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 wit