S. cerevisiae

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 - 32
A member of the Ran-binding protein family, Yrb2p, is involved in nuclear protein export; Taura T et al.; Yeast cells mutated in YRB2, which encodes a nuclear protein with similarity to other Ran-binding proteins, fail to export nuclear export signal (NES)-containing proteins including HIV Rev out of the nucleus . Unlike Xpo1p/Crm1p/exportin, an NES receptor, Yrb2p does not shuttle between the nucleus and the cytoplasm but instead remains inside the nucleus . However, by both biochemical and genetic criteria, Yrb2p interacts with Xpo1p and not with other members of the importin/karyopherin beta superfamily . Moreover, the Yrb2p region containing nucleoporin-like FG repeats is important for NES-mediated protein export . Taken together, these data suggest that Yrb2p acts inside the nucleus to mediate the action of Xpo1p in at least one of several nuclear export pathways.

J Exp Med, 1998 Jun 15, 187(12), 2081 - 9
Ku70 is required for late B cell development and immunoglobulin heavy chain class switching; Manis JP et al.; Immunoglobulin (Ig) heavy chain (HC) class switch recombination (CSR) is a late B cell process that involves intrachromosomal DNA rearrangement . Ku70 and Ku80 form a DNA end-binding complex required for DNA double strand break repair and V(D)J recombination . Ku70(-/-) (K70T) mice, like recombination activating gene (RAG)-1- or RAG-2-deficient (R1T or R2T) mice, have impaired B and T cell development at an early progenitor stage, which is thought to result at least in part from defective V(D)J recombination (Gu, Y., K.J . Seidl, G.A . Rathbun, C . Zhu, J.P . Manis, N . van der Stoep, L . Davidson, H.L . Cheng, J.M . Sekiguchi, K . Frank, et al . 1997 . Immunity . 7:653-665; Ouyang, H., A . Nussenzweig, A . Kurimasa, V.C . Soares, X . Li, C . Cordon-Cardo, W . Li, N . Cheong, M . Nussenzweig, G . Iliakis, et al . 1997 . J . Exp . Med . 186:921-929) . Therefore, to examine the potential role of Ku70 in CSR, we generated K70T mice that carry a germline Ig HC locus in which the JH region was replaced with a functionally rearranged VH(D)JH and Ig lambda light chain transgene (referred to as K70T/HL mice) . Previously, we have shown that B cells from R1T or R2T mice carrying these rearranged Ig genes (R1T/HL or R2T/HL mice) can undergo CSR to IgG isotypes (Lansford, R., J . Manis, E . Sonoda, K . Rajewsky, and F . Alt . 1998 . Int . Immunol . 10:325-332) . K70T/HL mice had significant numbers of peripheral surface IgM+ B cells, which generated serum IgM levels similar to those of R2T/HL mice . However, in contrast to R2T/HL mice, K70T/HL mice had no detectable serum IgG isotypes . In vitro culture of K70T/HL B cells with agents that induce CSR in normal or R2T/HL B cells did lead to the induction of germline CH transcripts, indicating that initial signaling pathways for CSR were intact in K70T/HL cells . However, treatment with such agents did not lead to detectable CSR by K70T/HL B cells, and instead, led to cell death within 72 h . We conclude that Ku70 is required for the generation of B cells that have undergone Ig HC class switching . Potential roles for Ku70 in the CSR process are discussed.

J Exp Med, 1998 Jun 15, 187(12), 2031 - 6
Nuclear factor of activated T cells (NFAT)-dependent transactivation regulated by the coactivators p300/CREB-binding protein (CBP); Garcia-Rodriguez C et al.; p300 and cAMP response element-binding protein (CREB)-binding protein (CBP) are members of a family of coactivators involved in the regulation of transcription and chromatin . We show that transcription factors of the nuclear factor of activated T cells (NFAT) family bind p300/CBP and recruit histone acetyltransferase activity from T cell nuclear extracts . The NH2-terminal transactivation domain of NFAT1 and the phospho-CREB- and E1A-binding sites of p300/CBP are involved in the interaction . The viral oncoprotein E1A inhibits NFAT-dependent transactivation in a p300-dependent manner . Recruitment of the coactivators p300/CBP by the transactivation domains of NFAT proteins is likely to play a critical role in NFAT-dependent gene expression during the immune response.

Nat Biotechnol, 1996 Nov, 14(11), 1579 - 83
Protein identification by solid phase microextraction-capillary zone electrophoresis-microelectrospray-tandem mass spectrometry; Figeys D et al.; We describe an analytical system for the rapid identification of proteins by correlation of tandem mass spectra with protein sequence databases . The system consists of an integrated solid phase microextraction/capillary zone electrophoresis peptide separation device that is connected through a microelectrospray ion source to a tandem mass spectrometer . The limits of detection are 660 amol of sample at a concentration limit of < 33 amol/microliters for peptide mass measurement, and < 10 fmol of sample, at a concentration limit of < 300 amol/microliters for peptide analysis by collision-induced dissociation . Using this system, we have identified low nanogram amounts of yeast proteins separated by high-resolution two-dimensional gel electrophoresis.

Biophys J, 1998 Jun, 74(6), 3190 - 7
Site-specific thermodynamics and kinetics of a coiled-coil transition by spin inversion transfer NMR; d'Avignon DA et al.; A 33-residue pseudo-wild-type GCN4 leucine zipper peptide is used to probe the equilibrium conformational population in proteins . 13Calpha-NMR shows that chain sites differ in structural content at a given temperature, and that two dimeric folded forms are evident at many sites . Spin inversion transfer experiments are reported bearing on the thermodynamics and kinetics of interconversion of the two dimeric folded forms (Fa <--> Fb) at the 13Calpha-labeled position L13 . At each temperature, at conditions wherein the population of unfolded chains is quite small, inversion of the Fa spins via a tuned Gaussian pi-pulse is followed by a time interval (tau), interrogation, and recording of the free induction decay . Fifteen such inversions, with varying tau, provide the time course for recovery of equilibrium magnetization after inversion . Similar experiments follow inversion of the Fb spins . Re-equilibration is known to be modulated by four first-order rate constants: two (T1a(-1) and T1b(-1)) for spin-lattice relaxation intrinsic to the respective sites, and two (kab and kba) for the conformational change . All four follow from joint, Bayesian analysis of all the data at each temperature . The equilibrium constant at each temperature for this local transition, determined simply from the equilibrium relative magnetizations at Fa and Fb sites, agrees well with the kinetic ratio kab/kba . The standard Gibbs energies, enthalpy, and entropy follow . Activation parameters, both ways, are accessible from the rate constants and suggest a transition state with high Gibbs energy and enthalpy, but with entropy between those of Fa and Fb.

Biophys J, 1998 Jun, 74(6), 2926 - 44
The sensor regions of VDAC are translocated from within the membrane to the surface during the gating processes; Song J et al.; The motion of the sensor regions in a mitochondrial voltage-gated channel called VDAC were probed by attaching biotin at specific locations and determining its ability to bind to added streptavidin . Site-directed mutagenesis was used to introduce single cysteine residues into Neurospora crassa VDAC (naturally lacks cysteine) . These were chemically biotinylated and reconstituted into planar phospholipid membranes . In the 19 sites examined, only two types of results were observed upon streptavidin addition: in type 1, channel conductance was reduced, but voltage gating could proceed; in type 2, channels were locked in a closed state . The result at type 1 sites is interpreted as streptavidin binding to sites in static regions close to the channel opening . The binding sterically interferes with ion flow . The result at type 2 sites indicates that these are located on a mobile domain and coincide with the previously identified sensor regions . The findings are consistent with closure resulting from the movement of a domain from within the transmembrane regions to the membrane surface . No single site was accessible to streptavidin from both membrane surfaces, indicating that the motion is limited . From the streptavidin-induced reduction in conductance at type 1 sites, structural information was obtained about the location of these sites.

Curr Biol, 1998 May 21, 8(11), 657 - 60
Telomere maintenance is dependent on activities required for end repair of double-strand breaks; Nugent CI et al.; Telomeres are functionally distinct from ends generated by chromosome breakage, in that telomeres, unlike double-strand breaks, are insulated from recombination with other chromosomal termini {1} . We report that the Ku heterodimer and the Rad50/Mre11/Xrs2 complex, both of which are required for repair of double-strand breaks {2-5}, have separate roles in normal telomere maintenance in yeast . Using epistasis analysis, we show that the Ku end-binding complex defined a third telomere-associated activity, required in parallel with telomerase {6} and Cdc13, a protein binding the single-strand portion of telomere DNA {7,8} . Furthermore, loss of Ku function altered the expression of telomere-located genes, indicative of a disruption of telomeric chromatin . These data suggest that the Ku complex and the Cdc13 protein function as terminus-binding factors, contributing distinct roles in chromosome end protection . In contrast, MRE11 and RAD50 were required for the telomerase-mediated pathway, rather than for telomeric end protection; we propose that this complex functions to prepare DNA ends for telomerase to replicate . These results suggest that as a part of normal telomere maintenance, telomeres are identified as double-strand breaks, with additional mechanisms required to prevent telomere recombination . Ku, Cdc13 and telomerase define three epistasis groups required in parallel for telomere maintenance.

Curr Biol, 1998 May 21, 8(11), R390 - 3
Membrane fusion: all done with SNAREpins?
Edwardson JM.
SNARE proteins are sufficient to fuse artificial membranes together . In the cell, vesicle transport may rely on fusion mediated by interaction between vesicle (v) and target (t) SNAREs, whereas the homotypic fusion of organelle biogenesis may be mediated by t-SNARE-t-SNARE interaction.

Curr Biol, 1998 May 21, 8(11), R373 - 5
Chromatin transcription: clearing the gridlock; Geiduschek EP; A factor has been purified that specifically facilitates RNA chain elongation in a chromatin context; the properties of this factor help to define the challenges that lie ahead in understanding how RNA polymerase manages to transcribe DNA that is packaged into chromatin.

Curr Biol, 1998 May 21, 8(11), R368 - 72
Nucleocytoplasmic transport: driving and directing transport; Cole CN et al.; Nucleocytoplasmic transport involves assembly and movement across the nuclear envelope of cargo-receptor complexes that interact with the small GTPase Ran . The asymmetric distribution of Ran regulator proteins, RanGAP1 and RCC1, provides the driving force and directionality for nuclear transport.

Plant Cell, 1998 Jun, 10(6), 877 - 88
Transposon tagging of the Defective embryo and meristems gene of tomato; Keddie JS et al.; The shoot and root apical meristems (SAMs and RAMs, respectively) of higher plants are mechanistically and structurally similar . This has led previously to the suggestion that the SAM and RAM represent modifications of a fundamentally homologous plan of organization . Despite recent interest in plant development, especially in the areas of meristem regulation, genes specifically required for the function of both the SAM and RAM have not yet been identified . Here, we report on a novel gene, Defective embryo and meristems (Dem), of tomato . This gene is required for the correct organization of shoot apical tissues of developing embryos, SAM development, and correct cell division patterns and meristem maintenance in roots . Dem was cloned using transposon tagging and shown to encode a novel protein of 72 kD with significant homology to YNV2, a protein of unknown function of Saccharomyces cerevisiae . Dem is expressed in root and shoot meristems and organ primordia but not in callus . The expression pattern of Dem mRNA in combination with the dem mutant phenotype suggests that Dem plays an important role within apical meristems.

J Biol Chem, 1998 Jun 19, 273(25), 15818 - 29
The vesicle transport protein Vps33p is an ATP-binding protein that localizes to the cytosol in an energy-dependent manner; Gerhardt B et al.; Molecular mechanisms of vesicle transport between the prevacuolar compartment and the vacuole in yeast or the lysosome in mammalian cells are poorly understood . To learn more about the specificity of this intercompartmental step, we have examined the subcellular localization of a SEC1 homologue, Vps33p, a protein implicated to function in transport between the prevacuolar compartment and the vacuole . Following short pulses, 80-90% of newly synthesized Vps33p cofractionated with a cytosolic enzyme marker after making permeabilized yeast cells . However, during a chase, 20-40% of Vps33p fractionated with permeabilized cell membranes in a time-dependent fashion with a half-time of approximately 40 min . Depletion of cellular ATP increased the association rate to a half-time of approximately 4 min and caused 80-90% of newly synthesized Vps33p to be associated with permeabilized cell membranes . The association of Vps33p with permeabilized cell membranes was reversible after restoring cells with glucose before permeabilization . The N-ethylmaleimide-sensitive fusion protein homologue, Sec18p, a protein with known ATP binding and hydrolysis activity, displayed the same reversible energy-dependent sedimentation characteristics as Vps33p . We determined that the photosensitive analog, 8-azido-{alpha-32P}ATP, could bind directly to Vps33p with low affinity . Interestingly, excess unlabeled ATP could enhance photoaffinity labeling of 8-azido-{alpha-32P}ATP to Vps33p, suggesting cooperative binding, which was not observed with excess GTP . Importantly, we did not detect significant photolabeling after deleting amino acid regions in Vps33p that show similarity to ATP interaction motifs . We visualized these events in living yeast cells after fusing the jellyfish green fluorescent protein (GFP) to the C terminus of full-length Vps33p . In metabolically active cells, the fully functional Vps33p-GFP fusion protein appeared to stain throughout the cytoplasm with one or two very bright fluorescent spots near the vacuole . After depleting cellular ATP, Vps33p-GFP appeared to localize with a punctate morphology, which was also reversible upon restoring cells with glucose . Overall, these data support a model where Vps33p cycles between soluble and particulate forms in an ATP-dependent manner, which may facilitate the specificity of transport vesicle docking or targeting to the yeast lysosome/vacuole.

EMBO J, 1998 May 1, 17(9), 2494 - 503
TRAPP, a highly conserved novel complex on the cis-Golgi that mediates vesicle docking and fusion; Sacher M et al.; We previously identified BET3 by its genetic interactions with BET1, a gene whose SNARE-like product acts in endoplasmic reticulum (ER)-to-Golgi transport . To gain insight into the function of Bet3p, we added three c-myc tags to its C-terminus and immunopurified this protein from a clarified detergent extract . Here we report that Bet3p is a member of a large complex ( approximately 800 kDa) that we call TRAPP (transport protein particle) . We propose that TRAPP plays a key role in the targeting and/or fusion of ER-to-Golgi transport vesicles with their acceptor compartment . The localization of Bet3p to the cis-Golgi complex, as well as biochemical studies showing that Bet3p functions on this compartment, support this hypothesis . TRAPP contains at least nine other constituents, five of which have been identified and shown to be highly conserved novel proteins.

Arch Biochem Biophys, 1998 Jun 1, 354(1), 102 - 10
An unassembled subunit of NAD(+)-dependent isocitrate dehydrogenase is insoluble and covalently modified; Gadde DM et al.; The NAD(+)-dependent isocitrate dehydrogenase of Saccharomyces cerevisiae is an octamer composed of four Idh1p subunits and four Idh2p subunits . Isocitrate dehydrogenase functions in the tricarboxylic acid cycle and has also been reported to bind to the 5' nontranslated region of mitochondrially encoded mRNAs . Mutants defective in either or both of these subunits are unable to grow on the nonfermentable carbon source, acetate, but will utilize glycerol or ethanol . Mutant strains lacking Idh2p maintain normal if not elevated levels of mitochondrial Idh1p . In addition to the mature unassembled Idh1p subunit, a complex of bands in the 85- to 170-kDa range (Idh1p-Cpx) is observed using NAD-IDH antiserum . Both Idh1p and Idh1p-Cpx are insoluble within the mitochondrion and are associated with the mitochondrial inner membrane . A histidine-tagged form of Idh1p was expressed in yeast strains . Chemical amounts of the Idh1p-Cpx could be purified from strains lacking Idh2p but not from strains containing normal levels of Idh2p . The data indicate that Idh1p-Cpx is an aggregated and cross-linked form of Idh1p that may be oxidized within the mitochondrion as a consequence of its aborted assembly.

J Biol Chem, 1998 Jun 26, 273(26), 16509 - 16
Recruitment of human TBP selectively activates RNA polymerase II TATA-dependent promoters; Majello B et al.; An increasing body of evidence suggests that eukaryotic activators stimulate polymerase II transcription by facilitating the assembly of the functional basal machinery at the promoter . Here we describe experiments that provide added support for the idea that recruitment of TATA-binding protein (TBP) is a rate-limiting step for transcription activation in mammalian cells . We found that, in human cell lines, recruitment of TBP to a promoter, as a GAL4-TBP fusion protein, can provide a substantial activation of transcription . Activation mediated by the hTBP, tethered to promoter DNA, is strictly dependent upon the presence of a functional TATA element, and it directs faithful transcription initiation . Interestingly, GAL4-hTBP activation was not observed from initiator (Inr) -dependent TATA-less promoters . These results suggest that TBP binding to DNA is not a rate-limiting step for the initial stages of TFIID recruitment to initiator-dependent TATA-less promoters . Finally, we provide evidence that synergy between GAL4-hTBP and defined transcription domains is restricted to activators, such as VP16 and Tat, which are likely to function at steps subsequent to the TFIID recruitment . These findings strengthen the idea that recruitment of TBP represents an important mechanism of activation of TATA-dependent promoters, and on the other hand, they suggest that TBP-DNA interactions are largely dispensable for specific transcription of initiator dependent TATA-less promoters.

J Biol Chem, 1998 Jun 26, 273(26), 16374 - 81
The import route of ADP/ATP carrier into mitochondria separates from the general import pathway of cleavable preproteins at the trans side of the outer membrane; Kubrich M et al.; The ADP/ATP carrier (AAC) of the mitochondrial inner membrane is synthesized in the cytosol without a cleavable presequence . The preprotein preferentially binds to the mitochondrial surface receptor Tom70 and joins the import pathway of presequence-carrying preproteins at the cis side of the outer membrane . Little is known about the translocation of the AAC across the outer membrane and where its import route separates from that of cleavable preproteins . Here we have characterized a translocation intermediate of AAC during transfer across the outer membrane . The major portion of the preprotein is exposed to the intermembrane space, while a short segment is still accessible to externally added protease . This intermediate can be quantitatively chased to the fully imported form in the inner membrane . Its accumulation depends on Tom7, but not on the intermembrane space domain of Tom22 in contrast to cleavable preproteins . Moreover, opening of the intermembrane space inhibits the import of AAC, but not that of cleavable preproteins into mitoplasts . We conclude that the import route of AAC diverges from the general import pathway of cleavable preproteins already at the trans side of the outer membrane.

Biochemistry, 1998 May 26, 37(21), 7834 - 43
The structure of the N-terminus of striated muscle alpha-tropomyosin in a chimeric peptide: nuclear magnetic resonance structure and circular dichroism studies; Greenfield NJ et al.; Tropomyosins (TMs) are highly conserved, coiled-coil, actin binding regulatory proteins found in most eukaryotic cells . The amino-terminal domain of 284-residue TMs is among the most conserved and functionally important regions . The first nine residues are proposed to bind to the carboxyl-terminal nine residues to form the "overlap" region between successive TMs, which bind along the actin filament . Here, the structure of the N-terminus of muscle alpha-TM, in a chimeric peptide, TMZip, has been solved using circular dichroism (CD) and two-dimensional proton nuclear magnetic resonance (2D 1H NMR) spectroscopy . Residues 1-14 of TMZip are the first 14 N-terminal residues of rabbit striated alpha-TM, and residues 15-32 of TMZip are the last 18 C-terminal residues of the yeast GCN4 transcription factor . CD measurements show that TMZip forms a two-stranded coiled-coil alpha-helix with an enthalpy of folding of -34 +/- 2 kcal/mol . In 2D1H NMR studies at 15 degrees C, pH 6.4, the peptide exhibits 123 sequential and medium range intrachain NOE cross peaks per chain, characteristic of alpha-helices extending from residue 1 to residue 29, together with 85 long-range NOE cross peaks arising from interchain interactions . The three-dimensional structure of TMZip has been determined using these data plus an additional 509 intrachain constraints per chain . The coiled-coil domain extends to the N-terminus . Amide hydrogen exchange studies, however, suggest that the TM region is less stable than the GCN4 region . The work reported here is the first atomic-resolution structure of any region of TM and it allows insight into the mechanism of the function of the highly conserved N-terminal domain.

Biochemistry, 1998 May 26, 37(21), 7733 - 40
Dissection of the sequence specificity of the Holliday junction endonuclease CCE1; Schofield MJ et al.; CCE1 is a Holliday (four-way DNA) junction-specific endonuclease which resolves mitochondrial DNA recombination intermediates in Saccharomycescerevisiae . The junction-resolving enzymes are a diverse class, widely distributed in nature from viruses to higher eukaryotes . In common with most other junction-resolving enzymes, the cleavage activity of CCE1 is nucleotide sequence-dependent . We have undertaken a systematic study of the sequence specificity of CCE1, using a single-turnover kinetic assay and a panel of synthetic four-way DNA junction substrates . A tetranucleotide consensus cleavage sequence 5'-ACT downward arrowA has been identified, with specificity residing mainly at the central CT dinucleotide . Equilibrium constants for CCE1 binding to four-way junctions are unaffected by sequence variations, suggesting that substrate discrimination occurs predominantly in the transition state complex . CCE1 cuts most efficiently at the junction center, but can also cleave the DNA backbone at positions one nucleotide 3' or 5' of the point of strand exchange, suggesting a significant degree of conformational flexibility in the CCE1:junction complex . Introduction of base analogues at single sites in four-way junctions has allowed investigation of the sequence specificity of CCE1 in finer detail . In particular, the N7 moiety of the guanine base-pairing with the cytosine of the consensus sequence appears to be crucial for catalysis . The functional significance of sequence specificity in junction-resolving enzymes is discussed.

J Cell Biol, 1998 May 18, 141(4), 887 - 94
The beta subunit of the Sec61 complex facilitates cotranslational protein transport and interacts with the signal peptidase during translocation; Kalies KU et al.; The Sec61 complex is the central component of the protein translocation apparatus of the ER membrane . We have addressed the role of the beta subunit (Sec61beta) during cotranslational protein translocation . With a reconstituted system, we show that a Sec61 complex lacking Sec61beta is essentially inactive when elongation and membrane targeting of a nascent chain occur at the same time . The translocation process is perturbed at a step where the nascent chain would be inserted into the translocation channel . However, if sufficient time is given for the interaction of the nascent polypeptide with the mutant Sec61 complex, translocation is almost normal . Thus Sec61beta kinetically facilitates cotranslational translocation, but is not essential for it . Using chemical cross-linking we show that Sec61beta not only interacts with subunits of the Sec61 complex but also with the 25-kD subunit of the signal peptidase complex (SPC25), thus demonstrating for the first time a tight interaction between the SPC and the Sec61 complex . Interestingly, the cross-links between Sec61beta and SPC25 and between Sec61beta and Sec61alpha depend on the presence of membrane-bound ribosomes, suggesting that these interactions are induced when translocation is initiated . We propose that the SPC is transiently recruited to the translocation site, thus enhancing its activity.

Mol Cell Biol, 1998 Jun, 18(6), 3580 - 5
The nucleic acid binding activity of bleomycin hydrolase is involved in bleomycin detoxification; Zheng W et al.; Yeast bleomycin hydrolase, Gal6p, is a cysteine peptidase that detoxifies the anticancer drug bleomycin . Gal6p is a dual-function protein capable of both nucleic acid binding and peptide cleavage . We now demonstrate that Gal6p exhibits sequence-independent, high-affinity binding to single-stranded DNA, nicked double-stranded DNA, and RNA . A region of the protein that is involved in binding both RNA and DNA substrates is delineated . Immunolocalization reveals that the Gal6 protein is chiefly cytoplasmic and thus may be involved in binding cellular RNAs . Variant Gal6 proteins that fail to bind nucleic acid also exhibit reduced ability to protect cells from bleomycin toxicity, suggesting that the nucleic acid binding activity of Gal6p is important in bleomycin detoxification and may be involved in its normal biological functions.

Mol Cell Biol, 1998 Jun, 18(6), 3502 - 8
Optimal activation of an endogenous gene by HOX11 requires the NH2-terminal 50 amino acids; Masson N et al.; The HOX11 homeobox gene was first identified through studies of the t(7;10) and t(10;14) chromosomal translocations of acute T-cell leukemia . In addition, analysis of Hox11-/- mice has demonstrated a critical role for this gene in murine spleen development . A possible mode of in vivo function for the HOX11 protein in these two situations is regulation of target genes following DNA binding via the homeodomain, but little is known about how HOX11 regulates transcription in vivo . By performing transcriptional studies in yeast and mammalian one-hybrid systems, a modular transcriptional transactivation region at the NH2 terminus of HOX11 has been functionally dissected from other parts of the protein . This NH2-terminal region includes the previously identified short conserved Hep motif, which itself activates transcription in one-hybrid assays . The importance of the NH2-terminal region for the function of HOX11 in vivo was assayed by activating a HOX11-dependent gene in NIH 3T3 cells . Activation of this gene was found to be dependent upon an intact homeodomain in HOX11, but maximal activation was obtained only when the NH2-terminal 50 amino acids of HOX11 was present, showing that this region of HOX11 is important for in vivo transcriptional control of a chromosomal target gene.

Mol Cell Biol, 1998 Jun, 18(6), 3445 - 54
A critical role for cyclin C in promotion of the hematopoietic cell cycle by cooperation with c-Myc; Liu ZJ et al.; Cyclin C, a putative G1 cyclin, was originally isolated through its ability to complement a Saccharomyces cerevisiae strain lacking the G1 cyclin gene CLN1-3 . Unlike cyclins D1 and E, the other two G1 cyclins obtained by the same approach and subsequently shown to play important roles during the G1/S transition, there is thus far no evidence to support the hypothesis that cyclin C is indeed critical for the promotion of cell cycle progression . In BAF-B03 cells, an interleukin 3 (IL-3)-dependent murine pro-B-cell line, cyclin C gene mRNA was induced at the G1/S phase upon IL-3 stimulation and reached a maximal level in the S phase . Enforced expression of exogenous cyclin C in this cell line failed to alter its growth properties . In the present study, we examined whether cyclin C is capable of cooperating with the cytokine-responsive immediate-early gene products c-Myc and c-Fos in the promotion of cell proliferation . We found that cyclin C is able to cooperate functionally with c-Myc, but not c-Fos, to induce both BAF-B03 cell proliferation in a cytokine-independent fashion and the formation of cell clusters . Furthermore, cyclin C was primarily responsible for the induction of cdc2 gene expression . Our data define a novel role for cyclin C in the regulation of both the G1/S and G2/M phases of the cell cycle, and this effect appears to be independent of the activity of CDK8 in the control of transcription.

Mol Cell Biol, 1998 Jun, 18(6), 3289 - 99
Cyclin partners determine Pho85 protein kinase substrate specificity in vitro and in vivo: control of glycogen biosynthesis by Pcl8 and Pcl10; Huang D et al.; In Saccharomyces cerevisiae, PHO85 encodes a cyclin-dependent protein kinase (Cdk) with multiple roles in cell cycle and metabolic controls . In association with the cyclin Pho80, Pho85 controls acid phosphatase gene expression through phosphorylation of the transcription factor Pho4 . Pho85 has also been implicated as a kinase that phosphorylates and negatively regulates glycogen synthase (Gsy2), and deletion of PHO85 causes glycogen overaccumulation . We report that the Pcl8/Pcl10 subgroup of cyclins directs Pho85 to phosphorylate glycogen synthase both in vivo and in vitro . Disruption of PCL8 and PCL10 caused hyperaccumulation of glycogen, activation of glycogen synthase, and a reduction in glycogen synthase kinase activity in vivo . However, unlike pho85 mutants, pcl8 pcl10 cells had normal morphologies, grew on glycerol, and showed proper regulation of acid phosphatase gene expression . In vitro, Pho80-Pho85 complexes effectively phosphorylated Pho4 but had much lower activity toward Gsy2 . In contrast, Pcl10-Pho85 complexes phosphorylated Gsy2 at Ser-654 and Thr-667, two physiologically relevant sites, but only poorly phosphorylated Pho4 . Thus, both the in vitro and in vivo substrate specificity of Pho85 is determined by the cyclin partner . Mutation of PHO85 suppressed the glycogen storage deficiency of snf1 or glc7-1 mutants in which glycogen synthase is locked in an inactive state . Deletion of PCL8 and PCL10 corrected the deficit in glycogen synthase activity in both the snf1 and glc7-1 mutants, but glycogen synthesis was restored only in the glc7-1 mutant strain . This genetic result suggests an additional role for Pho85 in the negative regulation of glycogen accumulation that is independent of Pcl8 and Pcl10.

Mol Cell Biol, 1998 Jun, 18(6), 3173 - 81
Role of the negative charges in the cytosolic domain of TOM22 in the import of precursor proteins into mitochondria; Nargang FE et al.; TOM22 is an essential mitochondrial outer membrane protein required for the import of precursor proteins into the organelles . The amino-terminal 84 amino acids of TOM22 extend into the cytosol and include 19 negatively and 6 positively charged residues . This region of the protein is thought to interact with positively charged presequences on mitochondrial preproteins, presumably via electrostatic interactions . We constructed a series of mutant derivatives of TOM22 in which 2 to 15 of the negatively charged residues in the cytosolic domain were changed to their corresponding amido forms . The mutant constructs were transformed into a sheltered Neurospora crassa heterokaryon bearing a tom22::hygromycin R disruption in one nucleus . All constructs restored viability to the disruption-carrying nucleus and gave rise to homokaryotic strains containing mutant tom22 alleles . Isolated mitochondria from three representative mutant strains, including the mutant carrying 15 neutralized residues (strain 861), imported precursor proteins at efficiencies comparable to those for wild-type organelles . Precursor binding studies with mitochondrial outer membrane vesicles from several of the mutant strains, including strain 861, revealed only slight differences from binding to wild-type vesicles . Deletion mutants lacking portions of the negatively charged region of TOM22 can also restore viability to the disruption-containing nucleus, but mutants lacking the entire region cannot . Taken together, these data suggest that an abundance of negative charges in the cytosolic domain of TOM22 is not essential for the binding or import of mitochondrial precursor proteins; however, other features in the domain are required.

J Biol Chem, 1998 May 15, 273(20), 12148 - 54
Characterization of human hect domain family members and their interaction with UbcH5 and UbcH7; Schwarz SE et al.; The hect domain protein family was originally identified by sequence similarity of its members to the C-terminal region of E6-AP, an E3 ubiquitin-protein ligase . Since the C terminus of E6-AP mediates thioester complex formation with ubiquitin, a necessary intermediate step in E6-AP-dependent ubiquitination, it was proposed that members of the hect domain family in general have E3 activity . The hect domain is approximately 350 amino acids in length, and we show here that the hect domain of E6-AP is necessary and sufficient for ubiquitin thioester adduct formation . Furthermore, the human genome encodes at least 20 different hect domain proteins, and in further support of the hypothesis that hect domain proteins represent a family of E3s, several of these are shown to form thioester complexes with ubiquitin . In addition, some hect domain proteins interact preferentially with UbcH5, whereas others interact with UbcH7, indicating that human hect domain proteins can be grouped into at least two classes based on their E2 specificity . Since E3s are thought to play a major role in substrate recognition, the presence of a large family of E3s should contribute to ensure the specificity and selectivity of ubiquitin-dependent proteolytic pathways.

EMBO J, 1998 May 1, 17(9), 2651 - 62
Dbp5p, a cytosolic RNA helicase, is required for poly(A)+ RNA export; Tseng SS et al.; The DBP5 gene encodes a putative RNA helicase of unknown function in the yeast Saccharomyces cerevisiae . It is shown here that Dbp5p is an ATP-dependent RNA helicase required for polyadenylated {poly(A)+} RNA export . Surprisingly, Dbp5p is present predominantly, if not exclusively, in the cytoplasm, and is highly enriched around the nuclear envelope . This observation raises the possibility that Dbp5p may play a role in unloading or remodeling messenger RNA particles (mRNPs) upon arrival in the cytoplasm and in coupling mRNP export and translation . The functions of Dbp5p are likely to be conserved, since its potential homologues can be found in a variety of eukaryotic cells.

Nucleic Acids Res, 1998 May 1, 26(9), 2098 - 104
The activation function 2 domain of hepatic nuclear factor 4 is regulated by a short C-terminal proline-rich repressor domain; Iyemere VP et al.; Hepatic nuclear factor 4 (HNF4) is a transcription factor whose expression is crucial for mouse embryonic development, for liver-specific gene expression and for the prevention of one form of maturity-onset diabetes of the young . Its domain structure has been defined previously and is similar to other members of the nuclear receptor superfamily . A repressor domain has now been localised to a region of 14 amino acids (residues 428-441) near the C-terminus of HNF4 and is sufficient by itself to repress the activity of the activation function 2 (AF2) domain . Multiple mutations within this repressor domain enhance activity . Interestingly, this repressor domain shares homology with a repressor domain in the progesterone receptor . In a detailed mutagenesis study of the AF2 core, we demonstrate that L 366, which is conserved in the AF2 core between HNF4 and a number of orphan nuclear receptors, is essential for the full activity of the AF2 domain . Furthermore, a double mutation of E 363 and L 366 suggests that these residues might act in a cooperative manner.

Curr Opin Struct Biol, 1998 Apr, 8(2), 177 - 85
Intermediate filament assembly: fibrillogenesis is driven by decisive dimer-dimer interactions; Herrmann H et al.; Intermediate filaments are built from one to several members of a multigene family encoding fibrous proteins that share a highly conserved hierarchic assembly plan for the formation of multistranded filaments from distinctly structured extended coiled coils . Despite the rather low primary sequence identity, intermediate filaments form apparently similar filaments with regard to their spatial dimensions and physical properties . Over the past few years, substantial progress has been made in the elucidation of the complex expression patterns and clinically relevant phenotypes of intermediate filaments . The key question of how these filaments assemble and what the molecular architecture of their distinct assembly intermediates comprises, however, has still not been answered to the extent that has been achieved for microfilaments and microtubules.

Nat Biotechnol, 1996 Apr, 14(4), 481 - 4
A fusion protein designed for noncovalent immobilization: stability, enzymatic activity, and use in an enzyme reactor; Stempfer G et al.; We have designed a new method for enzyme immobilization using a fusion protein of yeast alpha-glucosidase containing at its C-terminus a polycationic hexa-arginine fusion peptide . This fusion protein can be directly adsorbed from crude cell extracts on polyanionic matrices in a specific, oriented fashion . Upon noncovalent immobilization by polyionic interactions, the stability of the fusion protein is not affected by pH-, urea-, or thermal-denaturation . Furthermore, the enzymatic properties (specific activity at increasing enzyme concentration, Michaelis constant, or activation energy of the enzymatic reaction) are not influenced by this noncovalent coupling . The operational stability of the coupled enzyme under conditions of continuous substrate conversion is, however, increased significantly compared to the soluble form . Fusion proteins containing polyionic peptide sequences are proposed as versatile tools for the production of immobilized enzyme catalysts.

Chem Biol Interact, 1998 May 1, 113(1), 39 - 50
Covalent binding of carbamazepine reactive metabolites to P450 isoforms present in the skin; Wolkenstein P et al.; Carbamazepine is an anticonvulsant associated with a high risk for severe cutaneous reactions . Upon metabolism by cytochrome P450, carbamazepine may produce reactive metabolites . We evaluated in vitro the covalent binding of carbamazepine reactive metabolites on human P450s and then the presence of these P450s in human epidermis . Carbamazepine reactive metabolites covalent binding to human liver microsomes involved P450 subfamilies 1A, 2C and 3A . Specific covalent binding to yeasts expressing different P450s showed that carbamazepine reactive metabolites bound specifically to P450 1A2 and 3A4 . We confirmed the constitutive presence of P450 3A in human epidermis and after induction with coaltar of P450 1A . Consequently, the production in epidermis of carbamazepine reactive metabolites is theoretically possible with formation of P450 adduct metabolites.

Biochim Biophys Acta, 1998 May 11, 1397(3), 325 - 30
U14snoRNAs of the fern, Asplenium nidus, contain large sequence insertions compared with those of higher plants; Leader DJ et al.; Northern analyses of U14snoRNAs in different plant species showed the expected hybridising band of approximately 120 nt in monocotyledonous and dicotyledonous angiosperms . In the lower plant, Bird's nest fern (Asplenium nidus), U14s were larger and three hybridising RNAs of approximately 190, 210 and 250 nt were observed . RT-PCR cloning of all three size variants using primers to the conserved 5' and 3' ends of higher plant U14snoRNAs showed large insertions in one of the plant-specific regions corresponding in position to the yeast U14-specific Y-domain . The insertions are pyrimidine-rich in their 5' halves and purine-rich in their 3' halves and are likely to be sequestered in stem structures consistent with the proposed model of U14snoRNA secondary structure . The 5' flanking regions of one of the fern U14 variants was generated by PCR and lacked classical plant snRNA promoter elements .

J Nutr, 1998 Jun, 128(6), 940 - 6
Mechanisms of decreased lipoprotein lipase activity in adipocytes of starved rats depend on duration of starvation; Lee JJ et al.; The aim of this study was to delineate the mechanisms by which varying periods of starvation decrease lipoprotein lipase (LPL) activity in rat adipose tissue . LPL mRNA levels and rates of LPL synthesis, degradation and secretion were compared in adipocytes from male rats that had been fed or starved for 1 or 3 d . The decreased LPL activity after 3 d of starvation (-76%) was explained mainly by a 50% decrease in the relative abundance of LPL mRNA levels (P < 0.05) and a parallel 50% decrease in relative rates of LPL biosynthesis (P < 0.05) . In contrast, starvation for 1 d decreased total LPL activity by 47% (P < 0.05) but did not affect LPL mRNA levels or relative rates of LPL biosynthesis . Pulse-chase studies demonstrated that 1 d of starvation increased the rate of degradation of newly synthesized LPL (P < 0.05) and markedly decreased its secretion into the medium (P < 0.05) . A decrease in overall protein synthesis also contributed to the decreased LPL activity after 1 and 3 d of starvation . We conclude that the relative importance of pre- and post-translational mechanisms in regulating adipose tissue LPL activity depends on the duration of starvation . During short-term starvation, degradation of newly synthesized LPL is an important determinant to its secretion from the adipocyte and hence its functional activity at the capillary endothelium.

J Mol Evol, 1998 Jun, 46(6), 721 - 8
Phylogenetic position of the Hexactinellida within the phylum Porifera based on the amino acid sequence of the protein kinase C from Rhabdocalyptus dawsoni; Kruse M et al.; Recent analyses of genes encoding proteins typical for multicellularity, especially adhesion molecules and receptors, favor the conclusion that all metazoan phyla, including the phylum Porifera (sponges), are of monophyletic origin . However, none of these data includes cDNA encoding a protein from the sponge class Hexactinellida . We have now isolated and characterized the cDNA encoding a protein kinase C, belonging to the C subfamily (cPKC), from the hexactinellid sponge Rhabdocalyptus dawsoni . The two conserved regions, the regulatory part with the pseudosubstrate site, the two zinc fingers, and the C2 domain, as well as the catalytic domain were used for phylogenetic analyses . Sequence alignment and construction of a phylogenetic tree from the catalytic domains revealed that the yeast Saccharomyces cerevisiae and the protozoan Trypanosoma brucei are at the base of the tree, while the hexactinellid R . dawsoni branches off first among the metazoan sequences; the other two classes of the Porifera, the Calcarea (the sequence from Sycon raphanus was used) and the Demospongiae (sequences from Geodia cydonium and Suberites domuncula were used), branch off later . The statistically robust tree also shows that the two cPKC sequences from the higher invertebrates Drosophila melanogaster and Lytechinus pictus are most closely related to the calcareous sponge . This finding was also confirmed by comparing the regulatory part of the kinase gene . We suggest, that (i) within the phylum Porifera, the class Hexactinellida diverged first from a common ancestor to the Calcarea and the Demospongiae, which both appeared later, and (ii) the higher invertebrates are more closely related to the calcareous sponges.

Nucleic Acids Res, 1998 Jul 1, 26(13), 3215 - 20
Influence of promoter potency on the transcriptional effects of YY1, SRF and Msx-1 in transient transfection analysis; Lee T et al.; Potent viral promoters/enhancers are often used to achieve high level expression of ectopic genes in transient transfection analysis . By using a GAL4-responsive transcription assay system, we show that the use of potent eukaryotic expression vectors can lead to biased transcriptional effects . Three functionally diverse transcription factors, YY1, SRF and Msx-1, were examined and each was found to exhibit a strong transrepression function in the context of the DNA binding domain of GAL4 when expressed from the cytomegalovirus (pCMV) or simian virus 40 (pSV) promoters/enhancers . An internal 15 amino acid domain of YY1 mediating transrepression in the viral promoter setting was identified . This GAL4-mediated transcriptional repression could, however, be completely relieved by using the yeast alcohol dehydrogenase promoter (pADH) to drive gene expression, which is approximately 100-fold weaker than canonical pCMV and pSV in cultured mammalian cells . In addition, low level expression achieved with the pADH vector unveiled the intrinsic transactivation functions of YY1 and SRF previously not observed with the GAL4 assay system . Our results highlight a potential pitfall in conventional pCMV- and pSV-based transfection assays and suggest that the use of a low level expression system may be preferable in most transcriptional analysis.

EMBO J, 1998 Jun 15, 17(12), 3363 - 71
Leptomycin B-sensitive nuclear export of MAPKAP kinase 2 is regulated by phosphorylation; Engel K et al.; To study the intracellular localization of MAPKAP kinase 2 (MK2), which carries a putative bipartite nuclear localization signal (NLS), we constructed a green fluorescent protein-MAPKAP kinase 2 fusion protein (GFP-MK2) . In transfected cells, this protein is located predominantly in the nucleus; unexpectedly, upon stress, it rapidly translocates to the cytoplasm . This translocation can be blocked by the p38 MAP kinase inhibitor SB203580, indicating its regulation by phosphorylation . Molecular mimicry of MK2 phosphorylation at T317 in GFP-MK2 led to a mutant which is located almost exclusively in the cytoplasm of the cell, whereas the mutant T317A shows no stress-induced redistribution . Since leptomycin B, which inhibits the interaction of exportin 1 with the Rev-type leucine-rich nuclear export signal (NES), blocks stress-dependent translocation of GFP-MK2, it is supposed that phosphorylation-induced export of the protein causes the translocation . We have identified the region responsible for nuclear export in MK2 which is partially overlapping with and C-terminal to the autoinhibitory motif . This region contains a cluster of hydrophobic amino acids in the characteristic spacing of a leucine-rich Rev-type NES which is necessary to direct GFP-MK2 to the cytoplasm . However, unlike the Rev-type NES, this region alone is not sufficient for nuclear export . The data obtained indicate that MK2 contains a constitutively active NLS and a stress-regulated signal for nuclear export . Keywords: nuclear export/nuclear import/protein phosphorylation/signal transduction/stress response

EMBO J, 1998 Jun 15, 17(12), 3269 - 76
Vam7p, a vacuolar SNAP-25 homolog, is required for SNARE complex integrity and vacuole docking and fusion; Ungermann C et al.; The vacuole v-t-SNARE complex is disassembled by Sec17p/alpha-SNAP and Sec18p/NSF prior to vacuole docking and fusion . We now report a functional characterization of the vacuolar SNARE Vam7p, a SNAP-25 homolog . Although Vam7p has no hydrophobic domains, it is tightly associated with the vacuolar membrane . Vam7p is a constituent of the vacuole SNARE complex and is released from this complex by the Sec17p/Sec18p/ATP-mediated priming of the vacuoles . Even in the absence of the vacuolar v-SNARE Nyv1p, a subcomplex which includes Vam7p and the t-SNARE Vam3p is preserved . Vam7p is necessary for the stability of the vacuolar SNARE complex, since vacuoles from mutants deleted in VAM7 do not have a Vam3p-Nyv1p complex . Furthermore, Vam7p alone, in the absence of Nyv1p and Vam3p, cannot mediate fusion with wild-type vacuoles, whereas vacuoles with only Nyv1p or Vam3p alone can fuse with wild-type vacuoles in the absence of the other two SNAREs . Thus, Vam7p is important for the stable assembly and efficient function of the vacuolar SNARE complex and maintenance of the vacuolar morphology . This functional characterization of Vam7p suggests a general role for SNAP-25 homologs, not only on the plasma membrane but along the secretory pathway.

Nat Struct Biol, 1998 Jun, 5(6), 484 - 91
A new DNA-binding motif in the Skn-1 binding domain-DNA complex; Rupert PB et al.; The DNA-binding domain of Skn-1, a developmental transcription factor that specifies mesoderm in C . elegans, is shown by X-ray crystallography to have a novel fold in which a compact, monomeric, four-helix unit organizes two DNA-contact elements . At the C-terminus, a helix extends from the domain to occupy the major groove of DNA in a manner similar to bZip proteins . Skn-1, however, lacks the leucine zipper found in all bZips . Additional contacts with the DNA are made by a short basic segment at the N-terminus of the domain, reminiscent of the 'homeodomain arm'.

Biol Chem, 1998 Apr-May, 379(4-5), 489 - 95
Chimeric restriction enzyme: Gal4 fusion to FokI cleavage domain; Kim YG et al.; Gal4, a yeast protein, activates transcription of genes required for metabolism of galactose and melibiose . It binds as a dimer to a consensus palindromic 17-base pair DNA sequence . It is a member of the third family of proteins that contain zinc-mediated peptide loops that interact specifically with nucleic acids . Gal4 has a very distinctive zinc coordination profile and mode of DNA-binding . Here, we report the creation of a novel site-specific endonuclease by linking the N-terminal 147 amino acids of Gal4 to the cleavage domain of FokI endonuclease . The fusion protein is active and under optimal conditions, binds to a 17 bp consensus DNA site and cleaves near this site . As expected, the cleavage occurs on either side of the consensus binding site(s).

J Investig Dermatol Symp Proc, 1996 Apr, 1(2), 128 - 35
Functional interactions among members of the Myc superfamily and potential relevance to cutaneous growth and development; Chin L et al.; Myc family oncoproteins function as sequence-specific transcription factors that are believed to regulate the expression of genes governing cellular growth, differentiation, and programmed cell death . Activities of Myc are countered by those of Mad and Mxi1, two related members of the Myc superfamily . Mad and Mxi1 compete with Myc for common elements and interact with putative transcriptional repressors . While the precise role of the Myc superfamily in cutaneous biology remains to be determined, findings from a number of organ systems suggest that the regulated expression and function of its members are intimately correlated with proper development and physiology . Reviewed here are current data on Myc superfamily function with references where relevant to cutaneous processes with the ultimate goal of providing a framework upon which these proteins can be exploited in gene therapeutic approaches for diseases of the skin, including neoplasia.

Mol Endocrinol, 1998 Jun, 12(6), 864 - 81
A regulatory role for RIP140 in nuclear receptor activation; Treuter E et al.; Transcriptional regulation of gene expression by nuclear receptors requires negatively and positively acting cofactors . Recent models for receptor activation propose that certain receptors in the absence of ligands can recruit corepressors while ligand binding results in conformational changes leading to the recruitment of coactivators . Previous work has established a coactivator role for the SRC-1 family members as well as an involvement of the coactivators CBP/p300 in nuclear receptor signaling . However, in addition to coactivators, ligand-activated nuclear receptors bind a number of different proteins that possibly serve other functions . Using peroxisome proliferator-activated receptor-alpha (PPAR alpha) as bait in a yeast two-hybrid screening, we have isolated nuclear factor RIP140 whose function in receptor activation is unclear . We now report a detailed characterization of RIP140 action with a focus on the retinoid X receptor (RXR) heterodimeric receptors PPAR and thyroid hormone receptor (TR) . We show that putative PPAR ligands enhance the interaction of RIP140 with the rat PPAR subtypes alpha and gamma in solution but not with PPAR/RXR heterodimers on DNA . However, RIP140 forms ternary complexes in the presence of RXR ligands . Similar experiments with TR support the high affinity of RIP140 to the RXR subunit and also suggest that either partner in the TR/RXR heterodimer can independently respond to ligand . Coactivation experiments in yeast and mammalian cells confirm the coactivator role for SRC-1, but not for RIP140 . We provide important evidence that the in vitro binding of RIP140 and SRC-1 to nuclear receptors is competitive . Since RIP140 generally down-regulates receptor activity in mammalian cells and specifically down-regulates coactivation mediated by SRC-1, we propose a model in which RIP140 indirectly regulates nuclear receptor AF-2 activity by competition for coactivators such as SRC-1.

J Cell Sci, 1998 Jul, 111 ( Pt 13), 1877 - 88
Three distinct steps in transport of vesicular stomatitis virus glycoprotein from the ER to the cell surface in vivo with differential sensitivities to GTP gamma S; Pepperkok R et al.; Microinjected GTP gamma S revealed three distinct steps in the exocytic transport of the temperature sensitive glycoprotein of vesicular stomatitis virus (ts-O45-G) from the ER to the cell surface in intact Vero cells . While COPII dependent export of ts-O45-G from the ER is blocked in cells injected with recombinant protein of a dominant mutant of SAR1a (SAR1a{H79G}) inhibited in GTP hydrolysis, neither injected GTP gamma S nor antibodies against beta-COP (anti-EAGE) interfere with this transport step significantly . In contrast, transport to the Golgi complex is blocked by 50 microM GTP gamma S, a dominant mutant of ARF1 (ARF1{Q71L}) inhibited in GTP hydrolysis, or microinjected anti-EAGE, but injected Sar1a{H79G}p has no effect . Microinjection of GTP gamma S or expression of ARF{Q71L} rapidly induces accumulation of COPI coated vesicular structures lacking ts-O45-G . Finally, transport of ts-O45-G from the trans-Golgi network (TGN) to the cell surface is inhibited only by high concentrations of GTP gamma S (500 microM) . Interestingly, this step is only partially brefeldin A sensitive, and injected antibodies against beta-COP and p200/myosin II, a TGN membrane associated protein, have no effect . These data provide first strong in vivo evidence for at least three distinct steps in the exocytic pathway of mammalian cells regulated by different sets of GTPases and coat proteins . COPII, but not COPI, is required for ER export of ts-O45-G . COPI plays a role in subsequent transport to the Golgi complex, and a so far unidentified GTP gamma S sensitive coat appears to be involved in transport from the TGN to the cell surface.

J Virol, 1998 Jul, 72(7), 5789 - 96
The duck hepatitis B virus polymerase is activated by its RNA packaging signal, epsilon; Tavis JE et al.; The epsilon stem-loop at the 5' end of the pregenomic RNA of the hepatitis B viruses is both the primary element of the RNA packaging signal and the origin of reverse transcription . We have previously presented evidence for a third essential role for epsilon, that of an essential cofactor in the maturation of the viral polymerase (J . E . Tavis and D . Ganem, J . Virol . 70:5741-5750, 1996) . In this case, binding of epsilon to the polymerase is proposed to induce a physical alteration to the polymerase that is needed for it to develop enzymatic activity . Three lines of evidence employing duck hepatitis B virus supporting this hypothesis are presented here . First, an unusual DNA polymerase activity employing exogenous RNAs (the trans reaction) that was originally discovered with recombinant duck hepatitis B virus polymerase expressed in Saccharomyces cerevisiae yeasts was shown to be an authentic property of the viral polymerase . The trans reaction was found to be template-dependent reverse transcription of the exogenous RNA . The trans reaction occurred independently of the hepadnavirus protein-priming mechanism, yet it was still strongly stimulated by epsilon . This directly demonstrates a role for epsilon in activation of the polymerase . Second, the reverse transcriptase domain of the polymerase was shown to be physically altered following binding to epsilon, as would be expected if the alteration was required for maturation of the polymerase to an enzymatically active form . Finally, analysis of 15 mutations throughout the duck hepatitis B virus polymerase demonstrated that the epsilon-dependent alteration to the polymerase was a prerequisite for DNA priming, reverse transcription, and the trans reaction.

Genes Dev, 1998 Jun 1, 12(11), 1665 - 77
The surveillance complex interacts with the translation release factors to enhance termination and degrade aberrant mRNAs; Czaplinski K et al.; The nonsense-mediated mRNA decay pathway is an example of an evolutionarily conserved surveillance pathway that rids the cell of transcripts that contain nonsense mutations . The product of the UPF1 gene is a necessary component of the putative surveillance complex that recognizes and degrades aberrant mRNAs . Recent results indicate that the Upf1p also enhances translation termination at a nonsense codon . The results presented here demonstrate that the yeast and human forms of the Upf1p interact with both eukaryotic translation termination factors eRF1 and eRF3 . Consistent with Upf1p interacting with the eRFs, the Upf1p is found in the prion-like aggregates that contain eRF1 and eRF3 observed in yeast {PSI+} strains . These results suggest that interaction of the Upf1p with the peptidyl release factors may be a key event in the assembly of the putative surveillance complex that enhances translation termination, monitors whether termination has occurred prematurely, and promotes degradation of aberrant transcripts.

Genes Dev, 1998 Jun 1, 12(11), 1638 - 51
The histone acetylase PCAF is a nuclear receptor coactivator; Blanco JC et al.; Whereas the histone acetylase PCAF has been suggested to be part of a coactivator complex mediating transcriptional activation by the nuclear hormone receptors, the physical and functional interactions between nuclear receptors and PCAF have remained unclear . Our efforts to clarify these relationships have revealed two novel properties of nuclear receptors . First, we demonstrate that the RXR/RAR heterodimer directly recruits PCAF from mammalian cell extracts in a ligand-dependent manner and that increased expression of PCAF leads to enhanced retinoid-responsive transcription . Second, we demonstrate that, in vitro, PCAF directly associates with the DNA-binding domain of nuclear receptors, independently of p300/CBP binding, therefore defining a novel cofactor interaction surface . Furthermore, our results show that dissociation of corepressors enables ligand-dependent PCAF binding to the receptors . This observation illuminates how a ligand-dependent receptor function can be propagated to regions outside the ligand-binding domain itself . On the basis of these observations, we suggest that PCAF may play a more central role in nuclear receptor function than previously anticipated.

J Pharmacol Exp Ther, 1998 Jun, 285(3), 1296 - 302
Both the immunosuppressant SR31747 and the antiestrogen tamoxifen bind to an emopamil-insensitive site of mammalian Delta8-Delta7 sterol isomerase; Paul R et al.; SR31747 is a novel agent that elicits immunosuppressive and anti-inflammatory effects . This drug was shown to inhibit Delta8-Delta7 sterol isomerase in yeast . To test whether this enzyme could also be an SR31747 target in mammals, the binding, antiproliferative and sterol biosynthesis inhibitory properties of various drugs were studied in recombinant sterol isomerase-producing yeast cells . Our results clearly show that SR31747 is a high affinity ligand of recombinant mammalian sterol isomerase (Kd = 1 nM) . Tridemorph, a sterol biosynthesis inhibitor that is widely used in agriculture as an antifungal agent, is also a powerful inhibitor of murine and human sterol isomerases (IC50 value in the nanomolar range) . Some drugs, like cis-flupentixol, trifluoperazine, 7-ketocholestanol and tamoxifen, inhibit SR31747 binding only with the mammalian enzymes, whereas other drugs, like haloperidol and fenpropimorph, are much more effective with the yeast enzyme than with the mammalian ones . Emopamil, a high affinity ligand of human sterol isomerase, is inefficient in inhibiting SR31747 binding to its mammalian target, suggesting that the SR31747 and emopamil binding sites on mammalian sterol isomerase do not overlap . In contrast, SR31747 binding inhibition by tamoxifen is very efficient and competitive (IC50 value in the nanomolar range), indicating that mammalian sterol isomerase contains a so-called antiestrogen binding site . Tamoxifen is found to selectively inhibit sterol biosynthesis at the sterol isomerase step in the cells that are producing the mammalian enzyme in place of their own sterol isomerase . Finally, we also show that tridemorph, a sterol biosynthesis inhibitor widely used in agriculture as an antifungal agent, is not selective of yeast Delta8-Delta7 sterol isomerase but is also highly efficient against murine Delta8-Delta7 sterol isomerase or human Delta8-Delta7 sterol isomerase . This observation contrasts with our already published results showing that fenpropimorph, another sterol isomerase inhibitor used in agriculture, is only poorly efficient against the mammalian enzymes.

Mol Biol Cell, 1998 Jun, 9(6), 1395 - 410
Distinct morphological phenotypes of cell fusion mutants; Gammie AE et al.; Cell fusion in yeast is the process by which two haploid cells fuse to form a diploid zygote . To dissect the pathway of cell fusion, we phenotypically and genetically characterized four cell fusion mutants, fus6/spa2, fus7/rvs161, fus1, and fus2 . First, we examined the complete array of single and double mutants . In all cases but one, double mutants exhibited stronger cell fusion defects than single mutants . The exception was rvs161Delta fus2Delta, suggesting that Rvs161p and Fus2p act in concert . Dosage suppression analysis showed that Fus1p and Fus2p act downstream or parallel to Rvs161p and Spa2p . Second, electron microscopic analysis was used to define the mutant defects in cell fusion . In wild-type prezygotes vesicles were aligned and clustered across the cell fusion zone . The vesicles were associated with regions of cell wall thinning . Analysis of Fus- zygotes indicated that Fus1p was required for the normal localization of the vesicles to the zone of cell fusion, and Spa2p facilitated their clustering . In contrast, Fus2p and Rvs161p appeared to act after vesicle positioning . These findings lead us to propose that cell fusion is mediated in part by the localized release of vesicles containing components essential for cell fusion.

Mol Biol Cell, 1998 Jun, 9(6), 1265 - 78
Immunoisolation and characterization of a subdomain of the endoplasmic reticulum that concentrates proteins involved in COPII vesicle biogenesis; Hobman TC et al.; Rubella virus E1 glycoprotein normally complexes with E2 in the endoplasmic reticulum (ER) to form a heterodimer that is transported to and retained in the Golgi complex . In a previous study, we showed that in the absence of E2, unassembled E1 subunits accumulate in a tubular pre-Golgi compartment whose morphology and biochemical properties are distinct from both rough ER and Golgi . We hypothesized that this compartment corresponds to hypertrophied ER exit sites that have expanded in response to overexpression of E1 . In the present study we constructed BHK cells stably expressing E1 protein containing a cytoplasmically disposed epitope and isolated the pre-Golgi compartment from these cells by cell fractionation and immunoisolation . Double label indirect immunofluorescence in cells and immunoblotting of immunoisolated tubular networks revealed that proteins involved in formation of ER-derived transport vesicles, namely p58/ERGIC 53, Sec23p, and Sec13p, were concentrated in the E1-containing pre-Golgi compartment . Furthermore, budding structures were evident in these membrane profiles, and a highly abundant but unknown 65-kDa protein was also present . By comparison, marker proteins of the rough ER, Golgi, and COPI vesicles were not enriched in these membranes . These results demonstrate that the composition of the tubular networks corresponds to that expected of ER exit sites . Accordingly, we propose the name SEREC (smooth ER exit compartment) for this structure.

J Biol Chem, 1998 Jun 12, 273(24), 15030 - 4
Mammalian prenylcysteine carboxyl methyltransferase is in the endoplasmic reticulum; Dai Q et al.; Prenylcysteine carboxyl methyltransferase (pcCMT) is the third of three enzymes that posttranslationally modify C-terminal CAAX motifs and thereby target CAAX proteins to the plasma membrane . Here we report the molecular characterization and subcellular localization of the first mammalian (human myeloid) pcCMT . The deduced amino acid sequence of mammalian pcCMT predicts a multiple membrane-spanning protein with homologies to the yeast pcCMT, STE14, and the mammalian band 3 anion transporter . The human gene complemented a ste14 mutant . pcCMT mRNAs were ubiquitously expressed in human tissues . An anti-pcCMT antiserum detected a 33-kDa protein in myeloid cell membranes . Ectopically expressed recombinant pcCMT had enzymatic activity identical to that observed in neutrophil membranes . Mammalian pcCMT was not expressed at the plasma membrane but rather restricted to the endoplasmic reticulum . Thus, the final enzyme in the sequence that modifies CAAX motifs is located in membranes topologically removed from the CAAX protein target membrane.

Hum Mol Genet, 1998 Jun, 7(6), 1053 - 7
Interaction between hamartin and tuberin, the TSC1 and TSC2 gene products; van Slegtenhorst M et al.; Tuberous sclerosis (TSC) is an autosomal dominant disorder caused by a mutation in either the TSC1 or TSC2 tumour suppressor gene . The disease is characterized by a broad phenotypic spectrum that can include seizures, mental retardation, renal dysfunction and dermatological abnormalities . TSC2 encodes tuberin, a putative GTPase activating protein for rap1 and rab5 . The TSC1 gene was recently identified and codes for hamartin, a novel protein with no significant homology to tuberin or any other known vertebrate protein . Here, we show that hamartin and tuberin associate physically in vivo and that the interaction is mediated by predicted coiled-coil domains . Our data suggest that hamartin and tuberin function in the same complex rather than in separate pathways.

J Clin Invest, 1998 May 1, 101(9), 1870 - 5
Fatty acid-induced beta cell hypersensitivity to glucose . Increased phosphofructokinase activity and lowered glucose-6-phosphate content; Liu YQ et al.; Diabetic states are characterized by a raised serum/islet level of long chain fatty acids and a lowered ED50 for glucose-induced insulin secretion . Prolonged culture (> 6 h) of islets with long chain fatty acids replicates the basal insulin hypersecretion . We examined this effect in rat islets cultured for 24 h with 0.25 mM oleate . Insulin secretion at 2.8 mM glucose was doubled in combination with a 60% lowered islet content of glucose-6-phosphate (G6P) . Investigation of the lowered G6P showed: (a) increased glucose usage from 0.5 to 100 mM glucose with identical values measured by {2-3H}glucose and {5-3H}glucose, (c) indicating little glucose- 6-phosphatase activity, (b) unchanged low pentose phosphate shunt activity, (c) 50% increased phosphofructokinase (PFK) Vmax, (d) a normal ATP/ADP ratio, and (e) unchanged fructose 2,6 bisphosphate content . Triacsin C, an inhibitor of fatty acyl-CoA synthetase, prevented the increase in PFK activity and the lowered G6P content . These results suggest that long chain acyl-CoA mediates the rise in PFK activity, which in turn lowers the G6P level . We speculate that the inhibition of hexokinase by G6P is thus attenuated, thereby causing the basal insulin hypersecretion.

Genome Res, 1998 Apr, 8(4), 354 - 61
Multiple members of a third subfamily of P-type ATPases identified by genomic sequences and ESTs; Halleck MS et al.; The Saccharomyces cerevisiae genome contains five P-type ATPases divergent from both of the well-known subfamilies of these membrane ion transporters . This newly recognized third subfamily can be further divided into four classes of genes with nearly equal relatedness to each other . Genes of this new subfamily are also present and expressed in multicellular organisms such as Caenorhabditis elegans and mammals; some, but not all, can be assigned to the classes identified in yeast . Different classes of genes and different genes within a class are expressed differentially in tissues of the mouse . The recently cloned gene for the mammalian aminophospholipid translocase belongs to this new subfamily, suggesting that other subfamily members may transport other lipids or lipid-like molecules from one leaflet of the membrane bilayer to the other.

Phys Med Biol, 1998 May, 43(5), 1075 - 90
Automatic cell electrorotation measurements: studies of the biological effects of low-frequency magnetic fields and of heat shock; Zhou XF et al.; A computer-aided automatic imaging technique has been developed for measuring the electrorotation spectra of up to 256 particles at the same time . This offers advantages over the conventional manual method, especially when rapidly acquired statistical data are necessary in investigations of the response of cells or test beads to chemical exposure, for example . We have applied this technique to investigate the biological effects of heat shock and low-frequency EM fields reported by others for yeast cells . Although heat shock effects were observed, no changes of the electrorotational behaviour could be detected after exposing the cells to 50 Hz, 8 and 80 microT fields . Although this does not rule out the possibility that the cells were influenced by the magnetic fields, it does limit the number of possible physicochemical changes that might have occurred to their cell walls and membranes.

Biochemistry, 1998 Jun 2, 37(22), 8233 - 43
Structural and functional analysis of the homing endonuclease PI-sceI by limited proteolytic cleavage and molecular cloning of partial digestion products; Pingoud V et al.; PI-SceI is a member of an unusual class of rare cutting homing endonucleases produced by an autocatalytic protein splicing from a precursor . To analyze the structural and functional domain organization of the endonuclease PI-SceI and to examine whether the DNA binding activity can be structurally separated from the catalytic activity, we performed limited proteolytic digestion experiments with various proteases . Two protease-resistant fragments spanning the N- and C-terminal halves of the nuclease were identified using different proteases which cleave the protein in the same region . Each fragment contains one of the two conserved LAGLIDADG motifs . The products of the limited proteolytic digests were shown to remain associated and to exhibit specific DNA binding but to be inactive in DNA cleavage . Different from what is observed with native PI-SceI, only one complex is formed as shown in an electrophoretic mobility shift assay . Expression clones for the N- and C-terminal protein fragments obtained by tryptic digestion were constructed, and the proteins PI-SceI-N and PI-SceI-C were purified . Only PI-SceI-N exhibits DNA binding activity . Bending experiments with PI-SceI-N, a mixture of PI-SceI-N and PI-SceI-C, as well as the products of the limited tryptic digest show that a DNA substrate with the full length recognition sequence is bent by 45; . This degree of bending is also observed with a DNA containing only the right side of the recognition sequence, corresponding to one of the DNA cleavage products of PI-SceI . Our results demonstrate that the N-terminal half of PI-SceI which lacks one of the two LAGLDADG motifs is able to bind to DNA specifically and to induce one of the distortions observed to occur in the process of DNA binding by PI-SceI . These results are discussed in light of the recently solved crystal structure of PI-SceI and used to refine a model for the mechanism of DNA binding and cleavage by PI-SceI.

Biochemistry, 1998 Jun 2, 37(22), 8080 - 7
Spectroscopic characterization of recombinant pea cytosolic ascorbate peroxidase: similarities and differences with cytochrome c peroxidase; Nissum M et al.; Recombinant pea cytosolic ascorbate peroxidase (APX) has been characterized by resonance Raman (RR) and electronic absorption spectroscopies . The ferric and ferrous forms together with the complexes with fluoride and imidazole have been studied and compared with the corresponding spectra of cytochrome c peroxidase (CCP) . Ferric APX at neutral pH is a mixture of 6- and 5-coordinate high-spin and 6-c low-spin hemes, the latter two species being dominant . The results suggest that the low-spin form derives from a water/hydroxo ligand bound to the heme iron and not from a strong internal ligand as observed in CCP at alkaline pH . Two Fe-Im stretching modes are identified, as in CCP, but the RR frequencies confirm a weaker His163-Asp208 hydrogen bond than in CCP, as suggested on the basis of the X-ray structure {Patterson, W . R., and Poulos, T . L . (1995) Biochemistry 34, 4331-4341} . The data show that CCP and APX have markedly different orientations of the vinyl substituents on the heme chromophore resulting from different steric constraints exerted by the protein matrix.

J Cell Biol, 1998 Jun 1, 141(5), 1107 - 19
Sec35p, a novel peripheral membrane protein, is required for ER to Golgi vesicle docking; VanRheenen SM et al.; SEC35 was identified in a novel screen for temperature-sensitive mutants in the secretory pathway of the yeast Saccharomyces cerevisiae ( . Genetics . 142:393-406) . At the restrictive temperature, the sec35-1 strain exhibits a transport block between the ER and the Golgi apparatus and accumulates numerous vesicles . SEC35 encodes a novel cytosolic protein of 32 kD, peripherally associated with membranes . The temperature-sensitive phenotype of sec35-1 is efficiently suppressed by YPT1, which encodes the rab-like GTPase required early in the secretory pathway, or by SLY1-20, which encodes a dominant form of the ER to Golgi target -SNARE-associated protein Sly1p . Weaker suppression is evident upon overexpression of genes encoding the vesicle-SNAREs SEC22, BET1, or YKT6 . The cold-sensitive lethality that results from deleting SEC35 is suppressed by YPT1 or SLY1-20 . These genetic relationships suggest that Sec35p acts upstream of, or in conjunction with, Ypt1p and Sly1p as was previously found for Uso1p . Using a cell-free assay that measures distinct steps in vesicle transport from the ER to the Golgi, we find Sec35p is required for a vesicle docking stage catalyzed by Uso1p . These genetic and biochemical results suggest Sec35p acts with Uso1p to dock ER-derived vesicles to the Golgi complex.

J Hum Genet, 1998, 43(2), 115 - 22
Cloning and chromosomal mapping of a novel ABC transporter gene (hABC7), a candidate for X-linked sideroblastic anemia with spinocerebellar ataxia; Shimada Y et al.; We isolated a novel human ATP-binding cassette (ABC) transporter cDNA, determined its nucleotide sequence, and designated it human ABC7 (hABC7) . The nucleotide sequence was highly homologous to the ATM1 gene in yeast, which encodes an ABC transporter (yAtm1p) located in the mitochondrial inner membrane . The deduced human product, a putative half-type transporter, consists of 752 amino acids that are 48.9% identical to those of yAtm1p . A computer-assisted protein structural and localization analysis revealed that the mitochondrial targeting signal of yAtm1p is conserved in the N-terminal region of the primary sequence of the hABC7 protein, and therefore this product is also likely to be located in the mitochondrial inner membrane . The evidence strongly suggests that the hABC7 gene is a counterpart of ATM1 and that its product is probably involved in heme transport . We mapped the hABC7 gene to chromosome Xq13.1-q13.3 by fluorescence in-situ hybridization . As band Xq13 has been implicated in X-linked sideroblastic anemia with spinocerebellar ataxia, hABC7 becomes a candidate gene for this heritable disorder.

Genetics, 1998 Jun, 149(2), 833 - 41
A high copy suppressor screen reveals genetic interactions between BET3 and a new gene . Evidence for a novel complex in ER-to-Golgi transport; Jiang Y et al.; The BET3 gene in the yeast Saccharomyces cerevisiae encodes a 22-kD hydrophilic protein that is required for vesicular transport between the ER and Golgi complex . To gain insight into the role of Bet3p, we screened for genes that suppress the growth defect of the temperature-sensitive bet3 mutant at 34 degrees . This high copy suppressor screen resulted in the isolation of a new gene, called BET5 . BET5 encodes an essential 18-kD hydrophilic protein that in high copy allows growth of the bet3-1 mutant, but not other ER accumulating mutants . This strong and specific suppression is consistent with the fact that Bet3p and Bet5p are members of the same complex . Using PCR mutagenesis, we generated a temperature-sensitive mutation in BET5 (bet5-1) that blocks the transport of carboxypeptidase Y to the vacuole and prevents secretion of the yeast pheromone alpha-factor at 37 degrees . The precursor forms of these proteins that accumulate in this mutant are indicative of a block in membrane traffic between the ER and Golgi apparatus . High copy suppressors of the bet5-1 mutant include several genes whose products are required for ER-to-Golgi transport (BET1, SEC22, USO1 and DSS4) and the maintenance of the Golgi (ANP1) . These findings support the hypothesis that Bet5p acts in conjunction with Bet3p to mediate a late stage in ER-to-Golgi transport . The identification of mammalian homologues of Bet3p and Bet5p implies that the Bet3p/Bet5p complex is highly conserved in evolution.

Genetics, 1998 Jun, 149(2), 677 - 92
Molecular organization of the 20S proteasome gene family from Arabidopsis thaliana; Fu H et al.; The 20S proteasome is the proteolytic complex in eukaryotes responsible for degrading short-lived and abnormal intracellular proteins, especially those targeted by ubiquitin conjugation . The 700-kD complex exists as a hollow cylinder comprising four stacked rings with the catalytic sites located in the lumen . The two outer rings and the two inner rings are composed of seven different alpha and beta polypeptides, respectively, giving an alpha7/beta7/beta7/alpha7 symmetric organization . Here we describe the molecular organization of the 20S proteasome from the plant Arabidopsis thaliana . From an analysis of a collection of cDNA and genomic clones, we identified a superfamily of 23 genes encoding all 14 of the Arabidopsis proteasome subunits, designated PAA-PAG and PBA-PBG for Proteasome Alpha and Beta subunits A-G, respectively . Four of the subunits likely are encoded by single genes, and the remaining subunits are encoded by families of at least 2 genes . Expression of the alpha and beta subunit genes appears to be coordinately regulated . Three of the nine Arabidopsis proteasome subunit genes tested, PAC1 (alpha3), PAE1 (alpha5) and PBC2 (beta3), could functionally replace their yeast orthologs, providing the first evidence for cross-species complementation of 20S subunit genes . Taken together, these results demonstrate that the 20S proteasome is structurally and functionally conserved among eukaryotes and suggest that the subunit arrangement of the Arabidopsis 20S proteasome is similar if not identical to that recently determined for the yeast complex.

Genetics, 1998 Jun, 149(2), 633 - 9
Activation of latent transgenes in Arabidopsis using a hybrid transcription factor; Guyer D et al.; A hybrid transcription factor comprising a fusion of the DNA-binding domain of Saccharomyces cerevisiae GAL4 and the transcription activation domain of maize C1 was expressed in stably transformed Arabidopsis . Additional transgenic lines were created containing test genes controlled by a synthetic promoter consisting of concatemeric copies of the cis-acting site recognized by GAL4 (UASG) fused to a minimal promoter . The GAL4/C1 effector line was crossed to two lines containing a synthetic promoter/GUS fusion . Both histochemical staining and GUS activity assays indicate strong activation of GUS expression was achieved only after crossing . The GAL4/C1 effector line was also crossed to 15 lines containing a synthetic promoter/antisense adenylosuccinate synthetase gene . Severely retarded growth, and in some cases lethality, was observed in 40% of the F1 lines . This system of activation by crossing is generally useful for activating expression of test transgenes.

J Biol Chem, 1998 Jun 5, 273(23), 14322 - 30
DNA ligase I selectively affects DNA synthesis by DNA polymerases delta and epsilon suggesting differential functions in DNA replication and repair; Mossi R et al.; The joining of single-stranded breaks in double-stranded DNA is an essential step in many important processes such as DNA replication, DNA repair, and genetic recombination . Several data implicate a role for DNA ligase I in DNA replication, probably coordinated by the action of other enzymes and proteins . Since both DNA polymerases delta and epsilon show multiple functions in different DNA transactions, we investigated the effect of DNA ligase I on various DNA synthesis events catalyzed by these two essential DNA polymerases . DNA ligase I inhibited replication factor C-independent DNA synthesis by polymerase delta . Our results suggest that the inhibition may be due to DNA ligase I interaction with proliferating cell nuclear antigen (PCNA) and not to a direct interaction with the DNA polymerase delta itself . Strand displacement activity by DNA polymerase delta was also affected by DNA ligase I . The DNA polymerase delta holoenzyme (composed of DNA polymerase delta, PCNA, and replication factor C) was inhibited in the same way as the DNA polymerase delta core, strengthening the hypothesis of a PCNA interaction . Contrary to DNA polymerase delta, DNA synthesis by DNA polymerase epsilon was stimulated by DNA ligase I in a PCNA-dependent manner . We conclude that DNA ligase I displays different influences on the two multipotent DNA polymerases delta and epsilon through PCNA . This might be of importance in the selective involvement in DNA transactions such as DNA replication and various mechanisms of DNA repair.

Nature, 1998 May 28, 393(6683), 389 - 92
Transcriptional activation independent of TFIIH kinase and the RNA polymerase II mediator in vivo; Lee D et al.; The carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II becomes multiply phosphorylated by protein kinases during early steps in the gene transcription cycle both in vivo and in vitro . In yeast, the major CTD kinase is a subunit of the general transcription factor TFIIH, and is encoded by an essential gene, KIN28 . Although the CTD and its phosphorylation are important for transcription, in vitro studies have challenged whether CTD phosphorylation is an absolutely required step . The general importance of CTD phosphorylation by Kin28 for transcription in yeast has been suggested because, for all genes tested, transcription is inhibited at the non-permissive temperature in temperature-sensitive kin28 mutants . However, using such a mutant and a copper-inducible targeted destruction method, we show here that transcription of certain genes can be highly induced even when cells lack Kin28 . We also show that transcription of these Kin28-independent genes is independent of Srb4 and Srb6, critical components of the CTD-associated transcriptional mediator complex . These results indicate that there are at least two distinct pathways for transcriptional activation: one is dependent on Kin28 and the mediator complex, and the other is not.

Nucleic Acids Res, 1998 May 15, 26(10), 2298 - 305
Effects of phosphate neutralization on the shape of the AP-1 transcription factor binding site in duplex DNA; Tomky LA et al.; Previous electrophoretic experiments suggest that the AP-1 site in duplex DNA bends in response to the pattern of amino acid charges distal to the basic region in bound bZIP proteins . The extent and direction of apparent DNA bending are consistent with the prediction that DNA will collapse locally upon asymmetric phosphate charge neutralization . To prove that asymmetric phosphate neutralization could produce the observed degree of DNA bending, the present experiments partially substitute anionic phosphate diesters in the AP-1 site with various numbers of neutral methylphosphonate linkages . DNA bending is induced toward the neutralized face of DNA . The degree of DNA bending induced by methylphosphonate substitution (approximately 3.5 degrees per neutralized phosphate) is comparable to that induced by GCN4 variants carrying increasing numbers of additional basic amino acids . It is plausible, therefore, that asymmetric phosphate neutralization is the cause of DNA bending in such complexes.

Nat Genet, 1998 Jun, 19(2), 187 - 91
Methylated DNA and MeCP2 recruit histone deacetylase to repress transcription; Jones PL et al.; CpG methylation in vertebrates correlates with alterations in chromatin structure and gene silencing . Differences in DNA-methylation status are associated with imprinting phenomena and carcinogenesis . In Xenopus laevis oocytes, DNA methylation dominantly silences transcription through the assembly of a repressive nucleosomal array . Methylated DNA assembled into chromatin binds the transcriptional repressor MeCP2 which cofractionates with Sin3 and histone deacetylase . Silencing conferred by MeCP2 and methylated DNA can be relieved by inhibition of histone deacetylase, facilitating the remodelling of chromatin and transcriptional activation . These results establish a direct causal relationship between DNA methylation-dependent transcriptional silencing and the modification of chromatin.

Plant Mol Biol, 1998 May, 37(1), 155 - 69
The maize retinoblastoma protein homologue ZmRb-1 is regulated during leaf development and displays conserved interactions with G1/S regulators and plant cyclin D (CycD) proteins; Huntley R et al.; Recent discoveries of plant retinoblastoma (Rb) protein homologues and D-type cyclins suggest that control of the onset of cell division in plants may have stronger parallels with mammalian G1/S controls than with yeasts . In mammals, the Rb protein interacts specifically with D-type cyclins and regulates cell proliferation by binding and inhibiting E2F transcription factors . However, the developmental role of Rb in plants and its potential interaction with cell cycle regulators is unknown . We show that the maize Rb homologue ZmRb-1 is temporally and spatially regulated during maize leaf development . ZmRb-1 is highly expressed in differentiating cells, but almost undetectable in proliferating cells . In vitro, both ZmRb-1 and human Rb bind all classes of plant D-type cyclins with the involvement of a conserved N-terminal Leu-x-Cys-x-Glu (LxCxE) Rb-interaction motif . This binding is strongly reduced by mutation of the conserved Cys-470 of ZmRb-1 . ZmRb-1 binds human and Drosophila E2F, and inhibits transcriptional activation of human E2F . We also show that ZmRb-1 is a good in vitro substrate for all human G1/S protein kinases . The functional conservation of proteins that control the G1/S transition in mammals and plants points to the existence of plant E2F homologues . We conclude that evolution of Rb and cyclin D proteins occurred after separation of the fungi from the higher eukaryotic lineage, but preceded the divergence of plant and animal kingdoms.

Plant Mol Biol, 1998 May, 37(1), 121 - 9
Isolation and characterization of a functional A-type cyclin from maize; Hsieh WL et al.; Cyclins are involved in the regulation of cell cycle progression in eukaryotes . We have isolated a cyclin cDNA clone, cycZm2w, from maize root tip cells, which fits best into group A2 of current plant cyclin gene classification schemes . The cDNA encodes a protein with a domain homologous to the cyclin box of mitotic cyclins . Complementation studies revealed that cycZm2w was able to rescue a budding yeast cyclin-deficient mutant (BF305-15d#21) . As expected, cycZm2w is expressed in organs of the maize plant that possess meristematic activity, but is especially prominent in the proliferating regions of the root apex.

Oncogene, 1998 Apr 30, 16(17), 2283 - 5
The BRCA2 is a histone acetyltransferase; Siddique H et al.; Patients carrying mutations in BRCA1 or BRCA2 tumor suppressor genes have shown to have high risk in developing breast and ovarian cancers . Two potential functions of BRCA2 were proposed which includes role in the regulation of transcription and also in DNA repair . Forty-five-amino acid region encoded by exon 3 of BRCA2 was shown to have transcriptional activation function . Recent studies of the several enzymes involved in acetylation and deacetylation of histone residues have revealed a possible relationship between gene transcriptional activation and histone acetylation . Since BRCA2 appear to function as a transcriptional factor, we have tested for Histone acetyl transferase (HAT) activity of BRCA2 . Here, we present evidence that BRCA2 has intrinsic HAT activity, which maps to the amino-terminal region of BRCA2 . Our results demonstrate that BRCA2 proteins acetylate primarily H3 and H4 of free histones . These observations suggest that HAT activity of BRCA2 may play an important role in the regulation of transcription and tumor suppressor function.

Biochem Biophys Res Commun, 1998 May 29, 246(3), 760 - 4
A protein phosphatase is involved in the inhibition of histone deacetylation by sodium butyrate; Cuisset L et al.; Treatment of cells with sodium butyrate is known to increase histone acetylation by inhibiting deacetylases . Here we have observed, in cultured hepatoma cells, that the potent serine-threonine phosphatase inhibitors, okadaic acid or calyculin A, inhibited phosphatase activity and concomitantly decreased the histone acetylation classically maintained by sodium butyrate . These results suggest that a protein phosphatase may mediate the sodium butyrate effect on deacetylases . Since we have previously found that such a protein would also mediate the sodium butyrate effect on gene expression, we propose that a phosphatase activity constitutes an early and essential step in the sodium butyrate-triggered signalling pathway.

EMBO J, 1998 May 15, 17(10), 2938 - 46
Progression through the spliceosome cycle requires Prp38p function for U4/U6 snRNA dissociation; Xie J et al.; The elaborate and energy-intensive spliceosome assembly pathway belies the seemingly simple chemistry of pre-mRNA splicing . Prp38p was previously identified as a protein required in vivo and in vitro for the first pre-mRNA cleavage reaction catalyzed by the spliceosome . Here we show that Prp38p is a unique component of the U4/U6.U5 tri-small nuclear ribonucleoprotein (snRNP) particle and is necessary for an essential step late in spliceosome maturation . Without Prp38p activity spliceosomes form, but arrest in a catalytically impaired state . Functional spliceosomes shed U4 snRNA before 5' splice-site cleavage . In contrast, Prp38p-defective spliceosomes retain U4 snRNA bound to its U6 snRNA base-pairing partner . Prp38p is the first tri-snRNP-specific protein shown to be dispensable for assembly, but required for conformational changes which lead to catalytic activation of the spliceosome.

EMBO J, 1998 May 15, 17(10), 2926 - 37
The DEAH-box protein PRP22 is an ATPase that mediates ATP-dependent mRNA release from the spliceosome and unwinds RNA duplexes; Wagner JD et al.; Of the proteins required for pre-mRNA splicing, at least four, the DEAH-box proteins, are closely related due to the presence of a central 'RNA helicase-like' region, and extended homology through a large portion of the protein . A major unresolved question is the function of these proteins . Indirect evidence suggests that several of these proteins are catalysts for important structural rearrangements in the spliceosome . However, the mechanism for the proposed alterations is presently unknown . We present evidence that PRP22, a DEAH-box protein required for mRNA release from the spliceosome, unwinds RNA duplexes in a concentration- and ATP-dependent manner . This demonstrates that PRP22 can modify RNA structure directly . We also show that the PRP22-dependent release of mRNA from the spliceosome is an ATP-dependent process and that recombinant PRP22 is an ATPase . Non-hydrolyzable ATP analogs did not substitute for ATP in the RNA-unwinding reaction, suggesting that ATP hydrolysis is required for this reaction . Specific mutation of a putative ATP phosphate-binding motif in the recombinant protein eliminated the ATPase and RNA-unwinding capacity . Significantly, these data suggest that the DEAH-box proteins act directly on RNA substrates within the spliceosome.

EMBO J, 1998 May 15, 17(10), 2886 - 93
The acetyltransferase activity of CBP stimulates transcription; Martinez-Balbas MA et al.; The CBP co-activator protein possesses an intrinsic acetyltransferase (AT) activity capable of acetylating nucleosomal histones, as well as other proteins such as the transcription factors TFIIE and TFIIF . In addition, CBP associates with two other TSs, P/CAF and SRC1 . We set out to establish whether the intrinsic AT activity of CBP contributes to transcriptional activation . We show that a region of CBP, encompassing the previously defined histone AT (HAT) domain, can stimulate transcription when tethered to a promoter . The stimulatory effect of this activation domain shows some promoter preference and is dependent on AT activity . Analysis of 14 point mutations reveals a direct correlation between CBP's ability to acetylate histones in vitro and to activate transcription in vivo . We also find that the HAT domains of CBP and P/CAF share sequence similarity . Four conserved motifs are identified, three of which are analogous to motifs A, B and D, found in other N-acetyltransferases . The fourth motif, termed E, is unique to CBP and P/CAF . Mutagenesis shows that all four motifs in CBP contribute to its HAT activity in vitro and its ability to activate transcription in vivo . These results demonstrate that the AT activity of CBP is directly involved in stimulating gene transcription . The identification of specific HAT domain motifs, conserved between CBP and P/CAF, should facilitate the identification of other members of this AT family.

EMBO J, 1998 May 15, 17(10), 2809 - 16
Evidence that the RdeA protein is a component of a multistep phosphorelay modulating rate of development in Dictyostelium; Chang WT et al.; We have isolated an insertional mutant of Dictyostelium discoideum that aggregated rapidly and formed spores and stalk cells within 14 h of development instead of the normal 24 h . We have shown by parasexual genetics that the insertion is in the rdeA locus and have cloned the gene . It encodes a predicted 28 kDa protein (RdeA) that is enriched in charged residues and is very hydrophilic . Constructs with the DNA for the c-Myc epitope or for the green fluorescent protein indicate that RdeA is not compartmentalized . RdeA displays homology around a histidine residue at amino acid 65 with members of the H2 module family of phosphotransferases that participate in multistep phosphoryl relays . Replacement of this histidine rendered the protein inactive . The mutant is complemented by transformation with the Ypd1 gene of Saccharomyces cerevisiae, itself an H2 module protein . We propose that RdeA is part of a multistep phosphorelay system that modulates the rate of development.

Nucleic Acids Res, 1998 May 15, 26(10), 2442 - 8
Heterologous complementation reveals that mutant alleles of QSR1 render 60S ribosomal subunits unstable and translationally inactive; Dick FA et al.; QSR1 is a highly conserved gene which encodes a 60S ribosomal subunit protein that is required for joining of large and small ribosomal subunits . In this report we demonstrate heterologous complementation of a yeast QSR1 deletion strain with both the human and corn homologs and show that the human and corn proteins are assembled into hybrid yeast/human and yeast/corn ribosomes . While the homologous genes complement lethality of the QSR1 deletion, they also result in a diminished growth rate . Analyses of the translation rates of ribosomes containing the human and corn proteins reveal a partial loss of function . Velocity gradient analyses of the hybrid ribosomes after exposure to high concentrations of salt indicate that the decreased activity is due to lability of the hybrid 60S subunits.

Nucleic Acids Res, 1998 May 15, 26(10), 2344 - 52
A differential response of wild type and mutant promoters to TFIIIB70 overexpression in vivo and in vitro; Sethy-Coraci I et al.; TFIIIB, the initiation factor for transcription by RNA polymerase III (pol III) is, in yeast, composed of three subunits: TBP, TFIIIB70/Brf1 and TFIIIB90 . To determine the extent to which each of these subunits is limiting for pol III transcription, the effect of overexpressing each subunit was assessed on the expression of wild-type and promoter mutant pol III genes both in vivo and in vitro . In vivo , we find that the synthesis of wild-type pol III genes is not limited to a significant extent by the level of any TFIIIB subunit . There is, however, a two-fold increase in the synthesis of the promoter mutant gene, sup9-e A19-supS1 , in strains overexpressing TFIIIB70 . The findings suggest that overexpression of TFIIIB70has a differential effect on the expression of pol III genes with strong versus weak promoters . In vitro transcription assays support this conclusion and reveal an inverse correlation between the transcriptional response to TFIIIB70overexpression and promoter strength . The individual TFIIIB subunits are nuclear by immunofluorescence and are calculated to have nuclear concentrations in the low micromolar range . In comparison, the factors are diluted 100-fold or more in whole cell extracts . This dilution accounts for the generally limiting nature of TFIIIB70in pol III gene transcription in vitro.

Stem Cells, 1998, 16(3), 208 - 17
Mobilization of peripheral blood progenitor cells by Betafectin PGG-Glucan alone and in combination with granulocyte colony-stimulating factor; Patchen ML et al.; Betafectin PGG-Glucan, a novel beta-(1,6) branched beta-(1,3) glucan purified from the cell walls of Saccharomyces cerevisiae, has been shown to synergize with myeloid growth factors in vitro and to enhance hematopoietic recovery in myelosuppressed mice and primates . Here we report that PGG-Glucan is also capable of mobilizing peripheral blood progenitor cells (PBPC) . PGG-Glucan (0.5 mg/kg to 16 mg/kg) was administered intravenously to C3H/HeN male mice and blood collected at times ranging from 30 min to seven days after injection . Based on granulocyte-macrophage colony-forming cell (GM-CFC) levels, peak mobilization occurred 30 min after a 2 mg/kg PGG-Glucan dose . At this time GM-CFC numbers in PGG-Glucan-treated mice were approximately fourfold greater than in saline-treated control mice . A second, smaller wave of GM-CFC mobilization (approximately twofold increase) also occurred on days 4 and 5 after PGG-Glucan treatment . Mobilization was not associated with the induction of alpha-chemokines, which have recently been reported to induce rapid progenitor cell mobilization . Competitive repopulation experiments performed in irradiated female C3H/HeN mice revealed that, at three months after transplantation, more male DNA was present in bone marrow, splenic, and thymic tissues from animals transplanted with cells obtained from mice 30 min after a 2 mg/kg PGG-Glucan dose than in tissues from animals transplanted with cells obtained from saline-treated mice . Additional experiments evaluated the mobilization effects of PGG-Glucan (2 mg/kg) administered to mice which had been pretreated for three consecutive days with G-CSF (125 microg/kg/day) . When blood was collected 30 min after PGG-Glucan treatment, the number of GM-CFC mobilized in combination-treated mice was additive between the number mobilized in mice treated with G-CSF alone and the number mobilized in mice treated with PGG-Glucan alone . These studies demonstrate that: A) PGG-Glucan can rapidly mobilize PBPC; B) the kinetic pattern of PGG-Glucan-induced mobilization is different from that of the CSFs; C) the reconstitutional potential of PGG-Glucan mobilized cells is greater than that of steady-state PBPC, and D) PGG-Glucan can enhance G-CSF-mediated PBPC mobilization.

Cell Signal, 1998 Apr, 10(4), 277 - 82
A somatostatin analogue induces translocation of Ku 86 autoantigen from the cytosol to the nucleus in colon tumour cells; Tovari J et al.; Flow cytometric and electron microscopic immunocytochemical studies have been performed in HT-29 human colon tumour cells in vitro, to determine and localise p86 Ku protein, which is a regulatory subunit of DNA-dependent kinase and a specific binding site for somatostatin . We have demonstrated that HT-29 cells contain p86 Ku and that the distribution between the cytoplasm and the nucleus is even . After administration of the somatostatin analogues Sandostatin and TT-232 to HT-29 cells, the p86 Ku content of the cytoplasmic compartment decreased in the first 4 h . An increase in the content of this protein in the nuclear compartment was observed at hour 1 followed by a decrease at hour 4 after treatment . Quantitative differences between the two analogues have been observed in this respect . The practical significance of these findings is discussed.

Biochemistry, 1998 May 12, 37(19), 6727 - 37
Nucleosome assembly on telomeric sequences; Rossetti L et al.; The organization of telomeric chromatin differs from that of bulk chromatin in some peculiar features, such as the unusually short nucleosomal spacing found in vertebrates . Telomeric DNAs are straight, since they consist mostly of 6-8-bp repeated sequences, therefore out of phase with the B DNA period . This feature should be of relevance in nucleosome formation, suggesting the usefulness of studying simple model systems of nucleosome assembly . We reconstituted nucleosomes in vitro, by using purified histone octamers and/or by octamer transfer from chicken erythrocyte nucleosomes, onto telomeric sequences from human, Arabidopsis thaliana, and Saccharomyces cerevisiae . All of these telomeres contain GGG and GGT triplets but are characterized by different repeat lengths (6, 7, and 8 bp) . The free energies involved in the association process are the highest among the biological sequences so far assayed, suggesting a main role of DNA flexibility in the assembly of telomeric chromatin . Digestion studies with DNase I, hydroxyl radicals, exonuclease III, and lambda exonuclease indicate that telomeric nucleosomes are characterized by multiple translational positioning without rotational phasing, whereas the telomeric DNA folding around the histone octamer shows the canonical periodicity of about 10.2 bp . The experimental results and a theoretical simulation of DNase I digestion indicate a multiple nucleosome positioning with the periodicity of telomeric DNA . This suggests a main role of local chemical recognition between telomeric sequences and the histone octamer in nucleosome assembly.

J Bacteriol, 1998 May, 180(10), 2590 - 8
Stimulation of transcription by mutations affecting conserved regions of RNA polymerase II; Archambault J et al.; Mutations that increase the low-level transcription of the Saccharomyces cerevisiae HIS4 gene, which results from deletion of the genes encoding transcription factors BAS1, BAS2, and GCN4, were isolated previously in SIT1 (also known as RPO21, RPB1, and SUA8), the gene encoding the largest subunit of RNA polymerase II (RNAPII) . Here we show that sit1 substitutions cluster in two conserved regions of the enzyme which form part of the active site . Six sit1 mutations, affect region F, a region that is involved in transcriptional elongation and in resistance to alpha-aminatin . Four sit1 substitutions lie in another region involved in transcriptional elongation, region D, which binds Mg2+ ions essential for RNA catalysis . One region D substitution is lethal unless suppressed by a substitution in region G and interacts genetically with PPR2, the gene encoding transcription elongation factor IIS . Some sit1 substitutions affect the selection of transcriptional start sites at the CYC1 promoter in a manner reminiscent of that of sua8 (sua stands for suppression of upstream ATG) mutations . Together with previous findings which indicate that regions D and G are in close proximity to the 3' end of the nascent transcript and that region F is involved in the translocation process, our results suggest that transcriptional activation by the sit1 mutations results from alteration of the RNAPII active center.

Proc Natl Acad Sci U S A, 1998 Apr 28, 95(9), 5187 - 92
A genetic system based on split-ubiquitin for the analysis of interactions between membrane proteins in vivo; Stagljar I et al.; A detection system for interactions between membrane proteins in vivo is described . The system is based on split-ubiquitin {Johnsson, N . & Varshavsky, A . (1994) Proc . Natl . Acad . Sci . USA 91, 10340-10344} . Interaction between two membrane proteins is detected by proteolytic cleavage of a protein fusion . The cleavage releases a transcription factor, which activates reporter genes in the nucleus . As a result, interaction between membrane proteins can be analyzed by the means of a colorimetric assay . We use membrane proteins of the endoplasmic reticulum as a model system . Wbp1p and Ost1p are both subunits of the oligosaccharyl transferase membrane protein complex . The Alg5 protein also localizes to the membrane of the endoplasmic reticulum, but does not interact with the oligosaccharyltransferase . Specific interactions are detected between Wbp1p and Ost1p, but not between Wbp1p and Alg5p . The new system might be useful as a genetic and biochemical tool for the analysis of interactions between membrane proteins in vivo.

Proc Natl Acad Sci U S A, 1998 Apr 28, 95(9), 5172 - 7
Homology-directed repair is a major double-strand break repair pathway in mammalian cells; Liang F et al.; Mammalian cells have been presumed to repair potentially lethal chromosomal double-strand breaks (DSBs) in large part by processes that do not require homology to the break site . This contrasts with Saccharomyces cerevisiae where the major DSB repair pathway is homologous recombination . Recently, it has been determined that DSBs in genomic DNA in mammalian cells can stimulate homologous recombination as much as 3 or 4 orders of magnitude, suggesting that homology-directed repair may play an important role in the repair of chromosomal breaks . To determine whether mammalian cells use recombinational repair at a significant level, we have analyzed the spectrum of repair events at a defined chromosomal break by using direct physical analysis of repair products . When an endonuclease-generated DSB is introduced into one of two direct repeats, homologous repair is found to account for 30-50% of observed repair events . Both noncrossover and deletional homologous repair products are detected, at approximately a 1:3 ratio . These results demonstrate the importance of homologous recombination in the repair of DSBs in mammalian cells . In the remaining observed repair events, DSBs are repaired by nonhomologous processes . The nonhomologous repair events generally result in small deletions or insertions at the break site, although a small fraction of events result in larger chromosomal rearrangements . Interestingly, in two insertions, GT repeats were integrated at one of the broken chromosome ends, suggesting that DSB repair can contribute to the spread of microsatellite sequences in mammalian genomes.

Proc Natl Acad Sci U S A, 1998 Apr 28, 95(9), 4947 - 52
Perturbation of nucleosome core structure by the SWI/SNF complex persists after its detachment, enhancing subsequent transcription factor binding; Cote J et al.; To investigate the mechanism of SWI/SNF action, we have analyzed the pathway by which SWI/SNF stimulates formation of transcription factor-bound nucleosome core complexes . We report here that the SWI/SNF complex binds directly to nucleosome cores and uses the energy of ATP hydrolysis to disrupt histone/DNA interactions, altering the preferred path of DNA bending around the histone octamer . This disruption occurs without dissociating the DNA from the surface of the histone octamer . ATP-dependent disruption of nucleosomal DNA by SWI/SNF generates an altered nucleosome core conformation that can persist for an extended period after detachment of the SWI/SNF complex . This disrupted conformation retains an enhanced affinity for the transcription factor GAL4-AH . Thus, ATP-dependent nucleosome core disruption and enhanced binding of the transcription factor can be temporally separated . These results indicate that SWI/SNF can act transiently in the remodeling of chromatin structure, even before interactions of transcription factors.

J Biol Chem, 1998 May 1, 273(18), 10901 - 7
A negative vitamin D response DNA element in the human parathyroid hormone-related peptide gene binds to vitamin D receptor along with Ku antigen to mediate negative gene regulation by vitamin D; Nishishita T et al.; We found that the human parathyroid hormone-related peptide (hPTHrP) gene contained a DNA element (nVDREhPTHrP) homologous to a negative vitamin D response element in the human parathyroid hormone gene . It bound to vitamin D receptor (VDR) but not retinoic acid Xalpha receptor (RXRalpha) in the human T cell line MT2 cells . VDR binding to this element was confirmed by the Southwestern assay combined with immunodepletion using anti-VDR monoclonal antibody, and this binding activity was repressed by 1,25-dihydroxyvitamin D3 . Such a repression was reversed by acid phosphatase treatment, suggesting that 1,25-dihydroxyvitamin D3 phosphorylates VDR to weaken its binding activity to nVDREhPTHrP . In electrophoretic mobility shift assay, we found anti-Ku antigen antibody specifically supershifted the MT2 nuclear proteinnVDREhPTHrP complex . The nVDREhPTHrP-bearing reporter plasmid produced vitamin D-dependent inhibition of the reporter activity in MT2 cells, which was markedly masked by the introduction of the Ku antigen expression vector in the antisense orientation . On the other hand, such a procedure did not perturb the vitamin D response element-mediated gene stimulation by vitamin D . These results indicate that nVDREhPTHrP interacts with Ku antigen in addition to VDR to mediate gene suppression by vitamin D.

J Biol Chem, 1998 May 1, 273(18), 10868 - 73
Gar1p binds to the small nucleolar RNAs snR10 and snR30 in vitro through a nontypical RNA binding element; Bagni C et al.; The nucleolar proteins Gar1p and fibrillarin possess a typical nucleolar glycine/arginine-rich domain and belong to ribonucleoprotein particles . Both proteins are essential for yeast cell growth and are required for pre-rRNA processing . In addition, Gar1p is involved in pre-rRNA pseudouridylation, whereas fibrillarin is required for pre-rRNA methylation . Gar1p and fibrillarin are each associated with a different subset of the small nucleolar RNAs (snoRNAs) . Gar1p is co-immunoprecipitated with the H/ACA family of snoRNAs, whereas fibrillarin is co-immunoprecipitated with the C/D family . However, attempts to demonstrate direct interactions between fibrillarin and snoRNAs have failed, and such interactions between Gar1p and the H/ACA snoRNAs had not yet been reported . Among the H/ACA snoRNAs associated with Gar1p, one can distinguish a large group of snoRNAs that are not essential in yeast and serve as guides for pseudouridine synthesis onto the pre-rRNA molecule . In contrast, the two snoRNAs snR10 and snR30 are required for normal cell growth and for pre-rRNA cleavage . We show here that Gar1p interacts in vitro directly and specifically with these two snoRNAs . Deletion analysis of Gar1p indicates that a major RNA binding element, which is extremely well conserved throughout evolution, lies in the middle of the protein . However, this domain alone binds poorly to the target RNAs and an accessory domain is required to restore efficient binding . The accessory domain can be either one of the glycine/arginine-rich domains or a second element of the core of the protein that is highly conserved between different species.

EMBO J, 1998 Apr 1, 17(7), 1996 - 2007
GPR1 encodes a putative G protein-coupled receptor that associates with the Gpa2p Galpha subunit and functions in a Ras-independent pathway; Xue Y et al.; The yeast RAS1 and RAS2 genes appear to be involved in control of cell growth in response to nutrients . Here we show that this growth control also involves a signal mediated by the heterotrimeric G protein alpha subunit homolog encoded by GPA2 . A GPA2 null allele conferred a severe growth defect on cells containing a null allele of RAS2, although either mutation alone had little effect on growth rate . A constitutive allele of GPA2 could stimulate growth of a strain lacking both RAS genes . Constitutive GPA2 conferred heat shock sensitivity on both wild-type cells and cells lacking RAS function, but had no effect in a strain containing a null allele of SCH9, which encodes a kinase related to protein kinase A . The GPR1 gene was isolated and was found to encode a protein with the characteristics of a G protein-coupled receptor . Double Deltagpr1 Deltaras2 mutants displayed a severe growth defect that was suppressed by expression of the constitutive allele of GPA2, confirming that GPR1 acts upstream of GPA2 . Gpr1p is expressed on the cell surface and requires sequences in the membrane-proximal region of its third cytoplasmic loop for function, as expected for a G protein-coupled receptor . GPR1 RNA was induced when cells were starved for nitrogen and amino acids . These results are consistent with a model in which the GPR1/GPA2 pathway activates the Sch9p kinase to generate a response that acts in parallel with that generated by the Ras/cAMP pathway, resulting in the integration of nutrient signals.

EMBO J, 1998 Apr 1, 17(7), 1847 - 59
Genetic selection of intragenic suppressor mutations that reverse the effect of common p53 cancer mutations; Brachmann RK et al.; Several lines of evidence suggest that the presence of the wild-type tumor suppressor gene p53 in human cancers correlates well with successful anti-cancer therapy . Restoration of wild-type p53 function to cancer cells that have lost it might therefore improve treatment outcomes . Using a systematic yeast genetic approach, we selected second-site suppressor mutations that can overcome the deleterious effects of common p53 cancer mutations in human cells . We identified several suppressor mutations for the V143A, G245S and R249S cancer mutations . The beneficial effects of these suppressor mutations were demonstrated using mammalian reporter gene and apoptosis assays . Further experiments showed that these suppressor mutations could override additional p53 cancer mutations . The mechanisms of such suppressor mutations can be elucidated by structural studies, ultimately leading to a framework for the discovery of small molecules able to stabilize p53 mutants.

Nucleic Acids Res, 1998 Apr 15, 26(8), 1857 - 62
The protein splicing domain of the homing endonuclease PI-sceI is responsible for specific DNA binding; Grindl W et al.; The homing endonuclease PI- Sce I consists of a protein splicing domain (I) and an endonucleolytic domain (II) . To characterize the two domains with respect to their contribution to DNA recognition we cloned, purified and characterized the isolated domains . Both domains have no detectable endonucleolytic activity . Domain I binds specifically to the PI- Sce I recognition sequence, whereas domain II displays only weak non-specific DNA binding . In the specific complex with domain I the DNA is bent to a similar extent as observed with the initial complex formed between PI- Sce I and DNA . Our results indicate that protein splicing domain I is also involved in recognition of the DNA substrate.

J Mol Biol, 1998 Mar 27, 277(2), 285 - 308
Highly conserved charge-pair networks in the mitochondrial carrier family; Nelson DR et al.; Selection for regain-of-function mutations in the yeast ADP/ATP carrier AAC2 has revealed an unexpected series of charge-pairs . Four of the six amino acids involved are found in the mitochondrial energy transfer motifs used to define this family of proteins . As such, the results found with the ADP/ATP carrier may apply to the family as a whole . Mitochondrial carriers are built from three homologous domains, each with the conserved motif PX(D,E)XX(K,R) . Neutralization of the conserved positive charges at K48, R152 or R252 in these motifs results in respiration defective yeast . Neutralization of the negative charges at D149 and D249 also make respiration defective yeast, though E45G or E45Q mutants are able to grow on glycerol . Regain of function occurs when a complementary charge is lost from another site in the molecule . This phenomenon has been observed independently eight times and thus is strong evidence for charge-pairs existing between the affected residues . Five different charge-pairs have been detected in the yeast AAC2 by this method and three more can be predicted based on homology between the domains . The highly conserved charge-pairs occurring within or between the three mitochondrial energy transfer signatures seem to be a critical feature of mitochondrial carrier structure, independent of the substrates transported . Conformational switching between alternative charge-pairs may constitute part of the basis for transport .

Am J Physiol, 1998 Jun, 274(6 Pt 1), C1429 - 52
Proteinase-activated receptors: novel mechanisms of signaling by serine proteases; Dery O et al.; Although serine proteases are usually considered to act principally as degradative enzymes, certain proteases are signaling molecules that specifically regulate cells by cleaving and triggering members of a new family of proteinase-activated receptors (PARs) . There are three members of this family, PAR-1 and PAR-3, which are receptors for thrombin, and PAR-2, a receptor for trypsin and mast cell tryptase . Proteases cleave within the extracellular NH2-terminus of their receptors to expose a new NH2-terminus . Specific residues within this tethered ligand domain interact with extracellular domains of the cleaved receptor, resulting in activation . In common with many G protein-coupled receptors, PARs couple to multiple G proteins and thereby activate many parallel mechanisms of signal transduction . PARs are expressed in multiple tissues by a wide variety of cells, where they are involved in several pathophysiological processes, including growth and development, mitogenesis, and inflammation . Because the cleaved receptor is physically coupled to its agonist, efficient mechanisms exist to terminate signaling and prevent uncontrolled stimulation . These include cleavage of the tethered ligand, receptor phosphorylation and uncoupling from G proteins, and endocytosis and lysosomal degradation of activated receptors.

Ann Rheum Dis, 1998 Feb, 57(2), 122 - 4
Antisense oligonucleotides targeting c-fos mRNA inhibit rheumatoid synovial fibroblast proliferation; Morita Y et al.; OBJECTIVE: To determine whether antisense oligonucleotides targeting c-fos mRNA have the ability to inhibit the growth of interleukin 1 (IL1) stimulated fibroblast-like cells from the synovium in rheumatoid arthritis (RA) . METHODS: Fibroblast-like cells established from RA synovium were stimulated by IL1 with antisense or sense oligonucleotides complementary to c-fos mRNA, and the proliferation of these cells was determined by 3H-thymidine incorporation . Effect of antisense oligonucleotides on expression of activator protein 1 (AP1) activity was evaluated using electrophoretic mobility shift assay . RESULTS: C-fos antisense oligonucleotides inhibited IL1 stimulated synovial fibroblast proliferation . The expression of AP1 activity induced by IL1 was suppressed by treatment with antisense oligonucleotides . CONCLUSION: These results suggest the feasibility of antisense strategies designed to suppress c-fos expression as therapeutic agents for RA.

Am J Physiol, 1998 May, 274(5 Pt 1), C1397 - 410
Differential regulation of single CFTR channels by PP2C, PP2A, and other phosphatases; Luo J et al.; Cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel activity declines rapidly when excised from transfected Chinese hamster ovary (CHO) or human airway cells because of membrane-associated phosphatase activity . In the present study, we found that CFTR channels usually remained active in patches excised from baby hamster kidney (BHK) cells overexpressing CFTR . Those patches with stable channel activity were used to investigate the regulation of CFTR by exogenous protein phosphatases (PP) . Adding PP2A, PP2C, or alkaline phosphatase to excised patches reduced CFTR channel activity by > 90% but did not abolish it completely . PP2B caused weak deactivation, whereas PP1 had no detectable effect on open probability (Po) . Interestingly, the time course of deactivation by PP2C was identical to that of the spontaneous rundown observed in some patches after excision . PP2C and PP2A had distinct effects on channel gating Po declined during exposure to exogenous PP2C (and during spontaneous rundown, when it was observed) without any change in mean burst duration . By contrast, deactivation by exogenous PP2A was associated with a dramatic shortening of burst duration similar to that reported previously in patches from cardiac cells during deactivation of CFTR by endogenous phosphatases . Rundown of CFTR-mediated current across intact T84 epithelial cell monolayers was insensitive to toxic levels of the PP2A inhibitor calyculin A . These results demonstrate that exogenous PP2C is a potent regulator of CFTR activity, that its effects on single-channel gating are distinct from those of PP2A but similar to those of endogenous phosphatases in CHO, BHK, and T84 epithelial cells, and that multiple protein phosphatases may be required for complete deactivation of CFTR channels.

Trends Biochem Sci, 1998 May, 23(5), 162 - 9
The danger of metabolic pathways with turbo design; Teusink B et al.; Many catabolic pathways begin with an ATP-requiring activation step, after which further metabolism yields a surplus of ATP . Such a 'turbo' principle is useful but also contains an inherent risk . This is illustrated by a detailed kinetic analysis of a paradoxical Saccharomyces cerevisiae mutant; the mutant fails to grow on glucose because of overactive initial enzymes of glycolysis, but is defective only in an enzyme (trehalose 6-phosphate synthase) that appears to have little relevance to glycolysis . The ubiquity of pathways that possess an initial activation step, suggests that there might be many more genes that, when deleted, cause rather paradoxical regulation phenotypes (i.e . growth defects caused by enhanced utilization of growth substrate).

Nucleic Acids Res, 1998 Jun 15, 26(12), 2948 - 54
Cloning of Drosophila GCN5: conserved features among metazoan GCN5 family members; Smith ER et al.; PCAF and hGCN5 are distinct human genes that encode proteins related to the yeast histone acetyltransferase and transcriptional adapter GCN5 . The PCAF protein shares extensive similarity with the 439 amino acids of yGCN5, but it has an approximately 350 amino acid N-terminal extension that interacts with the transcriptional co-activator p300/CBP . Adenoviral protein E1a can disrupt PCAF-CBP interactions and prevent PCAF-dependent cellular differentiation . In this report, we describe the cloning and initial characterization of a Drosophila homolog of yGCN5 . In addition to the homology to yGCN5, the Drosophila protein shares sequencesimilarity with the N-terminal portion of human PCAF that is involved in binding to CBP . In the course of characterizing dGCN5, we have discovered that hGCN5 also contains an N-terminal extension with significant similarity to PCAF . Interestingly, in the case of the h GCN5 gene, alternative splicing may regulate the production of full-length hGCN5 . The presence of the N-terminal domain in a Drosophila GCN5 homolog and both human homologs suggests that it was part of the ancestral form of metazoan GCN5.

J Biol Chem, 1998 May 29, 273(22), 13776 - 80
Alterations in the GAL4 DNA-binding domain can affect transcriptional activation independent of DNA binding; Corton JC et al.; The GAL4 protein belongs to a large class of fungal transcriptional activator proteins encoding within their DNA-binding domains (DBD) six cysteines that coordinate two atoms of zinc (the Zn2Cys6 domain) . In an effort to characterize the interactions between the Zn2Cys6 class transcriptional activator proteins and their DNA-binding sites, we have replaced in the full-length GAL4 protein small regions of the Zn2Cys6 domain with the analogous regions of another Zn2Cys6 protein called PPR1 an activator of pyrimidine biosynthetic genes . Alterations between the first and third cysteines abolished binding to GAL4 (upstream activation sequence of GAL (UASG)) or PPR1 (upstream acitvation sequence of UAS) DNA-binding sites and severely reduced transcriptional activation in yeast . In contrast, alterations between the third and fourth cysteines had only minor effects on binding to UASG but led to substantial decreases in activation in both yeast and a mammalian cell line . In the crystal structure of the GAL4 DBD-UASG complex (Marmorstein, R., Carey, M., Ptashne, M., and Harrison, S . C . (1992) Nature 356, 408-414), this region is facing away from the DNA, making it likely that there exists within the GAL4 DBD an accessible domain important in activation.

J Biol Chem, 1998 May 29, 273(22), 13681 - 92
Identification of a candidate human spectrin Src homology 3 domain-binding protein suggests a general mechanism of association of tyrosine kinases with the spectrin-based membrane skeleton; Ziemnicka-Kotula D et al.; Spectrin is a widely expressed protein with specific isoforms found in erythroid and nonerythroid cells . Spectrin contains an Src homology 3 (SH3) domain of unknown function . A cDNA encoding a candidate spectrin SH3 domain-binding protein was identified by interaction screening of a human brain expression library using the human erythroid spectrin (alphaI) SH3 domain as a bait . Five isoforms of the alphaI SH3 domain-binding protein mRNA were identified in human brain . Mapping of SH3 binding regions revealed the presence of two alphaI SH3 domain binding regions and one Abl-SH3 domain binding region . The gene encoding the candidate spectrin SH3 domain-binding protein has been located to human chromosome 10p11.2 --> p12 . The gene belongs to a recently identified family of tyrosine kinase-binding proteins, and one of its isoforms is identical to e3B1, an eps8-binding protein (Biesova, Z., Piccoli, C., and Wong, W . T . (1997)Oncogene 14, 233-241) . Overexpression of the green fluorescent protein fusion of the SH3 domain-binding protein in NIH3T3 cells resulted in cytoplasmic punctate fluorescence characteristic of the reticulovesicular system . This fluorescence pattern was similar to that obtained with the anti-human erythroid spectrin alphaI SigmaI/betaI SigmaI antibody in untransfected NIH3T3 cells; in addition, the anti-alphaI SigmaI/betaI SigmaI antibody also stained Golgi apparatus . Immunofluorescence obtained using antibodies against alphaI SigmaI/++betaI SigmaI spectrin and Abl tyrosine kinase but not against alphaII/betaII spectrin colocalized with the overexpressed green fluorescent protein-SH3-binding protein . Based on the conservation of the spectrin SH3 binding site within members of this protein family and published interactions, a general mechanism of interactions of tyrosine kinases with the spectrin-based membrane skeleton is proposed.

Prostaglandins Leukot Essent Fatty Acids, 1998 Mar, 58(3), 169 - 76
Detection of acyl-CoA synthetase, acyl-CoA:lysophospholipid acyltransferase and phospholipase A2 activities in non-pregnant and pregnant guinea-pig uterine tissues; Norman SJ et al.; Acyl-CoA synthetase (ACS), acyl-CoA:lysophospholipid acyltransferase (ACLAT) and phospholipase (PL) A2 activities were detected in guinea-pig endometrium on days 7 and 15 of the cycle, and on days 15, 29 and 36 of pregnancy . Ovariectomy of non-pregnant animals resulted in an increase in the apparent activities of these three enzymes which was reversed by treatment with oestradiol and/or progesterone . ACS, ACLAT and PLA2 activities were detected in day 15 conceptuses, and in the placenta, sub-placenta, chorion and amnion on days 29 and 36 of pregnancy . Apparent activities of the enzymes were generally higher in the fetal membranes than in the placental tissue . This study has established that the enzymes involved in turnover of arachidonic acid in phospholipids are present in tissues in the non-pregnant and pregnant guinea-pig uterus . The higher apparent activities of enzymes (ACS and ACLAT) involved in arachidonic acid uptake compared to the enzyme (PLA2) involved in arachidonic acid release is in agreement with there being very low concentrations of free arachidonic acid in tissues.

Curr Opin Genet Dev, 1998 Apr, 8(2), 254 - 9
Chromosome dynamics: the SMC protein family; Jessberger R et al.; Recent evidence suggests that members of the structural maintenance of chromosomes (SMC) protein family are involved in a much broader spectrum of chromosome and DNA metabolic reactions than was originally thought . Other than their role in chromosome condensation, SMC proteins are essential for sister chromatid cohesion and gene dosage compensation, and are involved in DNA recombination . This diversity of function is achieved both through the formation of different heterodimers and through their participation in higher-order protein complexes adapted to achieve specific ends.

Curr Opin Genet Dev, 1998 Apr, 8(2), 212 - 8
Centromeres: proteins, protein complexes, and repeated domains at centromeres of simple eukaryotes; Clarke L; Similarities exist among components of simple and complex centromeres that may not have been expected on the basis of wide variation in size and sequence organization of centromeric DNAs among eukaryotes . Support is growing in systems from fungi to Drosophila for a model of centromere assembly and activation that is dependent on a particular underlying chromatin structure but not necessarily on a specific DNA sequence.

Curr Opin Genet Dev, 1998 Apr, 8(2), 200 - 11
Recombination at work for meiosis; Smith KN et al.; In sexually reproducing organisms, homologous recombination increases genetic diversity in gametes and ensures proper chromosome segregation . Recent publications have provided details of the molecular intermediates and proteins involved, the control of the distribution of recombination events at the chromosomal level, and the surveillance mechanisms that coordinate recombination with the meiotic cell cycle.

Curr Opin Genet Dev, 1998 Apr, 8(2), 173 - 8
Covalent modifications of histones: expression from chromatin templates; Davie JR; Recent advances highlight the involvement of histone acetyltransferases in transcriptional activation and histone deacetylases in transcriptional repression . Transcription factors loaded onto regulatory DNA elements may recruit either coactivators with histone acetyltransferase activity or corepressors associated with histone deacetylases . The recruited enzymes may either acetylate or deacetylate proximal nucleosomal histones or nonhistone chromosomal proteins.

Anal Chem, 1998 May 15, 70(10), 2050 - 9
Identification of phosphorylation sites in proteins separated by polyacrylamide gel electrophoresis; Zhang X et al.; We report a fast, sensitive, and robust procedure for the identification of precise phosphorylation sites in proteins separated by polyacrylamide gel electrophoresis by a combination of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF) and online capillary liquid chromatography electrospray tandem ion trap mass spectrometry (LC/ESI/MS/MS) . With this procedure, a single phosphorylation site was identified on as little as 20 ng (500 fmol) of the baculovirus-expressed catalytic domain of myosin I heavy-chain kinase separated by gel electrophoresis . The phosphoprotein is digested in the gel with trypsin, and the resulting peptides are extracted with > 60% yield and analyzed by MALDI/TOF before and after digestion with a phosphatase to identify the phosphopeptides . The phosphopeptides are then separated and fragmented in an on-line LC/ESI ion trap mass spectrometer to identify the precise phosphorylation sites . This procedure eliminates any off-line HPLC separation and minimizes sample handling . The use of MALDI/TOF and LCQ, two types of mass spectrometers that are widely available to the biological community, will make this procedure readily accessible to biologists . We applied this technique to identify two autophosphorylation sites and to assign at least another 12 phosphorylation sites to two tryptic peptides in a series of experiments using a gel slice containing only 200 ng (3 pmol) of human double-stranded RNA-activated protein kinase expressed in a mutant strain of the yeast Saccharomyces cerevisiae.

Methods, 1998 Apr, 14(4), 367 - 79
Ectopic gene expression in Drosophila using GAL4 system; Phelps CB et al.; Expressing a gene in cells in which it is not normally active is a powerful way of determining its function . The GAL4 system allows the selective expression of any cloned gene in a wide variety of cell- and tissue-specific patterns in Drosophila . A promoter (or enhancer) directs expression of the yeast transcriptional activator GAL4 in a particular pattern, and GAL4 in turn directs transcription of the GAL4-responsive (UAS) target gene in an identical pattern . The system's key feature is that the GAL4 gene and UAS-target gene are initially separated into two distinct transgenic lines . In the GAL4 line, the activator protein is present, but has no target gene to activate . In the UAS-target gene line, the target gene is silent because the activator is absent . It is only when the GAL4 line is crossed to the UAS-target gene line that the target gene is turned on in the progeny . In this article we describe, in detail, how to generate and characterize GAL4 lines and how to prepare UAS-target gene lines . Vector maps are provided for pGaTB, P{GawB}, and pP{UAST} . In addition, we consider the range of UAS-reporters currently available and review several new modifications of the GAL4 system.

Plant Cell, 1998 May, 10(5), 837 - 47
14-3-3 proteins are part of an abscisic acid-VIVIPAROUS1 (VP1) response complex in the Em promoter and interact with VP1 and EmBP1; Schultz TF et al.; Protein-DNA complexes were formed when nuclear extracts from embryogenic rice suspension cultures or maize embryos were incubated with an abscisic acid-VIVIPAROUS1 (VP1) response element (Em1a) from the Em promoter . Monoclonal antibodies generated to GF14, a 14-3-3 protein from plants, resulted in gel retardation of the Em1a-protein complexes . Antibodies generated to the C and N termini of GF14 detected protein isoforms in rice nuclear and cytoplasmic extracts, but no differences in distribution of the GF14 isoforms were recognized between the nucleus and cytoplasm or when abscisic acid-treated and untreated tissues were compared . When recombinant GF14 fusion proteins from rice were added to nuclear extracts, novel complexes were formed that required the dimerization domain of GF14 . Chemical cross-linking showed that GF-14 interacted with the basic leucine zipper factor EmBP1, which binds specifically to Em1a, and with VP1, which transactivates Em through Em1a . GF14 proteins from rice were shown to interact with VP1 in yeast through the dimerization domain of GF14 . Our results indicated that GF14 interacts with both site-specific DNA binding proteins (i.e., EmBP1) and tissue-specific regulatory factors (i.e., VP1) and may provide a structural link between VP1 and the Em1a transcriptional complex.

J Morphol, 1998 Jun, 236(3), 209 - 21
Hemocytes of the palaemonids Macrobrachium rosenbergii and M . acanthurus, and of the penaeid Penaeus paulensis; Gargioni R et al.; The hemocytes of two palaemonids and one penaeid were characterized using light and transmission electron microscopy (TEM) . The blood cells in all three species were classified as hyaline hemocytes (HH), small granule hemocytes (SGH), and large granule hemocytes (LGH) . The HH are unstable hemocytes with a characteristic high nucleo-cytoplasmic ratio . Their cytoplasm appears particularly dense and has from few to numerous granules that often exhibit a typical striated substructure . In both palaemonids, the great majority of the HH contain numerous granules, whereas in Penaeus paulensis, a small number of these cells have few or no granules . The cytoplasm of some HH of the penaeid exhibits typical electron-dense deposits . The granulocytes, LGH and SGH, contain abundant electron-dense granules that are usually smaller in the SGH . In both hemocyte types, the cytosol, but not the granules, is rich in carbohydrates (PAS positive) and numerous vesicles contain acid phosphatase (Gomori reactive) . In all studied shrimps, the SGH and LGH were actively phagocytic when examined on blood cell monolayers incubated with the yeast Saccharomyces cerevisiae . A few mitotic figures (less than 1%) were observed in the granulocytes of P . paulensis, but not in the palaemonids . SGH is the main circulating blood cell type in both palaemonids, whereas HH is predominant in the penaeid . Based on morphological and functional features, it appears that the hyaline and the granular hemocytes of the three shrimp species represent different cell lineages.

FEBS Lett, 1998 May 8, 427(2), 247 - 51
Proteolytic cleavage of HsRad51 during apoptosis; Flygare J et al.; The Rad51 gene of Saccharomyces cerevisiae is required for genetic recombination and recombinational repair of DNA strand breaks . In higher eukaryotes Rad51 is essential for embryonic development, and is involved in cell proliferation and DNA repair . Here we show that human Rad51 (HsRad51) is proteolytically cleaved during apoptosis in two T-lymphocyte cell lines, Jurkat and PFI-285 . Apoptosis was induced by camptothecin or anti-Fas monoclonal antibody (anti-Fas mAb) . HsRad51 was cleaved with similar kinetics as human poly(ADP-ribose) polymerase (HsPARP) after treatment with either agent . The time course of cleavage coincided with internucleosomal DNA fragmentation . The HsRad51 fragments observed in apoptotic cells were identical to those generated from in vitro translated (IVT) HsRad51 exposed to activated Jurkat S-100 extract in a cell-free system . In each case, cleavage of HsRad51 was abolished by acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO) . However, cleavage of IVT HsRad51 could not be demonstrated using purified caspase-2, -3 or -6 to -10, and the identity of the responsible protease thus remains to be determined . In summary, we have shown that HsRad51 belongs to a group of repair proteins, including PARP and DNA-dependent protein kinase, which are specifically cleaved during the execution phase of apoptosis.

FEBS Lett, 1998 May 8, 427(2), 236 - 40
The interacting RNA polymerase II subunits, hRPB11 and hRPB3, are coordinately expressed in adult human tissues and down-regulated by doxorubicin; Fanciulli M et al.; We previously isolated the human RPB11 cDNA, encoding the 13.3 kDa subunit of RNA polymerase II, and demonstrated that expression of this subunit is modulated by doxorubicin . Using hRPB11 as bait in a yeast two-hybrid system, two cDNA variants encoding a second RNA polymerase II subunit, hRPB3, have now been isolated and characterized . These two hRPB3 mRNA species differed in 3' UTR region length, the longer transcript containing the AU-rich sequence motif that mediates mRNA degradation . Both hRPB11 and hRPB3 transcripts share a similar pattern of distribution in human adult tissues, with particularly high levels in both heart and skeletal muscle, and the expression of both is down-regulated by doxorubicin as found previously for the hRPB11 subunit . Taken together, these findings suggest that the interaction between hRPB3 and hRPB11 is fundamental for their function and that this heterodimer is involved in doxorubicin toxicity.

Arch Biochem Biophys, 1998 May 15, 353(2), 331 - 6
Cloning and characterization of the CYP2D1-binding protein, retinol dehydrogenase; Imaoka S et al.; A CYP2D1-binding protein, 29 k-protein (p29), has been isolated and its N-terminal amino acid sequence has been reported (Ohishi et al . (1993) Biochim . Biophys . Acta 1158, 227-236) . In this study, p29 cDNA was isolated by PCR with oligonucleotide probes designed from the N-terminal amino acid sequence and p29 was found to be a microsomal retinol dehydrogenase, a member of the short-chain alcohol dehydrogenase family which metabolize hydroxysteroids and prostaglandins . CYP2D1 and p29 were expressed in Saccharomyces cerevisiae to characterize these proteins . CYP2D1 had an absorption maximum at 448 nm in a CO-reduced form . Expressed p29 in yeast cells was detected with anti-p29 antibody . Solubilized CYP2D1 and p29 from yeast microsomes were mixed and applied to an anti-CYP2D1 antibody-binding column . Both proteins were retained in the column and eluted with glycine buffer (pH 2.8) . However, when applied alone, p29 was not retained in the column . The findings indicated that CYP2D1 bound tightly with p29 . Catalytic activities of p29 expressed in yeast were investigated . p29 had retinal reductase activity in the presence of NADPH . Addition of CYP2D1 and NADPH-P450 reductase increased the retinal reductase activity of p29 . These findings suggest that the complex of CYP2D1, p29, and NADPH-P450 reductase has an important role in the metabolism of retinoids.

J Biol Chem, 1998 Apr 24, 273(17), 10349 - 54
A phosphatidylinositol 3-kinase and phosphatidylinositol transfer protein act synergistically in formation of constitutive transport vesicles from the trans-Golgi network; Jones SM et al.; Current evidence suggests that phosphatidylinositol (PI) kinases and phosphatidylinositol transfer protein (PITP) are involved in driving vesicular traffic from yeast and mammalian trans-Golgi network (TGN) . We have tested the interaction between these cytosolic proteins in an assay that measures the formation of constitutive transport vesicles from the TGN in a hepatocyte cell-free system . This reaction is dependent on a novel PI 3-kinase, and we now report that, under conditions of limiting cytosol, purified PI 3-kinase and PITP functionally cooperate to drive exocytic vesicle formation . This synergy was observed with both yeast and mammalian PITPs, and it also extended to the formation of PI 3-phosphate . These collective findings indicate that the PI 3-kinase and PITP synergize to form a pool of PI 3-phosphate that is essential for formation of exocytic vesicles from the hepatocyte TGN.

Anal Biochem, 1998 May 15, 259(1), 89 - 97
Lysophosphatidylglycerol: a novel effective detergent for solubilizing and purifying the cystic fibrosis transmembrane conductance regulator; Huang P et al.; Similar to the recombinant cystic fibrosis transmembrane conductance regulator (CFTR) expressed in Sf9 insect cells, underglycosylated CFTR expressed in yeast is not effectively solubilized by a variety of commonly used detergents, requiring instead harsh alkali and SDS treatments, which would denature most proteins . Moreover, solubilized CFTR has a strong tendency to aggregate and form high-molecular-weight aggregates during subsequent purification . We report here that the mild detergent, lysophosphatidylglycerol (LPG), is a very effective detergent for solubilizing the CFTR expressed in both yeast and Sf9 insect cells . LPG solubilizes nearly 100% of the CFTR in yeast in the absence of NaCl and none in the presence of 1 M NaCl . It is also very potent in preventing aggregation of the CFTR during subsequent purification . Exploiting these characteristics, a rapid simple procedure for the purification of functional recombinant CFTR expressed in yeast has been developed . It includes selective CFTR solubilization in the presence and the absence of NaCl followed by nickel-chelate chromatography of His-tagged CFTR . The CFTR produced by this procedure is about 70% pure . Purified CFTR molecules were reconstituted into liposomes and then fused to planar lipid bilayers for single-channel recording . The reconstituted CFTR exhibits regulatory chloride channel activities with a slope conductance of 7.1 pS and a reversal potential of -32 mV . The effectiveness and simplicity of this new purification procedure for the CFTR should greatly facilitate a variety of biochemical and biophysical studies of this important protein . Furthermore, the potency of LPG in solubilizing the notoriously intractable underglycosylated CFTR suggest that this detergent may be useful for solubilizing the CFTR from other sources and for other difficult membrane proteins as well.

J Steroid Biochem Mol Biol, 1998 Feb, 64(3-4), 129 - 35
Random mutagenesis of human estrogen receptor ligand binding domain identifies mutations that decrease sensitivity to estradiol and increase sensitivity to a diphenol indene-ol compound: basis for a regulatable expression system; Miller N et al.; We have used low fidelity polymerase chain reaction amplification to generate mutations in the human estrogen receptor ligand binding domain (LBD) . Screening of libraries of mutants in yeast revealed a variety of phenotypic changes including decreased responsiveness to estradiol and increased responsiveness to synthetic compounds . Identification of the mutations responsible for these phenotypic changes indicated discrete regions of the LBD that are important for human estrogen receptor function . Cumulative rounds of mutagenesis and screening allowed us to produce a mutant estrogen receptor that was of reversed specificity as compared with the wild type LBD, in that it was more responsive to a diphenol indene-ol than to estradiol . This mutant may form the basis of a useful regulatable expression system in mammalian cells.

Protein Sci, 1998 May, 7(5), 1214 - 20
Synthesis, physicochemical characterization, and crystallization of a putative retro-coiled coil; Liu N et al.; An artificial HIV enhancer-binding polypeptide has recently been dimerized by covalently linking it to the leucine zipper motif of the yeast transcriptional activator GCN4 (Liu N et al., 1997, Eur Biophys J 25:399-403) . Although it seemed that the dimerization of this peptide could be best achieved by the use of the retro sequence of the leucine zipper, this approach was not implemented in the original construct . As the first step toward the synthesis of a basic region-retro leucine zipper HIV enhancer-binding fusion protein, we have now prepared the retro version of the leucine zipper (r-LZ35) and performed initial physicochemical characterization . Circular dichroism and sedimentation equilibrium studies showed that, at concentrations < 100 microM, the retro peptide was an unstructured monomer . At higher concentrations, however, the monomer was in equilibrium with a tetramer and, at 1 mM, the retro peptide was almost fully helical . N-terminal extension of the retro peptide by the tripeptide Cys-Gly-Gly resulted in a 38-residue polypeptide that could be covalently dimerized by forming a disulfide bond between two chains to give the peptide (r-LZ38)2 . Even in the low micromolar concentration range peptide (r-LZ38)2 formed a stable, noncovalent, helical dimer as revealed by circular dichroism and sedimentation equilibrium in the presence and absence of guanidinium chloride . (r-LZ38)2 has been crystallized and X-ray structural analysis is under way . The disulfide-crosslinked retro-leucine zipper may lend itself to interesting protein structural studies, including protein design . The present work also highlights the structural and functional potential of retro proteins in general.

Cell, 1998 May 15, 93(4), 649 - 60
Nuclear access and action of notch in vivo; Struhl G et al.; The Drosophila Notch (N) gene encodes a conserved single-pass transmembrane receptor that transduces extracellular signals controlling cell fate . Here, we present evidence that the intracellular domain of Notch gains access to the nucleus in response to ligand, possibly through a mechanism involving proteolytic cleavage and release from the remainder of the protein . In addition, our results suggest that signal transduction by Notch depends on the ability of the intracellular domain, particularly the portion containing the CDC10 repeats, to reach the nucleus and to participate in the transcriptional activation of downstream target genes.

Cell, 1998 May 15, 93(4), 505 - 18
The Drosophila Fab-7 chromosomal element conveys epigenetic inheritance during mitosis and meiosis; Cavalli G et al.; Polycomb group (PcG) and trithorax group (trxG) gene products are responsible for the maintenance of repressed and active expression patterns of many developmentally important regulatory genes including the homeotic genes . In Drosophila embryos, Polycomb protein and the trxG protein GAGA factor colocalize at the Fab-7 DNA element of the bithorax complex . In transgenic lines, the Fab-7 element induces extensive silencing on a flanking GAL4-driven lacZ reporter and mini-white genes . However, a short single pulse of GAL4 during embryogenesis is sufficient to release PcG-dependent silencing from the transgene . Such an activated state of Fab-7 is mitotically inheritable through development and can be transmitted in a GAL4-independent manner to the subsequent generations through female meiosis . Thus, Fab-7 is a switchable chromosomal element, which can convey memory of epigenetically determined active and repressed chromatin states.

J Biochem (Tokyo), 1998 Jun, 123(6), 1010 - 6
Mitochondria-targeting sequence, a multi-role sorting sequence recognized at all steps of protein import into mitochondria; Omura T; The intracellular sorting of newly synthesized precursor proteins (preproteins) to mitochondria depends on the "mitochondria-targeting sequence" (MTS), which is located at the amino termini of the preproteins . MTS is required, however, not only for targeting newly synthesized preproteins to mitochondria, but also for all the following steps along the mitochondrial protein import pathway . MTS of nascent preproteins is first recognized by a cytoplasmic molecular chaperone, MSF, and then by Tom70 and Tom20 of the mitochondrial outer membrane receptor complex, Tom5 and Tom40 of the outer membrane protein translocation machinery, Tim23 of the inner membrane protein translocation machinery, and finally the processing peptidase, MPP, in the matrix . MTS is a multi-role sorting sequence which specifically interacts with various components along the mitochondrial protein import pathway . Recognition of MTS at multiple steps during the import of preproteins may contribute to the strict sorting of proteins destined for mitochondria.

Proc Natl Acad Sci U S A, 1998 May 26, 95(11), 6361 - 6
Chaperone-facilitated copper binding is a property common to several classes of familial amyotrophic lateral sclerosis-linked superoxide dismutase mutants; Corson LB et al.; Mutations in Cu, Zn superoxide dismutase (SOD1) cause the neurodegenerative disease familial amyotrophic lateral sclerosis from an as-yet-unidentified toxic property(ies) . Analysis in Saccharomyces cerevisiae of a broad range of human familial amyotrophic lateral sclerosis-linked SOD1 mutants (A4V, G37R, G41D, H46R, H48Q, G85R, G93C, and I113T) reveals one property common to these mutants (including two at residues that coordinate the catalytic copper): Each does indeed bind copper and scavenge oxygen-free radicals in vivo . Neither decreased copper binding nor decreased superoxide scavenging activity is a property shared by all mutants . The demonstration that shows that all mutants tested do bind copper under physiologic conditions supports a mechanism of SOD1 mutant-mediated disease arising from aberrant copper-mediated chemistry catalyzed by less tightly folded (and hence less constrained) mutant enzymes . The mutant enzymes also are shown to acquire the catalytic copper in vivo through the action of CCS, a specific copper chaperone for SOD1, which in turn suggests that a search for inhibitors of this SOD1 copper chaperone may represent a therapeutic avenue.

Proc Natl Acad Sci U S A, 1998 May 26, 95(11), 6212 - 6
Unexpected homology between inducible cell wall protein QID74 of filamentous fungi and BR3 salivary protein of the insect Chironomus; Rey M et al.; A gene, qid74, of mycoparasitic filamentous fungus Trichoderma harzianum and its allies encodes a cell wall protein that is induced by replacing glucose in the culture medium with chitin (simulated mycoparasitism conditions) . Because no trace of this gene can be detected in related species such as Gibberella fujikuroi and Saccharomyces cerevisiae, the qid74 gene appears to have arisen de novo within the genus Trichoderma . Qid74 protein, 687 residues long, is now seen as highly conserved tandem repeats of the 59-residue-long unit . This unit itself, however, may have arisen as tandem repeats of the shorter 13-residue-long basic unit . Within the genus Trichoderma, the amino acid sequence of Qid74 proteins has been conserved in toto . The most striking is the fact that Qid74 shares 25.3% sequence identity with the carboxyl-terminal half of the 1,572-residue-long BR3 protein of the dipteran insect Chironomus tentans . BR3 protein is secreted by the salivary gland of each aquatic larva of Chironomus to form a tube to house itself . Furthermore, the consensus sequence derived from these 59-residue-long repeating units resembles those of epidermal growth factor-like domains found in divergent invertebrate and vertebrate proteins as to the positions of critical cysteine residues and homology of residues surrounding these cysteines.

Proc Natl Acad Sci U S A, 1998 May 26, 95(11), 5865 - 71
Highly specific protein sequence motifs for genome analysis; Nevill-Manning CG et al.; We present a method for discovering conserved sequence motifs from families of aligned protein sequences . The method has been implemented as a computer program called EMOTIF . Given an aligned set of protein sequences, EMOTIF generates a set of motifs with a wide range of specificities and sensitivities . EMOTIF also can generate motifs that describe possible subfamilies of a protein superfamily . A disjunction of such motifs often can represent the entire superfamily with high specificity and sensitivity . We have used EMOTIF to generate sets of motifs from all 7,000 protein alignments in the BLOCKS and PRINTS databases . The resulting database, called IDENTIFY , contains more than 50,000 motifs . For each alignment, the database contains several motifs having a probability of matching a false positive that range from 10(-10) to 10(-5) . Highly specific motifs are well suited for searching entire proteomes, while generating very few false predictions . IDENTIFY assigns biological functions to 25-30% of all proteins encoded by the Saccharomyces cerevisiae genome and by several bacterial genomes . In particular, IDENTIFY assigned functions to 172 of proteins of unknown function in the yeast genome.

Curr Biol, 1998 Apr 9, 8(8), 441 - 51
The DEAH-box splicing factor Prp16 unwinds RNA duplexes in vitro; Wang Y et al.; BACKGROUND: During pre-mRNA splicing, dynamic rearrangement of RNA secondary structure within the spliceosome is crucial for intron recognition and formation of the catalytic core . Splicing factors belonging to the DExD/DExH-box family of RNA-dependent ATPases are thought to have a central role in directing these rearrangements by unwinding RNA helices . Proof of this hypothesis has, however, been conspicuously lacking . RESULTS: Prp16 is a DEAH-box protein that functions in the second step of splicing in vitro . Using various RNA duplexes as substrate, we have shown that Prp16 has an ATP-dependent RNA unwinding activity . This activity is independent of sequence in either the single-stranded or duplexed regions of the RNA substrate . A mutation (prp16-1) near the ATP-binding motif of Prp16 inhibits both the RNA-dependent ATPase activity and the ATP-dependent RNA unwinding activity . CONCLUSIONS: Our findings provide strong biochemical evidence that Prp16 can disrupt a duplexed RNA structure on the spliceosome . Because the purified protein lacks sequence specificity in unwinding RNA duplexes, targeting of the unwinding activity of Prp16 in the spliceosome is likely to be determined by other interacting protein factors . The demonstration of unwinding activity will also help our understanding of how the fidelity of branchpoint recognition is controlled by Prp16.

Curr Biol, 1998 Apr 9, 8(8), R262 - 5
Mitochondrial import: crossing the aqueous intermembrane space; Pfanner N; Mitochondrial protein import follows a general pathway for preproteins with amino-terminal presequences . The discovery of novel import components has now revealed a distinct pathway for translocation of hydrophobic proteins across the intermembrane space and into the inner membrane.

J Mol Biol, 1998 Apr 10, 277(4), 763 - 9
Human T cell cyclophilin18 binds to thiol-specific antioxidant protein Aop1 and stimulates its activity; Jaschke A et al.; Cyclophilins (CyPs) define a family of proteins binding to the immunosuppressive drug cyclosporin A (CsA) . They are evolutionary highly conserved proteins being present in both pro- and eukaryotes and in different subcellular locations . CyPs possess enzymatic activity, namely peptidyl-prolyl cis-trans isomerase (PPIase) activity and are involved in cellular protein folding and protein interactions . Here we describe a novel interaction of human T cell cyclophilin18 (hCyP18) . Abundant cytosolic hCyP18 binds to the thiol-specific antioxidant protein Aop1 and stimulates its enzymatic activity . Aop1 belongs to a family of proteins thought to be involved in defense of oxidative stress . The interaction of both proteins seem to be specific, since other PPIases do not have any stimulatory effect on Aop1 .

Nucleic Acids Res, 1998 Apr 1, 26(7), 1576 - 87
SnoRNA-guided ribose methylation of rRNA: structural features of the guide RNA duplex influencing the extent of the reaction; Cavaille J et al.; Eukaryotic rRNAs contain a large number of ribose-methylated nucleotides of elusive function which are confined to the universally conserved rRNA domains . Ribose methylation of these nucleotides is directed by a large family of small trans -acting guide RNAs, called box C/D antisense snoRNAs . Each snoRNA targets precisely one of the nucleotides to be methylated within the pre-rRNA sequence, through transient formation of a 10-21 bp regular RNA duplex around the modification site . In this study we have analyzed how different features of the double-stranded RNA guide structure affect the extent of site-specific ribose methylation, by co-expressing an appropriate RNA substrate and its cognate tailored snoRNA guide in transfected mouse cells . We show that an increased GC content of the duplex can make up for the inhibitory effects of a helix truncation or for the presence of helix irregularities such as a mismatched pair or a bulge nucleotide . However, some helix irregularities dramatically inhibit the reaction and are not offset by further stabilization of the duplex . Overall, the RNA duplex tolerates a much larger degree of irregularity than anticipated, even in the immediate vicinity of the methylation site, which offers new prospects in the search for additional snoRNA guides . Accordingly, a few snoRNA-like sequences of uncertain status detected in the yeast Saccharomyces cerevisiae genome now appear as likely bona fide ribose methylation guides.

Nucleic Acids Res, 1998 Apr 1, 26(7), 1551 - 9
Interaction of Ku protein and DNA-dependent protein kinase catalytic subunit with nucleic acids; Dynan WS et al.; The Ku protein-DNA-dependent protein kinase system is one of the major pathways by which cells of higher eukaryotes respond to double-strand DNA breaks . The components of the system are evolutionarily conserved and homologs are known from a number of organisms . The Ku protein component binds directly to DNA ends and may help align them for ligation . Binding of Ku protein to DNA also nucleates formation of an active enzyme complex containing the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) . The interaction between Ku protein, DNA-PKcs and nucleic acids has been extensively investigated . This review summarizes the results of these biochemical investigations and relates them to recent molecular genetic studies that reveal highly characteristic repair and recombination defects in mutant cells lacking Ku protein or DNA-PKcs.

Genes Dev, 1998 May 15, 12(10), 1525 - 38
Spalten, a protein containing Galpha-protein-like and PP2C domains, is essential for cell-type differentiation in Dictyostelium; Aubry L et al.; We have identified a novel gene, Spalten (Spn) that is essential for Dictyostelium multicellular development . Spn encodes a protein with an amino-terminal domain that shows very high homology to Galpha-protein subunits, a highly charged inter-region, and a carboxy-terminal domain that encodes a functional PP2C . Spn is essential for development past the mound stage, being required cell autonomously for prestalk gene expression and nonautonomously for prespore cell differentiation . Mutational analysis demonstrates that the PP2C domain is the Spn effector domain and is essential for Spn function, whereas the Galpha-like domain is required for membrane targeting and regulation of Spn function . Moreover, Spn carrying mutations in the Galpha-like domain that do not affect membrane targeting but affect specificity of guanine nucleotide binding in known GTP-binding proteins are unable to fully complement the spn- phenotype, suggesting that the Galpha-like domain regulates Spn function either directly or indirectly by mediating its interactions with other proteins . Our results suggest that Spn encodes a signaling molecule with a novel Galpha-like regulatory domain.

Genes Dev, 1998 May 15, 12(10), 1515 - 24
The ATP-dependent PIM1 protease is required for the expression of intron-containing genes in mitochondria; van Dyck L et al.; The ATP-dependent PIM1 protease, a Lon-like protease localized in the mitochondrial matrix, is required for mitochondrial genome integrity in yeast . Cells lacking PIM1 accumulate lesions in the mitochondrial DNA (mtDNA) and therefore lose respiratory competence . The identification of a multicopy suppressor, which stabilizes mtDNA in the absence of PIM1, enabled us to characterize novel functions of PIM1 protease during mitochondrial biogenesis . The synthesis of mitochondrially encoded cytochrome c oxidase subunit I (CoxI) and cytochrome b (Cob) is impaired in pim1 mutants containing mtDNA . PIM1-mediated proteolysis is required for the translation of mature COXI mRNA . Moreover, deficiencies in the splicing of COXI and COB transcripts, which appear to be restricted to introns encoding mRNA maturases, were observed in cells lacking the PIM1 gene . Transcripts of COXI and COB genes harboring multiple introns are degraded in the absence of PIM1 . These results establish multiple, essential functions of the ATP-dependent PIM1 protease during mitochondrial gene expression.

Genes Dev, 1998 May 15, 12(10), 1409 - 14
Phosphorylation of spliceosomal protein SAP 155 coupled with splicing catalysis; Wang C et al.; The U2 snRNP component SAP 155 contacts pre-mRNA on both sides of the branch site early in spliceosome assembly and is therefore positioned near or at the spliceosome catalytic center . We have isolated a cDNA encoding human SAP 155 and identified its highly related Saccharomyces cerevisiae homolog (50% identity) . The carboxy-terminal two-thirds of SAP 155 shows the highest conservation and is remarkably similar to the regulatory subunit A of the phosphatase PP2A . Significantly, SAP 155 is phosphorylated concomitant with or just after catalytic step one, making this the first example of a protein modification tightly regulated with splicing catalysis.

J Exp Biol, 1998 Apr, 201(Pt 8), 1163 - 75
Structure/function of oxygen-regulated isoforms in cytochrome c oxidase; Burke PV et al.; Eukaryotic cytochrome c oxidases are complex oligomeric membrane proteins composed of subunit polypeptides encoded by both nuclear and mitochondrial genomes . While the mitochondrially encoded subunits are encoded by unique genes, some of the nuclear-encoded subunits are encoded by multigene families . The isoforms produced by these multigene families are tissue-specific and/or developmentally regulated in mammals and environmentally regulated in lower eukaryotes . Isoforms for one of the subunits, V, in the yeast Saccharomyces cerevisiae and one of the subunits, VII, in the slime mold Dictyostelium discoideum are regulated differentially by oxygen concentration . Extensive studies with the yeast subunit V isoforms have revealed that the genes for these proteins are switched on or off at very low oxygen concentrations (0.5-1 micromol l-1 O2) and that they affect the catalytic properties of holocytochrome c oxidase differentially . By altering an internal step in electron transfer between heme a and the binuclear reaction center (composed of heme a3 and CuB), the 'hypoxic' isoform, Vb, enhances the catalytic constant three- to fourfold relative to the 'aerobic' isoform, Va . Modeling studies suggest that this occurs by an interaction between transmembrane helix VII of subunit I and the transmembrane helix in subunit V . The inverse regulation of these two isoforms allows cells to assemble different types of holoenzyme isoenzymes in response to oxygen concentration . Oxygen also regulates the level of transcription of the genes for the other nuclear-coded subunits of yeast cytochrome c oxidase and affects the level of two of the mitochondrially encoded subunits (I and II) post-transcriptionally . Thus, the level of cytochrome c oxidase activity that is produced at different oxygen tensions in yeast is determined in part by the number of holoenzyme molecules that are assembled and in part by the oxygen-regulated isoforms of subunit V . The possibility that this type of control exists in other organisms is considered.

Neuroreport, 1998 Apr 20, 9(6), 955 - 9
Apoptosis and expression of DNA repair proteins in ischaemic brain injury in man; Love S et al.; We examined the timing of apoptosis and the expression of the DNA repair proteins poly(ADP-ribose) polymerase (PARP) and Ku80 in sections of frontal and temporal lobes from patients who had suffered severe brain ischaemia due to a cardiac arrent . In situ end-labelling (ISEL) was used to detect apoptotic cells, and immunohistochemistry to assess PARP and Ku80 . ISEL of scattered neurons and glia was demonstrable predominantly during the first 24 h after ischaemia . PARP and Ku80 immunoreactivity increased markedly after cerebral ischaemia, PARP particularly in the regions of greatest susceptibility to hypoxic injury: the CA1 field of the hippocampus and the depths of neocortical sulci . The up-regulation of PARP is in keeping with experimental observations concerning the key role of this enzyme in mediating ischaemic cell death.

Genetics, 1998 May, 149(1), 101 - 16
A screen for dynein synthetic lethals in Aspergillus nidulans identifies spindle assembly checkpoint genes and other genes involved in mitosis; Efimov VP et al.; Cytoplasmic dynein is a ubiquitously expressed microtubule motor involved in vesicle transport, mitosis, nuclear migration, and spindle orientation . In the filamentous fungus Aspergillus nidulans, inactivation of cytoplasmic dynein, although not lethal, severely impairs nuclear migration . The role of dynein in mitosis and vesicle transport in this organism is unclear . To investigate the complete range of dynein function in A . nidulans, we searched for synthetic lethal mutations that significantly reduced growth in the absence of dynein but had little effect on their own . We isolated 19 sld (synthetic lethality without dynein) mutations in nine different genes . Mutations in two genes exacerbate the nuclear migration defect seen in the absence of dynein . Mutations in six other genes, including sldA and sldB, show a strong synthetic lethal interaction with a mutation in the mitotic kinesin bimC and, thus, are likely to play a role in mitosis . Mutations in sldA and sldB also confer hypersensitivity to the microtubule-destabilizing drug benomyl . sldA and sldB were cloned by complementation of their mutant phenotypes using an A . nidulans autonomously replicating vector . Sequencing revealed homology to the spindle assembly checkpoint genes BUB1 and BUB3 from Saccharomyces cerevisiae . Genetic interaction between dynein and spindle assembly checkpoint genes, as well as other mitotic genes, indicates that A . nidulans dynein plays a role in mitosis . We suggest a model for dynein motor action in A . nidulans that can explain dynein involvement in both mitosis and nuclear distribution.

J Biol Chem, 1998 May 22, 273(21), 13189 - 96
A genetic screen for aminophospholipid transport mutants identifies the phosphatidylinositol 4-kinase, STT4p, as an essential component in phosphatidylserine metabolism; Trotter PJ et al.; In an effort to understand molecular mechanisms of intracellular lipid transport, we have focused upon specific events required for de novo aminophospholipid synthesis in the yeast Saccharomyces cerevisiae . A genetic system for examining the steps between phosphatidylserine (PtdSer) synthesis in the endoplasmic reticulum and its transport to and decarboxylation by PtdSer decarboxylase 2 in the Golgi/vacuole has been developed . We have isolated a mutant, denoted pstB1, that accumulates PtdSer and has diminished phosphatidylethanolamine formation despite normal PtdSer decarboxylase 2 activity . The lesion in PtdSer metabolism is consistent with a defect in interorganelle lipid transport . A genomic DNA clone that complements the mutation was isolated, and sequencing revealed that the clone contains the STT4 gene, encoding a phosphatidylinositol 4-kinase . The pstB1 mutant exhibits a defect in Stt4p-type phosphatidylinositol 4-kinase activity, and direct gene replacement indicates that STT4 is the defective gene in the mutant . Creation of an STT4 null allele (stt4Delta::HIS3) demonstrates the gene is essential . These results provide evidence that implicates phosphoinositides in the regulation of intracellular aminophospholipid transport.

J Biol Chem, 1998 May 22, 273(21), 13119 - 28
Regulation of protein phosphatase 2A activity by caspase-3 during apoptosis; Santoro MF et al.; Although the available evidence suggests that whereas the caspase family plays a major role in apoptosis, they are not the sole stimulators of death . A random yeast two-hybrid screen of a lymphocyte cDNA library (using caspase-3 as the bait) found an interaction between caspase-3 and the regulatory subunit Aalpha of protein phosphatase 2A . This protein was found to be a substrate for caspase-3, but not caspase-1, and could compete effectively against either a protein or synthetic peptide substrate . In Jurkat cells induced to undergo apoptosis with anti-Fas antibody, protein phosphatase 2A (PP2A) activity increased 4.5-fold after 6 h . By 12 h, the regulatory Aalpha subunit could no longer be detected in cell lysates . There was no change in the amount of the catalytic subunit . The effects on PP2A could be prevented by the caspase family inhibitors acetyl-Asp-Glu-Val-Asp (DEVD) aldehyde or Ac-DEVD fluoromethyl ketone . The mitogen-activated protein (MAP) kinase pathway is regulated by PP2A . At 12 h after the addition of anti-Fas antibody, a decrease in the amount of the phosphorylated forms of MAP kinase was observed . Again, this loss of activated MAP kinase could be prevented by the addition of DEVD-cho or DEVD-fmk . These data are consistent with a pathway whereby induction of apoptosis activates caspase-3 . This enzyme then cleaves the regulatory Aalpha subunit of PP2A, increasing its activity . These data show that the activated PP2A will then effect a change in the phosphorylation state of the cell . These data provide a link between the caspases and signal transduction pathways.

J Biol Chem, 1998 May 22, 273(21), 13058 - 64
Murine cell line SX9 bearing a mutation in the dna-pkcs gene exhibits aberrant V(D)J recombination not only in the coding joint but also in the signal joint; Fukumura R et al.; We established the radiosensitive cell line SX9 from mammary carcinoma cell line FM3A . In SX9 cells a defect of DNA-dependent protein kinase (DNA-PK) activity was suggested . Additionally, a complementation test suggested that the SX9 cell line belongs to a x-ray cross-complementing group (XRCC) 7 . Isolation and sequence analyses of DNA-dependent protein kinase catalytic subunit (dna-pkcs) cDNA in SX9 cells disclosed nucleotide "T" (9572) to "C" transition causing substitution of amino acid residue leucine (3191) to proline . Interestingly, the mutation occurs in one allele, and transcripts of the dna-pkcs expressed exclusively from mutated allele . V(D)J recombination assay using extrachromosomal vector revealed the defects of not only coding but also signal joint formation . The frequency of the signal joint decreased to approximately one-tenth and the fidelity drastically decreased to 12 . 2% as compared with the normal cell line . To confirm the responsibility of the dna-pkcs gene for abnormal V(D)J recombination in SX9, the full-length dna-pkcs gene was introduced into SX9 . As a result, restoration of V(D)J recombination by wild type dna-pkcs cDNA was observed . SX9 is a novel dna-pkcs-deficient cell line.

J Biol Chem, 1998 May 22, 273(21), 12952 - 9
Characterization and cloning of a Dictyostelium Ste20-like protein kinase that phosphorylates the actin-binding protein severin; Eichinger L et al.; After receiving an external stimulus Dictyostelium amoebae are able to rearrange their actin cytoskeleton within seconds, and phosphorylation is a prime candidate for quick modification of cytoskeletal components . We isolated a kinase from cytosolic extracts that specifically phosphorylated severin, a Ca2+-dependent F-actin fragmenting protein . In gel filtration chromatography severin kinase eluted with a molecular mass of about 300 kDa and contained a 62-kDa component whose autophosphorylation caused a mobility shift in SDS-polyacrylamide gel electrophoresis and stimulated phosphorylation of severin . Severin kinase activity could be specifically precipitated with antibodies raised against the 62-kDa polypeptide . Phosphorylation of severin was strongly reduced in the presence of Ca2+, indicating additional regulation at the substrate level . Peptide sequencing and cloning of the cDNA demonstrated that the 62-kDa protein belongs to the Ste20p- or p21-activated protein kinase family . It is most closely related to the germinal center kinase subfamily with its N-terminal positioned catalytic domain followed by a presumptive regulatory domain at the C terminus . The presence of a Ste20-like severin kinase in Dictyostelium suggests a direct signal transduction from the plasma membrane to the cytoskeleton by phosphorylation of actin-binding proteins.

J Biol Chem, 1998 May 22, 273(21), 12935 - 42
Functional interactions among the subunits of replication factor C potentiate and modulate its ATPase activity; Podust VN et al.; Replication factor C (RF-C), a complex of five subunits, and several subassemblies of RF-C, representing intermediates along the proposed protein assembly pathway (Podust, V . N., and Fanning, E . (1997) J . Biol . Chem . 272, 6303-6310), were expressed in insect cells using baculoviruses encoding individual subunits (p140, p40, p38, p37, and p36) . Purified proteins were analyzed for ATPase activity to assess the role of individual subunits in ATP hydrolysis . His-tagged p40 contained low ATPase activity, but tagged p37 and p36 did not . Complexes of p40.p37.p36 bearing a His tag on any subunit displayed DNA-stimulated ATPase activity, in agreement with a recent report (Cai, J., Gibbs, E., Uhlmann, F., Philips, B., Yao, N., O'Donnell, M . , and Hurwitz, J . (1997) J . Biol . Chem . 272, 18974-18981) . In contrast, complex p38.p37.p36-his displayed no ATPase, suggesting that p40 is essential for ATPase activity . Although p38 was not required for ATPase activity, the activity of the p40-his.p38.p37 . p36 complex was more salt-resistant than that of the p40-his.p37.p36 complex . The p140 subunit further increased the specific ATPase activity of RF-C complex by enhancing its stimulation by DNA . Taken together, the data indicate that all five RF-C subunits constitute ATPase activity, although the contributions of the individual subunits differ . Predicted ATP-binding domains of all five subunits were mutated to assess the importance of multiple ATP-binding sites of RF-C . In each case, the Lys of the conserved P-loop motif was replaced by Glu . The ATP-binding domain of p38 was found to be dispensable for the activity of the five-subunit RF-C in polymerase delta DNA synthesis . In contrast, mutation of the ATP-binding domains in other RF-C subunits impaired RF-C assembly, function, or both.

Novartis Found Symp, 1998, 214, 114 - 26; discussion 126-32
Nuclear organization and silencing: trafficking of Sir proteins; Gasser SM et al.; In budding yeast genes integrated near telomeres succumb to a variegated pattern of gene repression that requires the silent information regulatory proteins Sir2p, Sir3p and Sir4p, which form a nucleosome-binding complex . Immunolocalization shows that the Sir proteins co-localize with the telomeric repeat binding protein Rap1p and with telomeric DNA in a limited number of foci near the periphery of interphase nuclei . All conditions tested so far that disrupt telomere proximal repression result in a dispersed staining pattern for Sir2p, Sir3p and Sir4p . Although the focal organization is clearly not sufficient for establishing repression, genetic studies suggest that the high local concentration of Sir proteins at telomeric foci facilitates the formation of repressed chromatin . In addition to its telomeric localization, Sir2p is shown by immunostaining and cross-linking to bind a subdomain of the nucleolus . In strains lacking an intact Sir4p, Sir3p also becomes concentrated in the nucleolus by a pathway requiring SIR2 and UTH4 . This unexpected localization correlates with observed effects of sir mutations on rDNA stability and longevity, defining a new site of action for silent information regulatory factors . We report a novel WD40 repeat-containing factor, Sif2p, that binds specifically to the Sir4p N-terminus . Like Sir1p and Uth4p, Sif2p antagonizes telomeric silencing by regulating an equilibrium between alternative assembly pathways at different subnuclear loci.

Biochem Biophys Res Commun, 1998 May 8, 246(1), 137 - 41
Non-histone chromosomal proteins HMG1 and 2 enhance ligation reaction of DNA double-strand breaks; Nagaki S et al.; DNA ligase IV in a complex with XRCC4 is responsible for DNA end-joining in repair of DNA double-strand breaks (DSB) and V(D)J recombination . We found that non-histone chromosomal high mobility group (HMG) proteins 1 and 2 enhanced the ligation of linearized pUC119 DNA with DNA ligase IV from rat liver nuclear extract . Intra-molecular and inter-molecular ligations of cohesive-ended and blunt-ended DNA were markedly stimulated by HMG1 and 2 . Recombinant HMG2-domain A, B, and (A + B) polypeptides were similarly, but non-identically, effective for the stimulation of DSB ligation reaction . Ligation of single-strand breaks (nicks) was only slightly activated by the HMG proteins . The DNA end-binding Ku protein singly or in combination with the catalytic component of DNA-dependent protein kinase (DNA-PK) as the DNA-PK holoenzyme was ineffective for the ligation of linearized pUC119 DNA . Although the stimulatory effect of HMG1 and 2 on ligation of DSB in vitro was not specific to DNA ligase IV, these results suggest that HMG1 and 2 are involved in the final ligation step in DNA end-joining processes of DSB repair and V(D)J recombination.

Biochem Biophys Res Commun, 1998 May 8, 246(1), 95 - 9
Tyrosine 1213 of Flt-1 is a major binding site of Nck and SHP-2; Igarashi K et al.; Vascular endothelial growth factor (VEGF) binds to its receptor tyrosine kinase Flt-1 and KDR/Flk-1 and stimulates their autophosphorylation . However, little is known about their downstream signal transduction properties . We examined the interactions of certain proteins with a SH2-domain with Flt-1 and KDR using the yeast two-hybrid system and found that Nck, SHP-2, PLC gamma, and PI3K p85 bind to Flt-1 . Extensive site-directed mutagenesis of Flt-1 revealed their major binding sites . Nck, SHP-2, and PI3K bind to Y1213 of Flt-1 . Nck also binds to Y1333 of Flt-1 . These results suggest that Nck, SHP-2, PLC gamma, and PI3K play important roles in Flt-1 signal transduction and that Y1213 of Flt-1 is a major binding site of PI3K, Nck, and SHP-2.

Radiats Biol Radioecol, 1997 Jul-Aug, 37(4), 475 - 81
{Current problems of neutron radiobiology}; Obaturov GM et al.; A brief review of up-date problems of neutron radiobiology, related to neutron therapy development and setting up of radiation safety standards for neutrons, is present . The main attention is paid to the effects of combined gamma-neutron irradiation, peculiarities of reactor neutrons biological action and new approaches in neutron capture therapy . On the basis of own and literature data the results of cellular and whole-body studies as well as the applicability of biophysical modelling for description and interpretation of experimental data are discussed.

Curr Genet, 1998 Apr, 33(4), 284 - 90
Trichoderma reesei prs12 encodes a stress- and unfolded-protein-response-inducible regulatory subunit of the fungal 26S proteasome; Goller SP et al.; We have cloned a gene, prs12, from the filamentous fungus Trichoderma reesei which encodes a fungal homologue of the mouse and Drosophila regulatory subunit 12 of the 26S proteasome (mov34) . Sequencing of both a genomic and a cDNA-clone predicts a 342-aa protein with high overall identity (56-68 %) to the homologous counterparts from human, mammals, Drosophila and Saccharomyces cerevisiae . The predicted protein contains several consensus sequences for phosphorylation, three of which are conserved in all published Prs12p homologues . Its C-terminus is rich in alternating K and E/D, and resembles a potential KEKE-motif . Prs12 exhibits a basal level of transcription during normal growth, but its expression is significantly increased over 60-120 min under conditions of stress evoked by the addition of cadmium ions and hygromycin B . It is also stimulated by the addition of tunicamycin and 2-mercaptoethanol, suggesting its regulation by the presence of unfolded proteins in the endoplasmic reticulum and by hygromycin B . Consistent with this behaviour, motifs in the prs12 5'-upstream sequences show sequence homology with the consensus sequences for general stress response, and for an ER traffic-response element.

EMBO J, 1998 Apr 15, 17(8), 2412 - 25
Regulation of DNA replication and repair proteins through interaction with the front side of proliferating cell nuclear antigen; Jonsson ZO et al.; The DNA polymerase accessory factor proliferating cell nuclear antigen (PCNA) has been caught in interaction with an ever increasing number of proteins . To characterize the sites and functions of some of these interactions, we constructed four mutants of human PCNA and analysed them in a variety of assays . By targeting loops on the surface of the PCNA trimer and changing three or four residues at a time to alanine, we found that a region including part of the domain-connecting loop of PCNA and loops on one face of the trimer, close to the C-termini, is involved in binding to all of the following proteins: DNA polymerase delta, replication factor C, the flap endonuclease Fen1, the cyclin dependent kinase inhibitor p21 and DNA ligase I . An inhibition of DNA ligation caused by the interaction of PCNA with DNA ligase I was found, and we show that DNA ligase I and Fen1 can inhibit DNA synthesis by DNA polymerase delta/PCNA . We demonstrate that PCNA must be located below a 5' flap on a forked template to stimulate Fen1 activity, and considering the interacting region on PCNA for Fen1, this suggests an orientation for PCNA during DNA replication with the C-termini facing forwards, in the direction of DNA synthesis.

EMBO J, 1998 Apr 15, 17(8), 2404 - 11
Ku80 is required for immunoglobulin isotype switching; Casellas R et al.; Isotype switching is the DNA recombination mechanism by which antibody genes diversify immunoglobulin effector functions . In contrast to V(D)J recombination, which is mediated by RAG1, RAG2 and DNA double-stranded break (DSB) repair proteins, little is known about the mechanism of switching . We have investigated the role of DNA DSB repair in switch recombination in mice that are unable to repair DSBs due to a deficiency in Ku80 (Ku80(-/-)) . B-cell development is arrested at the pro-B cell stage in Ku80(-/-) mice because of abnormalities in V(D)J recombination, and there are no mature B cells . To reconstitute the B-cell compartment in Ku80(-/-) mice, pre-rearranged VB1-8 DJH2 (mu i) and V3-83JK2 (kappa i) genes were introduced into the Ku80(-/-) background (Ku80(-/-)mu i/+kappa i/+) . Ku80(-/-)mu i/+ kappai/+ mice develop mature mIgM+ B cells that respond normally to lipopolysaccharide (LPS) or LPS plus interleukin-4 (IL-4) by producing specific germline Ig constant region transcripts and by forming switch region-specific DSBs . However, Ku80(-/-)mu i/+kappa i/+ B cells are unable to produce immunoglobulins of secondary isotypes, and fail to complete switch recombination . Thus, Ku80 is essential for switch recombination in vivo, suggesting a significant overlap between the molecular machinery that mediates DNA DSB repair, V(D)J recombination and isotype switching.

EMBO J, 1998 Apr 15, 17(8), 2224 - 34
Caspase-mediated activation and induction of apoptosis by the mammalian Ste20-like kinase Mst1; Graves JD et al.; Mst1 is a ubiquitously expressed serine-threonine kinase, homologous to the budding yeast Ste20, whose physiological regulation and cellular function are unknown . In this paper we show that Mst1 is specifically cleaved by a caspase 3-like activity during apoptosis induced by either cross-linking CD95/Fas or by staurosporine treatment . CD95/Fas-induced cleavage of Mst1 was blocked by the cysteine protease inhibitor ZVAD-fmk, the more selective caspase inhibitor DEVD-CHO and by the viral serpin CrmA . Caspase-mediated cleavage of Mst1 removes the C-terminal regulatory domain and correlates with an increase in Mst1 activity in vivo, consistent with caspase-mediated cleavage activating Mst1 . Overexpression of either wild-type Mst1 or a truncated mutant induces morphological changes characteristic of apoptosis . Furthermore, exogenously expressed Mst1 is cleaved, indicating that Mst1 can activate caspases that result in its cleavage . Kinase-dead Mst1 did not induce morphological alterations and was not cleaved upon overexpression, indicating that Mst1 must be catalytically active in order to mediate these effects . Mst1 activates MKK6, p38 MAPK, MKK7 and SAPK in co-transfection assays, suggesting that Mst1 may activate these pathways . Our findings suggest the existence of a positive feedback loop involving Mst1, and possibly the SAPK and p38 MAPK pathways, which serves to amplify the apoptotic response.

EMBO J, 1998 Apr 15, 17(8), 2208 - 14
A novel protein modification pathway related to the ubiquitin system; Liakopoulos D et al.; Ubiquitin conjugation is known to target protein substrates primarily to degradation by the proteasome or via the endocytic route . Here we describe a novel protein modification pathway in yeast which mediates the conjugation of RUB1, a ubiquitin-like protein displaying 53% amino acid identity to ubiquitin . We show that RUB1 conjugation requires at least three proteins in vivo . ULA1 and UBA3 are related to the N- and C-terminal domains of the E1 ubiquitin-activating enzyme, respectively, and together fulfil E1-like functions for RUB1 activation . RUB1 conjugation also requires UBC12, a protein related to E2 ubiquitin-conjugating enzymes, which functions analogously to E2 enzymes in RUB1-protein conjugate formation . Conjugation of RUB1 is not essential for normal cell growth and appears to be selective for a small set of substrates . Remarkably, CDC53/cullin, a common subunit of the multifunctional SCF ubiquitin ligase, was found to be a major substrate for RUB1 conjugation . This suggests that the RUB1 conjugation pathway is functionally affiliated to the ubiquitin-proteasome system and may play a regulatory role.

EMBO J, 1998 Apr 15, 17(8), 2196 - 207
Mtr10p functions as a nuclear import receptor for the mRNA-binding protein Npl3p; Senger B et al.; MTR10, previously shown to be involved in mRNA export, was found in a synthetic lethal relationship with nucleoporin NUP85 . Green fluorescent protein (GFP)-tagged Mtr10p localizes preferentially inside the nucleus, but a nuclear pore and cytoplasmic distribution is also evident . Purified Mtr10p forms a complex with Npl3p, an RNA-binding protein that shuttles in and out of the nucleus . In mtr10 mutants, nuclear uptake of Npl3p is strongly impaired at the restrictive temperature, while import of a classic nuclear localization signal (NLS)-containing protein is not . Accordingly, the NLS within Npl3p is extended and consists of the RGG box plus a short and non-repetitive C-terminal tail . Mtr10p interacts in vitro with Gsp1p-GTP, but with low affinity . Interestingly, Npl3p dissociates from Mtr10p only by incubation with Ran-GTP plus RNA . This suggests that Npl3p follows a distinct nuclear import pathway and that intranuclear release from its specific import receptor Mtr10p requires the cooperative action of both Ran-GTP and newly synthesized mRNA.

EMBO J, 1998 Apr 15, 17(8), 2156 - 65
Initial docking of ER-derived vesicles requires Uso1p and Ypt1p but is independent of SNARE proteins; Cao X et al.; ER-to-Golgi transport in yeast may be reproduced in vitro with washed membranes, purified proteins (COPII, Uso1p and LMA1) and energy . COPII coated vesicles that have budded from the ER are freely diffusible but then dock to Golgi membranes upon the addition of Uso1p . LMA1 and Sec18p are required for vesicle fusion after Uso1p function . Here, we report that the docking reaction is sensitive to excess levels of Sec19p (GDI), a treatment that removes the GTPase, Ypt1p . Once docked, however, vesicle fusion is no longer sensitive to GDI . In vitro binding experiments demonstrate that the amount of Uso1p associated with membranes is reduced when incubated with GDI and correlates with the level of membrane-bound Ypt1p, suggesting that this GTPase regulates Uso1p binding to membranes . To determine the influence of SNARE proteins on the vesicle docking step, thermosensitive mutations in Sed5p, Bet1p, Bos1p and Sly1p that prevent ER-to-Golgi transport in vitro at restrictive temperatures were employed . These mutations do not interfere with Uso1p-mediated docking, but block membrane fusion . We propose that an initial vesicle docking event of ER-derived vesicles, termed tethering, depends on Uso1p and Ypt1p but is independent of SNARE proteins.

Biochem J, 1997 Nov 1, 327 ( Pt 3), 625 - 35
Furin: a mammalian subtilisin/Kex2p-like endoprotease involved in processing of a wide variety of precursor proteins; Nakayama K; Limited endoproteolysis of inactive precursor proteins at sites marked by paired or multiple basic amino acids is a widespread process by which biologically active peptides and proteins are produced within the secretory pathway in eukaryotic cells . The identification of a novel family of endoproteases homologous with bacterial subtilisins and yeast Kex2p has accelerated progress in understanding the complex mechanisms underlying the production of the bioactive materials . Seven distinct proprotein convertases of this family (furin, PC2, PC1/PC3, PC4, PACE4, PC5/PC6, LPC/PC7/PC8/SPC7) have been identified in mammalian species, some having isoforms generated via alternative splicing . The family has been shown to be responsible for conversion of precursors of peptide hormones, neuropeptides, and many other proteins into their biologically active forms . Furin, the first proprotein convertase to be identified, has been most extensively studied . It has been shown to be expressed in all tissues and cell lines examined and to be mainly localized in the trans-Golgi network, although some proportion of the furin molecules cycle between this compartment and the cell surface . This endoprotease is capable of cleaving precursors of a wide variety of proteins, including growth factors, serum proteins, including proteases of the blood-clotting and complement systems, matrix metalloproteinases, receptors, viral-envelope glycoproteins and bacterial exotoxins, typically at sites marked by the consensus Arg-Xaa-(Lys/Arg)-Arg sequence . The present review covers the structure and function of mammalian subtilisin/Kex2p-like proprotein convertases, focusing on furin (EC 3.4.21.85).

FEBS Lett, 1998 Apr 10, 426(1), 57 - 61
Repulsive interparticle interactions in a denatured protein solution revealed by small angle neutron scattering; Receveur V et al.; In order to investigate the effect of concentration in biological processes such as protein folding, small angle neutron scattering measurements were used to determine the second virial coefficient of solutions of both native and strongly denatured phosphoglycerate kinase and the radius of gyration of the protein at zero concentration . The value of the second virial coefficient is a good probe of the non-ideality of a solution . The present results show that the unfolding of the protein leads to a drastic change in the repulsive intermolecular interactions . We conclude that these interactions are due mainly to the behaviour of the denatured polypeptide chain as an excluded volume polymer.

Genomics, 1998 Apr 15, 49(2), 327 - 30
Cloning, expression, and chromosomal localization of human long-chain fatty acid-CoA ligase 4 (FACL4); Cao Y et al.; Long-chain fatty acid-CoA ligase (also called fatty acid acyl-CoA synthetase) plays an essential role in lipid biosynthesis and fatty acid degradation . We report herein the cDNA cloning of the human long-chain fatty acid-CoA ligase 4 from a brain library . The cDNA encodes a functional long-chain fatty acid-CoA ligase that shows preference for arachidonic acid as substrate . We also studied the tissue distribution of gene expression by Northern hybridization . Human placenta, brain, testis, ovary, spleen, and adrenal cortex have the highest levels of expression of the long-chain fatty acid-CoA ligase 4, whereas the GI system has the lowest . Finally, this gene was localized to chromosome Xq23 in human by FISH analysis.

Science, 1998 May 8, 280(5365), 915 - 8
Identification of non-heme diiron proteins that catalyze triple bond and epoxy group formation; Lee M et al.; Acetylenic bonds are present in more than 600 naturally occurring compounds . Plant enzymes that catalyze the formation of the Delta12 acetylenic bond in 9-octadecen-12-ynoic acid and the Delta12 epoxy group in 12,13-epoxy-9-octadecenoic acid were characterized, and two genes, similar in sequence, were cloned . When these complementary DNAs were expressed in Arabidopsis thaliana, the content of acetylenic or epoxidated fatty acids in the seeds increased from 0 to 25 or 15 percent, respectively . Both enzymes have characteristics similar to the membrane proteins containing non-heme iron that have histidine-rich motifs.

J Cell Biol, 1998 Apr 20, 141(2), 349 - 58
Cytoplasmic tail phosphorylation of the alpha-factor receptor is required for its ubiquitination and internalization; Hicke L et al.; G protein-coupled (GPC) receptors are phosphorylated in response to ligand binding, a modification that promotes receptor desensitization or downregulation . The alpha-factor pheromone receptor (Ste2p) of Saccharomyces cerevisiae is a GPC receptor that is hyperphosphorylated and ubiquitinated upon binding alpha-factor . Ubiquitination triggers Ste2p internalization into the endocytic pathway . Here we demonstrate that phosphorylation of Ste2p promotes downregulation by positively regulating ubiquitination and internalization . Serines and a lysine are essential elements of the Ste2p SINNDAKSS internalization signal that can mediate both constitutive and ligand-stimulated endocytosis . The SINNDAKSS serines are required for receptor phosphorylation which, in turn, facilitates ubiquitination of the neighboring lysine . Constitutive phosphorylation is required to promote constitutive internalization, and is also a prerequisite for ligand-induced phosphorylation at or near the SINNDAKSS sequence . Mutants defective in yeast casein kinase I homologues are unable to internalize alpha-factor, and do not phosphorylate or ubiquitinate the receptor, indicating that these kinases play a direct or indirect role in phosphorylating the receptor . Finally, we provide evidence that the primary function of phosphorylation controlled by the SINNDAKSS sequence is to trigger receptor internalization, demonstrating that phosphorylation-dependent endocytosis is an important mechanism for the downregulation of GPC receptor activity.

J Cell Biol, 1998 Apr 20, 141(2), 335 - 48
The genomic sequences bound to special AT-rich sequence-binding protein 1 (SATB1) in vivo in Jurkat T cells are tightly associated with the nuclear matrix at the bases of the chromatin loops; de Belle I et al.; Special AT-rich sequence-binding protein 1 (SATB1), a DNA-binding protein expressed predominantly in thymocytes, recognizes an ATC sequence context that consists of a cluster of sequence stretches with well-mixed A's, T's, and C's without G's on one strand . Such regions confer a high propensity for stable base unpairing . Using an in vivo cross-linking strategy, specialized genomic sequences (0.1-1 . 1 kbp) that bind to SATB1 in human lymphoblastic cell line Jurkat cells were individually isolated and characterized . All in vivo SATB1-binding sequences examined contained typical ATC sequence contexts, with some exhibiting homology to autonomously replicating sequences from the yeast Saccharomyces cerevisiae that function as replication origins in yeast cells . In addition, LINE 1 elements, satellite 2 sequences, and CpG island-containing DNA were identified . To examine the higher-order packaging of these in vivo SATB1-binding sequences, high-resolution in situ fluorescence hybridization was performed with both nuclear "halos" with distended loops and the nuclear matrix after the majority of DNA had been removed by nuclease digestion . In vivo SATB1-binding sequences hybridized to genomic DNA as single spots within the residual nucleus circumscribed by the halo of DNA and remained as single spots in the nuclear matrix, indicating that these sequences are localized at the base of chromatin loops . In human breast cancer SK-BR-3 cells that do not express SATB1, at least one such sequence was found not anchored onto the nuclear matrix . These findings provide the first evidence that a cell type-specific factor such as SATB1 binds to the base of chromatin loops in vivo and suggests that a specific chromatin loop domain structure is involved in T cell-specific gene regulation.

J Biol Chem, 1998 Apr 17, 273(16), 9577 - 85
The guanylyltransferase domain of mammalian mRNA capping enzyme binds to the phosphorylated carboxyl-terminal domain of RNA polymerase II; Ho CK et al.; We have conducted a biochemical and genetic analysis of mouse mRNA capping enzyme (Mce1), a bifunctional 597-amino acid protein with RNA triphosphatase and RNA guanylyltransferase activities . The principal conclusions are as follows: (i) the mammalian capping enzyme consists of autonomous and nonoverlapping functional domains; (ii) the guanylyltransferase domain Mce1(211-597) is catalytically active in vitro and functional in vivo in yeast in lieu of the endogenous guanylyltransferase Ceg1; (iii) the guanylyltransferase domain per se binds to the phosphorylated RNA polymerase II carboxyl-terminal domain (CTD), whereas the triphosphatase domain, Mce1(1-210), does not bind to the CTD; and (iv) a mutation of the active site cysteine of the mouse triphosphatase elicits a strong growth-suppressive phenotype in yeast, conceivably by sequestering pre-mRNA ends in a nonproductive complex or by blocking access of the endogenous yeast triphosphatase to RNA polymerase II . These findings contribute to an emerging model of mRNA biogenesis wherein RNA processing enzymes are targeted to nascent polymerase II transcripts through contacts with the CTD . The phosphorylation-dependent interaction between guanylyltransferase and the CTD is conserved from yeast to mammals.

J Biol Chem, 1998 Apr 17, 273(16), 9365 - 8
A novel protein distinguishes between quiescent and activated forms of the type I transforming growth factor beta receptor; Charng MJ et al.; Transforming growth factor beta (TGFbeta) signal transduction is mediated by two receptor Ser/Thr kinases acting in series, type II TGFbeta receptor (TbetaR-II) phosphorylating type I TGFbeta receptor (TbetaR-I) . Because the failure of interaction cloning, thus far, to identify bona fide TbetaR-I substrates might reasonably have been due to the use of inactive TbetaR-I as bait, we sought to identify molecules that interact specifically with active TbetaR-I, employing the triple mutation L193A,P194A,T204D in a yeast two-hybrid system . The Leu-Pro substitutions prevent interaction with FK506-binding protein 12 (FKBP12), whose putative function in TGFbeta signaling we have previously disproved; the charge substitution at Thr204 constitutively activates TbetaR-I . Unlike previous screens using wild-type TbetaR-I, where FKBP12 predominated, none of the resulting colonies encoded FKBP12 . A novel protein was identified, TbetaR-I-associated protein-1 (TRAP-1), that interacts in yeast specifically with mutationally activated TbetaR-I, but not wild-type TbetaR-I, TbetaR-II, or irrelevant proteins . In mammalian cells, TRAP-1 was co-precipitated only by mutationally activated TbetaR-I and ligand-activated TbetaR-I, but not wild-type TbetaR-I in the absence of TGFbeta . The partial TRAP-1 protein that specifically binds these mutationally and ligand-activated forms of TbetaR-I can inhibit signaling by the native receptor after stimulation with TGFbeta or by the constitutively activated receptor mutation, as measured by a TGFbeta-dependent reporter gene . Thus, TRAP-1 can distinguish activated forms of the receptor from wild-type receptor in the absence of TGFbeta and may potentially have a functional role in TGFbeta signaling.

J Biol Chem, 1998 Apr 17, 273(16), 9353 - 6
Interaction of Drosophila inhibitors of apoptosis with thick veins, a type I serine/threonine kinase receptor for decapentaplegic; Oeda E et al.; Decapentaplegic (Dpp) is a Drosophila member of bone morphogenetic proteins, which belong to the transforming growth factor-beta superfamily . Members of this family regulate a variety of biological processes such as cell proliferation, morphogenesis, immune response, and apoptosis . Dpp plays a critical role in many aspects of Drosophila development . Members of the transforming growth factor-beta superfamily bind to two different types of serine/threonine kinase receptors, termed type I and type II . Type I receptors act as downstream components of type II receptors in the receptor complexes . Therefore, intracellular proteins that interact with the type I receptors are likely to play important roles in signaling . Several proteins have been identified through protein-protein interaction screenings . We identified Drosophila inhibitor of apoptosis (DIAP) 1 as an interacting protein of a Dpp type I receptor, Thick veins (Tkv) . DIAP1 associates with Tkv in vivo . The binding region in DIAP1 is mapped to its C-terminal RING finger region . DIAP2, another Drosophila member of the inhibitor of apoptosis protein family, also interacts with Tkv in vivo . These data suggest that DIAP1 and DIAP2 may be involved, possibly as negative regulators, in the Dpp signaling pathway, which leads to cell apoptosis.

Hum Mol Genet, 1998 May, 7(5), 847 - 53
Acyl-CoA:dihydroxyacetonephosphate acyltransferase: cloning of the human cDNA and resolution of the molecular basis in rhizomelic chondrodysplasia punctata type 2; Ofman R et al.; Rhizomelic chondrodysplasia punctata (RCDP) is a genetic disorder which is clinically characterized by rhizomelic shortening of the upper extremities, typical dysmorphic facial appearance, congenital contractures and severe growth and mental retardation . Patients with RCDP can be subdivided into three subgroups based on biochemical analyses and complementation studies . The largest subgroup contains patients with mutations in the PEX7 gene encoding the PTS2 receptor . This results in multiple peroxisomal abnormalities which includes a deficiency of acyl-CoA:dihydroxyacetonephosphate acyltransferase (DHAPAT), alkyl-dihydroxyacetonephosphate synthase (alkyl-DHAP synthase), peroxisomal 3-ketoacyl-CoA thiolase and phytanoyl-CoA hydroxylase, although there are differences in the extent of the deficiencies observed . Patients in the two other subgroups have been reported to be either deficient in the activity of DHAPAT (RCDP type 2) or alkyl-DHAP synthase (RCDP type 3) while no other abnormalities could be observed . To examine whether the gene encoding DHAPAT is mutated in patients with RCDP type 2, we determined the N-terminal amino acid sequence of the enzyme isolated from human placenta . Using this sequence as a query, we identified a 2040 bp open reading frame (ORF) in the human database of expressed sequence tags . Expression of this ORF in the yeast Saccharomyces cerevisiae showed that we have identified the DHAPAT cDNA . The deduced amino acid sequence revealed no PTS2 consensus sequence . In contrast DHAPAT appears to contain a putative PTS1 at the extreme C-terminus . All RCDP type 2 patients analyzed were found to contain mutations in their DHAPAT cDNA . This demonstrates that RCDP type 2 is the result of mutations in DHAPAT.

Plant Physiol, 1998 Apr, 116(4), 1299 - 305
The phytochrome response of the Lemna gibba NPR1 gene is mediated primarily through changes in abscisic acid levels; Weatherwax SC et al.; Two important signaling systems involved in the growth and development of plants, those triggered by the photoreceptor phytochrome and the hormone abscisic acid (ABA), are involved in the regulation of expression of the NPR1 gene of Lemna gibba . We previously demonstrated that phytochrome action mediates changes in ABA levels in L . gibba, correlating with changes in gene expression evoked by stimulation of the phytochrome system . We have now further characterized phytochrome- and ABA-mediated regulation of L . gibba NPR1 gene expression using a transient particle bombardment assay, demonstrating that regulatory elements controlling responses to both stimuli reside within 156 nucleotides upstream of the transcription start . Linker scan (LS) analysis of the region from -156 to -70 was used to identify two specific requisite and nonredundant cis-acting promoter elements between -143 to -135 (LS2) and -113 to -101 (LS5) . Mutation of either of these elements resulted in a coordinate loss of regulation by phytochrome and ABA . This suggests that, unlike the L . gibba Lhcb2*1 promoter, in which phytochrome and ABA regulatory elements are separable, the phytochrome response of the L . gibba NPR1 gene can be attributed to alterations in ABA levels.

Plant Physiol, 1998 May, 117(1), 85 - 90
Tomato fructokinases exhibit differential expression and substrate regulation
Kanayama Y, Granot D, Dai N, Petreikov M, Schaffer A, Powell A, Bennett AB.
Two divergent genes encoding fructokinase, Frk1 and Frk2, have been previously shown to be expressed in tomato (Lycopersicon esculentum L.) and have now been further characterized with regard to their spatial expression and the enzymic properties of the encoded proteins . Frk1 and Frk2 mRNA levels were coordinately induced by exogenous sugar, indicating that both belong to the growing class of sugar-regulated genes . However, in situ hybridization indicated that Frk1 and Frk2 were expressed in a spatially distinct manner, with Frk2 mRNA primarily localized in cells of the fruit pericarp, which store starch, and Frk1 mRNA distributed ubiquitously in pericarp tissue . To evaluate the biochemical characteristics of the products of the Frk1 and Frk2 genes, each cDNA was expressed in a mutant yeast (Saccharomyces cerevisiae) line defective in hexose phosphorylation and unable to grow on glucose or fructose (Fru) . Both Frk1 and Frk2 proteins expressed in yeast conferred the ability to grow on Fru and exhibited fructokinase activity in vitro . Although both Frk1 and Frk2 both utilized Fru as a substrate, only Frk2 activity was inhibited at high Fru concentrations . These results indicate that Frk2 can be distinguished from Frk1 by its sensitivity to substrate inhibition and by its temporal and spatial pattern of expression, which suggests that it plays a primary role in plant cells specialized for starch storage.

Trends Genet, 1998 Apr, 14(4), 151 - 5
The riddle of MAP kinase signaling specificity; Madhani HD et al.; Cells encounter an enormous variety of signals in their environments and must respond to each stimulus appropriately with changes in their genetic programs . Many of these external signals are transduced by a highly conserved eukaryotic signaling mechanism, the mitogen-activated protein kinase (MAPK) cascade . How are the myriad of inputs transduced accurately so that each evokes a specific response? One mechanism would be to have a distinct MAPK cascade for each signal; however, the situation appears to be more complicated.

Clin Chem Lab Med, 1998 Feb, 36(2), 83 - 6
An enzymatic method for the determination of free fatty acids in serum/plasma; Kiziltunc A et al.; Estimation of serum free fatty acids (FFA) in serum based on the formation of inorganic phosphate has been simplified by eliminating complex stages . The principle of the present method is based on breakdown of pyrophosphate, formed by thioesterification of free fatty acids with ATP and CoA with the aid of acyl-CoA synthetase (EC 6.2.1.3) to inorganic phosphate . This is measured using the reaction with molybdate . The reaction equations are as follows: {formula: see text} The recovery of free fatty acids was 96% . The interferences of citrate, phosphatidylserine, succinate, ascorbic acid and lecithin were between 0.5 and 2% . The correlation between the new enzymatic and the classic enzymatic method was 0.966 . The lower detection limit was 0.018 mmol/l . The method was linear between 0.02 and 2.0 mmol/l . The within-assay and between-assay imprecision (CV) of control sera was 5.5% and 8%, respectively.

Nucleic Acids Res, 1998 Jun 1, 26(11), 2580 - 5
Function of the C-terminal transactivation domain of human heat shock factor 2 is modulated by the adjacent negative regulatory segment; Yoshima T et al.; DNA binding of heat shock factor 2 (HSF2) is induced during hemin-induced differentiation of human erythroleukemia cell line K562 . To identify the transcriptional activation and the regulatory domains of HSF2, we constructed a series of deletion derivatives fused to the yeast GAL4 DNA binding domain and analyzed their transactivation activity . A minimal transactivation domain of HSF2 was localized to the C-terminus (residues 472-536), as in HSF1, although amino acid sequence similarity for these regions was rather limited and the potential transactivation ability was about 25% that of HSF1 . The transactivation mediated by this region of HSF2 was found to be negatively regulated by the adjacent 18 amino acid segment (residues 428-445) under normal conditions . Furthermore, the latter segment, when fused to the GAL4 activation domain, markedly inhibited GAL4 activity . Extract containing most derivatives of HSF2 retaining this segment exhibited doublet or triplet bands in gel mobility shift assays with heat shock element-containing DNA, suggesting possible involvement of some factors interacting with that segment in the negative regulation . Another putative transactivation domain and two negative regulatory regions were also localized within the internal region.

J Biochem (Tokyo), 1998 Apr, 123(4), 722 - 32
Identification of the protein import components of the rat mitochondrial inner membrane, rTIM17, rTIM23, and rTIM44; Ishihara N et al.; We cloned rat liver mitochondrial 18.1, 21.9, and 51.0 kDa proteins with a significant structural homology to the components of the translocase of the yeast mitochondrial inner membrane, Tim17, Tim23, and Tim44 . The 18.1 and 21.9 kDa proteins were synthesized as mature forms having four potential transmembrane segments and localized to the mitochondrial inner membrane . The 51.0 kDa protein is a precursor having a presequence of approximately 6 kDa which is cleaved during import into the mitochondria . The mature 45 kDa protein is located in the matrix, both in a soluble form and in a membrane-bound, alkali-extractable form . Immunofluorescence microscopy confirmed the location of all three proteins in the mitochondria . Antibodies against the 21.9 kDa protein, but not those against the 18.1 and 51.0 kDa proteins, inhibited the precursor import into the mitoplasts in vitro . Immunoprecipitation indicated that all three proteins interacted with the protein in transit to the matrix . Immunoprecipitation also revealed that the 18.1 kDa protein formed a complex with the 21.9 kDa protein and the 45 kDa protein with mHsp70; the latter complex was dissociated in an ATP- or ADP-dependent manner and the reaction was impeded by AMP-PNP or inorganic phosphate . These assays thus demonstrated the 18.1, 21.9, and 45 kDa proteins to be the translocator components of the rat mitochondrial inner membrane and, therefore, the functional homologues of Tim17, Tim23, and Tim44, respectively.

Nat Genet, 1998 May, 19(1), 32 - 8
X-linked dyskeratosis congenita is caused by mutations in a highly conserved gene with putative nucleolar functions; Heiss NS et al.; X-linked recessive dyskeratosis congenita (DKC) is a rare bone-marrow failure disorder linked to Xq28 . Hybridization screening with 28 candidate cDNAs resulted in the detection of a 3' deletion in one DKC patient with a cDNA probe (derived from XAP101) . Five different missense mutations in five unrelated patients were subsequently identified in XAP101, indicating that it is the gene responsible for X-linked DKC (DKC1) . DKC1 is highly conserved across species barriers and is the orthologue of rat NAP57 and Saccharomyces cerevisiae CBF5 . The peptide dyskerin contains two TruB pseudouridine (psi) synthase motifs, multiple phosphorylation sites, and a carboxy-terminal lysine-rich repeat domain . By analogy to the function of the known dyskerin orthologues, involvement in the cell cycle and nucleolar function is predicted for the protein.

Cell, 1998 May 1, 93(3), 349 - 59
Zip2, a meiosis-specific protein required for the initiation of chromosome synapsis; Chua PR et al.; We describe the identification and characterization of the Saccharomyces cerevisiae ZIP2 gene, which encodes a novel meiosis-specific protein essential for synaptonemal complex formation . In the zip2 mutant, chromosomes are homologously paired but not synapsed . The Zip2 protein localizes to discrete foci on meiotic chromosomes; these foci correspond to sites of convergence between paired homologs that are believed to be sites of synapsis initiation . Localization of Zip2p requires the initiation of meiotic recombination . In a mutant defective in double-strand break repair, Zip2p colocalizes with proteins involved in double-strand break formation and processing . We propose that Zip2p promotes the initiation of chromosome synapsis and that localization of Zip2p to sites of interhomolog recombination ensures synapsis between homologous chromosomes.

Radiat Res, 1998 May, 149(5), 440 - 5
Wortmannin inhibits repair of DNA double-strand breaks in irradiated normal human cells; Okayasu R et al.; Wortmannin, a specific inhibitor of PI-3 kinase, was recently found to be an effective radiosensitizer in cells of various human and murine cell lines . Another study indicated that wortmannin inhibited repair of DNA double-strand breaks (DSBs) in irradiated Chinese hamster ovary cells using the neutral elution assay . To further clarify the mechanism behind radiosensitization by wortmannin, we have studied DSB repair in gamma-irradiated normal human fibroblasts using pulsed-field gel electrophoresis . The rejoining of DSBs in irradiated cells was significantly inhibited when 20 microM or more of wortmannin was added to the cells . The colony formation assay in cultures treated with wortmannin showed that the radiosensitization occurred in a manner that was dependent on the drug concentration . However, significant sensitization was observed only with a concentration of wortmannin of 20 microM or higher, reflecting the results of DSB rejoining studies . No marked reduction in plating efficiencies was observed for cells treated with wortmannin alone . The studies of the levels of expression of DNA-dependent protein kinase (DNA-PK) indicated that, while there were no significant changes in expression of Ku protein, the expression of the DNA-PK catalytic subunit (DNA-PKcs) was reduced markedly in cultures treated with wortmannin using an antibody against the C-terminus region of DNA-PKcs . In addition, no reduction in the levels of expression of DNA-PKcs was observed in cells treated with wortmannin using an antibody which recognizes a mid-region of this large protein . These results together with those of related studies suggest that wortmannin radiosensitizes normal human cells by inhibiting DSB repair and that this inhibition is a consequence of an inactivation of kinase activity and/or a structural change caused by binding of wortmannin to the C-terminus region of DNA-PKcs.

Biochem Biophys Res Commun, 1998 Apr 28, 245(3), 900 - 5
A putative tumor suppressor, TSG101, acts as a transcriptional suppressor through its coiled-coil domain; Watanabe M et al.; TSG101 is thought as a putative tumor suppressor gene, and mutations of this gene were recently found in 7 of 15 breast cancer patients, though the physiological function remains to be elucidated . In this report, we showed that TSG101 protein acts as a transcriptional suppressor for estrogen receptor (ER) as well as other members of the nuclear hormone receptor super-family, VP16, and on its own . The basal promoter activity was also inhibited by TSG101 . The suppression of transcription by TSG101 protein required its coiled-coil domain, which is also shown to be required for the tumor suppressive function . Expressed TSG101 protein did not have any histone acetylase nor deacetylase activity, which certain transcriptional co-factors have . The requirement of the same domain in the TSG101 protein for transcriptional suppression and in the tumor suppression indicates a possibility that transcriptional suppression of TSG101 is related to its tumor suppression.

Proc Natl Acad Sci U S A, 1998 May 12, 95(10), 5521 - 6
Persistence of an alternate chromatin structure at silenced loci in the absence of silencers; Cheng TH et al.; In Saccharomyces cerevisiae, genes placed near telomeres or the silent HML and HMR mating-type loci are transcriptionally repressed by a heterochromatin-like structure . We have generated nonreplicating DNA rings by recombination in vivo to examine the role of chromosomal context on transcriptional repression . Specifically, recombination at HMR was used to produce rings that lacked the E and I silencers . An altered level of DNA supercoiling was observed in these rings but not in comparable rings from derepressed loci . Our results indicate that a repressive chromatin structure persists in an extrachromosomal environment immediately following removal of the cis-acting control elements . Examination of both chromatin footprints and DNA sequence dependence revealed that changes in nucleosome number could account for the topology shifts . Upon continued cell growth, the differences in supercoiling were lost and transcriptional competence was restored . These results show that silencers are required for sustained persistence of repressive chromatin structure, even in the absence of DNA replication.

Curr Biol, 1998 Apr 23, 8(9), R299 - 302
Peroxisome biogenesis: back to the endoplasmic reticulum?
Kunau WH, Erdmann R.
Proteins are targeted to the membrane and matrix of peroxisomes by distinct pathways . Recent observations suggest a further route: a subset of peroxisomal membrane proteins might be targeted first to the endoplasmic reticulum, and from there to peroxisomes by vesicle-mediated transport.

Development, 1998 Jun, 125(12), 2263 - 71
Identifying loci required for follicular patterning using directed mosaics; Duffy JB et al.; We have developed a 'directed mosaic' system in Drosophila by using the GAL4 system to control the expression of the yeast recombinase, FLP, in a spatial and temporal fashion . By directing FLP expression, we show that it is possible to efficiently and specifically target loss-of-function studies for vital loci to the developmental pathway of interest . A simple F1 adult phenotypic screen demonstrated that most adult tissues can be analyzed with this approach . Using GAL4 lines expressed during oogenesis, we have refined the system to examine the roles of vital loci in the development of the follicular epithelium . We have identified essential genes involved in egg chamber organization, cell migration and cell shape . Further, we have used this technique to gain insights into the role of the Drosophila EGF receptor pathway in establishing the egg axes . Finally, using different UAS-FLP, GAL4 and existing FRT lines, we have built stocks that permit the analysis of approximately 95% of the genome in follicular mosaics.

Curr Biol, 1998 Mar 26, 8(7), R238 - 40
The exosome: a versatile RNA processing machine; Decker CJ; A conserved complex of multiple 3'-to-5' exonucleases has been discovered recently in eukaryotes . This essential complex, the exosome, is required for diverse RNA processing events.

Biochemistry, 1998 Apr 21, 37(16), 5666 - 72
Kinetic studies of sequence-specific binding of GCN4-bZIP peptides to DNA strands immobilized on a 27-MHz quartz-crystal microbalance; Okahata Y et al.; Specific protein-DNA interaction was studied quantitatively by using a highly sensitive 27-MHz quartz-crystal microbalance (QCM) . Biotinylated DNA double strands (21 bp, having a CRE site of 5'ATGACGTCAT3') were immobilized on an avidin-bound QCM surface, and sequence-specific binding of bZIP 56-mer peptides (having both the basic region for binding and the leucine zipper region for dimerization) to the DNA strand on the QCM was observed . The binding amount (Deltam) at the nanogram level and kinetic parameters such as association constants (Ka) and binding and dissociation rate constants (k1 and k-1) could be obtained from time courses of QCM frequency decreases . A bZIP peptide as a dimer was observed to bind sequence-specifically to one DNA strand having a CRE site . Ka values of ss-bZIP, in which the leucine-zipper region of bZIP was substituted by a Cys-Cys linkage, were largely decreased, and the sequence selectivity also disappeared . Ka values obtained by the QCM method showed good agreement with those obtained from the conventional gel mobility shift assay or from circular dichroism spectrum changes . When the specific sequence of the CRE site of DNA strands was partly changed, Ka values decreased by about a half due to the increase of the dissociation rate constant (k-1) independent of the binding rate constant (k1).

J Comput Biol, 1998 Spring, 5(1), 101 - 11
Expectation and variance of true and false fragment matches in DNA restriction mapping; Siegel AF et al.; Consider a DNA mapping project in which overlap of clones is inferred from multiple complete restriction enzyme digests . Each enzyme cuts each clone randomly into fragments whose lengths are determined with some error . Clones that share fragments with matching lengths could contain a region of overlap . However, common fragment lengths may be due to random coincidence leading to a false overlap declaration . Although the probability of false fragment matching is small, a mapping project involves a large number of clone comparisons . Consequently, erroneous fragment matches can be a serious problem . We use a geometrical probability approach to develop exact integral formulas and first-order approximations for the expected number and variance of classes of fragment pairs that will be identified falsely as matching . We also find exact formulas for the expected value, and variance of the number of true fragment matches . These formulas are useful in comparing different mapping strategies.

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