S. cerevisiae

Proc Natl Acad Sci U S A, 1998 Apr 14, 95(8), 4350 - 5
Human PEX1 cloned by functional complementation on a CHO cell mutant is responsible for peroxisome-deficient Zellweger syndrome of complementation group I; Tamura S et al.; The peroxisome biogenesis disorders (PBDs), including Zellweger syndrome (ZS) and neonatal adrenoleukodystrophy (NALD), are autosomal recessive diseases caused by defects in peroxisome assembly, for which at least 10 complementation groups have been reported . We have isolated a human PEX1 cDNA (HsPEX1) by functional complementation of peroxisome deficiency of a mutant Chinese hamster ovary (CHO) cell line, ZP107, transformed with peroxisome targeting signal type 1-tagged "enhanced" green fluorescent protein . This cDNA encodes a hydrophilic protein (Pex1p) comprising 1,283 amino acids, with high homology to the AAA-type ATPase family . A stable transformant of ZP107 with HsPEX1 was morphologically and biochemically restored for peroxisome biogenesis . HsPEX1 expression restored peroxisomal protein import in fibroblasts from three patients with ZS and NALD of complementation group I (CG-I), which is the highest-incidence PBD . A CG-I ZS patient (PBDE-04) possessed compound heterozygous, inactivating mutations: a missense point mutation resulting in Leu-664 --> Pro and a deletion of the sequence from Gly-634 to His-690 presumably caused by missplicing (splice site mutation) . Both PBDE-04 PEX1 cDNAs were defective in peroxisome-restoring activity when expressed in the patient fibroblasts as well as in ZP107 cells . These results demonstrate that PEX1 is the causative gene for CG-I peroxisomal disorders.

Proc Natl Acad Sci U S A, 1998 Apr 14, 95(8), 4188 - 92
The human U5-200kD DEXH-box protein unwinds U4/U6 RNA duplices in vitro; Laggerbauer B et al.; Splicing of nuclear precursors of mRNA (pre-mRNA) involves dynamic interactions between the RNA constituents of the spliceosome . The rearrangement of RNA-RNA interactions, such as the unwinding of the U4/U6 duplex, is believed to be driven by ATP-dependent RNA helicases . We recently have shown that spliceosomal U5 small nuclear ribonucleoproteins (snRNPs) from HeLa cells contain two proteins, U5-200kD and U5-100kD, which share homology with the DEAD/DEXH-box families of RNA helicases . Here we demonstrate that purified U5 snRNPs exhibit ATP-dependent unwinding of U4/U6 RNA duplices in vitro . To identify the protein responsible for this activity, U5 snRNPs were depleted of a subset of proteins under high salt concentrations and assayed for RNA unwinding . The activity was retained in U5 snRNPs that contain the U5-200kD protein but lack U5-100kD, suggesting that the U5-200kD protein could mediate U4/U6 duplex unwinding . Finally, U5-200kD was purified to homogeneity by glycerol gradient centrifugation of U5 snRNP proteins in the presence of sodium thiocyanate, followed by ion exchange chromatography . The RNA unwinding activity was found to reside exclusively with the U5-200kD DEXH-box protein . Our data raise the interesting possibility that this RNA helicase catalyzes unwinding of the U4/U6 RNA duplex in the spliceosome.

Proc Natl Acad Sci U S A, 1998 Apr 14, 95(8), 4164 - 9
Disruption of cellular translational control by a viral truncated eukaryotic translation initiation factor 2alpha kinase homolog; Dever TE et al.; Phosphorylation of eukaryotic translation initiation factor 2alpha (eIF2alpha) is a common cellular mechanism to limit protein synthesis in stress conditions . Baculovirus PK2, which resembles the C-terminal half of a protein kinase domain, was found to inhibit both human and yeast eIF2alpha kinases . Insect cells infected with wild-type, but not pk2-deleted, baculovirus exhibited reduced eIF2alpha phosphorylation and increased translational activity . The negative regulatory effect of human protein kinase RNA-regulated (PKR), an eIF2alpha kinase, on virus production was counteracted by PK2, indicating that baculoviruses have evolved a unique strategy for disrupting a host stress response . PK2 was found in complex with PKR and blocked kinase autophosphorylation in vivo, suggesting a mechanism of kinase inhibition mediated by interaction between truncated and intact kinase domains.

J Biol Chem, 1998 Apr 10, 273(15), 9179 - 87
The mammalian numb phosphotyrosine-binding domain . Characterization of binding specificity and identification of a novel PDZ domain-containing numb binding protein, LNX; Dho SE et al.; Numb is a phosphotyrosine-binding (PTB) domain-containing protein implicated in the control of cell fate decisions during development . A modified two-hybrid screen in yeast was used to identify Numb PTB domain-interacting proteins important for Numb function . Here we report the identification of a novel protein, LNX, which interacts specifically with the Numb PTB domain . Two differentially expressed LNX messages encode overlapping proteins with predicted molecular masses of 80 kDa (LNX) and 70 kDa (LNX-b) . LNX and LNX-b contain unique amino-terminal sequences and share four PDZ domains . The unique amino-terminal region of LNX includes a RING finger domain . The Numb PTB domain binding region of LNX was mapped to the sequence motif LDNPAY, found in both protein isoforms . Mutational analysis of LNX and peptide competition experiments showed that phosphorylation of the tyrosine residue within this motif was not required for binding to the Numb PTB domain . Finally, we also provide evidence that tyrosine phosphorylation of the LDNPAY sequence motif in LNX could generate a binding site for the phosphorylation-dependent binding of other PTB domain-containing proteins such as SHC . We speculate that LNX may be important for clustering PTB-containing proteins with functionally related transmembrane proteins in specific membrane compartments.

J Biol Chem, 1998 Apr 10, 273(15), 9085 - 93
Alteration of the midpoint potential and catalytic activity of the rieske iron-sulfur protein by changes of amino acids forming hydrogen bonds to the iron-sulfur cluster; Denke E et al.; The crystal structure of the bovine Rieske iron-sulfur protein indicates a sulfur atom (S-1) of the iron-sulfur cluster and the sulfur atom (Sgamma) of a cysteine residue that coordinates one of the iron atoms form hydrogen bonds with the hydroxyl groups of Ser-163 and Tyr-165, respectively . We have altered the equivalent Ser-183 and Tyr-185 in the Saccharomyces cerevisiae Rieske iron-sulfur protein by site-directed mutagenesis of the iron-sulfur protein gene to examine how these hydrogen bonds affect the midpoint potential of the iron-sulfur cluster and how changes in the midpoint potential affect the activity of the enzyme . Eliminating the hydrogen bond from the hydroxyl group of Ser-183 to S-1 of the cluster lowers the midpoint potential of the cluster by 130 mV, and eliminating the hydrogen bond from the hydroxyl group of Tyr-185 to Sgamma of Cys-159 lowers the midpoint potential by 65 mV . Eliminating both hydrogen bonds has an approximately additive effect, lowering the midpoint potential by 180 mV . Thus, these hydrogen bonds contribute significantly to the positive midpoint potential of the cluster but are not essential for its assembly . The activity of the bc1 complex decreases with the decrease in midpoint potential, confirming that oxidation of ubiquinol by the iron-sulfur protein is the rate-limiting partial reaction in the bc1 complex, and that the rate of this reaction is extensively influenced by the midpoint potential of the iron-sulfur cluster.

J Biol Chem, 1998 Apr 10, 273(15), 9041 - 9
Folding a WD repeat propeller . Role of highly conserved aspartic acid residues in the G protein beta subunit and Sec13; Garcia-Higuera I et al.; The beta subunit of the heterotrimeric G proteins that transduce signals across the plasma membrane is made up of an amino-terminal alpha-helical segment followed by seven repeating units called WD (Trp-Asp) repeats that occur in about 140 different proteins . The seven WD repeats in Gbeta, the only WD repeat protein whose crystal structure is known, form seven antiparallel beta sheets making up the blades of a toroidal propeller structure (Wall, M . A., Coleman, D . E., Lee, E., Iniguez-Lluhi, J . A., Posner, B . A., Gilman, A . G., and Sprang, S . R . (1995) Cell 83, 1047-1058; Sondek, J., Bohm, A., Lambright, D . G., Hamm, H . E., and Sigler, P . B . (1996) Nature 379, 369-374) . It is likely that all proteins with WD repeats form a propeller structure . Alignment of the sequence of 918 unique WD repeats reveals that 85% of the repeats have an aspartic acid (D) residue (not the D of WD) in the turn connecting beta strands b and c of each putative propeller blade . We mutated each of these conserved Asp residues to Gly individually and in pairs in Gbeta and in Sec13, a yeast WD repeat protein involved in vesicular traffic, and then analyzed the ability of the mutant proteins to fold in vitro and in COS-7 cells . In vitro, most single mutant Gbeta subunits fold into Gbetagamma dimers more slowly than wild type to a degree that varies with the blade . In contrast, all single mutants form normal amounts of Gbetagamma in COS-7 cells, although some dimers show subtle local distortions of structure . Most double mutants assemble poorly in both systems . We conclude that the conserved Asp residues are not equivalent and not all are essential for the folding of the propeller structure . Some may affect the folding pathway or the affinity for chaperonins . Mutations of the conserved Asp in Sec13 affect folding equally in vitro and in COS-7 cells . The repeats that most affected folding were not at the same position in Sec13 and Gbeta . Our finding, both in Gbeta and in Sec13, that no mutation of the conserved Asp entirely prevents folding suggests that there is no obligatory folding order for each repeat and that the folding order is probably not the same for different WD repeat proteins, or even necessarily constant for the same protein.

J Biol Chem, 1998 Apr 10, 273(15), 9007 - 12
Interactions of the borna disease virus P, N, and X proteins and their functional implications; Schwemmle M et al.; Borna disease virus (BDV) causes persistent central nervous system infection and behavioral disturbances in warm-blooded animals . Protein interaction studies were pursued to gain insight into the functions of the putative nucleoprotein (N), phosphoprotein (P), atypical glycoprotein (gp18), and X protein (X) of BDV . Coimmunoprecipitation experiments indicated that N and P, and P and X, form complexes in infected cells . Two-hybrid analyses confirmed interactions between P and P, P and X, and P and N, but not between P and gp18, N and gp18, X and gp18, or X and N . Analysis of P truncation mutants identified three nonoverlapping regions important for oligomerization (amino acids (aa) 135-172), and binding to X (aa 33-115) or N (aa 197-201) . Coexpression of X stimulated oligomerization of P but decreased N-P complex formation . Immunocytochemistry of transfected noninfected CHO cells demonstrated that the distribution of X is dependent upon the presence of P-X expressed alone was found predominantly in the cytoplasm whereas coexpression of X and P resulted in nuclear localization . Immunocytochemistry of infected cells revealed nuclear colocalization of P and X . Interactions of P, N, and X may have implications for regulation of BDV transcription/replication and ribonucleoprotein assembly.

J Biol Chem, 1998 Apr 10, 273(15), 8867 - 74
Coordinated regulation of the tyrosine phosphorylation of Cbl by Fyn and Syk tyrosine kinases; Deckert M et al.; Cross-linking of the T cell antigen receptor (TCR)-CD3 complex induces rapid tyrosine phosphorylation and activation of Src (Lck and Fyn) and Syk (Syk and Zap-70) family protein tyrosine kinases (PTKs) which, in turn, phosphorylate multiple intracellular substrates . Cbl is a prominent PTK substrate suggesting a pivotal role for it in early signal transduction events . However, the regulation of Cbl function and tyrosine phosphorylation in T cells by upstream PTKs remains poorly understood . In the present study, we used genetic and biochemical approaches to demonstrate that Cbl directly interacts with Syk and Fyn via its N-terminal and C-terminal regions, respectively . Tyr-316 of Syk was required for the interaction with Cbl as well as for the maximal tyrosine phosphorylation of Cbl . However, both wild-type Syk and Y316F-mutated Syk phosphorylated equally well the C-terminal fragment of Cbl in vivo, suggesting the existence of an alternative, N terminus-independent mechanism for the Syk-induced tyrosine phosphorylation of Cbl . This mechanism appears to involve Fyn, since, in addition to its association with the C-terminal region of Cbl, Fyn also associated with Syk and enhanced the Syk-induced tyrosine phosphorylation of Cbl . These findings implicate Fyn as an adaptor protein that facilitates the interaction between Syk and Cbl, and suggest that Src and Syk family PTKs coordinately regulate the tyrosine phosphorylation of Cbl.

J Cell Biol, 1998 Apr 6, 141(1), 143 - 53
The 13-kD FK506 binding protein, FKBP13, interacts with a novel homologue of the erythrocyte membrane cytoskeletal protein 4.1; Walensky LD et al.; We have identified a novel generally expressed homologue of the erythrocyte membrane cytoskeletal protein 4.1, named 4.1G, based on the interaction of its COOH-terminal domain (CTD) with the immunophilin FKBP13 . The 129-amino acid peptide, designated 4.1G-CTD, is the first known physiologic binding target of FKBP13 . FKBP13 is a 13-kD protein originally identified by its high affinity binding to the immunosuppressant drugs FK506 and rapamycin (Jin, Y., M.W . Albers, W.S . Lane, B.E . Bierer, and S.J . Burakoff . 1991 . Proc . Natl . Acad . Sci . USA . 88:6677- 6681); it is a membrane-associated protein thought to function as an ER chaperone (Bush, K.T., B.A . Henrickson, and S.K . Nigam . 1994 . Biochem . J . {Tokyo} . 303:705-708) . We report the specific association of FKBP13 with 4.1G-CTD based on yeast two-hybrid, in vitro binding and coimmunoprecipitation experiments . The histidyl-proline moiety of 4.1G-CTD is required for FKBP13 binding, as indicated by yeast experiments with truncated and mutated 4.1G-CTD constructs . In situ hybridization studies reveal cellular colocalizations for FKBP13 and 4.1G-CTD throughout the body during development, supporting a physiologic role for the interaction . Interestingly, FKBP13 cofractionates with the red blood cell homologue of 4.1 (4.1R) in ghosts, inside-out vesicles, and Triton shell preparations . The identification of FKBP13 in erythrocytes, which lack ER, suggests that FKBP13 may additionally function as a component of membrane cytoskeletal scaffolds.

J Cell Biol, 1998 Apr 6, 141(1), 61 - 70
Cargo selection by the COPII budding machinery during export from the ER; Aridor M et al.; Cargo is selectively exported from the ER in COPII vesicles . To analyze the role of COPII in selective transport from the ER, we have purified components of the mammalian COPII complex from rat liver cytosol and then analyzed their role in cargo selection and ER export . The purified mammalian Sec23-24 complex is composed of an 85-kD (Sec23) protein and a 120-kD (Sec24) protein . Although the Sec23-24 complex or the monomeric Sec23 subunit were found to be the minimal cytosolic components recruited to membranes after the activation of Sar1, the addition of the mammalian Sec13-31 complex is required to complete budding . To define possible protein interactions between cargo and coat components, we recruited either glutathione-S-transferase (GST)-tagged Sar1 or GST- Sec23 to ER microsomes . Subsequently, we solubilized and reisolated the tagged subunits using glutathione-Sepharose beads to probe for interactions with cargo . We find that activated Sar1 in combination with either Sec23 or the Sec23-24 complex is necessary and sufficient to recover with high efficiency the type 1 transmembrane cargo protein vesicular stomatitis virus glycoprotein in a detergent-soluble prebudding protein complex that excludes ER resident proteins . Supplementing these minimal cargo recruitment conditions with the mammalian Sec13-31 complex leads to export of the selected cargo into COPII vesicles . The ability of cargo to interact with a partial COPII coat demonstrates that these proteins initiate cargo sorting on the ER membrane before budding and establishes the role of GTPase-dependent coat recruitment in cargo selection.

Mol Biol Cell, 1998 Apr, 9(4), 945 - 56
Swi5 controls a novel wave of cyclin synthesis in late mitosis; Aerne BL et al.; We have shown previously that the Swi5 transcription factor regulates the expression of the SIC1 Cdk inhibitor in late mitosis . This suggests that Swi5 might control other genes with roles in ending mitosis . We identified a gene with a Swi5-binding site in the promoter that encoded a protein with high homology to Pcl2, a cyclin-like protein that associates with the Cdk Pho85 . This gene, PCL9, is indeed regulated by Swi5 in late M phase, the only cyclin known to be expressed at this point in the cell cycle . The Pcl9 protein is associated with a Pho85-dependent protein kinase activity, and the protein is unstable with peak levels occurring in late M phase . PCL2 is already known to be expressed in late G1 and we find that, in addition, it is also regulated by Swi5 in telophase . The expression of PCL2 and PCL9 at this stage of the cell cycle implies a role for the Pho85 Cdk at the end of mitosis . Consistent with this a synthetic interaction was observed between pho85delta and strains deleted for SIC1, SWI5, and SPO12 . These and other studies support the notion that the M/G1 switch is a major cell cycle transition.

Gene, 1998 Mar 16, 209(1-2), 149 - 56
cDNA cloning and expression analysis of the murine ribonuclease L inhibitor; Benoit De Coignac A et al.; The 2-5A/RNase L system is one of the pathways induced by interferon (IFN) . It plays a major role in the antiviral and antiproliferative activities of IFNs . Recently, we have shown that the activity of the RNase L could be inhibited by a proteic inhibitor, the RNase L Inhibitor (RLI) . Human RLI (Hu-RLI) was cloned and characterized . We describe here the isolation and characterization of the cDNA encoding the murine RLI (Mu-RLI) . Hu-RLI and Mu-RLI protein have 98% amino acid identity . Mu-RLI is functionally homologous to Hu-RLI, and all the structural features and amino acid sequence motifs of Hu-RLI are conserved in Mu-RLI . Moreover, reticulocyte lysate translated Mu-RLI protein is also able to inhibit 2-5A binding on 2-5A-dependent RNAse-L . Northern blot analysis revealed that Mu-RLI cDNA hybridizes with one mRNA of 3.5 kb except for the testis where two mRNA of 3.5 and 2.1 kb, respectively, are detected, suggesting a tissue-specific regulation.

Hum Mol Genet, 1998 Apr, 7(4), 679 - 84
Specific interaction between the XNP/ATR-X gene product and the SET domain of the human EZH2 protein; Cardoso C et al.; Mutations in the XNP gene result in different inherited disorders, including the ATR-X syndrome which is characterized by mental retardation (MR) associated with alpha-thalaessemia . Amino acid sequence analysis revealed that the XNP protein is a new member of the SNF2-like family, which comprises numerous members involved in a broad range of biological functions: transcriptional regulation, DNA repair and chromosome segregation . Since experiments on fibroblasts from ATR-X patients have provided no evidence for either a DNA repair defect or abnormal chromosome breakage or segregation, it seems more likely that the XNP protein is somehow involved in regulation of gene expression . Recent genetic and biochemical studies have led to the emerging concept that SNF2-like proteins are components of a large protein complex which may exert its functions by modulating chromatin structure . To investigate whether XNP could mediate the activity of gene-specific activators through chromatin remodelling, we performed a yeast two-hybrid analysis using XNP and several human heterochromatin-associated proteins . We found a specific interaction between the XNP and the EZH2 proteins . In light of these observations, we discuss how the XNP protein may regulate gene transcription at the chromatin level.

RNA, 1998 May, 4(5), 594 - 602
Chimeric rRNAs containing the GTPase centers of the developmentally regulated ribosomal rRNAs of Plasmodium falciparum are functionally distinct; Velichutina IV et al.; The human malaria parasite, Plasmodium falciparum, maintains at least two distinct types, A and S, of developmentally controlled ribosomal RNAs . To investigate specific functions associated with these rRNAs, we replaced the Saccharomyces cerevisiae GTPase domain of the 25S rRNA with GTPase domains corresponding to the Plasmodium A- and S-type 28S rRNAs . The A-type rRNA differs in a single nonconserved base pair from the yeast GTPase domain . The S-type rRNA GTPase domain has three additional changes in highly conserved residues, making it unique among all known rRNA sequences . The expression of either A- or S-type chimeric rRNA in yeast increased translational accuracy . Yeast containing only A-type chimeric rRNA and no wild-type yeast rRNA grew at the wild-type level . In contrast, S-type chimeric rRNA severely inhibited growth in the presence of wild-type yeast rRNA, and caused lethality in the absence of the wild-type yeast rRNA . We show what before could only be hypothesized, that the changes in the GTPase center of ribosomes present during different developmental stages of Plasmodium species can result in fundamental changes in the biology of the organism.

Genes Cells, 1998 Jan, 3(1), 9 - 15
Interplay between positive and negative elongation factors: drawing a new view of DRB; Yamaguchi Y et al.; DRB is a classic inhibitor of transcription by RNA polymerase II (pol II) . Although it has been demonstrated that DRB inhibits the elongation step of transcription, its mode of action has been elusive . DRB also markedly inhibits human immunodeficiency virus (HIV) transcription, by targeting the elongation which is enhanced by the HIV-encoded transactivator Tat . Two factors essential for DRB action have recently been identified . These factors, positive transcription elongation factor b (P-TEFb) and DRB sensitivity-inducing factor (DSIF), positively and negatively regulate pol II elongation, and are likely to be relevant to the function of Tat . In this review, we summarize the recent findings on these factors, and discuss a possible model for the molecular mechanism of DRB action.

Neuron, 1998 Apr, 20(4), 683 - 91
SynGAP: a synaptic RasGAP that associates with the PSD-95/SAP90 protein family; Kim JH et al.; The PSD-95/SAP90 family of proteins has recently been implicated in the organization of synaptic structure . Here, we describe the isolation of a novel Ras-GTPase activating protein, SynGAP, that interacts with the PDZ domains of PSD-95 and SAP102 in vitro and in vivo . SynGAP is selectively expressed in brain and is highly enriched at excitatory synapses, where it is present in a large macromolecular complex with PSD-95 and the NMDA receptor . SynGAP stimulates the GTPase activity of Ras, suggesting that it negatively regulates Ras activity at excitatory synapses . Ras signaling at the postsynaptic membrane may be involved in the modulation of excitatory synaptic transmission by NMDA receptors and neurotrophins . These results indicate that SynGAP may play an important role in the modulation of synaptic plasticity.

Bull Mem Acad R Med Belg, 1997, 152(6), 247 - 63
{Prions and the problems they raise}; Burny A; A prion is an "infectious" protein . Most probably, prions play a major role, direct or indirect, in the propagation of neurodegenerative diseases such as spongiform encephalopathies . By extension, the term prion is also used to explain several cases of dominant cytoplasmic heredity known in the yeast Saccharomyces cerevisiae . Several recent publications, briefly discussed, suggest that amyloid fibrils (aggregated prions) appear late in some experimental neuropathies, long after the disease symptoms . The present uncertainty deals with the presence or not of a second component besides the prion to make up the infections agent . As such, the prion theory raises major problems about the chemistry of protein folding . A major contribution in prion research is urgent and mandatory.

J Cell Sci, 1998 Jun, 111 ( Pt 11), 1555 - 66
Role of fungal dynein in hyphal growth, microtubule organization, spindle pole body motility and nuclear migration; Inoue S et al.; Cytoplasmic dynein is a microtubule-associated motor protein with several putative subcellular functions . Sequencing of the gene (DHC1) for cytoplasmic dynein heavy chain of the filamentous ascomycete, Nectria haematococca, revealed a 4,349-codon open reading frame (interrupted by two introns) with four highly conserved P-loop motifs, typical of cytoplasmic dynein heavy chains . The predicted amino acid sequence is 78.0% identical to the cytoplasmic dynein heavy chain of Neurospora crassa, 70.2% identical to that of Aspergillus nidulans and 24.8% identical to that of Saccharomyces cerevisiae . The genomic copy of DHC1 in N . haematococca wild-type strain T213 was disrupted by inserting a selectable marker into the central motor domain . Mutants grew at 33% of the wild-type rate, forming dense compact colonies composed of spiral and highly branched hyphae . Major cytological phenotypes included (1) absence of aster-like arrays of cytoplasmic microtubules focused at the spindle pole bodies of post-mitotic and interphase nuclei, (2) limited post-mitotic nuclear migration, (3) lack of spindle pole body motility at interphase, (4) failure of spindle pole bodies to anchor interphase nuclei, (5) nonuniform distribution of interphase nuclei and (6) small or ephemeral Spitzenkorper at the apices of hyphal tip cells . Microtubule distribution in the apical region of tip cells of the mutant was essentially normal . The nonuniform distribution of nuclei in hyphae resulted primarily from a lack of both post-mitotic nuclear migration and anchoring of interphase nuclei by the spindle pole bodies . The results support the hypothesis that DHC1 is required for the motility and functions of spindle pole bodies, normal secretory vesicle transport to the hyphal apex and normal hyphal tip cell morphogenesis.

FEBS Lett, 1998 Feb 13, 423(1), 49 - 52
Role of UEV-1A, a homologue of the tumor suppressor protein TSG101, in protection from DNA damage; Thomson TM et al.; The open reading frame YGL087c in the budding yeast Saccharomyces cerevisiae genome encodes a polypeptide highly similar to the human UEV (ubiquitin-conjugating E2 enzyme variant) proteins, which have been proposed to belong to a family of putative dominant negative ubiquitin regulators . Deletion of the YGL087c open reading frame yields viable cells which are sensitive to UV irradiation or methyl methanesulfonate, but not to hydroxyurea . This phenotype is reminiscent of that of rad mutants and suggests that the YGL087c-encoded protein functions in a process related to tolerance to DNA damage . We also show that the mutant phenotype is fully complemented by expression of the human UEV-1A cDNA and we propose that UEV-1 proteins could also have a role in protecting higher eukaryotic cells from DNA damaging agents.

Anesthesiology, 1998 Apr, 88(4), 1076 - 84
TOK1 is a volatile anesthetic stimulated K+ channel; Gray AT et al.; BACKGROUND: Volatile anesthetic agents can activate the S channel, a baseline potassium (K+) channel, of the marine mollusk Aplysia . To investigate whether cloned ion channels with electrophysiologic properties similar to the S channel (potassium selectivity, outward rectification, and activation independent of voltage) also are modulated by volatile anesthetic agents, the authors expressed the cloned yeast ion channel TOK1 (tandem pore domain, outwardly rectifying K+ channel) in Xenopus oocytes and studied its sensitivity to volatile agents . METHODS: Standard two-electrode voltage and patch clamp recording methods were used to study TOK1 channels expressed in Xenopus oocytes . RESULTS: Studies with two-electrode voltage clamp at room temperature showed that halothane, isoflurane, and desflurane increased TOK1 outward currents by 48-65% in barium Frog Ringer's perfusate . The concentrations at which 50% potentiation occurred (EC50 values) were in the range of 768-814 microM (0.016-0.044 atm) and had a rank order of potency in atm in which halothane > isoflurane > desflurane . The potentiation of TOK1 by volatile anesthetic agents was rapid and reversible (onset and offset, 1-20 s) . In contrast, the nonanesthetic 1,2-dichlorohexafluorocyclobutane did not potentiate TOK1 currents in concentrations up to five times the MAC value predicted by the Meyer-Overton hypothesis based on oil/gas partition coefficients . Single TOK1 channel currents were recorded from excised outside-out patches . The single channel open probability increased as much as twofold in the presence of isoflurane and rapidly returned to the baseline values on washout . Volatile anesthetic agents did not alter the TOK1 single channel current-voltage (I-V) relationship, however, suggesting that the site of action does not affect the permeation pathway of the channel . CONCLUSION: TOK1 is a potassium channel that is stimulated by volatile anesthetic agents . The concentrations over which potentiation occurred (EC50 values) were higher than those commonly used in clinical practice (approximately twice MAC).

Clin Cancer Res, 1998 Mar, 4(3), 683 - 91
Effect of pyrazoloacridine (NSC 366140) on DNA topoisomerases I and II; Adjei AA et al.; Pyrazoloacridine (PA), an acridine congener with an unknown mechanism of action, has shown selective activity against solid tumor cells, cytotoxicity in noncycling and hypoxic cells, and promising antitumor activity in Phase I clinical trials . In the present study, the effect of PA on topoisomerase (topo) activity was evaluated using yeast strains lacking functional topo I or II, mammalian cell nuclear extracts, purified samples of mammalian topo I and topo II, and intact mammalian tissue culture cells . Clonogenic assays revealed that PA cytotoxicity in yeast strains was unaffected by selective loss of topo I or topo II activity . On the other hand, enzyme assays revealed that 2-4 microM PA abolished the catalytic activity of both topo I and topo II in vitro . In contrast to topotecan and etoposide, PA did not stabilize covalent topo-DNA complexes . Instead, PA inhibited topotecan-induced stabilization of covalent topo I-DNA complexes and etoposide-induced stabilization of topo II-DNA complexes in vitro and in intact cells . Consistent with these results, colony-forming assays indicated that short-term PA exposure inhibited the cytotoxicity of topotecan and etoposide, whereas prolonged PA exposure was itself toxic to these cells . Accumulation studies revealed that PA was concentrated as much as 250-fold in drug-treated cells, resulting in intranuclear concentrations that far exceeded those required to inhibit topo I and topo II . Collectively, these results not only suggest that PA can target both topo I and topo II at clinically achievable concentrations but also indicate that its mechanism is distinct from topo I and topo II poisons presently licensed for clinical use.

Biochem J, 1998 May 15, 332 ( Pt 1), 243 - 50
Mutational analysis of the domain structure of mouse protein phosphatase 2Cbeta; Kusuda K et al.; The structures of five distinct isoforms of mammalian protein phosphatase 2Cbeta (PP2Cbeta-1, -2, -3, -4 and -5) have previously been found to differ only at their C-terminal regions . In the present study, we performed mutational analysis of recombinant mouse PP2Cbeta-1 to determine the functional domains of the molecule and elucidate the biochemical significance of the structural differences in the isoforms . Differences in affinity for {32P}phosphohistone but not for {32P}phosphocasein were observed among the five PP2Cbeta isoforms . Deletion of 12 amino acids from the C-terminal end, which form a unique sequence for PP2Cbeta-1, caused a 35% loss of activity against {32P}phosphohistone but no loss of activity against {32P}phosphocasein . Deletion of up to 78 amino acids from this end did not cause any further alteration in activity, whereas deletion of 100 amino acids totally eliminated the activity against both {32P}phosphohistone and {32P}phosphocasein . On the other hand, deletion of 11 amino acids from the N-terminal end caused a 97% loss of enzyme activity, and further deletions caused a total loss of activity . Substitution of any of the six specific amino acids among 16 tested in this study, which were located among the 250 N-terminal residues, caused 98-100% loss of enzyme activity . Among these amino acids, three (Glu-38, -60 and -243) have recently been reported to be essential for the binding of metal ions in the catalytic site of the PP2C molecule {Das, Helps, Cohen and Barford (1996) EMBO J . 15, 6798-6809} . These observations indicate that PP2Cbeta is composed of at least two distinct functional domains, an N-terminal catalytic domain of about 310 amino acids and the remaining C-terminal domain, which is involved in determination of substrate specificity.

J Cell Sci, 1998 Mar, 111 ( Pt 6), 737 - 47
Oligomerization of the extracellular domain of Boss enhances its binding to the Sevenless receptor and its antagonistic effect on R7 induction; Sevrioukov EA et al.; In the developing compound eye of Drosophila, neuronal differentiation of the R7 photoreceptor cell is induced by the interaction of the receptor tyrosine kinase Sevenless with its ligand Bride of sevenless (Boss), which is expressed on the neighboring R8 cell . Boss is an unusual ligand of a receptor tyrosine kinase: it is composed of a large extracellular domain, a transmembrane domain with seven membrane-spanning segments and a cytoplasmic tail . Expression of a monomeric, secreted form of the extracellular domain of Boss is not sufficient for Sevenless activation, and instead acts as a weak antagonist . Because oligomerization appears to be a critical step in the activation of receptor tyrosine kinases, we used oligomerized forms of the Boss extracellular domain to test their ability to bind to Sevenless in vivo and restore R7 induction in vivo . Oligomerization was achieved by fusion to the leucine zipper of the yeast transcription factor GCN4 or to the tetramerization helix of Lac repressor . Binding of these multivalent proteins to Sevenless could be detected in vitro by immunoprecipitation of cross-linked ligand/receptor complexes and in vivo by receptor-dependent ligand localization . However, neither R8-specific or ubiquitous expression of multivalent Exboss ligands rescued the boss phenotype . Instead, these ligands acted as competitive inhibitors for wild-type Boss protein and thereby suppressed R7 induction . Therefore the role of the transmembrane or cytoplasmic domains of Boss in the activation of the Sev receptor cannot be replaced by oligomerization.

Science, 1998 Apr 24, 280(5363), 593 - 6
Formation of a preinitiation complex by S-phase cyclin CDK-dependent loading of Cdc45p onto chromatin; Zou L et al.; Cdc45p, a protein essential for initiation of DNA replication, associates with chromatin after "start" in late G1 and during the S phase of the cell cycle . Binding of Cdc45p to chromatin depends on Clb-Cdc28 kinase activity as well as functional Cdc6p and Mcm2p, which suggests that Cdc45p associates with the prereplication complex after activation of S-phase cyclin-dependent kinases (CDKs) . As indicated by the timing and the CDK dependence, binding of Cdc45p to chromatin is crucial for commitment to initiation of DNA replication . During S phase, Cdc45p physically interacts with minichromosome maintenance (MCM) proteins on chromatin; however, dissociation of Cdc45p from chromatin is slower than that of MCMs, which indicates that the proteins are released by different mechanisms.

Oncol Rep, 1998 May-Jun, 5(3), 585 - 9
Differential transcriptional activation by the N-terminal region of BRCA1 splice variants BRCA1a and BRCA1b; Cui JQ et al.; The breast and ovarian cancer susceptibility gene BRCA1, is a nuclear phosphoprotein which functions as a tumor suppressor in human breast cancer cells . BRCA1 protein contains an amino-terminal zinc finger motif and a carboxy-terminal acidic region . Recently, the carboxy-terminal region of BRCA1 and the amino-terminal region of BRCA2 proteins were shown to function as transactivation domains when fused to GAL4 DNA binding domain . We have recently isolated and characterized two new naturally occurring variants of BRCA1 (BRCA1a/p110 and BRCA1b/p100) which are phosphoproteins containing phosphotyrosine that associate with E2F transcriptional factors, cyclins and cyclin dependent kinases indicating a role for BRCA1 proteins in cell-cycle regulation . Here we show for the first time that the amino-terminal region of BRCA1a (BNT) but not BRCA1b can also function as a transcriptional activator when fused to GAL4 DNA binding domain . Thus, BRCA1/1a proteins contain two autonomous transcriptional activation domains, one at the amino-terminal region (BNT) and the other at the carboxy-terminal region (BCT) . BRCA1b retains only the BCT domain since it has lost part of the potential BNT domain as a result of alternative splicing . Our results also suggest the presence of an inhibitory domain at the carboxy terminal region of BRCA1 and BRCA1a proteins (BID) . Thus, BRCA1b protein may function as a dominant negative variant that could regulate the transcriptional activity of BRCA1/BRCA1a proteins and hence may serve as a marker for identifying individuals with greater potential for developing breast cancer . It may be possible that loss of transcriptional activation or protein-protein interactions in patients with mutations in the amino terminal zinc finger domain could deprive the cell of an important mechanism for regulating cell proliferation leading to the development of breast cancer.

Genes Dev, 1998 Apr 1, 12(7), 943 - 55
A CBP/p300 homolog specifies multiple differentiation pathways in Caenorhabditis elegans; Shi Y et al.; Mammalian p300 and CBP are related transcriptional cofactors that possess histone acetyltransferase activity . Inactivation of CBP/p300 is critical for adenovirus E1A to induce oncogenic transformation and to inhibit differentiation, suggesting that these proteins are likely to play a role in cell growth and differentiation . Here we show that a Caenorhabditis elegans gene closely related to CBP/p300, referred to as cbp-1, is required during early embryogenesis to specify several major differentiation pathways . Inhibition of cbp-1 expression causes developmental arrest of C . elegans embryos with no evidence of body morphogenesis but with nearly twice the normal complement of embryonic cells . Mesodermal, endodermal, and hypodermal cells appear to be completely absent in most embryos, however, all of the embryos exhibit evidence of neuronal differentiation . Our analysis of this phenotype suggests a critical role for CBP-1 in promoting all non-neuronal pathways of somatic differentiation in the C . elegans embryo . In contrast, we show that C . elegans genes related to components of a conserved mammalian histone deacetylase, appear to have a role in repressing somatic differentiation . Our findings suggest a model in which CBP-1 may activate transcription and differentiation in C . elegans by directly or indirectly antagonizing a repressive effect of histone deacetylase.

J Biol Chem, 1998 Apr 3, 273(14), 8040 - 7
Sorting of D-lactate dehydrogenase to the inner membrane of mitochondria . Analysis of topogenic signal and energetic requirements; Rojo EE et al.; D-Lactate dehydrogenase (D-LD) is located in the inner membrane of mitochondria . It spans the membrane once in an Nin-Cout orientation with the bulk of the protein residing as a folded domain in the intermembrane space . D-LD is synthesized as a precursor with an N-terminal cleavable presequence and is imported into the mitochondria in a Deltapsi-dependent, but mt-Hsp70-independent manner . Upon import in vitro D-LD folds in the intermembrane space to attain a conformation indistinguishable from endogenous D-LD . Sorting of D-LD to the inner membrane is directed by a composite topogenic signal consisting of the hydrophobic transmembrane segment and a cluster of charged amino acids C-terminal to it . We propose a model for the mode of operation of the sorting signal of D-LD . This model also accounts for the driving force of translocation across the outer membrane, in the apparent absence of mt-Hsp70-dependent assisted import and involves the folding of the D-LD in the intermembrane space.

J Biol Chem, 1998 Apr 3, 273(14), 7835 - 42
DNA structural elements required for ERCC1-XPF endonuclease activity; de Laat WL et al.; The heterodimeric complex ERCC1-XPF is a structure-specific endonuclease responsible for the 5' incision during mammalian nucleotide excision repair (NER) . Additionally, ERCC1-XPF is thought to function in the repair of interstrand DNA cross-links and, by analogy to the homologous Rad1-Rad10 complex in Saccharomyces cerevisiae, in recombination between direct repeated DNA sequences . To gain insight into the role of ERCC1-XPF in such recombinational processes and in the NER reaction, we studied in detail the DNA structural elements required for ERCC1-XPF endonucleolytic activity . Recombinant ERCC1-XPF, purified from insect cells, was found to cleave stem-loop substrates at the DNA junction in the absence of other proteins like replication protein A, showing that the structure-specific endonuclease activity is intrinsic to the complex . Cleavage depended on the presence of divalent cations and was optimal in low Mn2+ concentrations (0.2 mM) . A minimum of 4-8 unpaired nucleotides was required for incisions by ERCC1-XPF . Splayed arm and flap substrates were also cut by ERCC1-XPF, resulting in the removal of 3' protruding single-stranded arms . All incisions occurred in one strand of duplex DNA at the 5' side of a junction with single-stranded DNA . The exact cleavage position varied from 2 to 8 nucleotides away from the junction . One single-stranded arm, protruding either in the 3' or 5' direction, was necessary and sufficient for correct positioning of incisions by ERCC1-XPF . Our data specify the engagement of ERCC1-XPF in NER and allow a more direct search for its specific role in recombination.

Proc Natl Acad Sci U S A, 1998 Mar 31, 95(7), 3455 - 60
Transcriptional sequencing: A method for DNA sequencing using RNA polymerase; Sasaki N et al.; We have developed a sequencing method based on the RNA polymerase chain termination reaction with rhodamine dye attached to 3'-deoxynucleoside triphosphate (3'-dNTP) . This method enables us to conduct a rapid isothermal sequencing reaction in <30 min, to reduce the amount of template required, and to do PCR direct sequencing without the elimination of primers and 2'-dNTP, which disturbs the Sanger sequencing reaction . An accurate and longer read length was made possible by newly designed four-color dye-3'-dNTPs and mutated RNA polymerase with an improved incorporation rate of 3'-dNTP . This method should be useful for large-scale sequencing in genome projects and clinical diagnosis.

Development, 1998 Mar, 125(6), 1049 - 57
Systematic gain-of-function genetics in Drosophila; Rorth P et al.; A modular misexpression system was used to carry out systematic gain-of-function genetic screens in Drosophila . The system is based on inducible expression of genes tagged by insertion of a P-element vector carrying a GAL4-regulated promoter oriented to transcribe flanking genomic sequences . To identify genes involved in eye and wing development, the 2300 independent lines were screened for dominant phenotypes . Among many novel genes, the screen identified known genes, including hedgehog and decapentaplegic, implicated in these processes . A genetic interaction screen for suppressors of a cell migration defect in a hypomorphic slow border cells mutant identified known genes with likely roles in tyrosine kinase signaling and control of actin cytoskeleton, among many novel genes . These studies demonstrate the ability of the modular misexpression system to identify developmentally important genes and suggest that it will be generally useful for genetic interaction screens.

Nature, 1998 Apr 23, 392(6678), 835 - 9
Structure of the calcium pump from sarcoplasmic reticulum at 8-A resolution; Zhang P et al.; The calcium pump from sarcoplasmic reticulum (Ca2+-ATPase) is typical of the large family of P-type cation pumps . These couple ATP hydrolysis with cation transport, generating cation gradients across membranes . Ca2+-ATPase specifically maintains the low cytoplasmic calcium concentration of resting muscle by pumping calcium into the sarcoplasmic reticulum; subsequent release is used to initiate contraction . No high-resolution structure of a P-type pump has yet been determined, although a 14-A structure of Ca2+-ATPase, obtained by electron microscopy of frozen-hydrated, tubular crystals, showed a large cytoplasmic head connected to the transmembrane domain by a narrow stalk . We have now improved the resolution to 8A and can discern ten transmembrane alpha-helices, four of which continue into the stalk On the basis of constraints from transmembrane topology, site-directed mutagenesis and disulphide crosslinking, we have made tentative assignments for these alpha-helices within the amino-acid sequence . A distinct cavity leads to the putative calcium-binding site, providing a plausible path for calcium release to the lumen of the sarcoplasmic reticulum.

Nature, 1998 Apr 23, 392(6678), 831 - 5
Transcriptional repression by UME6 involves deacetylation of lysine 5 of histone H4 by RPD3; Rundlett SE et al.; The histone deacetylase RPD3 can be targeted to certain genes through its interaction with DNA-binding regulatory proteins . RPD3 can then repress gene transcription . In the yeast Saccharomyces cerevisiae, association of RPD3 with the transcriptional repressors SIN3 and UME6 results in repression of reporter genes containing the UME6-binding site . RPD3 can deacetylate all histone H4 acetylation sites in cell extracts . However, it is unknown how H4 proteins located at genes near UME6-binding sites are affected, nor whether the effect of RPD3 is localized to the promoter regions . Here we study the mechanism by which RPD3 represses gene activity by examining the acetylation state of histone proteins at UME6-regulated genes . We used antibodies specific for individual acetylation sites in H4 to immunoprecipitate chromatin fragments . A deletion of RPD3 or SIN3, but not of the related histone-deacetylase gene HDA1, results in increased acetylation of the lysine 5 residue of H4 in the promoters of the UME6-regulated INO1, IME2 and SPO13 genes . As increased acetylation of this residue is not merely a consequence of gene transcription, acetylation of this site may be essential for regulating gene activity.

RNA, 1998 Feb, 4(2), 205 - 14
ATP is a cofactor of the Upf1 protein that modulates its translation termination and RNA binding activities; Weng Y et al.; The nonsense-mediated mRNA decay pathway decreases the abundance of mRNAs that contain premature termination codons and prevents suppression of nonsense alleles . The UPF1 gene in the yeast Saccharomyces cerevisiae was shown to be a trans-acting factor in this decay pathway . The Upf1p demonstrates RNA-dependent ATPase, RNA helicase, and RNA binding activities . The results presented here investigate the binding affinity of the Upf1p for ATP and the consequences of ATP binding on its affinity for RNA . The results demonstrate that the Upf1p binds ATP in the absence of RNA . Consistent with this result, the TR800AA mutant form of the Upf1p still bound ATP, although it does not bind RNA . ATP binding also modulates the affinity of Upf1p for RNA . The RNA binding activity of the DE572AA mutant form of the Upf1p, which lacks ATPase activity, still bound ATP as efficiently as the wild-type Upf1p and destabilized the Upf1p-RNA complex . Similarly, ATPgammaS, a nonhydrolyzable analogue of ATP, interacted with Upf1p and promoted disassociation of the Upf1p-RNA complex . The conserved lysine residue (K436) in the helicase motif Ia in the Upf1p was shown to be critical for ATP binding . Taken together, these findings formally prove that ATP can bind Upf1p in the absence of RNA and that this interaction has consequences on the formation of the Upf1p-RNA complex . Further, the results support the genetic evidence indicating that ATP binding is important for the Upf1p to increase the translation termination efficiency at a nonsense codon . Based on these findings, a model describing how the Upf1p functions in modulating translation and turnover and the potential insights into the mechanism of the Upf1p helicase will be discussed.

Oncogene, 1998 Mar 26, 16(12), 1625 - 31
The EWS/ATF1 fusion protein contains a dispersed activation domain that functions directly; Pan S et al.; Naturally occurring chromosomal fusion of the Ewings Sarcoma Oncogene (EWS) to distinct cellular transcription factors, produces aberrant transcriptional activators that function as dominant oncogenes . In Malignant Melanoma of Soft Parts the N-terminal region of EWS is fused to C-terminal region of the cAMP-inducible transcription factor ATF1 . The EWS/ATF1 fusion protein binds to ATF sites present in cAMP-responsive promoters via the ATF1 bZIP domain and activates transcription constitutively in a manner that is dependent on an activation domain (EAD) present in EWS . To further define the requirements for trans-activation we have performed mutational analysis of EWS/ATF1 in mammalian cells and report several new findings . First, trans-activation by EWS/ATF1 does not require dimerisation with other ATF family members present in mammalian cells . Second, in contrast to the earlier suggestion of an allosteric role, the EAD can act directly . Third, determinants of trans-activation are dispersed throughout the EAD and cooperate synergistically to produce potent trans-activation . We also report that the region of EWS containing the EAD can activate transcription in Yeast . This latter finding might enable a genetic approach to understanding the mechanism of transcriptional activation by EWS and development of high-throughput screens for EWS inhibitors.

Oncogene, 1998 Mar 26, 16(12), 1553 - 60
Human normal peripheral blood B-lymphocytes are deficient in DNA-dependent protein kinase activity due to the expression of a variant form of the Ku86 protein; Muller C et al.; The heterodimeric Ku protein, which comprises a 86 kDa (Ku86) amd a 70 kDa (Ku70) subunits, is an abundant nuclear DNA-binding protein which binds in vitro to DNA termini without sequence specificity . Ku is the DNA-targeting component of the large catalytic sub-unit of the DNA-dependent protein kinase complex (DNA-PK{CS}), that plays a critical role in mammalian double-strand break repair and lymphoid V(D)J recombination . By using electrophoretic mobility shift assays, we demonstrated that in addition to the major Ku x DNA complex usually detected in cell line extracts, a second complex with faster electrophoretic mobility was observed in normal peripheral blood lymphocytes (PBL) extracts . The presence of this faster migrating complex was restricted to B cells among the circulating lymphocyte population . Western blot analysis revealed that B cells express a variant form of the Ku86 protein with an apparent molecular weight of 69 kDa, and not the 86 kDa- full-length protein . Although the heterodimer Ku70/variant-Ku86 binds to DNA-ends, this altered form of the Ku heterodimer has a decreased ability to recruit the catalytic component of the complex, DNA-PK(CS), which contributes to an absence of detectable DNA-PK activity in B cells . These data provide a molecular basis for the increased sensitivity of B cells to ionizing radiation and identify a new mechanism of regulation of DNA-PK activity that operates in vivo.

Cell, 1998 Apr 17, 93(2), 263 - 75
COPII-coated vesicle formation reconstituted with purified coat proteins and chemically defined liposomes; Matsuoka K et al.; COPII vesicle formation requires only three coat assembly subunits: Sar1p, Sec13/31p, and Sec23/24p . PI 4-phosphate or PI 4,5-bisphosphate is required for the binding of these proteins to liposomes . The GTP-bound form of Sar1p recruits Sec23/24p to the liposomes as well as to the ER membranes, and this Sar1p-Sec23/24p complex is required for the binding of Sec13/31p . Ultrastructural analysis shows that the binding of COPII coat proteins to liposomes results in coated patches, coated buds, and coated vesicles of 50-90 nm in diameter . Budding proceeds without rupture of the donor liposome or vesicle product . These observations suggest that the assembly of the COPII coat on the ER occurs by a sequential binding of coat proteins to specific lipids and that this assembly promotes the budding of COPII-coated vesicles.

Indian J Exp Biol, 1997 Dec, 35(12), 1261 - 72
Autoantigen Ku and its role in multiple cellular processes; Ghosh AK; Ku is a DNA binding protein composed of 70 and 80 kDa subunits which was discovered as autoantigen in a patient with scleroderma-polymyositis overlap syndrome . Ku can bind to the end of DNA and also to some internal sequences . Ku-autoantigen acts as a potential transcription factor for several RNA polymerase II genes and RNA polymerase I gene . Ku is also associated with DNA-dependent protein kinase and involved in V(D)J recombination and DNA break repair mechanisms . Ku may be involved in replication, helicase activity and cell signaling . Therefore, Ku-autoantigen is a very important cellular factor which plays important role in the multiple cellular processes.

Mol Cell Biol, 1998 May, 18(5), 2502 - 13
Posttranslational regulation of Ty1 retrotransposition by mitogen-activated protein kinase Fus3; Conte D Jr et al.; Ty1 retrotransposons in Saccharomyces cerevisiae are maintained in a state of transpositional dormancy . We isolated a mutation, rtt100-1, that increases the transposition of genomic Ty1 elements 18- to 56-fold but has little effect on the transposition of related Ty2 elements . rtt100-1 was shown to be a null allele of the FUS3 gene, which encodes a haploid-specific mitogen-activated protein kinase . In fus3 mutants, the levels of Ty1 RNA, protein synthesis, and proteolytic processing were not altered relative to those in FUS3 strains but steady-state levels of TyA, integrase, and reverse transcriptase proteins and Ty1 cDNA were all increased . These findings suggest that Fus3 suppresses Ty1 transposition by destabilizing viruslike particle-associated proteins . The Fus3 kinase is activated through the mating-pheromone response pathway by phosphorylation at basal levels in naive cells and at enhanced levels in pheromone-treated cells . We demonstrate that suppression of Ty1 transposition in naive cells requires basal levels of Fus3 activation . Substitution of conserved amino acids required for activation of Fus3 derepressed Ty1 transposition . Moreover, epistasis analyses revealed that components of the pheromone response pathway that act upstream of Fus3, including Ste4, Ste5, Ste7, and Ste11, are required for the posttranslational suppression of Ty1 transposition by Fus3 . The regulation of Ty1 transposition by Fus3 provides a haploid-specific mechanism through which environmental signals can modulate the levels of retrotransposition.

Mol Cell Biol, 1998 May, 18(5), 2923 - 31
Molecular evolution allows bypass of the requirement for activation loop phosphorylation of the Cdc28 cyclin-dependent kinase; Cross FR et al.; Many protein kinases are regulated by phosphorylation in the activation loop, which is required for enzymatic activity . Glutamic acid can substitute for phosphothreonine in some proteins activated by phosphorylation, but this substitution (T169E) at the site of activation loop phosphorylation in the Saccharomyces cerevisiae cyclin-dependent kinase (Cdk) Cdc28p blocks biological function and protein kinase activity . Using cycles of error-prone DNA amplification followed by selection for successively higher levels of function, we identified mutant versions of Cdc28p-T169E with high biological activity . The enzymatic and biological activity of the mutant Cdc28p was essentially normally regulated by cyclin, and the mutants supported normal cell cycle progression and regulation . Therefore, it is not a requirement for control of the yeast cell cycle that Cdc28p be cyclically phosphorylated and dephosphorylated . These CDC28 mutants allow viability in the absence of Cak1p, the essential kinase that phosphorylates Cdc28p-T169, demonstrating that T169 phosphorylation is the only essential function of Cak1p . Some growth defects remain in suppressed cak1 cdc28 strains carrying the mutant CDC28 genes, consistent with additional nonessential roles for CAK1.

Mol Cell Biol, 1998 May, 18(5), 2697 - 711
Dimerization by translation initiation factor 2 kinase GCN2 is mediated by interactions in the C-terminal ribosome-binding region and the protein kinase domain; Qiu H et al.; The protein kinase GCN2 stimulates translation of the transcriptional activator GCN4 in yeast cells starved for amino acids by phosphorylating translation initiation factor 2 . Several regulatory domains, including a pseudokinase domain, a histidyl-tRNA synthetase (HisRS)-related region, and a C-terminal (C-term) segment required for ribosome association, have been identified in GCN2 . We used the yeast two-hybrid assay, coimmunoprecipitation analysis, and in vitro binding assays to investigate physical interactions between the different functional domains of GCN2 . A segment containing about two thirds of the protein kinase (PK) catalytic domain and another containing the C-term region of GCN2 interacted with themselves in the two-hybrid assay, and both the PK and the C-term domains could be coimmunoprecipitated with wild-type GCN2 from yeast cell extracts . In addition, in vitro-translated PK and C-term segments showed specific binding in vitro to recombinant glutathione S-transferase (GST)-PK and GST-C-term fusion proteins, respectively . Wild-type GCN2 could be coimmunoprecipitated with a full-length LexA-GCN2 fusion protein from cell extracts, providing direct evidence for dimerization by full-length GCN2 molecules . Deleting the C-term or PK segments abolished or reduced, respectively, the yield of GCN2-LexA-GCN2 complexes . These results provide in vivo and in vitro evidence that GCN2 dimerizes through self-interactions involving the C-term and PK domains . The PK domain showed pairwise in vitro binding interactions with the pseudokinase, HisRS, and C-term domains; additionally, the HisRS domain interacted with the C-term region . We propose that physical interactions between the PK domain and its flanking regulatory regions and dimerization through the PK and C-term domains both play important roles in restricting GCN2 kinase activity to amino acid-starved cells.

Mol Cell Biol, 1998 May, 18(5), 2688 - 96
Ski6p is a homolog of RNA-processing enzymes that affects translation of non-poly(A) mRNAs and 60S ribosomal subunit biogenesis; Benard L et al.; We mapped and cloned SKI6 of Saccharomyces cerevisiae, a gene that represses the copy number of the L-A double-stranded RNA virus, and found that it encodes an essential 246-residue protein with homology to a tRNA-processing enzyme, RNase PH . The ski6-2 mutant expressed electroporated non-poly(A) luciferase mRNAs 8- to 10-fold better than did the isogenic wild type . No effect of ski6-2 on expression of uncapped or normal mRNAs was found . Kinetics of luciferase synthesis and direct measurement of radiolabeled electroporated mRNA indicate that the primary effect of Ski6p was on efficiency of translation rather than on mRNA stability . Both ski6 and ski2 mutants show hypersensitivity to hygromycin, suggesting functional alteration of the translation apparatus . The ski6-2 mutant has normal amounts of 40S and 60S ribosomal subunits but accumulates a 38S particle containing 5'-truncated 25S rRNA but no 5.8S rRNA, apparently an incomplete or degraded 60S subunit . This suggests an abnormality in 60S subunit assembly . The ski6-2 mutation suppresses the poor expression of the poly(A)- viral mRNA in a strain deficient in the 60S ribosomal protein L4 . Thus, a ski6 mutation bypasses the requirement of the poly(A) tail for translation, allowing better translation of non-poly(A) mRNA, including the L-A virus mRNA which lacks poly(A) . We speculate that the derepressed translation of non-poly(A) mRNAs is due to abnormal (but full-size) 60S subunits.

Mol Cell Biol, 1998 May, 18(5), 2629 - 39
Cooperative Pho2-Pho4 interactions at the PHO5 promoter are critical for binding of Pho4 to UASp1 and for efficient transactivation by Pho4 at UASp2; Barbaric S et al.; The activation of the PHO5 gene in Saccharomyces cerevisiae in response to phosphate starvation critically depends on two transcriptional activators, the basic helix-loop-helix protein Pho4 and the homeodomain protein Pho2 . Pho4 acts through two essential binding sites corresponding to the regulatory elements UASp1 and UASp2 . Mutation of either of them results in a 10-fold decrease in promoter activity, and mutation of both sites renders the promoter totally uninducible . The role of Pho4 appears relatively straightforward, but the mechanism of action of Pho2 had remained elusive . By in vitro footprinting, we have recently mapped multiple Pho2 binding sites adjacent to the Pho4 sites, and by mutating them individually or in combination, we now show that each of them contributes to PHO5 promoter activity . Their function is not only to recruit Pho2 to the promoter but to allow cooperative binding of Pho4 together with Pho2 . Cooperativity requires DNA binding of Pho2 to its target sites and Pho2-Pho4 interactions . A Pho4 derivative lacking the Pho2 interaction domain is unable to activate the promoter, but testing of UASp1 and UASp2 individually in a minimal CYC1 promoter reveals a striking difference between the two UAS elements . UASp1 is fully inactive, presumably because the Pho4 derivative is not recruited to its binding site . In contrast, UASp2 activates strongly in a Pho2-independent manner . From in vivo footprinting experiments and activity measurements with a promoter variant containing two UASp2 elements, we conclude that at UASp2, Pho2 is mainly required for the ability of Pho4 to transactivate.

Mol Cell Biol, 1998 May, 18(5), 2492 - 501
Regulation of cell size by glucose is exerted via repression of the CLN1 promoter; Flick K et al.; Yeast cells are keenly sensitive to the availability and quality of nutrients . Addition of glucose to cells growing on a poorer carbon source elicits a cell cycle delay during G1 phase and a concomitant increase in the cell size . The signal is transduced through the RAS-cyclic AMP pathway . Using synchronized populations of G1 cells, we show that the increase in cell size required for budding depends upon CLN1 but not other G1 cyclins . This delay in cell cycle initiation is associated specifically with transcriptional repression of CLN1 . CLN2 is not repressed . Repression of CLN1 is not limited to the first cycle following glucose addition but occurs in each cell cycle during growth on glucose . A 106-bp fragment of the CLN1 promoter containing the three MluI cell cycle box (MCB) core elements responsible for the majority of CLN1-associated upstream activation sequence activity is sufficient to confer glucose-induced repression on a heterologous reporter . A mutant CLN2 promoter that is rendered dependent upon its three MCB core elements due to inactivation of its Swi4-dependent cell cycle box (SCB) elements is also repressed by glucose . The response to glucose is partially suppressed by inactivation of SWI4, but not MBP1, which is consistent with the dependence of MCB core elements upon the SCB-binding transcription factor (SBF) . We suggest that differential regulation of CLN1 and CLN2 by glucose results from differences in the capacity of SBF to activate transcription driven by SCB and MCB core elements . Finally, we show that transcriptional repression is sufficient to explain the cell cycle delay that occurs in response to glucose.

J Mol Biol, 1998 Mar 13, 276(5), 887 - 902
The activation specificities of wild-type and mutant Gcn4p in vivo can be different from the DNA binding specificities of the corresponding bZip peptides in vitro; Suckow M et al.; Single amino acid substitutions which previously have been shown to alter the DNA binding specificity of a Gcn4p bZip peptide in vitro were transformed to full length Gcn4p, and activation of a test promoter carrying various palindromic and pseudo-palindromic binding sites was measured . All mutations were found to have different phenotypes, and the first change-of-specificity mutants for Gcn4p in vivo are described . The comparison of plasmids encoding no protein or a particular Gcn4p mutant with broadened activation specificity in gcn4 and gcn4 acr1 genetic backgrounds revealed three new DNA targets of the yeast Acr1p repressor . Surprisingly, we found the activation specificities Gcn4p and the mutants tested in vivo to be generally different from DNA binding specificities of the corresponding bZip peptides in vitro . Especially, the proteins respond differently, in vitro and in vivo, on changes in half site spacing of the DNA binding sites . We present data which largely exclude that the differences between in vivo and in vitro-derived results are due to differences in protein structure, or to the presence of competing protein factors in the yeast cell . We conclude that the differences between in vitro and in vivo-derived results are caused by differences in the degree of flexibility of the target DNA sequences in vitro and in vivo.

Somat Cell Mol Genet, 1997 Jul, 23(4), 237 - 47
Rare microsatellite polymorphisms in the DNA repair genes XRCC1, XRCC3 and XRCC5 associated with cancer in patients of varying radiosensitivity; Price EA et al.; DNA repair defects might contribute both to cancer progression and to the extreme reactions to radiotherapy observed in approximately 5% of patients . Polymorphic microsatellites in three DNA repair genes, XRCC1, XRCC3 and XRCC5, were analyzed for possible linkage to cancer status or clinical radiosensitivity . XRCC1, 3 and 5 proteins are involved in single-strand DNA break rejoining, recombinational repair, and double-strand DNA break rejoining respectively . Mendelianly inherited microsatellite polymorphisms in these genes were analyzed in three groups: volunteers with no cancer history; radiosensitive cancer patients; cancer patients with acceptable reactions to radiotherapy . Rare heterozygous alterations in all three gene regions were found solely in the cancer subpopulation . Association testing between these rare polymorphisms and cancer status revealed a significant association for XRCC1 (P = 0.005), and XRCC3 (P = 0.004) . There was also an association between these polymorphisms and clinical radiosensitivity for XRCC1 (P = 0.03), and XRCC3 (P = 0.005).

Science, 1998 Apr 10, 280(5361), 284 - 6
Conservation of substrate recognition mechanisms by tRNA splicing endonucleases; Fabbri S et al.; Accuracy in transfer RNA (tRNA) splicing is essential for the formation of functional tRNAs, and hence for gene expression, in both Eukaryotes and Archaea . The specificity for recognition of the tRNA precursor (pre-tRNA) resides in the endonuclease, which removes the intron by making two independent endonucleolytic cleavages . Although the eukaryal and archaeal enzymes appear to use different features of pre-tRNAs to determine the sites of cleavage, analysis of hybrid pre-tRNA substrates containing eukaryal and archaeal sequences, described here, reveals that the eukaryal enzyme retains the ability to use the archaeal recognition signals . This result indicates that there may be a common ancestral mechanism for recognition of pre-tRNA by proteins.

Science, 1998 Apr 10, 280(5361), 279 - 84
Crystal structure and evolution of a transfer RNA splicing enzyme; Li H et al.; The splicing of transfer RNA precursors is similar in Eucarya and Archaea . In both kingdoms an endonuclease recognizes the splice sites and releases the intron, but the mechanism of splice site recognition is different in each kingdom . The crystal structure of the endonuclease from the archaeon Methanococcus jannaschii was determined to a resolution of 2.3 angstroms . The structure indicates that the cleavage reaction is similar to that of ribonuclease A and the arrangement of the active sites is conserved between the archaeal and eucaryal enzymes . These results suggest an evolutionary pathway for splice site recognition.

EMBO J, 1998 Mar 16, 17(6), 1819 - 28
Components of the Ku-dependent non-homologous end-joining pathway are involved in telomeric length maintenance and telomeric silencing; Boulton SJ et al.; In the budding yeast, Saccharomyces cerevisiae, genes in close proximity to telomeres are subject to transcriptional silencing through the process of telomere position effect (TPE) . Here, we show that the protein Ku, previously implicated in DNA double-strand break (DSB) repair and in telomeric length maintenance, is also essential for telomeric silencing . Furthermore, using an in vivo plasmid rejoining assay, we demonstrate that SIR2, SIR3 and SIR4, three genes shown previously to function in TPE, are essential for Ku-dependent DSB repair . As is the case for Ku-deficient strains, residual repair operating in the absence of the SIR gene products ensues through an error-prone DNA repair pathway that results in terminal deletions . To identify novel components of the Ku-associated DSB repair pathway, we have tested several other candidate genes for their involvement in DNA DSB repair, telomeric maintenance and TPE . We show that TEL1, a gene required for telomeric length maintenance, is not required for either DNA DSB repair or TPE . However, RAD50, MRE11 and XRS2 function both in Ku-dependent DNA DSB repair and in telomeric length maintenance, although they have no major effects on TPE . These data provide important insights into DNA DSB repair and the linkage of this process to telomere length homeostasis and transcriptional silencing.

EMBO J, 1998 Mar 16, 17(6), 1569 - 76
Biogenesis of Tim23 and Tim17, integral components of the TIM machinery for matrix-targeted preproteins; Kaldi K et al.; We analysed the import pathway of Tim23 and of Tim17, components of the mitochondrial import machinery for matrix-targeted preproteins . Tim23 contains two independent import signals . One is located within the first 62 amino acid residues of the hydrophilic domain that, in the assembled protein, is exposed to the intermembrane space . This signal mediates translocation of Tim23 across the outer membrane independently of the membrane potential, DeltaPsi . A second import signal is located in the C-terminal membrane-integrated portion of Tim23 . It mediates translocation across the outer membrane and insertion into the inner membrane in a strictly DeltaPsi-dependent fashion . Structurally, Tim17 is related to Tim23 but lacks a hydrophilic domain . It contains an import signal in the C-terminal half and its import requires DeltaPsi . The DeltaPsi-dependent import signals of Tim23 and Tim17 are located at corresponding sites in these two homologous proteins . They exhibit features reminiscent of the positively charged N-terminal presequences of matrix-targeted precursors . Import of Tim23 and its insertion into the inner membrane requires Tim22 but not functional Tim23 . Thus, biogenesis of the Tim23.17 complex depends on the Tim22 complex, which is the translocase identified as mediating the import of carrier proteins.

Biol Chem, 1998 Mar, 379(3), 301 - 9
Directionality of polypeptide transfer in the mitochondrial pathway of chaperone-mediated protein folding; Heyrovska N et al.; Protein folding in mitochondria depends on the functional cooperation of the Hsp70 and Hsp60 chaperone systems, at least for a subset of mitochondrial polypeptides . As suggested previously, Hsp70 and Hsp60 act sequentially . However, recent proposals that the chaperonin Hsp60 functions by releasing substrate protein in an unfolded state would predict a lateral partitioning of folding intermediates between chaperone systems . Firefly luciferase, carrying a mitochondrial targeting signal, was used as a model protein to analyze the degree of coupling and the directionality of substrate transfer between the Hsp70 and Hsp60 chaperones . In vitro, Hsp60 binds unfolded luciferase with high affinity but is unable to promote its folding, whereas the Hsp70 system assists the folding of luciferase efficiently . Upon import into yeast mitochondria, luciferase interacted first with Hsp70 . Surprisingly, most of the protein subsequently accumulated in a complex with Hsp60 and never reached the native state . Import into mitochondria that lack a functional Hsp60 did not result in increased folding, but in the aggregation of luciferase . Thus, in intact organelles the two chaperone systems do not function independently in de novo folding of aggregation-sensitive proteins but rather act in an ordered pathway with substrate transfer predominantly in the direction from Hsp70 to Hsp60.

Oncol Res, 1997, 9(11-12), 623 - 7
TNF-alpha induction of A1 expression in human cancer cells; Pang XP et al.; A1, a member of the Bcl-2 gene family, was originally identified as a hemopoietic-specific early response gene . Later it was found that A1 was overexpressed in human stomach cancer tissues and was induced by tumor necrosis factor-alpha (TNF-alpha) in human vascular endothelial cells . However, its expression in human cancer cells has not been well characterized . In the present study, we examined the expression of A1, as well as the antioxidant manganous superoxide dismutase (MnSOD), in four human thyroid carcinoma cell lines, two human pancreatic carcinoma cell lines, and two human prostate carcinoma cell lines . A1 mRNA was expressed in all four thyroid carcinoma cell lines . TNF-alpha induced A1 in a time- and dose-dependent manner . In contrast, A1 mRNA was not detectable in the pancreatic and prostate carcinoma cell lines in the presence or absence of TNF-alpha . However, TNF-alpha induced manganous superoxide dismutase (MnSOD) mRNA in all the cell lines tested . Furthermore, an agonist antibody to the p55 TNF-alpha receptor induced A1, but the agonist antibody against p75 TNF-alpha receptor did not have this effect . The results indicate that A1 is expressed in human thyroid carcinoma cells and TNF-alpha induces A1 through the p55 TNF-alpha receptor-mediated pathway.

Ann Med, 1997 Dec, 29(6), 483 - 91
Mitochondrial deafness; Jacobs HT; Hearing impairment is a common disorder, largely genetic in origin, and showing classical features of a heterogeneous genetic disease . Up to 100 independently acting nuclear genes are involved in the disorder, of which around 30 have been mapped, but only a handful identified . Mutations in mitochondrial DNA also play a significant role in both syndromic and nonsyndromic sensorineural hearing impairment . Environmental agents such as aminoglycoside antibiotics and as yet unidentified nuclear genes interact with mitochondrial mutations in the expression of auditory phenotypes . The spectrum of different mitochondrial mutations associated with hearing impairment, taken together with mechanistic studies at the molecular level, suggests that the pathogenic process involves the accumulation of abnormal translation products inside mitochondria, in sensitive cells of the auditory system . This leads to a prediction of the involvement of a novel class of nuclear genes in hearing impairment, namely those with roles in 'mitochondrial protein quality control'.

Inflammation, 1998 Apr, 22(2), 145 - 59
Differential production of chemokines by phagocytosing rat neutrophils and macrophages; al-Mokdad M et al.; In this study, rat neutrophils and macrophages produced cytokine-induced neutrophil chemoattractants (CINCs) and rat macrophage inflammatory protein-1 alpha (MIP-1 alpha) in different patterns during phagocytosis of heat-killed yeast cells in vitro . The cultured supernatants of the phagocytosing rat neutrophils and macrophages had chemotactic activities toward neutrophils, and the chemotactic potencies were markedly inhibited by anti-CINCs IgGs or/and anti-MIP-1 alpha IgG, suggesting that CINCs and MIP-1 alpha are major neutrophil chemoattractants produced by the phagocytosing neutrophils and macrophages . Dexamethasone suppressed the production of CINCs and MIP-1 alpha by the phagocytosing cells in a dose-dependent manner . Our results demonstrate significant differences in the production of CINCs and MIP-1 alpha by neutrophils and macrophages during phagocytosis of yeast cells and thus may suggest the different contribution of each chemokine to neutrophil recruitment in the processes of inflammation in rats.

Genetics, 1998 Apr, 148(4), 1813 - 20
The fluffy gene of Neurospora crassa encodes a Gal4p-type C6 zinc cluster protein required for conidial development; Bailey LA et al.; Neurospora crassa fluffy (fl) mutants are unable to produce macroconidia . We cloned the fl gene to determine its role in regulating conidiation . A cosmid clone containing fl was identified by complementation . The sequence of fl revealed that it encodes a Gal4p-type C6 zinc cluster protein with greatest similarity to the N . crassa NIT4 protein that regulates genes required for nitrate utilization . Analysis of several fl mutant alleles demonstrated that null mutants are blocked in the budding phase of development required to produce conidiophores . fl mRNA is transiently induced just prior to the developmental commitment to budding growth . This timing of fl expression is consistent with a role for FL protein in activation of the previously characterized conidiation-specific (con) genes, con-6 and con-10 . These data suggest that FL acts as a developmentally regulated transcription factor required for conidiophore morphogenesis.

Genetics, 1998 Apr, 148(4), 1743 - 61
Posttranslational inhibition of Ty1 retrotransposition by nucleotide excision repair/transcription factor TFIIH subunits Ssl2p and Rad3p; Lee BS et al.; rtt4-1 (regulator of Ty transposition) is a cellular mutation that permits a high level of spontaneous Ty1 retrotransposition in Saccharomyces cerevisiae . The RTT4 gene is allelic with SSL2 (RAD25), which encodes a DNA helicase present in basal transcription (TFIIH) and nucleotide excision repair (NER) complexes . The ssl2-rtt (rtt4-1) mutation stimulates Ty1 retrotransposition, but does not alter Ty1 target site preferences, or increase cDNA or mitotic recombination . In addition to ssl2-rtt, the ssl2-dead and SSL2-1 mutations stimulate Ty1 transposition without altering the level of Ty1 RNA or proteins . However, the level of Ty1 cDNA markedly increases in the ssl2 mutants . Like SSL2, certain mutations in another NER/TFIIH DNA helicase encoded by RAD3 stimulate Ty1 transposition . Although Ssl2p and Rad3p are required for NER, inhibition of Ty1 transposition is independent of Ssl2p and Rad3p NER functions . Our work suggests that NER/TFIIH subunits antagonize Ty1 transposition posttranslationally by inhibiting reverse transcription or destabilizing Ty1 cDNA.

Curr Opin Lipidol, 1998 Apr, 9(2), 119 - 23
Acyl CoA:cholesterol acyltransferase genes and knockout mice; Farese RV Jr; Acyl coenzyme A:cholesterol acyltransferase (ACAT) (EC 2.3.1.26) is an enzyme, located in the endoplasmic reticulum of many types of cells, that catalyzes cholesterol ester formation from cholesterol and fatty acyl CoA substrates . Sterol esterification by ACAT or homologous enzymes is conserved in evolution dating back to yeast . The recent cloning of a human cDNA for ACAT, together with genome sequencing projects, has led to the identification of an ACAT gene family and provided molecular tools for determining ACAT's functions in vivo . Summarized here is the current knowledge concerning the molecular genetics of ACAT.

FEBS Lett, 1998 Mar 27, 425(2), 305 - 9
cDNA cloning and mRNA distribution of a mouse very long-chain acyl-CoA synthetase; Berger J et al.; The interaction of the adrenoleukodystrophy protein (ALDP), mutated in the peroxisomal disorder X-linked adrenoleukodystrophy, and the very long-chain acyl-CoA synthetase (VLACS), the enzyme whose function is missing in this disease, remains obscure . As a first step to studying this interaction in wild type versus ALDP-deficient mice, we have cloned a VLACS cDNA from mouse liver . The 1860 bp open reading frame encodes a 620 amino acid protein with a predicted molecular mass of 70.3 kDa . By Northern blot analysis, a 2.6 kbp VLACS mRNA was highly abundant in liver and kidney and present at low levels in brain and testes . By RT-PCR VLACS mRNA was also detected in heart and lung but remained undetectable in skeletal muscle and spleen . In contrast to the peroxisomal beta-oxidation marker acyl-CoA oxidase, whose mRNA level steadily increases during brain development, the VLACS transcript was found at a constant low level from embryo through adulthood, suggesting that additional isoforms may exist in brain.

Plant Cell Physiol, 1998 Feb, 39(2), 247 - 53
Characterization of delta 9 acyl-lipid desaturase homologues from Arabidopsis thaliana; Fukuchi-Mizutani M et al.; Two cDNAs, ADS1 and ADS2, were isolated from Arabidopsis . These cDNAs encoded proteins homologous to delta 9 acyl-lipid desaturases of cyanobacteria and acyl-CoA desaturases of yeast and mammals . The expression of ADS1 and ADS2 was organ-dependent . Cold temperature up-regulated the ADS2 expression, whereas it down-regulated the ADS1 expression.

Curr Opin Lipidol, 1998 Apr, 9(2), 99 - 102
CaaX converting enzymes; Ashby MN; Proteins that contain a carboxyl-terminal CaaX motif undergo post-translational processing involving prenylation, endoproteolysis and methylesterification . Two yeast genes, AFC1 and RCE1, which are candidates for genes encoding CaaX converting enzymes, were recently identified . Rce1p is required for the full penetrance of the activated Ras2pval19 phenotype in yeast, indicating its possible utility as a new target in Ras-based malignancies . Advances in our current understanding of CaaX convertases and the functional importance of CaaX proteolysis are discussed.

J Virol, 1998 May, 72(5), 4149 - 56
Identification of a novel cellular TPR-containing protein, SGT, that interacts with the nonstructural protein NS1 of parvovirus H-1; Cziepluch C et al.; The nonstructural protein NS1 of autonomous parvoviruses is essential for viral DNA amplification and gene expression and is also the major cytopathic effector of these viruses . NS1 acts as nickase, helicase, and ATPase and upregulates P38-driven transcription of the capsid genes . We report here the identification of a novel cellular protein that interacts with NS1 from parvovirus H-1 and which we termed SGT, for small glutamine-rich tetratricopeptide repeat (TPR)-containing protein . The cDNA encoding full-length SGT was isolated through a two-hybrid screen with, as bait, the truncated NS1dlC69 polypeptide, which lacks the C-terminal transactivation domain of NS1 . Full-length NS1 and SGT interacted in the two-hybrid system and in an in vitro interaction assay . Northern blot analysis revealed one major transcript of about 2 kb that was present in all rat tissues investigated . Rat sgt cDNA coded for 314 amino acids, and the protein migrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular mass of 34 kDa . SGT could be detected in both the nucleus and the cytoplasm of rat cells, as determined by indirect immunofluorescence analysis and Western blotting of fractionated cellular extracts with an affinity-purified antiserum raised against recombinant SGT (AC1.1) . In H-1 virus-infected rat and human cells, compared to mock-infected controls, differences in the migration of SGT polypeptides were revealed after Western blot analysis of total cellular extracts . Moreover, the transient expression of NS proteins was sufficient to induce SGT modification . These results show that cellular SGT, which we have identified as an NS1-interacting protein, is modified by parvovirus infection as well as NS expression.

J Virol, 1998 May, 72(5), 3560 - 70
Identification of regions of poliovirus 2BC protein that are involved in cytotoxicity; Barco A et al.; The expression of poliovirus 2BC protein in yeast and mammalian cells leads to a number of metabolic and morphological alterations, such as growth inhibition, intracellular membrane proliferation, blockade of the exocytic pathway, and enhanced membrane permeability . Yeast cells that express poliovirus 2BC in an inducible manner were used to identify the regions of 2BC implicated in the modifications of these cellular functions . Several 2BC deletion mutants were generated to define the minimal portion of 2BC required to alter these activities . Additional deletion mutants that were obtained by random mutagenesis followed by selection in yeast cells provided new insights into the structure and mechanism of action of 2BC . The activity responsible for membrane proliferation is located in 2C, while the activities responsible for membrane permeabilization and inhibition of the exocytic pathway are located in 2B . Several regions of 2B and 2C required for the different functions of 2BC were identified . Thus, the integrity of the N termini of both 2B and 2C is necessary for 2BC-induced cytotoxicity . It is also possible to separate the different cellular alterations provoked by 2BC by the use of several 2BC variants . Deletion of amino acids 52 to 65 in 2B generates a 2BC deletion variant, 2bC deltaAvrII, that still blocks yeast growth but is unable to enhance membrane permeability or to inhibit the exocytic pathway . On the other hand, 2Bcl28*.32b and 2Bcl28*.3c, which contain only 73 and 77 amino acids of 2B, interfere with yeast division and enhance membrane permeability but affect the exocytic pathway only weakly and do not induce membrane proliferation . Our findings indicate that Saccharomyces cerevisiae represents a useful model system to analyze the functions of poliovirus 2BC and show the feasibility of separating the activities assigned to this protein.

Prog Cell Cycle Res, 1997, 3, 99 - 108
The regulation of cyclin-dependent kinase inhibitors (CKIs); Peter M; Inhibitors of cyclin-dependent kinases (CKIs) play key roles in coordinating cell proliferation and development . They also function to control critical cell cycle transitions and as effectors of checkpoint pathways . The activity of CKIs is tightly controlled through the cell cycle and in response to various signals . Regulation generally affects the levels or availability of the CKIs rather than changing their intrinsic activities . Mechanisms controlling CKI function include the regulation of transcription, translation and proteolysis . In addition some signals appear to induce sequestration of CKIs within the cells, thereby changing their ability to interact with specific targets.

Prog Cell Cycle Res, 1996, 2, 107 - 14
The family of polo-like kinases; Golsteyn RM et al.; Here we discuss members of a new family of serine/threonine protein kinases with a likely role in cell cycle control . These kinases are referred to as polo-like kinases, after the prototypic founding member of the family, the polo gene product of Drosophila melanogaster . The polo kinase was originally identified in mutants that display abnormal mitotic spindle organization . Subsequently, potential homologues of Drosophila polo have been identified in yeasts (Cdc5p in Saccharomyces cerevisiae; plo1+ in Schizosaccharmoyces pombe) and in mammals (polo-like kinase 1; Plk1) . Genetic and biochemical studies suggest that polo, Cdc5p and plo1+ may be required for mitotic spindle organization and, possibly, for cytokinesis . Likewise, the patterns of expression, activity and subcellular localization of Plk1 strongly suggest that this mammalian kinase functions also during mitosis, possibly in spindle assembly and function . In addition to Plk1, however, more distantly related members of the polo-like kinase family have been identified in mammalian cells, and the available data are consistent with the idea that some of these may act earlier in the cell cycle, possibly during G1 . If this hypothesis is correct, different members of the polo-like kinase family would act at several points during the cell cycle, reminiscent of the behaviour of Cdk/cyclin complexes.

Prog Cell Cycle Res, 1996, 2, 83 - 90
DNA replication licensing factor; Chong JP et al.; DNA Replication Licensing Factor (RLF) is an essential activity required to restrict the duplication of genomic DNA to precisely once per cell cycle . Recent fractionation of RLF activity from Xenopus egg extracts has resulted in the identification of two essential components, RLF-B and RLF-M . RLF-M has been purified to homogeneity and has been shown to consist of a complex of proteins in the MCM/P1 family . RLF-B is still unidentified, but possible candidates for this activity have been identified in yeast . Elucidation of the RLF mechanism will provide important insights into the way that chromosome replication is controlled.

Prog Cell Cycle Res, 1995, 1, 229 - 40
The regulation and functions of cdk7; Shuttleworth J; cdk7 started its life rather anonymously as a kinase called MO15, identified during a search for cDNA's which encode protein kinases related to cdc2 . For several years its function remained obscure, but during the last 18 months MO15 has revealed itself as the catalytic subunit of cdk activating kinase, associating with at least two other subunits including a new cyclin, cyclin H . MO15(cdk7) has therefore been established paradoxically as both a new member and a regulator of the cyclin dependent kinase family . New evidence now suggests that cdk7 is also involved in the processes of transcription initiation and DNA repair, associating with the general transcription factor TFIIH . The engima of cdk7 is likely to remain for a while yet, and perhaps even more surprises are in store.

Prog Cell Cycle Res, 1995, 1, 173 - 85
The Cdc28 inhibitor p40SIC1; Mendenhall MD et al.; Sic1 inhibits the activity of Cdc28.Clb5 complexes in late G1, creating a delay between cell cycle commitment and S phase initiation . The ultimate purpose of this delay is unknown but loss of Sic1 activity negatively affects genomic stability and cellular viability . Sic1 levels are controlled by periodic changes in transcription rates and protein stability . The latter control is mediated through the Cdc34 ubiquitin transferase and, possibly, Cdc28.Cln activity . Possible roles of Sic1 in the G1/S and the M/G1 transitions are discussed.

Prog Cell Cycle Res, 1995, 1, 149 - 62
Regulation of cell cycle progression following DNA damage; Hensey C et al.; DNA damage causes an arrest in cell cycle progression . Checkpoints, which monitor the state of the DNA, exist throughout the cycle and negatively regulate cell cycle transitions when damage is detected . The molecular basis of how these checkpoints are activated, and interact with the cell cycle machinery, is just beginning to be understood . Studies in yeast have identified a number of genes involved in a G2 DNA damage checkpoint, while in mammalian cells a G1 checkpoint has been extensively studied.

Prog Cell Cycle Res, 1995, 1, 53 - 71
Mechanism of action of rapamycin: new insights into the regulation of G1-phase progression in eukaryotic cells; Wiederrecht GJ et al.; The immunosuppressant drug, rapamycin (RAP), is a potent inhibitor of IL-2-dependent T-cell proliferation . The antiproliferative effect of RAP is mediated through the formation of an active complex with its cytosolic receptor protein, FKBP12 . The molecular target of the FKBP12.RAP complex is a putative lipid kinase termed the mammalian Target Of Rapamycin (mTOR) . This review will discuss recent findings suggesting that mTOR is a novel regulator of G1- to S-phase progression in eukaryotic cells.

Prog Cell Cycle Res, 1995, 1, 33 - 52
MAP kinase-dependent pathways in cell cycle control; Pelech SL et al.; Mitogen-activated protein kinases such as Erk1 and Erk2 serve as a paradigm for a growing family of proline-directed protein kinases that mediate entry, progression and exit from the cell cycle in diverse eukaryotic cells . These enzymes function within highly conserved modules of sequentially activating protein kinases that transduce signals from diverse extracellular stimuli . In vertebrates, at least three distinct kinases modules have been characterized . Mitogens induce the sequential activation of the kinases Raf1-->Mek1-->Erk2-->Rsk via the G-protein Ras . Stress factors stimulate c-Jun activation through a related kinase pathway involving Mekk-->Sek-->SAPK c-Jun, and hsp27 phosphorylation via the MKK3-->Hog-->MAPKAPK-2 hsp27 route . Genetic and biochemical studies, for example from budding yeast, imply the existence of several related protein kinase modules that can operate in parallel or within integrated systems.

Prog Cell Cycle Res, 1995, 1, 9 - 19
The role of RB in cell cycle control; Hatakeyama M et al.; The retinoblastoma protein is an inhibitor of cell cycle progression from the G1 to the S phase of the cell cycle . It acts through its ability to interact with cellular target molecules such as E2F transcription factors . The function of pRB is negatively regulated by a cell-cycle dependent phosphorylation catalyzed by cyclin-dependent kinases in the late G1 cell cycle phase . Recent evidence indicates that this pRB inactivation is a key molecular event leading to the S-phase commitment at the G1 restriction point in the cell cycle . Deregulated inactivation of pRB in G1 phase may be a universal mechanism underlying cellular transformation.

Prog Cell Cycle Res, 1995, 1, 1 - 8
Checkpoints in the cell cycle from a modeler's perspective; Tyson JJ et al.; The cell division cycle is a complex process by which cells grow and divide into two viable daughter cells . So that mistakes are not made in this crucial replication process, cells stop at one or more "checkpoints" in the cycle to query their internal state and external conditions, before proceeding to the next stage of the cycle . In this paper we study some simple mathematical models of cell cycle arrest in G1 ("Start") and G2 . Our models help to relate the molecular mechanisms of these checkpoints with physiological properties of the cell cycle.

J Immunol, 1998 Jan 1, 160(1), 107 - 11
Long-lived B cells are distinguished by elevated expression of A1; Tomayko MM et al.; Only 5% of the 15 million B cells formed daily reach the long-lived peripheral B cell pool, presumably reflecting both negative and positive selection . These selective events occur primarily during late stages of differentiation in the marrow and periphery, when newly formed B cells bear surface IgM (sIgM), but differ from mature B cells in their expression of heat-stable Ag (CD24), B220 (CD45), and sIgD . Because genes of the Bcl-2 family influence longevity, we compared the expression of Bcl-2, Bax, and A1 among immature vs mature peripheral B cells using semiquantitative reverse-transcriptase PCR . While the levels of both Bcl-2 and Bax mRNA remain constant in these two populations, A1 expression is strikingly up-regulated among mature B cells . In addition, A1 expression is low among pro- and pre-B cells, as well as in immature (sIgM+) marrow B cells . Together, these data indicate that A1 mRNA expression is low at all stages of B cell development before final maturation in the periphery and, unlike other Bcl-2 family members whose expression changes little after marrow egress, A1 is up-regulated 10-fold as cells are recruited into the long-lived peripheral B cell pool.

Structure, 1998 Mar 15, 6(3), 363 - 76
The structure of SAICAR synthase: an enzyme in the de novo pathway of purine nucleotide biosynthesis; Levdikov VM et al.; BACKGROUND: The biosynthesis of key metabolic components is of major interest to biologists . Studies of de novo purine synthesis are aimed at obtaining a deeper understanding of this central pathway and the development of effective chemotherapeutic agents . Phosphoribosylaminoimidazolesuccinocarboxamide (SAICAR) synthase catalyses the seventh step out of ten in the biosynthesis of purine nucleotides . To date, only one structure of an enzyme involved in purine biosynthesis has been reported: adenylosuccinate synthetase, which catalyses the first committed step in the synthesis of AMP from IMP . RESULTS: We report the first three-dimensional structure of a SAICAR synthase, from Saccharomyces cerevisiae . It is a monomer with three domains . The first two domains consist of antiparallel beta sheets and the third is composed of two alpha helices . There is a long deep cleft made up of residues from all three domains . Comparison of SAICAR synthases by alignment of their sequences reveals a number of conserved residues, mostly located in the cleft . The presence of two sulphate ions bound in the cleft, the structure of SAICAR synthase in complex with ATP and a comparison of this structure with that of other ATP-dependent proteins point to the interdomain cleft as the location of the active site . CONCLUSIONS: The topology of the first domain of SAICAR synthase resembles that of the N-terminal domain of proteins belonging to the cyclic AMP-dependent protein kinase family . The fold of the second domain is similar to that of members of the D-alanine:D-alanine ligase family . Together these enzymes form a new superfamily of mononucleotide-binding domains . There appears to be no other enzyme, however, which is composed of the same combination of three domains, with the individual topologies found in SAICAR synthase.

Biochem Cell Biol, 1997, 75(5), 601 - 12
Domains of retinoid signalling and neurectodermal expression of zebrafish otx1 and goosecoid are mutually exclusive; Joore J et al.; Retinoid signalling plays an important role in embryonic pattern formation . Excess of retinoic acid during gastrulation results in axial defects in vertebrate embryos, suggesting that retinoids are involved in early anteroposterior patterning . To study retinoid signalling in zebrafish embryos, we developed a novel method to detect endogenous retinoids in situ in embryos, using a fusion protein of the ligand inducible transactivation domain of a retinoic acid receptor and a heterologous DNA binding domain . Using this method, we show that retinoid signalling is localized in zebrafish embryos in the region of the embryonic shield, and towards the end of gastrulation in a posterior dorsal domain . To investigate the relationships between the spatial distribution of retinoid signalling and the regulation of retinoid target genes, we studied the downregulation by retinoic acid of two genes expressed in anterior regions of the embryo, goosecoid and otx1 . These experiments show that expression of both genes is strongly downregulated in the anterior neurectoderm of zebrafish embryos treated with retinoic acid, whereas mesendodermal expression is only mildly affected . Interestingly, a significant downregulation of goosecoid expression by retinoic acid was observed only during midgastrulation but not in earlier stages . In agreement with these results, spatial expression of goosecoid and otx1 does not overlap with the region of retinoid signalling in the late gastrula . Our data support the hypothesis that a localized retinoid signal is involved in axial patterning during early development, at least in part through the repression of anterior genes in posterior regions of the embryo . Furthermore, our data suggest that the action of retinoids is spatially as well as temporally regulated in the developing embryo.

Biochim Biophys Acta, 1998 Mar 12, 1402(1), 95 - 101
Phosducin-like protein (PhLP), a regulator of G beta gamma function, interacts with the proteasomal protein SUG1; Barhite S et al.; Phosducin-like protein (PhLP) and phosducin are highly homologous proteins that interact with the beta gamma subunits of guanine nucleotide binding proteins . While phosducin has a well-characterized role in retinal signal transduction, PhLP function remains unclear . To further understand the function of PhLP, we have examined other potential protein:protein interactions with PhLP using the yeast two-hybrid system . PhLP was found to interact with a mouse homologue of the yeast SUG1, a subunit of the 26S proteasome which may also indirectly modulate transcription . This interaction was further confirmed by an in vitro binding assay and co-immunoprecipitation of the two proteins in overexpression studies . Inhibition of proteasome function by lactacystin led to accumulation of high molecular weight, ubiquitin-immunoreactive protein precipitated by PhLP antiserum . We suggest that PhLP/SUG1 interaction may target PhLP for proteasomal degradation.

J Immunol, 1997 Dec 15, 159(12), 6052 - 60
Linkage of LMP, TAP, and RING3 with Mhc class I rather than class II genes in the zebrafish; Takami K et al.; The LMP2 and LMP7 genes code for subunits of the proteasome, a multimeric enzymatic complex that degrades proteins into peptides . The two subunits replace corresponding constitutively expressed subunits during the immune response . Some of the peptides generated by the proteasome in the cytosol are transported by the products of the TAP1 and TAP2 genes into the lumen of the endoplasmic reticulum and are loaded onto the assembling MHC class I molecules . In mammals, the LMP2, LMP7, TAP1, and TAP2 genes reside in the class II region of the Mhc, closely linked to the RING3 gene . In the present study we identified, cloned, and sequenced the LMP, TAP2, and RING3 genes of the zebrafish, Danio rerio . We identified variants of these genes and used them in a segregation analysis of haploid embryos derived from heterozygous mothers . The analysis revealed that in zebrafish, the LMP2, LMP7, TAP12, and RING3 loci are closely linked but, in contrast to mammals, the LMP/TAP/RING3 cluster resides not in the Mhc class II but in the class I region . We also confirmed that in the zebrafish, the class I and class II regions are not linked to each other . In this species, therefore, the LMP/TAP/RING3 genes are clustered with the class I genes on a chromosome that apparently does not contain any class II genes . The linkage of LMP/TAP/RING3/class I may be the original and the LMP/TAP/RING3/class II a derived arrangement of these genes.

Biochemistry, 1998 Mar 31, 37(13), 4510 - 7
Retroviral envelope glycoprotein processing: structural investigation of the cleavage site; Moulard M et al.; Proteolytic activation of retroviral envelope glycoprotein precursors occurs at the carboxyl side of a consensus motif consisting of the amino acid sequence (Arg/Lys)-Xaa-(Arg/Lys)-Arg . Synthetic peptides spanning the processing sites of HIV-1/2 and SIV glycoprotein precursors were examined for their ability to be cleaved by the subtilisin-like endoproteases kexin and furin . To determine the potential role of secondary structure on proteolytic activation, we examined the secondary structure of synthetic peptides by circular dichroism and NMR spectroscopy . The results indicate that (i) the peptides were correctly cleaved by kexin and furin and therefore could be used as specific substrates for the purification and characterization of the lymphocyte endoprotease(s) responsible for proteolytic processing of precursors; (ii) the regions surrounding the cleavage sites could be characterized by their flexibility in aqueous solutions . However, a loop has been shown to be a determinant for the specificity of the interaction between the enzyme and its substrate as determined by molecular modeling . Furthermore, we determine and propose a possible structure of the cleavage site which fits to the active site of the modeled furin.

J Biol Chem, 1998 Mar 27, 273(13), 7776 - 81
Grb2 and its apoptotic isoform Grb3-3 associate with heterogeneous nuclear ribonucleoprotein C, and these interactions are modulated by poly(U) RNA; Romero F et al.; Grb2 is an adaptor molecule comprising one Src homology (SH) 2 and two SH3 domains . This protein has a natural isoform named Grb3-3 with a deletion within the SH2 domain . Numerous evidence points to a functional connection between SH2- and SH3-containing proteins and molecules implicated in RNA biogenesis . In this context, we have examined the binding of Grb2 and Grb3-3 to heterogeneous nuclear ribonucleoprotein (hnRNP) C . By the use of an in vivo genetic approach and through in vitro experiments, we furnish evidence that both Grb2 and Grb3-3 interact with hnRNP C proteins . Subcellular fractionation studies clearly show that Grb2 is partially localized in the nucleus . In addition, coimmunoprecipitation experiments demonstrate that Grb2.hnRNP C complexes exist in intact hematopoietic cells . The carboxyl-terminal SH3 domains of Grb2 and Grb3-3 are primarily responsible for the association with hnRNP C . However, although the proline-rich motif of hnRNP C is involved in the interaction with Grb2, it is not in the binding to Grb3-3 . Furthermore, poly(U) RNA inhibits the association of Grb2 with hnRNP C, whereas it enhances the interaction between Grb3-3 and hnRNP C . These findings suggest that the Grb2/Grb3-3-hnRNP C interactions might fulfill different biological functions.

J Biol Chem, 1998 Mar 27, 273(13), 7210 - 21
Dissection of the transactivation function of the transcription factor encoded by the eye developmental gene PAX6; Tang HK et al.; PAX6 is a transcription activator that regulates eye development in animals ranging from Drosophila to human . The C-terminal region of PAX6 is proline/serine/threonine-rich (PST) and functions as a potent transactivation domain when attached to a heterologous DNA-binding domain of the yeast transcription factor, GAL4 . The PST region comprises 152 amino acids encoded by four exons . The transactivation function of the PST region has not been defined and characterized in detail by in vitro mutagenesis . We dissected the PST domain in two independent systems, a heterologous system using a GAL4 DNA-binding site and the native system of PAX6 . Our data consistently showed that in both systems all four constituent exons of the PST domain are responsible for the transactivation function . The four exon fragments act synergistically to stimulate transcription, although none of them can function individually as an independent transactivation domain . Combinations of two or more exon fragments can reconstitute substantial transactivation activity when fused to the DNA-binding domain of GAL4, but they surprisingly do not produce much activity in the context of native PAX6, although the mutant PAX6 proteins are stable and their DNA-binding function remains unaffected . Our data suggest that these mutants may antagonize the wild-type PAX6 activity by competing for target DNA-binding sites . We conclude that the PAX6 protein contains an unusually large transactivation domain that is evolutionarily conserved to a high degree and that its full transactivation activity relies on the synergistic action of the four exon fragments.

Genes Dev, 1998 Mar 15, 12(6), 797 - 805
Histone deacetylase activity of Rpd3 is important for transcriptional repression in vivo; Kadosh D et al.; Eukaryotic organisms from yeast to human contain a multiprotein complex that includes Rpd3 histone deacetylase and Sin3 corepressor . The Sin3-Rpd3 complex, when recruited to promoters by specific DNA-binding proteins, can direct transcriptional repression of specific classes of target genes . It has been proposed that the histone deacetylase activity of Rpd3 is important for repression, but direct evidence is lacking . Here, we describe four Rpd3 derivatives with mutations in evolutionarily invariant histidine residues in a putative deacetylation motif . These Rpd3 mutants lack detectable histone deacetylase activity in vitro, but interact normally with Sin3 in vivo . In yeast cells, these catalytically inactive mutants are defective for transcriptional repression . They retain some residual Rpd3 function in vivo, however, suggesting that repression by the Sin3-Rpd3 complex may not be attributable exclusively to its intrinsic histone deacetylase activity . Finally, we show that a human Rpd3 homolog can interact with yeast Sin3 and repress transcription when artificially recruited to a promoter . These results suggest that the histone deacetylase activity of Rpd3 is important, but perhaps not absolutely required, for transcriptional repression in vivo.

J Cell Biol, 1998 Mar 23, 140(6), 1347 - 56
Vid24p, a novel protein localized to the fructose-1, 6-bisphosphatase-containing vesicles, regulates targeting of fructose-1,6-bisphosphatase from the vesicles to the vacuole for degradation; Chiang MC et al.; Glucose regulates the degradation of the key gluconeogenic enzyme, fructose-1,6-bisphosphatase (FBPase), in Saccharomyces cerevisiae . FBPase is targeted from the cytosol to a novel type of vesicle, and then to the vacuole for degradation when yeast cells are transferred from medium containing poor carbon sources to fresh glucose . To identify proteins involved in the FBPase degradation pathway, we cloned our first VID (vacuolar import and degradation) gene . The VID24 gene was identified by complementation of the FBPase degradation defect of the vid24-1 mutant . Vid24p is a novel protein of 41 kD and is synthesized in response to glucose . Vid24p is localized to the FBPase-containing vesicles as a peripheral membrane protein . In the absence of functional Vid24p, FBPase accumulates in the vesicles and fails to move to the vacuole, suggesting that Vid24p regulates FBPase targeting from the vesicles to the vacuole . FBPase sequestration into the vesicles is not affected in the vid24-1 mutant, indicating that Vid24p acts after FBPase sequestration into the vesicles has occurred . Vid24p is the first protein identified that marks the FBPase-containing vesicles and plays a critical role in delivering FBPase from the vesicles to the vacuole for degradation.

J Clin Invest, 1998 Mar 15, 101(6), 1379 - 88
The 86-kD subunit of Ku autoantigen mediates homotypic and heterotypic adhesion of multiple myeloma cells; Teoh G et al.; Previous studies have shown that triggering multiple myeloma (MM) cells via CD40 induces IL-6-mediated autocrine growth as well as increased expression of cell surface adhesion molecules including CD11a, CD11b, CD11c, and CD18 . In this study, we generated the 5E2 mAb which targets an antigen that is induced upon CD40 ligand (CD40L) activation of MM cells . Immunofluorescence, immunoprecipitation, and protein sequencing studies identified the target antigen of 5E2 mAb as the 86-kD subunit of the Ku autoantigen . We demonstrate that increased cell surface expression of Ku on CD40L-treated cells is due to migration of Ku from the cytoplasm to the cell surface membrane . Moreover, cell surface Ku on CD40L-treated MM cells mediates homotypic adhesion of tumor cells, as well as heterotypic adhesion of tumor cells to bone marrow stromal cells and to human fibronectin; and 5E2 mAb abrogates IL-6 secretion triggered by tumor cell adherence to bone marrow stromal cells . These data suggest that CD40L treatment induces a shift of Ku from the cytoplasm to the cell surface, and are the first to show that Ku functions as an adhesion molecule . They further suggest that cell surface Ku may play a role in both autocrine and paracrine IL-6-mediated MM cell growth and survival.

Nucleic Acids Res, 1998 Mar 15, 26(6), 1544 - 5
In vivo mapping of nucleosomes using psoralen-DNA crosslinking and primer extension; Wellinger RE et al.; By the use of psoralen crosslinking and primer extension, a method was developed which allows the analysis of chromatin structure in vivo . Using a yeast minichromosome, >9 nucleosomes were mapped with a resolution of at least +/-30 bp.

Nucleic Acids Res, 1998 Mar 15, 26(6), 1401 - 7
Determinants of the position of a Flp-induced DNA bend; Luetke KH et al.; The Flp site-specific recombinase from Saccharomyces cerevisiae induces DNA bending upon interaction with the Flp recognition target (FRT) site . The minimal FRT site is comprised of two inverted binding elements which flank a central core region . Binding of a single monomer of Flp to DNA induces a DNA bend of 60 degrees . The position of this bend differed depending on whether the substrate contained a single binding element or a two-element FRT site . In the present work we tested and disproved a model in which a single Flp monomer interacts with both symmetry elements of a single FRT site . Likewise, we showed that a model in which a Flp monomer dissociates from a singly occupied FRT site and reassociates with the unbound element of another singly occupied FRT site during electrophoresis, does not account for the apparent shift in the position of the bend centre . It seems that the movement of a Flp monomer between the a and b elements of one FRT site during electrophoresis accounts for this anomaly . The position of the DNA bend resulting from the association of a Flp monomer with the FRT site is also influenced by the DNA sequences flanking the site . We conclude that attempts to measure the bend centre of a complex of one Flp molecule bound to a DNA containing two binding elements give misleading results . The position of the bend is more accurately measured in the presence of a single binding element.

Brain Res Mol Brain Res, 1998 Jan, 53(1-2), 33 - 40
Cloning of a gene, YT521, for a novel RNA splicing-related protein induced by hypoxia/reoxygenation; Imai Y et al.; To elucidate the role of astrocytes in the stress response of the central nervous system to ischemia, early gene expression was examined in rat cultured astrocytes after the exposure to hypoxia/reoxygenation, and we have previously cloned a novel RNA binding protein, RA301, from the reoxygenated astrocytes . Furthermore, we have now cloned a new gene for RA301 binding protein, termed YT521, by a yeast two-hybrid screening technique to explore RA301 functions . The YT521 cDNA is about 3200 bp long with an open reading frame encoding 712 amino acids . This amino acid sequence contains arginine-aspartic acid-glutamic acid rich region and glutamic acid rich one, and has a low degree of homology with RNA binding proteins such as U1-70k . Northern blot analysis revealed that YT521 mRNA expression was up-regulated in reoxygenated astrocytes . Induction of YT521 mRNA was mediated by endogenously generated reactive oxygen species, as it was suppressed by treatment of the cells with diphenyl iodonium which blocks oxygen-free radical formation by astrocytes . These expression patterns resembled those of RA301 mRNA . Far Western blot analysis showed that YT521 protein was not only interacting with RA301 protein, but also with SC35 and SF2, both of which are splicing factors . These results suggest that YT521 is a novel candidate for RNA splicing-related protein.

J Cell Sci, 1998 Feb, 111 ( Pt 3), 295 - 301
The Kar3p and Kip2p motors function antagonistically at the spindle poles to influence cytoplasmic microtubule numbers; Huyett A et al.; Microtubules provide the substrate for intracellular trafficking by association with molecular motors of the kinesin and dynein superfamilies . Motor proteins are generally thought to function as force generating units for transport of various cargoes along the microtubule polymer . Recent work suggests additional roles for motor proteins in changing the structure of the microtubule network itself . We report here that in the budding yeast Saccharomyces cerevisiae microtubule motors have antagonistic effects on microtubule numbers and lengths . As shown previously, loss of the Kar3p motor stimulates cytoplasmic microtubule growth while loss of Kip2p leads to a sharp reduction in cytoplasmic microtubule numbers . Loss of both the Kip2p and Kar3p motors together in the same cell produces an intermediate phenotype, suggesting that these two motors act in opposition to control cytoplasmic microtubule density . A Kip2p-GFP fusion from single gene expression is most concentrated at the spindle poles, as shown previously for an epitope tagged Kar3p-HA, suggesting both of these motors act from the minus ends of the microtubules to influence microtubule numbers.

Eur J Biochem, 1998 Mar 15, 252(3), 513 - 9
Biosynthesis of C18 polyunsaturated fatty acids in microsomal membrane preparations from the filamentous fungus Mucor circinelloides; Jackson FM et al.; The biosynthesis of C18 polyunsaturated fatty acids has been studied in the fungus Mucor circinelloides . Microsomal membrane preparations contained delta9, delta12 and delta6 desaturase activities . The delta9 desaturase exhibited characteristics similar to those of the animal and yeast delta9 desaturases in being membrane bound and utilising stearoyl-CoA as substrate . Cytochrome b5 (a soluble form lacking the 20-amino-acid hydrophobic C-terminus) stimulated desaturation and was identified as a major cytochrome component of the membranes . A high ferricyanide reductase activity (indicative of NADH:cytochrome b5 reductase activity) coupled to inhibition by cyanide further supported the similarity with the mammalian and yeast enzymes . Time-course studies with radiolabelled oleoyl-CoA showed that the oleate {18:1(9)} was transferred to position sn-2 of phosphatidylcholine (PtdCho) and was desaturated to linoleoyl-PtdCho . Removal of the excess oleoyl-CoA from the membranes prior to addition of reductant confirmed that oleoyl-PtdCho is a substrate for the delta12 desaturase . The entry of oleate at this position of the phospholipid was facilitated by the activity of lyso-PtdCho:acyl-CoA acyltransferase (LPCAT), which readily transferred oleate from oleoyl-CoA to lyso-PtdCho . Desaturation of oleate at the sn-1 position of PtdCho was also demonstrated after the entry of oleate in to the phospholipid by the enzymes of the Kennedy pathway . Thus oleate at sn-1 and sn-2 positions served as substrate for the delta12 desaturase and is consistent with observations in oil seed tissues . LPCAT activity was substantially higher than that observed with lysophosphatidylethanolamine:acyl-CoA acyltransferase (LPEAT) indicating that oleate is less effectively channelled to phosphatidylethanolamine for linoleate synthesis . No desaturation on phosphatidylinositol could be demonstrated . Delta6 desaturase utilised linoleate at the sn-2 position of exogenously supplied PtdCho presented to the membranes in the presence of reductant . Thus, the entry of substrates into PtdCho via LPCAT and the synthesis of linoleate {18:2(9,12)} and gamma-linolenate {18:3(6,9,12)} on this phospholipid is similar to that reported for oil seed membranes.

Mol Cell Biochem, 1998 Mar, 180(1-2), 117 - 28
Palmitate oxidation by the mitochondria from volume-overloaded rat hearts; Christian B et al.; In this work, an attempt was made to identify the reasons of impaired long-chain fatty acid utilization that was previously described in volume-overloaded rat hearts . The most significant data are the following: (1) The slowing down of long-chain fatty acid oxidation in severely hypertrophied hearts cannot be related to a feedback inhibition of carnitine palmitoyltransferase I from an excessive stimulation of glucose oxidation since, because of decreased tissue levels of L-carnitine, glucose oxidation also declines in volume-overloaded hearts . (2) While, in control hearts, the estimated intracellular concentrations of free carnitine are in the range of the respective Km of mitochondrial CPT I, a kinetic limitation of this enzyme could occur in hypertrophied hearts due to a 40% decrease in free carnitine . (3) The impaired palmitate oxidation persists upon the isolation of the mitochondria from these hearts even in presence of saturating concentrations of L-carnitine . In contrast, the rates of the conversion of both palmitoyl-CoA and palmitoylcarnitine into acetyl-CoA are unchanged . (4) The kinetic analyses of palmitoyl-CoA synthase and carnitine palmitoyltransferase I reactions do not reveal any differences between the two mitochondrial populations studied . On the other hand, the conversion of palmitate into palmitoylcarnitine proves to be substrate inhibited already at physiological concentrations of exogenous palmitate . The data presented in this work demonstrate that, during the development of severe cardiac hypertrophy, a fragilization of the mitochondrial outer membrane may occur . The functional integrity of this membrane seems to be further deteriorated by increasing concentrations of free fatty acids which gives rise to an impaired cooperation between palmitoyl-CoA synthase and carnitine palmitoyltransferase I . In intact myocardium, the utilization of the in situ generated palmitoyl-CoA can be further slowed down by decreased intracellular concentrations of free carnitine.

Cell, 1998 Apr 3, 93(1), 81 - 91
Human T cell leukemia virus type 1 oncoprotein Tax targets the human mitotic checkpoint protein MAD1; Jin DY et al.; In searching for cellular targets of the HTLV-I oncoprotein Tax, we identified TXBP181, which we characterized as the human homolog of yeast mitotic checkpoint MAD1 protein . Evidence supporting TXBP181 as HsMAD1 includes sequence conservation with yeast MAD1, hyperphosphorylation during S/G2/M phases and upon treatment of cells with nocodazole, and binding to HsMAD2 . HsMAD1 functions as a homodimer . It localizes to the centrosome during metaphase and to the spindle midzone and the midbody during anaphase and telophase . Expression of either Tax or a transdominant-negative TXBP181 results in multinucleated cells, a phenotype consistent with a loss of HsMAD1 function . We propose a model of viral transformation in which Tax targets TXBP181, thereby abrogating a mitotic checkpoint.

Genomics, 1998 Mar 15, 48(3), 330 - 40
Identification and characterization of two putative human arginine methyltransferases (HRMT1L1 and HRMT1L2); Scott HS et al.; RNA-binding proteins such as heterogeneous nuclear ribonucleoproteins (hnRNPs), which contain the bulk of methylated arginine residues in eukaryotic cells, play many essential roles in the metabolism of nuclear pre-mRNA . Arginine methyltransferase activity has also been implicated in signal transduction events with components of the cellular growth and viral response pathways . We recently characterized a single yeast hnRNP methyltransferase (HMT1) . We now present the identification and characterization of two putative human arginine methyltransferases termed HRMT1L1 and HRMT1L2 . In addition to methyltransferase similarities, the N-terminal region of the HRMT1L1 protein contains an Src homology 2 domain . HRMT1L1 maps to a YAC containing the telomere of chromosome 21q . Three alternatively spliced HRMT1L2 transcripts with variable 5'-ends were observed, encoding proteins of 343, 347, and 361 amino acids, respectively . HRMT1L2 maps to human chromosome 19q . Recombinant HRMT1L2 protein encoded by the most common 5'-variant exhibited methyltransferase activity in vitro . Furthermore, in vivo activity was demonstrated by complementation of a yeast HMT1 mutant strain . The identification of highly conserved Hmt1p human homologues that function in yeast indicates that analyses of this class of enzymes in yeast may be directly applicable to higher eukaryotes . The possible roles of HRMT1L1 and HRMT1L2 in human disease are currently unknown.

Biochim Biophys Acta, 1998 Apr 1, 1397(1), 21 - 6
Molecular analysis of a glucose-regulated gene (grp78) of Neurospora crassa; Techel D et al.; The nucleotide sequence of the gene encoding the glucose-regulated protein 78 (GRP78) of Neurospora crassa was determined . The ORF codes for a protein of 662 amino acids (72 kDa) and belongs to the heat shock protein 70 (hsp70) gene family, which is characterized by three HSP70 'signature sequences' . The grp78 gene contains 5 introns . The protein carries the ER retention signal HDEL at its carboxy terminus and is most homologous to the KAR2/GRP78 protein of Saccharomyces cerevisiae (78%) and to KAR2/BiP of Yarrowia lipolytica (76%) . The expression of grp78 is constitutive and can be enhanced by starvation, treatment with tunicamycin, the calcium ionophore A23187 or elevated temperatures (40 degrees C) . An uninterrupted ORF was found on the reverse cDNA strand of grp78 . The putative peptide shows 47% homology to the NAD-specific glutamate dehydrogenase of Achlya klebsiana.

Chem Biol, 1998 Mar, 5(3), 119 - 33
Inhibition of major-groove-binding proteins by pyrrole-imidazole polyamides with an Arg-Pro-Arg positive patch; Bremer RE et al.; BACKGROUND: Gene-specific targeting of any protein-DNA complex by small molecules is a challenging goal at the interface of chemistry and biology . Polyamides containing N-methylimidazole and N-methylpyrrole amino acids are synthetic ligands that have an affinity and specificity for DNA comparable to many naturally occurring DNA-binding proteins . It has been shown that an eight-ring hairpin polyamide targeted to a specific minor-groove contact within a transcription factor binding site can inhibit protein-DNA binding and gene transcription . Polyamides and certain major-groove-binding proteins have been found to co-occupy the DNA helix, however . To expand the number of genes that can be targeted by pyrrole/imidazole polyamides, we set out to develop a class of polyamides that can selectively inhibit major-groove-binding proteins . RESULTS: An eight-ring hairpin polyamide conjugated to a carboxy-terminal Arg-Pro-Arg tripeptide was designed to deliver a positive residue to the DNA backbone and interfere with protein-phosphate contacts . Gel mobility shift analysis demonstrated that a polyamide hairpin-Arg-Pro-Arg binding in the minor groove selectively inhibits binding of the transcription factor GCN4 (222-281) in the adjacent major groove . Substitution within the Arg-Pro-Arg revealed that each residue was required for optimal GCN4 inhibition . CONCLUSIONS: A pyrrole-imidazole polyamide that binds to a predetermined site in the DNA minor groove and delivers a positive patch to the DNA backbone can selectively inhibit a DNA-binding protein that recognizes the adjacent major groove . A subtle alteration of the DNA microenvironment targeted to a precise location within a specific DNA sequence could achieve both gene-specific and protein-specific targeting.

J Biol Chem, 1998 Mar 20, 273(12), 6968 - 75
Genetic selection of mammalian adenylyl cyclases insensitive to stimulation by Gsalpha; Zimmermann G et al.; We describe the development of a genetic system allowing for the isolation of mutant mammalian adenylyl cyclases defective in their responses to G protein subunits, thus allowing for the identification of structural elements within the cyclase that are responsible for the recognition of these regulators . Expression of mammalian type V adenylyl cyclase in a cyclase-deleted yeast strain can conditionally complement the lethal phenotype of this strain . Type V adenylyl cyclase-expressing yeast grow only when the cyclase is activated by coexpression of Gsalpha or addition of forskolin to the medium; however, growth arrest is observed in the presence of both activators or under basal conditions . Utilizing this genetic system, we have isolated 25 adenylyl cyclase mutants defective in their response to Gsalpha . Sequence analysis and biochemical characterization of these mutants have identified residues in both cytoplasmic domains of the cyclase that are involved in the specific binding of and regulation by Gsalpha.

J Biol Chem, 1998 Mar 20, 273(12), 6951 - 9
Identification of a mitochondrial Na+/H+ exchanger; Numata M et al.; The electroneutral exchange of protons for Na+ and K+ across the mitochondrial inner membrane contributes to organellar volume and Ca2+ homeostasis . The molecular nature of these transporters remains unknown . In this report, we characterize a novel gene (YDR456w; renamed NHA2) in Saccharomyces cerevisiae whose deduced protein sequence is homologous to members of the mammalian Na+/H+ exchanger gene family . Fluorescence microscopy showed that a Nha2-green fluorescent protein chimera colocalizes with 4',6-diamidino-2-phenylindole staining of mitochondrial DNA . To assess the function of Nha2, we deleted the NHA2 gene by homologous disruption and found that benzamil-inhibitable, acid-activated 22Na+ uptake into mitochondria was abolished in the mutant strain . It also showed retarded growth on nonfermentable carbon sources and severely reduced survival during the stationary phase of the cell cycle compared with the parental strain, consistent with a defect in aerobic metabolism . Sequence comparisons revealed that Nha2 has highest identity to a putative Na+/H+ exchanger homologue (KIAA0267; renamed NHE6) in humans . Northern blot analysis demonstrated that NHE6 is ubiquitously expressed but is most abundant in mitochondrion-rich tissues such as brain, skeletal muscle, and heart . Fluorescence microscopy showed that a NHE6-green fluorescent protein chimera also accumulates in mitochondria of transfected HeLa cells . These data indicate that NHA2 and NHE6 encode homologous Na+/H+ exchangers and suggest they may be important for mitochondrial function in lower and higher eukaryotes, respectively.

J Biol Chem, 1998 Mar 20, 273(12), 6868 - 77
Human ZFM1 protein is a transcriptional repressor that interacts with the transcription activation domain of stage-specific activator protein; Zhang D et al.; Stage-specific activator protein (SSAP) is the transcription factor responsible for the activation of the sea urchin late H1 gene at the mid-blastula stage of embryogenesis . SSAP contains an extremely potent transcription activation domain that functions 4-5-fold better than VP16 in a variety of mammalian cell lines . We used the two-hybrid screening technique to identify human cDNAs from an HL60 cell-derived cDNA library that encode proteins that interact with the transcription activation domain of SSAP . One of these cDNAs encodes ZFM1, a protein previously identified at the locus linked to multiple endocrine neoplasia type 1 (MEN1) and as presplicing factor SF1 . Functional assays establish the ZFM1 protein as a transcriptional repressor . ZFM1 protein represses Gal4-GQC-mediated transcription, and this activity requires both a repression domain found in the N-terminal 137 amino acids of the protein, as well as a GQC interaction region . The physiological significance of repression mediated by ZFM1 comes from the ability of its specific repression domain to function when fused to Gal4 and tethered to promoters containing Gal4 binding sites . The activity is unique in that activated but not basal transcription levels are affected.

J Biol Chem, 1998 Mar 20, 273(12), 6763 - 8
Dimerization and autoprocessing of the Nedd2 (caspase-2) precursor requires both the prodomain and the carboxyl-terminal regions; Butt AJ et al.; Nedd2 (caspase-2) is a cysteine protease of the caspase family that has been demonstrated to play a role in the apoptotic pathway . The 51-kDa precursor of Nedd2 undergoes cleavage into two subunits following various apoptotic stimuli . In this study, we have investigated the dimerization of the Nedd2 precursor (pro-Nedd2) in Saccharomyces cerevisiae and its self-processing activity in vivo . We demonstrate that the expression of pro-Nedd2 in yeast cells results in processing of the precursor . A catalytically inactive pro-Nedd2 mutant dimerized in yeast, and the dimerization required both the prodomain and the carboxyl-terminal residues . Aspartate mutants that block the removal of the p14/p12 subunits, but not the wild-type Nedd2, were shown to dimerize in yeast cells, suggesting that dimerization occurs prior to processing . In vitro processing of pro-Nedd2 by recombinant active Nedd2 defined the aspartate residues that are crucial for processing to occur . Both the in vivo and in vitro processing of pro-Nedd2 directly correlated with its ability to induce cell death in transient overexpression experiments.

J Biol Chem, 1998 Mar 20, 273(12), 6679 - 88
Enhancement of estrogen receptor transcriptional activity by the coactivator GRIP-1 highlights the role of activation function 2 in determining estrogen receptor pharmacology; Norris JD et al.; The human estrogen receptor (ER) contains two major activation functions (AFs) responsible for its transcriptional activity . One of these, activation function 2 (AF-2), located within the hormone-binding domain (HBD), has been shown to mediate the ligand-dependent transcriptional activity of ER as well as other members of the nuclear receptor superfamily . Recently, proteins interacting with the HBD of several nuclear receptors have been cloned . One of these proteins, glucocorticoid receptor interacting protein (GRIP-1), has been shown to interact with ER and was originally hypothesized to mediate its transcriptional activity through AF-2 . However, we find in this study that the transcriptional activity of ER, containing mutations in the AF-2 core sequence, can be enhanced by coexpression of the coactivator GRIP-1, suggesting that this protein may not rely solely on the AF-2 domain for interaction . We propose, therefore, that the HBD of ER either contains multiple binding sites that are necessary for association with GRIP-1 or, alternatively, that this coactivator contacts the receptor in an undetermined region within the HBD . Importantly, these studies demonstrate also that mutations or deletion of AF-2 alter the ligand pharmacology of the receptor such that ER loses the ability to discriminate between agonists and antagonists . Interestingly, on these mutant receptors GRIP-1 still functions as a coactivator independent of the nature of the bound ligand . It is likely, therefore, that the C-terminal AF-2 domain may function as a molecular switch allowing the wild-type receptor to discriminate between agonists and antagonists as well as providing a surface with which associated proteins can interact.

J Biol Chem, 1998 Mar 20, 273(12), 6591 - 4
Complex formation of SMAP/KAP3, a KIF3A/B ATPase motor-associated protein, with a human chromosome-associated polypeptide; Shimizu K et al.; We have recently isolated SMAP (Smg GDS-associated protein; Smg GDS: small G protein GDP dissociation stimulator) as a novel Smg GDS-associated protein, which has Armadillo repeats and is phosphorylated by Src tyrosine kinase . SMAP is a human counterpart of mouse KAP3 (kinesin superfamily-associated protein) that is associated with mouse KIF3A/B (a kinesin superfamily protein), which functions as a microtubule-based ATPase motor for organelle transport . We isolated here a SMAP-interacting protein from a human brain cDNA library, identified it to be a human homolog of Xenopus XCAP-E (Xenopus chromosome-associated polypeptide), a subunit of condensins that regulate the assembly and structural maintenance of mitotic chromosomes, and named it HCAP (Human chromosome-associated polypeptide) . Tissue and subcellular distribution analyses indicated that HCAP was ubiquitously expressed and highly concentrated in the nuclear fraction, where SMAP and KIF3B were also present . SMAP was extracted as a ternary complex with HCAP and KIF3B from the nuclear fraction in the presence of Mg-ATP . The results suggest that SMAP/KAP3 serves as a linker between HCAP and KIF3A/B in the nucleus, and that SMAP/KAP3 plays a role in the interaction of chromosomes with an ATPase motor protein.

J Cell Biol, 1998 Mar 9, 140(5), 1063 - 74
Vac8p, a vacuolar protein with armadillo repeats, functions in both vacuole inheritance and protein targeting from the cytoplasm to vacuole; Wang YX et al.; During each cell cycle, the yeast vacuole actively partitions between mother and daughter cells . This process requires actin, profilin, an unconventional myosin (Myo2p), and Vac8p . A mutant yeast strain, vac8, is defective in vacuole inheritance, specifically, in early vacuole migration . Vac8p is a 64-kD protein found on the vacuole membrane, a site consistent with its role in vacuole inheritance . Both myristoylation and palmitoylation are required for complete Vac8p localization . Interestingly, whereas myristoylation of Vac8p is not required for vacuole inheritance, palmitoylation is essential . Thus, palmitoylation appears to play a more direct role in vacuole inheritance . Most of the VAC8 sequence encodes 11 armadillo (Arm) repeats . Arm repeats are thought to mediate protein-protein interactions, and many Arm proteins have multiple functions . This is also true for Vac8p . In addition to its role in early vacuole inheritance, Vac8p is required to target aminopeptidase I from the cytoplasm to the vacuole . Mutant analysis demonstrates that Vac8p functions separately in these two processes . Vac8p cosediments with actin filaments . Vac8p is related to beta-catenin and plakoglobin, which connect a specific region of the plasma membrane to the actin cytoskeleton . In analogy, Vac8p may link the vacuole to actin during vacuole partitioning.

Biosystems, 1998 Feb, 45(2), 165 - 76
The role of the genetic code in generating new coding sequences inside existing genes; Cebrat S et al.; The genetic code has a very interesting property--it generates an open reading frame (ORF) inside a coding sequence, in a specific phase of the antisense strand with much higher probability than in the random DNA sequences . Furthermore, these antisense ORFs (A-ORFs) possess the same features as real genes--the asymmetry in the nucleotide composition at the first and second positions in codons . About two thirds of the 2997 overlapping ORFs in the yeast genome possess this feature . Thus, the question arises: has this feature of the genetic code been exploited in the evolution of genes? We have searched the FASTA data bases for homologies with the antisense translation products of a specific class of genes and we have found some sequences with relatively high homology . Many of them have scores which could be randomly found in the searched data bases with a probability lower than 10(-6) . We conclude that some genes could arise by positioning a copy of the original gene under a promoter in the opposite direction in such a way that both, the original gene and its copy initially use the same nucleotides in the third, degenerated positions in codons.

Protein Sci, 1998 Mar, 7(3), 706 - 19
High throughput protein characterization by automated reverse-phase chromatography/electrospray tandem mass spectrometry; Ducret A et al.; We describe an integrated workstation for the automated, high-throughput, and conclusive identification of proteins by reverse-phase chromatography electrospray ionization tandem mass spectrometry . The instrumentation consists of a refrigerated autosampler, a submicrobore reverse-phase liquid chromatograph, and an electrospray triple quadrupole mass spectrometer . For protein identification, enzymatic digests of either homogeneous polypeptides or simple protein mixtures were generated and loaded into the autosampler . Samples were sequentially injected every 32 min . Ions of eluting peptides were automatically selected by the mass spectrometer and subjected to collision-induced dissociation . Following each run, the resulting tandem mass spectra were automatically analyzed by SEQUEST, a program that correlates uninterpreted peptide fragmentation patterns with amino acid sequences contained in databases . Protein identification was established by SEQUEST_SUMMARY a program that combines the SEQUEST scores of peptides originating from the same protein and ranks the cumulative results in a short summary . The workstation's performance was demonstrated by the unattended identification of 90 proteins from the yeast Saccharomyces cerevisiae, which were separated by high-resolution two-dimensional PAGE . The system was found to be very robust and identification was reliably and conclusively established for proteins if quantities exceeding 1-5 pmol were applied to the gel . The level of automation, the throughput, and the reliability of the results suggest that this system will be useful for the many projects that require the characterization of large numbers of proteins.

Protein Sci, 1998 Mar, 7(3), 545 - 55
Subunit asymmetry in the three-dimensional structure of a human CuZnSOD mutant found in familial amyotrophic lateral sclerosis; Hart PJ et al.; The X-ray crystal structure of a human copper/zinc superoxide dismutase mutant (G37R CuZnSOD) found in some patients with the inherited form of Lou Gehrig's disease (FALS) has been determined to 1.9 angstroms resolution . The two SOD subunits have distinct environments in the crystal and are different in structure at their copper binding sites . One subunit (subunit{intact}) shows a four-coordinate ligand geometry of the copper ion, whereas the other subunit (subunit{broken}) shows a three-coordinate geometry of the copper ion . Also, subunit(intact) displays higher atomic displacement parameters for backbone atoms ((B) = 30 +/- 10 angstroms2) than subunit(broken) ((B) = 24 +/- 11 angstroms2) . This structure is the first CuZnSOD to show large differences between the two subunits . Factors that may contribute to these differences are discussed and a possible link of a looser structure to FALS is suggested.

Mech Ageing Dev, 1998 Jan 30, 100(2), 197 - 208
Cell proliferation and ku protein expression in ageing humans; Frasca D et al.; Previous studies on DNA repair in ageing have demonstrated increased frequencies of single and double strand breaks in lymphocytes from elderly subjects and, as a consequence, decreased efficiency in DNA replication . We have investigated the relationship between cell proliferation and the nuclear expression of ku protein in a human population of 43 subjects of different ages . Ku is an heterodimeric protein composed of two subunits of 70 and 80 kDa, which is involved in the early steps of DNA damage recognition . In the present study, PBL from subjects of different ages were PHA-activated to evaluate the stimulation index and the production of Th1- and Th2-type cytokines . Moreover, nuclear extracts were obtained from activated lymphocytes to evaluate by a gel retardation assay the presence and the functional activity of the heterodimer ku 70/80 . Our results indicate that ageing affects the mitotic responsiveness and cytokine production to a significant extent, but only marginally the expression of ku 70/80 . These findings suggest that the age-related impairment in DNA repair mechanisms are only in part related to the reduced expression of ku protein able to recognize DNA damage.

FEBS Lett, 1998 Mar 20, 425(1), 14 - 8
Diffusion-controlled DNA recognition by an unfolded, monomeric bZIP transcription factor; Berger C et al.; Basic leucine zipper (bZIP) transcription factors are dimers that recognize mainly palindromic DNA sites . It has been assumed that bZIP factors have to form a dimer in order to bind to their target DNA . We find that DNA binding of both monomeric and dimeric bZIP transcription factor GCN4 is diffusion-limited and that, therefore, the rate of dimerization of the bZIP domain does not affect the rate of DNA recognition and GCN4 need not dimerize in order to bind to its specific DNA site . The results have implications for the mechanism by which bZIP transcription factors find their target sites for transcriptional regulation.

Biochim Biophys Acta, 1998 Feb 17, 1382(2), 243 - 8
Characterization of the monovalent and divalent cation requirements for the xenobiotic carboxylic acid: CoA ligases of bovine liver mitochondria; Vessey DA et al.; The XL-I, XL-II and XL-III forms of xenobiotic/medium-chain fatty acid: CoA ligase were found to be inactive toward benzoate in the absence of either monovalent or divalent cations . The absolute requirement for monovalent cation was satisfied by either K+, Rb+, or NH4+ . Na+ only supported a very low rate . Varying the nature of the anion had only a minor effect . For XL-I and XI-II, the optimum concentration of K+ was 50 mM; higher (physiologic) concentrations led to a decrease in activity . K+ did not inhibit XL-III . The absolute requirement for divalent cation was satisfied by Mg2+ or Mn2+, or to a lesser extent by Co2+ or Fe2+ . For the XL-I and XL-II, excess uncomplexed Mg2+ or Mn2+ decreased the rate; the optimum concentration of Mn2+ was approximately the same as the concentration of ATP in the assay, and the optimum concentration of Mg2+ was approximately double the concentration of ATP in the assay . This is consistent with the concept that the divalent cation is required to complex with ATP and with the known stability constants for the ATP complexes of these two divalent cations . XL-III was not inhibited by uncomplexed divalent cations . Uncomplexed ATP was a moderate inhibitor of XL-I and XL-II, and a weak inhibitor of XL-III . The data indicate that in vivo benzoate conjugation is K+ and Mg2+ dependent, and that the cation effects are complex and differ for XL-I and XL-II as compared with XL-III.

Cancer Res, 1998 Apr 1, 58(7), 1460 - 8
Chinese hamster ovary cells resistant to the topoisomerase II catalytic inhibitor ICRF-159: a Tyr49Phe mutation confers high-level resistance to bisdioxopiperazines; Sehested M et al.; Anticancer drugs targeted to the nuclear enzyme DNA topoisomerase II are classified as poisons that lead to DNA breaks or catalytic inhibitors that appear to completely block enzyme activity . To examine the effects of the bisdioxopiperazine class of catalytic inhibitors to topoisomerase II, we investigated a Chinese hamster ovary (CHO) subline selected for resistance to ICRF-159 (CHO/159-1) . Topoisomerase IIalpha content in CHO/159-1 cells was reduced by 40-50%, compared to wild-type CHO cells, whereas the beta isoform was increased by 10-20% in CHO/159-1 cells . However, the catalytic activity of topoisomerase II in nuclear extracts from CHO/159-1 cells was unchanged, as was its inhibition by the topoisomerase II poison etoposide (VP-16) . No inhibition of topoisomerase II catalytic activity by ICRF-187 was seen in CHO/159-1 cells up to 500 microM, whereas inhibition was evident at 50 microM in wild-type CHO cells . VP-16-mediated DNA single-strand breaks and cytotoxicity were similar in the two sublines . ICRF-187 could abrogate these VP-16 effects in the wild-type line but had no effect in CHO/159-1 cells . Western blots of topoisomerase IIalpha after incubation of CHO cells with ICRF-187 demonstrated a marked band depletion, whereas this effect was completely lacking in CHO/159-1 cells, and an equal effect of VP-16 was observed in both lines . These data imply that the CHO/159-1 topoisomerase IIalpha lacks sensitivity to bisdioxopiperazines and that the mechanism of resistance in this cell line does not confer cross-resistance to topoisomerase II poisons, suggesting that mutations conferring resistance to bisdioxopiperazines can occur at sites distinct from those responsible for resistance to complex stabilizing agents . Accordingly, CHO/159-1 cDNA showed two heterozygous mutations in the proximal NH2-terminal part of topoisomerase IIalpha (Tyr49Phe and delta 309Gln-Gln-Ile-Ser-Phe313), which is in contrast to those induced by topoisomerase II poisons, which cluster further downstream . Site-directed mutagenesis and transformation of the homologous Tyr50Phe coding mutation in human topoisomerase IIalpha in a temperature-conditional yeast system demonstrated a high-level resistance to ICRF-193, compared to cells expressing wild-type cDNA, but none toward the poisons VP-16 or amsacrine, thus confirming that the Tyr50Phe mutation confers specific resistance to bisdioxopiperazines . Thus, these results indicate that the region of the protein involved in ATP-binding also plays a critical role in sensitivity to bisdioxopiperazines, a result consistent with the known requirement for the formation of an ATP-bound closed clamp for bisdioxopiperazine activity . These results may enable a more precise understanding of the interaction of topoisomerase II-directed drugs with their target enzyme.

J Infect Dis, 1998 Apr, 177(4), 1132 - 5
Persistence of humoral response against sporozoite and blood-stage malaria antigens 7 years after a brief exposure to Plasmodium vivax; Braga EM et al.; The persistence of malarial antibodies was evaluated in subjects living in a rural community (in Minas Gerais State, Brazil) briefly exposed to a Plasmodium vivax malaria outbreak outside of the area in which malaria was endemic . Transmission was interrupted by treatment of all patients and their relatives and/or neighbors, although the latter had neither symptoms nor blood parasites . Antibodies to P . vivax antigens (recombinant proteins from sporozoites {rPvCS} and from blood stages {rPv200}) were measured in parallel by ELISA with sera collected at two time points after transmission . Anti-rPvCS IgG antibodies were positive in approximately 40% and 20% of the subjects 8 months and 7 years after exposure, respectively . Anti-rPv200 IgG was first detected in 61% of the subjects who had had malarial symptoms and remained positive in 47% after 7 years . Among the prophylactically treated group, anti-rPv200 IgG was detected in only 28% after 8 months . The levels of both antibodies decreased with time in all positive subjects.

Curr Biol, 1998 Feb 26, 8(5), 267 - 78
Functional binding between Gbeta and the LIM domain of Ste5 is required to activate the MEKK Ste11; Feng Y et al.; BACKGROUND: In the budding yeast Saccharomyces cerevisiae, the pheromones that induce haploid cells of opposite cell types to mate activate the Gbeta and Ggamma subunits of a heterotrimeric G protein . These subunits signal through the PAK kinase Ste20 to activate a mitogen-activated protein (MAP) kinase cascade comprising the MEKK Ste11, the MEK Ste7 and two MAP kinases, Fus3 and Kss1 . The pathway requires Ste5, a scaffold protein that tethers the MAP kinase cascade enzymes into a high molecular weight complex . Ste5 is thought to associate with Gbeta in a pheromone-independent manner, but it is not known if this interaction affects signaling . RESULTS: A ste5C180A mutant - which expresses Ste5 disrupted in the LIM domain, a putative metal-binding motif that has been proposed to be essential for Ste5 oligomerization - could not transmit the pheromone signal from Gbeta through Ste20 to Ste11 . The Ste5C180A protein was impaired in binding Gbeta, although it could oligomerize, bind Ste11, Ste7 and Fus3, facilitate the basal activation of Ste11, and relay the Ste11 signal to MAP kinases . Ste5 bound to Gbeta in a pheromone-dependent manner and preferentially associated with a phosphorylated form of Gbeta in wild-type and ste20Delta, but not in ste5C180A, strains . CONCLUSIONS: Pheromone induces binding of Gbeta to Ste5 through its LIM domain . This binding is essential for activation of Ste11 and is distinct from the ability of Ste5 to oligomerize or to serve as a scaffold and relay the signal from Ste11 to the MAP kinases . Pheromone also induces Ste5-dependent phosphorylation of Gbeta.

Genes Dev, 1998 Mar 1, 12(5), 679 - 91
Arginine methylation facilitates the nuclear export of hnRNP proteins; Shen EC et al.; Eukaryotic mRNA processing and export is mediated by various heterogeneous nuclear ribonucleoproteins (hnRNPs) . Many of these hnRNPs are methylated on arginine residues . In the yeast, Saccharomyces cerevisiae, the predominant enzyme responsible for arginine methylation is Hmt1p . Hmt1p methylates both Npl3p and Hrp1p, which are shuttling hnRNPs involved in mRNA processing and export . Here, we employ an in vivo nuclear export assay to show that arginine methylation is important for the nuclear export of these hnRNPs . Both Npl3p and Hrp1p fail to exit the nucleus in cells lacking Hmt1p, and overexpression of Hmt1p enhances Npl3p export . The export of a novel hnRNP-like protein, Hrb1p, which does not bind poly(A)+ RNA, however, is not affected by the lack of methylation . Furthermore, we find a genetic relationship between Hmt1p and cap-binding protein 80 (CBP80) . Together, these findings establish that one biological role for arginine methylation is in facilitating the export of certain hnRNPs out of the nucleus.

Genes Dev, 1998 Mar 1, 12(5), 640 - 53
Critical residues for histone acetylation by Gcn5, functioning in Ada and SAGA complexes, are also required for transcriptional function in vivo; Wang L et al.; Several previously known transcription cofactors have been demonstrated in vitro recently to be histone acetyltransferases and deacetyltransferases, suggesting that remodeling of chromatin through histone acetylation plays a fundamental role in gene regulation . Clear evidence has not yet been obtained, however, to demonstrate that histone acetylation is required for gene activation in vivo . In this study we performed an alanine-scan mutagenesis through the HAT (histone acetyltransferase) domain identified previously by deletion mapping in recombinant yeast Gcn5 . We identified multiple substitution mutations that eliminated completely Gcn5's ability to potentiate transcriptional activation in vivo . Strikingly, each of these mutations was also critical for free and nucleosomal histone acetylation by Gcn5 functioning within the native yeast HAT complexes, Ada, and SAGA . Moreover, the growth phenotypes of these mutations as measured by colony size and liquid growth assay closely tracked transcription and HAT activities . In contrast, mutations that did not affect in vivo function of Gcn5 were able to acetylate histones . These data argue strongly that acetylation is required for gene regulation by Gcn5 in vivo, and support previous arguments that nucleosomal histones are among the physiological substrates of acetylation by Gcn5.

J Biol Chem, 1998 Mar 13, 273(11), 6183 - 9
Identification of a novel cytoplasmic protein that specifically binds to nuclear localization signal motifs; Li S et al.; Active transport of proteins into the nucleus is mediated by interaction between the classical nuclear localization signals (NLSs) of the targeted proteins and the NLS receptor (importin) complex . This nuclear transport system is highly regulated and conserved in eukaryotes and is essential for cell survival . Using a fragment of BRCA1 containing the two NLS motifs as a bait for yeast two-hybrid screening, we have isolated four clones, one of which is importin alpha . Here we characterize one of the other clones identified, BRAP2, which is a novel gene and expressed as a 2-kilobase mRNA in human mammary epithelial cells and some but not all tissues of mice . The isolated full-length cDNA encodes a novel protein containing 600 amino acid residues with pI 6.04 . Characteristic motifs of C2H2 zinc fingers and leucine heptad repeats are present in the middle and C-terminal regions of the protein, respectively . BRAP2 also shares significant homology with a hypothetical protein from yeast Saccharomyces cerevisiae, especially in the zinc finger region . Antibodies prepared against the C-terminal region of BRAP2 fused to glutathione S-transferase specifically recognize a cellular protein with a molecular size of 68 kDa, consistent with the size of the in vitro translated protein . Cellular BRAP2 is mainly cytoplasmic and binds to the NLS motifs of BRCA1 with similar specificity to that of importin alpha in both two-hybrid assays in yeast and glutathione S-transferase pull-down assays in vitro . Other motifs such as the SV40 large T antigen NLS motif and the bipartite NLS motif found in mitosin are also recognized by BRAP2 . Similarly, the yeast homolog of BRAP2 also binds to these NLS motifs in vitro . These results imply that BRAP2 may function as a cytoplasmic retention protein and play a role in regulating transport of nuclear proteins.

J Biol Chem, 1998 Mar 13, 273(11), 6177 - 82
Binding of Rad51p to DNA . Interaction of Rad51p with single- and double-stranded DNA; Namsaraev EA et al.; Like RecA, Saccharomyces cerevisiae Rad51p promotes strand exchange between circular single-stranded DNA (ssDNA) and linear double-stranded DNA (dsDNA) . We have investigated several parameters characteristic of the interaction of Rad51p with ssDNA and dsDNA, particularly the effects of the nucleotide cofactors ATP and ADP and the analogs adenosine 5'-O-(thiotriphosphate) (ATPgammaS) and adenylyl-imidodiphosphate (AMP-PNP) . Rad51p binding to both 1-N6-ethenoadenosine and 3-N4-ethenocytidine ssDNA (epsilonDNA) and dsDNA requires the presence of Mg2+ and ATP; no binding occurs in the presence of ADP, AMP-PNP, or ATPgammaS . Binding of Rad51p to dsDNA also requires ATP; ADP is ineffective, whereas ATPgammaS and AMP-PNP are considerably less able to promote binding and only at elevated concentrations of Rad51p . ATP binding, not ATP hydrolysis, is required for Rad51p binding to DNA . The Kd values for ATP for promoting binding of Rad51p to ssDNA and dsDNA are 1 and 3 microM, respectively . Rad51p binding occurs with a stoichiometry of one monomer of Rad51p per approximately 6.3 nucleotides of epsilonDNA and approximately 3.3 base pairs of dsDNA . Once formed, Rad51p . ssDNA complexes are stable so long as sufficient ATP levels are maintained . ATP hydrolysis causes dissociation of Rad51p from DNA . Moreover, the preformed complex is stable in the presence of a 10-fold excess of ADP or AMP-PNP over ATP . ATPgammaS, however, in the same -fold excess over ATP causes dissociation of the Rad51p . ssDNA complex.

J Biol Chem, 1998 Mar 6, 273(10), 5979 - 87
Reconstitution of recombinant human replication factor C (RFC) and identification of an RFC subcomplex possessing DNA-dependent ATPase activity; Ellison V et al.; Replication factor C (RFC) is a five-subunit protein complex required for coordinate leading and lagging strand DNA synthesis during S phase and DNA repair in eukaryotic cells . It functions to load the proliferating cell nuclear antigen (PCNA), a processivity factor for polymerases delta and epsilon, onto primed DNA templates . This process, which is ATP-dependent, is carried out by 1) recognition of the primer terminus by RFC () binding to and disruption of the PCNA trimer, and then 3) topologically linking the PCNA to the DNA . In this report, we describe the purification and properties of recombinant human RFC expressed in Sf9 cells from baculovirus expression vectors . Like native RFC derived from 293 cells, recombinant RFC was found to support SV40 DNA synthesis and polymerase delta DNA synthesis in vitro and to possess an ATPase activity that was highly stimulated by DNA and further augmented by PCNA . Assembly of RFC was observed to involve distinct subunit interactions in which both the 36- and 38-kDa subunits interacted with the 37-kDa subunit, and the 40-kDa subunit interacted with the 36-kDa subunit-37-kDa subunit subcomplex . The 140-kDa subunit was found to require interactions primarily with the 38- and 40-kDa subunits for incorporation into the complex . In addition, a stable subcomplex lacking the 140-kDa subunit, although defective for DNA replication, was found to possess DNA-dependent ATPase activity that was not responsive to the addition of PCNA.

J Biol Chem, 1998 Mar 6, 273(10), 5878 - 84
Molecular characterization of the 50- and 57-kDa subunits of the bovine vacuolar proton pump; Zhou Z et al.; The vacuolar type proton-translocating ATPase of clathrin-coated vesicles is composed of two large domains: an extramembranous catalytic sector and a transmembranous proton channel . In addition, two polypeptides of 50 and 57 kDa have been found to co-purify with the pump . These proteins, termed SFD (sub-fifty-eight-kDa dimer) activate ATPase activity of the enzyme and couple ATPase activity to proton flow (Xie, X.-S., Crider, B.P., Ma, Y.-M., and Stone, D . K . (1994) J . Biol . Chem . 269, 28509-25815) . It has also been reported that the clathrin-coated vesicle proton pump contains AP50, a 50-kDa component of the AP-2 complex responsible for the assembly of clathrin-coated pits, and that AP50 is essential for function of the proton pump (Liu, Q., Feng, Y., and Forgac, M . (1994) J . Biol . Chem . 269, 31592-31597) . We demonstrate through the use of anti-AP50 antibody, identical to that of the latter study, that hydroxylapatite chromatography removes AP50 from impure proton pump preparations and that purified proton pump, devoid of AP50, is fully functional . To determine the true molecular identity of SFD, both the 50- and 57-kDa polypeptides were directly sequenced . A polymerase chain reaction-based strategy was used to screen a bovine brain cDNA library, yielding independent full-length clones (SFD-4A and SFD-21); these were identical in their open reading frames and encoded a protein with a predicted mass of 54,187 Da . The SFD-21 clone was then used in a reverse transcription-polymerase chain reaction-based strategy to isolate a related, but distinct, transcript present in bovine brain mRNA . The nucleotide and predicted amino acid sequences of this isolate are identical to SFD-21 except that the isolate contains a 54-base pair insert in the open reading frame, resulting in a protein with a predicted mass of 55,933 Da . Both clones had 16% identity to VMA13 of Saccharomyces cerevisiae . No sequence homology between the SFD clones and AP50 was detectable . Anti-peptide antibodies were generated against an epitope common to the two proteins and to the unique 18-amino acid insert of the larger protein . The former reacted with both components of native SFD, whereas the latter reacted only with the 57-kDa component . We term the 57- and 50-kDa polypeptides SFDalpha and SFDbeta, respectively.

J Biol Chem, 1998 Mar 6, 273(10), 5780 - 4
Selective uncoupling of RGS action by a single point mutation in the G protein alpha-subunit; DiBello PR et al.; Heterotrimeric G proteins function as molecular relays, shuttling between cell surface receptors and intracellular effectors that propagate a signal . G protein signaling is governed by the rates of GTP binding (catalyzed by the receptor) and GTP hydrolysis . RGS proteins (regulators of G protein signaling) were identified as potent negative regulators of G protein signaling pathways in simple eukaryotes and are now known to act as GTPase-activating proteins (GAPs) for G protein alpha-subunits in vitro . It is not known, however, if Galpha GAP activity is responsible for the regulatory action of RGS proteins in vivo . We describe here a Galpha mutant in yeast (gpa1(sst)) that phenotypically mimics the loss of its cognate RGS protein (SST2) . The gpa1(sst) mutant is resistant to an activated allele of SST2 in vivo and is unresponsive to RGS GAP activity in vitro . The analogous mutation in a mammalian Gqalpha is also resistant to RGS action in transfected cells . These mutants demonstrate that RGS proteins act through Galpha and that RGS-GAP activity is responsible for their desensitizing activity in cells . The Galphasst mutant will be useful for uncoupling RGS-mediated regulation from other modes of signal regulation in whole cells and animals.

Biochemistry, 1998 Mar 3, 37(9), 3078 - 85
The bis(naphthalimide) DMP-840 causes cytotoxicity by its action against eukaryotic topoisomerase II; Nitiss JL et al.; DMP 840 ((R,R)-2,2'-{1,2-ethanediylbis{imino(1-methyl-2, 1-ethanediyl)}-bis(5-nitro-1H-benz{de}isoquinoline-1,3(2H)-dione} dimethanesulfonate) is a novel bis(naphthalimide) that has shown promising antitumor activity in a variety of preclinical model systems . The compound binds to DNA with high affinity and intercalates, but the mechanism of cell killing has not been elucidated . We have used yeast strains to test whether DMP-840 is active against either topoisomerase I or II . We found that temperature-sensitive top2 mutants resistant to etoposide or amsacrine also confer resistance to DMP-840 . In addition, cells overexpressing yeast topoisomerase II were hypersensitive to the drug . By contrast, top1 deletions rendered cells hypersensitive to the drug . These results strongly suggest that DMP-840 acts against eukaryotic topoisomerase II and kills cells by converting the enzyme into a cellular poison . We verified that DMP-840 is active against eukaryotic topoisomerase II by demonstrating that the drug stimulates formation of a cleavage complex with purified yeast topoisomerase II in vitro . We also demonstrated that the drug is active against human topoisomerase II by showing that expression of human topoisomerase II restored sensitivity of resistant yeast cells to DMP-840 . We have also directly demonstrated that DMP-840 acts as a poison against purified human topoisomerase II alpha . Taken together, these results indicate that DMP-840 acts like other intercalating topoisomerase II poisons; it kills eukaryotic cells by stabilizing the cleavage complex of topoisomerase II with DNA.

Proc Natl Acad Sci U S A, 1998 Mar 3, 95(5), 2586 - 91
A splicing variant of a death domain protein that is regulated by a mitogen-activated kinase is a substrate for c-Jun N-terminal kinase in the human central nervous system; Zhang Y et al.; The mitogen-activated kinase activating death domain protein (MADD) that is differentially expressed in neoplastic vs . normal cells (DENN) was identified as a substrate for c-Jun N-terminal kinase 3, the first demonstration of such an activity for this stress-activated kinase that is predominantly expressed in the brain . A splice isoform was identified that is a variant of MADD . A protein identical to MADD has been reported to be expressed differentially in neoplastic vs . normal cells and is termed "DENN." We demonstrated differential effects on DENN/MADD in a stressed vs . basal environment . Using in situ hybridization, we localized both the substrate and the kinase to large pyramidal neurons in the human hippocampus . It was interesting that, in four of four patients with neuropathologically confirmed acute hypoxic changes, we detected a unique translocation of DENN/MADD to the nucleolus . These changes were apparent only in neurons sensitive to hypoxia . Moreover, in those cells, translocation of the substrate was accompanied by nuclear translocation of JNK3 . These findings place DENN/MADD and JNK in important hypoxia insult-induced intracellular signaling pathways . Our conclusions are important for future studies for understanding these stress-activated mechanisms.

Proc Natl Acad Sci U S A, 1998 Mar 3, 95(5), 2498 - 502
Fatty acid-induced beta cell apoptosis: a link between obesity and diabetes; Shimabukuro M et al.; Like obese humans, Zucker diabetic fatty (ZDF) rats exhibit early beta cell compensation for insulin resistance (4-fold beta cell hyperplasia) followed by decompensation (>50% loss of beta cells) . In prediabetic and diabetic ZDF islets, apoptosis measured by DNA laddering is increased 3- and >7-fold, respectively, compared with lean ZDF controls . Ceramide, a fatty acid-containing messenger in cytokine-induced apoptosis, was significantly increased (P < 0.01) in prediabetic and diabetic islets . Free fatty acids (FFAs) in plasma are high (>1 mM) in prediabetic and diabetic ZDF rats; therefore, we cultured prediabetic islets in 1 mM FFA . DNA laddering rose to 19.6% vs . 4.6% in lean control islets, preceded by an 82% increase in ceramide . C2-Ceramide without FFA induced DNA laddering, but fumonisin B1, a ceramide synthetase inhibitor, completely blocked FFA-induced DNA laddering in cultured ZDF islets . {3H}Palmitate incorporation in {3H}ceramide in ZDF islets was twice that of controls, but {3H}palmitate oxidation was 77% less . Triacsin C, an inhibitor of fatty acyl-CoA synthetase, and troglitazone, an enhancer of FFA oxidation in ZDF islets, both blocked DNA laddering . These agents also reduced inducible nitric oxide (NO) synthase mRNA and NO production, which are involved in FFA-induced apoptosis . In ZDF obesity, beta cell apoptosis is induced by increased FFA via de novo ceramide formation and increased NO production.

Proc Natl Acad Sci U S A, 1998 Mar 3, 95(5), 2400 - 5
Overexpression of the SUP45 gene encoding a Sup35p-binding protein inhibits the induction of the de novo appearance of the {PSI+} prion; Derkatch IL et al.; {PSI+}, a non-Mendelian element found in some strains of Saccharomyces cerevisiae, is presumed to be the manifestation of a self-propagating prion conformation of eRF3 (Sup35p) . Translation termination factor eRF3 enhances the activity of release factor eRF1 (Sup45p) . As predicted by the prion model, overproduction of Sup35p induces the de novo appearance of {PSI+} . However, another non-Mendelian determinant, {PIN+}, is required for this induction . We now show that SUP45 overexpression inhibits the induction of {PSI+} by Sup35p overproduction in {PIN+} strains, but has no effect on the propagation of {PSI+} or on the {PIN} status of the cells . We also show that SUP45 overexpression counteracts the growth inhibition usually associated with overexpression of SUP35 in {PSI+} strains . We argue that excess Sup45p inhibits {PSI+} seed formation . Because Sup45p complexes with Sup35p, we hypothesize that excess Sup45p may sequester Sup35p, thereby reducing the opportunity for Sup35p conformational flips and/or self-interactions leading to prion formation . This in vivo yeast result is reminiscent of the in vitro finding by investigators of Alzheimer disease that apolipoprotein E inhibits amyloid nucleation, but does not reduce seeded growth of amyloid.

Proc Natl Acad Sci U S A, 1998 Mar 3, 95(5), 2227 - 32
Brome mosaic virus RNA replication protein 1a dramatically increases in vivo stability but not translation of viral genomic RNA3; Janda M et al.; Brome mosaic virus (BMV), a positive-strand RNA virus in the alphavirus-like superfamily, encodes two RNA replication proteins: 1a, which contains a helicase-like domain and a domain implicated in RNA capping, and 2a, which contains a polymerase-like domain . To further explore their functions, we expressed 1a and 2a individually and together in yeast also expressing replicatable transcripts of BMV genomic RNA3 . Complementing prior results that 1a and 2a are required jointly for positive-strand RNA synthesis, both also were required for negative-strand RNA synthesis . Nevertheless, in the absence of 2a, 1a expression increased the accumulation of DNA-derived RNA3 transcripts 8-fold . Increased accumulation was specific for RNA3: none of a diverse set of yeast mRNAs tested showed increased accumulation in the presence of 1a . Increased RNA3 accumulation was not due to increased DNA transcription, but to a 20- to 40-fold increase in the in vivo half-life of RNA3 from 5-10 min in the absence of 1a to more than 3 hr in the presence of 1a . Although (1a+2a)-dependent RNA replication selectively amplified the natural viral 5' end from among multiple transcription starts of DNA-derived RNA3 transcripts, 1a-induced stabilization affected all RNA3 transcripts, without specificity for the precise viral 5' end . Increased RNA3 accumulation did not increase expression of a directly translatable, 5'-proximal gene in RNA3, implying that 1a-induced stabilization blocked rather than facilitated RNA3 translation . These and other results suggest that the striking, 1a-induced stabilization of RNA3 may reflect an interaction involved in recruiting viral RNA templates into RNA replication while diverting them from the competing processes of translation and degradation.

Genomics, 1998 Feb 1, 47(3), 350 - 8
FACL4, a new gene encoding long-chain acyl-CoA synthetase 4, is deleted in a family with Alport syndrome, elliptocytosis, and mental retardation; Piccini M et al.; We observed a family in which two boys were diagnosed with Alport syndrome, elliptocytosis, and mental retardation and carried a large deletion of the Xq22.3-q23 region, encompassing the COL4A5 gene . This suggests the possibility of a new contiguous gene syndrome . In an attempt to characterize the genes contributing to this complex phenotype, we have isolated a gene encoding a new long-chain acyl-CoA synthetase (FACL4 or LACS4) from the region deleted in these patients . Among several ESTs identified by searching the human gene map database maintained at the National Center for Biotechnology Information, using the map position as a query, only one was deleted in the patients . RACE products containing the entire ORF were subsequently generated . Northern blot analysis showed a 5-kb mRNA expressed in several tissues except for liver and lung . Brain shows a longer transcript, possibly reflecting the use of a brain-specific upstream ATG start codon . FACL4 encodes a predicted protein product of 670 amino acids (711 in brain), with a remarkable level of conservation compared to the rat acyl-CoA synthetases ACS4 and brain-specific ACS3 protein sequences . We are investigating the possibility that the absence of this enzyme may play a role in the development of mental retardation or other signs associated with Alport syndrome in the family .

Biochem J, 1998 Feb 15, 330 ( Pt 1), 277 - 86
Signalling pathways of an insulin-mimetic phosphoinositolglycan-peptide in muscle and adipose tissue; Kessler A et al.; A novel phosphoinositolglycan-peptide (PIG-P) from the yeast Saccharomyces cerevisiae potently mimicks insulin action on glucose transport and metabolism in rat muscle and adipose tissue . The aim of the present study was to elucidate the cellular signalling pathways of this insulin-mimetic compound . Rapid onset and reversibility of PIG-P action on glucose transport were observed in isolated adipocytes with a half-time of transport stimulation of 6-8 min (insulin less than 5 min) . Combined treatment with PIG-P and insulin indicated additive stimulation of glucose transport at submaximal concentrations and non-additive action of both agents at maximal doses . The tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) was markedly increased in response to PIG-P in rat cardiomyocytes without any effect on the tyrosine phosphorylation of the insulin receptor beta-subunit . PIG-P action in these cells was accompanied by phosphorylation/dephosphorylation of several proteins with molecular masses of 15-30 kDa, a response not detected with insulin . Downstream signalling of IRS-1 was then analysed by monitoring IRS-1-associated phosphatidylinositol 3-kinase (PI 3-kinase) activity in cardiomyocytes . A stable (2 and 15 min incubation with PIG-P) 7-fold stimulation corresponding to about 50% of insulin action could be detected . Increased tyrosine phosphorylation of IRS-1 and enhanced PI 3-kinase activity in response to PIG-P independent of the insulin receptor was also observed in isolated adipocytes . Involvement of PI 3-kinase in PIG-P action was subsequently confirmed by the dose-dependent inhibition of PIG-P-activated glucose transport in rat diaphragm and adipocytes by the PI 3-kinase inhibitors wortmannin and LY294002 . These data suggest divergent upstream signalling by insulin and PIG-P involving phosphoproteins not affected by insulin . However, PIG-P and insulin action converge at the level of IRS-1 inducing insulin-independent PI 3-kinase-mediated signalling to glucose transport.

Biochem J, 1998 Feb 15, 330 ( Pt 1), 181 - 7
In vitro analysis of UV-damage-induced inhibition of replication; Drissi R et al.; We investigated DNA-damage-induced inhibition of replication by using an in vitro system, with which both replication and repair can be examined simultaneously . The system contains non-irradiated simian virus 40 (SV40) origin-containing DNA, UV-irradiated circular duplex DNA lacking an SV40 origin, and cell extracts that support both replication and repair activities . Using this system, we show that replication is significantly inhibited in the presence of UV-irradiated, but not non-irradiated, DNA and, to a lesser extent, repair activity is also inhibited by the presence of replication activity . In contrast, replication activity was not affected by UV-damaged DNA when the reactions were carried out with purified replication proteins, suggesting that protein factor(s) in the cell extracts are involved in the inhibition of replication that is triggered by DNA damage . Inhibition was efficiently reversed by the combined actions of proteins involved in both repair and replication, suggesting that the inhibition of replication observed in our system may be caused by the recruitment of replication proteins to damaged DNA sites.

Curr Biol, 1998 Jan 29, 8(3), 177 - 80
Reconstitution of human telomerase activity in vitro; Beattie TL et al.; Telomerase is a ribonucleoprotein enzyme complex that adds single-stranded telomere DNA to chromosome ends {1} . The RNA component of telomerase contains the template for telomeric DNA addition and is essential for activity {1,2} . Telomerase proteins have been identified in ciliates, yeast and mammals {3-12} . In Saccharomyces cerevisiae, the Est2 protein is homologous to the 123 kDa reverse transcriptase subunit of Euplotes telomerase, and is essential for telomerase activity {8} . In humans, telomerase activity is associated with the telomerase RNA hTR {13}, the telomerase RNA-binding protein TP1/TLP1 {5,12} and the TP2 protein encoded by the human EST2 homolog {12} (also known as TRT1, hEST2 or TCS1 {9-11}) . The minimal complex sufficient for activity is, however, unknown . We have reconstituted human telomerase activity in reticulocyte lysates and find that only exogenous hTR and TP2 are required for telomerase activity in vitro . Recognition of telomerase RNA by TP2 was species specific, and nucleotides 10-159 of hTR were sufficient for telomerase activity . Telomerase activity immunoprecipitated from the reticulocyte lysate contained hTR and recombinant TP2 . Substitution of conserved amino acid residues in the reverse transcriptase domain of TP2 completely abolished telomerase activity . We suggest that TP2 and hTR might represent the minimal catalytic core of human telomerase.

Curr Opin Genet Dev, 1998 Feb, 8(1), 36 - 42
Proteolysis and the G1-S transition: the SCF connection; Krek W; Temporal control of ubiquitin-proteasome mediated protein degradation is critical for normal G1 and S phase progression . Recent work has shown that central to the temporal control mechanism is a relationship between newly identified E3 ubiquitin protein ligases, designated SCFs (Skp1-cullin-F-box protein ligase complexes), which confer substrate specificity on ubiquitination reactions and the activities of protein kinases that phosphorylate substrates destined for destruction at specific sites, thereby converting them into preferred targets for ubiquitin modification catalyzed by SCFs . The constituents of SCFs are members of evolutionary conserved protein families . SCF-based ubiquitination pathways may play a key role in diverse biological processes, such as cell proliferation, differentiation and development.

Mol Gen Genet, 1998 Feb, 257(4), 442 - 51
A genetic screen for elements of the network that regulates neurogenesis in Drosophila; Rottgen G et al.; The development of external sensory organs on the notum of Drosophila is promoted by the proneural genes achaete and scute . Their activity defines proneural cell clusters in the wing imaginal disc . Ectopic expression, under control of the GAL4 system, of the proneural gene lethal of scute (l'sc) causes the development of ectopic bristles . Persistent ectopic expression of l'sc is not sufficient to impose a neural fate on any given cell . This implies that mutual inhibition, mediated by the Notch signaling pathway, occurs among the cells of the ectopic proneural cluster . Consequently, the dominant, quantifiable phenotype associated with ectopic expression of l'sc is modified by mutations in genes known to be involved in neurogenesis . This phenotype has been utilized to screen for dominant enhancers and suppressors that modify the number of ectopic bristles . In this way, about 100,000 progeny of EMS or X-ray-treated flies have been analyzed to identify autosomal genes involved in regulation of the neural fate . In addition 1200 chromosomes carrying lethal P-element insertions were screened for modifiers . Besides mutations in genes expected to modify the phenotype, we have isolated mutations in six genes not known so far to be involved in neurogenesis.

Mol Cell Biol, 1998 Apr, 18(4), 2298 - 308
Shc and Enigma are both required for mitogenic signaling by Ret/ptc2; Durick K et al.; Ret/ptc2 is a constitutively active, oncogenic form of the c-Ret receptor tyrosine kinase . Like the other papillary thyroid carcinoma forms of Ret, Ret/ptc2 is activated through fusion of the Ret tyrosine kinase domain to the dimerization domain of another protein . Investigation of requirements for Ret/ptc2 mitogenic activity, using coexpression with dominant negative forms of Ras and Raf, indicated that these proteins are required for mitogenic signaling by Ret/ptc2 . Because activation of Ras requires recruitment of Grb2 and SOS to the plasma membrane, the subcellular distribution of Ret/ptc2 was investigated, and it was found to localize to the cell periphery . This localization was mediated by association with Enigma via the Ret/ptc2 sequence containing tyrosine 586 . Because Shc interacts with MEN2 forms of Ret, and because phosphorylation of Shc results in Grb2 recruitment and subsequent signaling through Ras and Raf, the potential interaction between Ret/ptc2 and Shc was investigated . The PTB domain of Shc also interacted with Ret/ptc2 at tyrosine 586, and this association resulted in tyrosine phosphorylation of Shc . Coexpression of chimeric proteins demonstrated that mitogenic signaling from Ret/ptc2 required both recruitment of Shc and subcellular localization by Enigma . Because Shc and Enigma interact with the same site on a Ret/ptc2 monomer, dimerization of Ret/ptc2 allows assembly of molecular complexes that are properly localized via Enigma and transmit mitogenic signals via Shc.

Mol Cell Biol, 1998 Apr, 18(4), 2252 - 61
Adf-1 is a nonmodular transcription factor that contains a TAF-binding Myb-like motif; Cutler G et al.; Adf-1 is an essential Drosophila melanogaster sequence-specific transactivator that binds the promoters of a diverse group of genes . We have performed a comprehensive mapping of the functional domains of Adf-1 to study the role of transactivators in the process of gene activation . Using a series of clustered point mutations and small deletions we have identified regions of Adf-1 required for DNA binding, dimerization, and activation . In contrast to most enhancer-binding factors, the Adf-1 activation regions are nonmodular and depend on an intact protein, including the Adf-1 DNA-binding domain, for activity . Like many transcriptional activators, Adf-1 contains a TFIID-binding domain that can interact with specific TAF subunits . Although TAFs are required for Adf-1-directed activation, TAF binding is not sufficient, suggesting that Adf-1 may direct multiple essential steps during activation . Interestingly, both the TAF-binding domain and the DNA-binding domain contain sequences homologous to those of the Myb family of DNA-binding domains . Thus, Adf-1 has evolved an unusual structure containing two versions of the Myb motif, one that binds DNA and one that binds proteins.

Mol Cell Biol, 1998 Apr, 18(4), 2240 - 51
The tomato Hsf system: HsfA2 needs interaction with HsfA1 for efficient nuclear import and may be localized in cytoplasmic heat stress granules; Scharf KD et al.; In heat-stressed (HS) tomato (Lycopersicon peruvianum) cell cultures, the constitutively expressed HS transcription factor HsfA1 is complemented by two HS-inducible forms, HsfA2 and HsfB1 . Because of its stability, HsfA2 accumulates to fairly high levels in the course of a prolonged HS and recovery regimen . Using immunofluorescence and cell fractionation experiments, we identified three states of HsfA2: (i) a soluble, cytoplasmic form in preinduced cultures maintained at 25 degrees C, (ii) a salt-resistant, nuclear form found in HS cells, and (iii) a stored form of HsfA2 in cytoplasmic HS granules . The efficient nuclear transport of HsfA2 evidently requires interaction with HsfA1 . When expressed in tobacco protoplasts by use of a transient-expression system, HsfA2 is mainly retained in the cytoplasm unless it is coexpressed with HsfA1 . The essential parts for the interaction and nuclear cotransport of the two Hsfs are the homologous oligomerization domain (HR-A/B region of the A-type Hsfs) and functional nuclear localization signal motifs of both partners . Direct physical interaction of the two Hsfs with formation of relatively stabile hetero-oligomers was shown by a two-hybrid test in Saccharomyces cerevisiae as well as by coimmunoprecipitation using tomato and tobacco whole-cell lysates.

Mol Cell Biol, 1998 Apr, 18(4), 2218 - 29
A role for CREB binding protein and p300 transcriptional coactivators in Ets-1 transactivation functions; Yang C et al.; The Ets-1 transcription factor plays a critical role in cell growth and development, but the means by which it activates transcription are still unclear (J . C . Bories, D . M . Willerford, D . Grevin, L . Davidson, A . Camus, P . Martin, D . Stehelin, F . W . Alt, and J . C . Borles, Nature 377:635-638, 1995; N . Muthusamy, K . Barton, and J . M . Leiden, Nature 377:639-642, 1995) . Here we show that Ets-1 binds the transcriptional coactivators CREB binding protein (CBP) and the related p300 protein (together referred to as CBP/p300) and that this interaction is required for specific Ets-1 transactivation functions . The Ets-1- and c-Myb-dependent aminopeptidase N (CD13/APN) promoter and an Ets-1-dependent artificial promoter were repressed by adenovirus E1A, a CBP/p300-specific inhibitor . Furthermore, Ets-1 activity was potentiated by CBP and p300 overexpression . The transactivation function of Ets-1 correlated with its ability to bind an N-terminal cysteine- and histidine-rich region spanning CBP residues 313 to 452 . Ets-1 also bound a second cysteine- and histidine-rich region of CBP, between residues 1449 and 1892 . Both Ets-1 and CBP/p300 formed a stable immunoprecipitable nuclear complex, independent of DNA binding . This Ets-1-CBP/p300 immunocomplex possessed histone acetyltransferase activity, consistent with previous findings that CBP/p300 is associated with such enzyme activity . Our results indicate that CBP/p300 may mediate antagonistic and synergistic interactions between Ets-1 and other transcription factors that use CBP/p300 as a coactivator, including c-Myb and AP-1.

Mol Cell Biol, 1998 Apr, 18(4), 2196 - 204
Snt309p, a component of the Prp19p-associated complex that interacts with Prp19p and associates with the spliceosome simultaneously with or immediately after dissociation of U4 in the same manner as Prp19p; Chen HR et al.; The yeast protein Prp19p is essential for pre-mRNA splicing and is associated with the spliceosome concurrently with or just after dissociation of U4 small nuclear RNA . In splicing extracts, Prp19p is associated with several other proteins in a large protein complex of unknown function, but at least one of these proteins is also essential for splicing (W.-Y . Tarn, C.-H . Hsu, K.-T . Huang, H.-R . Chen, H.-Y . Kao, K.-R . Lee, and S.-C . Cheng, EMBO J . 13:2421-2431, 1994) . To identify proteins in the Prp19p-associated complex, we have isolated trans-acting mutations that exacerbate the phenotypes of conditional alleles of prp19, using the ade2-ade3 sectoring system . A novel splicing factor, Snt309p, was identified through such a screen . Although the SNT309 gene was not essential for growth of Saccharomyces cerevisiae under normal conditions, yeast cells containing a null allele of the SNT309 gene were temperature sensitive and accumulated pre-mRNA at the nonpermissive temperature . Far-Western blot analysis revealed direct interaction between Prp19p and Snt309p . Snt309p was shown to be a component of the Prp19p-associated complex by Western blot analysis . Immunoprecipitation studies demonstrated that Snt309p was also a spliceosomal component and associated with the spliceosome in the same manner as Prp19p during spliceosome assembly . These results suggest that the functions of Prp19p and Snt309p in splicing may require coordinate action of these two proteins.

Mol Cell Biol, 1998 Apr, 18(4), 2130 - 42
Functions of the N- and C-terminal domains of human RAP74 in transcriptional initiation, elongation, and recycling of RNA polymerase II; Lei L et al.; Transcription factor IIF (TFIIF) cooperates with RNA polymerase II (pol II) during multiple stages of the transcription cycle including preinitiation complex assembly, initiation, elongation, and possibly termination and recycling . Human TFIIF appears to be an alpha2beta2 heterotetramer of RNA polymerase II-associating protein 74- and 30-kDa subunits (RAP74 and RAP30) . From inspection of its 517-amino-acid (aa) sequence, the RAP74 subunit appears to comprise separate N- and C-terminal domains connected by a flexible loop . In this study, we present functional data that strongly support this model for RAP74 architecture and further show that the N- and C-terminal domains and the central loop of RAP74 have distinct roles during separate phases of the transcription cycle . The N-terminal domain of RAP74 (minimally aa 1 to 172) is sufficient to deliver pol II into a complex formed on the adenovirus major late promoter with the TATA-binding protein, TFIIB, and RAP30 . A more complete N-terminal domain fragment (aa 1 to 217) strongly stimulates both accurate initiation and elongation by pol II . The region of RAP74 between aa 172 and 205 and a subregion between aa 170 and 178 are critical for both accurate initiation and elongation, and mutations in these regions have similar effects on initiation and elongation . Based on these observations, RAP74 appears to have similar functions in initiation and elongation . The central region and the C-terminal domain of RAP74 do not contribute strongly to single-round accurate initiation or elongation stimulation but do stimulate multiple-round transcription in an extract system.

Mol Cell Biol, 1998 Apr, 18(4), 2045 - 54
Expansions and contractions in a tandem repeat induced by double-strand break repair; Paques F et al.; Repair of a double-strand break (DSB) in yeast can induce very frequent expansions and contractions in a tandem array of 375-bp repeats . These results strongly suggest that DSB repair can be a major source of amplification of tandemly repeated sequences . Most of the DSB repair events are not associated with crossover . Rearrangements appear in 50% of these repaired recipient molecules . In contrast, the donor template nearly always remains unchanged . Among the rare crossover events, similar rearrangements are found . These results cannot readily be explained by the gap repair model of Szostak et al . (J . W . Szostak, T . L . Orr-Weaver, R . J . Rothstein, and F . W . Stahl, Cell 33:25-35, 1983) but can be explained by synthesis-dependent strand annealing (SDSA) models that allow for crossover . Support for SDSA models is provided by a demonstration that a single DSB repair event can use two donor templates located on two different chromosomes.

Mol Cell Biol, 1998 Apr, 18(4), 1967 - 77
Protein serine/threonine phosphatase Ptc2p negatively regulates the unfolded-protein response by dephosphorylating Ire1p kinase; Welihinda AA et al.; Cells respond to the accumulation of unfolded proteins in the endoplasmic reticulum (ER) by increasing the transcription of the genes encoding ER-resident chaperone proteins . Ire1p is a transmembrane protein kinase that transmits the signal from unfolded proteins in the lumen of the ER by a mechanism that requires oligomerization and trans-autophosphorylation of its cytoplasmic-nucleoplasmic kinase domain . Activation of Ire1p induces a novel spliced form of HAC1 mRNA that produces Hac1p, a transcription factor that is required for activation of the transcription of genes under the control of the unfolded-protein response (UPR) element . Searching for proteins that interact with Ire1p in Saccharomyces cerevisiae, we isolated PTC2, which encodes a serine/threonine phosphatase of type 2C . The Ptc2p interaction with Ire1p is specific, direct, dependent on Ire1p phosphorylation, and mediated through a kinase interaction domain within Ptc2p . Ptc2p dephosphorylates Ire1p efficiently in an Mg2+-dependent manner in vitro . PTC2 is nonessential for growth and negatively regulates the UPR pathway . Strains carrying null alleles of PTC2 have a three- to fourfold-increased UPR and increased levels of spliced HAC1 mRNA . Overexpression of wild-type Ptc2p but not catalytically inactive Ptc2p reduces levels of spliced HAC1 mRNA and attenuates the UPR, demonstrating that the phosphatase activity of Ptc2p is required for regulation of the UPR . These results demonstrate that Ptc2p downregulates the UPR by dephosphorylating Ire1p and reveal a novel mechanism of regulation in the UPR pathway upstream of the HAC1 mRNA splicing event.

Mol Cell Biol, 1998 Apr, 18(4), 1835 - 43
Multiple developmental requirements of noisette, the Drosophila homolog of the U2 snRNP-associated polypeptide SP3a60; Meyer V et al.; We report the cloning of the noisette gene (noi), which encodes the Drosophila melanogaster ortholog of a U2 snRNP-associated splicing factor, SF3a60 (SAP61) in humans and PRP9p in Saccharomyces cerevisiae . Antibodies raised against human SF3a60 recognized NOI in flies, showing a nuclear localization in all the stages examined, including the embryo, the dividing cells of imaginal discs, and the larval polyploid nuclei . NOI is expressed in somatic and germinal cells of both male and female gonads . By mobilization of P transposons, we have generated a large number of noi mutations . Complete loss of function resulted in lethality at the end of embryogenesis, without obvious morphological defects . Hypomorphic alleles revealed multiple roles of noi for the survival and differentiation of male germ cells, the differentiation of female germ cells, and the development of several adult structures.

Mol Cell Biol, 1998 Apr, 18(4), 1774 - 82
SWI-SNF complex participation in transcriptional activation at a step subsequent to activator binding; Ryan MP et al.; The SWI-SNF complex in yeast and related complexes in higher eukaryotes have been implicated in assisting gene activation by overcoming the repressive effects of chromatin . We show that the ability of the transcriptional activator GAL4 to bind to a site in a positioned nucleosome is not appreciably impaired in swi mutant yeast cells . However, chromatin remodeling that depends on a transcriptional activation domain shows a considerable, although not complete, SWI-SNF dependence, suggesting that the SWI-SNF complex exerts its major effect at a step subsequent to activator binding . We tested this idea further by comparing the SWI-SNF dependence of a reporter gene based on the GAL10 promoter, which has an accessible upstream activating sequence and a nucleosomal TATA element, with that of a CYC1-lacZ reporter, which has a relatively accessible TATA element . We found that the GAL10-based reporter gene showed a much stronger SWI-SNF dependence than did the CYC1-lacZ reporter with several different activators . Remarkably, transcription of the GAL10-based reporter by a GAL4-GAL11 fusion protein showed a nearly complete requirement for the SWI-SNF complex, strongly suggesting that SWI-SNF is needed to allow access of TFIID or the RNA polymerase II holoenzyme . Taken together, our results demonstrate that chromatin remodeling in vivo can occur by both SWI-SNF-dependent and -independent avenues and suggest that the SWI-SNF complex exerts its major effect in transcriptional activation at a step subsequent to transcriptional activator-promoter recognition.

J Chromatogr A, 1998 Feb 6, 795(2), 199 - 210
Direct measurements of convective fluid velocities in superporous agarose beads; Gustavsson PE et al.; Superporous agarose beads contain two sets of pores, diffusion pores and so-called superpores or flow pores, in which the chromatographic flow can transport substances to the interior of each individual bead {Gustavsson and Larsson, J . Chromatogr . A 734 (1996) 231} . The existence of pore flow may be proven indirectly by the chromatographic performance of beads but it has never been directly demonstrated in a chromatographic bed . In this report, pore flow was directly measured by following the movement of micro-particles (dyed yeast cells) in a packed bed . The passage of the micro-particles through the superpores and through the interstitial pores was followed by a microscope/video camera focused on beads which were situated four layers from the glass wall . The video recordings were subsequently used to determine the convective fluid velocities in both the superpores and the interstitial pores . Experiments were carried out with three different bead size ranges, all of which contained superporous beads having an average superpore diameter of 30 microns . The superpore fluid velocity as % of interstitial fluid velocity was determined to be 2-5% for columns packed with 300-500-micron beads (3% average value), 6-12% for columns packed with 180-300-micron beads (7% average value) and 11-24% for columns packed with 106-180-micron beads (17% average value) . These data were compared to and found to agree with theoretically calculated values based on the Kozeny-Carman equation . In order to observe and accurately measure fluid velocities within a chromatographic bed, special techniques were adopted . Also, precautions were made to ensure that the experimental conditions used were representative of normal chromatography runs.

Nucleic Acids Symp Ser, 1997, (36), 64 - 7
Mpp10p, a new protein component of the U3 snoRNP required for processing of 18S rRNA precursors; Baserga SJ et al.; We have used the yeast, Saccharomyces cerevisiae, as a model system to identify a new protein component of the U3 small nucleolar ribonucleoprotein (snoRNP) and to study its role in pre-rRNA processing . Mpp10p, which we have cloned and characterized from humans, mice and yeast, is a 110 kDa protein in yeast . Antibodies to it immunoprecipitate the U3 snoRNA, indicating that it is a U3 snoRNP component . MPP10 is an essential gene . Depletion of Mpp10p by repression of MPP10 expressed from a conditional promoter causes slow growth . Analysis of pre-rRNA processing in the depleted cells indicates that Mpp10p is required for processing at the 3 U3 snoRNA-dependent sites in the pre-rRNA (A0, A1, A2) . Truncations of terminal charged domains create Mpp10 proteins that confer slow growth at 30 degrees C and/or at 22 degrees C . Analysis of pre-rRNA processing in these mutants indicates a deficiency in processing at only two of the U3 snoRNA-dependent sites in the pre-rRNA (A1 and A2) . Therefore truncated Mpp10 proteins are able to separate the function of the U3 snoRNP into a requirement for it in distinct processing events.

Nucleic Acids Res, 1998 Mar 1, 26(5), 1329 - 36
Selection of novel, specific single-stranded DNA sequences by Flp, a duplex-specific DNA binding protein; Zhu XD et al.; Flp is a member of the integrase family of site-specific recombinases . Flp is known to be a double-stranded (ds)DNA binding protein that binds sequence specifically to the 13 bp binding elements in the FRT site (Flprecognitiontarget) . We subjected a random pool of oligonucleotides to the in vitro binding site selection method and have unexpectedly recovered a series of single-stranded oligonucleotides to which Flp binds with high affinity . These single-stranded oligonucleotides differ in sequence from the duplex FRT site . The minimal length of the oligonucleotides which is active is 29 nt . This single strand-specific DNA binding activity is located in the same C-terminal 32 kDa domain of Flp in which the site-specific dsDNA binding activity resides . Competition studies suggest that the apparent affinity of Flp for single-stranded oligonucleotide is somewhat less than for a complete duplex FRT site but greater than for a single duplex 13 bp binding element . We have also shown that Cre, another member of the integrase family of site-specific recombinases, also exhibits single-stranded DNA binding similar to that of Flp.

Nucleic Acids Res, 1998 Mar 1, 26(5), 1301 - 8
Probing structural elements in RNA using engineered disulfide cross-links; Maglott EJ et al.; Three analogs of unmodified yeast tRNAPhe, each possessing a single disulfide cross-link, have been designed and synthesized . One cross-link is between G1 and C72 in the amino acid acceptor stem, a second cross-link is in the central D region of yeast tRNAPhe between C11 and C25 and the third cross-link bridges U16 and C60 at the D loop/T loop interface . Air oxidation to form the cross-links is quantitative and analysis of the cross-linked products by native and denaturing PAGE, RNase T1 mapping, Pb(II) cleavage, UV cross-linking and thermal denaturation demonstrates that the disulfide bridges do not alter folding of the modified tRNAs relative to the parent sequence . The finding that cross-link formation between thiol-derivatized residues correlates with the position of these groups in the crystal structure of native yeast tRNAPhe and that the modifications do not significantly perturb native structure suggests that this methodology should be applicable to the study of RNA structure, conformational dynamics and folding pathways.

Nucleic Acids Res, 1998 Mar 1, 26(5), 1191 - 7
Multiple receptor interaction domains of GRIP1 function in synergy; Schmidt S et al.; Nuclear hormone receptors are exerting their effect on transcription by interacting with basal factors of the transcription machinery and/or by recruiting intermediary factors, such as the mouse protein GRIP1 . GRIP1 is one of the recently identified coactivators for nuclear hormone receptors . Upon interaction with the hormone-binding domain of the receptors, GRIP1 increases their transcriptional activity . Here we show that GRIP1 contains at least two receptor-interacting regions using the hormone-binding domain of several receptors as bait in the yeast two-hybrid assay . GRIP1 interacts in a hormone-dependent manner with the C-termini of nuclear hormone receptors such as GRalpha, TRalpha, TRbeta, RARalpha and RXRalpha but not with v-ErbA . GRIP1 contains several LXXLL motifs which were shown to be required for receptor interaction . A protein fragment containing all of the three LXXLL motifs, but having the activation domain deleted, is able to repress the transcriptional activity of human TRbeta, whereas a region harbouring only one LXXLL motif fails to do so . A protein fragment with two LXXLL motifs exhibits an intermediate modulation of the TRbeta transactivation . While one motif seems to be sufficient for receptor interaction, more than one motif is needed for functional interference.

Nucleic Acids Res, 1998 Mar 1, 26(5), 1179 - 84
Isolation and characterization of RAD51C, a new human member of the RAD51 family of related genes; Dosanjh MK et al.; The yeast and human RAD51 genes encode strand-transfer proteins that are thought to be involved in both recombinational repair of DNA damage and meiotic recombination . In yeast, the Rad51 family of related proteins also includes Rad55, Rad57 and Dmc1 . In mammalian cells, five genes in this family have been identified (HsRAD51, XRCC2, XRCC3, RAD51B/hREC2 and HsDMC1), and here we report the isolation of the sixth member, RAD51C . RAD51C was originally identified by a computer screen of the EST database . A full-length approximately 1.3 kb cDNA clone has been isolated that encodes a protein of 376 aa, having a 18-26% aa identity with other human Rad51 family members . RAD51C includes a previously mapped sequenced-tagged site location near the end of chromosome 17q . The RAD51C transcript is expressed in various human tissues, with highest level of expression in testis, followed by heart muscle, spleen and prostate . Yeast two-hybrid experiments indicate that the Rad51C protein binds to two other members of the Rad51 protein family (Xrcc3 and Rad51B) but not to itself . These findings suggest that Rad51C may function similarly to the yeast Rad55 or Rad57 proteins, rather than as a Rad51 functional homolog.

DNA Seq, 1997, 7(6), 337 - 47
The isolation, localization and characterization of the QM homolog in Drosophila melanogaster; Nguyen-Yue YH et al.; A fragment of 443 bp was amplified from a lambda ZAPII Drosophila central nervous system (CNS) cDNA library using minimally degenerate primers to very conserved regions of the QM gene . This fragment was used as a probe to screen the lambda ZAPII Drosophila CNS cDNA library . Two clones of the Drosophila QM homolog (pDQM-7A1 and pDQM-2B1), each containing the complete coding region, were isolated . The 5'-UTR of this gene was obtained by RACE PCR and ligated to the coding sequence to produce a the full-length copy of the Drosophila QM homolog (DQM) cDNA . The DQM cDNA measures 746 nucleotides in length and encodes a polypeptide of 218 residues . The amino acid sequence shows 76.1 percent identity with human QM and 69.1 percent identity with QSR1, the yeast homolog of QM . Unlike the human or mouse genome which contains multiple copies of the QM gene, the Drosophila genome has only a single copy as indicated by genomic Southern blot analysis . In situ hybridization confirms the presence of a single copy of DQM in the Drosophila genome and localizes it to the left arm of the third chromosome at the end of region 80A (80A-4).

Clin Nephrol, 1998 Feb, 49(2), 69 - 73
Demonstration of DNA replication factor C in human glomerular lesions; Komatsu T et al.; Glomerulosclerosis is a common pathological finding in many human glomerular diseases that ultimately leads to end-stage kidney disease . The regulatory mechanism that controls mesangial proliferation as well as accumulation of mesangial matrix, however, is not known . Recently, a protein factor (MSW) which binds to the specific sequence of the promoter of the alpha 1 and alpha 2 type IV collagen genes was cloned . MSW was found to be identical to a large subunit (Alp145) of DNA replication factor C . These findings suggest that MSW may have important functions in mesangial cell proliferation and type IV collagen synthesis, both of which are prominent findings in glomerulosclerosis . In the present study, we report that augmented expression of MSW protein in human glomerular diseases that exhibit glomerulosclerosis (IgA nephropathy, membranoproliferative glomerulonephritis, and focal glomerulosclerosis) . Minimal expression of MSW protein was observed in human glomerular diseases that rarely show glomerulosclerosis (membranous nephropathy, and minimal change nephrotic syndrome) . There was a significant correlation between the levels of MSW expression and type IV collagen expression . Elevated expressions of both proliferating cell nuclear antigen and MSW were also observed in most patients with proliferative glomerular diseases . These studies suggest that MSW protein plays a regulatory role in the development of mesangial cell proliferation and matrix expansion during progression of glomerular injuries.

Gene, 1998 Feb 27, 208(2), 207 - 13
Cloning of a cDNA from Arabidopsis thaliana homologous to the human XPB gene; Ribeiro DT et al.; The human gene XPB, defective in xeroderma pigmentosum patients complementation group B, encodes a DNA helicase involved in several DNA metabolic pathways, including DNA repair and transcription . The high conservation of this gene has allowed the cloning of homologs in various species, such as mouse, yeast and Drosophila . Not much information on the molecular basis of nucleotide excision repair in plants is available, but these organisms may have similar mechanisms to other eukaryotes . A homolog of XPB was isolated in Arabidopsis thaliana by using polymerase chain reaction (PCR) with degenerate oligonucleotides based on protein domains which are conserved among several species . Screening of an Arabidopsis cDNA library led to the identification and isolation of a cDNA clone with 2670 bp encoding a predicted protein of 767 amino acids, denoted araXPB . Genomic analysis indicated that this is a nuclear single copy gene in plant cells . Northern blot with the cDNA probe revealed a major transcript which migrated at approx . 2,800 b, in agreement with the size of the cDNA isolated . The araXPB protein shares approximately 50% identical and 70% conserved amino acids with the yeast and human homologs . The plant protein maintains all the functional domains found in the other proteins, including nuclear localization signal, DNA-binding domain and helicase motifs, suggesting that it might also act as part of the RNA transcription apparatus, as well as nucleotide excision repair in plant cells.

Biochim Biophys Acta, 1998 Mar 4, 1396(1), 67 - 87
Isolation of an Alu repetitive DNA binding protein and effect of CpG methylation on binding to its recognition sequence; Cox GS et al.; The structure, expression, and evolution of Alu repetitive DNA elements have been extensively studied, but the role of these sequences in the function of primate genomes has yet to be elucidated . The contribution of Alu repetitive sequences (ARS) to the structure, maintenance, or expression of the human genome is undoubtedly mediated by one or more DNA binding proteins . As part of a larger study in this laboratory to define the molecular mechanisms that result in de-repression of the glycoprotein hormone alpha-subunit (GPH alpha) gene in a variety of tumor cell types, it was found that the gene was hypermethylated in a variety of cell lines that produce alpha-subunit at high levels and significantly less methylated in cell lines where the gene is unexpressed or expressed at low levels . This is in sharp contrast to the majority of genes examined in this regard, which show an inverse correlation between methylation and expression . The analysis was extended to a group of clones isolated from a single cell line (HeLa) that were differentially methylated over the GPH alpha gene and exhibited a 400-fold range in its expression . These analyses demonstrated that methylation of a small number of CpG dinucleotides correlated with high level expression of the gene . Two of these sites are imbedded in oppositely oriented Alu repeats located in the 5'-flanking DNA and second intron . The upstream site was examined in some detail . DNase I footprint analysis demonstrated that the protein protects a region encompassing the sequence 5'-TTGAACCCGGGAG-3', and electrophoretic gel mobility shift analysis demonstrated specific binding of a protein to an oligonucleotide containing the DNase footprint sequence . Chromatography of nuclear extracts on Sephacryl S-200, heparin--agarose, and oligonucleotide--Sepharose produced an apparently homogeneous preparation of the 50-53 kDa DNA-binding protein as judged by silver staining of sodium dodecylsulfate polyacrylamide gels . The affinity-purified material was enriched 15- to 18,000-fold over crude nuclear extracts . Binding of this protein to an oligonucleotide containing the DNase-protected sequence was severely inhibited when CpG dinucleotide in the Msp I recognition site was methylated on either the sense or antisense strands . Based on its properties, this protein has been termed MeSABp50 for methylation-sensitive Alu binding protein of 50 kDa.

Genes Chromosomes Cancer, 1998 Mar, 21(3), 217 - 22
Multiple possible sites of BRCA2 interacting with DNA repair protein RAD51; Katagiri T et al.; To investigate the biological consequences of aberrant BRCA2 protein during mammary carcinogenesis, we attempted to identify proteins that normally interact with BRCA2 . By using a yeast two-hybrid system with a hybrid protein that contained residues 639-1,508 of BRCA2 protein fused to the GAL4 DNA-binding domain, we isolated five independent cDNA clones that encoded parts of RAD51 protein, a human homolog of bacterial RecA . In vitro experiments using anti-RAD51 antibody confirmed interaction of BRCA2 with RAD51 . The RAD51-binding region of BRCA2 detected in the present study was distinct from the region reported recently . Further studies using smaller portions of BRCA2 defined at least two additional RAD51-binding domains, residues 982-1,066 and 1,139-1,266 . Our results suggest that BRCA2 can interact with RAD51 through multiple sites of BRCA2 and that control of mitotic and meiotic recombination and/or of genomic integrity through binding to RAD51 may be a crucial mechanism by which BRCA2 suppresses abnormal proliferation of mammary cells.

Eur J Biochem, 1998 Feb 15, 252(1), 162 - 8
Negative control of the Mig1p repressor by Snf1p-dependent phosphorylation in the absence of glucose; Ostling J et al.; Mig1p, a zinc-finger protein that is related to the Krox/Egr, Wilms' tumor and Sp1 proteins, mediates glucose repression in the yeast Saccharomyces cerevisiae . Mig1p is inactive in the absence of glucose, and this inhibition is dependent on the Snf1p (Cat1p) protein kinase . The regulation is mediated by an internal part of Mig1p, and it can be transferred to a Mig1-viral protein 16 (VP16) fusion protein that functions as an activator {Ostling, J., Carlberg, M . & Ronne, H . (1996) Mol . Cell . Biol . 16, 753-761} . We have used Mig1-VP16 to identify three target sites for phosphorylation that mediate Snf1p-dependent inhibition of its activity in the absence of glucose . Two of the sites, Ser278 and Ser311, fit the consensus sequence for phosphorylation by the kinase Snf1p, as determined in vitro . However, a third phosphorylated site, Ser108, does not resemble a Snf1p site . We tested the effect of deleting residues 181-245, which contain two conserved alanine-leucine-serine motifs . We found that the deletion produces a partially constitutive activator, indicating that this region plays a general negative role in regulating Mig1p.

Int Rev Cytol, 1998, 182, 69 - 109
The role of the dynactin complex in intracellular motility; Holleran EA et al.; Dynactin is a multisubunit complex that binds to the minus-end-directed microtubule motor cytoplasmic dynein and may provide a link between the motor and its cargo . Results from genetic studies in Saccharomyces cerevisiae, Neurospora crassa, Aspergillus nidulans, and Drosophila have suggested that cytoplasmic dynein and dynactin function in the same cellular pathways . p150Glued, a vertebrate homologue of the Drosophila gene Glued, is the largest polypeptide in the dynactin complex with multiple protein interactions . Centractin, the most abundant dynactin subunit polypeptide, forms an actin-like filament at the base of the complex . Studies on dynamitin, the 50-kDa dynactin subunit, predict a role for dynactin in mitotic spindle assembly . Other subunits of dynactin have also been cloned and characterized; these studies have provided insight into the role of the complex in essential cellular processes.

Biochemistry, 1998 Mar 17, 37(11), 3711 - 22
Destabilized PCNA trimers suppress defective Rfc1 proteins in vivo and in vitro; Beckwith WH et al.; Replication factor C (RFC) and the proliferating cell nuclear antigen (PCNA) are two essential DNA polymerase accessory proteins that are required for numerous aspects of DNA metabolism including DNA replication, DNA repair, and telomere metabolism . PCNA is a homotrimeric ring-shaped sliding DNA clamp that can facilitate DNA replication by tethering DNA polymerase delta or DNA polymerase epsilon to the DNA template . RFC is the 5-subunit multiprotein complex that loads PCNA onto DNA at primer-template junctions in an ATP-dependent reaction . All five of the RFC subunits share a set of related sequences (RFC boxes) that include nucleotide-binding consensus sequences . We report here that a mutation in the gene encoding the large subunit of yeast RFC gives rise to DNA metabolism defects that can be observed in vivo and in vitro . The rfc1-1 substitution (D513N) lies within the widely conserved RFC box VIII consensus sequence and results in phenotypes including DNA replication defects, increased sensitivity to DNA damaging agents, and elongated telomeres . Mutant Rfc1-1 complexes exhibit in vitro DNA replication defects that are sensitive to ATP concentrations, and these defects can be suppressed by mutant PCNA proteins which contain substitutions that destabilize the homotrimeric sliding DNA clamp.

Biochemistry, 1998 Mar 10, 37(10), 3386 - 91
Interplay between S1 and S4 subsites in Kex2 protease: Kex2 exhibits dual specificity for the P4 side chain; Rockwell NC et al.; The yeast Kex2 protease is the prototype of a family of eukaryotic proprotein processing proteases that includes PC1, PC2, and furin . The catalytic domains of these enzymes are homologous to the degradative serine proteases of the subtilisin family . Kex2 exhibits optimal activity toward substrates with Lys or Arg at P2 and Arg at P1 (Lys-Arg or Arg-Arg cleavage sites) . However, mammalian proprotein processing proteases such as furin exhibit more stringent requirements for basic residues at P4 than at P2 . Here we demonstrate that Kex2 protease also recognizes P4, with dual specificity for aliphatic and basic residues . Recognition of P4 is even more readily apparent in substrates having a poor P1 residue (Lys) . Kinetic analysis of a series of otherwise identical fluorogenic substrates with Lys at P1 and different residues at P4 indicates that large, aliphatic P4 residues increase kcat/KM by 100-fold . However, smaller residues or acidic residues at P4 do not . P4 Arg also confers efficient cleavage on such a substrate, but the uncharged isostere of Arg, citrulline, does not . Kex2 may thus possess distinct subsites that recognize aliphatic or basic P4 side chains . Although a favorable P4 residue can partially compensate for the defects in kcat and kcat/KM seen with Lys in place of Arg at P1, this substitution resulted in a change in rate-determining step for all substrates examined . As previously seen in the case of subtilisin, effects of substitutions at the P1 and P4 positions were not independent, suggesting that interplay between these two positions is a common feature of substrate specificity for both processing proteases and degradative enzymes of the subtilisin superfamily.

Biochemistry, 1998 Mar 10, 37(10), 3337 - 50
Mutational analysis of the P-glycoprotein first intracellular loop and flanking transmembrane domains; Kwan T et al.; The role of individual intracellular (IC) loops linking transmembrane (TM) domains in P-glycoprotein (P-gp) function remains largely unknown . The high degree of sequence conservation of these regions in the P-gp family and other ABC transporters suggests an important role in a common mechanism of action of these proteins . To gain insight into this problem, we have randomly mutagenized a portion of TM2, the entire IC1 loop, TM3, the entire extracellular loop (EC2), and part of TM4, and analyzed the effect of such mutations on P-gp function . Random mutagenesis was carried out using Taq DNA polymerase and dITP under conditions of low polymerase fidelity, and the mutagenized segments were reintroduced in the full length mdr3 cDNA by homologous recombination in the yeast Saccharomyces cerevisiae strain JPY201 . The biological activity of mutant P-gp variants was analyzed in yeast by their ability to confer cellular resistance to the antifungal drug FK506 and the peptide ionophore valinomycin, and by their ability to complement the yeast Ste6 gene and restore mating in a yeast strain bearing a null mutation {Raymond, M., et al . (1992) Science 256, 232-4} at this locus . The analysis of 782 independent yeast transformants allowed the identification of 49 independent mutants bearing single amino acid substitutions in the mutagenized segment resulting in an altered P-gp function . The mutants could be phenotypically classified into two major groups, those that resulted in partial or complete overall loss of function and those that seemed to affect substrate specificity . Several of the mutants affecting overall activity mapped in IC1; in particular we identified a segment of four consecutive mutation sensitive residues (TRLT, positions 169-172) with such a phenotype . On the other hand, we identified a cluster of mutants affecting substrate specificity within the short EC2 segment and in the adjacent portion of the neighboring TM4 domain . Expression and partial purification of a representative subset of these mutants showed that in all but two cases, loss of function was associated with loss of drug-induced ATPase activity of P-gp . Therefore, it appears that TM domains, IC and EC loops, are structurally and functionally tightly coupled in the process of drug stimulatable ATPase characteristic of P-gp.

Mol Gen Genet, 1998 Feb, 257(3), 255 - 63
The Aspergillus nidulans sulphur regulatory gene sconB encodes a protein with WD40 repeats and an F-box; Natorff R et al.; The Aspergillus nidulans gene sconB, one of the four identified genes controlling sulphur metabolite repression, was cloned and analysed . It encodes a polypeptide of 678 amino acids containing seven WD repeats characteristic of the large WD40 family of eukaryotic regulatory proteins . The SCONB protein has nuclear localisation signals and is very similar to the Neurospora crassa SCON2 and Saccharomyces cerevisiae Met30 proteins, both of which are involved in the regulation of sulphur metabolism . The N . crassa scon-2 gene complements the sconB2 mutation . All three proteins also contain a newly identified motif, the F-box, found in a number of eukaryotic regulatory proteins . This motif is responsible, at least in some cases, for ubiquitin-mediated proteolysis . The sconB transcript is derepressed under sulphur limitation conditions and partly repressed by high methionine.

Structure, 1998 Feb 15, 6(2), 195 - 210
The allosteric regulation of pyruvate kinase by fructose-1,6-bisphosphate; Jurica MS et al.; BACKGROUND: Yeast pyruvate kinase (PK) catalyzes the final step in glycolysis . The enzyme therefore represents an important control point and is allosterically activated by fructose-1,6-bisphosphate (FBP) . In mammals the enzyme is found as four different isozymes with different regulatory properties: two of these isozymes are produced by alternate splicing . The allosteric regulation of PK is directly related to proliferation of certain cell types, as demonstrated by the expression of an allosterically regulated isozyme in tumor cells . A model for the allosteric transition from the inactive (T) state to the active (R) state has been proposed previously, but until now the FBP-binding site had not been identified . RESULTS: We report here the structures of PK from yeast complexed with a substrate analog and catalytic metal ions in the presence and absence of bound FBP . The allosteric site is located 40 A from the active site and is entirely located in the enzyme regulatory (C) domain . A phosphate-binding site for the allosteric activator is created by residues encoded by a region of the gene corresponding to the alternately spliced exon of mammalian isozymes . FBP activation appears to induce several conformational changes among active-site sidechains through a mechanism that is most likely to involve significant domain motions, as previously hypothesized . CONCLUSIONS: The structure and location of the allosteric activator site agrees with the pattern of alternate genetic splicing of the PK gene in multicellular eukaryotes that distinguishes between a non-regulated isozyme and the regulated fetal isozymes . The conformational differences observed between the active sites of inactive and fully active PK enzymes is in agreement with the recently determined thermodynamic mechanism of allosteric activation through a 'metal relay' that increases the affinity of the enzyme for its natural phosphoenolpyruvate substrate.

Curr Opin Struct Biol, 1998 Feb, 8(1), 19 - 25
Novel site-specific DNA endonucleases; Aggarwal AK et al.; Site-specific hydrolysis of DNA is common to many biological processes . Three new structures, FokI, I-CreI and PI-SceI, were reported in the past year, providing the first view of type IIs endonucleases and homing endonucleases . Together, they reveal an extraordinary set of new mechanisms by which endonucleases target the hydrolysis of specific DNA sequences.

Science, 1998 Mar 13, 279(5357), 1733 - 7
Roles for ORC in M phase and S phase; Dillin A et al.; The origin recognition complex (ORC), a six-subunit protein, functions as the replication initiator in the yeast Saccharomyces cerevisiae . Initiation depends on the assembly of the prereplication complex in late M phase and activation in S phase . One subunit of ORC, Orc5p, was required at G1/S and in early M phase . Asynchronous cells with a temperature-sensitive orc5-1 allele arrested in early M phase . In contrast, cells that were first synchronized in M phase, shifted to the restrictive temperature, and then released from the block arrested at the G1/S boundary . The G1/S arrest phenotype could not be suppressed by introducing wild-type Orc5p during G1 . Although all orc2 and orc5 mutations were recessive in the conventional sense, this dominant phenotype was shared with other orc5 alleles and an orc2 allele . The dominant inhibition to cell-cycle progression exhibited by the orc mutants was restricted to the nucleus, suggesting that chromosomes with mutant ORC complexes were capable of sending a signal that blocked initiation on chromosomes containing functional origins.

Biochemistry, 1998 Feb 24, 37(8), 2282 - 90
Essential binding and functional domains of human bleomycin hydrolase; Koldamova RP et al.; Bleomycin hydrolase (BH) is unusual among cysteine proteinases because it appears to form multihomomeric structures, inactivates the antitumor glycopeptide bleomycin, and contains a unique C-terminal amino acid sequence . We now demonstrate intrinsic endopeptidase activity associated with human BH (hBH) using artificial substrates and intracellular dimerization of hBH using a yeast two-hybrid assay . To determine domains important for homomeric interactions and catalysis, we constructed N- and C-terminal deletion mutants and identified an N-terminal region (hBH1-82) that interacted with two nonoverlaping hBH domains: one near the N-terminus (hBH14-103) and another neighboring the C-terminus (hBH358-455) . In vitro hBH aggregated with a molecular mass of 235 kD corresponding to a homotetramer and the C-terminus was critical for this oligomerization since no tetramers were found when the last 40 amino acids were deleted . The penultimate 8 amino acids, which constitute a unique and highly conserved bleomycin hydrolase-like domain (BHYD), were essential for BH and aminopeptidase activity but not for endopeptidase activity or oligomer formation . Thus, the C-terminus of hBH has two independent roles controlling both the catalytic activity and oligomerization of hBH.

J Biol Chem, 1998 Feb 27, 273(9), 4972 - 5
Evaluation of GAL4/TATA in vivo . Induction of transgene expression by adenovirally mediated gene codelivery; Fang B et al.; A synthetic GAL4-responsive promoter consisting of five GAL4-binding sites and a TATA box (GAL4/TATA) was evaluated for its transcriptional activity in an adenoviral backbone using luciferase as the reporter . Basal luciferase activities in vitro were the same for cells infected with either adenovirus-containing luciferase cDNA driven by GAL4/TATA (Ad/GT-Luc) or adenovirus-containing luciferase cDNA not driven by a promoter (Ad/PO-Luc) . In vitro induction of GAL4/TATA by coinfection of cells with adenovirus expressing the GAL4/VP16 fusion protein (Ad/3-phosphoglycerate kinase (PGK)-GV16) was dose-dependent and reached as high as 4 x 10(4)- to 9 x 10(4)-fold above basal levels when GAL4/TATA and GAL4/VP16 were delivered at a ratio of 10:1 . In vivo studies in Balb/c mice showed no detectable luciferase activities in liver or other tissues examined in mice infused with either Ad/GT-Luc or Ad/PO-Luc . High levels of luciferase activity were, however, elicited when animals were infused with Ad/GT-Luc and Ad/PGK-GV16 . Together, these results suggest that combination of the GAL4 gene regulatory system with adenovirally mediated in vivo gene delivery may be applicable to the in vivo evaluation of promoter activities and in vivo targeting of gene expression.

J Biol Chem, 1998 Feb 27, 273(9), 4957 - 66
Regulation of Munc-18/syntaxin 1A interaction by cyclin-dependent kinase 5 in nerve endings; Shuang R et al.; The Munc-18-syntaxin 1A complex has been postulated to act as a negative control on the regulated exocytotic process because its formation blocks the interaction of syntaxin with vesicle SNARE proteins . However, the formation of this complex is simultaneously essential for the final stages of secretion as evidenced by the necessity of Munc-18's homologues in Saccharomyces cerevisiae (Sec1p), Drosophila (ROP), and Caenorhabditis elegans (Unc-18) for proper secretion in these organisms . As such, any event that regulates the interaction of these two proteins is important for the control of secretion . One candidate for such regulation is cyclin-dependent kinase 5 (Cdk5), a member of the Cdc2 family of cell division cycle kinases that has recently been copurified with Munc-18 from rat brain . The present study shows that Cdk5 bound to its neural specific activator p35 not only binds to Munc-18 but utilizes it as a substrate for phosphorylation . Furthermore, it is demonstrated that Munc-18 that has been phosphorylated by Cdk5 has a significantly reduced affinity for syntaxin 1A . Finally, it is shown that Cdk5 can also bind to syntaxin 1A and that a complex of Cdk5, p35, Munc-18, and syntaxin 1A can be fashioned in the absence of ATP and promptly disassembled upon the addition of ATP . These results suggest a model in which p35-activated Cdk5 becomes localized to the Munc-18-syntaxin 1A complex by its affinity for both proteins so that it may phosphorylate Munc-18 and thus permit the positive interaction of syntaxin 1A with upstream protein effectors of the secretory mechanism.

EMBO J, 1998 Feb 16, 17(4), 1107 - 19
Nup116p and nup100p are interchangeable through a conserved motif which constitutes a docking site for the mRNA transport factor gle2p; Bailer SM et al.; Nup116p and Nup100p are highly related yeast GLFG nucleoporins, but only Nup116p is stoichiometrically bound to Gle2p, a previously identified mRNA export factor . A short Gle2p-binding sequence within Nup116p (GLEBS; residues 110-166) is sufficient and necessary to anchor Gle2p at the nuclear pores, whereas the carboxy-terminal domain of Nup116p mediates its own nuclear pore complex (NPC) association . The GLEBS is evolutionarily conserved and found in rat/Xenopus Nup98 and an uncharacterized Caenorhabditis elegans ORF, but is absent from Nup100p . When the GLEBS is deleted from Nup116p, Gle2p dissociates from the nuclear envelope and clusters of herniated nuclear pores form . When the GLEBS is inserted into Nup100p, Nup100p-GLEBS complements both the thermosensitive and NPC-herniated phenotype of nup116- cells, and Gle2p is retargeted concomitantly to the NPCs . Thus, the in vivo function of Gle2p is strictly coupled to the short GLEBS within Nup116p which links this putative mRNA transport factor to the nuclear pores.

EMBO J, 1998 Feb 16, 17(4), 1096 - 106
The NOT proteins are part of the CCR4 transcriptional complex and affect gene expression both positively and negatively; Liu HY et al.; The CCR4 transcriptional regulatory complex consisting of CCR4, CAF1, DBF2 and other unidentified factors is one of several groups of proteins that affect gene expression . Using mass spectrometry, we have identified the 195, 185 and 116 kDa species which are part of the CCR4 complex . The 195 and 185 kDa proteins were found to be NOT1 and the 116 kDa species was identical to NOT3 . NOT1, 2, 3 and 4 proteins are part of a regulatory complex that negatively affects transcription . All four NOT proteins were found to co-immunoprecipitate with CCR4 and CAF1, and NOT1 co-purified with CCR4 and CAF1 through three chromatographic steps in a complex estimated to be 1.2x10(6) Da in size . Mutations in the NOT genes affected many of the same genes and processes that are affected by defects in the CCR4 complex components, including reduction in ADH2 derepression, defective cell wall integrity and increased sensitivity to monoand divalent ions . Similarly, ccr4, caf1 and dbf2 alleles negatively regulated FUS1-lacZ expression, as do defects in the NOT genes . These results indicate that the NOT proteins are physically and functionally part of the CCR4 complex which forms a unique and novel complex that affects transcription both positively and negatively.

EMBO J, 1998 Feb 16, 17(4), 985 - 95
epsilon-COP is a structural component of coatomer that functions to stabilize alpha-COP; Duden R et al.; We isolated a novel yeast alpha-COP mutant, ret1-3, in which alpha-COP is degraded after cells are shifted to a restrictive temperature . ret1-3 cells cease growth at 28 degrees C and accumulate the ER precursor of carboxypeptidase Y (p1 CPY) . In a screen for high copy suppressors of these defects, we isolated the previously unidentified yeast epsilon-COP gene . epsilon-COP (Sec28p) overproduction suppresses the defects of ret1-3 cells up to 34 degrees C, through stabilizing levels of alpha-COP . Surprisingly, cells lacking epsilon-COP (sec28 Delta) grow well up to 34 degrees C and display normal trafficking of carboxypeptidase Y and KKXX-tagged proteins at a permissive temperature . epsilon-COP is thus non-essential for yeast cell growth, but sec28 Delta cells are thermosensitive . In sec28 Delta cells shifted to 37 degrees C, wild-type alpha-COP (Ret1p) levels diminish rapidly and cells accumulate p1 CPY; these defects can be suppressed by alpha-COP overproduction . Mutant coatomer from sec28 Delta cells behaves as an unusually large protein complex in gel filtration experiments . The sec28 Delta mutation displays allele-specific synthetic-lethal interactions with alpha-COP mutations: sec28 Delta ret1-3 double mutants are unviable at all temperatures, whereas sec28 Delta ret1-1 double mutants grow well up to 30 degrees C . Our results suggest that a function of epsilon-COP is to stabilize alpha-COP and the coatomer complex.

EMBO J, 1998 Feb 16, 17(4), 952 - 66
A novel protein complex promoting formation of functional alpha- and gamma-tubulin; Geissler S et al.; We describe the identification of GIM1/YKE2, GIM2/PAC10, GIM3, GIM4 and GIM5 in a screen for mutants that are synthetically lethal with tub4-1, encoding a mutated yeast gamma-tubulin . The cytoplasmic Gim proteins encoded by these GIM genes are present in common complexes as judged by co-immunoprecipitation and gel filtration experiments . The disruption of any of these genes results in similar phenotypes: the gim null mutants are synthetically lethal with tub4-1 and super-sensitive towards the microtubule-depolymerizing drug benomyl . All except Deltagim4 are cold-sensitive and their microtubules disassemble at 14 degrees C . The Gim proteins have one function related to alpha-tubulin and another to Tub4p, supported by the finding that the benomyl super-sensitivity is caused by a reduced level of alpha-tubulin while the synthetic lethality with tub4-1 is not . In addition, GIM1/YKE2 genetically interacts with two distinct classes of genes, one of which is involved in tubulin folding and the other in microtubule nucleation . We show that the Gim proteins are important for Tub4p function and bind to overproduced Tub4p . The mammalian homologues of GIM1/YKE2 and GIM2/PAC10 rescue the synthetically lethal phenotype with tub4-1 as well as the cold-sensitivity and benomyl super-sensitivity of the yeast deletion mutants . We suggest that the Gim proteins form a protein complex that promotes formation of functional alpha- and gamma-tubulin.

EMBO J, 1998 Feb 16, 17(4), 877 - 85
The first step of glycosylphosphatidylinositol biosynthesis is mediated by a complex of PIG-A, PIG-H, PIG-C and GPI1; Watanabe R et al.; Biosynthesis of glycosylphosphatidylinositol (GPI) is initiated by transfer of N-acetylglucosamine (GlcNAc) from UDP-GlcNAc to phosphatidylinositol (PI) . This chemically simple step is genetically complex because three genes are required in both mammals and yeast . Mammalian PIG-A and PIG-C are homologous to yeast GPI3 and GPI2, respectively; however, mammalian PIG-H is not homologous to yeast GPI1 . Here, we report cloning of a human homolog of GPI1 (hGPI1) and demonstrate that four mammalian gene products form a protein complex in the endoplasmic reticulum membrane . PIG-L, which is involved in the second step in GPI synthesis, GlcNAc-PI de-N-acetylation, did not associate with the isolated complex . The protein complex had GPI-GlcNAc transferase (GPI-GnT) activity in vitro, but did not mediate the second reaction . Bovine PI was utilized approximately 100-fold more efficiently than soybean PI as a substrate, and lyso PI was a very inefficient substrate . These results suggest that GPI-GnT recognizes the fatty acyl chains of PI . The unusually complex organization of GPI-GnT may be relevant to selective usage of PI and/or regulation.

Biochem Biophys Res Commun, 1998 Mar 17, 244(2), 384 - 9
Interaction of the second coding exon of Tat with human EF-1 delta delineates a mechanism for HIV-1-mediated shut-off of host mRNA translation; Xiao H et al.; HIV-1 Tat has pleiotropic functions . While its most studied role is to activate transcription from the retroviral long terminal repeat (LTR)-promoter, Tat also has functions as a secretable growth factor, a T-cell activator, and an inducer of cellular apoptosis, amongst others . For its transcriptional function, the first coding exon of Tat appears wholly sufficient; however, lentiviruses (HIVs and SIVs) maintain and conserve a second coding exon for Tat . While the function(s) of the second exon of Tat has remained largely unknown, its integrity in lentiviral genomes suggests biological importance, possibly a role in non-transcriptional activities . To understand better the biology of the second exon of Tat in HIV-1 infection of cells, we have searched for cellular proteins that bind specifically to this protein domain . Here, we report that the human translation elongation factor 1-delta (EF-1 delta) binds to the second exon of HIV-1 Tat . Interaction between Tat and EF-1 delta dramatically reduces the efficiency of the translation of cellular, but not viral, mRNAs . These findings suggest that a non-transcriptional activity of Tat modulates cellular protein synthesis, thereby affecting the metabolism of host cells.

Biochem Biophys Res Commun, 1998 Mar 17, 244(2), 353 - 9
Molecular cloning and characterization of a novel protein kinase C-interacting protein with structural motifs related to RBCC family proteins; Tokunaga C et al.; A novel protein kinase C (PKC)-interacting protein was identified by the yeast two-hybrid screening using the regulatory domain of PKC beta I as a bait . The protein contained several structural motifs such as two putative coiled-coil regions, a RING-finger, a B-box, and a B-box-like motif in the order from NH2- to COOH-terminals . The molecular organization of the protein resembles the structure of the RBCC protein family proteins which usually have a RING-finger, a B-box, and a coiled-coil region . Therefore, the protein identified was designated as RBCK1 (RBCC protein interacting with PKC 1) . Northern blot analysis showed that RBCK1 gene is expressed ubiquitously among rat tissues . RBCK1 protein associated with PKC beta I and PKC zeta when coexpressed in cultured mammalian cells . By the polymerase chain reaction-assisted DNA-binding site selection and the electrophoretic mobility shift assay, RBCK1 protein was shown to bind to several DNA fragments containing TGG-rich sequences . When the yeast GAL4 DNA-binding domain fused RBCK1 protein was expressed in COS-7 cells harboring the luciferase gene placed under a synthetic promoter containing GAL4-binding sites, the fusion protein showed enhanced transcriptional activity comparing with the GAL4 DNA-binding domain . These results suggest that RBCK1 protein might be a transcription factor that has a role in the signaling pathway through PKC.

Biophys J, 1998 Mar, 74(3), 1484 - 91
Insertion of telomere repeat sequence decreases plasmid DNA condensation by cobalt (III) hexaammine; Schnell JR et al.; Telomere repeat sequence (TRS) DNA is found at the termini of most eukaryotic chromosomes . The sequences are highly repetitive and G-rich (e.g., {C(1-3)A/TG(1-3)}n for the yeast Saccharomyces cerevisiae) and are packaged into nonnucleosomal protein-DNA structures in vivo . We have used total intensity light scattering and electron microscopy to monitor the effects of yeast TRS inserts on in vitro DNA condensation by cobalt (III) hexaammine . Insertion of 72 bp of TRS into a 3.3-kb plasmid depresses condensation as seen by light scattering and results in a 22% decrease in condensate thickness as measured by electron microscopy . Analysis of toroidal condensate dimensions suggests that the growth stages of condensation are inhibited by the presence of a TRS insert . The depression in total light scattering intensity is greater when the plasmid is linearized with the TRS at an end (39-49%) than when linearized with the TRS in the interior (18-22%) . Circular dichroism of a 95-bp fragment containing the TRS insert gives a spectrum that is intermediate between the A-form and B-form, and the anomalous condensation behavior of the TRS suggests a noncanonical DNA structure . We speculate that under conditions in which the plasmid DNA condenses, the telomeric insert assumes a helical geometry that is similar to the A-form and is incompatible with packing into the otherwise B-form lattice of the condensate interior.

Biophys J, 1998 Mar, 74(3), 1346 - 57
Vectorially oriented monolayers of the cytochrome c/cytochrome oxidase bimolecular complex; Edwards AM et al.; Vectorially oriented monolayers of yeast cytochrome c and its bimolecular complex with bovine heart cytochrome c oxidase have been formed by self-assembly from solution . Both quartz and Ge/Si multilayer substrates were chemical vapor deposited with an amine-terminated alkylsiloxane monolayer that was then reacted with a hetero-bifunctional cross-linking reagent, and the resulting maleimide endgroup surface then provided for covalent interactions with the naturally occurring single surface cysteine 102 of the yeast cytochrome c . The bimolecular complex was formed by further incubating these cytochrome c monolayers in detergent-solubilized cytochrome oxidase . The sequential formation of such monolayers and the vectorially oriented nature of the cytochrome oxidase was studied via meridional x-ray diffraction, which directly provided electron density profiles of the protein(s) along the axis normal to the substrate plane . The nature of these profiles is consistent with previous work performed on vectorially oriented monolayers of either cytochrome c or cytochrome oxidase alone . Furthermore, optical spectroscopy has indicated that the rate of binding of cytochrome oxidase to the cytochrome c monolayer is an order of magnitude faster than the binding of cytochrome oxidase to an amine-terminated surface that was meant to mimic the ring of lysine residues around the heme edge of cytochrome c, which are known to be involved in the binding of this protein to cytochrome oxidase.

Biophys J, 1998 Mar, 74(3), 1203 - 14
Using experimental information to produce a model of the transmembrane domain of the ion channel phospholamban; Herzyk P et al.; Molecular models of the transmembrane domain of the phospholamban pentamer have been generated by a computational method that uses the experimentally measured effects of systematic single-site mutations as a guiding force in the modeling procedure . This method makes the assumptions that 1) the phospholamban transmembrane domain is a parallel five-helix bundle, and 2) nondisruptive mutation positions are lipid exposed, whereas 3) disruptive or partially disruptive mutations are not . Our procedure requires substantially less computer time than systematic search methods, allowing rapid assessment of the effects of different experimental results on the helix arrangement . The effectiveness of the approach is investigated in test calculations on two helix-dimer systems of known structure . Two independently derived sets of mutagenesis data were used to define the restraints for generating models of phospholamban . Both resulting models are left-handed, highly symmetrical pentamers . Although the overall bundle geometry is very similar in the two models, the orientation of individual helices differs by approximately 50 degrees, resulting in different sets of residues facing the pore . This demonstrates how differences in restraints can have an effect on the model structures generated, and how the violation of these restraints can identify inconsistent experimental data.

Acta Biochim Pol, 1997, 44(3), 591 - 600
Interaction of HIV Tat model peptides with tRNA and 5S rRNA; Giel-Pietraszuk M et al.; New data are presented on the interaction of model synthetic peptides containing an arginine-rich region of human immunodeficiency virus (HIV-Tat), with native RNA molecules: tRNA(Phe) of Saccharomyces cerevisiae and 5S rRNA from Lupinus luteus . Both RNA species form complexes with the Tat1 (GRKKRRQRRRA) and Tat2 (GRKKRRQRRRAPQDSQTHQASLSKQPA) peptides, as shown by electrophoretic gel shift and RNase footprint assays, and CD measurements . The nucleotide sequence UGGG located in the dihydrouridine loop of tRNAPhe as well as in the loop D of 5S rRNA is specifically protected against RNases . Our data indicate direct interactions of guanine of RNA moieties with arginine residues . These interactions seem similar to those observed in DNA-protein complexes, but different from those previously observed in the TAR RNA-Tat complexes.

Gene, 1998 Jan 30, 207(2), 127 - 34
Nucleotide sequence of an alternative glucoamylase-encoding gene (glaB) expressed in solid-state culture of Aspergillus oryzae; Hata Y et al.; The DNA (glaB) and a cDNA-encoding glucoamylase produced in solid-state culture of Aspergillus oryzae were cloned using oligodeoxyribonucleotide probes derived from internal amino acid sequences of the enzyme . Comparison of the nucleotide sequences of a genomic DNA fragment with its cDNA showed the glaB gene carried three exons interrupted by two introns and had an open reading frame encoding 493 aa residues . The 5'-flanking region had a TATA box at nt -87 from the start codon and two putative CAAT sequences at nt -276 and -288 . The glaB gene shared 57% homology at the aa level with the glaA gene which was cloned previously from A . oryzae . Interestingly, the glucoamylase encoded by the glaB gene had no C-terminal domain such as that proposed to have starch binding activity in Aspergillus glucoamylases . Introduction of cDNA of the glaB gene to Saccharomyces cerevisiae caused the secretion of active glucoamylase to culture medium and introduction of the glaB gene to A . oryzae increased glucoamylase productivity in solid-state culture . Northern blot analysis showed the glaB gene was expressed in solid-state culture, but not in submerged culture.

Gene, 1998 Jan 19, 207(1), 61 - 9
Characterization of NOT5 that encodes a new component of the Not protein complex; Oberholzer U et al.; The yeast HIS3 gene has two core promoters: TC, a TATA-less element and TR, a canonical TATA element . Four genes encode global negative regulators of transcription that preferentially repress TC-dependent transcription: NOT1 (CDC39), NOT2 (CDC36), NOT3 and NOT4 (SIG1, MOT2) . Genetic and biochemical experiments suggest that the products of these genes are associated in a complex and regulate TFIID function . In this paper, we describe a new gene, NOT5, that also represses transcription of the HIS3 TATA-less promoter preferentially and encodes a protein whose N-terminal region is 44% identical to that of Not3p . Our results indicate that NOT5 is involved in Not function and encodes a product that is physically associated with the other Not proteins . First, overexpression of NOT3 or NOT4 suppresses mutations in NOT5 . Secondly, mutations in NOT4 are synthetically lethal with mutations in NOT5 . Thirdly, NOT5 interacts with NOT1 and NOT3 in the two-hybrid assay . Finally, Not1p, Not3p and Not4p co-immunoprecipitate with Not5p.

Gene, 1998 Jan 19, 207(1), 1 - 7
Uncoupling protein-3: a muscle-specific gene upregulated by leptin in ob/ob mice; Liu Q et al.; We identified and partially characterized another member of the uncoupling protein termed UCP3 . Human and mouse UCP3 protein sequences are 86% identical to each other, and 73% and 59% identical to UCP2 and UCP1, respectively . Expression of human UCP3 in yeast resulted in a drastic decrease of mitochondria membrane potential . Northern analysis showed that UCP3 was highly expressed in skeletal muscle in human, rat, and mouse . Mapping of UCP3 placed it to the same chromosomal region of UCP2 in both human and mouse, a region that is linked to obesity and hyperinsulinemia . Furthermore, adenovirus-mediated leptin expression in obese ob/ob mice led to increased expression of UCP3 in skeletal muscle . The data indicate that UCP3 encodes a muscle-specific uncoupling protein that may play an important role in the regulation of energy expenditure and development of obesity.

Chromosome Res, 1998 Jan, 6(1), 59 - 63
The acetylation patterns of histones H3 and H4 along Vicia faba chromosomes are different; Belyaev ND et al.; The acetylation pattern of H3 was studied on field bean chromosomes by means of indirect immunofluorescence using polyclonal antibodies recognizing H3 isoforms acetylated at lysine positions 9/18, 14 and 23 . H3 was found to be hypoacetylated at lysine residues 9/18 and 14 within the heterochromatic regions composed of tandem repetitive Fok-I elements . Hyperacetylation of these residues was observed at the nucleolar organizing region (NOR) and in heterochromatic regions composed of repeats other than Fok-I elements . In contrast, H4 was underacetylated (H4.Ac5, 8, 12) or uniformly acetylated (H4.Ac16) at all heterochromatic regions, and acetylated above the average at all four lysines only within the NOR . Acetylation of lysine-23 of H3 was uniform, except for the NOR that showed no fluorescence . Inhibition of deacetylase during and after replication of heterochromatin by trichostatin A had no influence on the acetylation status of H3 but mediated an increase in acetylation of lysines 5, 12 and 16 of H4 above the average in the field bean heterochromatin . Thus, the chromosomal acetylation patterns of H4 and H3 of this species revealed common and divergent features . Whereas the acetylation level of H4 correlates well with the potential transcriptional activity and inversely with the time of replication of defined chromatin domains of Vicia faba, this is not generally true for H3.

Jpn J Cancer Res, 1998 Jan, 89(1), 33 - 9
Frameshift mutations of the hMSH6 gene in human leukemia cell lines; Hosoya N et al.; Defects in DNA mismatch repair mechanisms, including frameshift mutations of the hMSH6 and hMSH3 genes at their (C)8 and (A)8 tracks, respectively, have been shown to be associated with human malignancies . To clarify the possible involvement of these mutations in hematopoietic malignancies, we screened a total of forty-four human leukemia and lymphoma cell lines for mutations in the hMSH6 and hMSH3 genes, as well as in other genes required for DNA replication or repair, by polymerase chain reaction single-strand conformation polymorphism analysis and sequencing analysis . Frameshift mutations at the (C)8 track of the hMSH6 gene were detected in two cell lines established from lymphoid leukemias . These two cell lines had no wild-type alleles, and both of them showed microsatellite instability . This is the first report that describes mutations and inactivation of the hMSH6 gene in hematological malignancies, suggesting that defects of the hMSH6 gene may be associated with development of hematological malignancies.

Dev Genet, 1998, 22(1), 74 - 84
The SAM domain of polyhomeotic, RAE28, and scm mediates specific interactions through conserved residues; Kyba M et al.; The SAM (sterile alpha motif) domain is a 65- to 70-amino acid sequence found in many diverse proteins whose functions range from signal transduction to transcriptional repression . We show that the SAM domain of the Drosophila Polycomb group protein, polyhomeotic (ph), is capable of binding to itself in vitro . We test a number of near relatives of the ph SAM domain from fruit fly, mouse, and yeast and show that all are capable of self-binding . Heterologous interactions are seen among a subset of SAM domains, including ph, Scm, and RAE28 . Several conserved amino acid residues were mutated in the ph SAM domain, and the effects on self-binding and heterologous association were demonstrated . L33, L41, and 162 are shown to be important determinants of the binding interface, while W1 and G50 are likely essential for the structure of the domain.

J Biochem (Tokyo), 1998 Jan, 123(1), 120 - 7
Ku antigen binds to Alu family DNA; Tsuchiya T et al.; The GC-rich segment containing GGAGGC (Alu core) is conserved within the RNA polymerase III (pol III) promoters of Alu family sequences . We have shown that the GGAGGC motif functions as a modulator of DNA replication as well as of transcription, and identified the proteins binding to the motif in human HeLa cells . In this study, the Alu core binding proteins were partially purified from human Raji cells by using an Alu core DNA affinity column . Both the proteins thus purified were implied to be subunits of Ku antigen based on the following criteria: The molecular weights of the proteins estimated on gel electrophoreses were 70 and 85 kDa, respectively, under denaturing conditions, while under non-denaturing conditions only one band was observed for the same sample at 150 kDa, probably representing hetero-dimer formed between the 70 and 85 kDa proteins . The sizes and the hetero-dimer formation are reminiscent of the 70 and 80 kDa subunits of Ku antigen (Ku-p70 and Ku-p80) . Moreover, the purified proteins were immunoreactive with anti-Ku antibodies, and the specific DNA-protein complex on the Alu core element was cancelled by the anti-Ku antibodies . The nucleoprotein complex showed the same clipping patterns as those of the complex between the Alu core element and an authentically purified Ku antigen after proteolytic cleavage with trypsin and chymotrypsin.

Curr Biol, 1998 Feb 12, 8(4), R121 - 3
Splicing: HACking into the unfolded-protein response; Shamu CE; Unfolded proteins in the endoplasmic reticulum of Saccharomyces cerevisiae trigger a specialized RNA splicing event that allows the subsequent translation of the Hac1p transcription factor . This splicing can be reconstituted with Ire1p, a transmembrane kinase that has a site-specific RNase activity, and tRNA ligase.

Fungal Genet Biol, 1998 Feb, 23(1), 81 - 94
Cloning and characterization of a general amino acid control transcriptional activator from the chestnut blight fungus Cryphonectria parasitica; Wang P et al.; We have cloned and characterized a homologue of the Neurospora crassa general amino acid control gene cpc-1 from the chestnut blight fungus Cryphonectria parasitica . The deduced amino acid sequence of C . parasitica CPC1 (cpCPC1) contains regions with significant homology to the transcriptional activation, DNA binding, and dimerization domains previously defined for N . crassa CPC1 (ncCPC1) and the equivalent "b-ZIP" transcription factor from Saccharomyces cerevisiae, GCN4 (scGCN4) . Treatment of C . parasitica with low levels of the protein synthesis inhibitor cycloheximide caused cpc-1 transcript levels to undergo a rapid, transient increase similar to that reported for the mammalian b-ZIP transactivators, c-Jun and c-Fos . Northern analysis also revealed that amino acid starvation of C . parasitica elicits an increase in cpc-1 transcript levels . Hypovirus infection did not affect this increase, although transcript accumulation for several amino acid biosynthetic genes was slightly diminished in the hypovirus-containing strain . Recombinant cpCPC1 specifically bound to the consensus DNA binding element (AP-1), 5'-A/GTGACTCAT-3', also located upstream of the C . parasitica cpc-1 coding region . Constitutive transgenic expression of a DNA binding defective cpCPC1 mutant impaired the ability of C . parasitica to adjust to amino acid starvation . Moreover, these transformants showed reduced ability to grow on host chestnut tissue . Our results define a general amino acid control transactivator in a plant pathogenic fungus and suggest that functional modulation of this factor can influence fungal virulence.

Insect Biochem Mol Biol, 1997 Nov, 27(11), 963 - 72
Partial characterization of a fatty acid desaturase gene in Drosophila melanogaster; Wicker-Thomas C et al.; The open reading frame of a gene encoding a protein homologous to vertebrate fatty acid desaturases has been characterized in Drosophila melanogaster . This is the first description of a desaturase sequence in insects . Two cDNAs were cloned: the combined 1.5 kb nucleotide sequences indicate that the gene encodes a polypeptide of 383 amino acid residues with a high sequence similarity to the well defined delta 9 desaturases from rat and yeast and desaturases from carp and tick . The Drosophila protein contains two histidine cluster motifs (HXXHH) and two hydrophobic regions which are conserved among all desaturases, whereas the amino and carboxy ends look more variable . The desaturase gene seems to be expressed in adults at a low level, with a greater prevalence in females than in males . Two genomic sequences have been amplified by polymerase chain reaction (PCR), and have a very similar open reading frame but a different organization; one with three introns and the other without introns . Both seem to correspond to two distinct genes and have been named "desat locus" . They are localized to a single locus, 87C, on the right arm of the third chromosome . The possible involvement of the desat product in the biosynthesis of D . melanogaster contact pheromones is discussed.

Nat Genet, 1998 Mar, 18(3), 271 - 5
The Hand1 bHLH transcription factor is essential for placentation and cardiac morphogenesis; Riley P et al.; The placenta and cardiovascular system are the first organ systems to form during mammalian embryogenesis . We show here that a single gene is critical for development of both . The Hand1 gene, previously called Hxt, eHAND and Thing1, encodes a basic helix-loop-helix (bHLH) transcription factor that starts to be expressed during pre-implantation development . After implantation, Hand1 expression is restricted to placental trophoblast cells and later to embryonic cardiac and neural crest cells . We generated Hand1-null mutant mice by gene targetting . Homozygous mutant embryos arrested by embryonic day (E) 7.5 of gestation with defects in trophoblast giant cell differentiation . This early mortality could be rescued by aggregation of mutant embryos with wild-type tetraploid embryos, which contribute wild-type cells to the trophoblast, but not the embryo . By E10.5, however, the Hand1-null fetuses derived from tetraploid chimaeras died due to cardiac failure . Their heart tubes showed abnormal looping and ventricular myocardial differentiation . Therefore, Hand1 is essential for differentiation of both trophoblast and cardiomyocytes, which are embryologically distinct cell lineages.

Virology, 1998 Feb 15, 241(2), 234 - 50
Chimeras containing influenza NS1 and HIV-1 Rev protein sequences: mechanism of their inhibition of nuclear export of Rev protein-RNA complexes; Chen Z et al.; Nuclear RNA export mediated by the HIV-1 Rev protein is inhibited by chimeric proteins in which the wild-type Rev protein is covalently linked to amino acid sequences of the NS1 protein of influenza A virus (NS1A protein), a protein that inhibits nuclear RNA export . These chimeric molecules function not only in cis but also in trans: they inhibit nuclear RNA export mediated by Rev protein molecules that are not covalently linked to the NS1A protein sequence . Here we show that inhibition occurs with a NS1-Rev chimera in which the 78 amino-terminal amino acids of the NS1A protein comprising its entire RNA-binding domain is deleted, thereby establishing that this carboxyl portion of the NS1A protein can function as an independent effector domain . The mechanism by which this NS1-Rev chimera inhibits Rev function in trans was determined . The Rev sequence in this chimera oligomerizes with Rev molecules in trans, and the resulting mixed oligomers are retained in the nucleus because the nuclear retention activity of the NS1 effector domain is dominant over the nuclear transport activity of the Rev effector domain . Binding of the FG-containing nucleoporin-like Rab protein to this NS1-Rev chimera, as measured in yeast two-hybrid assays, is much stronger than that to the Rev protein itself, yet nuclear export does not occur in the presence of the chimera . Unexpectedly, the introduction of specific mutations into the NS1A portion of this NS1-Rev chimera not only restores Rev-mediated unclear export of RNA but also eliminates detectable Rab binding, indicating that this nuclear export can occur without detectable Rab binding . A different NS1-Rev chimera, one in which the NS1A protein is full-length but contains a mutated RNA-binding domain, effectively inhibits Rev-mediated nuclear export of RNA without blocking the nuclear export of the Rev protein, indicating that nuclear export of the carrier Rev protein can be uncoupled from nuclear export of its passenger RNA.

Blood, 1998 Mar 1, 91(5), 1793 - 801
Analysis of DNA binding proteins associated with hemin-induced transcriptional inhibition . The hemin response element binding protein is a heterogeneous complex that includes the Ku protein; Reddy SV et al.; Hemin inhibits transcription of the tartrate resistant acid phosphatase (TRAP) gene . Using deletion mutagenesis of the mouse TRAP 5'-flanking region, we previously identified a 27-bp DNA segment containing a central GAGGC tandem repeat sequence (the hemin response element {HRE}), which bound nuclear proteins (hemin response element binding proteins {HREBPs}) from hemin-treated cells and appeared to be responsible for mediating transcriptional inhibition in response to hemin . We now have used affinity binding to HRE-derivatized beads to identify four HREBP components with apparent molecular masses of 133-, 90-, 80-, and 37-kD, respectively . The 80- and 90-kD components correspond to the p70 and p80/86 subunits of Ku antigen (KuAg) as documented by partial amino acid microsequencing of tryptic digests and immunologic reactivity . Based on reactivity of the HREBP gel shift band with antibodies to the redox factor protein (ref1) in shift Western experiments, it is shown that the 37-kD component represents ref1 . The 133-kD component appeared to be a unique protein . KuAg participation in HREBP complexes was specific as it was present in HREBPs bound to HRE microcircles . Results of depletion/reconstitution experiments suggested that KuAg does not bind alone or directly to HRE DNA, but does so only in conjunction with the 133- and/or 37-kD proteins . We conclude that HREBP is a heterogeneous complex composed of KuAg, ref1, and a unique 133-kD protein . We speculate that the role of heme may be to promote interactions among these components, thereby facilitating HRE binding and downregulation of hemin responsive genes.

J Cell Biol, 1998 Feb 23, 140(4), 873 - 83
Smy1p, a kinesin-related protein that does not require microtubules; Lillie SH et al.; We have previously reported that a defect in Myo2p, a myosin in budding yeast (Saccharomyces cerevisiae), can be partially corrected by overexpression of Smy1p, which is by sequence a kinesin-related protein (Lillie, S.H., and S.S . Brown . 1992 . Nature . 356:358- 361) . Such a functional link between putative actin- and microtubule-based motors is surprising, so here we have tested the prediction that Smy1p indeed acts as a microtubule-based motor . Unexpectedly, we found that abolition of microtubules by nocodazole does not interfere with the ability of Smy1p to correct the mutant Myo2p defect, nor does it interfere with the ability of Smy1p to localize properly . In addition, other perturbations of microtubules, such as treatment with benomyl or introduction of tubulin mutations, do not exacerbate the Myo2p defect . Furthermore, a mutation in SMY1 strongly predicted to destroy motor activity does not destroy Smy1p function . We have also observed a genetic interaction between SMY1 and two of the late SEC mutations, sec2 and sec4 . This indicates that Smy1p can play a role even when Myo2p is wild type, and that Smy1p acts at a specific step of the late secretory pathway . We conclude that Smy1p does not act as a microtubule-based motor to localize properly or to compensate for defective Myo2p, but that it must instead act in some novel way.

J Cell Biol, 1998 Feb 23, 140(4), 751 - 65
gp25L/emp24/p24 protein family members of the cis-Golgi network bind both COP I and II coatomer; Dominguez M et al.; Abstract . Five mammalian members of the gp25L/ emp24/p24 family have been identified as major constituents of the cis-Golgi network of rat liver and HeLa cells . Two of these were also found in membranes of higher density (corresponding to the ER), and this correlated with their ability to bind COP I in vitro . This binding was mediated by a K(X)KXX-like retrieval motif present in the cytoplasmic domain of these two members . A second motif, double phenylalanine (FF), present in the cytoplasmic domain of all five members, was shown to participate in the binding of Sec23 (COP II) . This motif is part of a larger one, similar to the F/YXXXXF/Y strong endocytosis and putative AP2 binding motif . In vivo mutational analysis confirmed the roles of both motifs so that when COP I binding was expected to be impaired, cell surface expression was observed, whereas mutation of the Sec23 binding motif resulted in a redistribution to the ER . Surprisingly, upon expression of mutated members, steady-state distribution of unmutated ones shifted as well, presumably as a consequence of their observed oligomeric properties.

Genes Dev, 1998 Feb 15, 12(4), 586 - 97
Nuclear localization of the C2H2 zinc finger protein Msn2p is regulated by stress and protein kinase A activity; Gorner W et al.; Msn2p and the partially redundant factor Msn4p are key regulators of stress-responsive gene expression in Saccharomyces cerevisiae . They are required for the transcription of a number of genes coding for proteins with stress-protective functions . Both Msn2p and Msn4p are Cys2His2 zinc finger proteins and bind to the stress response element (STRE) . In vivo footprinting studies show that the occupation of STREs is enhanced in stressed cells and dependent on the presence of Msn2p and Msn4p . Both factors accumulate in the nucleus under stress conditions, such as heat shock, osmotic stress, carbon-source starvation, and in the presence of ethanol or sorbate . Stress-induced nuclear localization was found to be rapid, reversible, and independent of protein synthesis . Nuclear localization of Msn2p and Msn4p was shown to be correlated inversely to cAMP levels and protein kinase A (PKA) activity . A region with significant homologies shared between Msn2p and Msn4p is sufficient to confer stress-regulated localization to a SV40-NLS-GFP fusion protein . Serine to alanine or aspartate substitutions in a conserved PKA consensus site abolished cAMP-driven nuclear export and cytoplasmic localization in unstressed cells . We propose stress and cAMP-regulated intracellular localization of Msn2p to be a key step in STRE-dependent transcription and in the general stress response.

Genes Dev, 1998 Feb 15, 12(4), 514 - 26
eIF2 independently binds two distinct eIF2B subcomplexes that catalyze and regulate guanine-nucleotide exchange; Pavitt GD et al.; eIF2B is a heteropentameric guanine-nucleotide exchange factor essential for protein synthesis initiation in eukaryotes . Its activity is inhibited in response to starvation or stress by phosphorylation of the alpha subunit of its substrate, translation initiation factor eIF2, resulting in reduced rates of translation and cell growth . We have used an in vitro nucleotide-exchange assay to show that wild-type yeast eIF2B is inhibited by phosphorylated eIF2 {eIF2(alphaP)} and to characterize eIF2B regulatory mutations that render translation initiation insensitive to eIF2 phosphorylation in vivo . Unlike wild-type eIF2B, eIF2B complexes with mutated GCN3 or GCD7 subunits efficiently catalyzed GDP exchange using eIF2(alphaP) as a substrate . Using an affinity-binding assay, we show that an eIF2B subcomplex of the GCN3, GCD7, and GCD2 subunits binds to eIF2 and has a higher affinity for eIF2(alphaP), but it lacks nucleotide-exchange activity . In contrast, the GCD1 and GCD6 subunits form an eIF2B subcomplex that binds equally to eIF2 and eIF2(alphaP) . Remarkably, this second subcomplex has higher nucleotide-exchange activity than wild-type eIF2B that is not inhibited by eIF2(alphaP) . The identification of regulatory and catalytic eIF2B subcomplexes leads us to propose that binding of eIF2(alphaP) to the regulatory subcomplex prevents a productive interaction with the catalytic subcomplex, thereby inhibiting nucleotide exchange.

Genes Dev, 1998 Feb 15, 12(4), 480 - 90
The Cdc7 protein kinase is required for origin firing during S phase; Bousset K et al.; The Cdc7p protein kinase plays an essential, but undefined, role promoting S phase in the budding yeast, Saccharomyces cerevisiae . Previous experiments have shown that the essential function of Cdc7 is executed near the G1-S boundary; after Start but before the elongation phase of DNA replication . Origins of DNA replication fire throughout S phase in budding yeast . Therefore, the G1-S transition is a cell-cycle event that precedes, and is distinct from, the activation of individual origins . Consequently, we have asked whether Cdc7 is only required for S-phase entry or if it plays a role during S phase in origin firing . In this article, we show that partial loss of Cdc7 function results in slow progression through S phase rather than slow entry into S phase and that Cdc7 is still required for the timely completion of S phase after a block to elongation with hydroxyurea . This is because Cdc7 is still required for the activation of late-firing origins after the hydroxyurea block . These experiments show that, rather than acting as a global regulator of the G1-S transition, Cdc7 appears to play a more direct role in the firing of replication origins during S phase.

Science, 1998 Feb 20, 279(5354), 1219 - 22
Identification of a cullin homology region in a subunit of the anaphase-promoting complex; Yu H et al.; The anaphase-promoting complex is composed of eight protein subunits, including BimE (APC1), CDC27 (APC3), CDC16 (APC6), and CDC23 (APC8) . The remaining four human APC subunits, APC2, APC4, APC5, and APC7, as well as human CDC23, were cloned . APC7 contains multiple copies of the tetratrico peptide repeat, similar to CDC16, CDC23, and CDC27 . Whereas APC4 and APC5 share no similarity to proteins of known function, APC2 contains a region that is similar to a sequence in cullins, a family of proteins implicated in the ubiquitination of G1 phase cyclins and cyclin-dependent kinase inhibitors . The APC2 gene is essential in Saccharomyces cerevisiae, and apc2 mutants arrest at metaphase and are defective in the degradation of Pds1p . APC2 and cullins may be distantly related members of a ubiquitin ligase family that targets cell cycle regulators for degradation.

Proc Natl Acad Sci U S A, 1998 Feb 17, 95(4), 1899 - 902
Molecular cloning and expression of the human delta7-sterol reductase; Moebius FF et al.; Inhibitors of the last steps of cholesterol biosynthesis such as AY9944 and BM15766 severely impair brain development . Their molecular target is the Delta7-sterol reductase (EC 1.3.1.21), suspected to be defective in the Smith-Lemli-Opitz syndrome, a frequent inborn disorder of sterol metabolism . Molecular cloning of the cDNA revealed that the human enzyme is a membrane-bound protein with a predicted molecular mass of 55 kDa and six to nine putative transmembrane segments . The protein is structurally related to plant and yeast sterol reductases . In adults the ubiquitously transcribed mRNA is most abundant in adrenal gland, liver, testis, and brain . The Delta7-sterol reductase is the ultimate enzyme of cholesterol biosynthesis in vertebrates and is absent from yeast . Microsomes from Saccharomyces cerevisiae strains heterologously expressing the human cDNA remove the C7-8 double bond in 7-dehydrocholesterol . The conversion to cholesterol depends on NADPH and is potently inhibited by AY9944 (IC50 0.013 microM), BM15766 (IC50 1.2 microM), and triparanol (IC50 14 microM) . Our work paves the way to clarify whether a defect in the delta7-sterol reductase gene underlies the Smith-Lemli-Opitz syndrome.

Proc Natl Acad Sci U S A, 1998 Feb 17, 95(4), 1607 - 12
Interaction of the HIV-1 rev cofactor eukaryotic initiation factor 5A with ribosomal protein L5; Schatz O et al.; It has previously been shown that interaction of eukaryotic initiation factor 5A (eIF-5A) with the Rev trans-activator protein of HIV-1 mediates the transport of unspliced or incompletely spliced viral mRNAs across the nuclear envelope . Consequently, mutants of eIF-5A block Rev function and thereby replication of HIV-1 in trans, indicating that eIF-5A is a crucial protein that connects the viral Rev regulator with cellular RNA transport systems . Here we show that the ribosomal protein L5, which is the central protein component of the 5S rRNA export system, is a cellular interaction partner of eIF-5A . Functional studies demonstrate that overexpression of L5 protein significantly enhances Rev activity . Furthermore, Rev nuclear export activity is inhibited in human somatic cells by antibodies that recognize eIF-5A or L5 . Our data suggest that the Rev export pathway shares components of a cellular transport system involved in the intracellular trafficking of polymerase III (5S rRNA) transcripts.

Proc Natl Acad Sci U S A, 1998 Feb 17, 95(4), 1495 - 9
Two chaperone sites in Hsp90 differing in substrate specificity and ATP dependence; Scheibel T et al.; The abundant molecular chaperone Hsp90 is a key regulator of protein structure in the cytosol of eukaryotic cells . Although under physiological conditions a specific subset of proteins is substrate for Hsp90, under stress conditions Hsp90 seems to perform more general functions . However, the underlying mechanism of Hsp90 remained enigmatic . Here, we analyzed the function of conserved Hsp90 domains . We show that Hsp90 possesses two chaperone sites located in the N- and C-terminal fragments, respectively . The C-terminal fragment binds to partially folded proteins in an ATP-independent way potentially regulated by cochaperones . The N-terminal domain contains a peptide binding site that seems to bind preferentially peptides longer than 10 amino acids . Peptide dissociation is induced by ATP binding . Furthermore, the antitumor drug geldanamycin both inhibits the weak ATPase of Hsp90 and stimulates peptide release . We propose that the existence of two functionally different chaperone sites together with a substrate-selecting set of cochaperones allows Hsp90 to guide the folding of a subset of target proteins and, at the same time, to exhibit general chaperone functions.

Proc Natl Acad Sci U S A, 1998 Feb 17, 95(4), 1410 - 5
Characterization of the ATF/CREB site and its complex with GCN4; Hockings SC et al.; We have studied DNA minicircles containing the ATF/CREB binding site for GCN4 by using a combination of cyclization kinetics experiments and Monte Carlo simulations . Cyclization rates were determined with and without GCN4 for DNA constructs containing the ATF/CREB site separated from a phased A-tract multimer bend by a variable length phasing adaptor . The cyclization results show that GCN4 binding does not significantly change the conformation of the ATF/CREB site, which is intrinsically slightly bent toward the major groove . Monte Carlo simulations quantitate the ATF/CREB site structure as an 8 degrees bend toward the major groove in a coordinate frame near the center of the site . The ATF/CREB site is underwound by 53 degrees relative to the related AP-1 site DNA . The effect of GCN4 binding can be modeled either as a decrease in the local flexibility, corresponding to an estimated 60% increase in the persistence length for the 10-bp binding site, or possibly as a small decrease (1 degrees) in intrinsic bend angle . Our results agree with recent electrophoretic and crystallographic studies and demonstrate that cyclization and simulation can characterize subtle changes in DNA structure and flexibility.

Nucleic Acids Res, 1998 Feb 15, 26(4), 1063 - 9
The C-terminal silencing domain of Rap1p is essential for the repression of ribosomal protein genes in response to a defect in the secretory pathway; Mizuta K et al.; We have previously shown that a functional secretory pathway is essential for continued ribosome synthesis in Saccharomyces cerevisiae . When a temperature-sensitive mutant defective in the secretory pathway is transferred to the non-permissive temperature, transcription of both rRNA genes and ribosomal protein genes is nearly abolished . In order to define the cis -acting element(s) of ribosomal protein genes sensitive to a defect in the secretory pathway, we have constructed a series of fusion genes containing the CYH2 promoter region, with various deletions, fused to lacZ . Each fusion gene for which transcription is detected is subject to the repression . Rap1p is the transcriptional activator for most ribosomal protein genes, as well as having an important role in silencing in the vicinity of telomeres and at the silent mating-type loci . To assess its role in the repression of transcription by the defect in the secretory pathway, we have introduced rap1 mutations . The replacement of wild-type Rap1p by Rap1p truncated at the C-terminal region caused substantial attenuation of the repression . Furthermore, we have demonstrated that the Rap1p-truncation affects the repression of TCM1 , encoding ribosomal protein L3, which has no Rap1p-binding site in its upstream regulatory region . These results suggest that the repression of transcription of ribosomal protein genes by a secretory defect is mediated through Rap1p, but does not require a Rap1p-binding site within the UAS.

Nucleic Acids Res, 1998 Feb 15, 26(4), 974 - 9
A central region of Ku80 mediates interaction with Ku70 in vivo; Cary RB et al.; Ku, the DNA binding component of DNA-dependent protein kinase (DNA-PK), is a heterodimer composed of 70 and 86 kDa subunits, known as Ku70 and Ku80 respectively . Defects in DNA-PK subunits have been shown to result in a reduced capacity to repair DNA double-strand breaks . Assembly of the Ku heterodimer is required to obtain DNA end binding activity and association of the DNA-PK catalytic subunit . The regions of the Ku subunits responsible for heterodimerization have not been clearly defined in vivo . A previous study has suggested that the C-terminus of Ku80 is required for interaction with Ku70 . Here we examine Ku subunit interaction using N- and C-terminal Ku80 deletions in a GAL4-based two-hybrid system and an independent mammalian in vivo system . Our two-hybrid study suggests that the central region of Ku80, not its C-terminus, is capable of mediating interaction with Ku70 . To determine if this region mediates interaction with Ku70 in mammalian cells we transfected xrs-6 cells, which lack endogenous Ku80, with epitope-tagged Ku80 deletions carrying a nuclear localization signal . Immunoprecipitation from transfected cell extracts revealed that the central domain identified by the GAL4 two-hybrid studies stabilizes and co-immunoprecipitates with endogenous xrs-6 Ku70 . The central interaction domain maps to the internally deleted regions of Ku80 in the mutant cell lines XR-V9B and XR-V15B . These findings indicate that the internally deleted Ku80 mutations carried in these cell lines are incapable of heterodimerization with Ku70.

Curr Biol, 1998 Jan 15, 8(2), 96 - 108
Nucleosomal DNA regulates the core-histone-binding subunit of the human Hat1 acetyltransferase; Verreault A et al.; BACKGROUND: In eukaryotic cells, newly synthesized histone H4 is acetylated at lysines 5 and 12, a transient modification erased by deacetylases shortly after deposition of histones into chromosomes . Genetic studies in Saccharomyces cerevisiae revealed that acetylation of newly synthesized histones H3 and H4 is likely to be important for maintaining cell viability; the precise biochemical function of this acetylation is not known, however . The identification of enzymes mediating site-specific acetylation of H4 at Lys5 and Lys12 may help explain the function of the acetylation of newly synthesized histones . RESULTS: A cDNA encoding the catalytic subunit of the human Hat1 acetyltransferase was cloned and, using specific antibodies, the Hat1 holoenzyme was purified from human 293 cells . The human enzyme acetylates soluble but not nucleosomal H4 at Lys5 and Lys12 and acetylates histone H2A at Lys5 . Unexpectedly, we found Hat1 in the nucleus of S-phase cells . Like its yeast counterpart, the human holoenzyme consists of two subunits: a catalytic subunit, Hat1, and a subunit that binds core histones, p46, which greatly stimulates the acetyltransferase activity of Hat1 . Both p46 and the highly related p48 polypeptide (the small subunit of human chromatin assembly factor 1; CAF-1) bind directly to helix 1 of histone H4, a region that is not accessible when H4 is in chromatin . CONCLUSIONS: We suggest that p46 and p48 are core-histone-binding subunits that target chromatin assembly factors, chromatin remodeling factors, histone acetyltransferases and histone deacetylases to their histone substrates in a manner that is regulated by nucleosomal DNA.

Hum Mol Genet, 1997 Dec, 6(13), 2205 - 12
Huntingtin-associated protein 1 (HAP1) interacts with the p150Glued subunit of dynactin; Engelender S et al.; Huntington's disease (HD) is an inherited neurodegenerative disease caused by expansion of a polyglutamine repeat in the HD protein huntingtin . Huntingtin's localization within the cell includes an association with cytoskeletal elements and vesicles . We previously identified a protein (HAP1) which binds to huntingtin in a glutamine repeat length-dependent manner . We now report that HAP1 interacts with cytoskeletal proteins, namely the p150 Glued subunit of dynactin and the pericentriolar protein PCM-1 . Structural predictions indicate that both HAP1 and the interacting proteins have a high probability of forming coiled coils . We examined the interaction of HAP1 with p150 Glued . Binding of HAP1 to p150 Glued (amino acids 879-1150) was confirmed in vitro by binding of p150 Glued to a HAP1-GST fusion protein immobilized on glutathione-Sepharose beads . Also, HAP1 co-immunoprecipitated with p150 Glued from brain extracts, indicating that the interaction occurs in vivo . Like HAP1, p150 Glued is highly expressed in neurons in brain and both proteins are enriched in a nerve terminal vesicle-rich fraction . Double label immunofluorescence experiments in NGF-treated PC12 cells using confocal microscopy revealed that HAP1 and p150 Glued partially co-localize . These results suggest that HAP1 might function as an adaptor protein using coiled coils to mediate interactions among cytoskeletal, vesicular and motor proteins . Thus, HAP1 and huntingtin may play a role in vesicle trafficking within the cell and disruption of this function could contribute to the neuronal dysfunction and death seen in HD.

Cell, 1998 Mar 6, 92(5), 611 - 20
Organelle membrane fusion: a novel function for the syntaxin homolog Ufe1p in ER membrane fusion; Patel SK et al.; The fusion of endoplasmic reticulum (ER) membranes in yeast does not require Sec18p/NSF and Sec17p, two proteins needed for docking of vesicles with their target membrane . Instead, ER membranes require a NSF-related ATPase, Cdc48p . Since both vesicular and organelle fusion events use related ATPases, we investigated whether both fusion events are also SNARE mediated . We present evidence that the fusion of ER membranes requires Ufe1p, a t-SNARE that localizes to the ER, but no known v-SNAREs . We propose that the Ufe1 protein acts in the dual capacity of an organelle membrane fusion-associated SNARE by undergoing direct t-t-SNARE and Cdc48p interactions during organelle membrane fusion as well as a t-SNARE for vesicular traffic.

Genetics, 1998 Feb, 148(2), 611 - 24
The RAD52 recombinational repair pathway is essential in pol30 (PCNA) mutants that accumulate small single-stranded DNA fragments during DNA synthesis; Merrill BJ et al.; To identify in vivo pathways that compensate for impaired proliferating cell nuclear antigen (PCNA or Pol30p in yeast) activity, we performed a synthetic lethal screen with the yeast pol30-104 mutation . We identified nine mutations that display synthetic lethality with pol30-104; three mutations affected the structural gene for the large subunit of replication factor C (rfc1), which loads PCNA onto DNA, and six mutations affected three members of the RAD52 epistasis group for DNA recombinational repair (rad50, rad52 and rad57) . We also found that pol30-104 displayed synthetic lethality with mutations in other members of the RAD52 epistasis group (rad51 and rad54), but not with mutations in members of the RAD3 nor the RAD6 epistasis group . Analysis of nine different pol30 mutations shows that the requirement for the RAD52 pathway is correlated with a DNA replication defect but not with the relative DNA repair defect caused by pol30 mutations . In addition, mutants that require RAD52 for viability (pol30-100, pol30-104, rfc1-1 and rth1delta) accumulate small single-stranded DNA fragments during DNA replication in vivo . Taken together, these data suggest that the RAD52 pathway is required when there are defects in the maturation of Okazaki fragments.

Biochem Biophys Res Commun, 1998 Feb 24, 243(3), 694 - 9
Direct interaction between the intracellular domains of bullous pemphigoid antigen 2 (BP180) and beta 4 integrin, hemidesmosomal components of basal keratinocytes; Aho S et al.; Bullous pemphigoid antigen 2 (BPAG2/BP180, also known as type XVII collagen) and alpha 6 beta 4 integrin are both transmembrane proteins and hemidesmosomal components of basal keratinocytes . In this study, using yeast two-hybrid system, we demonstrate direct protein-protein interaction between the intracellular domains of BP180 and the beta 4 integrin subunit . Detailed analysis revealed that a bait construct spanning amino acids 13-89 of BP180 contained sufficient information for the protein protein interaction, but further deletion of 13 amino-terminal amino acids, which eliminates a predicted beta-sheet, abolished the interaction . The intracellular domain of the beta 4 integrin subunit contains two pairs of fibronectin type III (FNIII) repeats separated by a connecting segment . Series of expression constructs, sequentially deleting each domain, revealed that the connecting segment, the second pair of FNIII repeats and the tail region of the beta 4 integrin subunit were necessary for the interaction with BP180 in yeast two-hybrid system.

Arch Biochem Biophys, 1998 Mar 1, 351(1), 53 - 9
Protein N-arginine methylation in adenosine dialdehyde-treated lymphoblastoid cells; Li C et al.; Protein arginine methyltransferase was recently identified to be associated with some proteins in signal transduction pathways . N-Arginine methylation in RNA binding proteins with arginine- and glycine-rich RGG motifs is known to be the major protein methylation in cells . Considering that arginine methylation might be involved in certain human disorders, we used human lymphoblastoid cells that can be easily prepared from lymphocytes as a model system to study the methylation . Lymphoblastoid cells grown in the presence of 20 microM indirect methyltransferase inhibitor adenosine dialdehyde (AdOx) for 72 h appeared to accumulate high levels of hypomethylated proteins for the endogenous protein methyltransferase or recombinant glutathion S-transferase-fused yeast arginine methyltransferase (RMT1) . Analysis of methyl-accepting polypeptides in AdOx-treated lymphoblastoid cells by SDS-PAGE and fluorography showed that many polypeptides between 29,000 and 90,000 Da were methylated by the endogenous methyltransferase . A few polypeptides could be methylated to a higher extent upon the addition of yeast GST-RMT1 fusion protein . A peptide (GGRGRGGGF) could compete for the majority of the methyl-accepting protein substrates in the AdOx-treated lymphoblastoid cell extracts, whether or not exogenous yeast RMT1 was included in the reaction . When the arginine residues in the peptide were replaced by lysine, no competition was observed . The results indicated that the protein methyl acceptors in lymphoblastoid cells share similar RGG motifs and that arginine residues should be the site of methylation.

FEBS Lett, 1998 Feb 6, 422(3), 359 - 62
The core domain of RGS16 retains G-protein binding and GAP activity in vitro, but is not functional in vivo; Chen C et al.; The regulators of G-protein signaling (RGS) family members contain a conserved region, the RGS domain, and are GTPase-activating proteins for many members of G-protein alpha-subunits . We report here that the core domain of RGS16 is sufficient for in vitro biochemical functions as assayed by its G-protein binding affinity and its ability to stimulate GTP hydrolysis by G alpha(o) protein . RGS16 also requires, in addition to the RGS domain, the divergent N-terminus for its biological function in the attenuation of pheromone signaling in yeast, whereas its C-terminus region is dispensable . Together with other evidence, these data support the notion that RGS proteins interact with other cellular factors and may serve to link specific G-proteins to different downstream effectors in G-protein-mediated signaling pathways.

J Biochem (Tokyo), 1997 Dec, 122(6), 1105 - 13
PSM, an insulin-dependent, pro-rich, PH, SH2 domain containing partner of the insulin receptor; Riedel H et al.; Insulin stimulation results in a considerable spectrum of cellular responses, only part of which have been firmly correlated with the activation of established insulin receptor (IR) targets such as IRS-1, IRS-2, and Shc . Many responses may be transduced by alternative direct IR targets, some of which may still be unknown, may act in parallel to but independently of IRS-1, IRS-2, and Shc, and may be members of the growing family of SH2 domain-containing signaling adaptors . An SH2 domain-coding region of a protein termed PSM was cloned based on its interaction with an activated IR cytoplasmic fragment in a yeast two-hybrid screen . When used as a hybridization probe this region led to the isolation of a protein-coding cDNA which is expressed with a wide tissue distribution and exists in several variant forms . A pleckstrin homology domain and three Pro-rich regions including a putative SH3 domain binding site were identified in addition to the SH2 domain in the deduced 756 amino acid sequence . They imply a role of PSM in tyrosine kinase and phosphatase-mediated signaling pathways . A similar sequence termed SH2-B had been reported in an earlier study, which may represent the rat homolog of PSM . A role of PSM specifically in insulin action is suggested by the interaction of its SH2 domain with an activated but not with an inactive catalytic fragment of the IR in the yeast two-hybrid system in vivo, by the insulin-dependent association of a glutathione S-transferase (GST) PSM SH2 domain fusion protein with purified IR in vitro, and by the insulin-dependent association of GST PSM SH2 with the IR in cell extracts . In contrast, PSM was not found to associate with the established IR substrate IRS-1 under any conditions and appears to act independently of IRS-1 . All of our findings are compatible with a putative role of PSM in insulin action.

Clin Chim Acta, 1998 Jan 12, 269(1), 77 - 89
An improved biotonometric method for determination of haemoglobin oxygen equilibrium curves; Ahola TO; A new biotonometric method for the determination of haemoglobin oxygen equilibrium curves is described . The procedure is based on the mixing of an oxygen-consuming organism, the yeast cell (Saccharomyces cerevisiae), with an oxidized blood/plasma mixture . The yeast cell consumes oxygen at a uniform rate, thus reducing the partial pressure of oxygen in the mixture . This in turn induces the dissociation of oxygen in a characteristic manner . The study of the whole blood samples from 26 healthy volunteer subjects gave the following results: p50 = 3.63 kPa +/- 0.23 kPa (mean +/- 1 S.D.); Hill slope n = 2.44 +/- 0.16; and CO2 Bohr factor = -0.47 +/- 0.11 . For the within-run imprecision the coefficients of variation for the different parameters were: p50 C.V . = 4.4%; Hill slope n C.V . = 4.7%; and CO2 Bohr factor C.V . = 19% . The determination can be carried out with simple equipment: a blood gas analyzer with a coupled recorder.

EMBO J, 1998 Feb 2, 17(3), 797 - 807
Sequence and structural elements of methylation guide snoRNAs essential for site-specific ribose methylation of pre-rRNA; Kiss-Laszlo Z et al.; Site-specific 2'-O-ribose methylation of eukaryotic rRNAs is guided by small nucleolar RNAs (snoRNAs) . The methylation guide snoRNAs carry long perfect complementaries to rRNAs . These antisense elements are located either in the 5' half or in the 3' end region of the snoRNA, and are followed by the conserved D' or D box motifs, respectively . An uninterrupted helix formed between the rRNA and the antisense element of the snoRNA, in conjunction with the adjacent D' or D box, constitute the recognition signal for the putative methyltransferase . Here, we have identified an additional essential box element common to methylation guide snoRNAs, termed the C' box . We show that the C' box functions in concert with the D' box and plays a crucial role in the methyltransfer reaction directed by the upstream antisense element and the D' box . We also show that an internal fragment of U24 methylation guide snoRNA, encompassing the upstream antisense element and the D' and C' box motifs, can support the site-specific methylation of rRNA . This strongly suggests that the C box of methylation guide snoRNAs plays an essential role in the methyltransfer reaction guided by the 3'-terminal antisense element and the D box of the snoRNA.

EMBO J, 1998 Feb 2, 17(3), 667 - 76
AIM-1: a mammalian midbody-associated protein required for cytokinesis; Terada Y et al.; Mitosis is a highly coordinated process that assures the fidelity of chromosome segregation . Errors in this process result in aneuploidy which can lead to cell death or oncogenesis . In this paper we describe a putative mammalian protein kinase, AIM-1 (Aurora and Ipl1-like midbody-associated protein), related to Drosophila Aurora and Saccharomyces cerevisiae Ipl1, both of which are required for chromosome segregation . AIM-1 message and protein accumulate at G2/M phase . The protein localizes at the equator of central spindles during late anaphase and at the midbody during telophase and cytokinesis . Overexpression of kinase-inactive AIM-1 disrupts cleavage furrow formation without affecting nuclear division . Furthermore, cytokinesis frequently fails, resulting in cell polyploidy and subsequent cell death . These results strongly suggest that AIM-1 is required for proper progression of cytokinesis in mammalian cells.

Proc Natl Acad Sci U S A, 1998 Feb 3, 95(3), 975 - 80
Mutational analysis of protein phosphatase 2C involved in abscisic acid signal transduction in higher plants; Sheen J; Protein phosphatase 2C (PP2C) is a class of ubiquitous and evolutionarily conserved serine/threonine PP involved in stress responses in yeasts, mammals, and plants . Here, I present mutational analysis of two Arabidopsis thaliana PP2Cs, encoded by ABI1 and AtPP2C, involved in the plant stress hormone abscisic acid (ABA) signaling in maize mesophyll protoplasts . Consistent with the crystal structure of the human PP2C, the mutation of two conserved motifs in ABI1, predicted to be involved in metal binding and catalysis, abolished PP2C activity . Surprisingly, although the DGH177-179KLN mutant lost the ability to be a negative regulator in ABA signaling, the MED141-143IGH mutant still inhibited ABA-inducible transcription, perhaps through a dominant interfering effect . Moreover, two G to D mutations near the DGH motif eliminated PP2C activity but displayed opposite effects on ABA signaling . The G174D mutant had no effect but the G180D mutant showed strong inhibitory effect on ABA-inducible transcription . Based on the results that a constitutive PP2C blocks but constitutive Ca2+-dependent protein kinases (CDPKs) activate ABA responses, the MED141-143IGH and G180D dominant mutants are unlikely to impede the wild-type PP2C and cause hyperphosphorylation of substrates . In contrast, these dominant mutants could trap cellular targets and prevent phosphorylation by PKs required for ABA signaling . The equivalent mutations in AtPP2C showed similar effects on ABA responses . This study suggests a mechanism for the action of dominant PP2C mutants that could serve as valuable tools to understand protein-protein interactions mediating ABA signal transduction in higher plants.

Proc Natl Acad Sci U S A, 1998 Feb 3, 95(3), 951 - 6
A translational repression assay procedure (TRAP) for RNA-protein interactions in vivo; Paraskeva E et al.; RNA-protein interactions are central to many aspects of cellular metabolism, cell differentiation, and development as well as the replication of infectious pathogens . We have devised a versatile, broadly applicable in vivo system for the analysis of RNA-protein interactions in yeast . TRAP (translational repression assay procedure) is based on the translational repression of a reporter mRNA encoding green fluorescent protein by an RNA-binding protein for which a cognate binding site has been introduced into the 5' untranslated region . Because protein binding to the 5' untranslated region can sterically inhibit ribosome association, expression of the cognate binding protein causes significant reduction in the levels of green fluorescent protein fluorescence . By using RNA-protein interactions with affinities in the micromolar to nanomolar range, we demonstrate the specificity of TRAP as well as its ability to recover the cDNA encoding a specific RNA-binding protein, which has been diluted 500,000-fold with unrelated cDNAs, by using fluorescence-activated cell sorting . We suggest that TRAP offers a strategy to clone RNA-binding proteins for which little else than the binding site is known, to delineate RNA sequence requirements for protein binding as well as the protein domains required for RNA binding, and to study effectors of RNA-protein interactions in vivo.

Nucleic Acids Res, 1998 Feb 1, 26(3), 839 - 46
Sp1 activation of a TATA-less promoter requires a species-specific interaction involving transcription factor IID; Emami KH et al.; Sp1 is a ubiquitous activator of numerous TATA-containing and TATA-less promoters within the human genome . This transcription factor is distinct from several other mammalian activators because it cannot stimulate transcription of reporter genes when ectopically expressed in Saccharomyces cerevisiae . Here we report that in cultured cells from Drosophila melanogaster human Sp1 efficiently activates transcription from synthetic promoters containing TATA boxes, but not from promoters that contain an initiator instead of a TATA box . The inability of Sp1 to activate initiator-mediated transcription did not result from inactivity of the consensus initiator element used for the experiments, as other initiator functions were conserved in Drosophila cells . Interestingly, a difference between the Drosophila and human TFIID complexes was found to be responsible for the selective inability of Sp1 to activate initiator-mediated transcription in Drosophila; in a complementation assay with a TFIID-depleted HeLa cell extract both the Drosophila and human TFIID complexes supported TATA-mediated transcription, but only the human complex supported initiator-mediated transcription . These results suggest that a species-specific interaction is required for activation of TATA-less promoters by Sp1, revealing a difference in transcriptional activation mechanisms between vertebrates and invertebrates.

Mol Biol Cell, 1998 Jan, 9(1), 209 - 22
Der3p/Hrd1p is required for endoplasmic reticulum-associated degradation of misfolded lumenal and integral membrane proteins; Bordallo J et al.; We have studied components of the endoplasmic reticulum (ER) proofreading and degradation system in the yeast Saccharomyces cerevisiae . Using a der3-1 mutant defective in the degradation of a mutated lumenal protein, carboxypeptidase yscY (CPY*), a gene was cloned which encodes a 64-kDa protein of the ER membrane . Der3p was found to be identical with Hrd1p, a protein identified to be necessary for degradation of HMG-CoA reductase . Der3p contains five putative transmembrane domains and a long hydrophilic C-terminal tail containing a RING-H2 finger domain which is oriented to the ER lumen . Deletion of DER3 leads to an accumulation of CPY* inside the ER due to a complete block of its degradation . In addition, a DER3 null mutant allele suppresses the temperature-dependent growth phenotype of a mutant carrying the sec61-2 allele . This is accompanied by the stabilization of the Sec61-2 mutant protein . In contrast, overproduction of Der3p is lethal in a sec61-2 strain at the permissive temperature of 25 degrees C . A mutant Der3p lacking 114 amino acids of the lumenal tail including the RING-H2 finger domain is unable to mediate degradation of CPY* and Sec61-2p . We propose that Der3p acts prior to retrograde transport of ER membrane and lumenal proteins to the cytoplasm where they are subject to degradation via the ubiquitin-proteasome system . Interestingly, in ubc6-ubc7 double mutants, CPY* accumulates in the ER, indicating the necessity of an intact cytoplasmic proteolysis machinery for retrograde transport of CPY* . Der3p might serve as a component programming the translocon for retrograde transport of ER proteins, or it might be involved in recognition through its lumenal RING-H2 motif of proteins of the ER that are destined for degradation.

Mol Biol Cell, 1998 Jan, 9(1), 131 - 41
Tyrosine phosphorylation regulates cell cycle-dependent nuclear localization of Cdc48p; Madeo F et al.; Cdc48p from Saccharomyces cerevisiae and its highly conserved mammalian homologue VCP (valosin-containing protein) are ATPases with essential functions in cell division and homotypic fusion of endoplasmic reticulum vesicles . Both are mainly attached to the endoplasmic reticulum, but relocalize in a cell cycle-dependent manner: Cdc48p enters the nucleus during late G1; VCP aggregates at the centrosome during mitosis . The nuclear import signal sequence of Cdc48p was localized near the amino terminus and its function demonstrated by mutagenesis . The nuclear import is regulated by a cell cycle-dependent phosphorylation of a tyrosine residue near the carboxy terminus . Two-hybrid studies indicate that the phosphorylation results in a conformational change of the protein, exposing the nuclear import signal sequence previously masked by a stretch of acidic residues.

Biotechnol Prog, 1998 Jan-Feb, 14(1), 79 - 87
Bioprocess fault detection by nonlinear multivariate analysis: application of an artificial autoassociative neural network and wavelet filter bank; Shimizu H et al.; A nonlinear multivariate analysis, artificial autoassociative neural network (AANN), was applied to bioprocess fault detection . In an optimal production process of a recombinant yeast with a temperature controllable expression system, faults in test cases with faulty temperature sensors and plasmid instability of recombinant cells could be detected by the AANN . Since the raw data of measured variables included high-frequency noise, a wavelet filter bank (WFB) was applied to noise elimination before training of the AANN . The filtering performance of the WFB was compared with those of some classical first-order digital filters . The filtered signals at several resolution scales by the WFB were employed as the training data of the AANN . The computing time and summation of square of errors in training were compared, and the appropriate degree of the noise filtering and the density of the training data of the AANN were discussed . The performance of the feature capturing by the AANN was compared with that by a linear multivariate analysis, principal component analysis . A J index defined in this paper, using inputs and outputs of the AANN, was used for fault detection successfully . The output of the first unit of the trained AANN functioned effectively for the discrimination of the data in the abnormal cases from the data in the normal cases.

Nature, 1998 Feb 26, 391(6670), 915 - 8
Group II intron splicing in vivo by first-step hydrolysis; Podar M et al.; Group I, group II and spliceosomal introns splice by two sequential transesterification reactions . For both spliceosomal and group II introns, the first-step reaction occurs by nucleophilic attack on the 5' splice junction by the 2' hydroxyl of an internal adenosine, forming a 2'-5' phosphodiester branch in the intron . The second reaction joins the two exons with a 3'-5' phosphodiester bond and releases intron lariat . In vitro, group II introns can self-splice by an efficient alternative pathway in which the first-step reaction occurs by hydrolysis . The resulting linear splicing intermediate participates in normal second-step reactions, forming spliced exon and linear intron RNAs . Here we show that the group II intron first-step hydrolysis reaction occurs in vivo in place of transesterification in the mitochondria of yeast strains containing branch-site mutations . As expected, the mutations block branching, but surprisingly still allow accurate splicing . This hydrolysis pathway may have been a step in the evolution of splicing mechanisms.

Nature, 1998 Feb 26, 391(6670), 912 - 5
Carrier protein import into mitochondria mediated by the intermembrane proteins Tim10/Mrs11 and Tim12/Mrs5; Sirrenberg C et al.; Import of nuclear-encoded precursor proteins into mitochondria and their subsequent sorting into mitochondrial subcompartments is mediated by translocase enzymes in the mitochondrial outer and inner membranes . Precursor proteins carrying amino-terminal targeting signals are translocated into the matrix by the integral inner membrane proteins Tim23 and Tim17 in cooperation with Tim44 and mitochondrial Hsp70 . We describe here the discovery of a new pathway for the transport of members of the mitochondrial carrier family and other inner membrane proteins that contain internal targeting signals . Two related proteins in the intermembrane space, Tim10/Mrs11 and Tim12/Mrs5, interact sequentially with these precursors and facilitate their translocation across the outer membrane, irrespective of the membrane potential . Tim10 and Tim12 are found in a complex with Tim22, which takes over the precursor and mediates its membrane-potential-dependent insertion into the inner membrane . This interaction of Tim10 and Tim12 with the precursors depends on the presence of divalent metal ions . Both proteins contain a zinc-finger-like motif with four cysteines and bind equimolar amounts of zinc ions.

Eur J Biochem, 1998 Jan 15, 251(1-2), 487 - 95
p66/p51 and p51/p51 recombinant forms of reverse transcriptase from human immunodeficiency virus type 1--interactions with primer tRNA(Lys3), initiation of cDNA synthesis, and effect of inhibitors; Dufour E et al.; Human immunodeficiency virus type-1 (HIV-1) reverse transcriptase (RT) initiates reverse transcription from tRNA(Lys3) . HIV-1 RT is a heterodimer consisting of two polypeptides, p66 and p51 . In this work, the possible role of each subunit of RT in the interaction with its natural primer tRNA(Lys3) was studied . Two recombinant forms of HIV-1 RT, heterodimer p66/p51 and homodimer p51/p51, were used . Previously we have expressed and purified recombinant RT p51/p51 which possesses DNA polymerase activity {El Dirani-Diab, R., Andreola, M . L., Nevinsky, G., Tharaud, D., Barr, P . J., Litvak, S . & Tarrago-Litvak, L . (1992) FEBS Lett . 301, 23-28} . Here we show that HIV-1 RT p51/p51 displays certain properties very similar to the p66/p51 recombinant enzyme . The homodimer was able to anneal tRNA(Lys3) to the primer-binding site of the HIV-1 RNA template leading to a functional complex capable of synthesizing cDNA . Further, the p51/p51 enzyme behaved like RT p66/p51 concerning the strong inhibition produced by a non-nucleoside RT inhibitor . These data show that for RT p51/p51, one of the subunits of the homodimer adopts a conformation similar to the catalytic subunit (p66) present in the heterodimeric form . Part of this work was devoted to the study of the complex between the recombinant forms of HIV-1 RT and its primer tRNA . Each enzymatic form was cross-linked to tRNA(Lys3) in the presence of a platinum derivative, giving different ribonucleoprotein complexes of molecular masses higher than 100 kDa, suggesting that primer tRNA may interact with both subunits in the heterodimeric enzyme . After RNase A treatment of the complex RT p66/p51 x tRNA, the label was mainly found to migrate with the p66 subunit, although some cross-linking was also found associated to the p51 subunit . These results show that the p66 and p51 subunits of RT interact with tRNA(Lys3) . Moreover, cross-linking of tRNA(Lys3) with HIV-1 RT p66/p51 in the presence of a DNA template containing the primer-binding-site sequence yielded an enzymatically active complex.

Eur J Biochem, 1998 Jan 15, 251(1-2), 9 - 18
Chromatin and transcription--how transcription factors battle with a repressive chromatin environment; Gregory PD et al.; The last year has seen much progress in our understanding of chromatin and transcription . Transcriptionally active chromatin has long been correlated with a higher level of histone acetlyation . The discovery of a nuclear histone acetyltransferase activity encoded by factors with a role in transcription raises the possibility that the cell is able to dynamically modulate the (local) level of histone acteylation, switching chromatin templates from inactive to transcriptionally active states . Furthermore, histone acetylation states have shown to play a role in determining the efficacy of transcriptionally silenced chromatin in both yeast and Drosophila . The advances in our knowledge regarding the role of the origin-recognition complex in the establishment of silencing, and the requirement for a locally concentrated zone of the silence information regulator proteins in the nucleus has provided insights into the complex architecture of silenced chromatin . The goal of understanding the mechanisms by which the cell is able to 'open' repressive chromatin structures has prompted the discovery of multiple chromatin remodelling activities . These large protein complexes identified from organisms as diverse as yeast, mouse, fly and man demonstrate the ubiquity and fundamental importance of the ability to perturb the structure of chromatin allowing transcription of the desired genes . These data provide the latest and potentially most significant demonstration of the importance of the nucleosome in the regulation of transcription.

Cell, 1998 Feb 20, 92(4), 475 - 87
Activation of SRF-regulated chromosomal templates by Rho-family GTPases requires a signal that also induces H4 hyperacetylation; Alberts AS et al.; Constitutively active forms of the small GTPases RhoA (RhoA.V14) and Cdc42 (Cdc42.V12) induce expression of extrachromosomal SRF reporter genes in microinjection experiments, but only Cdc42.V12 can efficiently activate a chromosomal template . Both SAPK/JNK-dependent or -independent signals can cooperate with RhoA.V14 to activate chromosomal SRF reporters, and it is SAPK/JNK activation by Cdc42.V12 that allows it to activate chromosomal templates . Cooperating signals can be bypassed by deacetylase inhibitors . Three findings show that histone H4 hyperacetylation is one target for cooperating signals, although it alone is not sufficient: (1) Cdc42.V12, but not RhoA.V14, induces H4 hyperacetylation; (2) cooperating signals use the same SAPK/JNK-dependent or -independent pathways to induce H4 hyperacetylation; (3) growth factor and stress stimuli induce substantial H4 hyperacetylation, detectable in reporter gene chromatin . These data establish a link between signal-regulated acetylation events and gene transcription.

Genes Cells, 1997 Nov, 2(11), 695 - 709
Molecular cloning of the cDNA for the catalytic subunit of plant DNA polymerase alpha and its cell-cycle dependent expression; Yokoi M et al.; BACKGROUND: DNA polymerase alpha has been studied in considerable detail in yeast and animals . Genetic and biochemical analyses reveal that this enzyme is composed of a heterotetramer and is necessary for replicon initiation and primer synthesis in lagging strand synthesis . In spite of the fact that modes of DNA replication in plants seem to be similar to those in other eukaryotes, very little is known about the biochemical components that participate in DNA replication of plants, including DNA polymerases . RESULTS: Using a 561-base pair DNA fragment, obtained by polymerase chain reaction amplification from a rice cDNA library as a probe, we isolated and sequenced a cDNA homologous to the cDNA for the catalytic subunit of rice DNA polymerase alpha . The encoded polypeptide has extensive homology with the catalytic subunit of DNA polymerase alpha from several species . Furthermore, when the cDNA was expressed in eukaryotic transcription/translation systems, the protein products showed DNA polymerase activity which was inhibited by a monoclonal antibody specific for DNA polymerase alpha . Using RNA gel blot analysis, we found that the levels of mRNA of the catalytic subunit of this enzyme is regulated during the cell-cycle in plant cells . CONCLUSION: This is the first report which describes the cDNA cloning of plant DNA polymerase . We conclude that the principal features of the DNA polymerase alpha catalytic subunit are conserved in plants.

Nature, 1998 Feb 12, 391(6668), 715 - 8
Rad23 links DNA repair to the ubiquitin/proteasome pathway; Schauber C et al.; Rad23 is an evolutionarily conserved protein that is important for nucleotide excision repair . A regulatory role has been proposed for Rad23 because rad23 mutants are sensitive to ultraviolet light but are still capable of incising damaged DNA . Here we show that Rad23 interacts with the 26S proteasome through an amino-terminal ubiquitin-like domain (UbL{R23}) . The carboxy terminus of Rad23 binds to the Rad4 DNA repair protein and creates a link between the DNA repair and proteasome pathways . The ultraviolet sensitivity caused by deletion of the UbL(R23) domain may therefore arise from its inability to interact with the proteasome . The fusion proteins glutathione S-transferase (GST)-Rad23 and Rad4-haemagglutinin (HA), and the proteasome subunits Cim3 and Cim5, cofractionate through consecutive chromatography steps . The ubiquitin-like domain of human Rad23 (UbL{HRB}) also interacts with the human proteasome . These results demonstrate that ubiquitin-like domains (UbLs) represent a new class of proteasome-interacting motifs.

Lijec Vjesn, 1997 Mar-Apr, 119(3-4), 103 - 5
{Pharmacologic study of the effects of the components of beer in vitro}; Zuskin E et al.; The effect of brewery dust extracts was studied on 28 isolated nonsensitized guinea pig tracheal smooth muscle . Dust extracts of barley, hops and brewery yeast were prepared in a concentration of 1:10 w/v . Biological activity (constriction of smooth muscle) of dust extracts was expressed as a percent of initial maximal carbacol contraction 10(-4) M . Pharmacological studies were performed by pretreating guinea pig tracheal tissue with drugs such as atropin (10(-6) M), pirilamin (10(-6) M), indometacin (10(-6) M) i verapamil (10(-5) M) . Constrictor effects of the dust extracts was significantly inhibited by atropine suggesting an interaction of the extracts with parasympathetic nerves . Inhibition of contraction of other mediators was less effective and varied with dust extract . Our data suggest that brewery dust extracts cause a dose related airway smooth muscle constriction by nonimmunological mechanisms involving cholinergic receptors and a variety of airway mediators.

Eur J Biochem, 1998 Feb 1, 251(3), 804 - 11
Studies on the effect of fucosylated and non-fucosylated finger/growth-factor constructs on the clearance of tissue-type plasminogen activator mediated by the low-density-lipoprotein-receptor-related protein; Camani C et al.; Previously we observed that the finger/growth factor (FG) region of tissue-type plasminogen activator (t-PA) blocked the low-density-lipoprotein-receptor-related protein (LRP)-mediated clearance of t-PA by rat hepatocytes . However, the concentrations needed were much higher than those for intact t-PA . The FG region was expressed in yeast and lacked the fucose on Thr61, which was reported to be important for efficient clearance of t-PA by human hepatoma cells . At position 83 it had a serine, whereas human t-PA has a free cysteine and rodent t-PA an arginine at this position . To understand the reason for the low efficacy of the FG protein we produced in CHO cells chimeric molecules composed of two FG modules linked to the Fc portion of human IgG1 (FG2-Fc) . Two variants were studied, one having Ser83, the other Arg83 . The two fucosylated FG2-Fc chimeras were compared with each other, with non-fucosylated FG and with intact t-PA with regard to their effect on the clearance of t-PA and t-PA x plasminogen-activator inhibitor type 1 (PAI-1) . For this comparison, LRP-specific clearance models were used . In rat hepatoma cells and in mouse embryonic fibroblasts (MEF-1) the clearance of t-PA and of t-PA x PAI-1 was inhibited more than 95% by receptor-associated protein, an inhibitor of LRP-mediated clearance, whereas no t-PA or t-PA x PAI-1 clearance was observed in LRP-deficient PEA-13 mouse embryonic fibroblasts . The Ser83 and Arg83 FG2-Fc chimeras were equally efficient inhibitors in these models . Their efficacies in inhibiting t-PA and t-PA x PAI-1 degradation (IC50 750 nM and 890 nM, respectively) were similar to those of non-fucosylated FG (IC50 1950 nM and 1560 nM) and 75-fold lower than that of intact t-PA (IC50 9.9 nM and 21.1 nM) . The results indicate that the presence of a serine or an arginine at position 83 and the presence of a fucose on Thr61 are not of major importance for the LRP-mediated clearance of t-PA.

Eur J Biochem, 1998 Feb 1, 251(3), 729 - 33
Identification of the lamina-associated-polypeptide-2-binding domain of B-type lamin; Furukawa K et al.; Lamina-associated polypeptide (LAP)2, which directly interacts with B-type lamins and chromosomes, is an integral membrane protein specifically distributed along the inner nuclear membrane of the nuclear envelope . Multiple regions of its large nucleoplasmic domain promote this localization, including the first (residues 1-296) and the second (residues 298-409) halves of the LAP2 N terminus . The second half is involved in LAP2 association with the nuclear lamina {Furukawa, K., Pante, N., Aebi, U . & Gerace, L . (1995) EMBO J . 14, 1626-1636} . In this study to further define its role, we examined which domain of B-type lamin interacts with LAP2 by means of a binding assay with bacterially expressed proteins and a yeast two-hybrid system . We found that amino acids in the region of residues 78-258 of the lamin B1 rod domain directly bound with LAP2 . The data suggest that LAP2 may modulate the assembly of nuclear lamins.

FEBS Lett, 1998 Jan 30, 422(2), 175 - 8
Adsorption of human lysozyme onto hydroxyapatite . Identification of its adsorbing site using site-directed mutagenesis; Aizawa T et al.; To elucidate hydroxyapatite-protein interaction, mutant human lysozymes in which the surface charge was modified by site-directed mutagenesis were used . Five mutant human lysozymes (K1A, K13A, K33A, R10A, R14A) were expressed in yeast . The chromatographic behavior of these lysozymes was studied with a HPLC hydroxyapatite column . Elution molarities of K1A and R14A mutants were greatly lowered . While Lys-13 and Arg-10 are located around Lys-1 and Arg-14, K13A and R10A mutants bound onto hydroxyapatite stronger than K1A and R14A mutants . In combination with an X-ray crystal structure of human lysozyme, it is concluded that the adsorbing site of human lysozyme is at the back of the active site and that Arg-14, Lys-1, Arg-10 and Lys-13 play important roles in binding.

Biochemistry, 1998 Feb 3, 37(5), 1336 - 43
Characterization of the RNA binding properties of Ku protein; Yoo S et al.; Ku protein, a heterodimer of 70 and 83 kDa polypeptides, is the regulatory component of the DNA-dependent protein kinase (DNA-PK) . Ku protein binds to DNA ends and is essential for DNA double-strand break repair and V(D)J recombination . Although there is some evidence that Ku protein also binds RNA, its RNA binding properties have not been systematically explored . In the present study, Ku-binding RNAs were identified using systematic evolution of ligands by exponential enrichment (SELEX) technology . These RNAs were assigned to three classes based on common sequence motifs . Most of the selected RNAs bound to Ku protein with a Kd < or = 2 nM, comparable to the affinity of DNA fragments for Ku protein under similar conditions . Many of the RNAs inhibited DNA-PK activity by competing with DNA for a common binding site in Ku protein . None of several RNAs that were tested activated DNA-PK in the absence of DNA . The identification of diverse RNAs that bind avidly to Ku protein is consistent with the idea that natural RNAs may serve as modulators of DNA-PK activity . Moreover, the RNAs identified in this study may have utility as tools for experimental manipulation of DNA double-strand break repair activity in cells and cell extracts.

J Biol Chem, 1998 Feb 6, 273(6), 3765 - 70
Functional expression of the menkes disease protein reveals common biochemical mechanisms among the copper-transporting P-type ATPases; Payne AS et al.; Menkes disease is a fatal neurodegenerative disorder of childhood caused by the absence or dysfunction of a putative P-type ATPase encoded on the X chromosome . To elucidate the function of the Menkes disease protein, a plasmid containing the open reading frame of the human Menkes disease gene was constructed and used to transform a strain of Saccharomyces cerevisiae deficient in CCC2, the yeast Menkes/Wilson disease gene homologue . ccc2Delta yeast are deficient in copper transport into the secretory pathway, and expression of a wild type human Menkes cDNA complemented this defect, as evidenced by the restoration of copper incorporation into the multicopper oxidase Fet3p . Site-directed mutagenesis demonstrated the essential role of four specific amino acids in this process, including a conserved histidine, which is the site of the most common disease mutation in the homologous Wilson disease protein . The expression of Menkes cDNAs with successive mutations of the conserved cysteine residues in the six amino-terminal MXCXXC metal binding domains confirmed the essential role of these cysteine residues in copper transport but revealed that each of these domains is not functionally equivalent . These data demonstrate that the Menkes disease protein functions to deliver copper into the secretory pathway of the cell and that this process involves biochemical mechanisms common to previously characterized members of this P-type ATPase family.

J Biol Chem, 1998 Feb 6, 273(6), 3476 - 83
14-3-3 proteins interact with specific MEK kinases; Fanger GR et al.; MEK (mitogen-activated protein kinase/extracellular signal-regulated kinase kinase) kinases (MEKKs) regulate c-Jun N-terminal kinase and extracellular response kinase pathways . The 14-3-3zeta and 14-3-3epsilon isoforms were isolated in a two-hybrid screen for proteins interacting with the N-terminal regulatory domain of MEKK3 . 14-3-3 proteins bound both the N-terminal regulatory and C-terminal kinase domains of MEKK3 . The binding affinity of 14-3-3 for the MEKK3 N terminus was 90 nM, demonstrating a high affinity interaction . 14-3-3 proteins also interacted with MEKK1 and MEKK2, but not MEKK4 . Endogenous 14-3-3 protein and MEKK1 and MEKK2 were similarly distributed in the cell, consistent with their in vitro interactions . MEKK1 and 14-3-3 proteins colocalized using two-color digital confocal immunofluorescence . Binding of 14-3-3 proteins mapped to the N-terminal 393 residues of 196-kDa MEKK1 . Unlike MEKK2 and MEKK3, the C-terminal kinase domain of MEKK1 demonstrated little or no ability to interact with 14-3-3 proteins . MEKK1, but not MEKK2, -3 or -4, is a caspase-3 substrate that when cleaved releases the kinase domain from the N-terminal regulatory domain . Functionally, caspase-3 cleavage of MEKK1 releases the kinase domain from the N-terminal 14-3-3-binding region, demonstrating that caspases can selectively alter protein kinase interactions with regulatory proteins . With regard to MEKK1, -2 and -3, 14-3-3 proteins do not appear to directly influence activity, but rather function as "scaffolds" for protein-protein interactions.

J Biol Chem, 1998 Feb 6, 273(6), 3375 - 80
Thyroid hormone response element architecture affects corepressor release from thyroid hormone receptor dimers; Olson DP et al.; Thyroid hormone receptors are ligand-modulated transcription factors that can repress or activate transcription depending upon the absence or presence of thyroid hormone and the nature of the hormone response element to which the receptors are bound . The ability of thyroid hormone receptors to repress transcription in the absence of ligand is thought to be due to associations with nuclear hormone receptor corepressors . Ligand binding by the thyroid hormone receptor is believed to dissociate these corepressors and recruit coactivators to promote transcription from target promoters . We hypothesize that variations in response element architecture may influence both the association and dissociation of corepressors from DNA-bound thyroid hormone receptors . Using a chimeric corepressor, we find that ligand alone does not fully relieve corepressor-mediated repression, particularly in the presence of thyroid hormone receptor and its heterodimerization partner, the retinoid X receptor . Interestingly, the steroid receptor coactivator 1 together with ligand is able to mediate full release of corepression, but this relief is dependent upon the architecture of the response element to which the nuclear receptor dimer-corepressor complex is bound . These studies suggest that other cellular factors in addition to ligand may be required for the release of corepressors from thyroid hormone receptor dimers.

J Biol Chem, 1998 Feb 6, 273(6), 3253 - 6
Identification of a Sec4p GTPase-activating protein (GAP) as a novel member of a Rab GAP family; Du LL et al.; A yeast open reading frame sharing homology with the two known yeast Rab GTPase-activating proteins (GAPs), Gyp6p and Gyp7p, was found in a data base search . We have named the gene containing this open reading frame GYP1 . Recombinant Gyp1p showed GAP activity on Sec4p, increasing both its steady-state rate and single turnover GTPase activity . Gyp1p also stimulated the GTPase activity of several other yeast Rab proteins including Ypt1p, Ypt7p, and Ypt51p but showed no GAP activity on Ypt6p and Ypt32p . Deletion of the GYP1 gene or overexpression of Gyp1p did not alter the growth rate of yeast . However, overexpression of Gyp1p was inhibitory in combination with a subset of secretory mutants including sec4-8 and several ypt1 mutants . This effect is probably due to the increase in GAP activity, which can be observed in a lysate from cells overexpressing Gyp1p . The finding that yeast Rab GAPs share homology with proteins in other species, such as Caenorhabditis elegans and human, suggests the existence of a conserved Rab GAP family.

J Biol Chem, 1998 Feb 6, 273(6), 3158 - 65
Cytochrome b5 augments the 17,20-lyase activity of human P450c17 without direct electron transfer; Auchus RJ et al.; In the biosynthesis of steroid hormones, P450c17 is the single enzyme that catalyzes both the 17alpha-hydroxylation of 21-carbon steroids and the 17,20-lyase activity that cleaves the C17-C20 bond to produce C19 sex steroids . Cytochrome b5 augments the 17,20-lyase activity of cytochrome P450c17 in vitro, but this has not been demonstrated in membranes, and the mechanism of this action is unknown . We expressed human P450c17, human P450-oxidoreductase (OR), and/or human cytochrome b5 in Saccharomyces cerevisiae and analyzed the 17alpha-hydroxylase and 17,20-lyase activities of the resulting yeast microsomes . Yeast expressing only P450c17 have 17alpha-hydroxylase and trace 17,20-lyase activities toward both Delta4 and Delta5 steroids . Coexpression of human OR with P450c17 increases the Vmax of both the 17alpha-hydroxylase and 17,20-lyase reactions 5-fold; coexpression of human b5 with P450c17 also increases the Vmax of the 17,20-lyase reactions but not of the 17alpha-hydroxylase reactions . Simultaneous expression of human b5 with P450c17 and OR, or addition of purified human b5 to microsomes from yeast coexpressing human P450c17 and OR, further increases the Vmax of the 17,20-lyase reaction without altering 17alpha-hydroxylase activity . Genetically engineered yeast and mixing experiments demonstrate that OR is both necessary and sufficient for microsomal 17,20-lyase activity . Addition of purified human holo-b5, apo-b5, or cytochrome c to microsomes containing both human P450c17 and OR demonstrate that the stimulatory action of b5 does not require electron transfer from b5 to P450c17 . These data suggest that human b5 acts principally as an allosteric effector that interacts primarily with the P450c17.OR complex to stimulate 17, 20-lyase activity.

J Biol Chem, 1998 Feb 6, 273(6), 3148 - 51
Elongation factor 2 as a novel target for selective inhibition of fungal protein synthesis; Justice MC et al.; Elongation factor 2 (EF2) is an essential protein catalyzing ribosomal translocation during protein synthesis and is highly conserved in all eukaryotes . It is largely interchangeable in translation systems reconstituted from such divergent organisms as human, wheat, and fungi . We have identified the sordarins as selective inhibitors of fungal protein synthesis acting via a specific interaction with EF2 despite the high degree of amino acid sequence homology exhibited by EF2s from various eukaryotes . In vitro reconstitution assays using purified components from human, yeast, and plant cells demonstrate that sordarin sensitivity is dependent on fungal EF2 . Genetic analysis of sordarin-resistant mutants of Saccharomyces cerevisiae shows that resistance to the inhibitor is linked to the genes EFT1 and EFT2 that encode EF2 . Sordarin blocks ribosomal translocation by stabilizing the fungal EF2-ribosome complex in a manner similar to that of fusidic acid . The fungal specificity of the sordarins, along with a detailed understanding of its mechanism of action, make EF2 an attractive antifungal target . These findings are of particular significance due to the need for new antifungal agents.

J Biol Chem, 1998 Feb 6, 273(6), 3132 - 5
Association of acyl-CoA synthetase-1 with GLUT4-containing vesicles; Sleeman MW et al.; GLUT4, the glucose transporter present in insulin-sensitive tissues, resides in intracellular vesicular structures and translocates to the cell surface in response to insulin . In an attempt to identify proteins present in these structures, GLUT4-enriched vesicles prepared from rat adipocytes treated with or without insulin were prepared by sucrose velocity gradient centrifugation and immunoadsorbed with anti-GLUT4 antibody . We report here the sequence identification by high performance liquid chromatography-ion trap mass spectrometry of a p75 protein band, long chain acyl-CoA synthetase-1, specifically present in immunoadsorbed GLUT4-containing vesicles but not in vesicles adsorbed by nonimmune serum . Acyl-CoA synthetase activity detected in GLUT4-enriched vesicles prepared by gradient centrifugation from insulin-treated adipocytes was decreased to about the same extent as GLUT4 protein . Additionally, immunoadsorbed GLUT4 vesicles were found to catalyze palmitoylation of proteins when incubated with labeled palmitate, a pathway that requires palmitate esterification with CoA . These data indicate that the insulin-sensitive membrane compartment that sequesters GLUT4 in fat cells contains long chain acyl-CoA synthetase-1 and its product fatty acyl-CoA, shown previously to be required for budding and fusion in membrane trafficking processes.

Chem Biol, 1997 Dec, 4(12), 961 - 8
Small molecule-dependent genetic selection in stochastic nanodroplets as a means of detecting protein-ligand interactions on a large scale; Borchardt A et al.; BACKGROUND: Understanding the cellular role of a protein often requires a means of altering its function, most commonly by mutating the gene encoding the protein . Alternatively, protein function can be altered directly using a small molecule that binds to the protein, but no general method exists for the systematic discovery of small molecule ligands . Split-pool synthesis provides a means of synthesizing vast numbers of small molecules . Synthetic chemists will soon be able to synthesize natural product-like substances by this method, so compatible screening methods that detect the activity of minute quantities of molecules among many inactive ones will be in demand . RESULTS: We describe two advances towards achieving the above goals . First, a technique is described that uses a simple spray gun to create 5000-8000 droplets randomly, each having a volume of 50-200 nanoliters . The individual 'nanodroplets' contain a controlled number of cells and many also contain individual synthesis beads . As small molecules can be photochemically released from the beads in a time-dependent manner, the concentration of ligands that the cells are exposed to can be controlled . The spatial segregation of nanodroplets prevents the mixing of compounds from other beads so the effects of each molecule can be assayed individually . Second, a small molecule-dependent genetic selection involving engineered budding yeast cells was used to detect intracellular protein-ligand interactions in nanodroplets . CONCLUSIONS: The technique described here should facilitate the discovery of new cell-permeable ligands, especially when combined with a positive selection assay that detects intracellular binding of small molecules to proteins . Using 'anchored combinatorial libraries', it may be possible to screen entire libraries of natural product-like molecules against the entire collection of proteins encoded within cDNA libraries in a single experiment.

Mol Cell Biol, 1998 Mar, 18(3), 1711 - 24
The Gcn4p activation domain interacts specifically in vitro with RNA polymerase II holoenzyme, TFIID, and the Adap-Gcn5p coactivator complex; Drysdale CM et al.; The Gcn4p activation domain contains seven clusters of hydrophobic residues that make additive contributions to transcriptional activation in vivo . We observed efficient binding of a glutathione S-transferase (GST)-Gcn4p fusion protein to components of three different coactivator complexes in Saccharomyces cerevisiae cell extracts, including subunits of transcription factor IID (TFIID) (yeast TAFII20 {yTAFII20}, yTAFII60, and yTAFII90), the holoenzyme mediator (Srb2p, Srb4p, and Srb7p), and the Adap-Gcn5p complex (Ada2p and Ada3p) . The binding to these coactivator subunits was completely dependent on the hydrophobic clusters in the Gcn4p activation domain . Alanine substitutions in single clusters led to moderate reductions in binding, double-cluster substitutions generally led to greater reductions in binding than the corresponding single-cluster mutations, and mutations in four or more clusters reduced binding to all of the coactivator proteins to background levels . The additive effects of these mutations on binding of coactivator proteins correlated with their cumulative effects on transcriptional activation by Gcn4p in vivo, particularly with Ada3p, suggesting that recruitment of these coactivator complexes to the promoter is a cardinal function of the Gcn4p activation domain . As judged by immunoprecipitation analysis, components of the mediator were not associated with constituents of TFIID and Adap-Gcn5p in the extracts, implying that GST-Gcn4p interacted with the mediator independently of these other coactivators . Unexpectedly, a proportion of Ada2p coimmunoprecipitated with yTAFII90, and the yTAFII20, -60, and -90 proteins were coimmunoprecipitated with Ada3p, revealing a stable interaction between components of TFIID and the Adap-Gcn5p complex . Because GST-Gcn4p did not bind specifically to highly purified TFIID, Gcn4p may interact with TFIID via the Adap-Gcn5p complex or some other adapter proteins . The ability of Gcn4p to interact with several distinct coactivator complexes that are physically and genetically linked to TATA box-binding protein can provide an explanation for the observation that yTAFII proteins are dispensable for activation by Gcn4p in vivo.

Mol Cell Biol, 1998 Mar, 18(3), 1692 - 700
Polymerase (Pol) III TATA box-binding protein (TBP)-associated factor Brf binds to a surface on TBP also required for activated Pol II transcription; Shen Y et al.; The TATA box-binding protein (TBP) plays an essential role in transcription by all three eukaryotic nuclear RNA polymerases, polymerases (Pol) I, II, and III . In each case, TBP interacts with class-specific TBP-associated factors (TAFs) to form class-specific transcription initiation factors . For yeast Pol III transcription, TBP associates with Brf (from TFIIB-related factor) and B", two Pol III TAFs, to form Pol III transcription factor TFIIIB . Here, we identify TBP surface residues that are required for interaction with yeast Pol III TAFs . Ninety-one human TBP surface residue mutants with radical substitutions were analyzed for the ability to form stable gel shift complexes with purified Brf and B" and for their activities for in vitro synthesis of yeast U6 snRNA . Mutations in a large positively charged epitope extending from the top (that is, on the surface opposite the DNA-facing "saddle" of TBP) and onto the side of the first TBP repeat inhibited binding to Brf (residues K181, L185, R186, E206, R231, L232, R235, K236, R239, Q242, K243, K249, and F250) . A triple-mutant TBP (R231E + R235E + R239S) had greatly reduced activity for yeast U6 snRNA gene transcription while remaining active for Pol II basal transcription . Similar results were observed when selected mutations were introduced into yeast TBP at equivalent positions . A C-terminal fragment of Brf lacking the region of homology with TFIIB retains the ability to bind TBP-DNA complexes (G . Kassavetis, C . Bardeleben, A . Kumar, E . Ramirez, and E . P . Geiduschek, Mol . Cell . Biol . 17:5299-5306, 1997); the same TBP mutations reduced binding by this fragment . Mutations in TBP residues that interact with TFIIB did not affect Brf binding or U6 gene transcription . These results indicate that Brf and TFIIB interact differently with TBP . An extensively overlapping epitope on the top surface of TBP was found previously to be required for activated Pol II transcription and has been hypothesized to interact with Pol II TAFs . Our results map the surface of TBP that interacts with Brf and suggest that Pol II and Pol III TAFs interact with the same surface of TBP.

Mol Cell Biol, 1998 Mar, 18(3), 1601 - 10
Interaction of an adenovirus E3 14.7-kilodalton protein with a novel tumor necrosis factor alpha-inducible cellular protein containing leucine zipper domains; Li Y et al.; Early region 3 (E3) of group C human adenoviruses (Ad) encodes several inhibitors of tumor necrosis factor alpha (TNF-alpha) cytolysis, including an E3 14.7-kDa protein (E3-14.7K) and a heterodimer containing two polypeptides of 10.4 and 14.5 kDa . To understand the mechanism by which the viral proteins inhibit TNF-alpha functions, the E3-14.7K protein was used to screen a HeLa cell cDNA library to search for interacting proteins in the yeast two-hybrid system . A novel protein containing multiple leucine zipper domains without any significant homology with any known protein was identified and has been named FIP-2 (for 14.7K-interacting protein) . FIP-2 interacted with E3-14.7K both in vitro and in vivo . It colocalized with Ad E3-14.7K in the cytoplasm, especially near the nuclear membrane, and caused redistribution of the viral protein . FIP-2 by itself does not cause cell death; however, it can reverse the protective effect of E3-14.7K on cell killing induced by overexpression of the intracellular domain of the 55-kDa TNF receptor or by RIP, a death protein involved in the TNF-alpha and Fas apoptosis pathways . Deletion analysis indicates that the reversal effect of FIP-2 depends on its interaction with E3-14.7K . Three major mRNA forms of FIP-2 have been detected in multiple human tissues, and expression of the transcripts was induced by TNF-alpha treatment in a time-dependent manner in two different cell lines . FIP-2 has consensus sequences for several potential posttranslational modifications . These data suggest that FIP-2 is one of the cellular targets for Ad E3-14.7K and that its mechanism of affecting cell death involves the TNF receptor, RIP, or a downstream molecule affected by either of these two molecules.

Mol Cell Biol, 1998 Mar, 18(3), 1506 - 16
The Mof2/Sui1 protein is a general monitor of translational accuracy; Cui Y et al.; Although it is essential for protein synthesis to be highly accurate, a number of cases of directed ribosomal frameshifting have been reported in RNA viruses, as well as in procaryotic and eucaryotic genes . Changes in the efficiency of ribosomal frameshifting can have major effects on the ability of cells to propagate viruses which use this mechanism . Furthermore, studies of this process can illuminate the mechanisms involved in the maintenance of the normal translation reading frame . The yeast Saccharomyces cerevisiae killer virus system uses programmed -1 ribosomal frameshifting to synthesize its gene products . Strains harboring the mof2-1 allele demonstrated a fivefold increase in frameshifting and prevented killer virus propagation . In this report, we present the results of the cloning and characterization of the wild-type MOF2 gene . mof2-1 is a novel allele of SUI1, a gene previously shown to play a role in translation initiation start site selection . Strains harboring the mof2-1 allele demonstrated a mutant start site selection phenotype and increased efficiency of programmed -1 ribosomal frameshifting and conferred paromomycin sensitivity . The increased frameshifting observed in vivo was reproduced in extracts prepared from mof2-1 cells . Addition of purified wild-type Mof2p/Sui1p reduced frameshifting efficiencies to wild-type levels . Expression of the human SUI1 homolog in yeast corrects all of the mof2-1 phenotypes, demonstrating that the function of this protein is conserved throughout evolution . Taken together, these results suggest that Mof2p/Sui1p functions as a general modulator of accuracy at both the initiation and elongation phases of translation.

Mol Cell Biol, 1998 Mar, 18(3), 1349 - 58
Repression of GCN5 histone acetyltransferase activity via bromodomain-mediated binding and phosphorylation by the Ku-DNA-dependent protein kinase complex; Barlev NA et al.; GCN5, a putative transcriptional adapter in humans and yeast, possesses histone acetyltransferase (HAT) activity which has been linked to GCN5's role in transcriptional activation in yeast . In this report, we demonstrate a functional interaction between human GCN5 (hGCN5) and the DNA-dependent protein kinase (DNA-PK) holoenzyme . Yeast two-hybrid screening detected an interaction between the bromodomain of hGCN5 and the p70 subunit of the human Ku heterodimer (p70-p80), which is the DNA-binding component of DNA-PK . Interaction between intact hGCN5 and Ku70 was shown biochemically using recombinant proteins and by coimmunoprecipitation of endogenous proteins following chromatography of HeLa nuclear extracts . We demonstrate that the catalytic subunit of DNA-PK phosphorylates hGCN5 both in vivo and in vitro and, moreover, that the phosphorylation inhibits the HAT activity of hGCN5 . These findings suggest a possible regulatory mechanism of HAT activity.

Mol Cell Biol, 1998 Mar, 18(3), 1303 - 11
The alpha chain of the nascent polypeptide-associated complex functions as a transcriptional coactivator; Yotov WV et al.; We report the characterization of clone 1.9.2, a gene expressed in mineralizing osteoblasts . Remarkably, clone 1.9.2 is the murine homolog of the alpha chain of the nascent polypeptide-associated complex (alpha-NAC) . Based on sequence similarities between alpha-NAC/1.9.2 and transcriptional regulatory proteins and the fact that the heterodimerization partner of alpha-NAC was identified as the transcription factor BTF3b (B . Wiedmann, H . Sakai, T . A . Davis, and M . Wiedmann, Nature 370:434-440, 1994), we investigated a putative role for alpha-NAC/ 1.9.2 in transcriptional control . The alpha-NAC/1.9.2 protein potentiated by 10-fold the activity of the chimeric activator GAL4/VP-16 in vivo . The potentiation was shown to be mediated at the level of gene transcription, because alpha-NAC/1.9.2 increased GAL4/VP-16-mediated mRNA synthesis without affecting the half-life of the GAL4/VP-16 fusion protein . Moreover, the interaction of alpha-NAC/1.9.2 with a transcriptionally defective mutant of GAL4/VP-16 was severely compromised . Specific protein-protein interactions between alpha-NAC/1.9.2 and GAL4/VP-16 were demonstrated by gel retardation, affinity chromatography, and protein blotting assays, while interactions with TATA box-binding protein (TBP) were detected by immunoprecipitation, affinity chromatography, and protein blotting assays . Based on these interactions that define the coactivator class of proteins, we conclude that the alapha-NAC/1.9.2 gene product functions as a transcriptional coactivator.

Mol Cell Biol, 1998 Mar, 18(3), 1201 - 12
Gal4p-mediated chromatin remodeling depends on binding site position in nucleosomes but does not require DNA replication; Xu M et al.; Biochemical studies have demonstrated decreased binding of various proteins to DNA in nucleosome cores as their cognate sites are moved from the edge of the nucleosome to the pseudodyad (center) . However, to date no study has addressed whether this structural characteristic of nucleosomes modulates the function of a transcription factor in living cells, where processes of DNA replication and chromatin modification or remodeling could significantly affect factor binding . Using a sensitive, high-resolution methyltransferase assay, we have monitored the ability of Gal4p in vivo to interact with a nucleosome at positions that are known to be inaccessible in nucleosome cores in vitro . Gal4p efficiently bound a single cognate site (UASG) centered at 41 bp from the edge of a positioned nucleosome, perturbing chromatin structure and inducing transcription . DNA binding and chromatin perturbation accompanying this interaction also occurred in the presence of hydroxyurea, indicating that DNA replication is not necessary for Gal4p-mediated nucleosome disruption . These data extend previous studies, which demonstrated DNA replication-independent chromatin remodeling, by showing that a single dimer of Gal4p, without the benefit of cooperative interactions that occur at complex wild-type promoters, is competent for invasion of a preestablished nucleosome . When the UASG was localized at the nucleosomal pseudodyad, relative occupancy by Gal4p, nucleosome disruption, and transcriptional activation were substantially compromised . Therefore, despite the increased nucleosome binding capability of Gal4p in cells, the precise translational position of a factor binding site in one nucleosome in an array can affect the ability of a transcriptional regulator to overcome the repressive influence of chromatin.

Mol Cell Biol, 1998 Mar, 18(3), 1181 - 9
Processing of the precursors to small nucleolar RNAs and rRNAs requires common components; Petfalski E et al.; The genes encoding the small nucleolar RNA (snoRNA) species snR190 and U14 are located close together in the genome of Saccharomyces cerevisiae . Here we report that these two snoRNAs are synthesized by processing of a larger common transcript . In strains mutant for two 5'-->3' exonucleases, Xrn1p and Rat1p, families of 5'-extended forms of snR190 and U14 accumulate; these have 5' extensions of up to 42 and 55 nucleotides, respectively . We conclude that the 5' ends of both snR190 and U14 are generated by exonuclease digestion from upstream processing sites . In contrast to snR190 and U14, the snoRNAs U18 and U24 are excised from the introns of pre-mRNAs which encode proteins in their exonic sequences . Analysis of RNA extracted from a dbr1-delta strain, which lacks intron lariat-debranching activity, shows that U24 can be synthesized only from the debranched lariat . In contrast, a substantial level of U18 can be synthesized in the absence of debranching activity . The 5' ends of these snoRNAs are also generated by Xrn1p and Rat1p . The same exonucleases are responsible for the degradation of several excised fragments of the pre-rRNA spacer regions, in addition to generating the 5' end of the 5.8S rRNA . Processing of the pre-rRNA and both intronic and polycistronic snoRNAs therefore involves common components.

Cell Mol Life Sci, 1998 Jan, 54(1), 21 - 31
Histone acetylation as an epigenetic determinant of long-term transcriptional competence; Turner BM; All four histones of the nucleosome core particle are subject to post-translational acetylation of selected lysine residues in their amino-terminal domains . The modification is ubiquitous and frequent . Steady-state levels of acetylation have been shown to vary from one part of the genome to another and to be maintained by a dynamic balance between the activities of two enzyme families, the histone acetyltransferases (HATs) and deacetylases (HDAs) . The recent demonstration that some at least of these enzymes are homologous to, or identical with, known regulators of transcription, has renewed interest in the involvement of histone acetylation in transcriptional control . Acetylation might influence the initiation and/or elongation phases of transcription in a chromatin context, possibly by regulating the accessibility of nucleosomal DNA to transcription factors or the displacement of histones by the progressing transcription complex . But there is also evidence to suggest that acetylation might be involved in the longer-term regulation of transcription, acting as a marker by which states of genetic activity or inactivity are maintained from one cell generation to the next . This review outlines the evidence for such a role, using centric heterochromatin and the dosage-compensated male X chromosome in Drosophila as model systems, and suggests possible mechanisms by which it might operate.

Cell Mol Life Sci, 1998 Jan, 54(1), 6 - 20
Linking histone acetylation to transcriptional regulation; Mizzen CA et al.; In eukaryotes, DNA is assembled with histones to form nucleosomes, the basic subunit of chromatin structure . The wrapping of DNA around histone octamers to form nucleosomal filaments and further folding of these filaments are necessary to contain eukaryotic genomes within nuclei . However, the dense packing of chromatin in nuclei and the association of DNA with histones restrict the access of proteins involved in gene transcription to DNA . Abundant biochemical data supports a long-standing correlation between histone acetylation and gene activation, suggesting that histone acetylation acts to enhance the access of transcription-associated proteins to DNA . However, despite this correlation, nuclear enzymes responsible for transcription-associated histone acetylation have been identified only recently . Here we review evidence suggesting that histone acetylation represents a major pathway for transcriptional regulation, and discuss possible roles for transcription-associated histone acetyltransferases in this regulation.

J Neurosci Res, 1998 Feb 1, 51(3), 328 - 38
Protein phosphatase type-2C isozymes present in vertebrate retinae: purification, characterization, and localization in photoreceptors; Klumpp S et al.; Posttranslational modification of proteins by kinases and phosphatases plays an important role in the regulation of cellular signaling in general and neurochemistry in particular . This also applies to vertebrate photoreceptors where phosphorylation of rhodopsin causes uncoupling from the signal transduction cascade . Functional activity of rhodopsin is restored after substitution of the bleached photopigment 11-cisretinal and by dephosphorylation of the opsin moiety . Phosphatases type-1 and type-2A have been identified in vertebrate retinae . Recently, we have shown by molecular cloning that two isozymes of protein phosphatase type-2C (PP2C, PPM) do exist in retinal tissue . In this report, we have purified PP2Calpha and PP2Cbeta from bovine retinae . Thirty to 40% of PP2C was recovered in the cytosolic fraction . Biochemical properties of native and heterologously expressed recombinant enzymes were similar . Enzymatic activity is strictly dependent on the presence of Mg2+ . Addition of Ca2+ ions inhibits Mg2+-sustained activity . Antiserum raised against a C-terminal peptide of PP2Cbeta specifically labeled the outer segments of rod photoreceptor cells . PP2C protein levels were significantly reduced in RCS rats, which develop age-dependent photoreceptor degeneration comparable to the hereditary disease retinitis pigmentosa . Although the retinal substrate(s) remain to be identified, the results suggest that PP2C modulates cellular components of the phototransduction machinery.

Biochemistry, 1998 Feb 17, 37(7), 1961 - 8
Importance of intramembrane carboxylic acids for occlusion of K+ ions at equilibrium in renal Na,K-ATPase; Nielsen JM et al.; Site-directed mutagenesis and assay of Rb+ and Tl+ occlusion in recombinant Na,K-ATPase from yeast were combined to establish structure-function relationships of amino acid side chains involved in high-affinity occlusion of K+ in the E2{2K} form . The wild-type yeast enzyme was capable of occluding 2 Rb+ or Tl+ ions/ouabain binding site or alpha 1 beta 1 unit with high apparent affinity (Kd(Tl+) = 7 +/- 2 microM), like the purified Na,K-ATPase from pig kidney . Mutations of Glu327(Gln,Asp), Asp804(Asn, Glu), Asp808(Asn, Glu) and Glu779(Asp) abolished high-affinity occlusion of Rb+ or Tl+ ions . The substitution of Glu779 for Gln reduced the occlusion capacity to 1 Tl+ ion/alpha 1 beta 1-unit with a 3-fold decrease of the apparent affinity for the ion (Kd(Tl+) = 24 +/- 8 microM) . These effects on occlusion were closely correlated to effects of the mutations on K0.5(K+) for K+ displacement of ATP binding . Each of the four carboxylate residues Glu327, Glu779, and Asp804 or Asp808 in transmembrane segments 4, 5, and 6 is therefore essential for high-affinity occlusion of K+ in the E2{2K} form . These residues either may engage directly in cation coordination or they may be important for formation or stability of the occlusion cavity.

Plant Mol Biol, 1998 Jan, 36(1), 125 - 36
Conserved Ser residues in the basic region of the bZIP-type transcription factor HBP-1a(17): importance in DNA binding and possible targets for phosphorylation; Meshi T et al.; HBP-1a(17) is representative of a group of plant bZIP-type transcription factors which includes HBP-1a proteins and G-box-binding factors . We found kinase activity in wheat nuclear extract that phosphorylated HBP-1a(17) . Experiments using recombinant HBP-1a(17) derivatives as substrates revealed that all three of the Ser residues in the basic region, Ser-261, Ser-265, and Ser-269, were phosphorylated in a Ca(2+)-stimulated manner . DNA-binding analysis of mutants with a Ser-to-Glu change, prepared to mimic the phosphorylated proteins, indicated that introduction of a negative charge at position 265 or 269 prevents HBP-1a(17) from binding DNA not only in the homodimer of mutants but also in heterodimers with a wild-type protein . It is therefore suggested that the phosphorylation regulates the function of HBP-1a(17) at least at the level of DNA binding . Since Ser-265 and Ser-269 are highly conserved among the plant bZIP-type factors known to date, a common Ca(2+)-mediated regulatory mechanism may exert an effect on the bZIP-type factors through phosphorylation of these conserved Ser residues.

Plant Mol Biol, 1998 Jan, 36(1), 23 - 31
Molecular cloning of a novel fimbrin-like cDNA from Arabidopsis thaliana; McCurdy DW et al.; Fimbrin is a 68-70 kDa actin-bundling protein in animal cells and lower eukaryotes that participates in diverse morphogenetic processes by cross-linking actin filaments into bundles . Here we report the cloning by degenerate polymerase chain reaction (PCR) of ATFIM1, a 2.3 kb cDNA from Arabidopsis thaliana that codes for a novel 76 kDa fimbrin-like polypeptide (AtFim1) . The predicted sequence of AtFim1 shares ca . 40% identity with nonplant fimbrins and contains two tandem repeats, each possessing a 27 amino acid region identified as a putative actin-binding domain in fimbrins and in a larger family of actin cross-linking proteins . Preceding the tandem repeats at the amino terminus of AtFim1 is a single-EF-hand-like domain with moderate homology to calmodulin-like calcium-binding proteins . AtFim1 differs from non-plant fimbrins, however, in that it contains an extended carboxy-terminal tail of ca . 65 amino acids . ATFIM1 is encoded by a single gene, although sequencing of two partial fimbrin-like expressed sequence tag (EST) clones indicates that Arabidopsis contains at least two fimbrin-like proteins . Northern blot analysis and reverse-transcription PCR (RT-PCR) demonstrated that ATFIM1 is expressed in all major organs examined (roots, leaves, stems, flowers and siliques) . This is the first report of the cloning of a full length plant gene that encodes a putative actin filament-bundling protein.

Plant Mol Biol, 1998 Mar, 36(4), 541 - 52
Changes in the levels of seven proteins involved in polypeptide folding and transport during endosperm development of two barley genotypes differing in storage protein localisation; Mogelsvang S et al.; The Russian barley cultivar Nevsky lacks gamma 3 hordein and accumulates most of its hordein in the lumen of the endoplasmic reticulum and only a minor portion in the vacuole . In wild type barley and all other temperate cereals, storage proteins are deposited in the vacuole . F1 crosses revealed that the Nevsky phenotype is recessive; but the extent of hordein accumulation in the endoplasmic reticulum in F2 endosperm lacking gamma 3 hordein was very much less than in the Nevsky parent . In order to study the Nevsky endosperm phenotype we have measured the levels of seven proteins and two mRNAs involved in protein folding in the ER lumen or ER to Golgi transport during endosperm development . The protein levels were unaltered in Nevsky as compared to the wild-type variety Bomi . When the levels of these seven proteins were correlated with the rate of hordein accumulation, four of these (HSP70, PDI, Sar1p and Sec18p) were consistently up-regulated with hordein synthesis . Accumulation of hordein in the endoplasmic reticulum appears to be determined by the absence of gamma 3 hordein, or the product of a gene closely linked to it, plus one or more other recessive genes.

Plant Mol Biol, 1998 Jan, 36(2), 323 - 8
Analysis of the isopentenyl diphosphate isomerase gene family from Arabidopsis thaliana; Campbell M et al.; Two Arabidopsis thaliana cDNAs (IPP1 and IPP2) encoding isopentenyl diphosphate isomerase (IPP isomerase) were isolated by complementation of an IPP isomerase mutant strain of Saccharomyces cerevisiae . Both cDNAs encode enzymes with an amino terminus that may function as a transit peptide for localization in plastids . At least 31 amino acids from the amino terminus of the IPP1 protein and 56 amino acids from the amino terminus of the IPP2 protein are not essential for enzymatic activity . Genomic DNA blot analysis confirmed that IPP1 and IPP2 are derived from a small gene family in A . thaliana . Based on northern analysis expression of both cDNAs occurs predominantly in roots of mature A . thaliana plants grown to the pre-flowering stage.

Mol Endocrinol, 1998 Feb, 12(2), 302 - 13
Nuclear receptor-binding sites of coactivators glucocorticoid receptor interacting protein 1 (GRIP1) and steroid receptor coactivator 1 (SRC-1): multiple motifs with different binding specificities; Ding XF et al.; The activity of the AF-2 transcriptional activation function of nuclear receptors (NR) is mediated by the partially homologous transcriptional coactivators, glucocorticoid receptor interacting protein 1 (GRIP1)/transcriptional intermediary factor 2 (TIF2) and steroid receptor coactivator 1 (SRC-1) . GRIP1 and SRC-1 bound nine different NRs and exhibited similar, but not identical, NR binding preferences . The most striking difference was seen with the androgen receptor, which bound well to GRIP1 but poorly to SRC-1 . GRIP1 and SRC-1 contain three copies of the NR binding motif LXXLL (called an NR Box) in their central regions . Mutation of both NR Box II and NR Box III in GRIP1 almost completely eliminated functional and binding interactions with NRs, indicating that these two sites are crucial for most of GRIP1's NR binding activity . Interactions of GRIP1 with the estrogen receptor were more strongly affected by mutations in NR Box II, whereas interactions with the androgen receptor and glucocorticoid receptor were more strongly affected by NR Box III mutations . One isoform of SRC-1 has an additional NR Box (NR Box IV) at its extreme C terminus with an NR-binding preference somewhat different from that of the central NR-binding domain of SRC-1 . GRIP1 has no NR Box in its C-terminal region and therefore no C-terminal NR-binding function . In summary, GRIP1 and SRC-1 have overlapping NR-binding preferences, but specific NRs display both coactivator and NR Box preferences that may contribute to the specificity of hormonal responses.

Trends Microbiol, 1998 Jan, 6(1), 35 - 40
Lipophosphoglycan (LPG) and the identification of virulence genes in the protozoan parasite Leishmania; Beverley SM et al.; Leishmania exploits several strategies to survive within the phagolysosome of vertebrate macrophages and be transmitted by sand fly vectors . Recent advances in functional genetic analysis provide a new avenue for identifying genes implicated in the infectious cycle of the parasite, such as those necessary for the synthesis and expression of the key surface glycoconjugate, lipophosphoglycan (LPG).

Genomics, 1998 Jan 15, 47(2), 310 - 3
Discovery of three novel G-protein-coupled receptor genes; O'Dowd BF et al.; We report here the molecular cloning, tissue distribution, and chromosomal localization of novel genes encoding G-protein-coupled receptors (GPCRs) . A search of a mouse database of expressed sequence tags revealed an EST partially encoding a GPCR, which was used to screen a mouse genomic library to obtain the translational open reading frame (ORF) . The resultant clone, GPR27, contained an intronless ORF, encoding a receptor of 379 amino acids . In an alternate strategy, human genomic DNA was subjected to polymerase chain reaction (PCR) amplification, using degenerate oligonucleotides based on GPR1 . Two PCR products partially encoding GPCRs were isolated and used to screen a genomic library to obtain the translational ORF . One of the resultant clones, GPR30, contained an intronless ORF encoding a receptor of 375 amino acids . The other clone, GPR35, also contained an intronless ORF encoding a receptor of 309 amino acids . Transcripts corresponding to GPR27 and GPR30 were detected in several areas of human and rat CNS, While GPR35 expression was detected only in the rat intestine . Through fluorescence in situ hybridization analysis the gene encoding GPR30 was localized to chromosome 7p22 and GPR35 to chromosome 2q37.3.

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