FEBS Lett, 1998 Jun 5, 429(1), 27 - 30 The carboxy-terminal domain of the receptor-associated protein binds to the Vps10p domain of sortilin; Tauris J et al.; Binding of the receptor-associated protein (RAP) to the newly identified putative sorting receptor, sortilin, was analyzed by surface plasmon resonance analysis of recombinant RAP and sortilin domains and compared with binding to megalin and low density lipoprotein receptor-related protein (LRP) . The data show that the RAP-binding site in sortilin is localized in the cysteine-rich lumenal part homologous to yeast vacuolar protein-sorting 10 protein (Vps10p), and the sortilin-binding site in RAP is localized in the carboxy-terminal domain III of the three homologous domains in RAP . Whereas sortilin bound only RAP domain III, megalin and LRP bound all RAP domains with the functional affinity order: domain III >domain I > domain II. FEBS Lett, 1998 Jun 5, 429(1), 17 - 20 Overexpression of hMTH1 mRNA: a molecular marker of oxidative stress in lung cancer cells; Kennedy CH et al.; Human MutT homologue (hMTH1) mRNA was overexpressed in SV-40-transformed non-tumorigenic human bronchial epithelial cells (BEAS-2B cells) and in 11 out of 12 human lung cancer cell lines relative to normal human bronchial epithelial cells . Expression levels of hMTH1 mRNA were inversely proportional to cellular levels of 8-oxo-deoxyguanosine . Together, these results suggest that hMTH1 gene expression may represent a molecular marker of oxidative stress that could ultimately be used to elucidate the temporal relationships between oxidative stress, genomic instability and the development of lung cancer. Cell, 1998 Jun 26, 93(7), 1125 - 34 LMA1 binds to vacuoles at Sec18p (NSF), transfers upon ATP hydrolysis to a t-SNARE (Vam3p) complex, and is released during fusion; Xu Z et al.; Vacuole fusion requires Sec18p (NSF), Sec17p (alpha-SNAP), Ypt7p (GTP binding protein), Vam3p (t-SNARE), Nyv1p (v-SNARE), and LMA1 (low Mr activity 1, a heterodimer of thioredoxin and I(B)2) . LMA1 requires Sec18p for saturable, high-affinity binding to vacuoles, and Sec18p "priming" ATPase requires both Sec17p and LMA1 . Either the sec18-1 mutation and deletion of I(B)2, or deletion of both I(B)2 and p13 (an I(B)2 homolog) causes a striking synthetic vacuole fragmentation phenotype . Upon Sec18p ATP hydrolysis, LMA1 transfers to (and stabilizes) a Vam3p complex . LMA1 is released from vacuoles in a phosphatase-regulated reaction . This LMA1 cycle explains how priming by Sec18p is coupled to t-SNARE stabilization and to fusion. Biochim Biophys Acta, 1998 Jun 29, 1385(2), 253 - 70 Structure-function relationships and flexible tetramer assembly in pyruvate decarboxylase revealed by analysis of crystal structures; Furey W et al.; The crystal structures of pyruvate decarboxylase from the yeast Saccharomyces uvarum and Saccharomyces cerevisiae have been determined at 2.4 and 2.3 A resolution, respectively . These structures provide details about the protein fold and domain assembly within subunits, about subunit assembly to form dimers and about dimer assembly to form tetramers . They also provide a clear picture of the active site centered on the thiamin diphosphate cofactor, and have allowed amino acids critical for catalysis and involved in stabilization of the unusual cofactor conformation to be identified . The structural information has enabled identification of the site of allosteric activation to be centered on Cys-221, and suggests that a six residue segment leading from the regulatory site to the catalytic site may be involved in transmission of a binding signal . The importance of several amino acids within this segment in the regulatory process, as well as some involved in stabilizing and activating the cofactor has been confirmed by analyzing the behavior of recombinant enzymes with single point mutations introduced at these sites . Additional structures have been determined for pyruvate decarboxylase in multiple crystal forms, some of which were obtained from crystals grown with known allosteric activators present in the media . Currently four distinct types of tetramers have been observed, with each showing a different mode of association of dimers to form the tetramers . In some of the cases involving the presence of allosteric activators drastic changes in the mode of dimer assembly to form tetramers is seen. Immunity, 1998 Jun, 8(6), 703 - 11 Cabin 1, a negative regulator for calcineurin signaling in T lymphocytes; Sun L et al.; Calcineurin plays a pivotal role in the T cell receptor (TCR)-mediated signal transduction pathway and serves as a common target for the immunosuppressants FK506 and cyclosporin A . We report the identification of a novel endogenous calcineurin binding protein named Cabin 1 that inhibits calcineurin-mediated signal transduction . The interaction between Cabin 1 and calcineurin is dependent on PKC activation . Overexpression of Cabin 1 or its N-terminal truncation mutants inhibits the transcriptional activation of calcineurin-responsive elements in the interleukin-2 promoter and blocks dephosphorylation of NF-AT upon T cell activation . These results suggest a negative regulatory role for Cabin 1 in calcineurin signaling and provide a possible mechanism of feedback inhibition of TCR signaling through cross-talk between protein kinases and calcineurin. J Inorg Biochem, 1998 Mar, 69(4), 293 - 303 A role for HEM2 in cadmium tolerance; Hunter TC et al.; A Candida glabrata cadmium-sensitive mutant partially defective in glutathione production and exhibiting a complete absence of phytochelatins was used to clone a gene required for Cd tolerance . Transformation of the Cd-sensitive mutant with a genomic library from the wild-type C . glabrata led to the cloning of a gene that restored Cd tolerance and formation of Cd-glutathione and Cd-phytochelatin complexes . The cloned gene showed high levels of nucleic acid and protein sequence homology to the HEM2 genes, encoding porphobilinogen synthases, from several sources . It was shown that the C, glabrata Cd-sensitive mutant indeed exhibited a significant reduction in porphobilinogen synthase levels . The cloned C . glabrata gene complemented a hem2 mutant of Saccharomyces cerevisiae and restored porphobilinogen synthase activity in the mutant . The Cd sensitive mutant predictably showed decreased levels of sulfite reductase that requires siroheme, a metabolite produced in the heme biosynthetic pathway . The addition of cysteine, but not methionine, increased glutathione levels and Cd tolerance of both the wild-type and the mutant strain . However, addition of hemin chloride and methionine together restored Cd tolerance indicating that heme was required for transsulfuration of homocysteine to cysteine. FEBS Lett, 1998 May 29, 428(3), 259 - 62 A novel importin alpha from rice, a component involved in the process of nuclear protein transport; Iwasaki T et al.; In eukaryotes, nuclear proteins that are transported into nuclei have nuclear localization signals (NLSs), which are recognized by proteins called importin alpha . We isolated a rice cDNA, #61L, and the corresponding gene that encodes a protein, which shows significant homology to the importin alpha . Although the encoded protein had only 23-27% amino acid identity to the importin alphas from various organisms including plants, the fusion protein with glutathione S-transferase showed a specific binding activity to the NLS of SV40 T-antigen . These results suggest that the rice #61L protein is a novel importin alpha in plants. Eur J Biochem, 1998 May 1, 253(3), 804 - 9 Molecular cloning, expression and characterization of an Ascaris inhibitor for pepsin and cathepsin E; Kageyama T; Molecular cloning of a cDNA for a pepsin inhibitor in the round worm, Ascaris suum, was achieved . The ORF was found to encode a 20-residue potential signal peptide and a 149-residue inhibitor moiety . Northern analysis showed the mRNA for the inhibitor to be expressed in the body wall and not in the viscera . To obtain the active inhibitor, we constructed a yeast expression vector, pYES2API, containing the inducible galactosidase promoter and a DNA fragment encoding a fusion protein of the yeast alpha-factor leader and the Ascaris pepsin inhibitor . The active inhibitor was secreted in the culture medium, the yield being approximately 3 mg x l(-1) x day(-1), and purified by a two-step procedure that included HPLC . The inhibitor inactivated pepsin A and cathepsin E almost completely at amounts equimolar with the enzymes, but was 100-fold less effective against pepsin C and did not act on cathepsin D and renin . Ki values for the inhibition of pepsin A and cathepsin E were in the nanomolar range below pH 5 . Since the inhibitor activity was lost by modification of specific Lys residues, including Lys110, an electrostatic interaction between these Lys residues and Asp/Glu residues of pepsin A or cathepsin E was thought to be essential for the inhibition. Eur J Biochem, 1998 May 1, 253(3), 734 - 42 The 58-kDa microspherule protein (MSP58), a nucleolar protein, interacts with nucleolar protein p120; Ren Y et al.; Protein p120 is a proliferation-related nucleolar protein which is detectable early in the G1 phase of the cell cycle and peaks early in the S phase . Most human malignant tumors contain much higher levels of protein p120 than normal resting cells . To identify p120-associated protein(s), a yeast two-hybrid screen was carried out using protein p120 as the bait . Two positive clones encoded portions of a novel protein, designated microspherule protein 58 kDa (MSP58) . MSP58 mRNA is 1.9 kb and encodes an approximately 58-kDa polypeptide of 462 amino acids as shown by Western blotting of HeLa nucleolar proteins . The mouse MSP58 homolog has 97% amino acid similarity to human MSP58, but no MSP58 homolog was found in the yeast genome . The MSP58 N-terminal region contains serine-rich clusters and its C-terminal region has a coiled-coil domain . In insect Sf9 cells, recombinant p120 and MSP58 proteins associated with each other, confirming the results of the yeast two-hybrid assay . Deletion mutations revealed that the binding of MSP58 to p120 required a previously unrecognized coiled-coil domain within the N-terminal region of p120 and the C-terminal region of MSP58 protein . Immunofluorescence indicated that the MSP58 protein is localized in microspherules in the nucleolus . Anti-MSP58 Ig labeled nucleolar 'caps' when HeLa cells were treated with actinomycin D . When the MSP58 protein was overexpressed in COS-7 cells, the nucleolus became irregularly enlarged, which suggests that MSP58 may affect the size and shape of the nucleolus. Int J Dev Biol, 1998, 42(3), 249 - 55 Probing for gene specificity in epithelial development; Schupbach T et al.; We surveyed a total of 228 random insertions of a P{GawB} element to determine the fraction of regulatory regions in the Drosophila genome that activate gene expression specifically in follicle cells versus producing more complex patterns of expression . We monitored the GAL4 expression encoded by this construct in the ovarian follicle cells by crossing the lines to a strain containing a lacZ reporter construct . Sixty four per cent of the insertions showed ovarian expression . To assess the specificity of this expression, 124 of the 228 lines were crossed to strains containing either an activated form of Armadillo, the Drosophila homolog of beta-catenin, or an activated form of Torpedo/Egfr, the Drosophila homolog of the Epidermal Growth Factor receptor, under the control of GAL4 target sites . The lethality and imaginal disc phenotypes observed in these crosses suggest that most random insertions cause GAL4 expression in a variety of tissues . Very few insertions appear to drive expression only in follicle cells . Although the activated form of Armadillo produced higher frequencies of lethality and disk phenotypes, expression in the follicle cell epithelium at later stages of oogenesis did not lead to a visible phenotype . This contrasts with the dorsalized phenotypes observed in the combination of the same GAL4 lines with the activated Torpedo construct. Genomics, 1998 Jun 1, 50(2), 241 - 50 cDNA cloning and characterization of a human proteasomal modulator subunit, p27 (PSMD9); Watanabe TK et al.; We have employed cDNA cloning to deduce the complete primary structure of a new subunit, designated p27, of the modulator trimer complex that stimulates the association of the PA700 regulator with the catalytic 20S proteasome to form the ATP-dependent active 26S proteasome . We found two distinct cDNAs encoding two highly homologous proteins except in the C-terminal region, which are termed tentatively p27-1 and p27-2 . The short p27-2 cDNA has a deletion of 65 bp near the 3'-end region of the long p27-1 cDNA, which encodes a large protein with an extended C-terminal region, designated p27-L, whereas the long p27-1 cDNA encodes a small protein named p27-S . The polypeptides of p27-L and p27-S consist of 223 and 209 amino acid residues with calculated molecular masses of 24,852 and 22,764 and isoelectric points of 6.50 and 5.28, respectively . Immunoblot analysis with anti-p27 antibody revealed that p27, together with two other ATPase components, TBP1 and p42, was associated with not only the modulator complex but also significantly with the 26S proteasome complex, suggesting that the three are common/sharing subunits in these two complexes . By the fluorescence in situ hybridization method, the p27 (PSMD9) gene was mapped to the q24.2-q24.3 band of human chromosome 12 . Computer-assisted homology analysis revealed the high sequence similarities of p27-L with a possible counterpart in Caenorhabditis elegans and Saccharomyces cerevisiae whose function is yet unknown, the yeast gene that is here termed NAS2 (non-ATPase subunit 2) . Disruption of NAS2 had no effect on cell viability, indicating that the subunit is not essential for proliferation of yeast cells. Genomics, 1998 Jun 1, 50(2), 222 - 8 Identification and characterization of the gene encoding a second proteolipid subunit of human vacuolar H(+)-ATPase (ATP6F); Nishigori H et al.; The proteolipid domain of vacuolar H(+)-ATPase (V-ATPase) plays a major role in H+ transport in microvesicles and other acidic organelles . We have cloned the second human proteolipid of the V-ATPase (designated hATP6F), a homologue of the Saccharomyces cerevisiae proteolipid VMA16, which is an essential subunit of yeast V-ATPase . hATP6F is a hydrophobic protein with five putative transmembrane segments, having 61% amino acid identity and 83% similarity to the yeast protein, except in the N-terminus, and contains a conserved glutamic acid residue (Glu98) that is essential for H(+)-transporting activity . The gene for hATP6F (gene symbol, ATP6F), which consists of eight exons and spans approximately 3.5 kb, was isolated and mapped to human chromosome band 1p32.3 and the region 10.81 cR centromeric of the STS marker SHGC36789 (LOD = 6.75) by fluorescence in situ hybridization and radiation hybrid mapping, respectively . This is the first evidence in human of the existence of a second gene encoding a distinct V-ATPase proteolipid. Proc Natl Acad Sci U S A, 1998 Jul 7, 95(14), 7904 - 8 PRT1 of Arabidopsis thaliana encodes a component of the plant N-end rule pathway; Potuschak T et al.; Mutants in the PRT1 gene of Arabidopsis thaliana are impaired in the degradation of a normally short-lived intracellular protein that contains a destabilizing N-terminal residue . Proteins bearing such residues are the substrates of an ubiquitin-dependent proteolytic system called the N-end rule pathway . The chromosomal position of PRT1 was determined, and the PRT1 gene was isolated by map-based cloning . The 45-kDa PRT1 protein contains two RING finger domains and one ZZ domain . No other proteins in databases match these characteristics of PRT1 . There is, however, a weak similarity to Rad18p of Saccharomyces cerevisiae . The RING finger domains have been found in a number of other proteins that are involved in ubiquitin conjugation, consistent with the proposed role of PRT1 in the plant N-end rule pathway. Proc Natl Acad Sci U S A, 1998 Jul 7, 95(14), 7898 - 903 The mouse and human genes encoding the recognition component of the N-end rule pathway; Kwon YT et al.; The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue . The N-end rule pathway is one proteolytic pathway of the ubiquitin system . The recognition component of this pathway, called N-recognin or E3, binds to a destabilizing N-terminal residue of a substrate protein and participates in the formation of a substrate-linked multiubiquitin chain . We report the cloning of the mouse and human Ubr1 cDNAs and genes that encode a mammalian N-recognin called E3alpha . Mouse UBR1p (E3alpha) is a 1,757-residue (200-kDa) protein that contains regions of sequence similarity to the 225-kDa Ubr1p of the yeast Saccharomyces cerevisiae . Mouse and human UBR1p have apparent homologs in other eukaryotes as well, thus defining a distinct family of proteins, the UBR family . The residues essential for substrate recognition by the yeast Ubr1p are conserved in the mouse UBR1p . The regions of similarity among the UBR family members include a putative zinc finger and RING-H2 finger, another zinc-binding domain . Ubr1 is located in the middle of mouse chromosome 2 and in the syntenic 15q15-q21.1 region of human chromosome 15 . Mouse Ubr1 spans approximately 120 kilobases of genomic DNA and contains approximately 50 exons . Ubr1 is ubiquitously expressed in adults, with skeletal muscle and heart being the sites of highest expression . In mouse embryos, the Ubr1 expression is highest in the branchial arches and in the tail and limb buds . The cloning of Ubr1 makes possible the construction of Ubr1-lacking mouse strains, a prerequisite for the functional understanding of the mammalian N-end rule pathway. Proc Natl Acad Sci U S A, 1998 Jul 7, 95(14), 7882 - 7 Control of interferon-tau gene expression by Ets-2; Ezashi T et al.; Expression of the multiple interferon-tau (IFN-tau) genes is restricted to embryonic trophectoderm of ruminant ungulate species for a few days in early pregnancy . The promoter regions of these genes are highly conserved . A proximal (bp -91 to -69) sequence has been implicated in controlling trophoblast-specific expression . Here it was used as a target for yeast one-hybrid screening of a day 13 conceptus cDNA library . Two transcription factors of the Ets family, Ets-2 and GABPalpha, were identified, consistent with the observation that active ovine IFN-tau genes contain a single 10-bp Ets motif (core: GGAA) in the proximal segment, whereas three known inactive ovine genes contain a mutated core motif (TGAA) . Cotransfection of a promoter- (-126 to +50) luciferase reporter construct from an active gene (bovineIFN-tau1; boIFNT1) and an Ets-2 expression plasmid in human JAr cells provided up to a 30-fold increase in reporter expression, whereas promoters from inactive genes were not transactivated . GABPalpha alone was ineffective and had only a approximately 2-fold positive effect when coexpressed with its partner GABPbeta . Other Ets-related transcription factors, which were not detected in the genetic screen, also provided a range of lesser transactivation effects . Coexpression of Ets-2 and activated Ras failed to transactivate the IFNT promoter greater than Ets-2 alone in JAr cells . The presence of Ets-2 in nuclei of embryonic trophectoderm was confirmed immunocytochemically . Together, these data suggest that Ets-2 plays a role in the transient expression of the nonvirally inducible IFNT genes. Int J Radiat Biol, 1998 May, 73(5), 469 - 74 Reduced joining of DNA double strand breaks with an abnormal mutation spectrum in rodent mutants of DNA-PKcs and Ku80; Tzung TY et al.; PURPOSE: To characterize further the contribution of the DNA-PK-dependent dsb repair pathway in mammalian cells . MATERIALS AND METHODS: The efficiency and fidelity of the joining of linear plasmids by DNA-PKcs-defective mouse cells (SCID) and Ku80-defective Chinese hamster ovary cells (xrs-6) was measured using either linear or circular replicating shuttle vector pZ189 . RESULTS: The authors found a 3.9-10.7-fold reduced joining of the DNA ends, as compared with wild-type cells . Mutation analysis of the joining site revealed that the joining process was not hypermutable in the mutated cells . However, the SCID and xrs-6 cells produced a different spectrum of mutations at the joining site with a significantly lower proportion of insertions or more complex mutations . CONCLUSIONS: The remaining joining ability of the mutant cells points to an alternative DNA-PK-independent pathway of dsb repair . Comparison of these data with similar data from yeast suggest that the postulated alternative pathway of dsb repair is at least as efficient and less error-prone in rodent cells. Eur J Biochem, 1998 May 15, 254(1), 111 - 6 Evidence of an overlap between the two half-sites of UAS1-B/CYC1--a new model for Cyp1p (Hap1p) DNA binding; Nait-Kaoudjt R et al.; Cyp1p (Hap1p) is a yeast transcriptional regulator belonging to the zinc-cluster family . CGGNNNTANCGG was identified by PCR selection as the DNA sequence allowing its optimal binding . Nevertheless, this sequence is not a consensus sequence, the simultaneous presence of the two CGGs and the TA generally not being found in the known natural Cyp1p targets . In fact, our previous studies showed that the mechanism of Cyp1p DNA binding was target dependent . Data concerning the binding of Cyp1p to the UAS1-B/CYC1 are presented here . This target, containing the CGGGGTTTACGG sequence, was found to present the particular ability of stabilizing the binding of only one molecule of some monomeric Cyp1p fragments . This property was used to investigate the actual contribution of the TT and CGG sequences in the binding of Cyp1p . Our results indicate that each CGG belongs to a different half-site and, in contrast to a previous hypothesis, that the T nucleotide located four bases downstream from the left CGG is essential for the binding of one monomer to each half-site . The two half-sites of the UAS1-B/CYC1 thus overlap. Curr Biol, 1998 Jun 18, 8(13), 750 - 60 The regulation of Cdc20 proteolysis reveals a role for APC components Cdc23 and Cdc27 during S phase and early mitosis; Prinz S et al.; BACKGROUND: In eukaryotic cells, a specialized proteolysis machinery that targets proteins containing destruction-box sequences for degradation and that uses a ubiquitin ligase known as the anaphase-promoting complex/cyclosome (APC) plays a key role in the regulation of mitosis . APC-dependent proteolysis triggers the separation of sister chromatids at the metaphase-anaphase transition and the destruction of mitotic cyclins at the end of mitosis . Recently, two highly conserved WD40-repeat proteins, Cdc20 and Cdh1/Hct1, have been identified as substrate-specific regulators for APC-dependent proteolysis in the budding yeast Saccharomyces cerevisiae . Here, we have investigated the cell cycle regulation of Cdc20 and Cdh1/Hct1 . RESULTS: Whereas the levels CDH1/HCT1 RNA and Cdh1/Hct1 protein are constant throughout the cell cycle, CDC20 RNA and Cdc20 protein are present only during late S phase and mitosis and Cdc20 protein is unstable throughout the entire cell cycle . The instability of Cdc20 depends on CDC23 and CDC27, which encode components of the APC . During the G1 phase, a destruction box within Cdc20 mediates its instability, but during S phase and mitosis, although Cdc20 destruction is still dependent on CDC23 and CDC27, it does not depend on the Cdc20 destruction box . CONCLUSIONS: There are remarkable differences in the regulation of Cdc20 and Cdh1/Hct1 . Furthermore, the APC activator Cdc20 is itself a substrate of the Cdc27 have a role in the degradation of Cdc20 during S Phase and early mitosis that is not mediated by its destruction box. Curr Biol, 1998 Jun 18, 8(13), R461 - 3 Meiosis: step-by-step through sporulation; Clancy MJ; A transcription factor, Ndt80p, has been identified that has a critical role in the pathway that controls meiosis--sporulation--in budding yeast . Ndt80p coordinately controls genes that mediate spore formation and progression through the two meiotic divisions; it may also be a target of a checkpoint control. Mol Cell, 1998 Jun, 1(7), 969 - 79 The 3' to 5' exonuclease activity of Mre 11 facilitates repair of DNA double-strand breaks; Paull TT et al.; MRE11 and RAD50 are known to be required for nonhomologous joining of DNA ends in vivo . We have investigated the enzymatic activities of the purified proteins and found that Mre11 by itself has 3' to 5' exonuclease activity that is increased when Mre11 is in a complex with Rad50 . Mre11 also exhibits endonuclease activity, as shown by the asymmetric opening of DNA hairpin loops . In conjunction with a DNA ligase, Mre11 promotes the joining of noncomplementary ends in vitro by utilizing short homologies near the ends of the DNA fragments . Sequence identities of 1-5 base pairs are present at all of these junctions, and their diversity is consistent with the products of nonhomologous end-joining observed in vivo. Mol Cell, 1998 Jun, 1(7), 939 - 48 The CRY1 blue light photoreceptor of Arabidopsis interacts with phytochrome A in vitro; Ahmad M et al.; Plants have at least two major photosensory receptors: phytochrome (absorbing primarily red/far-red light) and cryptochrome (absorbing blue/UV-A light); considerable physiological and genetic evidence suggests some form of communication or functional dependence between the receptors . Here, we demonstrate in vitro, using purified recombinant photoreceptors, that Arabidopsis CRY1 and CRY2 (cryptochrome) are substrates for phosphorylation by a phytochrome A-associated kinase activity . Several mutations within the CRY1 C terminus lead to reduced phosphorylation by phytochrome preparations in vitro . Yeast two-hybrid interaction studies using expressed C-terminal fragments of CRY1 and phytochrome A from Arabidopsis confirm a direct physical interaction between both photoreceptors . In vivo labeling studies and specific mutant alleles of CRY1, which interfere with the function of phytochrome, suggest the possible relevance of these findings in vivo. Mutat Res, 1998 Mar 30, 413(3), 205 - 17 Isolation of a novel metabolizing system enriched in phase-II enzymes for short-term genotoxicity bioassays; Paolini M et al.; Murine S9 liver fractions isolated from mice fed 7.5 g kg-1 2(3)-tert-Butyl-4-hydroxyanisole (BHA) for 3 weeks were tested to determine: (a) the profile of both phase-I and phase-II xenobiotic metabolizing enzymes; (b) their ability to induce in vitro covalent binding of some precarcinogens to calf thymus DNA; and (c) their activation in a standard genetic toxicology assay . With regard to phase-I pathway, the S9 fraction expressed various cytochrome P-450-(CYP) (classes 1A1, 1A2, 2B1, 2E1, and 3A)-dependent biotransformation enzymes at levels comparable with those present in murine control liver . For post-oxidative enzymes, the S9 expressed high levels of glutathione S-transferases (up to 12-fold increase), glutathione S-epoxide-transferase (up to 2.6-fold), UDP-glucuronosyl transferase (up to 5.3-fold) and epoxide hydrolase (up to 2.6-fold) activities, as compared to untreated mice . The in vitro DNA binding of the precarcinogenic agents {14C}-1,4-dichlorobenzene, {14C}-1,2-dichlorobenzene and {14C}-1,4-dibromobenzene, mediated by BHA-induced cytosol and/or microsomal preparation, showed an increase in specific activity comparable to that observed with phase-I (PB/beta NF) induced S9 . In some instances, covalent binding was even more elevated using the BHA-induced systems as compared with traditional S9 fractions . For example, cytosol derived from BHA-administered mice was able to induce a significant binding to calf thymus DNA up to 26.2-fold increase for {14C}-1,4-dichlorobenzene, while cytosol from PB/beta NF was not . A high mutagenic response on diploid D7 strain of Saccharomyces cerevisiae as exemplified by a marked induction of mitotic gene conversion and point (reverse) mutation confirmed that BHA-derived S9 fractions activate precarcinogens to final genotoxins . Because a number of chemicals are activated by either oxidative or post-oxidative enzymes, the use of metabolizing biosystems, with an enhanced phase-II pathway, together with classical S9 fractions, can improve the sensitivity of the assay in detecting unknown genotoxins. Gene, 1998 Jul 3, 214(1-2), 139 - 46 Novel testis-specific protein that interacts with heat shock factor 2; Yoshima T et al.; Although heat shock factor 2 (HSF2) binds to heat shock element (HSE) constitutively during differentiation, development and spermatogenesis, little is known about the nature and mechanism of transcriptional control of heat shock genes by HSF2 . We screened a human testis cDNA library for proteins that can associate with HSF2 by the yeast two-hybrid system, and isolated clones encoding a novel protein, designated HSF2 binding protein (HSF2BP), that associates with HSF2 in vitro and in vivo and is specifically expressed in testis . The interaction seemed to occur between the trimerization domain of HSF2 and the amino terminal hydrophilic region of HSF2BP that comprises two leucine zipper motifs . HSF2BP may therefore be involved in modulating HSF2 activation in testis. J Biol Chem, 1998 Jul 10, 273(28), 17749 - 55 GTPase activating specificity of RGS12 and binding specificity of an alternatively spliced PDZ (PSD-95/Dlg/ZO-1) domain; Snow BE et al.; Regulator of G-protein signaling (RGS) proteins increase the intrinsic guanosine triphosphatase (GTPase) activity of G-protein alpha subunits in vitro, but how specific G-protein-coupled receptor systems are targeted for down-regulation by RGS proteins remains uncharacterized . Here, we describe the GTPase specificity of RGS12 and identify four alternatively spliced forms of human RGS12 mRNA . Two RGS12 isoforms of 6.3 and 5.7 kilobases (kb), encoding both an N-terminal PDZ (PSD-95/Dlg/ZO-1) domain and the RGS domain, are expressed in most tissues, with highest levels observed in testis, ovary, spleen, cerebellum, and caudate nucleus . The 5.7-kb isoform has an alternative 3' end encoding a putative C-terminal PDZ domain docking site . Two smaller isoforms, of 3.1 and 3.7 kb, which lack the PDZ domain and encode the RGS domain with and without the alternative 3' end, respectively, are most abundantly expressed in brain, kidney, thymus, and prostate . In vitro biochemical assays indicate that RGS12 is a GTPase-activating protein for Gi class alpha subunits . Biochemical and interaction trap experiments suggest that the RGS12 N terminus acts as a classical PDZ domain, binding selectively to C-terminal (A/S)-T-X-(L/V) motifs as found within both the interleukin-8 receptor B (CXCR2) and the alternative 3' exon form of RGS12 . The presence of an alternatively spliced PDZ domain within RGS12 suggests a mechanism by which RGS proteins may target specific G-protein-coupled receptor systems for desensitization. J Biol Chem, 1998 Jul 10, 273(28), 17720 - 5 Inhibition of hGrb10 binding to the insulin receptor by functional domain-mediated oligomerization; Dong LQ et al.; hGrb10 is a newly identified Src homology 2 (SH2) and pleckstrin homology (PH) domain-containing protein that binds to autophosphorylated receptor tyrosine kinases, including the insulin and insulin-like growth factor receptors . To identify potential downstream proteins that interact with hGrb10, we screened a yeast two-hybrid cDNA library using the full-length hGrb10gamma as bait . A fragment of hGrb10, which included the IPS (insert between the PH and SH2 domain) and the SH2 domains, was found to bind with high affinity to the full-length protein . The interaction between the IPS/SH2 domain and the full-length hGrb10 was further confirmed by in vitro glutathione S-transferase fusion protein binding studies . Gel filtration assays showed that hGrb10 underwent tetramerization in mammalian cells . The interaction involved at least two functional domains, the IPS/SH2 region and the PH domain, both of which interacted with the NH2-terminal amino acid sequence of hGrb10gamma (hGrb10gamma DeltaC, residues 4-414) . Competition studies showed that hGrb10gamma DeltaC inhibited the binding of hGrb10 to the tyrosine-phosphorylated insulin receptor, suggesting that this region may play a regulatory role in hGrb10/insulin receptor interaction . We present a model for hGrb10 tetramerization and its potential role in receptor tyrosine kinase signal transduction. J Biol Chem, 1998 Jul 10, 273(28), 17713 - 9 Interaction of eye protein kinase C and INAD in Drosophila . Localization of binding domains and electrophysiological characterization of a loss of association in transgenic flies; Adamski FM et al.; Drosophila eye-specific protein kinase C (eye-PKC) is involved in light adaptation and deactivation . eye-PKC, NORPA (phospholipase Cbeta), and transient-receptor-potential (TRP) (calcium channel) are integral components of a signal transduction complex organized by INAD, a protein containing five PDZ domains . We previously demonstrated the direct association between the third PDZ domain of INAD with TRP in addition to the carboxyl-terminal half of INAD with the last three residues of NORPA . In this work, the molecular interaction between eye-PKC and INAD is defined via the yeast two-hybrid and ligand overlay assays . We show that the second PDZ domain of INAD interacts with the last three residues in the carboxyl-terminal tail of eye-PKC, Thr-Ile-Ile . The association between eye-PKC and INAD is disrupted by an amino acid substitution (Ile-700 to Asp) at the final residue of eye-PKC . In flies lacking endogenous eye-PKC (inaCp215), normal visual physiology is restored upon expression of wild-type eye-PKC, whereas the eye-PKCI700D mutant is completely inactive . Flies homozygous for inaCp209 and InaDp215, a mutation that causes a loss of the INAD-TRP association, were generated . These double mutants display a more severe response inactivation than either of the single mutants . Based on these findings, we conclude that the in vivo activity of eye-PKC depends on its association with INAD and that the sensitivity of photoreceptors is cooperatively regulated by the presence of both eye-PKC and TRP in the signaling complex. J Biol Chem, 1998 Jul 10, 273(28), 17463 - 8 Zinc cluster proteins Leu3p and Uga3p recognize highly related but distinct DNA targets; Noel J et al.; Members of the family of fungal zinc cluster DNA-binding proteins possess 6 highly conserved cysteines that bind to two zinc atoms forming a structure (Zn2Cys6) that is required for recognition of specific DNA sequences . Many zinc cluster proteins have been shown to bind as homodimers to a pair of CGG triplets oriented either as direct (CGG NX CGG), inverted (CGG NX CCG), or everted repeats (CCG NX CGG), where N indicates nucleotides . Variation in the spacing between the CGG triplets also contributes to the diversity of sites recognized . For example, Leu3p binds to the everted sequence CCG N4 CGG with a strict requirement for a 4-base pair spacing . Here, we show that another member of the family, Uga3p, recognizes the same DNA motif as Leu3p . However, these transcription factors have distinct DNA targets . We demonstrate that additional specificity of binding is provided by nucleotides located between the two everted CGG triplets . Altering the 4 nucleotides between to the two everted CGG triplets switches the specificity from a Uga3p site to a Leu3p site in both in vitro and in vivo assays . Thus, our results identify a new mechanism that expands the repertoire of DNA targets of the family of zinc cluster proteins . These experiments provide a model for discrimination between targets of zinc cluster proteins. Br Poult Sci, 1998 May, 39(2), 245 - 50 Comparison of the utilisation of palm kernel meal, brewers' dried grains and maize offal by broiler chicks; Onifade AA et al.; 1 . Palm kernel meal (PKM), brewers dried grains (BDG) and maize offal (MO) were included in broiler diets, each at 100, 150 or 200 g/kg; the diets were fed up to 35 d of age . 2 . Overall food intake and weight gain decreased in the order BDG, PKM and MO . There were, however, significant interactions between the test ingredients and dietary concentrations in all the growth responses . Food intakes increased with the dietary concentrations of each test ingredient, but the increase was greater for BDG than PKM or MO . For weight gain, at 100 g/kg, the final body weights of the chicks fed on the diets with BDG and MO were similar, and those of chicks fed on the diet with PKM slightly lower . However, at 200 g/kg, growth rate of chicks fed on the BDG and PKM diets were similar while those of chicks fed on the MO diet was 7% lower . Efficiency of food utilisation was similar at 100 g/kg for all the ingredients and decreased as their concentrations increased; however, the decrease was considerably less for the PKM than for the MO and BDG diets . 3 . Broilers fed on the BDG-based diets voided most excreta followed by those fed on the PKM and MO diets; excreta water content was highest from birds fed on the MO diets followed by the PKM and BDG diets . Apparent retention of dry matter was similar with all the test ingredients, but it decreased only significantly at 200 g/kg dietary concentration . The rate of passage was faster with the PKM diets followed by the MO and BDG diets; it was increased at 200 g/kg dietary concentration of the test ingredients. EMBO J, 1998 Jul 1, 17(13), 3786 - 95 DNA ligase I is recruited to sites of DNA replication by an interaction with proliferating cell nuclear antigen: identification of a common targeting mechanism for the assembly of replication factories; Montecucco A et al.; In mammalian cells, DNA replication occurs at discrete nuclear sites termed replication factories . Here we demonstrate that DNA ligase I and the large subunit of replication factor C (RF-C p140) have a homologous sequence of approximately 20 amino acids at their N-termini that functions as a replication factory targeting sequence (RFTS) . This motif consists of two boxes: box 1 contains the sequence IxxFF whereas box 2 is rich in positively charged residues . N-terminal fragments of DNA ligase I and the RF-C large subunit that contain the RFTS both interact with proliferating cell nuclear antigen (PCNA) in vitro . Moreover, the RFTS of DNA ligase I and of the RF-C large subunit is necessary and sufficient for the interaction with PCNA . Both subnuclear targeting and PCNA binding by the DNA ligase I RFTS are abolished by replacement of the adjacent phenylalanine residues within box 1 . Since sequences similar to the RFTS/PCNA-binding motif have been identified in other DNA replication enzymes and in p21(CIP1/WAF1), we propose that, in addition to functioning as a DNA polymerase processivity factor, PCNA plays a central role in the recruitment and stable association of DNA replication proteins at replication factories. EMBO J, 1998 Jul 1, 17(13), 3747 - 57 The snoRNA box C/D motif directs nucleolar targeting and also couples snoRNA synthesis and localization; Samarsky DA et al.; Most small nucleolar RNAs (snoRNAs) fall into two families, known as the box C/D and box H/ACA snoRNAs . The various box elements are essential for snoRNA production and for snoRNA-directed modification of rRNA nucleotides . In the case of the box C/D snoRNAs, boxes C and D and an adjoining stem form a vital structure, known as the box C/D motif . Here, we examined expression of natural and artificial box C/D snoRNAs in yeast and mammalian cells, to assess the role of the box C/D motif in snoRNA localization . The results demonstrate that the motif is necessary and sufficient for nucleolar targeting, both in yeast and mammals . Moreover, in mammalian cells, RNA is targeted to coiled bodies as well . Thus, the box C/D motif is the first intranuclear RNA trafficking signal identified for an RNA family . Remarkably, it also couples snoRNA localization with synthesis and, most likely, function . The distribution of snoRNA precursors in mammalian cells suggests that this coupling is provided by a specific protein(s) which binds the box C/D motif during or rapidly after snoRNA transcription . The conserved nature of the box C/D motif indicates that its role in coupling production and localization of snoRNAs is of ancient evolutionary origin. EMBO J, 1998 Jul 1, 17(13), 3726 - 37 Processing of a dicistronic small nucleolar RNA precursor by the RNA endonuclease Rnt1; Chanfreau G et al.; Small nucleolar RNAs (snoRNAs) are intron encoded or expressed from monocistronic independent transcription units, or, in the case of plants, from polycistronic clusters . We show that the snR190 and U14 snoRNAs from the yeast Saccharomyces cerevisiae are co-transcribed as a dicistronic precursor which is processed by the RNA endonuclease Rnt1, the yeast ortholog of bacterial RNase III . RNT1 disruption results in a dramatic decrease in the levels of mature U14 and snR190 and in accumulation of dicistronic snR190-U14 RNAs . Addition of recombinant Rnt1 to yeast extracts made from RNT1 disruptants induces the chase of dicistronic RNAs into mature snoRNAs, showing that dicistronic RNAs correspond to functional precursors stalled in the processing pathway . Rnt1 cleaves a dicistronic transcript in vitro in the absence of other factors, separating snR190 from U14 . Thus, one of the functions of eukaryotic RNase III is, as for the bacterial enzyme, to liberate monocistronic RNAs from polycistronic transcripts. EMBO J, 1998 Jul 1, 17(13), 3692 - 703 A specialized form of RNA polymerase I, essential for initiation and growth-dependent regulation of rRNA synthesis, is disrupted during transcription; Milkereit P et al.; Only a small proportion (<2%) of RNA polymerase I (pol I) from whole-cell extracts appeared to be competent for specific initiation at the ribosomal gene promoter in a yeast reconstituted transcription system . Initiation-competent pol I molecules were found exclusively in salt-resistant complexes that contain the pol I-specific initiation factor Rrn3p . Levels of initiation-competent complexes in extracts were independent of total Rrn3p content and varied with the growth state of the cells . Although extracts from stationary phase cells contained substantial amounts of Rrn3p and pol I, they lacked the pol I-Rrn3p complex and were inactive in promoter-dependent transcription . Activity was restored by adding purified pol I-Rrn3p complex to extracts from stationary phase cells . The pol I-Rrn3p complex dissociated during transcription and lost its capacity for subsequent reinitiation in vitro, suggesting a stoichiometric rather than a catalytic activity in initiation . We propose that the formation and disruption of the pol I-Rrn3p complex reflects a molecular switch for regulating rRNA synthesis and its growth rate-dependent regulation. EMBO J, 1998 Jul 1, 17(13), 3597 - 607 Aut2p and Aut7p, two novel microtubule-associated proteins are essential for delivery of autophagic vesicles to the vacuole; Lang T et al.; AUT2 and AUT7, two novel genes essential for autophagocytosis in the yeast Saccharomyces cerevisiae were isolated . AUT7 was identified as a low copy suppressor of autophagic defects in aut2-1 cells . Aut7p is a homologue of the rat microtubule-associated protein (MAP) light chain 3 (LC3) . Aut2p and Aut7p interact physically . Aut7p is attached to microtubules via Aut2p, which interacts with tubulins Tub1p and Tub2p . aut2- and aut7-deleted cells are unable to deliver autophagic vesicles and the precursor of aminopeptidase I to the vacuole . Double membrane-layered autophagosome-like vesicles accumulate in the cytoplasm of these cells . Our findings suggest that microtubules and an attached protein complex of Aut2p and Aut7p are involved in the delivery of autophagic vesicles to the vacuole. EMBO J, 1998 Jul 1, 17(13), 3542 - 55 Elongation and clustering of glycosomes in Trypanosoma brucei overexpressing the glycosomal Pex11p; Lorenz P et al.; Kinetoplastid protozoa confine large parts of glycolysis within glycosomes, which are microbodies related to peroxisomes . We cloned the gene encoding the second most abundant integral membrane protein of Trypanosoma brucei glycosomes . The 24 kDa protein is very basic and hydrophobic, with two predicted transmembrane domains . It is targeted to peroxisomes when expressed in mammalian cells and yeast . The protein is a functional homologue of Pex11p from Saccharomyces cerevisiae: pex11Delta mutants, which are defective in peroxisome proliferation, can be complemented by the trypanosome gene . Sequence conservation is significant in the N- and C-terminal domains of all putative Pex11p homologues known, from trypanosomes, yeasts and mammals . Several lines of evidence indicate that these domains are oriented towards the cytosol . TbPex11p can form homodimers, like its yeast counterpart . The TbPEX11 gene is essential in trypanosomes . Inducible overexpression of the protein in T.brucei bloodstream forms causes growth arrest, the globular glycosomes being transformed to clusters of long tubules filling significant proportions of the cytoplasm . Reduced expression results in trypanosomes with fewer, but larger, organelles. Biochemistry, 1998 Jun 30, 37(26), 9586 - 94 Role of important hydrophobic amino acids in the interaction between the glucocorticoid receptor tau 1-core activation domain and target factors; Almlof T et al.; In this work, we determined how altered-function mutants affecting hydrophobic residues within the tau 1-core activation domain of the human glucocorticoid receptor (GR) influence its physical interaction with different target proteins of the transcriptional machinery . Screening of putative target proteins showed that the tau 1-core can interact with the C-terminal part of the CREB-binding protein (CBP) . In addition, the previously identified interactions of the tau 1-core with the TATA-binding protein (TBP) and the Ada2 adaptor protein were localized to the C- and N-terminal regions of these proteins, respectively . A panel of mutations within the tau 1-core that either decrease or increase activation potential was used to probe the interaction of the tau 1-core domain with TBP, Ada2, and CBP . We found that the pattern of effects caused by the mutations was similar for each of the interactions and that the effects on binding generally reflected effects on gene activation potential . Thus, the predominant effect of the mutations appears to influence a property of the tau 1-core that is common to all three interactions, rather than properties that are differentially required by each of the target factor interactions, individually . Such a property could be the ability of the domain to adopt a folded conformation that is generally necessary for interaction with target factors . We have also shown that TBP, Ada2, and CBP can interact with both the tau 1-core and the GR ligand-binding domain, offering a possible mechanism for synergistic interaction between the tau 1-core and other receptor activation domains . However, other target proteins (e.g., RIP140, and SRC-1), which interact with the GR C terminus, did not show significant interactions with the tau 1-core under our conditions. Biochem Biophys Res Commun, 1998 Jun 29, 247(3), 833 - 7 Differential expression of human histone deacetylase mRNAs in response to immune cell apoptosis induction by trichostatin A and butyrate; Dangond F et al.; The reversible acetylation of histones by histone deacetylases (HDACs) and acetyltransferases (HATs) plays a fundamental role in gene transcription . We previously showed that HDAC mRNA is upregulated in immune cells upon PHA-induced activation . Little is known, however, about the differential regulation of HDAC mRNAs by the HDAC inhibitors Trichostatin A (TSA) and butyrate, agents known to block proliferation and induce apoptosis . We report that apoptosis-inducing concentrations of TSA and butyrate upregulate the expression of HDAC mRNAs in a differential manner and act synergistically with PHA to induce HDAC expression, suggesting the presence of independent HDAC regulatory mechanisms . Moreover, we show that HDAC inhibitor-induced apoptosis is associated with early abrogation of gamma-IFN production by Th1 lymphocytes and with p53 mRNA downregulation . Our findings highlight the dynamic interplay of cell cycle-, activation- and apoptosis-related proteins in association with time-dependent expression of HDACs and are suggestive of different specific roles for these enzymes. Br J Cancer, 1998 Jun, 77 Suppl 4, 18 - 20 Advances in the management of malignancy-associated hyperuricaemia; Mahmoud HH et al.; Acute tumour lysis syndrome (ATLS) is a metabolic derangement (hyperuricaemia, hyperphosphataemia, hyperkalaemia and hypocalcaemia) associated with lymphoproliferative malignancies . The nature and severity of the metabolic alterations are variable . Major complications are oliguric acute renal failure and delays in initiating chemotherapy . Current management of ATLS includes hydration, alkalinization, diuretics, when indicated, and the reduction of uric acid levels using allopurinol or urate oxidase . Allopurinol inhibits xanthine oxidase, an enzyme that catalyses the conversion of hypoxanthine and xanthine to uric acid . Urate oxidase (Uricozyme), a naturally occurring proteolytic enzyme in many mammals, degrades uric acid to allantoins, which are ten times more soluble than uric acid and easily eliminated by the kidneys . Recently, Sanofi Research isolated a recombinant urate oxidase (SR29142) as a cDNA clone from Aspergillus flavus, expressed in the yeast strain Saccharomyces cerevisiae . Preclinical studies have documented its biological effects as a urolytic enzyme . Twenty-eight healthy male volunteers received SR29142, and a rapid decline of uric acid below measurable levels was seen within 4 h in all patients receiving a dose of more than 0.10 mg kg(-1) . Currently, SR29142 is undergoing clinical studies in both Europe and the USA in patients with acute leukaemias or B-cell non-Hodgkin's lymphoma to demonstrate its efficacy and safety in this population of patients at highest risk of developing ATLS or its life-threatening sequelae. Int Immunol, 1998 May, 10(5), 631 - 7 Multiple gene duplication and expression of mouse bcl-2-related genes, A1; Hatakeyama S et al.; Here we report the genomic cloning and characterization of the murine A1 genes, which belong to the bcl-2 gene family . Southern analysis indicated the existence of at least four A1 genes in the murine genome and four different A1 genes, designated A1-a, -b, -c and -d, were cloned from the murine genomic library . The A1-a, -b and -d genes consisted of two exons, whereas the A1-c gene contained 1 bp insertion in the coding region which may result in an aberrant and truncated protein by frame-shift . With the exception of A1-c, the coding regions among A1 genes are highly conserved at >97% at the nucleotide level and at >96% at the amino acid level . A1-a, -b and -d genes appeared to be expressed specifically in organs containing many neutrophils . In neutrophils, A1-a, -b and -d transcripts were detected at a comparable level . Our data suggest that the multiple A1 genes in mice were generated by gene duplication and each of them may function as anti-apoptotic molecules in neutrophils. FEBS Lett, 1998 May 22, 428(1-2), 23 - 6 Staurosporine-sensitive protein phosphorylation is required for postreplication DNA repair in human cells; Svetlova MP et al.; DNA repair is an important factor of stability of pro- and eukaryotic genomes which plays a central role in mutagenesis and carcinogenesis . Genetic control of nucleotide excision repair (NER) in mammalian cells is well studied, but little is known about molecular mechanisms of postreplication repair (PRR) which allows bypass of base lesions in template strands after DNA replication . In Saccharomyces cerevisiae PRR is controlled by the RAD61RAD18 pathway which involves POL30 gene encoding proliferating cell nuclear antigen (PCNA), and in human cells PCNA is known to be closely associated with the newly replicated chromatin where PRR probably takes place . In UV-irradiated human cells distinct PCNA foci may be detected in some cells which accumulate phosphorylated breast cancer susceptibility protein BRCA1 and another protein BARD1 . Human PCNA is also known to be phosphorylated after UV-irradiation . In this study we found that the known inhibitor of protein kinases staurosporine supresses PRR in NER-deficient cells which is consistent with the view that BRCA1 and PCNA are required for PRR . We also have shown that the distinct PCNA foci in UV-irradiated NER-deficient cells are actually associated with the newly replicated chromatin . Since RAD18 protein is not essential for normal DNA replication and directly controls PRR in yeast, we analysed whether this protein as well as its human homologs (HR18A and HR18B) have common domains with BRCA1 and BARD1 . It is found that HR18A has a subregion of homology to BARD1 and HR18A-to BRCA1 . Taken together the results indicate that BRCA1 and BARD1 may be involved in PRR in human cells. Arch Virol, 1998, 143(5), 981 - 96 In vivo interactions among rotavirus nonstructural proteins; Gonzalez RA et al.; The rotavirus genome encodes six nonstructural (NS) proteins, five of which (NSP1, NSP2, NSP3, NSP5, and NSP6) have been suggested to be involved in a variety of events, such as genome replication, regulation of gene expression, and gene assortment . These NS proteins have been found to be associated with replication complexes that are precursors of the viral core, however, little information is available about the intermolecular interactions that may exist among them . Using the yeast two-hybrid system, which allows the detection of protein-protein interactions in vivo, all possible combinations among the rotavirus NS proteins were tested, and several interactions were observed . NSP1 interacted with the other four proteins tested; NSP3 associated with itself; and NSP5 was found to form homodimers and to interact with NSP6 . Co-immunoprecipitation of proteins from rotavirus-infected cells, using hyperimmune sera monospecific for the NS proteins, showed the same interactions for NSP1 as those observed in yeast . Immunofluorescence co-localization analysis of virus-infected epithelial cells revealed that the intracellular distribution of proteins that were seen to interact in yeast had patterns of distribution that would allow such intermolecular interactions to occur . These findings should contribute to the understanding of the role these proteins play in different aspects of the virus replication cycle. Curr Genet, 1998 Jun, 33(6), 395 - 405 Functional analysis of different regions of the positive-acting CYS3 regulatory protein of Neurospora crassa; Coulter KR et al.; In the filamentous fungus Neurospora crassa during conditions of sulfur limitation, CYS3, a major positive-acting regulatory protein, turns on the expression of an entire set of genes which encode permeases and enzymes involved in the acquisition of sulfur from environmental sources . CYS3 functions as a homodimeric protein and possesses a b-Zip domain that confers sequence-specific DNA binding . Expression of various hybrid GAL4-CYS3 fusion proteins in yeast was used to detect regions involved in gene activation . An amino-terminal serine/threonine-rich domain of CYS3 alone strongly activated expression of beta-galactosidase, the yeast reporter . Moreover, mutant CYS3 proteins with amino-acid substitutions in this region that showed increased expression in Neurospora also displayed an enhanced activation potential in yeast . The cys-3 gene of the exotic N . crassa Mauriceville strain and of N . intermedia were cloned and demonstrated to be functional for gene activation and for sulfur-mediated regulation by complementation of a loss-of-function cys-3 mutation . The amino-terminal serine/threonine-rich region is highly conserved in these two CYS3 proteins, in agreement with the possibility that it serves as the activation domain . Surprisingly, an extended promoter region of the cys-3 gene in the Mauriceville strain and in N . intermedia was very well conserved with that of the standard N . crassa gene, including the presence of three CYS3-binding sites possibly involved in autogenous control . Results are presented which indicate that synthesis of the CYS3 regulatory protein is highly regulated and can be detected in the nucleus of cells subjected to sulfur de-repression, but is not found in the nucleus or the cytoplasm of S-repressed cells . The amino-acid substitutions of the CYS3 protein present in a temperature-sensitive cys-3 mutant and in a second-site revertant of a cys-3 null mutation are presented and are shown to affect their DNA-binding activities. Mol Med, 1998 May, 4(5), 299 - 323 Convergence and divergence of the signaling pathways for insulin and phosphoinositolglycans; Muller G et al.; Phosphoinositolglycan molecules isolated from insulin-sensitive mammalian tissues have been demonstrated in numerous in vitro studies to exert partial insulin-mimetic activity on glucose and lipid metabolism in insulin-sensitive cells . However, their ill-defined structures, heterogeneous nature, and limited availability have prohibited the analysis of the underlying molecular mechanism . Phosphoinositolglycan-peptide (PIG-P) of defined and homogeneous structure prepared in large scale from the core glycan of a glycosyl-phosphatidylinositol-anchored membrane protein from Saccharomyces cerevisiae has recently been shown to stimulate glucose transport as well as a number of glucose-metabolizing enzymes and pathways to up to 90% (at 2 to 10 microns) of the maximal insulin effect in isolated rat adipocytes, cardiomyocytes, and diaphragms (G . Muller et al., 1997, Endocrinology 138: 3459-3476) . Consequently, we used this PIG-P for the present study in which we compare its intracellular signaling with that of insulin . The activation of glucose transport by both PIG-P and insulin in isolated rat adipocytes and diaphragms was found to require stimulation of phosphatidylinositol (PI) 3-kinase but to be independent of functional p70S6kinase and mitogen-activated protein kinase . The increase in glycerol-3-phosphate acyltransferase activity in rat adipocytes in response to PIG-P and insulin was dependent on both PI 3-kinase and p70S6kinase . This suggest that the signaling pathways for PIG-P and insulin to glucose transport and metabolism converage at the level of PI 3-kinase . A component of the PIG-P signaling pathway located up-stream of PI 3-kinase was identified by desensitization of isolated rat adipocytes for PIG-P action by combined treatment with trypsin and NaCl under conditions that preserved cell viability and the insulin-mimetic activity of sodium vanadate but completely blunted the insulin response . Incubation of the cells with either trypsin or NaCl alone was ineffective . The desensitized adipocytes were reconstituted for stimulation of lipogenesis by PIG-P by addition of the concentrated trypsin/salt extract . The reconstituted adipocytes exhibited 65-75% of the maximal PIG-P response and similar EC50 values for PIG-P (2 to 5 microns) compared with control cells . A proteinaceous N-ethylmaleimide (NEM)-sensitive component contained in the trypsin/salt extract was demonstrated to bind in a functional manner to the adipocyte plasma membrane of desensitized adipocytes via bipolar interactions . An excess of trypsin/salt extract inhibited PIG-P action in untreated adipocytes in a competitive fashion compatible with a receptor function for PIG-P of this protein . The presence of the putative PIG-P receptor protein in detergent-insoluble complexes prepared from isolated rat adipocytes suggests that caveolae/detergent-insoluble complexes of the plasma membrane may play a role in insulin-mimetic signaling by PIG-P . Furthermore, treatment of isolated rat diaphragms and adipocytes with PIG-P as well as with other agents exerting partially insulin-mimetic activity, such as PI-specific phospholipase C (PLC) and the sulfonylurea glimepiride, triggered tyrosine phosphorylation of the caveolar marker protein caveolin, which was apparently correlated with stimulation of lipogenesis . Strikingly, in adipocytes subjected to combined trypsin/salt treatment, PIG-P, PI-specific PLC, and glimepiride failed completely to provoke insulin-mimetic effects . A working model is presented for a signaling pathway in insulin-sensitive cells used by PIG(-P) molecules which involves GPI structures, the trypsin/salt- and NEM-sensitive receptor protein for PIG-P, and additional proteins located in caveolae/detergent-insoluble complexes. J Biol Chem, 1998 Jul 3, 273(27), 16935 - 45 PRMT 3, a type I protein arginine N-methyltransferase that differs from PRMT1 in its oligomerization, subcellular localization, substrate specificity, and regulation; Tang J et al.; Methylation is one of the many post-translational modifications that modulate protein function . Although asymmetric NG,NG-dimethylation of arginine residues in glycine-arginine-rich domains of eucaryotic proteins, catalyzed by type I protein arginine N-methyltransferases (PRMT), has been known for some time, members of this enzyme class have only recently been cloned . The first example of this type of enzyme, designated PRMT1, cloned because of its ability to interact with the mammalian TIS21 immediate-early protein, was then shown to have protein arginine methyltransferase activity . We have now isolated rat and human cDNA orthologues that encode proteins with substantial sequence similarity to PRMT1 . A recombinant glutathione S-transferase (GST) fusion product of this new rat protein, named PRMT3, asymmetrically dimethylates arginine residues present both in the designed substrate GST-GAR and in substrate proteins present in hypomethylated extracts of a yeast rmt1 mutant that lacks type I arginine methyltransferase activity; PRMT3 is thus a functional type I protein arginine N-methyltransferase . However, rat PRMT1 and PRMT3 glutathione S-transferase fusion proteins have distinct enzyme specificities for substrates present in both hypomethylated rmt1 yeast extract and hypomethylated RAT1 embryo cell extract . TIS21 protein modulates the enzymatic activity of recombinant GST-PRMT1 fusion protein but not the activity of GST-PRMT3 . Western blot analysis of gel filtration fractions suggests that PRMT3 is present as a monomer in RAT1 cell extracts . In contrast, PRMT1 is present in an oligomeric complex . Immunofluorescence analysis localized PRMT1 predominantly to the nucleus of RAT1 cells . In contrast, PRMT3 is predominantly cytoplasmic. Biochem Biophys Res Commun, 1998 Jun 18, 247(2), 530 - 5 Synergistic activation of transcription by physiologically unrelated transcription factors through cooperative DNA-binding; Vashee S et al.; Most eukaryotic promoters contain binding sites for several different transcription factors, which often act synergistically . Mechanistically, synergy is ascribed either to cooperative DNA-binding of the factors to the promoter or to some type of "multiple contact" mechanism in which each activator performs a different task in stimulating the transcription machinery . Here, it is shown that the yeast activators Gal4 and Put3 bind to DNA cooperatively in vivo and can activate transcription synergistically from certain synthetic promoters . Normally, Gal4 and Put3 bind to completely different promoters and activate physiologically unrelated sets of genes and it is extremely unlikely that they have evolved direct protein-protein contacts . These studies add to a growing body of evidence that binding of proteins to nearby sites in chromatin is intrinsically cooperative and suggest that many examples of synergy ascribed to multiple contact mechanisms may instead involve non-traditional cooperative DNA-binding . Biochem Biophys Res Commun, 1998 Jun 18, 247(2), 271 - 6 Phagocyte NADPH oxidase p67-phox possesses a novel carboxylterminal binding site for the GTPases Rac2 and Cdc42; Faris SL et al.; Rac GTPases regulate activation of the phagocyte NADPH oxidase, a multi-component enzyme complex that produces superoxide in response to host infection . GTP-bound Rac binds to the cytosol protein p67-phox enabling it to participate in oxidase assembly . Details of this interaction are poorly understood . Previous studies showed that Rac/p67-phox binding is GTP-dependent and that several Rac1 mutants lost the ability to activate the oxidase even though they still bound p67-phox . Using two hybrid and blot overlay binding methods, we identified a novel binding site in the p67-phox C-terminus that binds Rac1, Rac2, and Cdc42, a related GTPase which does not activate the oxidase . Binding was independent of the GDP/GTP state . We also showed that GTP-Cdc42 binds p67-phox N-terminus similar to GTP-Rac . Therefore, Rac binding to p67-phox is not synonymous with NADPH oxidase activation, and Rac probably participates in other steps of oxidase activation in addition to binding p67-phox . Biochem Biophys Res Commun, 1998 Jun 18, 247(2), 255 - 60 A novel relative of the very-long-chain acyl-CoA synthetase and fatty acid transporter protein genes with a distinct expression pattern; Berger J et al.; Based on its relationship to very-long-chain acyl-CoA synthetase (VLACS), we have cloned and identified a novel, VLACS-related (VLACSR) cDNA from mouse liver . The 2067-bp open reading frame encodes a 689-amino-acid protein with a predicted molecular mass of 76.2 kDa . The carboxy-terminal 500 amino acids of VLACSR show 48% identity and 70% similarity to mouse VLACS and 43% identity and 60% similarity to mouse fatty acid transporter (FATP), respectively . In addition, a partial cDNA of the human VLACSR ortholog was identified . By Northern blot analysis, a 2.6-kb VLACS mRNA was highly abundant only in mouse liver . Low levels of shorter mRNAs were present in brain, lung, testes, and spleen (2.5 kb) and in skeletal muscle (2.2 kb) . In heart, but not in kidney, transcripts undetectable by Northern blot analysis could be demonstrated by RT PCR . Southern blot analysis indicated single-copy VLACSR genes in the mouse and human genomes . Curr Opin Cell Biol, 1998 Jun, 10(3), 373 - 83 Co-activators and co-repressors in the integration of transcriptional responses; Torchia J et al.; The nuclear hormone receptors are DNA binding transcription factors that are regulated by binding of ligands, switching them from an inactive or repressive state to gene-activating functions . Recent evidence supports the hypothesis that many nuclear receptors switch, in a ligand-dependent manner, between binding of a multicomponent co-repressor complex containing histone deacetyltransferase activity, and binding of a co-activator complex containing factors with histone acetyltransferase activity that are further regulated by diverse signal transduction pathways . The identification of these limiting co-repressor and co-activator complexes and their specific interaction motifs, in concert with solution of the structures of the receptor ligand-binding domain in apo (empty) and ligand bound forms, indicates a common molecular mechanism by which these factors activate and repress gene transcription. Curr Opin Cell Biol, 1998 Jun, 10(3), 332 - 8 Telomeres, the nucleolus and aging; Johnson FB et al.; Reactivation of telomerase in cultured human cells extends their replicative life span beyond the Hayflick limit . How telomere shortening triggers cell senescence and whether it contributes to aging in vivo are under investigation . Studies in yeast have revealed another site critical to cellular aging: the nucleolus . The accumulation of ribosomal DNA circles is a cause of aging in this organism . The possible relevance of this mechanism to human aging is also being considered. Curr Opin Cell Biol, 1998 Jun, 10(3), 304 - 10 New systems for replicating DNA in vitro; Pasero P et al.; Current paradigms for the regulation of genomic DNA replication in eukaryotes are derived primarily from cell fusion experiments, yeast genetics, and from in vitro assays in Xenopus egg extracts . Initially, many aspects seemed irreconcilably different among the various organisms and model systems . In the past year, however, divergent approaches have arrived at a consensus on how the cell cycle regulates the initiation of DNA replication . All major players appear to be conserved from yeast to vertebrates, yet the important challenge of reconstituting eukaryotic replication from purified components remains . Three novel in vitro assays that replicate nuclear templates bring us closer to this goal. J Chromatogr A, 1998 May 8, 806(1), 187 - 97 Comparison of DNA migrations in two clamped homogeneous electric field chambers of different sizes . Relation between sample thickness and electrophoresis time; Lopez-Canovas L et al.; We present here a method to compare the mathematical descriptions of DNA migration per pulse as a function of pulse time . It is based on obtaining robust estimates and variances of DNA reorientation time, migration velocities during and after DNA reorientation; and on the statistical comparisons of these estimates . We demonstrated an equal description for the migration per pulse of each DNA molecule separated under identical conditions in clamped homogeneous electric field (CHEF) and miniCHEF chambers . However, miniCHEF resolved the patterns in shorter times, because it uses thinner samples . The relationship between sample thickness and CHEF run time is also presented. Mutat Res, 1998 Jun 5, 401(1-2), 1 - 10 Increased transcription decreases the spontaneous mutation rate at the thymidine kinase locus in human cells; Lippert MJ et al.; Transcription increases DNA repair efficiency and modulates the distribution of certain types of DNA damage . Furthermore, increased transcription level stimulates spontaneous mutation rate in yeast . We explored whether transcription level affects spontaneous mutation rate in human cells . We first developed two thymidine kinase (tk) inducible human cell lines using the Gal4-Estrogen receptor system . In our TK6i-G3 and G9 tk heterozygous cell lines, the active tk allele is linked to an inducible promoter element . Tk mRNA is induced following treatment with estrogen . Spontaneous mutation rate was significantly decreased in human cell lines after induction in contrast to the report in yeast . Thus, humans may have evolved different or additional mechanisms to deal with transcription related spontaneous mutagenesis . Chromosoma, 1998 Jun, 107(3), 211 - 5 Two separate conserved domains of eukaryotic DNA topoisomerase I bind to each other and reconstitute enzymatic activity; Park H et al.; The two-hybrid system was used to identify proteins that interact with the central conserved domain of Saccharomyces cerevisiae DNA topoisomerase I . Several different C-terminal domain-containing fragments of topoisomerase I, none of which overlapped with the central domain, were identified as specific interacting polypeptides . Coexpression of these two domains in yeast partially complemented the growth defects of top1-top2ts and top1-hpr1 mutants . Moreover, an in vitro assay showed that some topoisomerase I enzymatic activity was restored to these mutants . The results demonstrate that the central domain of topoisomerase I interacts with the C-terminal domain of the protein and that these two domains reconstitute enzymatic activity in vivo, even when expressed as separate polypeptides. Biochem J, 1998 Jul 1, 333 ( Pt 1), 151 - 8 Targeting and assembly of the oxoglutarate carrier: general principles for biogenesis of carrier proteins of the mitochondrial inner membrane; Palmisano A et al.; We have studied the targeting and assembly of the 2-oxoglutarate carrier (OGC), an integral inner-membrane protein of mitochondria . The precursor of OGC, synthesized without a cleavable presequence, is transported into mitochondria in an ATP- and membrane potential-dependent manner . Import of the mammalian OGC occurs efficiently into both mammalian and yeast mitochondria . Targeting of OGC reveals a clear dependence on the mitochondrial surface receptor Tom70 (the 70 kDa subunit of the translocase of the outer mitochondrial membrane), whereas a cleavable preprotein depends on Tom20 (the 20 kDa subunit), supporting a model of specificity differences of the receptors and the existence of distinct targeting pathways to mitochondria . The assembly of minute amounts of OGC imported in vitro to the dimeric form can be monitored by blue native electrophoresis of digitonin-lysed mitochondria . The assembly of mammalian OGC and fungal ADP/ATP carrier occurs with high efficiency in both mammalian and yeast mitochondria . These findings indicate a dynamic behaviour of the carrier dimers in the mitochondrial inner membrane and suggest a high conservation of the assembly reactions from mammals to fungi. Biochem J, 1998 Jul 1, 333 ( Pt 1), 65 - 9 Changing nucleosome positions in vivo through modification of the DNA rotational information; Di Marcotullio L et al.; The effects of the rotational information of DNA in determining the in vivo localization of nucleosomal core particles (ncps) have been studied in the Saccharomyces cerevisiae 5 S rRNA repeat gene . The distribution of the phased series of flexibility signals present in this DNA has been altered by inserting in its centre a 25 bp tract . The effects of such alteration on the in vivo distribution of the helically phased, alternatively located ncps have been determined relative to a reference 21 bp insertion mutant . The results show that the answers provided in vitro and in vivo by the yeast 5 S rRNA gene sequence to specific modifications of the DNA rotational frame are similar, thus pointing to the relevance of DNA rotational information in vivo. EMBO J, 1998 Jun 1, 17(11), 3155 - 67 Essential and redundant functions of histone acetylation revealed by mutation of target lysines and loss of the Gcn5p acetyltransferase; Zhang W et al.; The Gcn5p histone acetyltransferase exhibits a limited substrate specificity in vitro . However, neither the specificity of this enzyme in vivo nor the importance of particular acetylated residues to transcription or cell growth are well defined . To probe these questions, we mutated specific lysines in the N-termini of histones H3 and H4 and examined the effects of these mutations in yeast strains with and without functional GCN5 . We found that in vivo, GCN5 is required either directly or indirectly for the acetylation of several sites in H3 and H4 in addition to those recognized by the recombinant enzyme in vitro . Moreover, in the absence of GCN5, cells accumulate in G2/M indicating that Gcn5p functions are important for normal cell-cycle progression . Mutation of K14 in H3, which serves as the major target of recombinant Gcn5p acetylation in vitro, confers a strong, synthetic growth defect in gcn5 cells . Synergistic growth defects were also observed in gcn5 cells carrying mutations in lysine pairs (K8/K16 or K5/K12) in histone H4 . Strikingly, simultaneous mutation of K14 in H3 and K8 and K16 in H4 to arginine, or deletion of either the H3 or the H4 N-terminal tail, results in the death of gcn5 cells . Mutation of these same three sites to glutamine is not lethal . Indeed, this combination of mutations largely bypasses the need for GCN5 for transcriptional activation by Gal4-VP16, supporting an important role for histone acetylation in Gcn5p-mediated regulation of transcription . Our data indicate that acetylation of particular lysines in histones H3 and H4 serves both unique and overlapping functions important for normal cell growth, and that a critical overall level of histone acetylation is essential for cell viability. EMBO J, 1998 Jun 1, 17(11), 3052 - 65 A homologue of Drosophila aurora kinase is oncogenic and amplified in human colorectal cancers; Bischoff JR et al.; Genetic and biochemical studies in lower eukaryotes have identified several proteins that ensure accurate segregation of chromosomes . These include the Drosophila aurora and yeast Ipl1 kinases that are required for centrosome maturation and chromosome segregation . We have identified two human homologues of these genes, termed aurora1 and aurora2, that encode cell-cycle-regulated serine/threonine kinases . Here we demonstrate that the aurora2 gene maps to chromosome 20q13, a region amplified in a variety of human cancers, including a significant number of colorectal malignancies . We propose that aurora2 may be a target of this amplicon since its DNA is amplified and its RNA overexpressed, in more than 50% of primary colorectal cancers . Furthermore, overexpression of aurora2 transforms rodent fibroblasts . These observations implicate aurora2 as a potential oncogene in many colon, breast and other solid tumors, and identify centrosome-associated proteins as novel targets for cancer therapy. EMBO J, 1998 Jun 1, 17(11), 2982 - 93 The Vps4p AAA ATPase regulates membrane association of a Vps protein complex required for normal endosome function; Babst M et al.; Vps4p is an AAA-type ATPase required for efficient transport of biosynthetic and endocytic cargo from an endosome to the lysosome-like vacuole of Saccharomyces cerevisiae . Vps4p mutants that do not bind ATP or are defective in ATP hydrolysis were characterized both in vivo and in vitro . The nucleotide-free or ADP-bound form of Vps4p existed as a dimer, whereas in the ATP-locked state, Vps4p dimers assembled into a decameric complex . This suggests that ATP hydrolysis drives a cycle of association and dissociation of Vps4p dimers/decamers . Nucleotide binding also regulated the association of Vps4p with an endosomal compartment in vivo . This membrane association required the N-terminal coiled-coil motif of Vps4p, but deletion of the coiled-coil domain did not affect ATPase activity or oligomeric assembly of the protein . Membrane association of two previously uncharacterized class E Vps proteins, Vps24p and Vps32p/Snf7p, was also affected by mutations in VPS4 . Upon inactivation of a temperature-conditional vps4 mutant, Vps24p and Vps32p/Snf7p rapidly accumulated in a large membrane-bound complex . Immunofluorescence indicated that both proteins function with Vps4p at a common endosomal compartment . Together, the data suggest that the Vps4 ATPase catalyzes the release (uncoating) of an endosomal membrane-associated class E protein complex(es) required for normal morphology and sorting activity of the endosome. Biochem J, 1998 Jun 1, 332 ( Pt 2), 439 - 49 N epsilon,N epsilon-dimethyl-lysine cytochrome c as an NMR probe for lysine involvement in protein-protein complex formation; Moore GR et al.; The reductively dimethylated derivatives of horse and yeast iso-1-ferricytochromes c have been prepared and characterized for use as NMR probes of the complexes formed by cytochrome c with bovine liver cytochrome b5 and yeast cytochrome c peroxidase . The electrostatic properties and structures of the derivatized cytochromes are not significantly perturbed by the modifications; neither are the electrostatics of protein-protein complex formation or rates of interprotein electron transfer . Two-dimensional 1H-13C NMR spectroscopy of the complexes formed by the derivatized cytochromes with cytochrome b5 and cytochrome c peroxidase has been used to investigate the number and identity of lysine residues of cytochrome c that are involved in interprotein interactions of cytochrome c . The NMR data are incompatible with simple static models proposed previously for the complexes formed by these proteins, but are consistent with the presence of multiple, interconverting complexes of comparable stability, consistent with studies employing Brownian dynamics to model the complexes . The NMR characteristics of the Nepsilon,Nepsilon-dimethyl-lysine groups, their chemical shift dispersion, oxidation state and temperature dependences and the possibility of chemical exchange phenomena are discussed with relevance to the utility of Nepsilon, Nepsilon-dimethyl-lysine's being a generally useful derivative for characterizing protein-protein complexes. Electrophoresis, 1998 May, 19(6), 1024 - 35 Correlation of acidic and basic carrier ampholyte and immobilized pH gradient two-dimensional gel electrophoresis patterns based on mass spectrometric protein identification; Nawrocki A et al.; Separation of proteins on either carrier ampholyte-based or immobilized pH gradient-based two-dimensional (2-D) gels gives rise to electrophoretic patterns that are difficult to compare visually . In this paper we have used matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) to determine the identities of 335 protein spots in these two 2-D gel systems, including a substantial number of basic proteins which had never been identified before . Proteins that were identified in both gel systems allowed us to cross-reference the gel patterns . Vector analysis of these cross-references demonstrated that there is no obvious pattern by which the mobility of a protein in one gel system can be used to predict its mobility in the other . Thus, as laboratories adopt the immobilized pH gradient-based 2-D gel systems, the only reliable means of translating the data gained with the carrier ampholyte-based gel system is to positively identify the proteins in both 2-D systems. Electrophoresis, 1998 May, 19(6), 998 - 1005 Protein identification using mass spectrometric information; Fenyo D et al.; In an effort to gain an understanding of the value of the information in different mass spectrometric measurements for protein identification, the genome of Saccharomyces cerevisiae was studied in silico . We calculate how constraining the knowledge of the mass of a proteolytic peptide is as a function of mass and mass accuracy . We also assess the value for protein identification of additional information concerning a proteolytic peptide, including the presence or absence of a given amino acid, the number of exchangeable hydrogens, the N-terminal sequence, and the masses of mass spectrometrically produced fragment ions . Knowledge of the relative value of these different constraints is useful in the design of efficient protein identification experiments . Finally, we describe a software tool, PepFrag, for searching protein and DNA sequence databases that can use different types of mass spectrometric information to restrict the search. Mol Cells, 1998 Apr 30, 8(2), 240 - 5 Byr4, a dosage-dependent regulator of cytokinesis in S . pombe, interacts with a possible small GTPase pathway including Spg1 and Cdc16; Jwa M et al.; Coordination between karyokinesis and cytokinesis in the cell division cycle is fundamental to a precise transmission of duplicated genome into dividing daughter cells . byr4, a previously isolated essential gene, affects the mitotic cell cycle and cytokinesis in S . pombe . Phenotypic analyses of the null alleles and the overexpression of byr4 suggest that byr4 is a dosage-dependent coordinator of karyokinesis and cytokinesis (Song et al., 1996) . In this study, the functional mechanisms of byr4 were investigated using a byr4 mutant that exhibits byr4 overexpression phenotypes in thiamine deficient media . Genetic suppression analyses of this byr4 mutant with other cytokinesis regulatory genes in S . pombe, cdc16, cdc7, cdc15, cdc14, and plo1, show that byr4 overexpression phenotypes are suppressed by the overexpression of cdc16 and cdc7, but not by plo1, cdc14, and cdc15 . Also, the basal expression of byr4 and cdc7 suppresses the temperature-sensitive cdc16 mutation . However, the basal expression of either byr4 or cdc16 does not suppress the temperature-sensitive cdc7 mutation . The results of these suppression tests suggest that byr4 genetically interacts with cdc16 and cdc7: byr4 functions at the same level with or downstream of cdc16 and upstream of cdc7 . In the present study, we also show that Byr4 interacts with Cdc16 and Spg1 in the yeast two-hybrid assays . Recent reports suggest a possible small GTPase pathway to regulate the timing of cytokinesis where Cdc16 functions as a GAP (GTPase activating protein), Spg1 as a GTPase, and Cdc7 as a downstream effector . Combined genetic and two-hybrid analyses of this study strongly suggest that Byr4 directly interacts with this possible small GTPase pathway including Cdc16, Spg1, and Cdc7 to regulate cytokinesis in S . pombe. Curr Opin Immunol, 1998 Jun, 10(3), 330 - 6 Mammalian target of rapamycin: immunosuppressive drugs uncover a novel pathway of cytokine receptor signaling; Abraham RT; Recent findings have significantly advanced our understanding of the mechanism by which the potent immunosuppressive drug rapamycin inhibits cytokine-dependent lymphocyte proliferation . The protein targeted by the immunophilin-rapamycin complex is a member of a newly defined family of phosphoinositide-3-kinase-related kinases . The rapamycin target protein functions as a protein kinase in a signal transduction pathway that regulates the synthesis of proteins required for cell-cycle progression in both lymphoid and nonlymphoid cells. Cell Adhes Commun, 1998 Jan, 5(1), 61 - 73 A conserved role for L1 as a transmembrane link between neuronal adhesion and membrane cytoskeleton assembly; Hortsch M et al.; The L1-family of cell adhesion molecules is involved in many important aspects of nervous system development . Mutations in the human L1-CAM gene cause a complicated array of neurological phenotypes; however, the molecular basis of these effects cannot be explained by a simple loss of adhesive function . Human L1-CAM and its Drosophila homolog neuroglian are rather divergent in sequence, with the highest degree of amino acid sequence conservation between segments of their cytoplasmic domains . In an attempt to elucidate the fundamental functions shared between these distantly related members of the L1-family, we demonstrate here that the extracellular domains of mammalian L1-CAMs and Drosophila neuroglian are both able to induce the aggregation of transfected Drosophila S2 cells in vitro . To a limited degree they even interact with each other in cell adhesion and neurite outgrowth assays . The cytoplasmic domains of human L1-CAM and neuroglian are both able to interact with the Drosophila homolog of the cytoskeletal linker protein ankyrin . Moreover the recruitment of ankyrin to cell-cell contacts is completely dependent on L1-mediated cell adhesion . These findings support a model of L1 function in which the phenotypes of human L1-CAM mutations results from a disruption of the link between the extracellular environment and the neuronal cytoskeleton. Curr Biol, 1998 Jun 4, 8(12), R422 - 4 Transcription: gene control by targeted histone acetylation; Imhof A et al.; A transcriptional regulator in yeast, Gcn5p, activates transcription by targeted acetylation of specific lysine residues in the amino-terminal tails of histones . This targeted modification is restricted to nucleosomes assembled on the promoters of Gcn5p-responsive genes. Environ Health Perspect, 1998 Jul, 106(7), 421 - 6 Aromatic hydrocarbon receptor polymorphism: development of new methods to correlate genotype with phenotype; Maier A et al.; Differential CYP1A1 inducibility, reflecting variations in aromatic hydrocarbon receptor (AHR) affinity among inbred mouse strains, is an important determinant of environmental toxicity . We took advantage of the Ahr polymorphism in C57BL/6 and DBA/2 mice to develop an oligonucleotide-hybridization screening approach for the rapid identification of DNA sequence differences between Ahr alleles . Oligonucleotides containing single-base changes at polymorphic sites were immobilized on a solid support and hybridized with C57BL/6 or DBA/2 AHR cDNA radiolabeled probes . The observed hybridization patterns demonstrate that this approach can be used to detect nucleotide differences in the Ahr coding region with very high accuracy . In parallel experiments, we used a yeast two-hybrid system to assess phenotypic differences in AHR function . AHR activation, as measured by beta-galactosidase reporter activity in Saccharomyces cerevisiae strain SFY526, was determined following treatment with varying doses of the AHR ligand beta-naphthoflavone (BNF) . We found that the C57BL/6 AHR has about a 15-fold higher affinity for BNF than the DBA/2 AHR, in much better agreement with results reported for whole-animal studies than the values observed by in vitro ligand-binding assays . Using C57BL/6 and DBA/2 AHR chimeric proteins, we also confirmed the previously reported observation that an A375V change is principally responsible for the high- to low-affinity AHR phenotype . There has been no straightforward method to reliably and reproducibly phenotype large numbers of humans for CYP1A1 inducibility or AHR affinity . Screening human AHR cDNAs by oligonucleotide-hybridization and yeast two-hybrid methodologies will be invaluable for the rapid and unequivocal determination of changes in DNA sequence and receptor-ligand affinities associated with human AHR polymorphisms. Mol Cell Biol, 1998 Jul, 18(7), 4377 - 84 Transcriptional regulation of the MDR1 gene by histone acetyltransferase and deacetylase is mediated by NF-Y; Jin S et al.; Recent studies have shown that the histone-modifying enzymes histone acetyltransferase (HAT) and histone deacetylase (HDAC) are involved in transcriptional activation and repression, respectively . However, little is known about the endogenous genes that are regulated by these enzymes or how specificity is achieved . In the present report, we demonstrate that HAT and HDAC activities modulate transcription of the P-glycoprotein-encoding gene, MDR1 . Incubation of human colon carcinoma SW620 cells in 100-ng/ml trichostatin A (TSA), a specific HDAC inhibitor, increased the steady-state level of MDR1 mRNA 20-fold . Furthermore, TSA treatment of cells transfected with a wild-type MDR1 promoter/luciferase construct resulted in a 10- to 15-fold induction of promoter activity . Deletion and point mutation analysis determined that an inverted CCAAT box was essential for this activation . Consistent with this observation, overexpression of p300/CREB binding protein-associated factor (P/CAF), a transcriptional coactivator with intrinsic HAT activity, activated the wild-type MDR1 promoter but not a promoter containing a mutation in the CCAAT box; deletion of the P/CAF HAT domain abolished activation . Gel shift and supershift analyses identified NF-Y as the CCAAT-box binding protein in these cells, and cotransfection of a dominant negative NF-Y expression vector decreased the activation of the MDR1 promoter by TSA . Moreover, NF-YA and P/CAF were shown to interact in vitro . This is the first report of a natural promoter that is modulated by HAT and HDAC activities in which the transcription factor mediating this regulation has been identified. Mol Cell Biol, 1998 Jul, 18(7), 4291 - 300 Human cyclin K, a novel RNA polymerase II-associated cyclin possessing both carboxy-terminal domain kinase and Cdk-activating kinase activity; Edwards MC et al.; The gene coding for human cyclin K was isolated as a CPR (cell-cycle progression restoration) gene by virtue of its ability to impart a Far- phenotype to the budding yeast Saccharomyces cerevisiae and to rescue the lethality of a deletion of the G1 cyclin genes CLN1, CLN2, and CLN3 . The cyclin K gene encodes a 357-amino-acid protein most closely related to human cyclins C and H, which have been proposed to play a role in regulating basal transcription through their association with and activation of cyclin-dependent kinases (Cdks) that phosphorylate the carboxyl-terminal domain (CTD) of the large subunit of RNA polymerase II (RNAP II) . Murine and Drosophila melanogaster homologs of cyclin K have also been identified . Cyclin K mRNA is ubiquitously expressed in adult mouse and human tissues, but is most abundant in the developing germ cells of the adult testis and ovaries . Cyclin K is associated with potent CTD kinase and Cdk kinase (CAK) activity in vitro and coimmunoprecipitates with the large subunit of RNAP II . Thus, cyclin K represents a new member of the "transcription" cyclin family which may play a dual role in regulating Cdk and RNAP II activity. Mol Cell Biol, 1998 Jul, 18(7), 4177 - 87 Cyclin-dependent kinase inhibitor p21 modulates the DNA primer-template recognition complex; Waga S et al.; The p21 protein, a cyclin-dependent kinase (CDK) inhibitor, is capable of binding to both cyclin-CDK and the proliferating cell nuclear antigen (PCNA) . Through its binding to PCNA, p21 can regulate the function of PCNA differentially in replication and repair . To gain an understanding of the precise mechanism by which p21 affects PCNA function, we have designed a new assay for replication factor C (RFC)-catalyzed loading of PCNA onto DNA, a method that utilizes a primer-template DNA attached to agarose beads via biotin-streptavidin . Using this assay, we showed that RFC remains transiently associated with PCNA on the DNA after the loading reaction . Addition of p21 did not inhibit RFC-dependent PCNA loading; rather, p21 formed a stable complex with PCNA on the DNA . In contrast, the formation of a p21-PCNA complex on the DNA resulted in the displacement of RFC from the DNA . The nonhydrolyzable analogs of ATP, adenosine-5'-O-(3-thiotriphosphate) (ATPgammaS) and adenyl-imidodiphosphate, each stabilized the primer recognition complex containing RFC and PCNA in the absence of p21 . RFC in the ATPgammaS-activated complex was no longer displaced from the DNA by p21 . We propose that p21 stimulates the dissociation of the RFC from the PCNA-DNA complex in a process that requires ATP hydrolysis and then inhibits subsequent PCNA-dependent events in DNA replication . The data suggest that the conformation of RFC in the primer recognition complex might change on hydrolysis of ATP . We also suggest that the p21-PCNA complex that remains attached to DNA might function to tether cyclin-CDK complexes to specific regions of the genome. Mol Cell Biol, 1998 Jul, 18(7), 4165 - 76 The capacity of polyomavirus enhancer binding protein 2alphaB (AML1/Cbfa2) to stimulate polyomavirus DNA replication is related to its affinity for the nuclear matrix; Chen LF et al.; The nuclear matrix is thought to play an important role in the DNA replication of eukaryotic cells, although direct evidence for such a role is still lacking . A nuclear matrix-associated transcription factor, polyomavirus (Py) enhancer binding protein 2alphaB1 (PEBP2alphaB1) (AML1/Cbfa2), was found to stimulate Py replication through its cognate binding site . The minimal replication activation domain (RAD) was identified between amino acid (aa) 302 and aa 371 by using a fusion protein containing the GAL4 DNA binding domain (GAL4-RAD) . In addition, the region showed affinity for the nuclear matrix and, on the basis of competition studies, binding activity for one or more proteins involved in the initiation of Py DNA replication . A leukemogenic chimeric protein, AML1/ETO(MTG8), which does not contain this region of PEBP2alphaB1/AML1, was also localized in the nuclear matrix fraction and competed for nuclear matrix association with PEBP2alphaB1 and GAL4-RAD . Moreover, AML1/ETO inhibited Py DNA replication stimulated by PEBP2alphaB1 and GAL4-RAD . The inhibition was specific for replication mediated by PEBP2alphaB1 and GAL4-RAD, and proportional to the degree of loss of these activators from the nuclear matrix, suggesting a requirement for nuclear matrix targeting in the stimulation of Py DNA replication by RAD . These results are the first to suggest a molecular link between the initiation of DNA replication and the nuclear matrix compartment. Mol Cell Biol, 1998 Jul, 18(7), 4012 - 22 Adenovirus E1B 19,000-molecular-weight protein activates c-Jun N-terminal kinase and c-Jun-mediated transcription; See RH et al.; Adenovirus E1B proteins (19,000-molecular-weight {19K} and 55K proteins) inhibit apoptosis and cooperate with adenovirus E1A to induce full oncogenic transformation of primary cells . The E1B 19K protein has previously been shown to be capable of activating transcription; however, the underlying mechanisms are unclear . Here, we show that adenovirus infection activates the c-Jun N-terminal kinase (JNK) and that the E1B gene products are necessary for adenovirus to activate JNK . In transfection assays, we show that the E1B 19K protein is sufficient to activate JNK and can strongly induce c-Jun-dependent transcription . Mapping studies show that the C-terminal portion of E1B 19K is necessary for induction of c-Jun-mediated transcription . Using dominant-negative mutants of several kinases upstream of JNK, we show that MEKK1 and MKK4, but not Ras, are involved in the induction of JNK activity by adenovirus infection . The same dominant-negative kinase mutants also block the ability of E1B 19K to induce c-Jun-mediated transcription . Taken together, these results suggest that E1B 19K may utilize the MEKK1-MKK4-JNK signaling pathway to activate c-Jun-dependent transcription and demonstrate a novel, kinase-activating activity of E1B 19K that may underlie its ability to regulate transcription. Mol Cell Biol, 1998 Jul, 18(7), 3681 - 91 Pheromone-dependent G1 cell cycle arrest requires Far1 phosphorylation, but may not involve inhibition of Cdc28-Cln2 kinase, in vivo; Gartner A et al.; In yeast, the pheromone alpha-factor acts as an antiproliferative factor that induces G1 arrest and cellular differentiation . Previous data have indicated that Far1, a factor dedicated to pheromone-induced cell cycle arrest, is under positive and negative posttranslational regulation . Phosphorylation by the pheromone-stimulated mitogen-activated protein (MAP) kinase Fus3 has been thought to enhance the binding of Far1 to G1-specific cyclin-dependent kinase (Cdk) complexes, thereby inhibiting their catalytic activity . Cdk-dependent phosphorylation events were invoked to account for the high instability of Far1 outside early G1 phase . To confirm any functional role of Far1 phosphorylation, we undertook a systematic mutational analysis of potential MAP kinase and Cdk recognition motifs . Two putative phosphorylation sites that strongly affect Far1 behavior were identified . A change of serine 87 to alanine prevents the cell cycle-dependent degradation of Far1, causing enhanced sensitivity to pheromone . In contrast, threonine 306 seems to be an important recipient of an activating modification, as substitutions at this position abolish the G1 arrest function of Far1 . Only the phosphorylated wild-type Far1 protein, not the T306-to-A substitution product, can be found in stable association with the Cdc28-Cln2 complex . Surprisingly, Far1-associated Cdc28-Cln2 complexes are at best moderately inhibited in immunoprecipitation kinase assays, suggesting unconventional inhibitory mechanisms of Far1. Science, 1998 Jun 19, 280(5371), 1943 - 5 A calcium sensor homolog required for plant salt tolerance; Liu J et al.; Excessive sodium (Na+) in salinized soils inhibits plant growth and development . A mutation in the SOS3 gene renders Arabidopsis thaliana plants hypersensitive to Na+-induced growth inhibition . SOS3 encodes a protein that shares significant sequence similarity with the calcineurin B subunit from yeast and neuronal calcium sensors from animals . The results suggest that intracellular calcium signaling through a calcineurin-like pathway mediates the beneficial effect of calcium on plant salt tolerance. J Cell Biol, 1998 Jun 15, 141(6), 1433 - 48 Differential nuclear translocation and transactivation potential of beta-catenin and plakoglobin; Simcha I et al.; beta-Catenin and plakoglobin are homologous proteins that function in cell adhesion by linking cadherins to the cytoskeleton and in signaling by transactivation together with lymphoid-enhancing binding/T cell (LEF/TCF) transcription factors . Here we compared the nuclear translocation and transactivation abilities of beta-catenin and plakoglobin in mammalian cells . Overexpression of each of the two proteins in MDCK cells resulted in nuclear translocation and formation of nuclear aggregates . The beta-catenin-containing nuclear structures also contained LEF-1 and vinculin, while plakoglobin was inefficient in recruiting these molecules, suggesting that its interaction with LEF-1 and vinculin is significantly weaker . Moreover, transfection of LEF-1 translocated endogenous beta-catenin, but not plakoglobin to the nucleus . Chimeras consisting of Gal4 DNA-binding domain and the transactivation domains of either plakoglobin or beta-catenin were equally potent in transactivating a Gal4-responsive reporter, whereas activation of LEF-1- responsive transcription was significantly higher with beta-catenin . Overexpression of wild-type plakoglobin or mutant beta-catenin lacking the transactivation domain induced accumulation of the endogenous beta-catenin in the nucleus and LEF-1-responsive transactivation . It is further shown that the constitutive beta-catenin-dependent transactivation in SW480 colon carcinoma cells and its nuclear localization can be inhibited by overexpressing N-cadherin or alpha-catenin . The results indicate that (a) plakoglobin and beta-catenin differ in their nuclear translocation and complexing with LEF-1 and vinculin; (b) LEF-1-dependent transactivation is preferentially driven by beta-catenin; and (c) the cytoplasmic partners of beta-catenin, cadherin and alpha-catenin, can sequester it to the cytoplasm and inhibit its transcriptional activity. J Cell Biol, 1998 Jun 15, 141(6), 1393 - 406 Mammalian p55CDC mediates association of the spindle checkpoint protein Mad2 with the cyclosome/anaphase-promoting complex, and is involved in regulating anaphase onset and late mitotic events; Kallio M et al.; We have investigated the function of p55CDC, a mammalian protein related to Cdc20 and Hct1/Cdh1 in Saccharomyces cerevisiae, and Fizzy and Fizzy-related in Drosophila . Immunofluorescence studies and expression of a p55CDC-GFP chimera demonstrate that p55CDC is concentrated at the kinetochores in M phase cells from late prophase to telophase . Some p55CDC is also associated with the spindle microtubules and spindle poles, and some is diffuse in the cytoplasm . At anaphase, the concentration of p55CDC at the kinetochores gradually diminishes, and is gone by late telophase . In extracts prepared from M phase, but not from interphase HeLa cells, p55CDC coimmunoprecipitates with three important elements of the M phase checkpoint machinery: Cdc27, Cdc16, and Mad2 . p55CDC is required for binding Mad2 with the Cdc27 and Cdc16 . Thus, it is likely that p55CDC mediates the association of Mad2 with the cyclosome/anaphase-promoting complex . Microinjection of anti-p55CDC antibody into mitotic mammalian cells induces arrest or delay at metaphase, and impairs progression of late mitotic events . These studies suggest that mammalian p55CDC may be part of a regulatory and targeting complex for the anaphase-promoting complex. Plant Physiol, 1998 Jun, 117(2), 407 - 15 An Arabidopsis VPS45p homolog implicated in protein transport to the vacuole; Bassham DC et al.; The Sec1p family of proteins is required for vesicle-mediated protein trafficking between various organelles of the endomembrane system . This family includes Vps45p, which is required for transport to the vacuole in yeast (Saccharomyces cerevisiae) . We have isolated a cDNA encoding a VPS45 homolog from Arabidopsis thaliana (AtVPS45) . The cDNA is able to complement both the temperature-sensitive growth defect and the vacuolar-targeting defect of a yeast vps45 mutant, indicating that the two proteins are functionally related . AtVPS45p is a peripheral membrane protein that associates with microsomal membranes . Sucrose-density gradient fractionation demonstrated that AtVPS45p co-fractionates with AtELP, a potential vacuolar protein sorting receptor, implying that they may reside on the same membrane populations . These results indicate that AtVPS45p is likely to function in the transport of proteins to the vacuole in plants. Genes Dev, 1998 Jun 15, 12(12), 1871 - 83 The checkpoint protein MAD2 and the mitotic regulator CDC20 form a ternary complex with the anaphase-promoting complex to control anaphase initiation; Fang G et al.; The spindle assembly checkpoint mechanism delays anaphase initiation until all chromosomes are aligned at the metaphase plate . Activation of the anaphase-promoting complex (APC) by binding of CDC20 and CDH1 is required for exit from mitosis, and APC has been implicated as a target for the checkpoint intervention . We show that the human checkpoint protein hMAD2 prevents activation of APC by forming a hMAD2-CDC20-APC complex . When injected into Xenopus embryos, hMAD2 arrests cells at mitosis with an inactive APC . The recombinant hMAD2 protein exists in two-folded states: a tetramer and a monomer . Both the tetramer and the monomer bind to CDC20, but only the tetramer inhibits activation of APC and blocks cell cycle progression . Thus, hMAD2 binding is not sufficient for inhibition, and a change in hMAD2 structure may play a role in transducing the checkpoint signal . There are at least three different forms of mitotic APC that can be detected in vivo: an inactive hMAD2-CDC20-APC ternary complex present at metaphase, a CDC20-APC binary complex active in degrading specific substrates at anaphase, and a CDH1-APC complex active later in mitosis and in G1 . We conclude that the checkpoint-mediated cell cycle arrest involves hMAD2 receiving an upstream signal to inhibit activation of APC. Genes Dev, 1998 Jun 15, 12(12), 1812 - 24 A stress response pathway from the endoplasmic reticulum to the nucleus requires a novel bifunctional protein kinase/endoribonuclease (Ire1p) in mammalian cells; Tirasophon W et al.; Eukaryotes respond to the presence of unfolded protein in the endoplasmic reticulum (ER) by up-regulating the transcription of genes encoding ER protein chaperones, such as BiP . We have isolated a novel human cDNA encoding a homolog to Saccharomyces cerevisiae Ire1p, a proximal sensor for this signal transduction pathway in yeast . The gene product hIre1p is a type 1 transmembrane protein containing a cytoplasmic domain that is highly conserved to the yeast counterpart having a Ser/Thr protein kinase domain and a domain homologous to RNase L . However, the luminal domain has extensively diverged from the yeast gene product . hIre1p expressed in mammalian cells displayed intrinsic autophosphorylation activity and an endoribonuclease activity that cleaved the 5' splice site of yeast HAC1 mRNA, a substrate for the endoribonuclease activity of yeast Ire1p . Overexpressed hIre1p was localized to the ER with particular concentration around the nuclear envelope and some colocalization with the nuclear pore complex . Expression of Ire1p mRNA was autoregulated through a process that required a functional hIre1p kinase activity . Finally, overexpression of wild-type hIre1p constitutively activated a reporter gene under transcriptional control of the rat BiP promoter, whereas expression of a catalytically inactive hIre1p acted in a trans-dominant-negative manner to prevent transcriptional activation of the BiP promoter in response to ER stress induced by inhibition of N-linked glycosylation . These results demonstrate that hIre1p is an essential proximal sensor of the unfolded protein response pathway in mammalian cells. Genes Dev, 1998 Jun 15, 12(12), 1787 - 800 A novel protein complex that interacts with the vitamin D3 receptor in a ligand-dependent manner and enhances VDR transactivation in a cell-free system; Rachez C et al.; Nuclear receptors transduce hormonal signals by binding directly to DNA target sites in promoters and modulating the transcription of linked genes . Receptor-mediated transactivation appears to be potentiated in response to ligand by a number of coactivators that may provide key interactions with components of the transcription preinitiation complex and/or alter chromatin structure . Here, we use the vitamin D3 receptor ligand-binding domain (VDR LBD) as an affinity matrix to identify components of a transcriptionally active nuclear extract that interact with VDR in response to ligand . We describe the purification of a complex of at least 10 VDR interacting proteins (DRIPs) ranging from 65 to 250 kD that associate with the receptor in a strictly 1,25-dihydroxyvitamin D3-dependent manner . These proteins also appear to interact with other, but not all, nuclear receptors, such as the thyroid hormone receptor . The DRIPs are distinct from known nuclear receptor coactivators, although like these coactivators, their interaction also requires the AF-2 transactivation motif of VDR . In addition, the DRIP complex contains histone acetyltransferase activity, indicating that at least one or more of the DRIPs may function at the level of nucleosomal modification . However, we show that the DRIPs selectively enhance the transcriptional activity of VDR on a naked DNA template utilizing a cell-free, ligand-dependent transcription assay . Moreover, this activity can be specifically depleted from the extract by liganded, but not unliganded, VDR-LBD . Overexpression of DRIP100 in vivo resulted in a strong squelching of VDR transactivation, suggesting the sequestration of other limiting factors, including components of the DRIP complex . These results demonstrate the existence of a new complex of novel functional nuclear receptor coactivators. Int J Immunopharmacol, 1997 Sep-Oct, 19(9-10), 607 - 9 Glucan as stimulator of hematopoiesis in normal and gamma-irradiated mice . A survey of the authors' results; Hofer M et al.; Glucan, a beta-1,3-linked polyglucose derived from the yeast Saccharomyces cerevisiae, is a broad spectrum enhancer of host defense mechanisms stimulating humoral and cell-mediated immunity . On the basis of these features, glucan has been tested by the authors' research group in experiments on gamma-irradiated mice . Two glucan forms, particulate and soluble, have been studied . Attention has been focused on various application regimens in relation to the time of irradiation (pre- or postirradiation application), the possibilities of using glucan in various radiation regimens (single or repeated irradiation), combined pharmacological therapy (joint administration of glucan with cystamine or inhibitors of prostaglandin synthesis), and on the negative side effects of therapy with glucan . Some studies included also experiments on unirradiated mice . The results have demonstrated the ability of glucan to influence positively the course of the acute radiation disease . Stimulation of hematopoiesis has been found to be the most important mechanism of glucan's radioprotective effects . In this communication, the results of 11 full-length articles are summarized and discussed. Proc Natl Acad Sci U S A, 1998 Jun 23, 95(13), 7508 - 13 Transdominant genetic analysis of a growth control pathway; Caponigro G et al.; Genetic selections that use proteinaceous transdominant inhibitors encoded by DNA libraries to cause mutant phenocopies may facilitate genetic analysis in traditionally nongenetic organisms . We performed a selection for random short peptides and larger protein fragments (collectively termed "perturbagens") that inhibit the yeast pheromone response pathway . Peptide and protein fragment perturbagens that permit cell division in the presence of pheromone were recovered . Two perturbagens were derived from proteins required for pheromone response, and an additional two were derived from proteins that may negatively influence the pheromone response pathway . Furthermore, three known components of the pathway were identified as probable perturbagen targets based on physical interaction assays . Thus, by selection for transdominant inhibitors of pheromone response, multiple pathway components were identified either directly as gene fragments or indirectly as the likely targets of specific perturbagens . These results, combined with the results of previous work {Holzmayer, T . A., Pestov, D . G . & Roninson, I . B . (1992) Nucl . Acids . Res . 20, 711-717; Whiteway, M., Dignard, D . & Thomas, D . Y . (1992) Proc . Natl . Acad . Sci . USA 89, 9410-9414; and Gudkov, A . V., Kazarov, A . R., Thimmapaya, R., Axenovich, S . A., Mazo, I . A . & Roninson, I . B . (1994) Proc . Natl . Acad . Sci . USA 91, 3744-3748}, suggest that transdominant genetic analysis of the type described here will be broadly applicable. Proc Natl Acad Sci U S A, 1998 Jun 23, 95(13), 7451 - 6 Human CUL1 forms an evolutionarily conserved ubiquitin ligase complex (SCF) with SKP1 and an F-box protein; Lyapina SA et al.; The SCF ubiquitin ligase complex of budding yeast triggers DNA replication by catalyzing ubiquitination of the S phase cyclin-dependent kinase inhibitor SIC1 . SCF is composed of three proteins-ySKP1, CDC53 (Cullin), and the F-box protein CDC4-that are conserved from yeast to humans . As part of an effort to identify components and substrates of a putative human SCF complex, we isolated hSKP1 in a two-hybrid screen with hCUL1, the closest human homologue of CDC53 . Here, we show that hCUL1 associates with hSKP1 in vivo and directly interacts with both hSKP1 and the human F-box protein SKP2 in vitro, forming an SCF-like particle . Moreover, hCUL1 complements the growth defect of yeast cdc53(ts) mutants, associates with ubiquitination-promoting activity in human cell extracts, and can assemble into functional, chimeric ubiquitin ligase complexes with yeast SCF components . Taken together, these data suggest that hCUL1 functions as part of an SCF ubiquitin ligase complex in human cells . Further application of biochemical assays similar to those described here can now be used to identify regulators/components of hCUL1-based SCF complexes, to determine whether the hCUL2-hCUL5 proteins also are components of ubiquitin ligase complexes in human cells, and to screen for chemical compounds that modulate the activities of the hSKP1 and hCUL1 proteins. Proc Natl Acad Sci U S A, 1998 Jun 23, 95(13), 7427 -