 Apr, 5(4), 759 - 67 The mitochondrial protein import motor: dissociation of mitochondrial hsp70 from its membrane anchor requires ATP binding rather than ATP hydrolysis; Horst M et al.; During protein import into mitochondria, matrix-localized mitochondrial hsp70 (mhsp70) interacts with the inner membrane protein Tim44 to pull a precursor across the inner membrane . We have proposed that the Tim44-mhsp70 complex functions as an ATP-dependent "translocation motor" that exerts an inward force on the precursor chain . To clarify the role of ATP in mhsp70-driven translocation, we tested the effect of the purified ATP analogues AMP-PNP and ATP gamma S on the Tim44-mhsp70 interaction . Both analogues mimicked ATP by causing dissociation of mhsp70 from Tim44 . ADP did not disrupt the Tim44-mhsp70 complex, but did block the ATP-induced dissociation of this complex . In the presence of ADP, mhsp70 can bind simultaneously to Tim44 and to a peptide substrate . These data are consistent with a model in which mhsp70 first hydrolyzes ATP, then associates tightly with Tim44 and a precursor protein, and finally undergoes a conformational change to drive translocation. J Steroid Biochem Mol Biol, 1996 Apr, 58(1), 3 - 12 Generation of estrogen receptor mutants with altered ligand specificity for use in establishing a regulatable gene expression system; Whelan J et al.; Considerable interest exists in developing an artificial system for the control of gene expression, based on the hormone binding domain (HBD) of steroid receptors . In this study we describe a yeast based approach which allows the identification of mutations within the HBD of steroid receptors, in this case the estrogen receptor, which result in altered specificity of the HBD with respect to its activation by ligands . Using this approach in yeast, we identified an estrogen receptor (HBD) mutant (His524 to Gln) whose activation by 17 beta-estradiol (E2) is significantly reduced while activation by a diphenol indene-ol compound (GR132706X) is increased, compared to the wild type estrogen receptor . When the activity of the mutant receptor was tested in mammalian cells the altered specificity was maintained . A chimeric transcription factor was constructed, in which the mutated estrogen receptor HBD was linked to the DNA binding domain of GAL4 and an 11 amino acid transcriptional activation domain of RelA . Reporter gene activation by this chimera was decreased in response to E2 and increased in response to GR132706X, as compared to the corresponding chimeric transcription factor containing the wild type estrogen receptor HBD . This approach should allow the development of a steroid receptor HBD based regulator of gene expression, whose activity is controlled specifically by a synthetic ligand, that would not affect the activity of endogenous steroid receptors. Yeast, 1996 Apr, 12(5), 493 - 9 The sequence of 12.8 kb from the left arm of chromosome XIV reveals a sigma element, a pro-tRNA and six complete open reading frames, one of which encodes a protein similar to the human leukotriene A4 hydrolase; Nasr F et al.; We have determined the nucleotide sequence of a 12.8 kb fragment from the left arm of chromosome XIV carried by the cosmid 14-16d . An analysis of the sequence reveals the presence of a sigma element, a pro-tRNA gene and eight open reading frames, six of which are complete . All of the eight open reading frames correspond to new genes . Of the eight new genes, two show strong similarities to a pair of new genes from chromosome IX, suggesting an ancestral duplication, and one gene encodes a protein similar to mammalian leukotriene A4 hydrolase. Mol Phylogenet Evol, 1996 Apr, 5(2), 414 - 22 Early evolution of the Metazoa and phylogenetic status of diploblasts as inferred from amino acid sequence of elongation factor-1 alpha; Kobayashi M et al.; To understand the early evolution of the Metazoa, it is necessary to determine the correct phylogenetic status of diploblastic animals (poriferan, cnidaria, and ctenophora) . Despite clasdistic studies of morphological characters and recent molecular phylogenetic studies, it remains uncertain whether diploblasts are monophyletic or paraphyletic, and how these three phyla of diploblasts are phylogenetically related . To obtain insight into these phylogenetic problems, we sequenced almost the entire nucleic acid sequence of elongation factor-1 alpha from a sponge, two cnidarians, a ctenophora, and a turbellarian . We then investigated the phylogenetic status of the diploblasts . We compared the amino acid sequences, nucleotide sequences at the first and second codon positions, and those at the second positions . Phylogenetic trees were inferred by neighbor-joining, maximum likelihood, and maximum parsimony, and they supported the monophyly of the Metazoa . However, the phylogenetic relationships of the diploblast groups were not significantly resolved, although the trees preferred the monopoly of the diploblasts. Biochem Mol Biol Int, 1996 Apr, 38(4), 791 - 9 Cloning and sequence analysis of a putative transcription factor (MTF1) gene from Mucor circinelloides; Mukhtar M et al.; We describe here the fortuitous cloning of a putative transcription factor gene (MTF1) from the dimorphic fungus Mucor circinelloides . Sequence analysis of MTF1 revealed an open reading frame (ORF) of 1059 nucleotides encoding a protein of M(r) 39601 . The deduced amino acid sequence from the ORF imparts two glutamine-rich stretches which are homologous to a number of transcription factors characterized previously from various organisms . A Southern blot analysis of Mucor genomic DNA digested with different restriction endonucleases and probed with the 1.9 kb EcoR1 fragment of the putative transcription factor gene shows a single copy number of the the gene . Northern analysis during morphogenetic changes in Mucor suggested constitutive expression of the gene. Glycobiology, 1996 Apr, 6(3), 265 - 70 A spectrophotometric assay for alpha-mannosidase activity; Scaman CH et al.; A simple and versatile spectrophotometric assay for alpha-mannosidase activity, which can be used with unlabelled natural substrate or synthetic substrates, was developed . The reducing mannose released from the substrate by the enzyme is quantitated using glucose oxidase, peroxidase and o-dianisidine . Using recombinant alpha 1,2-mannosidase obtained from Saccharomyces cerevisiae and Man9, GlcNAc, the spectrophotometric assay yielded values of 0.3 mM for Km and 15 mU/microgram for V(max), which are comparable to those obtained using the traditional radiochemical assay . The assay was also used to evaluate some alternative oligosaccharides as substrates for the enzyme . Man5-O(CH2)8-COOCH3 shows potential as an alternative synthetic substrate as the enzyme retained its specificity for a single alpha 1,2-mannose residue . Kinetic results suggest that the lower 1,3 linked arm of Man9GlcNAc is more critically involved in substrate recognition than the upper 1,6 linked arm. Curr Biol, 1996 Apr 1, 6(4), 446 - 54 A mechanism of Bud1p GTPase action suggested by mutational analysis and immunolocalization; Michelitch M et al.; BACKGROUND: Yeast cells polarize, bud, and divide in either of two genetically programmed patterns: axial or bipolar . The Saccharomyces cerevisiae gene BUD1 (also known as RSR1) encodes a Ras-related GTPase critical for selection of a bud sites in these patterns . To distinguish between possible mechanisms of Bud1p action, we have examined the function and subcellular localization of Bud1p in a variety of mutant situations . RESULTS: Bud1p has 57% identity to H-ras, except for an 81 amino-acid insertion near the carboxyl terminus . Mutation of the proposed BUD1 effector domain produces a protein which can neither support normal patterns of budding nor interact with CDC24, which encodes a likely Bud1p effector . A version of Bud1p deleted for the 81 amino-acid unique region is essentially wild-type . Immunofluorescence and cell fractionation indicate that Bud1p remains associated with the membrane throughout its GTPase cycle . Both potential effectors of Bud1p, Bem1p and Cdc24p, are also membrane associated even in the absence of Bud1p, suggesting that Bud1p is not required to dock these proteins from the cytosol but may couple these proteins and others within the plane of the plasma membrane . CONCLUSIONS: Based upon observations reported here and elsewhere, we propose a novel mechanism of Bud1p GTPase action . Like Ras, Bud1p GTPase is constitutively associated with the plasma membrane; however, concentrated activities of Bud5p GDP-GTP exchange factor and Bud2p GTPase-activating protein at the future bud site promote rapid cycling of Bud1p between GTP- and GDP-bound conformations in a spatially restricted manner . Local GTPase cycling serves to efficiently nucleate complexes between polarity establishment functions that direct cytoskeletal polarization towards the bud site. Curr Opin Genet Dev, 1996 Apr, 6(2), 176 - 84 Special HATs for special occasions: linking histone acetylation to chromatin assembly and gene activation; Brownell JE et al.; Post-translational acetylation of the core histone amino-terminal tails has long been associated with both chromatin assembly and the regulation of gene expression . The recent identification and cloning of histone acetyltransferase genes represents a significant breakthrough in our understanding of how specific acetylation states are established . Ongoing characterization of these enzymes and their molecular cohorts supports a direct role for acetylation in a signaling pathway that modulates chromatin structure to create new patterns of transcription. Curr Opin Genet Dev, 1996 Apr, 6(2), 164 - 70 Regulation of gene expression by nucleosomes; Svaren J et al.; During the past year, the characterization of mechanisms and factors capable of disrupting nucleosomes during transcriptional activation has been a recurrent theme in studies which address the contribution of nucleosome structure to gene regulation . In vivo studies using yeast and Drosophila together with biochemical purification schemes using nucleosome perturbation assays have provided evidence for the existence of multiprotein complexes that are able to alleviate nucleosome repression . At the same time, new insights into the mechanism of heterochromatin formation have been gained, which have direct links to nucleosome structure. Trends Biochem Sci, 1996 Apr, 21(4), 140 - 5 The biology of HMG-CoA reductase: the pros of contra-regulation; Hampton R et al.; Hydroxymethylglutaryl-CoA reductase (HMG-R) is a key enzyme in the mevalonate pathway, from which thousands of molecules are derived including cholesterol and prenyl moieties . The regulation of HMG-R is complex and includes feedback control, cross-regulation by independent bio-chemical processes and contra-regulation of separate isozymes . From studies in yeast, these separate modes of regulation can be considered in an integrated fashion. Trends Biochem Sci, 1996 Apr, 21(4), 122 - 6 Post-translational protein translocation: not all hsc70s are created equal; Brodsky JL; Hsc70s in the yeast endoplasmic reticulum (ER) and mitochondria interact with membrane-associated components of the translocation machinery and are required for post-translational protein import . Although it has been proposed that the mitochondrial and ER machines function similarly, a variety of experiments suggest that BiP, the ER hsc70, might play a more elaborate role. Curr Eye Res, 1996 Apr, 15(4), 363 - 9 Peptide hydrolysis in lens: role of leucine aminopeptidase, aminopeptidase III, prolyloligopeptidase and acylpeptidehydrolase; Sharma KK et al.; The distribution of leucine aminopeptidase, aminopeptidase III, prolyloligopeptidase and acylpeptidehydrolase activities in different regions of a bovine lens was determined and correlated with the distribution of crystallin fragments (measured as < 18 kDa protein) and water-insoluble proteins in the same lens . A gradient of activity was observed for all the peptidases tested, with the highest specific activity present in the cortical fibers which decreased to one half or below in the inner cortical fibers and nucleus . An inverse correlation between peptidase activities and the amount of crystallin fragments was observed in different regions of the lens . However, a direct correlation between the water-insoluble protein content and the crystallin fragments was observed in all fibers of the same lens . The amount of crystallin fragments and the amount of water-insoluble proteins increased from 2.7% and 8% in the outer cortical fibers to 13% and 68% in the nucleus of the same lens . The water-insoluble fraction from both cortical and nuclear fibers however displayed 4-5 fold more crystallin fragments compared to that present in the water-soluble fraction of the same preparation . When the bovine lens cortical and nuclear extracts were tested for their ability to hydrolyze the peptide substrate, Ile-Ser-bradykinin, the cortical extract was found to be at least ten times superior to the nuclear extract . Prior inactivation of prolyloligopeptidase and other serine proteases by diisopropylfluorophosphate however diminished the ability of the cortical extract to hydrolyze peptide substrates . Bovine lens cortical extract was able to completely hydrolyze alpha-melanocyte stimulating hormone as well as N-Acetyl-Met-Asp-Arg-Val-Leu-Ser-Arg-Tyr showing the presence of active acylpeptidehydrolase facilitating the complete hydrolysis of N-terminally blocked peptides . The human lens extract was found to contain both diisopropylfluorophosphate sensitive and resistant enzymes capable of hydrolyzing peptide substrates. Mol Cell Biol, 1996 Apr, 16(4), 1832 - 41 Characterization of an essential Orc2p-associated factor that plays a role in DNA replication; Hardy CF; The Saccharomyces cerevisiae Orc2 protein is a subunit of the origin recognition complex, ORC, which binds in a sequence-specific manner to yeast origins of DNA replication . With screens for orc2-1 synthetic lethal mutations and Orc2p two-hybrid interactors, a novel Orc2p-associated factor (Oaf1p) was identified . OAF1 is essential, its gene product is localized to the nucleus, and an oaf1 temperature-sensitive mutant arrests as large budded cells with a single nucleus . The mutant oaf1-2, isolated in the synthetic lethal screen, loses plasmids containing a single origin of DNA replication at a high rate, but it maintains plasmids carrying multiple potential origins of DNA replication . In addition, the OAF1 gene product tagged with the hemagglutinin antigen epitope binds to a DNA affinity column containing covalently linked tandem repeats of an essential origin element . These results suggest a role for OAFI in the initiation of DNA replication . Mutant alleles of cdc7 and cdc14 were also isolated in the orc2-1 synthetic lethal screen . Cdc7p, like Oaf1p, also interacts with Orc2p in two-hybrid assays. Mol Cell Biol, 1996 Apr, 16(4), 1746 - 58 Role of negative regulation in promoter specificity of the homologous transcriptional activators Ace2p and Swi5p; Dohrmann PR et al.; The Ace2p and Swi5p zinc finger proteins have nearly identical DNA-binding domains, yet in vivo they activate transcription of different genes, CTS1 and HO . We now demonstrate that Ace2p and Swi5p recognize sites in the CTS1 and HO promoters with the same affinities, raising the question of how promoter specificity is achieved by these proteins with similar DNA-binding domains . It has been previously shown that Swi5p binds to the HO promoter cooperatively with the Pho2p (Base2p/Grf10p) homeodomain protein, and we now show that Ace2p does not interact with Pho2p . Analysis of CTS1 promoter fragments inserted into a heterologous promoter identify a sequence 90 bp away from the Ace2p binding sites which is required to prevent activation by Swi5p through these binding sites . These results suggest that a regulatory protein bound to the CTS1 promoter is needed to prevent Swi5p from activating CT1S expression . A genetic screen was conducted to identify suppressor mutations which allow CTS1 expression in the absence of the Ace2p activator . The nce3 mutation suppresses the ace2 defect in CTS1 expression only if the strain contains a functional SWI5 gene, suggesting that NCE3 normally functions to prevent Swi5p from activating CTS1 . The role of negative regulators such as NCE3, as well as the previously described SIN5 gene, in determining the promoter specificity of homologous activators is discussed. Mol Cell Biol, 1996 Apr, 16(4), 1641 - 8 Transcriptional corepression in vitro: a Mot1p-associated form of TATA-binding protein is required for repression by Leu3p; Wade PA et al.; Signals from transcriptional activators to the general mRNA transcription apparatus are communicated by factors associated with RNA polymerase II or the TATA-binding protein (TBP) . Currently, little is known about how gene-specific transcription repressors communicate with RNA polymerase II . We have analyzed the requirements for repression by the saccharomyces cerevisiae Leu3 protein (Leu3p) in a reconstituted transcription system . We have identified a complex form of TBP which is required for communication of the repressing signal . This TFIID-like complex contains a known TBP-associated protein, Mot1p, which has been implicated in the repression of a subset of yeast genes by genetic analysis . Leu3p-dependent repression can be reconstituted with purified Mot1p and recombinant TBP . In addition, a mutation in the Mot1 gene leads to partial derepression of the Leu3p-dependent LEU2 promoter . These in vivo and in vitro observations define a role for Mot1p as a transcriptional corepressor. Mol Cell Biol, 1996 Apr, 16(4), 1519 - 26 Ku86 defines the genetic defect and restores X-ray resistance and V(D)J recombination to complementation group 5 hamster cell mutants; Errami A et al.; X-ray-sensitive hamster cells in complementation groups 4, 5, 6, and 7 are impaired for both double-strand break repair and V(D)J recombination . Here we show that in two mutant cell lines (XR-V15B and XR-V9B) from group 5, the genetic defects are in the gene encoding the 86-kDa subunit of the Ku autoantigen, a nuclear protein that binds to the double-stranded DNA ends . These mutants express Ku86 mRNA containing deletions of 138 and 252 bp, respectively, and the encoded proteins contain internal, in-frame deletions of 46 and 84 amino acids . Two X-ray-resistant revertants of XR-V15B expressed two Ku86 transcripts, one with and one without the deletion, suggesting that reversion occurred by activation of a silent wild-type allele . Transfection of full-length cDNA encoding hamster Ku86 into XR-V15B cells resulted in a complete rescue of DNA-end-binding (DEB) activity and Ku70 levels, suggesting that Ku86 stabilizes the Ku70 polypeptide . In addition, cells expressing wild-type levels of DEB activity were fully rescued for X-ray resistance and V(D)J recombination, whereas cells expressing lower levels of DEB activity were only partially rescued . Thus, Ku is an essential component of the pathway(s) utilized for the resolution of DNA double-strand breaks induced by either X rays or V(D)J recombination, and mutations in the Ku86 gene are responsible for the phenotype of group 5 cells. J Virol, 1996 Apr, 70(4), 2481 - 9 Epstein-Barr virus nuclear antigen 3C is a powerful repressor of transcription when tethered to DNA; Bain M et al.; The expression of Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA3C) is essential for the activation and immortalization of human B lymphocytes by EBV . EBNA3C consists of 992 amino acids and includes a potential bZIP motif and regions rich in acidic, proline, and glutamine residues . Thus, EBNA3C resembles several trans regulators of gene expression . It has recently been shown that a fragment of EBNA3C can activate reporter gene expression when fused to the DNA-binding domain of GAL4 (D . Marshall and C . Sample, J . Virol . 69:3624-3630,1995) . Although EBNA3C binds DNA, a specific site for EBNA3C binding has not been identified; to test the ability of full-length EBNA3C to regulate transcription, EBNA3C (amino acids 11 to 992) was fused to the DNA-binding domain of GAL4 . We show that this fusion protein does not transactivate but rather is a potent repressor of reporter gene expression . Repression is dependent on the dose of GAL4-EBNA3C and on the presence of GAL4-binding sites within reporter plasmids . Repression is not restricted to B cells nor is it species or promoter specific . Repression is independent of the location of the GAL4-binding sites relative to the transcription start site . A fragment of EBNA3C (amino acids 280 to 525) which represses expression in a manner which is nearly identical to that of the full-length protein has been identified; this fragment is rich in acidic and proline residues . A second, less potent repressor region located C terminal to amino acids 280 to 525 has also been identified; this domain is rich in proline and glutamine residues . We also show binding of EBNA3C, in vitro, to the TATA-binding protein component of TFIID, and this suggests a mechanism by which EBNA3C may communicate with the basal transcription complex. J Virol, 1996 Apr, 70(4), 2124 - 31 Identification and characterization of a small modular domain in the herpes simplex virus host shutoff protein sufficient for interaction with VP16; Schmelter J et al.; The herpes simplex virus transactivator VP16 and the virion host shutoff protein vhs are viral structural components that direct the activation of immediate-early gene expression and the arrest of host protein synthesis, respectively, during an infection . Recent studies show that VP16 and vhs physically interact with each other in vitro and in infected cells, suggesting that their respective regulatory functions are coupled . In this report, we used the yeast two-hybrid system and affinity chromatography with purified VP16 fusion proteins to precisely map a region in vhs that directs interaction with VP16 . Deletion analysis of vhs demonstrated that a 21-amino-acid-long domain spanning residues 310 to 330 (PAAGGTEMRVSWTEILTQQIA) was sufficient for directing complex formation with VP16 in vivo and in vitro when fused to a heterologous protein . Site-directed mutagenesis of this region identified tryptophan 321 as a crucial determinant for interaction with VP16 in vitro and in vivo and additional residues that are important for stable complex formation in vitro . These findings indicate that vhs residues 310 to 330 constitute an independent and modular binding interface that is recognized by VP16. DNA Cell Biol, 1996 Apr, 15(4), 297 - 304 Isolation and characterization of BART-1: A novel balloon angioplasty responsive transcript in rat carotid arteries; Autieri MV et al.; In previous studies, differential display analysis of balloon angioplasty-damaged rat carotid arteries identified a temporally expressed partial cDNA termed RC9 . This message is undetectable in undamaged vessels, reaches maximal levels 3 days post-procedure, and reduces to half-maximal expression by 14 days post angioplasty . Using RC9 to screen a cDNA library, we now report the isolation and characterization of a full-length clone, termed BART-1 . BART-1 is 98% homologous to RC9 and shows the same mRNA expression pattern as RC9 in rat carotid arteries subject to balloon angioplasty . Northern analysis of various rat tissues reveals tissue specificity and possible differential processing . Neither the nucleic acid nor amino acid sequences demonstrate similarity to previously reported expressed sequences . Predicted amino acid analysis reveals two strongly hydrophilic and one hydrophobic region, suggesting a type II integral membrane protein . The cDNA sequence hybridized to genomic DNA from a variety of species, suggesting evolutionary conservation . Thus, BART-1 mRNA appears to represent an inducible, tissue-specific transcript encoding a putative integral membrane protein transiently expressed in response to vascular trauma. Mol Carcinog, 1996 Apr, 15(4), 270 - 5 Complete sequence analysis of a gene (OS-9) ubiquitously expressed in human tissues and amplified in sarcomas; Su YA et al.; Amplification and overexpression of genes involved in cellular growth control occur frequently in human tumors . Using a chromosome microdissection-based hybrid-selection strategy, we recently identified two novel genes (OS-9 and OS-4) within 12q13-15, a region frequently amplified in human cancers . We now report further characterization of the full-length OS-9 cDNA sequence consists of 2785 bp from which an open reading frame (ORF) with 667 amino-acid residues was deduced, The predicted polypeptide was water soluble and acidic . We also demonstrate that the OS-9 gene encoded a 2.8-kb mRNA transcribed in all 16 human tissues examined, suggesting that OS-9 is ubiquitously expressed in human tissues . OS-9 was co-amplified with CDK4 in three of five sarcoma tissues . Homology analysis of the amino-acid sequence reveals significant similarities between OS-9 and two ORFs deduced from genomic sequences in Caenorhabditis elegans and Saccharomyces cerevisiae . The region of similarity extended over 200 residues (approximately one-third of each ORF), and eight cysteines were conserved in all three ORFs . These observations suggest that this region comprises a functional domain present in a novel evolutionarily conserved gene family defined by OS-9. Curr Genet, 1996 Apr, 29(5), 490 - 5 Endoglucanase II (EGII) of Penicillium janthinellum: cDNA sequence, heterologous expression and promotor analysis; Mernitz G et al.; The cDNA coding for the endoglucanase EGII of P . janthinellum was cloned and sequenced . The open reading frame comprises 1230 nucleotides and the deduced amino-acid sequence shows an overall homology of 63% with the T . reesei egl2 . The cellulose-binding domain of EGII represents a typical member of the A family of cellulases . The egl2 gene is only induced by cellulose or cellobiose and not by sophorose . A promotor fragment including 1 kb was cloned and sequenced . Three major transcription startpoints were identified . Five motifs matching the binding site of the carbon-catabolite repressor CREA of A . nidulans were detected . Their potential implication in repression was analyzed by bandshift assays. Plant J, 1996 Apr, 9(4), 491 - 503 A sugar transporter from Medicago truncatula: altered expression pattern in roots during vesicular-arbuscular (VA) mycorrhizal associations; Harrison MJ; A cDNA clone encoding a hexose transporter has been isolated from a library prepared from Medicago truncatula roots colonized by the mycorrhizal fungus Glomus versiforme . The clone (Mtst1) represents a M . truncatula gene and expression studies in yeast indicate that the encoded protein transports glucose and fructose but not sucrose . Transcripts corresponding to Mtst1 are expressed in leaves, stems and roots of M . truncatula, with the highest levels of expression in roots . In the roots, Mtst1 transcripts were detected in two distinct locations; the phloem fiber cells of the vascular tissue, and the cells of the root tip . Mtst1 expression in the roots is regulated in response to colonization by G . versiforme; transcript levels increased two- to fourfold in both M . truncatula and M . sativa following colonization by G . versiforme but did not increase during the unsuccessful interaction between G . versiforme and a M . sativa myc- mutant, suggesting that the increase in Mtst1 transcripts in the successful mycorrhizal interaction is correlated with internal growth of the fungus and potentially with a functioning symbiosis . Mtst1 transcripts were also detected in the cortical cells of the mycorrhizal root, specifically in areas of the root that were highly colonized by the mycorrhizal fungus . Thus, the formation of a symbiotic association with a VA mycorrhizal fungus is accompanied by a change in the cell type-specific expression of a transporter that potentially functions to supply sugars to root cells critically involved in the symbiotic association. FASEB J, 1996 Apr, 10(5), 637 - 42 Tec protein-tyrosine kinase is an effector molecule of Lyn protein-tyrosine kinase; Mano H et al.; The Tec family is a recently emerging subfamily among nonreceptor type protein-tyrosine kinases (PTKs) consisting of Tec, Txk, Btk, Bmx, and Itk/Tsk/Emt . They have a long amino-terminal unique region containing a pleckstrin homology domain and a Tec-homology domain . We could previously show that, through the Tec-homology domain, Tec is bound to Lyn kinase both in vitro and in vivo . Because Tec is coexpressed with Lyn in many hematopoietic cell types, it has been intriguing to investigate the biological role of the Tec-Lyn association . Here we demonstrate that Lyn can phosphorylate tyrosine residues of the Tec protein, and thereby activate Tec in 3T3 fibroblasts . However, coexpression of Tec has little effect on the phospho-tyrosine-contents of Lyn . By using the in vitro kinase assay and the yeast system, we could prove that the Tec protein is a direct substrate of the Lyn kinase both in vitro and in vivo . From this evidence we conclude that Tec acts downstream of Lyn in intracellular signaling pathways . This is a novel case where one PTK is phosphorylated and regulated by another. Eur J Biochem, 1996 Apr 1, 237(1), 153 - 8 C23 interacts with B23, a putative nucleolar-localization-signal-binding protein; Li YP et al.; The human protein C23 (nucleolin) is a major nucleolar protein . Its interactions with other proteins were studied with the two-hybrid system which identified nucleolar protein B23 (nucleophosmin) as being associated with C23 . Both proteins were co-immunoprecipitated from HeLa cell nuclear extract by either monoclonal anti-C23 or monoclonal anti-B23 . Binding studies utilizing deletion mutants indicated that the binding of C23 and B23 involves specific motifs . In addition to an approximately 46-amino-acid-binding domain in B23 (amino acids 194-239), amino acids 540-628 of C23 were required for binding; this region of C23 is required for the nucleolar localization . In addition, nucleolar protein p120 was also found to be co-immunoprecipitated with B23 . A fragment of p120 containing a functional nucleolar localization signal bound to the truncated binding domain of B23, as did C23 . These results suggest that the interaction of C23 and B23 may represent a nucleolar-targeting mechanism in which B23 acts as a nucleolar-localization signal-binding protein. Virology, 1996 Apr 1, 218(1), 43 - 51 Homotypic interaction and multimerization of hepatitis C virus core protein; Matsumoto M et al.; Hepatitis C virus (HCV) core protein constitutes a viral nucleocapsid and may possess multiple functions . In this study, we demonstrated the homotypic interaction and multimerization of HCV core protein in vitro and in vivo . By using a yeast two-hybrid system, we showed that the amino-terminal hydrophilic portion (amino acids 1-115) of the core protein could interact with itself . Deletion analysis mapped the interacting domain within amino acid residues 36-91 . The homotypic interaction of the core protein was also confirmed by in vitro protein-protein blotting assay using the recombinant HCV core proteins and by its binding to the glutathione S-transferase core fusion protein . The biological significance of the core protein self-interactions was demonstrated by the detection of multimeric forms of the core protein in mammalian cells . The domain responsible for multimerization was determined to be within the amino-terminal hydrophilic region (amino acids 1-115) . Both the membrane-bound and the free core proteins exist in dimeric and multimeric forms, suggesting that multimerization of the HCV core protein occurred at an early stage of viral assembly and that the multimer forms may be involved in multiple functions of the core protein. Nucleic Acids Res, 1996 Apr 1, 24(7), 1260 - 6 In vitro generation of a circular exon from a linear pre-mRNA transcript; Schindewolf C et al.; Recent findings have firmly established the existence of circular exons in vivo . We were interested in the possible splicing mechanism by which these unusual mRNA molecules could be created in vitro, though no biological relevance has been attached to their existence as yet . In this report we demonstrate that a modified synthetic linear yeast ACT1 transcript whose sequence begins with the 3'-part of its original intron, is continued by 247 nt of exon sequence and terminates with the 5'-part of its intron will generate a circular exon when introduced to standard in vitro splicing reactions in whole cell splice extracts from Saccharomyces cerevisiae . The formation of a circular exon was found to be independent of specific circular or secondary structures of the pre-mRNA transcript . We hypothesize that circular exons which are found in vivo may be generated from pre-mRNAs which derive from rare events of transcription initiation within an intron. EMBO J, 1996 Apr 1, 15(7), 1658 - 65 Synergistic enhancement of both initiation and elongation by acidic transcription activation domains; Blair WS et al.; The effects of activation domain synergy on transcription initiation and elongation have been examined utilizing a system that permits the targeting of a defined number of activation modules to promoter DNA . As predicted, incremental increases in targeted activation potential were found to result in corresponding increases in transcription initiation . Surprisingly, however, transcriptional processivity, and hence mRNA synthesis, required a threshold level of activation domain synergy that exceeded the level required for at least modest levels of transcription initiation . The degree to which transcriptional processivity was enhanced was shown to depend on the quantity of activation modules targeted to the promoter DNA, rather than the quality . While the RNA-sequence specific HIV-1 Tat trans-activator was also shown to enhance processivity in this assay system, Tat differed from DNA-sequence specific activation domains in exerting a more dramatic effect on the efficiency of transcript elongation. J Cell Biol, 1996 Apr, 133(2), 269 - 80 The sorting sequence of the peroxisomal integral membrane protein PMP47 is contained within a short hydrophilic loop; Dyer JM et al.; No targeting sequence for peroxisomal integral membrane proteins has yet been identified . We have previously shown that a region of 67 amino acids is necessary to target Pmp47, a protein that spans the membrane six times, to peroxisomes . This region comprises two membrane spans and the intervening loop . We now demonstrate that the 20 amino acid loop, which is predicted to face the matrix, is both necessary and sufficient for peroxisomal targeting . Sufficiency was demonstrated with both chloramphenicol acetyltransferase and green fluorescent protein as carriers . There is a cluster of basic amino acids in the middle of the loop that we predict protrudes from the membrane surface into the matrix by a flanking stem structure . We show that the targeting signal is composed of this basic cluster and a block of amino acids immediately down-stream from it. J Bacteriol, 1996 Apr, 178(7), 2136 - 40 Nucleotide excision repair gene function in short-sequence recombination; Bailis AM et al.; Mutations in several nucleotide excision repair genes were found to affect the efficiency of recombination between short DNA sequences in Saccharomyces cerevisiae . These effects could be due to observed changes in the processing of recombination intermediates. J Cell Biol, 1996 Apr, 133(1), 111 - 24 Role of calmodulin and Spc110p interaction in the proper assembly of spindle pole body compenents; Sundberg HA et al.; Previously we demonstrated that calmodulin binds to the carboxy terminus of Spc110p, an essential component of the Saccharomyces cerevisiae spindle pole body (SPB), and that this interaction is required for chromosome segregation . Immunoelectron microscopy presented here shows that calmodulin and thus the carboxy terminus of Spc110p localize to the central plaque . We created temperature-sensitive SPC110 mutations by combining PCR mutagenesis with a plasmid shuffle strategy . The temperature-sensitive allele spc110-220 differs from wild type at two sites . The cysteine 911 to arginine mutation resides in the calmodulin-binding site and alone confers a temperature-sensitive phenotype . Calmodulin overproduction suppresses the temperature sensitivity of spc110-220 . Furthermore, calmodulin levels at the SPB decrease in the mutant cells at the restrictive temperature . Thus, calmodulin binding to Spc110-220p is defective at the nonpermissive temperature . Synchronized mutant cells incubated at the nonpermissive temperature arrest as large budded cells with a G2 content of DNA and suffer considerable lethality . Immunofluorescent staining demonstrates failure of nuclear DNA segregation and breakage of many spindles . Electron microscopy reveals an aberrant nuclear structure, the intranuclear microtubule organizer (IMO), that differs from a SPB but serves as a center of microtubule organization . The IMO appears during nascent SPB formation and disappears after SPB separation . The IMO contains both the 90-kD and the mutant 110-kD SPB components . Our results suggest that disruption of the calmodulin Spc110p interaction leads to the aberrant assembly of SPB components into the IMO, which in turn perturbs spindle formation. Cell Tissue Res, 1996 Apr, 284(1), 77 - 85 Studies on the structure of ocellar photoreceptor cells of Drosophila melanogaster with special reference to subrhabdomeric cisternae; Yoon CS et al.; We studied the structure of ocellar photoreceptor cells of Drosophila melanogaster, particularly the subrhabdomeric cisternae which our previous studies have shown to be essential structures for turnover of photoreceptive membranes in compound eyes . Each ocellus contained elongated photoreceptor cells with rhabdomeres positioned distally . In the subrhabdomeric regions, endocytotic invaginations were frequently observed, suggesting active turnover of photoreceptive membranes . In the vicinity of the photoreceptive microvilli, membranous structures similar to the subrhabdomeric cisternae in compound eyes were observed . These membranous structures were immunopositive for the rdgB protein, a phosphatidylinositol transfer protein that is localized to the subrhabdomeric cisternae in compound eyes . The ocellar photoreceptor cells of the retinal degeneration mutants (rdgA,B) were also studied . In these mutants, retinal degeneration has been reported to start, in compound eyes, with the disappearance of the subrhabdomeric cisternae . We found that the ocellar subrhabdomeric cisternae also disappear during the initial stage of retinal degeneration . From these observations, we conclude that the mechanism of photoreceptive membrane turnover in ocellar photoreceptor cells involves the rdgB and probably the rdgA proteins which are associated with subrhabdomeric cisternae, as is the case for photoreceptive membrane turnover in compound eyes. Yeast, 1996 Mar 30, 12(4), 411 - 3 Physical mapping of a centromere-proximal region of chromosome IV-L defines the placement of genes USO1, MBP1, PSA1 and SLC1; Gardner DC et al.; A physical map of a 14.5 kb region close to the centromere on the left arm of chromosome IV of Saccharomyces cerevisiae is presented . This map has been constructed by restriction analysis of a clone from a YCp50 genomic library and by use of pre-existing and new sequence data from this region . The map reveals the following gene order (reading from the most centromere-distal to the most centromere-proximal locus): USO1/INT1-MBP1-PSA1-SLC1-YLA1 and defines the size of the open reading frames and intergenic regions. J Biol Chem, 1996 Mar 29, 271(13), 7774 - 80 Arabidopsis thaliana contains two differentially expressed farnesyl-diphosphate synthase genes; Cunillera N et al.; The enzyme farnesyl-diphosphate synthase (FPS; EC 2.5.1.1/EC 2.5.1.10) catalyzes the synthesis of farnesyl diphosphate (FPP) from isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) . This reaction is considered to be a rate-limiting step in isoprenoid biosynthesis . Southern blot analysis indicates that Arabidopsis thaliana contains at least 2 genes (FPS1 and FPS2) encoding FPS . The FPS1 and FPS2 genes have been cloned and characterized . The two genes have a very similar organization with regard to intron positions and exon sizes and share a high level of sequence similarity, not only in the coding region but also in the intronic sequences . Northern blot analysis showed that FPS1 and FPS2 have a different pattern of expression . FPS1 mRNA accumulates preferentially in roots and inflorescences, whereas FPS2 mRNA is predominantly expressed in inflorescences . The cDNA corresponding to the FPS1 gene was isolated by functional complementation of a mutant yeast strain defective in FPS activity (Delourme, D., Lacroute, F., and Karst, F . (1994) Plant Mol . Biol . 26, 1867-1873) . By using a reverse transcription-polymerase chain reaction strategy we have cloned the cDNA corresponding to the FPS2 gene . Analysis of the FPS2 cDNA sequence revealed an open reading frame encoding a protein of 342 amino acid residues with a predicted molecular mass of 39,825 Da . FPS1 and FPS2 isoforms share an overall amino acid identity of 90.6% . Arabidopsis FPS2 was able to rescue the lethal phenotype of an ERG20-disrupted yeast strain . We demonstrate that FPS2 catalyzes the two successive condensations of IPP with both DMAPP and geranyl diphosphate leading to FPP . The significance of the occurrence of different FPS isoforms in plants is discussed in the context of the complex organization of the plant isoprenoid pathway. J Biol Chem, 1996 Mar 29, 271(13), 7654 - 8 The nuclear localization signal of lymphoid enhancer factor-1 is recognized by two differentially expressed Srp1-nuclear localization sequence receptor proteins; Prieve MG et al.; Proteins are directed to the nucleus by their nuclear localization sequences (NLSs) in a multistep process . The first step, which is to dock the NLS-containing protein to the nuclear pore, is carried out in part by a recently identified NLS receptor named Srp1/importin-alpha . Using the high mobility group (HMG) DNA binding domain of human lymphoid enhancer factor-1 (hLEF-1) as bait in a yeast two-hybrid screen, we have identified two different mouse Srp1 proteins (pendulin/importin-alpha and mSrp1) that each bind to a 9-amino acid sequence in hLEF-1 called the B box . We show that the B box of hLEF-1, a region essential for high affinity DNA binding, is also necessary and sufficient for nuclear localization, lending support to the model that NLSs can function both in nuclear transport and DNA binding . Pendulin and mSrp1 are the mouse homologues of hRch1/hSrp1alpha/importin-alpha and hSrp1/karyopherin alpha/NPI-1, respectively, and show considerable sequence divergence from each other . We find a surprising and significant difference in the expression pattern of pendulin and mSrp1 mRNA, suggesting that these two Srp1 proteins are distinguishable in function as well as sequence. J Biol Chem, 1996 Mar 29, 271(13), 7440 - 4 Proapoptotic protein Bax heterodimerizes with Bcl-2 and homodimerizes with Bax via a novel domain (BH3) distinct from BH1 and BH2; Zha H et al.; Most members of the Bcl-2 protein family of apoptosis regulating proteins contain two evolutionarily conserved domains, termed BH1 and BH2 . Both BH1 and BH2 in the Bcl-2 protein are required for its function as an inhibitor of cell death and for heterodimerization with the proapoptotic protein Bax . In this report, we mapped the region in Bax required for heterodimerization with Bcl-2 and homodimerization with Bax, using yeast two-hybrid and in vitro protein-protein interaction assays . Neither the BH1 nor the BH2 domain of Bax was required for binding to the wild-type Bcl-2 and Bax proteins . Moreover, Bax (deltaBH1) and Bax (deltaBH2) mutant proteins bound efficiently to themselves and each other, further confirming the lack of requirement for BH1 and BH2 for Bax/Bax homodimerization . Bax/Bax homodimerization was not dependent on the inclusion of the NH2-terminal 58 amino acids of the Bax protein in each dimerization partner, unlike Bcl-2/Bcl-2 homodimers which involve head-to-tail interactions between the region of Bcl-2 where BH1 and BH2 resides, and an NH2-terminal domain in Bcl-2 that contains another domain BH4 which is conserved among antiapoptotic members of the Bcl-2 family . Similarly, heterodimerization with Bcl-2 occurred without the NH2-terminal domain of either Bax or Bcl-2, suggesting a tail-to-tail interaction . The essential region in Bax required for both homodimerization with Bax and heterodimerization with Bcl-2 was mapped to residues 59-101 . This region in Bax contains a stretch of 15 amino acids that is highly homologous in several members of the Bcl-2 protein family, suggesting the existence of a novel functional domain which we have termed BH3 . Deletion of this 15-amino acid region abolished the ability of Bax to dimerize with itself and to heterodimerize with Bcl-2 . The findings suggest that the structural features of Bax and Bcl-2 that allow them to participate in homo-and heterodimerization phenomena are markedly different, despite their amino-acid sequence similarity. J Biol Chem, 1996 Mar 29, 271(13), 7265 - 8 Phosphorylation of Munc-18/n-Sec1/rbSec1 by protein kinase C: its implication in regulating the interaction of Munc-18/n-Sec1/rbSec1 with syntaxin; Fujita Y et al.; Munc-18/n-Sec1/rbSec1 interacts with syntaxin and this interaction inhibits the association of vesicle-associated membrane protein (VAMP)/synaptobrevin and synaptosomal-associated protein of 25 kDa (SNAP-25) with syntaxin . Syntaxin, VAMP, and SNAP-25 serve as soluble N-ethylmaleimide-sensitive fusion protein attachment protein (SNAP) receptors essential for docking and/or fusion of synaptic vesicles with the presynaptic plasma membrane . Genetic analyses in yeast, Caenorhabditis elegans, and Drosophila suggest that Munc-18 is essential for vesicle transport . On the other hand, protein kinase C (PKC) stimulates Ca2+-dependent exocytosis in various types of secretory cells . However, the modes of action of Munc-18 and PKC in vesicle transport have not been clarified . Here, we show that recombinant Munc-18 is phosphorylated by conventional PKC in a Ca2+- and phospholipid-dependent manner in a cell-free system . About 1 mol of phosphate is maximally incorporated into 1 mol of Munc-18 . The major phosphorylation sites are Ser306 and Ser313 . The Munc-18 complexed with syntaxin is not phosphorylated . The PKC-catalyzed phosphorylation of Munc-18 inhibits its interaction with syntaxin . These results suggest that the PKC-catalyzed phosphorylation of Munc-18 plays an important role in regulating the interaction of Munc-18 with syntaxin and thereby the docking and/or the fusion of synaptic vesicles with the presynaptic plasma membrane. Science, 1996 Mar 29, 271(5257), 1858 - 60 Inhibition of HIV-1 replication in lymphocytes by mutants of the Rev cofactor eIF-5A; Bevec D et al.; Eukaryotic initiation factor 5A(eIF-5A) is a cellular cofactor require d for the function of the human immunodeficiency virus type-1 (HIV-1) Rev trans-activator protein . The majority of a set of eIF-5A mutants did not support growth of yeast cells having an inactivated genomic copy of eIF-5A, indicating that the introduced mutation eliminated eIF-5A activity . Two nonfunctional mutants, eIF-5AM13 and eIF-5AM14, retained their binding capacity for the HIV-1 Rev response element:Rev complex . Both mutants were constitutively expressed in human T cells . When these T cells were infected with replication-competent HIV-1, virus replication was inhibited . The eIF-5AM13 and eIF5AM14 proteins blocked Rev trans-activation and Rev-mediated nuclear export. Biochem Biophys Res Commun, 1996 Mar 27, 220(3), 963 - 8 The role of the unique motifs in the amino-terminal region of PKN on its enzymatic activity; Kitagawa M et al.; The yeast two-hybrid system and in vitro binding assay were carried out to characterize the interaction between the amino-terminal and carboxyl-terminal region of PKN . It was revealed that the amino-terminal region containing the regulatory domain associated with the carboxyl-terminal catalytic region . A synthetic peptide, corresponding to the amino acid residues of PKN from 39 to 53, with substitution of isoleucine46 with serine was shown to become a potent substrate for PKN, and its wild type synthetic peptide inhibited the phosphorylation by PKN . These results suggest that the amino-terminal region of PKN contains the pseudosubstrate sequence and acts as an autoinhibitory region. Biochem Biophys Res Commun, 1996 Mar 27, 220(3), 911 - 5 Multiple domains participate in distance-independent LAZ3/BCL6-mediated transcriptional repression; Albagli O et al.; The LAZ3/BCL6 gene implicated in diffuse large cell lymphomas encodes a transcriptional repressor containing Kruppel-fike zinc fingers . It harbours at its N-terminus a conserved protein/protein interaction motif, the BTB/POZ domain, which is also an autonomous transcriptional repression domain . We demonstrate here using several GAL4-LAZ3/BCL6 chimeras that the BTB/POZ domain plays an important but not exclusive role as its deletion gives rise to a GAL4 chimera that mediates significant, albeit reduced, transcriptional repression . Moreover, the repressive effect mediated either by LAZ3/BCL6 or by the isolated domains occurs with unaltered efficiency even at long distance (1.6 Kbp), ruling out steric hindrance mechanisms . Finally, though the absence of a TATA box appears to weaken this activity, it is largely promoter independent . Taken together, our results demonstrate that multiple domains participate in the promoter and distance-independent LAZ3/BCL6-mediated transcriptional repression. Biochem Biophys Res Commun, 1996 Mar 27, 220(3), 843 - 7 The alpha-subunit of the mammalian guanine nucleotide-exchange factor eIF-2B is essential for catalytic activity in vitro; Craddock BL et al.; Eukaryotic initiation factor (eIF)-2B, the guanine nucleotide exchange factor for eIF-2, consists of five distinct subunits in both mammals and the yeast Saccharomyces cerevisiae . The exchange reaction mediated by eIF-2B can be regulated by phosphorylation of eIF-2 on its alpha-subunit . This represents a key control point in the initiation of translation . The functions of the individual subunits of the eIF-2B complex remain unclear . Mutational analysis in Saccharomyces cerevisiae suggested that the smallest subunit (the alpha) is dispensable for exchange, but required for the inhibition of eIF-2B by eIF-2(alphaP) . Here we present evidence that, in mammalian cells, eIF-2Balpha is essential for the activity of the complex, since preparations of eIF-2B lacking this subunit are not active in nucleotide exchange in vitro, although the complex still contains the beta, gamma, delta and epsilon subunits. Biochim Biophys Acta, 1996 Mar 27, 1311(1), 53 - 63 Identification of a 200 kDa polypeptide as type 3 phosphatidylinositol 4-kinase from bovine brain by partial protein and cDNA sequencing; Gehrmann T et al.; Two phosphatidylinositol 4-kinase isozymes, type 3 and type 2, have been separated on hydroxylapatite after solubilizing bovine brain microsomes with Triton X-114 . Employing a newly developed renaturation procedure following SDS-PAGE, we demonstrate that a 200 kDa polypeptide carries the enzyme activity of this type 3 isoform . Chromatography on hydroxylapatite, Heparin-Sepharose, Superdex 200 and finally SDS-PAGE results in an approximately 30,000-fold purification . Tryptic peptides generated from the 200 kDa polypeptide after SDS-PAGE have been sequenced and the obtained data have been used for constructing and synthesizing degenerated oligonucleotides . Polymerase chain reaction as well as screening of cDNA libraries allowed several clones to be isolated from which a 4.7 kb contiguous sequence can be built up . The open reading frame covers 4.4 kb with a 0.3 kb untranslated 3' end which yields a deduced amino acid sequence of 1,467 amino acids . The C-terminal part of ca . 300 amino acids represents the catalytic domain . Sequence alignment of this domain with the mammalian counterpart, the human type 2 phosphatidylinositol 4-kinase, the yeast kinases STT4 and PIK1, as well as with the catalytic domains of bovine, human, mouse and yeast phosphatidylinositol 3-kinases reveals a high degree of identity: 26 of these approximately 300 amino acids are invariable in all of these eight catalytic domains . Five motifs indicate nuclear localization and DNA binding properties of the enzyme . Two leucine zipper motifs (amino acids 358-386, 862-882) are detectable . Furthermore, a helix loop helix motif (amino acids 716-729) as well as two nuclear localization signals (amino acids 838-854, 345-349) indicate the presence of the type 3 isoform in the nucleus. J Biol Chem, 1996 Mar 22, 271(12), 7141 - 6 Differential ability to form the G protein betagamma complex among members of the beta and gamma subunit families; Yan K et al.; We have determined the relative abilities of several members of the G protein beta and gamma subunit families to associate with each other using the yeast two-hybrid system . We show first that the mammalian beta1 and gamma3 fusion proteins form a complex in yeast and that formation of the complex activates the reporter gene for beta-galactosidase . Second, the magnitude of reporter activity stimulated by various combinations of beta and gamma subunit types varies widely . Third, the reporter activity evoked by a particular combination of beta and gamma subunit types is not correlated with the expression levels of these subunit types in the yeast cells . Finally, the reporter activity shows a direct relationship with the amount of hybrid betagamma complex formed in the cell as determined by immunoprecipitation . These results suggest that different beta and gamma subunit types interact with each other with widely varying abilities, and this in combination with the level of expression of a subunit type in a mammalian cell determines which G protein will be active in that cell . The strong preference of all gamma subunit types for the beta1 subunit type explains the preponderence of this subunit type in most G proteins. Biochem Pharmacol, 1996 Mar 22, 51(6), 751 - 8 Effects of chain length and sulphur position of thia fatty acids on their incorporation into phospholipids in 7800 C1 hepatoma cells and isolated rat hepatocytes, and their effects on fatty acid composition of phospholipids; Wu P et al.; Incorporation of thia fatty acids and their effects on the fatty acid composition in phospholipids has been investigated in 7800 C1 hepatoma cells and cultured hepatocytes . 3-Thia fatty acids of chain lengths from dodecyl-to hexadecyl-thioacetic acid were incorporated into phospholipids during a 3-day incubation . Longer and shorter 3-thia fatty acids were barely detectable . Tetradecylthioacetic acid, 3-thia stearate, and their delta9- desaturated derivatives were maximally incorporated into whole-cell phospholipids . The amount of tetradecylthioacetic acid incorporated into phospholipids of hepatoma cells remained almost identical in cells cultured for 3 days or adapted over a period of 1 year . Delta9-desaturated metabolites of long chain thia fatty acids (C13-to C16-S-acetic acid) were identified by GC-MS in phospholipids . 3-Thia stearate appeared to be the best substrated for delta9 desaturase . Incubation of hepatoma cells with thia fatty acids led to alterations in the amount of normal fatty acids in total phospholipids . The amounts of 16:0 and 18:1 decreased and 18:2 (n-6) and 20:5 (n-3) increased . Changes in the normal fatty acid composition of phospholipids were seen both with thia acids incorporated into phospholipids and those not incorporated . These effects, therefore, may be only partially dependent on displacement of normal fatty acids by thia fatty acids . Morris 7800 C1 hepatoma cell acyl-CoA synthetase (ACS) and peroxisomal acyl-CpA oxidase (ACO) were induced by thia fatty acids of all chain lengths, and with the sulphur atom(s) in different positions . Control experiments with hepatocytes revealed a similar incorporation of thia fatty acids in these physiologically more normal cells. Cell, 1996 Mar 22, 84(6), 863 - 74 Crystal structure and mutational analysis of the human CDK2 kinase complex with cell cycle-regulatory protein CksHs1; Bourne Y et al.; The 2.6 Angstrom crystal structure for human cyclin-dependent kinase 2(CDK2) in complex with CksHs1, a human homolog of essential yeast cell cycle-regulatory proteins suc1 and Cks1, reveals that CksHs1 binds via all four beta strands to the kinase C-terminal lobe . This interface is biologically critical, based upon mutational analysis, but far from the CDK2 N-terminal lobe, cyclin, and regulatory phosphorylation sites . CDK2 binds the Cks single domain conformation and interacts with conserved hydrophobic residues plus His-60 and Glu-63 in their closed beta-hinge motif conformation . The beta hinge opening to form the Cks beta-interchanged dimer sterically precludes CDK2 binding, providing a possible mechanism regulating CDK2-Cks interactions . One face of the complex exposes the sequence-conserved phosphate-binding region on Cks and the ATP-binding site on CDK2, suggesting that CKs may target CDK2 to other phosphoproteins during the cell cycle. Nature, 1996 Mar 21, 380(6571), 265 - 8 Sequence-specific DNA binding by Ku autoantigen and its effects on transcription; Giffin W et al.; DNA-dependent protein kinase (DNA-PK) has been implicated in several nuclear processes including transcription, DNA replication, double-stranded DNA break repair, and V(D)J recombination . Linkage of kinase and substrate on DNA in cis is required for efficient phosphorylation . Recruitment of DNA-PK to DNA is by Ku autoantigen, a DNA-end-binding protein required for DNA-PK catalytic activity . Although Ku is known to translocate along naked DNA, how DNA-end binding by Ku might lead to DNA-PK-mediated phosphorylation of sequence-specific DNA-binding proteins in vivo has not been obvious . Here we report the identification of Ku as a transcription factor that recruits DNA-PK directly to specific DNA sequences . NRE1 (negative regulatory element 1) is a DNA sequence element (-394/ -381) in the long terminal repeat of mouse mammary tumour virus (MMTV) that is important for repressing inappropriate viral expression . We show that direct binding of Ku/DNA-PK to NRE1 represses glucocorticoid-induced MMTV transcription. Biochemistry, 1996 Mar 19, 35(11), 3625 - 35 Mutagenesis of transmembrane domain 11 of P-glycoprotein by alanine scanning; Hanna M et al.; The biochemical and genetic analyses of P-glycoprotein (P-gp) have indicated that the membrane-associated regions of P-gp play an important role in drug recognition and drug transport . Predicted transmembrane domain 11 (TM11) maps near a major drug binding site revealed by photoaffinity labeling, and mutations in this domain alter the substrate specificity of P-gp . To investigate further the role of TM11 in P-gp function in general, and substrate specificity in particular, each of the 21 residues of TM11 of the P-gp isoform encoded by the mouse mdr3 gene was independently mutated to alanine, or to glycine in the case of endogenous alanines . After transfection and overexpression in Chinese hamster ovary cells, pools of stable transfectants were analyzed for qualitative or quantitative deviations from the profile of resistance to vinblastine, adriamycin, colchicine, and actinomycin D displayed by the wild-type protein . While mutations at eight of the positions had no effect on P-gp function, 13 mutants showed a 2-10-fold reduction of activity against one of the four drugs tested . Although the phenotype of individual mutants was varied, replacements at most mutation-sensitive positions seemed to affect the drug resistance profiles rather than the overall activity of the mutant P-gp . When TM11 was projected in a alpha-helical configuration, the distribution of deleterious and neutral mutations was not random but segregated with a more hydrophobic (mutation-insensitive) face and a more hydrophilic (mutation-sensitive) face of a putative amphipathic helix . The alternate clustering pattern of deleterious vs neutral mutations in TM11 together with the altered drug resistance profile of deleterious mutants suggest that the more hydrophilic face of the TM11 helix may play an important structural or functional role in drug recognition and transport by P-gp . Finally, the conservation of the two residues most sensitive to mutations (Y949 and Y953) in TM11, and in the homologous TM5, of all mammalian P-gps and also in other ABC transporters, suggests that these residues and domains may play an important role in structural as well as mechanistic aspects common to this family of proteins. Proc Natl Acad Sci U S A, 1996 Mar 19, 93(6), 2296 - 301 Human protein Sam68 relocalization and interaction with poliovirus RNA polymerase in infected cells; McBride AE et al.; A HeLa cDNA expression library was screened for human polypeptides that interacted with the poliovirus RNA-dependent RNA polymerase, 3D, using the two-hybrid system in the yeast Saccharomyces cerevisiae . Sam68 (Src-associated in mitosis, 68 kDa) emerged as the human cDNA that, when fused to a transcriptional activation domain, gave the strongest 3D interaction signal with a LexA-3D hybrid protein . 3D polymerase and Sam68 coimmunoprecipitated from infected human cell lysates with antibodies that recognized either protein . Upon poliovirus infection, Sam68 relocalized from the nucleus to the cytoplasm, where poliovirus replication occurs . Sam68 was isolated from infected cell lysates with an antibody that recognizes poliovirus protein 2C, suggesting that it is found on poliovirus-induced membranes upon which viral RNA synthesis occurs . These data, in combination with the known RNA- and protein-binding properties of Sam68, make Sam68 a strong candidate for a host protein with a functional role in poliovirus replication. FEBS Lett, 1996 Mar 18, 382(3), 313 - 8 Non-histone protein 1 (NHP1) is a member of the Ku protein family which is upregulated in differentiating mouse myoblasts and human promyelocytes; Oderwald H et al.; We have previously purified and characterized a ubiquitous non-histone protein (NHP1) which has a high affinity (Kd 10(-11) M) for different avian vitellogenin gene sequences containing CpGs (Hughes et al . (1989) Biochemistry 28, 9137-9142; Hughes and Jost (1989) Nucleic Acids Res . 17, 8511-8520) . Here we show by microsequencing that the peptides derived from the purified p75 and p85 subunits of NHP1 from HeLa cells have between 64 and 100% identity with the human Ku autoantigen . During the differentiation of human HL-60 promyelocytes there is an increase in the amount of p85 subunit protein whereas the level of the p75 subunit is unchanged . In differentiating mouse G8 myoblasts there is, however, an upregulation of both the p75 and p85 subunits and of the p85 mRNA . An inhibition of mouse myoblast differentiation by either cAMP, 3-aminobenzamide or sodium butyrate abolishes the upregulation of the p85 subunit . In G8 myoblasts chemical, or physical stress by UV light or X-rays does not upregulate the level of the p85 subunit . The possible involvement of NHP1 in the active demethylation of bifilarly methylated DNA will be discussed. Yeast, 1996 Mar 15, 12(3), 267 - 72 Identification of ASK10 as a multicopy activator of Skn7p-dependent transcription of a HIS3 reporter gene; Page N et al.; Recent evidence has demonstrated that the yeast Skn7p appears to act as a 'response regulator' in a eukaryotic 'two-component' signal transduction pathway . A search to identify possible regulators of the SKN7 mediated 'two-component' regulatory system has identified Ask10p as a novel potential transcription factor . The ASK10 sequence has been deposited in GenBank with Accession Number U27209. Eur J Biochem, 1996 Mar 15, 236(3), 856 - 61 Determinants in the presequence of cytochrome b2 for import into mitochondria and for proteolytic processing; Klaus C et al.; Determinants in a mitochondrial targeting signal for import and processing were analyzed by introducing deletions into the presequence of cytochrome b2 . The matrix targeting signal and the signal recognized by the mitochondrial processing peptidase were found to be separate . The signal for import into the matrix is located at the N-terminus within a stretch of 20 amino acid residues that has the potential to form a positively charged, amphipathic alpha-helix . The mitochondrial processing peptidase cleaves after residue 31 and recognizes a short sequence motif around the scissile bond . In the context of a presequence, the cleavage site is accessible for the processing peptidase . At a different location or in a different context, the cleavage site motif is still specifically recognized but processed with lower efficiency . The matrix targeting signal may help to present the cleavage site motif to the mitochondrial processing peptidase. EMBO J, 1996 Mar 15, 15(6), 1403 - 11 RNA editing in bryophytes and a molecular phylogeny of land plants; Malek O et al.; RNA editing has been observed to date in all groups of vascular plants, but not in bryophytes . Its occurrence was therefore assumed to correlate with the evolution of tracheophytes . To gain more insight into both the phylogeny of early land plants and the evolution of mitochondrial RNA editing we have investigated a number of vascular and non-vascular plant species . Contrary to the belief that editing is absent from bryophytes, here we report mitochondrial RNA editing in cox3 mRNA of the liverwort Pellia epiphylla, the mosses Tetraphis pellucida and Ceratodon purpureus and the hornwort Anthroceros crispulus . RNA editing in plants consequently predates the evolution of tracheophytes . Editing is also found in the eusporangiate ferns Ophioglossum petiolatum and Angiopteris palmiformis, the whisk fern Tmesipteris elongata and the gnetopsid Ephedra gerardiana, but was not detected in Gnetum gnemon.cox3 mRNA of the lycopsid Isoetes lacustris shows the highest frequency of RNA editing ever observed in a plant, with 39% of all cytidine residues converted to uridines . The frequency of RNA editing correlates with the genomic GC content rather than with the phylogenetic position of a species . Phylogenetic trees derived from the slowly evolving mitochondrial sequences find external support from the assessments of classical systematics. J Biol Chem, 1996 Mar 15, 271(11), 6206 - 11 Molecular cloning and sequencing of the cytostatic G protein-activated protein kinase PAK I; Jakobi R et al.; The serine/threonine protein kinase PAK I (p2l-activated protein kinase), a ubiquitous multipotential protein kinase of 58-60 kDa, has been shown to have cytostatic properties . Data from our laboratory show that PAK I is highly active in oocytes and quiescent and serum-starved cells, and injection of active PAK I into one blastomere of two-cell frog embryos inhibits cleavage of the injected blastomere . To clone the cDNA encoding PAK I, purified peptides from rabbit PAK I were sequenced, degenerate oligonucleotides were used to isolate PAK I clones from a rabbit spleen library, and the 5'-terminus was obtained by polymerase chain reaction . The entire cDNA sequence extends over 4471 nucleotides, with an open reading frame for a protein of 524 residues and a 3'-noncoding region of 2826 nucleotides . Clones with the same open reading frame but with 3'-noncoding regions of 1055 and 2478 nucleotides were isolated, suggesting the generation of different transcripts by alternative termination of transcription . The amino acid sequence of PAK I shows high homology to the p2l-activated protein kinases from human placenta and rat brain and to yeast STE20 . PAK I is activated by Cdc42(GTP) . The PAK enzymes have been proposed to regulate the stress-activated protein kinase (also known as the Jun kinase) signaling pathway (Coso, O . A., Chiariello, M., Yu, J.-C., Teramoto, H., Crespo, P., Xu, N., Miki, T., and Gutkind, J . S . (1995) Cell 81, 1137-1146; Minden, A., Lin, A., Claret, F.-X., Abo, A., and Karin, M . (1995) Cell 81, 1147-1157). J Biol Chem, 1996 Mar 15, 271(11), 6164 - 71 Ubiquinol-cytochrome c oxidoreductase . The redox reactions of the bis-heme cytochrome b in ubiquinone-sufficient and ubiquinone-deficient systems; Matsuno-Yagi A et al.; Antimycin and myxothiazol are stoichiometric inhibitors of complex III (ubiquinol-cytochrome c oxidoreductase), exerting their highest degree of inhibition at I mol each/mol of complex III monomer . Phenomenologically, however, they each inhibit three steps in the redox reaction of the bis-heme cytochrome b in submitochondrial particles (SMP), and all three inhibitions are incomplete to various extents . (i) In SMP, reduction of hemes bH and bL by NADH or succinate is inhibited when the particles are treated with both antimycin and myxothiazol . Each inhibitor alone allows reduced bH and bL to accumulate, indicating that each inhibits the reoxidation of these hemes . (E)-Methyl-3-methoxy-2-(4')-trans-stilbenyl)acrylatc in combination with antimycin or 2-n-heptyl-4-hydroxyquinoline-N-oxide in combination with myxothiazol causes less inhibition of b reduction than the combination of antimycin and myxothiazol . (ii) Reoxidation of reduced b, is inhibited by either antimycin or myxothiazol (or 2-n-heptyl-4-hydroxyquinoline-N-oxide, (E)-methyl-3-methoxy-2-(4'-trans-stilbenyl)acrylate, or stigmatellin) . (iii) Reoxidation of reduced bH is also inhibited by any one of these reagents . These inhibitions are also incomplete, and reduced bL is oxidized through the leaks allowed by these inhibitors at least 10 times faster than reduced bH . Heme bH can be reduced in SMP via cytochrome c, and the Rieske iron-sulfur protein by ascorbate and faster by ascorbate + TMPD (N,N,N',N'-tetramethyl-p-phenylenediamine) . Energization of SMP by the addition of ATP affords reduction of bL as well . Reverse electron transfer to bH and bL is inhibited partially by myxothiazol, much more by antimycin . Ascorbate + TMPD also reduce bH in ubiquinone-extracted SMP in which the molar ratio of ubiquinone to cytochrome b has been reduced 200-fold from 12.5 to aproximately 0.06 . Reconstitution of the extracted particles with ubiquinone-10 restores substrate oxidation but does not improve the rate or the extent of b, reduction by ascorbate + TMPD . These reagents also partially reduce cytochrome b in SMP from a ubiquinone-deficient yeast mutant . The above results are discussed in relation to the Q-cycle hypothesis. J Biol Chem, 1996 Mar 15, 271(11), 5980 - 3 Insulin receptor substrate-2 binds to the insulin receptor through its phosphotyrosine-binding domain and through a newly identified domain comprising amino acids 591-786; Sawka-Verhelle D et al.; We compared the interaction between the insulin receptor (IR) and the IR substrate (IRS) proteins IRS-1 and IRS-2) using the yeast two-hybrid system . Both IRS proteins interact specifically with the cytoplasmic portion of the IR and the related insulin-like growth factor-I receptor, and these interactions require receptor tyrosine kinase activity . Alignment of IRS-1 and IRS-2 revealed two conserved domains at the NH2 terminus, called IH1PH and IH2PTB, which resemble a pleckstrin homology (PH) domain and a phosphotyrosine binding (PTB) domain, respectively . The IH2PTB binds to the phosphorylated NPXY motif (Tyr-960) in the activated insulin receptor, providing a specific mechanism for the interaction between the receptor and IRS-1 . Although the IH2PTB of IRS-2 also interacts with the NPEY motif of the insulin receptor, it is not essential for the interaction between the insulin receptor and IRS-2 in the yeast two-hybrid system . IRS-2 contains another interaction domain between residues 591 and 786, which is absent in IRS-1 . This IRS-2-specific domain is independent of the IH2PTB and does not require the NPEY motif; however, it requires a functional insulin receptor kinase and the presence of three tyrosine phosphorylation sites in the regulatory loop (Tyr-1146, Tyr-1150, and Tyr-1151) . Importantly, this novel domain mediates the association between IRS-2 and insulin receptor lacking the NPXY motif and may provide a mechanism by which the stoichiometry of regulatory loop autophosphorylation enhances IRS-2 phosphorylation. J Clin Invest, 1996 Mar 15, 97(6), 1417 - 21 Autoantibodies to DNA-dependent protein kinase . Probes for the catalytic subunit; Suwa A et al.; DNA-dependent protein kinase (DNA-PK) is an important nuclear enzyme which consists of a catalytic subunit known as DNA-PKcs and a regulatory component identified as the Ku autoantigen . In the present study, we surveyed 312 patients in a search for this specificity . 10 sera immunoprecipitated a large polypeptide which exactly comigrated with DNA-PKcs in SDS-PAGE . Immunoblot analysis demonstrated that this polypeptide was recognizable by a rabbit antiserum specific for DNA-PKcs . Although the patient sera did not bind to biochemically purified DNA-PKcs in immunoblots or ELISA, they were able to deplete DNA-PK catalytic activity from extracts of HeLa cells in a dose-dependent manner . We conclude that these antibodies should be useful probes for studies which aim to define the role of DNA-PK in cells . Since six sera simultaneously contained antibodies to the Ku protein, these studies suggest that relatively intact forms of DNA-PK complex act as autoantigenic particles in selected patients. Science, 1996 Mar 15, 271(5255), 1597 - 601 Rapid degradation of the G1 cyclin Cln2 induced by CDK-dependent phosphorylation; Lanker S et al.; Cyclins regulate the major cell cycle transitions in eukaryotes through association with cyclin-dependent protein kinases (CDKs) . In yeast, G1 cyclins are essential, rate-limiting activators of cell cycle initiation . G1-specific accumulation of one G1 cyclin, Cln2, results from periodic gene expression coupled with rapid protein turnover . Site-directed mutagenesis of CLN2 revealed that its phosphorylation provides a signal that promotes rapid degradation . Cln2 phosphorylation is dependent on the Cdc28 protein kinase, the CDK that it activates . These findings suggest that Cln2 is rendered self-limiting by virtue of its ability to activate its cognate CDK subunit. Science, 1996 Mar 15, 271(5255), 1589 - 92 Coordination of three signaling enzymes by AKAP79, a mammalian scaffold protein; Klauck TM et al.; Multivalent binding proteins, such as the yeast scaffold protein Sterile-5, coordinate the location of kinases by serving as platforms for the assembly of signaling units . Similarly, in mammalian cells the cyclic adenosine 3',5'-monophosphate-dependent protein kinase (PKA) and phosphatase 2B {calcineurin (CaN)} are complexed by an A kinase anchoring protein, AKAP79 . Deletion analysis and binding studies demonstrate that a third enzyme, protein kinase C (PKC), binds AKAP79 at a site distinct from those bound by PKA or CaN . The subcellular distributions of PKC and AKAP79 were similar in neurons . Thus, AKAP79 appears to function as a scaffold protein for three multifunctional enzymes. Genes Dev, 1996 Mar 15, 10(6), 725 - 39 Alternative outcomes in assembly of promoter complexes: the roles of TBP and a flexible linker in placing TFIIIB on tRNA genes; Joazeiro CA et al.; Saccharomyces cerevisiae transcription factor (TF) IIIB, a TATA-binding protein (TBP)-containing multisubunit factor, recruits RNA polymerase (Pol) III for multiple rounds of transcription . TFIIIC is an assembly factor for TFIIIB on TATA-less tRNA gene promoters . To investigate the role of TBP-DNA interactions in tRNA gene transcription, we generated sequence substitutions in the SUP4 tRNATyr gene TFIIIB binding site . Purified transcription proteins were used to analyze the selection of transcription initiation sites and the physical structures of the protein complexes formed on these mutant genes . We show that the association of TFIIIB with tRNA genes proceeds through an initial step of binding-site selection that is codirected by its TBP subunit and by TFIIIC . TFIIIB is assembled in a predominantly metric manner with regard to box A, the start site-proximal binding site of TFIIIC, but TFIIIC opens a window within which wild-type TBP can select the TFIIIB-binding site . Despite its clear preference for AT-rich sequences, TBP can mediate TFIIIB assembly at diverse DNA sequences, including stretches containing only G and C . However, a mutant TBP, m3, which recognizes TATAAA and TGTAAA and is active for Pol III transcription, utilizes other sequences only poorly . We also show that alternative alignments between DNA-bound TFIIIB and TFIIIC are possible, implying a remarkably flexible linkage, and suggest that Tfc4, the TFIIIB-assembling subunit of TFIIIC, could be responsible for such elasticity . The relevance of these findings to alternative initiation of Pol II- and other Pol III-transcribed genes is discussed. J Biol Chem, 1996 Mar 8, 271(10), 5671 - 9 Mammalian vesicle trafficking proteins of the endoplasmic reticulum and Golgi apparatus; Hay JC et al.; Vesicle traffic propagates and maintains distinct subcellular compartments and routes secretory products from their site of synthesis to their final destinations . As a basis for the specificity of vesicular transport reactions, each step in the secretory pathway appears to be handled by a distinct set of evolutionarily conserved proteins . Mammalian proteins responsible for vesicle trafficking at early steps in the secretory pathway are not well understood . In this report, we describe rat sec22 (rsec22) and rat bet1 (rbet1), mammalian sequence homologs of yeast proteins identified as mediators of endoplasmic reticulum-to-Golgi protein transport . rsec22 and rbet1 were expressed widely in mammalian tissues, as anticipated for proteins involved in fundamental membrane trafficking reactions . Recombinant rsec22 and rbet1 proteins behaved as integral membrane components of 28 and 18 kDa, respectively, consistent with their primary structures, which contain a predicted transmembrane domain at or near the carboxyl terminus . Recombinant rsec22 and rbet1 had distinct subcellular localizations, with rsec22 residing on endoplasmic reticulum membranes and rbet1 found on Golgi membranes . Studies with brefeldin A and nocodazole indicated that rbet1 function might involve interaction with or retention in the intermediate compartment . The distinct localizations of rsec22 and rbet1 may reflect their participation in opposite directions of membrane flow between the endoplasmic reticulum and Golgi apparatus. Biophys Chem, 1996 Mar 7, 59(1-2), 179 - 84 A graph-topological approach to recognition of pattern and similarity in RNA secondary structures; Benedetti G et al.; Secondary and tertiary RNA structures play an important role in many biological processes . Therefore the necessity arises to find similar higher-order structures for different but functionally homologous RNA sequences . We propose here a graph-topological approach to the problem, which shows two main features: simplified graph representation which allows the recognition of similarity of RNA secondary structures with the same branching look despite minor differences . This allows comparison among foldings from different sequences, and "pruning" of the secondary structures not shared by all the sequences since the early stages of the search . (b) The graph representation is encoded by the Randic topological index, and the search for the folding similarity is reduced to checking the identity of single numbers . These characteristics make this approach significantly different, less depending on empirical criteria, and less computationally heavy then previous methods, where the folding consensus has been measured by an alignment procedure or correlation of strings representing the secondary structures . Some U2 snRNA and viroid sequences are studied by this approach, which is imbedded in our previous search method based on genetic algorithms. Nature, 1996 Mar 7, 380(6569), 82 - 5 A mammalian SRB protein associated with an RNA polymerase II holoenzyme; Chao DM et al.; A large multisubunit complex containing RNA polymerase II, general transcription factors and SRB regulatory proteins initiates transcription of class II genes in yeast cells . The SRB proteins are a hallmark of this RNA polymerase II holoenzyme as they are found only in this complex, where they contribute to the response to regulators . We have now isolated a human homologue of the yeast SRB7 gene and used antibodies against human SRB7 protein to purify and characterize a mammalian RNA polymerase II holoenzyme containing the general transcription factors TFIIE and TFIIH . This holoenzyme is more responsive to transcriptional activators than core RNA polymerase II when assayed in the presence of coactivators. Proc Natl Acad Sci U S A, 1996 Mar 5, 93(5), 1781 - 5 Observation via one-dimensional 13Calpha NMR of local conformational substates in thermal unfolding equilibria of a synthetic analog of the GCN4 leucine zipper; Lovett EG et al.; Synthesis of a 33-residue, capped leucine zipper analogous to that in GCN4 is reported . Histidine and arginine residues are mutated to lysine to reduce the unfolding temperature . CD and ultracentrifugation studies indicate that the molecule is a two-stranded coiled coil under benign conditions . Versions of the same peptide are made with 99% 13Calpha at selected sites . One-dimensional 13C NMR spectra are assigned by inspection and used to study thermal unfolding equilibria over the entire transition from 8 to 73 degrees C . Spectra at the temperature extremes establish the approximate chemical shifts for folded and unfolded forms at each labeled site . Resonances for each amino acid appear at both locations at intermediate T, indicating that folded and unfolded forms interconvert slowly (> >2 ms) on the NMR time scale . Moreover, near room temperature, the structured form's resonance is double at several, but not all, sites, indicating at least two slowly interconverting, structured, local conformational substates . Analysis of the dynamics is possible . For example, near room temperature at the Val-9, Ala-24, and Gly-31 positions, the equilibrium constant for interconversion of the two structured forms is near unity and the time scale is > or= 10-20 ms. Biosci Biotechnol Biochem, 1996 Mar, 60(3), 496 - 7 Increase in activity for the C-terminal Pro-X bond by site-directed mutagenesis of Gly137 to Ala in carboxypeptidase Z; Ratanakhanokchai K et al.; A mutant carboxypeptidase Z from Absidia zychae in which Gly137 was replaced by Ala by site-directed mutagenesis was constructed and expressed in Saccharomyces cerevisiae YPH250 . The mutant enzyme hydrolyzed C-terminal Pro-X bonds (X = amino acid) more efficiently than the wild-type enzyme and sequentially released amino acids from the C-termini of oligopeptides. Trends Biochem Sci, 1996 Mar, 21(3), 102 - 6 The role of MCM/P1 proteins in the licensing of DNA replication; Chong JP et al.; The DNA replication licensing system ensures that eukaryotic chromosomes replicate precisely once per cell cycle . A central component of the licensing system, RLF-M, has recently been shown to consist of a complex of Mcm/P1 proteins . This result allows us to integrate data about the MCM/P1 family obtained in different eukaryotes, ranging from yeast to man, into a general picture of the way that chromosome replication is controlled. J Nat Prod, 1996 Mar, 59(3), 283 - 5 Bioactive steroidal alkaloids from Solanum umbelliferum; Kim YC et al.; Bioassay-directed fractionation of the MeOH extract of Solanum umbelliferum afforded solasodine (1), O-acetylsolasodine (2), and solasodine 3-O-beta-D- glucopyranoside (3) . Alkaloids 1 and 2 exhibited significant activity toward DNA repair-deficient yeast mutants, whereas 3 and the synthetic analogues N-acetylsolasodine (4) and N,O-diacetylsolasodine (5) were found to be inactive . Compounds 2 and 3 are new natural products. Transgenic Res, 1996 Mar, 5(2), 105 - 113 A system for tissue-specific copper-controllable gene expression in transgenic plants: nodule-specific antisense of aspartate aminotransferase-P2; Mett VL et al.; A vector system, based on copper controllable gene expression, has been developed to give control over place as well as time of expression of an introduced gene . This system consists of two elements: (1) the yeast ace1 gene encoding a metallo-regulatory transcription factor, ACE1, under control of either an organ-specific or a constitutive promoter; and (2) a gene of interest under control of a chimaeric promoter consisting of the 46 bp TATA fragment of the CaMV 35S RNA promoter linked to four repeats of the ACE1 binding site . The functioning of the system in an organ-specific manner was tested in nodulated Lotus corniculatus plants which consisted of non-transformed shoots plus transformed hairy root tissue 'wild-type tops/transgenic roots' . After addition of copper ions to the plant nutrient solution, beta-glucuronidase (GUS) expression was visualized either specifically in nodules or in both roots and nodules when the ace1 gene was placed under control of the nod45 promoter or the CaMV 35S RNA promoter, respectively . The nodule-specific system was used to express antisense constructs of aspartate aminotransferase-P2 in transgenic Lotus corniculatus plants . When expression was induced by the addition of copper ions to the plant nutrient solution aspartate aminotransferase-P2 activity declined dramatically, and a decrease of up to 90% was observed in nodule asparagine concentration. Chem Res Toxicol, 1996 Mar, 9(2), 426 - 33 Induction of terata in hamsters by solanidane alkaloids derived from Solanum tuberosum; Gaffield W et al.; The potential induction of terata by solanidanes has been of public health concern since a report in 1972 hypothesized that certain birth defects in humans could be attributed to ingestion of blighted potatoes . The potential teratogenicity of solanidane alkaloids from potatoes and tomatoes in domestic livestock had been considered even earlier . In the present report, oral administration of the steroidal alkaloid glycosides alpha-solanine and alpha-chaconine and their aglycone solanidine is shown to induce craniofacial malformations (exencephaly, encephalocele, and anophthalmia) in Syrian hamsters . All three alkaloids, that were either isolated or obtained by hydrolysis from Solanum tuberosum (var . Kennebec) sprouts, possessed the 22-(R),25(S)-configuration in the indolizidine moiety with no other isomers present . Toxicity constraints precluded administration of dosages high enough to induce statistically significant levels of terata in litters dosed with alpha-chaconine and permitted the attainment of only marginal statistical significance for alpha-solanine . However, malformation induction at p < 0.005 was observed in litters upon dosing both the nontoxic aglycone solanidine and the derivative solanidine N-oxide at higher levels . The relatively high teratogenicity of nontoxic solanidine, compared to the glycosides, demonstrates that terata induction by solanidanes is not due to maternal toxicity nor is the oligosaccharide portion of steroidal alkaloid glycosides required to facilitate passage of the teratogen to the fetus . The teratogenicity of solanidine N-oxide, a putative metabolite, suggests that N-oxidation is not an effective mammalian detoxification pathway . Relative teratogenic potencies (RTP) were assigned to solanidanes by conversion of literature data to equimolar doses compared to the powerful Veratrum teratogen jervine and the nonteratogenic spirosolane tomatidine . RTP values are as follows: jervine (100), 22(S),-25(R)-solanidanes (50), alpha-chaconine (43), alpha-solanine (32), 22(R),25(S)-solanidine (32), solanidine N-oxide (32), 5 alpha,6-dihydrosolanidine (9), and tomatidine (0). Cell Growth Differ, 1996 Mar, 7(3), 319 - 26 Overexpression of mouse p140 subunit of replication factor C accelerates cellular proliferation; Jaharul Haque S et al.; Screening of a murine cDNA expression library with an IFN-stimulated response element (ISRE), as a recognition site DNA probe, resulted in the isolation of a cDNA encoding a polypeptide of 1145 amino acids designated ISRE-binding factor-1 . This was subsequently shown to be identical to the M(r) 140,000 subunit of replication factor C (RFC) . RFC is required, along with the proliferating cell nuclear antigen and DNA polymerase delta, for the synthesis of the leading strand during DNA replication . RFC exhibits a structure-specific DNA-binding activity that has been localized to its M(r) 140,000 subunit (p140) . Sequence-specific binding of this polypeptide to the ISRE occurs only with low affinity . Based on DNA-binding activity of the truncated RFC-p140 encoded by the partial cDNA isolated, the DNA-binding domain of this polypeptide was mapped to a region encoded by amino acids 366 to 540 . Transfection of NIH 3T3 cells with an expression plasmid containing murine RFC-p140 driven by cytomegalovirus early promoter led to the establishment of stable cell lines that expressed a 2.5- to 3.0-fold higher RFC-p140 protein level in comparison with control cells . The stable clones exhibited significantly accelerated cell proliferation, indicating that RFC-p140 is the limiting subunit of an active RFC complex in normal cells. Biochem Mol Biol Int, 1996 Mar, 38(3), 635 - 43 Role of PM-ATPase, amino acid transport and free amino acid pool in the salt stress of, Candida membranefaciens; Khaware RK et al.; The salt tolerant yeast Candida membranefaciens exhibited a pleiotropic modification in response to high NaCl stress . The in vivo specific activity of Plasma Membrane-ATPase (PM-ATPase) of 1.35 M NaCl adapted cells was enhanced at the mid-log phase . The enhancement in the PM-ATPase activity was NaCl specific as cells stressed with identical concentration of KCl did not have any effect on PM-ATPase . The NaCl specific enhancement in the PM-ATPase activity was associated with decreased Km . Studies on H+ efflux correlated with the results of PM-ATPase . However, in vitro incubation of the enzyme with exogenously added salts like NaCl and KCl invariably inhibited enzyme activity by 70-90% in a dose dependent manner to suggest that in vivo effects of the salts on PM-ATPase were different from the in vitro effects . C . membranefaciens showed a higher intracellular levels of glutamate and aspartate in presence of 1.35 M NaCl which may impart osmoprotection to the stressed cells . It was interesting to observe that the transport activities of aspartate and glutamate were not enhanced according to their relative proportion in the total pool of free amino acids . Instead, transport of these and other amino acids (except lysine and arginine) showed a drastic reduction (upto 90%) in the 1.35 M NaCl grown cells. Biophys J, 1996 Mar, 70(3), 1198 - 213 Calcium and cell cycle progression: possible effects of external perturbations on cell proliferation; Baran I; Exit from the phase of cellular division appears to be driven by a calcium signal that triggers a cascade of events leading to the completion of mitosis . Here we propose a model that relates the dynamics of cytosolic calcium to progression through mitosis, G1 and G2 phases of the cell cycle . To this end, the assumption has been made that the transient rise ir cytosolic calcium concentration during mitosis is induced by inositol(1,4,5)triphosphate (IP3), which in turn is released at high levels of mitosis-promoting factor (MPF) . On this basis, a system of ordinary differential equations is proposed to simulate the evolution of ten cell-cycle-specific molecular species, including cyclins A and B, MPF, IP3, Ca2+, the CaMKII holoenzyme, and the ubiquitination complex . The influence on the cell proliferation capacity exerted by external perturbations, like calcium microinjections, depletion of intracellular calcium stores, electromagnetic fields, or stimulation/inhibition of different calcium currents through the plasma membrane, can be studied by appropriate modulation of the parameters involved in the signal transduction pathway. Biophys J, 1996 Mar, 70(3), 1096 - 104 Factors influencing accuracy of computer-built models: a study based on leucine zipper GCN4 structure; Shen L et al.; A three-dimensional model of the leucine zipper GCN4 built from its amino acid sequence had been reported previously by us . When the two alternative x-ray structures of the GCN4 dimer became available, the root mean square (r.m.s.) shifts between our model and the structures were determined as approximately 2.7 A on all atoms . These values are similar to the r.m.s . shift of 2.8 A between the two GCN4 structures in the different crystal forms (C2 and P2(1)2(1)2(1)) . CONGEN conformational searches were run to better understand the conditions that may determine the preference of different conformers in different environments and to test the sensitivity of our current modeling techniques . With a judicious choice of CONGEN search parameters, the backbone r.m.s . deviation impr