J Cell Biol, 1999 May 31, 145(5), 961 - 72 The J-related segment of tim44 is essential for cell viability: a mutant Tim44 remains in the mitochondrial import site, but inefficiently recruits mtHsp70 and impairs protein translocation; Merlin A et al.; Tim44 is a protein of the mitochondrial inner membrane and serves as an adaptor protein for mtHsp70 that drives the import of preproteins in an ATP-dependent manner . In this study we have modified the interaction of Tim44 with mtHsp70 and characterized the consequences for protein translocation . By deletion of an 18-residue segment of Tim44 with limited similarity to J-proteins, the binding of Tim44 to mtHsp70 was weakened . We found that in the yeast Saccharomyces cerevisiae the deletion of this segment is lethal . To investigate the role of the 18-residue segment, we expressed Tim44Delta18 in addition to the endogenous wild-type Tim44 . Tim44Delta18 is correctly targeted to mitochondria and assembles in the inner membrane import site . The coexpression of Tim44Delta18 together with wild-type Tim44, however, does not stimulate protein import, but reduces its efficiency . In particular, the promotion of unfolding of preproteins during translocation is inhibited . mtHsp70 is still able to bind to Tim44Delta18 in an ATP-regulated manner, but the efficiency of interaction is reduced . These results suggest that the J-related segment of Tim44 is needed for productive interaction with mtHsp70 . The efficient cooperation of mtHsp70 with Tim44 facilitates the translocation of loosely folded preproteins and plays a crucial role in the import of preproteins which contain a tightly folded domain. J Cell Biol, 1999 May 31, 145(5), 933 - 50 The unstable F-box protein p58-Ctf13 forms the structural core of the CBF3 kinetochore complex; Russell ID et al.; Kinetochores are smaller and more accessible experimentally in budding yeast than in any other eukaryote . Believing that simple and complex kinetochores have important structural and functional properties in common, we characterized the structure of CBF3, the essential centromere-binding complex that initiates kinetochore formation in Saccharomyces cerevisiae . We find that the four subunits of CBF3 are multimeric in solution: p23(Skp1) and p58(Ctf13) form a heterodimer, and p64(Cep3) and p110(Ndc10) form homodimers . Subcomplexes involving p58 and each of the other CBF3 subunits can assemble in the absence of centromeric DNA . In these subcomplexes, p58 appears to function as a structural core mediating stable interactions among other CBF3 proteins . p58 has a short half-life in yeast, being subject to ubiquitin-dependent proteolysis, but we find that it is much more stable following association with p64 . We propose that p23(Skp1)-p58-p64 complexes constitute the primary pool of active p58 in yeast cells . These complexes can either dissociate, reexposing p58 to the degradation pathway, or can bind to p110 and centromeric DNA, forming a functional CBF3 complex in which p58 is fully protected from degradation . This pathway may constitute an editing mechanism preventing the formation of ectopic kinetochores and ensuring the fidelity of chromosome segregation. Biochemistry, 1999 May 18, 38(20), 6369 - 73 Remarkable stabilization of zwitterionic intermediates may account for a billion-fold rate acceleration by thiamin diphosphate-dependent decarboxylases; Jordan F et al.; When the E91D variant of apo-yeast pyruvate decarboxylase (EC 4.1.1 . 1) is exposed to C2alpha-hydroxybenzylthiamin diphosphate, this putative intermediate is partitioned on the enzyme between release of the benzaldehyde product (as evidenced by regeneration of active enzyme) and dissociation of the proton at C2alpha to form the enamine-C2alpha-carbanion intermediate . While the pKa (the negative log of the acid dissociation constant) for this dissociation is approximately 15.4 in water, formation of the enamine at pH 6.0 on the enzyme indicates a >9 unit pKa suppression by the enzyme environment . The dramatic stabilization of this zwitterionic enamine intermediate at the active center is sufficient to account for as much as a 10(9)-fold rate acceleration on the enzyme . This "solvent" effect could be useful for achieving the bulk of the rate acceleration provided by the protein over and above that afforded by the coenzyme on all thiamin diphosphate-dependent 2-oxo acid decarboxylases. Plant Mol Biol, 1999 Mar, 39(4), 721 - 8 An example of intron junctional sliding in the gene families encoding squalene monooxygenase homologues in Arabidopsis thaliana and Brassica napus; Schafer UA et al.; Sequences of three Arabidopsis thaliana and two Brassica napus cDNAs encoding squalene monooxygenase homologues (Sqp1 and Sqp2) are reported . Southern analysis confirmed that these cDNAs are derived from small gene families in both species . Expression analysis indicates that Sqp1 genes in B . napus are strongly expressed in leaves but not roots or developing seeds . Comparison of cDNA and genomic sequences indicate that the 3' splice site of an intron in these genes has undergone junctional sliding . The evolutionary significance of this phenomenon is discussed. FEBS Lett, 1999 Apr 30, 450(1-2), 149 - 53 Antigen recognition by conformational selection; Berger C et al.; Conformational adaptation between antigen and antibody can modulate the antibody specificity . The phenomenon has often been proposed to result from an 'induced fit', which implies that the binding reaction induces a conformational change in the antigen and the antibody . Thus, an 'induced fit' requires initial complex formation followed by a conformational change in the complex . However, an antibody may select those antigen molecules that happen to be in a fitting conformational state . This leads to the same end result as an induced fit . Here, we demonstrate conformational selection by a single chain antibody fragment, raised against a random coil variant of the leucine zipper domain of transcription factor GCN4, when it cross-reacts with the wild-type dimeric leucine zipper . Kinetic and equilibrium data show that the single chain antibody fragment fragment selects monomeric peptides from the population in equilibrium with the leucine zipper dimer. J Biol Chem, 1999 Jun 4, 274(23), 16487 - 93 Cloning and characterization of a human genotoxic and endoplasmic reticulum stress-inducible cDNA that encodes translation initiation factor 1(eIF1(A121/SUI1)); Sheikh MS et al.; We report the cloning and characterization of a DNA damage-inducible (DDI) transcript DDI A121 . The full-length human DDI A121 cDNA contains an open reading frame of 113 amino acids, corresponding to a protein of 12.7 kDa . The deduced amino acid sequence of A121 shows high homology to the yeast translation initiation factor (eIF) sui1 and also exhibits perfect identity to the partial sequence of recently purified human eIF1 . Expression of human A121 corrected the mutant sui1 phenotype in yeast, demonstrating that human A121 encodes a bona fide translation initiation factor that is equivalent to yeast sui1p . The mammalian A121/SUI1 gene exhibits two transcripts (1.35 kilobases and 0.65 kilobases) containing a common coding region but differing in their 3'-untranslated region . The long and short A121/SUI1 mRNAs are differentially regulated by genotoxic and endoplasmic reticulum stress . The genotoxic stress induction of A121/SUI1 mRNA is conserved in both humans and rodents and occurs in a p53-independent manner . Our identification of a stress-inducible cDNA that encodes eIF1 suggests that modulation of translation initiation appears to occur during cellular stress and may represent an important adaptive response to genotoxic as well as endoplasmic reticulum stress. J Biol Chem, 1999 Jun 4, 274(23), 16279 - 86 Putative reaction intermediates in Crm1-mediated nuclear protein export; Floer M et al.; We discovered several novel interactions between proteins involved in Crm1-mediated nuclear export of the nuclear export signal containing human immunodeficiency virus type 1 protein Rev . First, a Rev/Crm1/RanGTP complex (where Ran is Ras-related nuclear protein) reacts with some nucleoporins (Nup42 and Nup159) but not others (NSP1, Nup116, and Nup1), forming a Nup/Crm1/RanGTP complex and concomitantly releasing Rev . Second, RanBP1 (or homologous proteins) can displace Nup and form a ternary RanBP1/RanGTP/Crm1 complex that can be disassembled by RanGAP via GTP hydrolysis . Third, and most surprisingly, RanBP1/RanGTP/Crm1 can be disassembled without GTP hydrolysis by the nucleotide exchange factor RanGEF . Recycling of a Ran/RanGEF complex by GTP and Mg2+ is stimulated by both Crm1 and Rev, allowing reformation of a Rev/Crm1/RanGTP complex . Based on these reactions we propose a model for Crm1-mediated export. J Biol Chem, 1999 Jun 4, 274(23), 16242 - 8 Physical and functional interactions of neuronal growth suppressor necdin with p53; Taniura H et al.; Necdin is expressed in virtually all postmitotic neurons, and ectopic expression of this protein suppresses cell proliferation . Necdin, like the retinoblastoma protein, interacts with cell cycle promoting proteins such as simian virus 40 large T antigen, adenovirus E1A, and the transcription factor E2F1 . Here we demonstrate that necdin interacts with the tumor suppressor protein p53 as well . The yeast two-hybrid and in vitro binding analyses revealed that necdin bound to a narrow region (amino acids 35-62) located between the MDM2-binding site and the proline-rich region in the amino-terminal domain of p53 . The electrophoretic mobility shift assay showed that necdin supershifted a complex between p53 and its binding DNA, implying that the p53-necdin complex is competent for DNA binding . In p53-deficient osteosarcoma SAOS-2 cells, necdin markedly suppressed p53-dependent activation of the p21/WAF promoter . Necdin and p53 inhibited cell growth in an additive manner as assessed by the colony formation of SAOS-2 cells, suggesting that necdin does not affect p53-mediated growth suppression . On the other hand, necdin inhibited p53-induced apoptosis of osteosarcoma U2OS cells . Thus, necdin can be a growth suppressor that targets p53 and modulates its biological functions in postmitotic neurons. Biochemistry, 1999 May 25, 38(21), 6846 - 54 Role of configurational gating in intracomplex electron transfer from cytochrome c to the radical cation in cytochrome c peroxidase; Mei H et al.; Electron transfer within complexes of cytochrome c (Cc) and cytochrome c peroxidase (CcP) was studied to determine whether the reactions are gated by fluctuations in configuration . Electron transfer in the physiological complex of yeast Cc (yCc) and CcP was studied using the Ru-39-Cc derivative, in which the H39C/C102T variant of yeast iso-1-cytochrome c is labeled at the single cysteine residue on the back surface with trisbipyridylruthenium(II) . Laser excitation of the 1:1 Ru-39-Cc-CcP compound I complex at low ionic strength results in rapid electron transfer from RuII to heme c FeIII, followed by electron transfer from heme c FeII to the Trp-191 indolyl radical cation with a rate constant keta of 2 x 10(6) s-1 at 20 degrees C . keta is not changed by increasing the viscosity up to 40 cP with glycerol and is independent of temperature . These results suggest that this reaction is not gated by fluctuations in the configuration of the complex, but may represent the elementary electron transfer step . The value of keta is consistent with the efficient pathway for electron transfer in the crystalline yCc-CcP complex, which has a distance of 16 A between the edge of heme c and the Trp-191 indole {Pelletier, H., and Kraut, J . (1992) Science 258, 1748-1755} . Electron transfer in the complex of horse Cc (hCc) and CcP was examined using Ru-27-Cc, in which hCc is labeled with trisbipyridylruthenium(II) at Lys-27 . Laser excitation of the Ru-27-Cc-CcP complex results in electron transfer from RuII to heme c FeII with a rate constant k1 of 2.3 x 10(7) s-1, followed by oxidation of the Trp-191 indole to a radical cation by RuIII with a rate constant k3 of 7 x 10(6) s-1 . The cycle is completed by electron transfer from heme c FeII to the Trp-191 radical cation with a rate constant k4 of 6.1 x 10(4) s-1 . The rate constant k4 decreases to 3.4 x 10(3) s-1 as the viscosity is increased to 84 cP, but the rate constants k1 and k3 remain the same . The results are consistent with a gating mechanism in which the Ru-27-Cc-CcP complex undergoes fluctuations between a major state A with the configuration of the hCc-CcP crystalline complex and a minor state B with the configuration of the yCc-CcP complex . The hCc-CcP complex, state A, has an inefficient pathway for electron transfer from heme c to the Trp-191 indolyl radical cation with a distance of 20.5 A and a predicted value of 5 x 10(2) s-1 for k4A . The observed rate constant k4 is thus gated by the rate constant ka for conversion of state A to state B, where the rate of electron transfer k4B is expected to be 2 x 10(6) s-1 . The temperature dependence of k4 provides activation parameters that are consistent with the proposed gating mechanism . These studies provide evidence that configurational gating does not control electron transfer in the physiological yCc-CcP complex, but is required in the nonphysiological hCc-CcP complex. Genes Dev, 1999 May 15, 13(10), 1276 - 88 Nbs1 potentiates ATP-driven DNA unwinding and endonuclease cleavage by the Mre11/Rad50 complex; Paull TT et al.; The Nijmegen breakage syndrome gene product (Nbs1) was shown recently to associate in vivo with the Mre11 and Rad50 proteins, which play pivotal roles in eukaryotic DNA double-strand break repair, meiotic recombination, and telomere maintenance . We show in this work that the triple complex of recombinant Nbs1, Mre11, and Rad50 proteins binds cooperatively to DNA and forms a distinct protein-DNA species . The Mre11/Rad50/Nbs1 complex displays several enzymatic activities that are not seen without Nbs1, including partial unwinding of a DNA duplex and efficient cleavage of fully paired hairpins . Unwinding and hairpin cleavage are both increased by the presence of ATP . On nonhairpin DNA ends, ATP controls a switch in endonuclease specificity that allows Mre11/Rad50/Nbs1 to cleave a 3'-protruding strand at a double-/single-strand transition . Mutational analysis demonstrates that Rad50 is responsible for ATP binding by the complex, but the ATP-dependent activities are expressed only with Nbs1 present. Am J Trop Med Hyg, 1999 May, 60(5), 749 - 51 Short report: Immunodiagnosis of human fascioliasis using recombinant Fasciola hepatica cathepsin L1 cysteine proteinase; O'Neill SM et al.; Our laboratory recently developed a diagnostic test (ELISA) for human fascioliasis based on the detection of serum IgG4 antibodies reactive with Fasciola hepatica cathepsin L1 (CL1) . In the present study, we have used recombinant CL1, generated by functional expression of the cDNA in Saccharomyces cerevisiae, in this immunodiagnostic test and compared its performance with native CL1 . Sera obtained from 64 individuals living in Cutusuma village in the northern Altiplano of Bolivia, a region with a high prevalence of human fascioliasis, were analyzed by the IgG4-ELISA . A highly statistically significant correlation (r2 = 0.751, P < 0.001) was demonstrated between the absorbances obtained using the recombinant and native proteins . These assays showed that 38 (59%) of the individuals tested were seropositive for fascioliasis, whereas only 26 of them were coprologically positive for F . hepatica eggs . All seronegative patients were also coprologically negative . Serum from individuals infected with schistosomiasis mansoni, cysticercosis, hydatidosis, and Chagas disease did not contain antibodies reactive with the recombinant or native CL1 . Therefore, recombinant CL1 shows excellent potential for the development of the first standardized assay for the sensitive and specific diagnosis of human fascioliasis . Finally, our data supports earlier reports on the high prevalence of human fascioliasis in the Bolivian Altiplano, which collectively suggest that the disease has been endemic there for more than a decade. Plant Mol Biol, 1999 Mar, 39(5), 933 - 43 Differential expression of two spermidine synthase genes during early fruit development and in vegetative tissues of pea; Alabadi D et al.; Two cDNAs from young pea fruits coding for functional spermidine synthases (EC 2.5.1.16) were isolated . The corresponding genes were named psSPDSYN1 and psSPDSYN2 . Both cDNAs complemented spe3delta gene when introduced into the Y480 strain of Saccharomyces cerevisiae, which is a null mutant for the spermidine synthase gene . psSPDSYN1 and psSPDSYN2 are regulated differentially . psSPDSYN1 is up-regulated early after fruit set whereas psSPDSYN2 is expressed later . Spermidine synthase activity was detected in pea ovaries, and correlates with the pattern of expression of psSPDSYN1 . In the pea plant, psSPDSYN1 is highly expressed in actively growing tissues, whereas the highest level of psSPDSYN2 mRNA was detected in fully elongated stem. Plant Mol Biol, 1999 Mar, 39(5), 891 - 906 Delta7-sterol-C5-desaturase: molecular characterization and functional expression of wild-type and mutant alleles; Husselstein T et al.; An Arabidopsis thaliana recessive monogenic mutant (ste1-1) presenting a deficiency of the delta7-sterol-C5(6)-desaturase step in the sterol pathway has been reported previously {12} . To further characterize ste1-1, Arabidopsis, Nicotiana tabacum and Homo sapiens cDNAs encoding delta7-sterol-C5(6)-desaturases were isolated and identified on the basis of their ability to restore ergosterol synthesis in erg3, a yeast null mutant whose gene encoding the delta7-sterol-C5(6)-desaturase was disrupted . Overexpression of the Arabidopsis cDNA driven by a 35S promoter in transgenic ste1-1 plants led to full complementation of the mutant . This result demonstrates that STE1 was the impaired component in the desaturation system . Four independent reverse transcriptions of ste1-1 RNA followed by polymerase chain reactions (RT-PCRs), yielded a single product . Alignment of the wild-type ORF with the RT-PCR derived ste1-1 ORF revealed a single amino acid substitution: Thr-114 in the wild-type is changed to Ile in ste1-1 . Expression in erg3 resulted in a 6-fold lowered efficiency of the ste1-1 ORF in complementing the yeast biosynthetic pathway when compared to the wild-type ORF . The presence of this mutation in the mutant ste1-1 genomic sequence (and no additional modification between ste1-1 and wild-type genes) demonstrates that the change of the Thr-114 to Ile is necessary and sufficient to create the leaky allele ste1-1 . The occurrence of a hydroxylated amino acid (Thr or Ser) at the position corresponding to Thr-114 in the five delta7-sterol-C5(6)-desaturases identified so far suggests that this amino acid is important for normal enzymatic function. Mutat Res, 1999 Apr 9, 433(3), 159 - 68 Analysis of intrachromosomal homologous recombination in mammalian cell, using tandem repeat sequences; Lambert S et al.; In all the organisms, homologous recombination (HR) is involved in fundamental processes such as genome diversification and DNA repair . Several strategies can be devised to measure homologous recombination in mammalian cells . We present here the interest of using intrachromosomal tandem repeat sequences to measure HR in mammalian cells and we discuss the differences with the ectopic plasmids recombination . The present review focuses on the molecular mechanisms of HR between tandem repeats in mammalian cells . The possibility to use two different orientations of tandem repeats (direct or inverted repeats) in parallel constitutes also an advantage . While inverted repeats measure only events arising by strand exchange (gene conversion and crossing over), direct repeats monitor strand exchange events and also non-conservative processes such as single strand annealing or replication slippage . In yeast, these processes depend on different pathways, most of them also existing in mammalian cells . These data permit to devise substrates adapted to specific questions about HR in mammalian cells . The effect of substrate structures (heterologies, insertions/deletions, GT repeats, transcription) and consequences of DNA double strand breaks induced by ionizing radiation or endonuclease (especially the rare-cutting endonuclease ISce-I) on HR are discussed . Finally, transgenic mouse models using tandem repeats are briefly presented. Plant J, 1999 Apr, 18(1), 97 - 104 Characterisation of a novel gene family of putative cyclic nucleotide- and calmodulin-regulated ion channels in Arabidopsis thaliana; Kohler C et al.; In plants, cyclic GMP is involved in signal transduction in response to light and gibberellic acid . For cyclic AMP, a potential role during the plant cell cycle was recently reported . However, cellular targets for cyclic nucleotides in plants are largely unknown . Here we report on the identification and characterisation of a new gene family in Arabidopsis, which share features with cyclic nucleotide-gated channels from animals and inward-rectifying K+ channels from plants . The identified gene family comprises six members (Arabidopsis thaliana cyclic nucleotide-gated channels, AtCNGC1-6) with significant homology among the deduced proteins . Hydrophobicity analysis predicted six membrane-spanning domains flanked by hydrophilic amino and carboxy termini . A putative cyclic nucleotide binding domain (CNBD) which contains several residues that are invariant in other CNBDs was located in the carboxy terminus . This domain overlaps with a predicted calmodulin (CaM) binding site, suggesting interaction between cyclic nucleotide and CaM regulation . We demonstrated interaction of the carboxy termini of AtCNGC1 and AtCNGC2 with CaM in yeast, indicating that the CaM binding sites are functional . Furthermore, it was shown that both AtCNGC1 and AtCNGC2 can partly complement the K(+)-uptake-deficient yeast mutant CY162 . Therefore, we propose that the identified genes constitute a family of plant cyclic nucleotide- and CaM-regulated ion channels. Plant J, 1999 Apr, 18(1), 23 - 32 Functional analysis of a Golgi-localized Kex2p-like protease in tobacco suspension culture cells; Jiang L et al.; Kex2p is the prototype of a Golgi-resident protease responsible for the processing of prohormones in yeast and mammalian cells . A Kex2p-like pathway was shown to be responsible for processing the fungal KP6 protoxin in transgenic tobacco plants . We previously described a chimeric integral membrane reporter protein that traffics through Golgi to the lytic prevacuole where it was proteolytically processed . As a first step to isolate and clone the Kex2p-like protease in plant cells, we designed and used a similar chimeric reporter protein containing Kex2 cleavage sites to assay the Kex2p-like activity and to determine its substrate specificity in tobacco cells . Here we demonstrate that the Kex2 cleavage sites of the reporter were specifically processed by a protease activity with a substrate specificity characteristic of yeast Kex2p . This Kex2p-like protease in tobacco cells is also a Golgi-resident enzyme . Thus, the reporter protein provides a biochemical marker for studying protein traffic through the Golgi in plant cells . These results additionally should allow the design of synthetic substrates for use in biochemical purification of the plant enzyme. Int J Mol Med, 1999 Jun, 3(6), 591 - 6 Dynamic redistribution of nuclear matrix proteins by adenovirus infection; Yu E et al.; We analyzed the redistribution of nuclear proteins, PML protein, Ku70/Ku80, a putative spliceosome associated protein, pNMM102 and a nucleolar proliferating antigen, p120 after adenovirus 5 (Ad5) infection using immunofluorescent staining . These proteins remained after in situ fractionation . PML was located at irregular bars 6 h after infection from fine dots of uninfected cells . Distribution pattern of PML was not changed throughout the course of infection . Internal nuclear matrix network composed of Ku protein became much coarser in interphase cells at 12 h after infection . Ku protein was clumped at 18 h after infection, where EIA protein was colocalized . Speckles and interconnecting fibrils recognized by monoclonal antibody NMM102 disappeared early after infection, and reappeared at 18 h after infection in various patterns . The number and staining intensity of p120 containing domains increased markedly in early replication phase, and their shape became irregular with a few fine dots . A few Ad5 infected cells revealed diffuse nucleoplasmic as well as nucleolar p120 in late replication phase . Redistribution of four different nuclear matrix proteins by Ad5 infection indicates that the nuclear matrix is dynamically involved in gene expression. Bioorg Med Chem Lett, 1999 May 3, 9(9), 1255 - 60 Isotope edited NMR studies of glycosidases: design and synthesis of a novel glycosidase inhibitor; Hines JV et al.; N-13C-methyl-deoxynojirimycin was synthesized and used in isotope-edited NMR studies to probe the binding site of an alpha-glucosidase . Results from this analysis led to the design and preparation of a novel alpha-glucosidase inhibitor, N-glycyl deoxynojirimycin. Proc Natl Acad Sci U S A, 1999 May 25, 96(11), 6523 - 8 Interaction of NPR1 with basic leucine zipper protein transcription factors that bind sequences required for salicylic acid induction of the PR-1 gene; Zhang Y et al.; The Arabidopsis thaliana NPR1 has been shown to be a key regulator of gene expression during the onset of a plant disease-resistance response known as systemic acquired resistance . The npr1 mutant plants fail to respond to systemic acquired resistance-inducing signals such as salicylic acid (SA), or express SA-induced pathogenesis-related (PR) genes . Using NPR1 as bait in a yeast two-hybrid screen, we identified a subclass of transcription factors in the basic leucine zipper protein family (AHBP-1b and TGA6) and showed that they interact specifically in yeast and in vitro with NPR1 . Point mutations that abolish the NPR1 function in A . thaliana also impair the interactions between NPR1 and the transcription factors in the yeast two-hybrid assay . Furthermore, a gel mobility shift assay showed that the purified transcription factor protein, AHBP-1b, binds specifically to an SA-responsive promoter element of the A . thaliana PR-1 gene . These data suggest that NPR1 may regulate PR-1 gene expression by interacting with a subclass of basic leucine zipper protein transcription factors. Proc Natl Acad Sci U S A, 1999 May 25, 96(11), 6371 - 6 Interaction between RGS7 and polycystin; Kim E et al.; Regulators of G protein signaling (RGS) proteins accelerate the intrinsic GTPase activity of certain Galpha subunits and thereby modulate a number of G protein-dependent signaling cascades . Currently, little is known about the regulation of RGS proteins themselves . We identified a short-lived RGS protein, RGS7, that is rapidly degraded through the proteasome pathway . The degradation of RGS7 is inhibited by interaction with a C-terminal domain of polycystin, the protein encoded by PKD1, a gene involved in autosomal-dominant polycystic kidney disease . Furthermore, membranous expression of C-terminal polycystin relocalized RGS7 . Our results indicate that rapid degradation and interaction with integral membrane proteins are potential means of regulating RGS proteins. Proc Natl Acad Sci U S A, 1999 May 25, 96(11), 6205 - 10 Spc29p is a component of the Spc110p subcomplex and is essential for spindle pole body duplication; Elliott S et al.; In yeast, microtubules are organized by the spindle pole body (SPB) . The SPB is a disk-like multilayered structure that is embedded in the nuclear envelope via its central plaque, whereas the outer and inner plaques are exposed to the cytoplasm and nucleoplasm, respectively . How the SPB assembles is poorly understood . We show that the inner/central plaque is composed of a stable SPB subcomplex, containing the gamma-tubulin complex-binding protein Spc110p, calmodulin, Spc42p, and Spc29p . Spc29p acts as a linker between the central plaque component Spc42p and the inner plaque protein Spc110p . Evidence is provided that the calmodulin-binding site of Spc110p influences the binding of Spc29p to Spc110p . Spc42p also was identified as a component of a cytoplasmic SPB subcomplex containing Spc94p/Nud1p, Cnm67p, and Spc42p . Spc29p and Spc42p may be part of a critical interface of nucleoplasmic and cytoplasmic assembled SPB subcomplexes that form during SPB duplication . In agreement with this, overexpressed Spc29p was found to be a nuclear protein, whereas Spc42p is cytoplasmic . In addition, an essential function of SPC29 during SPB assembly is indicated by the SPB duplication defect of conditional lethal spc29(ts) cells and by the genetic interaction of SPC29 with CDC31 and KAR1, two genes that are involved in SPB duplication. Proc Natl Acad Sci U S A, 1999 May 25, 96(11), 6193 - 8 Multistep regulation of DNA replication by Cdk phosphorylation of HsCdc6; Jiang W et al.; We have characterized HsCdc6, a human protein homologous to the budding yeast Cdc6p that is essential for DNA replication . We show that, unlike Cdc6p, the levels of HsCdc6 protein remain constant throughout the cell cycle in human cells . However, phosphorylation of HsCdc6 is regulated during the cell cycle . HsCdc6 is an excellent substrate for Cdk2 in vitro and is phosphorylated in vivo at three sites (Ser-54, Ser-74, and Ser-106) that are phosphorylated by Cdk2 in vitro, strongly suggesting that HsCdc6 is an in vivo Cdk substrate . HsCdc6 is nuclear in G1, but translocates to the cytoplasm at the start of S phase via Crm1-dependent export . An HsCdc6A1A2A3 mutant, which mimics unphosphorylated HsCdc6, is exclusively nuclear, and its expression inhibits initiation of DNA replication . An HsCdc6E1E2E3 mutant, which mimics phosphorylated HsCdc6, is exclusively cytoplasmic and is not associated with the chromatin/nuclear matrix fraction . Based on these results, we propose that phosphorylation of HsCdc6 by Cdks regulates DNA replication of at least two steps: first, by promoting initiation of DNA replication and, second, through nuclear exclusion preventing DNA rereplication. Proc Natl Acad Sci U S A, 1999 May 25, 96(11), 6031 - 6 Observation of strange kinetics in protein folding; Sabelko J et al.; Highly nonexponential folding kinetics in aqueous solution have been observed during temperature jump-induced refolding of two proteins, yeast phosphoglycerate kinase and a ubiquitin mutant . The observations are most easily interpreted in terms of downhill folding, which posits a heterogeneous ensemble of structures en route to the folded state . The data are also reconciled with exponential kinetics measured under different experimental conditions and with titration experiments indicating cooperative folding. Proc Natl Acad Sci U S A, 1999 May 25, 96(11), 6020 - 4 Chemistry for the analysis of protein-protein interactions: rapid and efficient cross-linking triggered by long wavelength light; Fancy DA et al.; Chemical cross-linking is a potentially useful technique for probing the architecture of multiprotein complexes . However, analyses using typical bifunctional cross-linkers often suffer from poor yields, and large-scale modification of nucleophilic side chains can result in artifactual results attributable to structural destabilization . We report here the de novo design and development of a type of protein cross-linking reaction that uses a photogenerated oxidant to mediate rapid and efficient cross-linking of associated proteins . The process involves brief photolysis of tris-bipyridylruthenium(II) dication with visible light in the presence of the electron acceptor ammonium persulfate and the proteins of interest . Very high yields of cross-linked products can be obtained with irradiation times of <1 second . This chemistry obviates many of the problems associated with standard cross-linking reagents. Blood, 1999 Jun 1, 93(11), 3803 - 10 Overexpression of A1, an NF-kappaB-inducible anti-apoptotic bcl gene, inhibits endothelial cell activation; Stroka DM et al.; A1 is an anti-apoptotic bcl gene that is expressed in endothelial cells (EC) in response to pro-inflammatory stimuli . We show that in addition to protecting EC from apoptosis, A1 inhibits EC activation and its associated expression of pro-inflammatory proteins by inhibiting the transcription factor nuclear factor (NF)-kappaB . This new anti-inflammatory function gives a broader dimension to the protective role of A1 in EC . We also show that activation of NF-kappaB is essential for the expression of A1 . Taken together, our data suggest that A1 downregulates not only the pro-apoptotic and pro-inflammatory response, but also its own expression, thus restoring a quiescent phenotype to EC. J Mol Biol, 1999 May 28, 289(1), 69 - 82 Genetic and structural characterization of the human mitochondrial inner membrane translocase; Bauer MF et al.; Translocation of nuclear-encoded mitochondrial preproteins is mediated by translocases in the outer and inner membranes . In the yeast Saccharomyces cerevisiae, translocation of preproteins into the matrix requires the membrane proteins Tim23, Tim17 and Tim44, which drive translocation in cooperation with mtHsp70 and its co-chaperone Mge1p . We have cloned and functionally analyzed the human homologues of Tim17, Tim23 and Tim44 . In contrast to yeast, two TIM17 genes were found to be expressed in humans . TIM44, TIM23 and TIM17a genes were mapped to chromosomes 19p13.2-p13.3, 10q11 . 21-q11.23 and 1q32 . The TIM17b gene mapped to Xp11.23, near the fusion point where an autosomal region was proposed to have been added to the "ancient" part of the X chromosome about 80-130 MY ago . The primary sequences of the two proteins, hTim17a and hTim17b, are essentially identical, significant differences being restricted to their C termini . They are ubiquitously expressed in fetal and adult tissues, and both show expression levels comparable to that of hTim23 . Biochemical characterization of the human Tim components revealed that hTim44 is localized in the matrix and, in contrast to yeast, only loosely associated with the inner membrane . hTim23 is organized into two distinct complexes in the inner membrane, one containing hTim17a and one containing hTim17b . Both TIM complexes display a native molecular mass of 110 kDa . We suggest that the structural organization of TIM23.17 preprotein translocases is conserved from low to high eukaryotes . Hum Mutat, 1999, 13(5), 351 - 61 Congenital hyperinsulinism: molecular basis of a heterogeneous disease; Meissner T et al.; Congenital hyperinsulinism (CHI) is a disease phenotype characterized by increased, usually irregular, insulin secretion leading to hypoglycemia, coma, and severe brain damage, left untreated . Hyperinsulinism may be caused by a range of biochemical disturbances and molecular defects . In pancreatic beta cells, insulin secretion is stimulated by closure of the ATP-dependent potassium channel (K(ATP) channel) . K(ATP) channel is a complex composed of at least two subunits: the sulfonylurea receptor SUR1 and Kir6.2, an inward rectifier K+ channel member . Mutations in both subunits have been identified in patients with the autosomal recessive form of hyperinsulinism, including 28 different mutations in the SUR1 gene and two mutations in the Kir6.2 gene . These mutations co-segregated with disease phenotype, also known as persistent hyperinsulinemic hypoglycemia of infancy (PHHI), and with attenuated K(ATP) channel function . Inadequately high insulin secretion in one family with an autosomal dominant mode of inheritance is caused by a mutation in the glucokinase gene, resulting in increased affinity of the enzyme for glucose . Five different mutations have been identified in the glutamate dehydrogenase gene, resulting in overactivity of this enzyme and causing a syndrome of hyperinsulinism and hyperammonemia . In 13 cases, hyperinsulinism was caused by one or more focal pancreatic lesions with specific loss of maternal alleles of the imprinted chromosome region 11p15 . In five patients, this loss of heterozygosity unmasked a paternally inherited recessive SUR1 mutation . The new molecular approaches in PHHI give further insight into the mechanism of pancreatic beta cell insulin secretion . The heterogeneous group of patients with CHI may now be classified according to their basic defects in the four different genes, with potential implications for a more specific treatment. Glycobiology, 1999 Jun, 9(6), 617 - 25 Ordered assembly of the asymmetrically branched lipid-linked oligosaccharide in the endoplasmic reticulum is ensured by the substrate specificity of the individual glycosyltransferases; Burda P et al.; The assembly of the lipid-linked core oligosaccharide Glc3Man9GlcNAc2, the substrate for N-linked glycosylation of proteins in the endoplasmic reticulum (ER), is catalyzed by different glycosyltransferases located at the membrane of the ER . We report on the identification and characterization of the ALG12 locus encoding a novel mannosyltransferase responsible for the addition of the alpha-1,6 mannose to dolichol-linked Man7GlcNAc2 . The biosynthesis of the highly branched oligosaccharide follows an ordered pathway which ensures that only completely assembled oligosaccharide is transferred from the lipid anchor to proteins . Using the combination of mutant strains affected in the assembly pathway of lipid-linked oligosaccharides and overexpression of distinct glycosyltransferases, we were able to define the substrate specificities of the transferases that are critical for branching . Our results demonstrate that branched oligosaccharide structures can be specifically recognized by the ER glycosyltransferases . This substrate specificity of the different transferases explains the ordered assembly of the complex structure of lipid-linked Glc3Man9GlcNAc2 in the endoplasmic reticulum. J Biol Chem, 1999 May 28, 274(22), 15937 - 46 A di-acidic (DXE) code directs concentration of cargo during export from the endoplasmic reticulum; Nishimura N et al.; Efficient export of vesicular stomatitis virus glycoprotein (VSV-G), a type I transmembrane protein, from the endoplasmic reticulum requires a di-acidic code (DXE) located in the cytosolic carboxyl-terminal tail (Nishimura, N., and Balch, W . E . (1997) Science 277, 556-558) . Mutation of the DXE code by mutation to AXA did not prevent VSV-G recruitment to pre-budding complexes formed in the presence of the activated form of the Sar1 and the Sec23/24 complex, components of the COPII budding machinery . However, the signal was required at a subsequent concentration step preceding vesicle fission . By using green fluorescence protein-tagged VSV-G to image movement in a single cell, we found that VSV-G lacking the DXE code fails to be concentrated into COPII vesicles . As a result, the normal 5-10-fold increase in the steady-state concentration of VSV-G in downstream pre-Golgi intermediates and Golgi compartments was lost . These results demonstrate for the first time that inactivation of the DXE signal uncouples early cargo selection steps from concentration into COPII vesicles . We propose that two sequential steps are required for efficient export from the endoplasmic reticulum. J Biol Chem, 1999 May 28, 274(22), 15883 - 91 Activation of the murine dihydrofolate reductase promoter by E2F1 . A requirement for CBP recruitment; Fry CJ et al.; The E2F family of heterodimeric transcription factors plays an important role in the regulation of gene expression at the G1/S phase transition of the mammalian cell cycle . Previously, we have demonstrated that cell cycle regulation of murine dihydrofolate reductase (dhfr) expression requires E2F-mediated activation of the dhfr promoter in S phase . To investigate the mechanism by which E2F activates an authentic E2F-regulated promoter, we precisely replaced the E2F binding site in the dhfr promoter with a Gal4 binding site . Using Gal4-E2F1 derivatives, we found that E2F1 amino acids 409-437 contain a potent core transactivation domain . Functional analysis of the E2F1 core domain demonstrated that replacement of phenylalanine residues 413, 425, and 429 with alanine reduces both transcriptional activation of the dhfr promoter and protein-protein interactions with CBP, transcription factor (TF) IIH, and TATA-binding protein (TBP) . However, additional amino acid substitutions for phenylalanine 429 demonstrated a strong correlation between activation of the dhfr promoter and binding of CBP, but not TFIIH or TBP . Finally, transactivator bypass experiments indicated that direct recruitment of CBP is sufficient for activation of the dhfr promoter . Therefore, we suggest that recruitment of CBP is one mechanism by which E2F activates the dhfr promoter. J Biol Chem, 1999 May 28, 274(22), 15751 - 6 Hepatitis C virus core protein binds to a DEAD box RNA helicase; Mamiya N et al.; Approximately 4 million Americans are infected with the hepatitis C virus (HCV), making it a major cause of chronic liver disease . Because of the lack of an efficient cell culture system, little is known about the interaction between HCV and host cells . We performed a yeast two-hybrid screen of a human liver cell cDNA library with HCV core protein as bait and isolated the DEAD box protein DBX . DBX has significant amino acid sequence identity to mouse PL10, an ATP-dependent RNA helicase . The binding of DBX to HCV core protein occurred in an in vitro binding assay in the presence of 1 M NaCl or detergent . When expressed in mammalian cells, HCV core protein and DBX were co-localized at the endoplasmic reticulum . In a mutant strain of Saccharomyces cerevisiae, DBX complemented the function of Ded1p, an essential DEAD box RNA helicase . HCV core protein inhibited the growth of DBX-complemented mutant yeast but not Ded1p-expressing yeast . HCV core protein also inhibited the in vitro translation of capped but not uncapped RNA . These findings demonstrate an interaction between HCV core protein and a host cell protein involved in RNA translation and suggest a mechanism by which HCV may inhibit host cell mRNA translation. Biochem Biophys Res Commun, 1999 May 27, 259(1), 133 - 5 No metal cofactor in orotidine 5'-monophosphate decarboxylase; Cui W et al.; Orotidine 5'-monophosphate decarboxylase (OMP decarboxylase, ODCase) is an important enzyme that catalyzes the final step of de novo pyrimidine nucleotide biosynthesis . The mechanism of this unique enzyme and whether metal ions play any role in catalysis have been topics of intense research interest . In this report, the role of Zn in ODCase was reexamined . Atomic absorption (AA) and X-ray absorption (XAS) spectroscopic studies did not detect zinc in active enzyme samples at high concentration . The XAS results also indicated the absence of other transition metal ions in ODCase . Nat Struct Biol, 1999 May, 6(5), 478 - 85 A folding transition and novel zinc finger accessory domain in the transcription factor ADR1; Bowers PM et al.; The region responsible for sequence-specific DNA binding by the transcription factor ADR1 contains two Cys2-His2 zinc fingers and an additional N-terminal proximal accessory region (PAR) . The N-terminal (non-finger) PAR is unstructured in the absence of DNA and undergoes a folding transition on binding the DNA transcription target site . We have used a set of HN-HN NOEs derived from a perdeuterated protein-DNA complex to describe the fold of ADR1 bound to the UAS1 binding site . The PAR forms a compact domain consisting of three antiparallel strands that contact A-T base pairs in the major groove . The three-strand domain is a novel fold among all known DNA-binding proteins . The PAR shares sequence homology with the N-terminal regions of other zinc finger proteins, suggesting that it represents a new DNA-binding module that extends the binding repertoire of zinc finger proteins. RNA, 1999 May, 5(5), 678 - 86 The 5' end of the 18S rRNA can be positioned from within the mature rRNA; Sharma K et al.; In yeast, the 5' end of the mature 18S rRNA is generated by endonucleolytic cleavage at site A1, the position of which is specified by two distinct signals . An evolutionarily conserved sequence immediately upstream of the cleavage site has previously been shown to constitute one of these signals . We report here that a conserved stem-loop structure within the 5' region of the 18S rRNA is recognized as a second positioning signal . Mutations predicted to either extend or destabilize the stem inhibited the normal positioning of site A1 from within the 18S rRNA sequence, as did substitution of the loop nucleotides . In addition, these mutations destabilized the mature 18S rRNA, indicating that recognition of the stem-loop structure is also required for 18S rRNA stability . Several mutations tested reduced the efficiency of pre-rRNA cleavage at site A1 . There was, however, a poor correlation between the effects of the different mutations on the efficiency of cleavage and on the choice of cleavage site, indicating that these involve recognition of the stem-loop region by distinct factors . In contrast, the cleavages at sites A1 and A2 are coupled and the positioning signals appear to be similar, suggesting that both cleavages may be carried out by the same endonuclease. J Urol, 1999 Jun, 161(6), 1973 - 5 Selective detection of inactivating mutations of the tumor suppressor gene p53 in bladder tumors; Pfister C et al.; OBJECTIVE: Mutations in the p53 gene are implicated in the pathogenesis of half of all human tumors . In bladder tumors they are usually detected by immunohistochemistry . The assumption underlying protein analysis is that high level p53 expression is a consequence of mutations, but numerous exceptions have been reported . We describe the detection of p53 mutations in bladder cancer using a functional assay in yeast . MATERIALS AND METHODS: The prospective study consisted of 60 consecutive patients with bladder tumors (7 pT0, 2 CIS, 23 pTa, 24 pT1 and 4 pT2) . High grade 3 was observed in primary carcinoma in situ, in 75% of pT1 tumors and in all pT2 tumors . The p53 mRNA extracted from endoscopic resection tissue was reverse transcribed and PCR-amplified . The transcriptional competence of the p53 cDNA was then tested in a yeast reporter strain . A simple functional assay was developed for p53 mutation in which human p53 is expressed in Saccharomyces cerevisiae which activates transcription of the ADE2 gene . Colonies containing wild type p53 are white and colonies containing mutant p53 are red . RESULTS: As this assay evaluates the critical biological function of p53, it can distinguish inactivating mutations from functionally silent mutations . In pTo and pTa bladder tumors, no p53 mutations were detected . In contrast, the functional assay permitted us to detect p53 mutations in 66% of patients with stage T1 tumors (72% of case of high grade 3) and in all cases with primary carcinoma in situ and in 4 cases of stage T2 tumors . CONCLUSION: This preliminary study demonstrates that this functional assay method is a simple and efficient procedure to detect p53 mutations in bladder cancers and suggests that p53 mutations seem to be associated with invasive bladder tumors. Hum Mol Genet, 1999 Jun, 8(6), 959 - 69 T-STAR/ETOILE: a novel relative of SAM68 that interacts with an RNA-binding protein implicated in spermatogenesis; Venables JP et al.; RBM is an RNA-binding protein encoded on the Y chromosome in mammals and is expressed only in the nuclei of male germ cells . Genetic evidence from infertile men implicates it in spermatogenesis, but its function is unknown . Of a number of potential partners for RBM identified by a yeast two-hybrid screen with testis cDNA, the most frequent isolates encoded a novel RNA-binding protein, termed T-STAR, that is closely related to SAM68, an Src-associated protein of unknown function . The mouse homologue was also cloned and designated etoile . It mapped to chromosome 15, while T-STAR mapped to the syntenic region on human chromosome 8 . T-STAR/etoile is expressed primarily in the testis; in rat germ cells, the expression of both T-STAR/etoile and SAM68 is regulated during meiosis . Transfection of T-STAR/etoile fused with green fluorescent protein into HeLa cells caused an accumulation of protein in a novel compartment of the nucleus, adjacent to the nucleolus but distinct from the peri-nucleolar compartment . RBM and other hnRNP G family members are candidate downstream targets for regulation by T-STAR/ETOILE and SAM68. Hum Mol Genet, 1999 Jun, 8(6), 947 - 57 Dentatorubral-pallidoluysian atrophy protein interacts through a proline-rich region near polyglutamine with the SH3 domain of an insulin receptor tyrosine kinase substrate; Okamura-Oho Y et al.; Dentatorubral-pallidoluysian atrophy (DRPLA) is an autosomal dominant neuro degrees enerative disorder associated with CAG/glutamine repeat expansion . While the DRPLA gene is ubiquitously expressed, neuron death occurs in specific anatomical areas of the brain . This predicts that the DRPLA protein interacts with other proteins and that these interactions may play a role in pathogenesis . Here, we describe a protein that binds to the DRPLA product . One of the clones isolated with a yeast two-hybrid system was identified as a human homolog of the insulin receptor tyrosine kinase substrate protein of 53 kDa (IRSp53) . The gene produced two mRNA forms by differential splicing and encoded 552 and 521 amino acids, respectively . The longer form was mainly expressed in the brain and the shorter one in other tissues . The products were phosphorylated upon stimulation of cultured cells with insulin or insulin-like growth factor 1 . Binding of the DRPLA protein to IRSp53 was ascertained by co-immunoprecipitation with antibodies and also by co-localization in perinuclear oval dots in cells expressing engineered constructs . A proline-rich region near the polyglutamine tract of the DRPLA protein and the SH3 domain of IRSp53 were involved in the binding . An extended polyglutamine tract significantly reduced binding ability in yeast cells, but not in in vitro binding assays . The identification of IRSp53 and other proteins detected by the yeast hybrid system predicts that DRPLA functions in a signal transduction pathway coupled with insulin/IGF-1. Anal Chem, 1999 May 1, 71(9), 1663 - 7 Sharp DNA bends as landmarks of protein-binding sites on straightened DNA; Yokota H et al.; We have developed a fluorescence-based method for mapping single or multiple protein-binding sites on straightened, large-size DNA molecules (> 5 kbp) . In the described method, protein-DNA complexes were straightened and immobilized on a flat surface using surface tension . A fraction of the immobilized complexes displayed a sharp DNA bend with two DNA segments extending from the apex . The presence of DNA-binding proteins at the apex was verified by atomic force microscopy . The position of protein binding relative to the ends of the DNA molecule was determined by measuring the length of two DNA segments using fluorescence microscopy . We demonstrate the potential of the fluorescence-based method to localize protein-binding sites on the DNA template and to evaluate relative binding affinity . The proposed protein-binding-site mapping technique is simple and easy to perform . Practical applications include screening for DNA-binding proteins and the localization of protein-binding sites on large segments of DNA. Plant Cell, 1999 May, 11(5), 937 - 48 Three functional transporters for constitutive, diurnally regulated, and starvation-induced uptake of ammonium into Arabidopsis roots; Gazzarrini S et al.; Ammonium and nitrate are the prevalent nitrogen sources for growth and development of higher plants . 15N-uptake studies demonstrated that ammonium is preferred up to 20-fold over nitrate by Arabidopsis plants . To study the regulation and complex kinetics of ammonium uptake, we isolated two new ammonium transporter (AMT) genes and showed that they functionally complemented an ammonium uptake-deficient yeast mutant . Uptake studies with 14C-methylammonium and inhibition by ammonium yielded distinct substrate affinities between </=0.5 and 40 microM . Correlation of gene expression with 15NH4+ uptake into plant roots showed that nitrogen supply and time of day differentially regulated the individual carriers . Transcript levels of AtAMT1;1, which possesses an affinity in the nanomolar range, steeply increased with ammonium uptake in roots when nitrogen nutrition became limiting, whereas those of AtAMT1;3 increased slightly, with AtAMT1;2 being more constitutively expressed . All three ammonium transporters showed diurnal variation in expression, but AtAMT1;3 transcript levels peaked with ammonium uptake at the end of the light period, suggesting that AtAMT1;3 provides a link between nitrogen assimilation and carbon provision in roots . Our results show that high-affinity ammonium uptake in roots is regulated in relation to the physiological status of the plant at the transcriptional level and by substrate affinities of individual members of the AMT1 gene family. J Cell Biol, 1999 May 17, 145(4), 659 - 72 LST1 is a SEC24 homologue used for selective export of the plasma membrane ATPase from the endoplasmic reticulum; Roberg KJ et al.; In Saccharomyces cerevisiae, vesicles that carry proteins from the ER to the Golgi compartment are encapsulated by COPII coat proteins . We identified mutations in ten genes, designated LST (lethal with sec-thirteen), that were lethal in combination with the COPII mutation sec13-1 . LST1 showed synthetic-lethal interactions with the complete set of COPII genes, indicating that LST1 encodes a new COPII function . LST1 codes for a protein similar in sequence to the COPII subunit Sec24p . Like Sec24p, Lst1p is a peripheral ER membrane protein that binds to the COPII subunit Sec23p . Chromosomal deletion of LST1 is not lethal, but inhibits transport of the plasma membrane proton-ATPase (Pma1p) to the cell surface, causing poor growth on media of low pH . Localization by both immunofluorescence microscopy and cell fractionation shows that the export of Pma1p from the ER is impaired in lst1Delta mutants . Transport of other proteins from the ER was not affected by lst1Delta, nor was Pma1p transport found to be particularly sensitive to other COPII defects . Together, these findings suggest that a specialized form of the COPII coat subunit, with Lst1p in place of Sec24p, is used for the efficient packaging of Pma1p into vesicles derived from the ER. Int Immunol, 1999 May, 11(5), 825 - 33 Mutational pattern of the nurse shark antigen receptor gene (NAR) is similar to that of mammalian Ig genes and to spontaneous mutations in evolution: the translesion synthesis model of somatic hypermutation; Diaz M et al.; The pattern of somatic mutations of shark and frog Ig is distinct from somatic hypermutation of Ig in mammals in that there is a bias to mutate GC base pairs and a low frequency of mutations . Previous analysis of the new antigen receptor gene in nurse sharks (NAR), however, revealed no bias to mutate GC base pairs and the frequency of mutation was comparable to that of mammalian IgG . Here, we analyzed 1023 mutations in NAR and found no targeting of the mechanism to any particular nucleotide but did obtain strong evidence for a transition bias and for strand polarity . As seen for all species studied to date, the serine codon AGC/T in NAR was a mutational hotspot . The NAR mutational pattern is most similar to that of mammalian IgG and furthermore both are strikingly akin to mutations acquired during the neutral evolution of nuclear pseudogenes, suggesting that a similar mechanism is at work for both processes . In yeast, most spontaneous mutations are introduced by the translesion synthesis DNA polymerase zeta (REV3) and in various DNA repair-deficient backgrounds transitions were more often REV3-dependent than were transversions . Therefore, we propose a model of somatic hypermutation where DNA polymerase zeta is recruited to the Ig locus . An excess of DNA glycosylases in germinal center reactions may further enhance the mutation frequency by a REV3-dependent mutagenic process known as imbalanced base excision repair. Mol Cell Biol, 1999 Jun, 19(6), 4516 - 24 Two prion-inducing regions of Ure2p are nonoverlapping; Maddelein ML et al.; Ure2p of Saccharomyces cerevisiae normally functions in blocking utilization of a poor nitrogen source when a good nitrogen source is available . The non-Mendelian genetic element {URE3} is a prion (infectious protein) form of Ure2p, so that overexpression of Ure2p induces the de novo appearance of infectious {URE3} . Earlier studies defined a prion domain comprising Ure2p residues 1 to 64 and a nitrogen regulation domain included in residues 66 to 354 . We find that deletion of individual runs of asparagine within the prion domain reduce prion-inducing activity . Although residues 1 to 64 are sufficient for prion induction, the fragment from residues 1 to 80 is a more efficient inducer of {URE3} . In-frame deletion of a region around residue 224 does not affect nitrogen regulation but does eliminate prion induction by the remainder of Ure2p . Larger deletions removing the region around residue 224 and more of the C-terminal part of Ure2p restore prion-inducing ability . A fragment of Ure2p lacking the original prion domain does not induce {URE3}, but surprisingly, further deletion of residues 151 to 157 and 348 to 354 leaves a fragment that can do so . The region from 66 to 80 and the region around residue 224 are both necessary for this second prion-inducing activity . Thus, each of two nonoverlapping parts of Ure2p is sufficient to induce the appearance of the {URE3} prion. Mol Cell Biol, 1999 Jun, 19(6), 4390 - 404 Btf, a novel death-promoting transcriptional repressor that interacts with Bcl-2-related proteins; Kasof GM et al.; The adenovirus E1B 19,000-molecular-weight (19K) protein is a potent inhibitor of apoptosis and cooperates with E1A to transform primary rodent cells . E1B 19K shows sequence and functional homology to the mammalian antiapoptotic gene product, Bcl-2 . Like Bcl-2, the biochemical mechanism of E1B 19K function includes binding to and antagonization of cellular proapoptotic proteins such as Bax, Bak, and Nbk/Bik . In addition, there is evidence that E1B 19K can affect gene expression, but whether this contributes to its antiapoptotic function has not been determined . In an effort to further understand the functions of E1B 19K, we screened for 19K-associated proteins by the yeast two-hybrid system . A novel protein, Btf (Bcl-2-associated transcription factor), that interacts with E1B 19K as well as with the antiapoptotic family members Bcl-2 and Bcl-xL but not with the proapoptotic protein Bax was identified . btf is a widely expressed gene that encodes a protein with homology to the basic zipper (bZip) and Myb DNA binding domains . Btf binds DNA in vitro and represses transcription in reporter assays . E1B 19K, Bcl-2, and Bcl-xL sequester Btf in the cytoplasm and block its transcriptional repression activity . Expression of Btf also inhibited transformation by E1A with either E1B 19K or mutant p53, suggesting a role in either promotion of apoptosis or cell cycle arrest . Indeed, the sustained overexpression of Btf in HeLa cells induced apoptosis, which was inhibited by E1B 19K . Furthermore, the chromosomal localization of btf (6q22-23) maps to a region that is deleted in some cancers, consistent with a role for Btf in tumor suppression . Thus, btf may represent a novel tumor suppressor gene residing in a unique pathway by which the Bcl-2 family can regulate apoptosis. Mol Cell Biol, 1999 Jun, 19(6), 4270 - 8 Cell cycle regulation of DNA replication initiator factor Dbf4p; Cheng L et al.; The precise duplication of eukaryotic genetic material takes place once and only once per cell cycle and is dependent on the completion of the previous mitosis . Two evolutionarily conserved kinases, the cyclin B (Clb)/cyclin-dependent kinase (Cdk/Cdc28p) and Cdc7p along with its interacting factor Dbf4p, are required late in G1 to initiate DNA replication . We have determined that the levels of Dbf4p are cell cycle regulated . Dbf4p levels increase as cells begin S phase and remain high through late mitosis, after which they decline dramatically as cells begin the next cell cycle . We report that Dbf4p levels are sensitive to mutations in key components of the anaphase-promoting complex (APC) . In addition, Dbf4p is modified in response to DNA damage, and this modification is dependent upon the DNA damage response pathway . We had previously shown that Dbf4p interacts with the M phase polo-like kinase Cdc5p, a key regulator of the APC late in mitosis . These results further link the actions of the initiator protein, Dbf4p, to the completion of mitosis and suggest possible roles for Dbf4p during progression through mitosis. Mol Cell Biol, 1999 Jun, 19(6), 4247 - 54 B-Cell coactivator OBF-1 exhibits unusual transcriptional properties and functions in a DNA-bound Oct-1-dependent fashion; Krapp A et al.; Eukaryotic transcriptional activators generally comprise both a DNA-binding domain that recognizes specific cis-regulatory elements in the target genes and an activation domain which is essential for transcriptional stimulation . Activation domains typically behave as structurally and functionally autonomous modules that retain their intrinsic activities when directed to a promoter by a variety of heterologous DNA-binding domains . Here we report that OBF-1, a B-cell-specific coactivator for transcription factor Oct-1, challenges this traditional view in that it contains an atypical activation domain that exhibits two unexpected functional properties when tested in the yeast Saccharomyces cerevisiae . First, OBF-1 by itself has essentially no intrinsic activation potential, yet it strongly synergizes with other activation domains such as VP16 and Gal4 . Second, OBF-1 exerts its effect in association with DNA-bound Oct-1 but is inactive when attached to a heterologous DNA-binding domain . These findings suggest that activation by OBF-1 is not obtained by simple recruitment of the coactivator to the promoter but requires interaction with DNA-bound Oct-1 to stimulate a step distinct from those regulated by classical activation domains. Mol Cell Biol, 1999 Jun, 19(6), 4134 - 42 Homology search and choice of homologous partner during mitotic recombination; Inbar O et al.; Homologous recombination is an important DNA repair mechanism in vegetative cells . During the repair of double-strand breaks, genetic information is transferred between the interacting DNA sequences (gene conversion) . This event is often accompanied by a reciprocal exchange between the homologous molecules, resulting in crossing over . The repair of DNA damage by homologous recombination with repeated sequences dispersed throughout the genome might result in chromosomal aberrations or in the inactivation of genes . It is therefore important to understand how the suitable homologous partner for recombination is chosen . We have developed a system in the yeast Saccharomyces cerevisiae that can monitor the fate of a chromosomal double-strand break without the need to select for recombinants . The broken chromosome is efficiently repaired by recombination with one of two potential partners located elsewhere in the genome . One of the partners has homology to the broken ends of the chromosome, whereas the other is homologous to sequences distant from the break . Surprisingly, a large proportion of the repair is carried out by recombination involving the sequences distant from the broken ends . This repair is very efficient, despite the fact that it requires the processing of a large chromosomal region flanking the break . Our results imply that the homology search involves extensive regions of the broken chromosome and is not carried out exclusively by sequences adjacent to the double-strand break . We show that the mechanism that governs the choice of homologous partners is affected by the length and sequence divergence of the interacting partners, as well as by mutations in the mismatch repair genes . We present a model to explain how the suitable homologous partner is chosen during recombinational repair . The model provides a mechanism that may guard the integrity of the genome by preventing recombination between dispersed repeated sequences. Mol Cell Biol, 1999 Jun, 19(6), 4065 - 78 Ku antigen-DNA conformation determines the activation of DNA-dependent protein kinase and DNA sequence-directed repression of mouse mammary tumor virus transcription; Giffin W et al.; Mouse mammary tumor virus (MMTV) transcription is repressed by DNA-dependent protein kinase (DNA-PK) through a DNA sequence element, NRE1, in the viral long terminal repeat that is a sequence-specific DNA binding site for the Ku antigen subunit of the kinase . While Ku is an essential component of the active kinase, how the catalytic subunit of DNA-PK (DNA-PKcs) is regulated through its association with Ku is only beginning to be understood . We report that activation of DNA-PKcs and the repression of MMTV transcription from NRE1 are dependent upon Ku conformation, the manipulation of DNA structure by Ku, and the contact of Ku80 with DNA . Truncation of one copy of the overlapping direct repeat that comprises NRE1 abrogated the repression of MMTV transcription by Ku-DNA-PKcs . Remarkably, the truncated element was recognized by Ku-DNA-PKcs with affinity similar to that of the full-length element but was unable to promote the activation of DNA-PKcs . Analysis of Ku-DNA-PKcs interactions with DNA ends, double- and single-stranded forms of NRE1, and the truncated NRE1 element revealed striking differences in Ku conformation that differentially affected the recruitment of DNA-PKcs and the activation of kinase activity. Mol Cell Biol, 1999 Jun, 19(6), 3951 - 7 A TATA-binding protein mutant defective for TFIID complex formation in vivo; Ranallo RT et al.; Using an intragenic complementation screen, we have identified a temperature-sensitive TATA-binding protein (TBP) mutant (K151L, K156Y) that is defective for interaction with certain yeast TBP-associated factors (TAFs) at the restrictive temperature . The K151L,K156Y mutant appears to be functional for RNA polymerase I (Pol I) and Pol III transcription, and it is capable of supporting Gal4-activated and Gcn4-activated transcription by Pol II . However, transcription from certain TATA-containing and TATA-less Pol II promoters is reduced at the restrictive temperature . Immunoprecipitation analysis of extracts prepared after culturing cells at the restrictive temperature for 1 h indicates that the K151L,K156Y derivative is severely compromised in its ability to interact with TAF130, TAF90, TAF68/61, and TAF25 while remaining functional for interaction with TAF60 and TAF30 . Thus, a TBP mutant that is compromised in its ability to form TFIID can support the response to Gcn4 but is defective for transcription from specific promoters in vivo. J Biol Chem, 1999 May 21, 274(21), 15284 - 91 Vps9p is a guanine nucleotide exchange factor involved in vesicle-mediated vacuolar protein transport; Hama H et al.; Vacuolar protein sorting (vps) mutants of Saccharomyces cerevisiae missort and secrete vacuolar hydrolases . The gene affected in one of these mutants, VPS21, encodes a member of the Sec4/Ypt/Rab family of small GTPases . Rab proteins play an essential role in vesicle-mediated protein transport . Using both yeast two-hybrid assays and chemical cross-linking, we have identified another VPS gene product, Vps9p, that preferentially interacts with a mutant form of Vps21p-S21N that binds GDP but not GTP . In vitro purified Vps9p was found to stimulate GDP release from Vps21p in a dose-dependent manner . Vps9p also stimulated GTP association as a result of facilitated GDP release . However, Vps9p did not stimulate guanine nucleotide exchange of GTP-bound Vps21p or GTP hydrolysis . We tested the ability of Vps9p to stimulate the intrinsic guanine nucleotide exchange activity of Rab5, which is a mammalian sequence homologue of Vps21p, and Ypt7p, which is another yeast Rab protein involved in vacuolar protein transport . Rab5, but not Ypt7p was responsive to Vps9p, which indicates that Vps9p recognizes sequence variation among Rab proteins . We conclude that Vps9p is a novel guanine nucleotide exchange factor that is specific for Vps21p/Rab5 . Since there are no obvious Vps9p sequence homologues in yeast, Vps9p may also possess unique regulatory functions required for vacuolar protein transport. J Biol Chem, 1999 May 21, 274(21), 15262 - 70 Interaction of insulin receptor substrate 3 with insulin receptor, insulin receptor-related receptor, insulin-like growth factor-1 receptor, and downstream signaling proteins; Xu P et al.; Insulin receptor substrates (IRS) mediate biological actions of insulin, growth factors, and cytokines . All four mammalian IRS proteins contain pleckstrin homology (PH) and phosphotyrosine binding (PTB) domains at their N termini . However, the molecules diverge in their C-terminal sequences . IRS3 is considerably shorter than IRS1, IRS2, and IRS4, and is predicted to interact with a distinct group of downstream signaling molecules . In the present study, we investigated interactions of IRS3 with various signaling molecules . The PTB domain of mIRS3 is necessary and sufficient for binding to the juxtamembrane NPXpY motif of the insulin receptor in the yeast two-hybrid system . This interaction is stronger if the PH domain or the C-terminal phosphorylation domain is retained in the construct . As determined in a modified yeast two-hybrid system, mIRS3 bound strongly to the p85 subunit of phosphatidylinositol 3-kinase . Although high affinity interaction required the presence of at least two of the four YXXM motifs in mIRS3, there was not a requirement for specific YXXM motifs . mIRS3 also bound to SHP2, Grb2, Nck, and Shc, but less strongly than to p85 . Studies in COS-7 cells demonstrated that deletion of either the PH or the PTB domain abolished insulin-stimulated phosphorylation of mIRS3 . Insulin stimulation promoted the association of mIRS3 with p85, SHP2, Nck, and Shc . Despite weak association between mIRS3 and Grb2, this interaction was not increased by insulin, and may not be mediated by the SH2 domain of Grb2 . Thus, in contrast to other IRS proteins, mIRS3 appears to have greater specificity for activation of the phosphatidylinositol 3-kinase pathway rather than the Grb2/Ras pathway. J Biol Chem, 1999 May 21, 274(21), 15041 - 5 Structure-function analyses of the ATX1 metallochaperone; Portnoy ME et al.; Saccharomyces cerevisiae Atx1p represents a member of the family of metallochaperone molecules that escort copper to distinct intracellular targets . Atx1p specifically delivers copper to the Ccc2p copper transporter in the Golgi . Additionally, when overproduced, Atx1p substitutes for superoxide dismutase 1 in preventing oxidative damage; however the mechanistic overlap between these functions is unresolved . The crystal structure of Atx1p has been solved recently . By examining a surface electrostatic potential distribution, multiple conserved lysines are revealed on one face of Atx1p . An additional conserved lysine (Lys65) lies in close proximity to the metal binding site . Through site-directed mutagenesis, residues in the metal binding region including Lys65 were found to be necessary for both copper delivery to Ccc2p and for Atx1p antioxidant activity . Copper trafficking to Ccc2p also relied on the lysine-rich face of Atx1p . Surprisingly however, elimination of these lysines did not inhibit the antioxidant activity of Atx1p . We provide evidence that Atx1p does not suppress oxidative damage by a metallochaperone mechanism but may directly consume superoxide . Purified Cu-Atx1p reacts noncatalytically with superoxide anion in vitro . We conclude that the copper-trafficking and antioxidant functions of Atx1p arise from chemically and structurally distinct attributes of this metallochaperone. J Biol Chem, 1999 May 21, 274(21), 14806 - 17 Molecular dissection of guanine nucleotide dissociation inhibitor function in vivo . Rab-independent binding to membranes and role of Rab recycling factors; Luan P et al.; Guanine nucleotide dissociation inhibitor (GDI) is an essential protein required for the recycling of Rab GTPases mediating the targeting and fusion of vesicles in the exocytic and endocytic pathways . Using site-directed mutagenesis of yeast GDI1, we demonstrate that amino acid residues required for Rab recognition in vitro are critical for function in vivo in Saccharomyces cerevisiae . Analysis of the effects of Rab-binding mutants on function in vivo reveals that only a small pool of recycling Rab protein is essential for growth, and that the rates of recycling of distinct Rabs are differentially sensitive to GDI . Furthermore, we find that membrane association of Gdi1p is Rab-independent . Mutant Gdi1 proteins unable to bind Rabs were able to associate with cellular membranes as efficiently as wild-type Gdi1p, yet caused a striking loss of the endogenous cytosolic Gdi1p-Rab pools leading to dominant inhibition of growth when expressed at levels of the normal, endogenous pool . These results demonstrate a potential role for a new recycling factor in the retrieval of Rab-GDP from membranes, and illustrate the importance of multiple effectors in regulating GDI function in Rab delivery and retrieval from membranes. J Biol Chem, 1999 May 21, 274(21), 14685 - 91 Identification of rabbit reticulocyte E217K as a UBC7 homologue and functional characterization of its core domain loop; Lin H et al.; The structural basis by which ubiquitin (Ub)-conjugating enzymes (E2s) determine substrate specificity remains unclear . We cloned rabbit reticulocyte E217K because unlike the similarly sized class I E2s, E214K and UBC4, it is unable to support ubiquitin-protein ligase (E3)-dependent conjugation to endogenous proteins . RNA analysis revealed that this E2 was expressed in all tissues tested, with higher levels in the testis . Analysis of testis RNA from rats of different ages showed that E217K mRNA was induced from days 15 to 30 . The predicted amino acid sequence indicates that E217K is a 19 . 5-kDa class I E2 but differs from other class I enzymes in possessing an insertion of 13 amino acids distal to the active site cysteine . E217K shows 74% amino acid identity with Saccharomyces cerevisiae UBC7, and therefore, we rename it mammalian UBC7 . Yeast UBC7 crystal structure indicates that this insertion forms a loop out of the otherwise conserved folding structure . Sequence analysis of E2s had previously suggested that this loop is a hypervariable region and may play a role in substrate specificity . We created mutant UBC7 lacking the loop (ubc7Deltaloop) and a mutant E214k with an inserted loop (E214k+loop) and characterized their biochemical functions . Ubc7Deltaloop had higher affinity for the E1-Ub thiol ester than native UBC7 and permitted conjugation of Ub to selected proteins in the testis but did not permit the broad spectrum E3-dependent conjugation to endogenous reticulocyte proteins . Surprisingly, E214k+loop was unable to accept Ub from ubiquitin-activating enzyme (E1) but was able to accept NEDD8 from E1 . E214k+loop was able to support conjugation of NEDD8 to endogenous reticulocyte proteins but with much lower efficiency than E214k . Thus, the loop can influence interactions of the E2 with charged E1 as well as with E3s or substrates, but the exact nature of these interactions depends on divergent sequences in the remaining conserved core domain. EMBO J, 1999 May 17, 18(10), 2756 - 63 A constitutively active G-protein-coupled receptor causes mating self-compatibility in the mushroom Coprinus; Olesnicky NS et al.; In the mushroom Coprinus cinereus, the multiallelic B mating type genes are predicted to encode a large family of seven-transmembrane domain receptors and CaaX-modified pheromones . We have shown that a single amino acid change Q229P in transmembrane domain VI of one receptor confers a self-compatible mating phenotype . Using a heterologous yeast assay, we have demonstrated that this C.cinereus pheromone receptor is a G-protein-coupled receptor and that the Q229P mutation is constitutively activating . A C.cinereus pheromone precursor was processed to an active species specifically in yeast MATa cells and activated the co-expressed wild-type receptor . Yeast cells expressing the wild-type receptor were used to test the activity of synthetic peptides, enabling us to predict the structure of the mature C.cinereus pheromone and to show that the Q229P mutation does not compromise normal receptor function. EMBO J, 1999 May 17, 18(10), 2707 - 21 Sister chromatid separation and chromosome re-duplication are regulated by different mechanisms in response to spindle damage; Alexandru G et al.; In yeast, anaphase entry depends on Pds1 proteolysis, while chromosome re-duplication in the subsequent S-phase involves degradation of mitotic cyclins such as Clb2 . Sequential proteolysis of Pds1 and mitotic cyclins is mediated by the anaphase-promoting complex (APC) . Lagging chromosomes or spindle damage are detected by surveillance mechanisms (checkpoints) which block anaphase onset, cytokinesis and DNA re-replication . Until now, the MAD and BUB genes implicated in this regulation were thought to function in a single pathway that blocks APC activity . We show that spindle damage blocks sister chromatid separation solely by inhibiting APCCdc20-dependent Pds1 proteolysis and that this process requires Mad2 . Blocking APCCdh1-mediated Clb2 proteolysis and chromosome re-duplication does not require Mad2 but a different protein, Bub2 . Our data imply that Mad1, Mad2, Mad3 and Bub1 regulate APCCdc20, whereas Bub2 regulates APCCdh1. Virology, 1999 May 10, 257(2), 341 - 51 Self-interaction of the herpes simplex virus type 1 regulatory protein ICP27; Zhi Y et al.; The herpes simplex virus type 1 (HSV-1) regulatory protein ICP27 is a nuclear phosphoprotein required for viral lytic infection, which acts partly at the posttranscriptional level to affect RNA processing and export . In the present study, we show that ICP27 can interact with itself in vivo . Immunofluorescent staining of cells expressing both an ICP27 mutant with a deletion of the major nuclear localization signal and wild-type ICP27 showed that the mutant protein was efficiently imported into the nucleus in the majority of the cotransfected cells, suggesting heterodimer formation between the wild-type and mutant proteins . Coimmunoprecipitation experiments using epitope-tagged wild-type ICP27 and a series of ICP27 mutants with deletions and insertions in important functional regions of the protein revealed that the C-terminal cysteine-histidine-rich zinc-finger-like region of ICP27 was required for the self-association . Furthermore the self-association was also shown in yeast using two-hybrid assays, and again, an intact C-terminal zinc-finger-like region was required for the interaction . This study provides biochemical evidence that ICP27 may function as a multimer in infected cells . Virology, 1999 May 10, 257(2), 330 - 40 Hepatitis C virus core protein interacts with a human DEAD box protein DDX3; Owsianka AM et al.; Several studies have implicated hepatitis C virus (HCV) core in influencing the expression of host genes . To identify cellular factors with a possible role in HCV replication and pathogenesis, we looked for cellular proteins that interact with the viral core protein . A human liver cDNA library was screened in a yeast two-hybrid assay to identify cellular proteins that bind to core . Several positive clones were isolated, one of which encoded the C-terminal 253 amino acids of a putative RNA helicase, a DEAD box protein designated DDX3 . Bacterially expressed glutathione-S-transferase-DDX3 fusion protein specifically pulled down in vitro translated and radiolabeled HCV core, confirming a direct interaction . Immunofluorescent staining of HeLa cells with a polyclonal antiserum showed that DDX3 is located predominantly in nuclear speckles and at low levels throughout the cytoplasm . In cells infected with a recombinant vaccinia virus expressing HCV structural proteins (core, E1, and E2), DDX3 and core colocalized in distinct spots in the perinuclear region of the cytoplasm . The regions of the proteins involved in binding were found by deletion analysis to be the N-terminal 59 amino acid residues of core and a C-terminal RS-like domain of DDX3 . The human DDX3 is a putative RNA helicase and a member of a highly conserved DEAD box subclass that includes murine PL10, Xenopus An3, and yeast Ded1 proteins . Their role in RNA metabolism or gene expression is unknown . The significance of core-helicase interaction in HCV replication and pathogenesis is discussed . Biochem Biophys Res Commun, 1999 May 19, 258(3), 572 - 3 RIP-JIP60 alters conformation of ribosomes in vivo; Dunaeva M et al.; Recently it has been demonstrated that expression putative ribosome-inactivating protein JIP 60 in transgenic tobacco plants leads to the depurination of large rRNA and inactivation of plant ribosomes . Treatment of ribosomes from transgenic tobacco plants with low concentration of alpha-sarcin resulted in the appearance of an rRNA specific fragment . No fragment was observed under the same conditions for ribosomes from wild type plants . The alteration of the comformation of ribosomes in transgenic tobacco due to the expression of JIP60 is assumed . Biochem Biophys Res Commun, 1999 May 10, 258(2), 482 - 9 Characterization of telomere-binding activity of replication factor C large subunit p140; Uchiumi F et al.; The large subunit of RFC (RFC p140) has been suggested to be associated with the 3'-end of elongating DNA primer and to recruit proliferating cell nuclear antigen (PCNA) onto DNA polymerase delta . Previously, we isolated a cDNA clone encoding a DNA-binding domain of RFC p140 as a telomeric repeat (TTAGGG)n binding protein . This domain was shown to have a specific affinity for the 5'-phosphate ends of a telomere repeat sequence . In order to investigate the structure and function of RFC p140, we constructed the full-length recombinant RFC p140 as well as N- and/or C-terminal deleted mutants and analyzed their telomere-binding activities . South-Western blot and gel mobility shift analyses revealed that deletion of the N- but not the C-terminal region enhances recognition of the telomeric repeat sequence and 5'-phosphate ends, suggesting the negative effect of the N-terminal region of the RFC p140 binding to the telomeric repeat . On the other hand, the C-terminal truncated RFC inhibits the telomerase activity more than the N-terminal-deleted and full-length RFC p140 . The inhibitory effect of RFC p140 on telomerase activity is completely diminished by both terminal deletions . Thus, a certain interaction of the N- and C-terminal regions is considered to be required for RFC p140 to suppress telomerase activity . Taken together, these results suggest that both telomeric repeat-binding and telomerase inhibitory activities of RFC p140 are finely regulated by the intrinsic N- and C-terminal regions . J Mol Biol, 1999 May 14, 288(4), 743 - 52 Helix capping in the GCN4 leucine zipper; Lu M et al.; Capping interactions associated with specific sequences at or near the ends of alpha-helices are important determinants of the stability of protein secondary and tertiary structure . We investigate here the role of the helix-capping motif Ser-X-X-Glu, a sequence that occurs frequently at the N termini of alpha helices in proteins, on the conformation and stability of the GCN4 leucine zipper . The 1.8 A resolution crystal structure of the capped molecule reveals distinct conformations, packing geometries and hydrogen-bonding networks at the amino terminus of the two helices in the leucine zipper dimer . The free energy of helix stabilization associated with the hydrogen-bonding and hydrophobic interactions in this capping structure is -1.2 kcal/mol, evaluated from thermal unfolding experiments . A single cap thus contributes appreciably to stabilizing the terminated helix and thereby the native state . These results suggest that helix capping plays a further role in protein folding, providing a sensitive connector linking alpha-helix formation to the developing tertiary structure of a protein . Arch Biochem Biophys, 1999 May 15, 365(2), 307 - 16 The Delta8-desaturase of Euglena gracilis: an alternate pathway for synthesis of 20-carbon polyunsaturated fatty acids; Wallis JG et al.; Desaturation of fatty acids is an important metabolic process . In mammals, 20-carbon and longer polyunsaturated fatty acids are not only incorporated into cellular membranes in a tissue-specific manner, but also serve as the precursors to synthesis of eicosanoid metabolic regulators . The processes of desaturation and elongation in human liver are well characterized, but an alternate Delta8 desaturation pathway that may be important in certain tissues or in cancer cells is less well examined . The Delta8-desaturase enzyme introduces a double bond at the 8-position in 20-carbon fatty acids that have an existing Delta11 unsaturation . We have isolated the first fatty acid Delta8-desaturase, from the protist Euglena gracilis, in order to explore this alternate pathway . A full-length cDNA was obtained after reverse transcription of mRNA purified from heterotrophically grown Euglena, followed by PCR amplification with primers degenerate to conserved histidine-rich regions of microsomal desaturases . The protein predicted from the cDNA sequence is highly homologous to Delta5 and Delta6 desaturases of Caenhorabditis elegans . When the cDNA was expressed in Saccharomyces cerevisiae, the yeast cultures readily desaturated appropriate 20-carbon fatty acids by inserting an additional double bond at the Delta8-position . The enzyme demonstrated a preference for substrates of metabolic significance, 20:3 Delta11,14,17 and 20:2 Delta11,14 . Cloning of a Delta8 fatty acid desaturase offers the opportunity to examine an alternate pathway of long chain fatty acid biosynthesis . Oncogene, 1999 Apr 1, 18(13), 2273 - 9 HOX11 interacts with CTF1 and mediates hematopoietic precursor cell immortalization; Zhang N et al.; HOX11 is a homeodomain-containing oncogenic transcription factor that immortalizes hematopoietic precursor cells . The mechanism by which HOX11 facilitates this initial step of leukemogenesis is, however, not well understood . We have used a DNA binding site selection assay to investigate cooperative DNA binding by HOX11 with other transcription factors . A consensus sequence was derived and identified as the binding site for the CCAAT-box-binding transcription factors (CTF) . HOX11 was shown to interact in vitro and in vivo with CTF1 . Retrovirus-mediated transduction of an antisense CTF1 cDNA dramatically reduced the proliferative capacity of HOX11-immortalized hematopoietic precursor cells . CTF1 is, therefore, the first HOX11 protein partner identified that plays an important role in hematopoietic precursor cell immortalization. Nucleic Acids Res, 1999 Jun 1, 27(11), 2393 - 9 Identification and analysis of the Arabidopsis thaliana BSH gene, a member of the SNF5 gene family; Brzeski J et al.; The multiprotein complexes involved in active dis-ruption of chromatin structure, homologous to yeast SWI/SNF complex, have been described for human and Drosophila cells . In all SWI/SNF-class complexes characterised so far, one of the key components is the SNF5-type protein . Here we describe the isolation of a plant (Arabidopsis thaliana ) cDNA encoding a 27 kDa protein which we named BSH, with high homology to yeast SNF5p and its human (INI1) and Drosophila (SNR1) counterparts as well as to other putative SNF5-type proteins from Caenorhabditis elegans, fish and yeast . With 240 amino acids, the Arabidopsis BSH is the smallest SNF5-type protein so far identified . When expressed in Saccharomyces cerevisiae, the gene for BSH partially complements the snf5 mutation . BSH is, however, unable to activate transcription in yeast when tethered to DNA . The gene for BSH occurs in single copy in the Arabidopsis genome and is ubiquitously expressed in the plant . Analysis of the whole cell and nuclear protein extracts with antibodies against recombinant BSH indicates that the protein is localised in nuclei . Transgenic Arabidopsis plants with markedly decreased physiological level of the BSH mRNA, resulting from the expression of antisense messenger, are viable but exhibit a distinctive phenotype characterised by bushy growth and flowers that are unable to produce seeds. Protein Eng, 1999 Apr, 12(4), 297 - 303 Modelling of the disulphide-swapped isomer of human insulin-like growth factor-1: implications for receptor binding; Gill R et al.; Insulin-like growth factor-1 (IGF-1) is a serum protein which unexpectedly folds to yield two stable tertiary structures with different disulphide connectivities; native IGF-1 {18-61,6-48,47-52} and IGF-1 swap {18-61,6-47, 48-52} . Here we demonstrate in detail the biological properties of recombinant human native IGF-1 and IGF-1 swap secreted from Saccharomyces cerevisiae . IGF-1 swap had a approximately 30 fold loss in affinity for the IGF-1 receptor overexpressed on BHK cells compared with native IGF-1.The parallel increase in dose required to induce negative cooperativity together with the parallel loss in mitogenicity in NIH 3T3 cells implies that disruption of the IGF-1 receptor binding interaction rather than restriction of a post-binding conformational change is responsible for the reduction in biological activity of IGF-1 swap . Interestingly, the affinity of IGF-1 swap for the insulin receptor was approximately 200 fold lower than that of native IGF-1 indicating that the binding surface complementary to the insulin receptor (or the ability to attain it) is disturbed to a greater extent than that to the IGF-1 receptor . A 1.0 ns high-temperature molecular dynamics study of the local energy landscape of IGF-1 swap resulted in uncoiling of the first A-region alpha-helix and a rearrangement in the relative orientation of the A- and B-regions . The model of IGF-1 swap is structurally homologous to the NMR structure of insulin swap and CD spectra consistent with the model are presented . However, in the model of IGF-1 swap the C-region has filled the space where the first A-region alpha-helix has uncoiled and this may be hindering interaction of Val44 with the second insulin receptor binding pocket. J Clin Microbiol, 1999 Jun, 37(6), 1985 - 93 Identification of medically relevant Trichosporon species based on sequences of internal transcribed spacer regions and construction of a database for Trichosporon identification; Sugita T et al.; The nucleotide sequences of the internal transcribed spacer (ITS) 1 and 2 regions in the rRNA gene were determined by directly sequencing PCR-amplified fragments for all of the species (17 species and five varieties) in the genus Trichosporon . Comparative sequence analysis suggests that six medically relevant species, T . asahii, T . asteroides, T . cutaneum, T . inkin, T . mucoides, and T . ovoides, can be readily identified by their ITS sequences . In addition, the sequence analysis showed that conspecific strains have fewer than 1% nucleotide differences in the ITS 1 and 2 regions overall . Molecular phylogenetic trees are also presented. J Clin Endocrinol Metab, 1999 May, 84(5), 1582 - 9 Noninsulinoma pancreatogenous hypoglycemia: a novel syndrome of hyperinsulinemic hypoglycemia in adults independent of mutations in Kir6.2 and SUR1 genes; Service FJ et al.; In adults, endogenous hyperinsulinemic hypoglycemia is almost invariably due to insulinoma . In these patients with insulinoma, neuroglycopenic episodes exclusively after meal ingestion and negative 72-h fasts are extraordinarily rare . We describe five adults with neuroglycopenic episodes from hyperinsulinemic hypoglycemia within 4 h of meal ingestion and negative 72-h fasts . Each had negative transabdominal ultrasonography, spiral computed tomographic scanning, and celiac axis angiography of the pancreas . However, all showed positive selective arterial calcium stimulation tests indicative of pancreatic beta-cell hyperfunction . At pancreatic exploration, no insulinoma was detected by intraoperative ultrasonography and complete mobilization and palpation of the pancreas . Moreover, the resected pancreata showed islet hypertrophy and nesidioblastosis, but no insulinoma . No definite disease-causing mutation was detected in Kir6.2 and SUR1 genes, which encode the subunits of the pancreatic ATP-sensitive potassium channel responsible for glucose-induced insulin secretion . Four patients who underwent gradient-guided partial pancreatectomy have been free of hypoglycemic symptoms for up to 3 yr follow-up; the other, who underwent a limited distal pancreatectomy, has had brief recurrence of symptoms . The unique clinical features and responses to dynamic testing in these adults with hyperinsulinemic hypoglycemia in the absence of insulinoma may constitute a new syndrome of postprandial hypoglycemia from diffuse beta-cell hyperfunction. Mol Gen Genet, 1999 Apr, 261(3), 452 - 62 NAD+-dependent glutamate dehydrogenase of the edible mushroom Agaricus bisporus: biochemical and molecular characterization; Kersten MA et al.; The NAD+-dependent glutamate dehydrogenase (NAD-GDH) of Agaricus bisporus, a key enzyme in nitrogen metabolism, was purified to homogeneity . The apparent molecular mass of the native enzyme is 474 kDa comprising four subunits of 116 kDa . The isoelectric point of the enzyme is about 7.0 . Km values for ammonium, 2-oxoglutarate, NADH, glutamate and NAD+ were 6.5, 3.5, 0.06, 37.1 and 0.046 mM, respectively . The enzyme is specific for NAD(H) . The gene encoding this enzyme (gdhB) was isolated from an A . bisporus H39 recombinant lambda phage library . The deduced amino acid sequence specifies a 1029-amino acid protein with a deduced molecular mass of 115,463 Da, which displays a significant degree of similarity with NAD-GDH of Saccharomyces cerevisiae and Neurospora crassa . The ORF is interrupted by fifteen introns . Northern analysis combined with enzyme activity measurements suggest that NAD-GDH from A . bisporus is regulated by the nitrogen source . NAD-GDH levels in mycelium grown on glutamate were higher than NAD-GDH levels in mycelium grown on ammonium as a nitrogen source . Combined with the kinetic parameters, these results suggest a catabolic role for NAD-GDH . However, upon addition of ammonium to the culture transcription of the gene is not repressed as strongly as that of the gene encoding NADP-GDH (gdhA) . To date, tetrameric NAD-GDHs with large subunits, and their corresponding genes, have only been isolated from a few species . This enzyme represents the first NAD-GDH of basidiomycete origin to be purified and is the first such enzyme from basidiomycetes whose sequence has been determined. Trends Cell Biol, 1999 May, 9(5), 193 - 8 Histone deacetylases: transcriptional repression with SINers and NuRDs; Ayer DE; The DNA in eukaryotic cells is packaged into chromatin, which functions as a boundary to the transcriptional activation process . The nucleosome is the basic repeating unit of chromatin . The purification and characterization of several chromatin-remodelling complexes and the demonstration that histone acetyltransferases and histone deacetylases are regulatory components of coactivator and corepressor complexes, respectively, demonstrates that the nucleosome is not simply a static architectural feature of chromatin but, rather, plays a dynamic and integral role in the regulation of gene expression . This review focuses primarily on histone deacetylases and deacetylase-containing complexes and their role in mediating transcriptional repression. Trends Biochem Sci, 1999 Apr, 24(4), 146 - 50 Interactions among pathways for phosphatidylcholine metabolism, CTP synthesis and secretion through the Golgi apparatus; Kent C et al.; Phosphatidylcholine is the major phospholipid in eukaryotic cells . It serves as a structural component of cell membranes and a reservoir of several lipid messengers . Recent studies in yeast and mammalian systems have revealed interrelationships between the two pathways of phosphatidylcholine metabolism, and between these pathways and those for CTP synthesis and secretion via the Golgi . These processes involve the regulation of the CDP-choline and phosphatidylethanolamine-methylation pathways of phosphatidylcholine synthesis, CTP synthetase, phospholipase D and the phospholipid-transfer protein Sec14p. Curr Opin Struct Biol, 1999 Apr, 9(2), 268 - 74 Microtubule-based motor function in mitosis; Heald R et al.; Microtubule-based motors are essential both for the proper assembly of the mitotic spindle and for chromosome segregation . Mitotic motors in the yeast Saccharomyces cerevisiae exhibit either overlapping or opposing activities in order to achieve proper spindle function, whereas the analysis of motors using vertebrate cytoplasmic extracts has revealed less functional redundancy . In several systems, biochemical, genetic and two-hybrid approaches have been used both to identify associated nonmotor proteins and to address the molecular mechanisms behind kinetochore movements during chromosome alignment and segregation. Curr Opin Struct Biol, 1999 Apr, 9(2), 250 - 9 Gamma-tubulin complexes and their interaction with microtubule-organizing centers; Wiese C et al.; Gamma-tubulin is as ubiquitous in eukaryotes as alpha- and beta-tubulin . Rather than forming part of the microtubule wall, however, gamma-tubulin is involved in microtubule nucleation . Although gamma-tubulin concentrates at microtubule-organizing centers, it also exists in a cytoplasmic complex whose size and complexity depends on the organism and cell type . In the past year, progress in understanding the functions of gamma-tubulin was made on two fronts: identifying the proteins that interact with gamma-tubulin and identifying the proteins that interact with the gamma-tubulin complex to tether it to the microtubule-organizing center. Curr Opin Genet Dev, 1999 Apr, 9(2), 140 - 7 Coactivator and corepressor complexes in nuclear receptor function; Xu L et al.; The nuclear hormone receptors constitute a large family of transcription factors . The binding of the hormonal ligands induces nuclear receptors to assume a configuration that leads to transcriptional activation . Recent studies of retinoic acid and thyroid hormone receptors revealed that, upon ligand binding, a histone deacetylase (HDAC)-containing complex is displaced from the nuclear receptor in exchange for a histone acetyltransferase (HAT)-containing complex . These observations suggest that ligand-dependent recruitment of chromatin-remodeling activity serves as a general mechanism underlying the switch of nuclear receptors from being transcriptionally repressive to being transcriptionally active. J Cell Biochem, 1999 Jun 1, 73(3), 390 - 9 Human p120ctn catenin: tissue-specific expression of isoforms and molecular interactions with BP180/type XVII collagen; Aho S et al.; Catenins, a family of structurally related proteins, are involved in epidermal keratinocyte cell-cell adhesion by interacting through their central Armadillo repeats with the intracellular domains of cadherins, transmembrane components of the adhesion junctions . p120ctn is a catenin expressed in different isoforms due to alternative splicing and multiple translation start sites . BP180 is a collagenous transmembrane protein (type XVII collagen) localized to hemidesmosomal attachment complexes in basal keratinocytes . In this study, we have delineated the molecular interaction between these two proteins utilizing the yeast two-hybrid system, which was confirmed by an in vitro protein-protein interaction assay . Specifically, it was shown that an amino-terminal segment of BP180 (aa . 13-25) contains the information necessary for binding to p120ctn isoforms 1-3, but not to the isoform 4, suggesting that the interacting domain is located immediately upstream from the Armadillo repeats and is encoded by exons 5 and 6, which are subject to alternative splicing only in a minority of transcripts . In addition to epidermal keratinocytes, p120ctn was shown to be expressed in a variety of adult and fetal tissues as well as in a number of human tumors . The expression pattern of various p120ctn transcripts, reflecting alternative splicing of the 5' exons, was strikingly similar between the corresponding adult and fetal tissues, while the expression patterns were discordant between certain tumors and their normal parental tissues, suggesting a functional role for the tissue-specific expression of the p120ctn isoforms . Finally, the tissue-specific expression of BP180 was shown to partially overlap with that of p120ctn, suggesting that the interaction of these two proteins may contribute to the modulation of cell-cell/matrix interactions in such tissues. Nat Genet, 1999 May, 22(1), 110 - 4 Synphilin-1 associates with alpha-synuclein and promotes the formation of cytosolic inclusions; Engelender S et al.; Parkinson disease (PD) is a neurodegenerative disease characterized by tremor, bradykinesia, rigidity and postural instability . Post-mortem examination shows loss of neurons and Lewy bodies, which are cytoplasmic eosinophilic inclusions, in the substantia nigra and other brain regions . A few families have PD caused by mutations (A53T or A30P) in the gene SNCA (encoding alpha-synuclein) . Alpha-synuclein is present in Lewy bodies of patients with sporadic PD, suggesting that alpha-synuclein may be involved in the pathogenesis of PD . It is unknown how alpha-synuclein contributes to the cellular and biochemical mechanisms of PD, and its normal functions and biochemical properties are poorly understood . To determine the protein-interaction partners of alpha-synuclein, we performed a yeast two-hybrid screen . We identified a novel interacting protein, which we term synphilin-1 (encoded by the gene SNCAIP) . We found that alpha-synuclein interacts in vivo with synphilin-1 in neurons . Co-transfection of both proteins (but not control proteins) in HEK 293 cells yields cytoplasmic eosinophilic inclusions. Nat Genet, 1999 May, 22(1), 98 - 101 Acetylated histones are associated with FMR1 in normal but not fragile X-syndrome cells; Coffee B et al.; Mutation of FMR1 results in fragile X mental retardation . The most common FMR1 mutation is expansion of a CGG repeat tract at the 5' end of FMR1, which leads to cytosine methylation and transcriptional silencing . Both DNA methylation and histone deacetylation have been associated with transcriptional inactivity . The finding that the methyl cytosine-binding protein MeCP2 binds to histone deacetylases and represses transcription in vivo supports a model in which MeCP2 recruits histone deacetylases to methylated DNA, resulting in histone deacetylation, chromatin condensation and transcriptional silencing . Here we demonstrate that the 5' end of FMR1 is associated with acetylated histones H3 and H4 in cells from normal individuals, but acetylation is reduced in cells from fragile X patients . Treatment of fragile X cells with 5-aza-2'-deoxycytidine (5-aza-dC) resulted in reassociation of acetylated histones H3 and H4 with FMR1 and transcriptional reactivation, whereas treatment with trichostatin A (TSA) led to almost complete acetylated histone H4 and little acetylated histone H3 reassociation with FMR1, as well as no detectable transcription . Our results represent the first description of loss of histone acetylation at a specific locus in human disease, and advance understanding of the mechanism of FMR1 transcriptional silencing. J Bacteriol, 1999 May, 181(10), 3136 - 43 SSB, encoding a ribosome-associated chaperone, is coordinately regulated with ribosomal protein genes; Lopez N et al.; Genes encoding ribosomal proteins and other components of the translational apparatus are coregulated to efficiently adjust the protein synthetic capacity of the cell . Ssb, a Saccharomyces cerevisiae Hsp70 cytosolic molecular chaperone, is associated with the ribosome-nascent chain complex . To determine whether this chaperone is coregulated with ribosomal proteins, we studied the mRNA regulation of SSB under several environmental conditions . Ssb and the ribosomal protein rpL5 mRNAs were up-regulated upon carbon upshift and down-regulated upon amino acid limitation, unlike the mRNA of another cytosolic Hsp70, Ssa . Ribosomal protein and Ssb mRNAs, like many mRNAs, are down-regulated upon a rapid temperature upshift . The mRNA reduction of several ribosomal protein genes and Ssb was delayed by the presence of an allele, EXA3-1, of the gene encoding the heat shock factor (HSF) . However, upon a heat shock the EXA3-1 mutation did not significantly alter the reduction in the mRNA levels of two genes encoding proteins unrelated to the translational apparatus . Analysis of gene fusions indicated that the transcribed region, but not the promoter of SSB, is sufficient for this HSF-dependent regulation . Our studies suggest that Ssb is regulated like a core component of the ribosome and that HSF is required for proper regulation of SSB and ribosomal mRNA after a temperature upshift. Genes Cells, 1999 Feb, 4(2), 77 - 85 The biochemistry and biological significance of nonhomologous DNA end joining: an essential repair process in multicellular eukaryotes; Lieber MR; Recent progress over the past year has provided new insights into the proteins involved in nonhomologous end joining . The assembly of Ku and DNA-dependent protein kinase at DNA ends is now understood in greater detail . Murine genetic knockouts for DNA ligase IV and XRCC4 are embryonic lethal, indicating that nonhomologous end joining is essential for viability . Interestingly, neurones, in addition to lymphocytes, are particularly vulnerable to an absence of NHEJ. Science, 1999 May 7, 284(5416), 977 - 80 Roles of phosphorylation sites in regulating activity of the transcription factor Pho4; Komeili A et al.; Transcription factors are often phosphorylated at multiple sites . Here it is shown that multiple phosphorylation sites on the budding yeast transcription factor Pho4 play distinct and separable roles in regulating the factor's activity . Phosphorylation of Pho4 at two sites promotes the factor's nuclear export and phosphorylation at a third site inhibits its nuclear import . Phosphorylation of a fourth site blocks the interaction of Pho4 with the transcription factor Pho2 . Multiple phosphorylation sites provide overlapping and partially redundant layers of regulation that function to efficiently control the activity of Pho4. Cell, 1999 Apr 30, 97(3), 313 - 24 Pch2 links chromatin silencing to meiotic checkpoint control; San-Segundo PA et al.; The PCH2 gene of Saccharomyces cerevisiae is required for the meiotic checkpoint that prevents chromosome segregation when recombination and chromosome synapsis are defective . Mutation of PCH2 relieves the checkpoint-induced pachytene arrest of the zip1, zip2, and dmc1 mutants, resulting in chromosome missegregation and low spore viability . Most of the Pch2 protein localizes to the nucleolus, where it represses meiotic interhomolog recombination in the ribosomal DNA, apparently by excluding the meiosis-specific Hop1 protein . Nucleolar localization of Pch2 depends on the silencing factor Sir2, and mutation of SIR2 also bypasses the zip1 pachytene arrest . Under certain circumstances, Sir3-dependent localization of Pch2 to telomeres also provides checkpoint function . These unexpected findings link the nucleolus, chromatin silencing, and the pachytene checkpoint. Cell, 1999 Apr 30, 97(3), 299 - 311 Ordered recruitment of transcription and chromatin remodeling factors to a cell cycle- and developmentally regulated promoter; Cosma MP et al.; Gene activation in eukaryotes requires chromatin remodeling complexes like Swi/Snf and histone acetylases like SAGA . How these factors are recruited to promoters is not yet understood . Using CHIP, we measured recruitment of Swi/Snf, SAGA, the repressor Ash1p, and transcription factors Swi5p and SBF to the HO endonuclease promoter as cells progress through the yeast cell cycle . Swi5p's entry into nuclei at the end of anaphase recruits Swi/Snf, which then recruits SAGA . These two factors then facilitate SBF's binding . Ash1p, which only accumulates in daughter cell nuclei, binds to HO soon after Swi5p and aborts recruitment of Swi/Snf, SAGA, and SBF . Swi5p remains at HO for only 5 min . Swi/Snf's and SAGA's subsequent persistence at HO is self sustaining and constitutes an "epigenetic memory" of HO's transient interaction with Swi5p. Radiat Res, 1999 May, 151(5), 540 - 9 Induction of DNA double-strand breaks by 1H and 4He lons in primary human skin fibroblasts in the LET range of 8 to 124 keV/microm; Frankenberg D et al.; Yields of DNA double-strand breaks were determined in primary human skin fibroblasts exposed to 1H and 4He ions at various linear energy transfers (LETs) and to 15 MeV electrons as the reference radiation . The values obtained for the relative biological effectiveness (RBE) were 2.03, 1.45 and 1.36 for 1H ions at LETs of 35, 23 and 7.9 keV/microm, respectively, and 1.2, 1.18, 1.38 and 1.31 for 4He ions at LETs of 124, 76, 35 and 27 keV/microm, respectively . The data were obtained using pulsed-field gel electrophoresis of DNA released from cells using the chromosomes of the yeast Saccharomyces cerevisiae as length markers and fitting the experimental mass distributions of fragmented DNA to those obtained by computer simulation of the random breakage of human chromosomes . The RBE values for induction of DSBs in mammalian cells cannot be fitted to a common RBE-LET relationship for electrons and 1H, 4He and light ions . Comparison of the RBEs for mammalian cells with the corresponding RBEs obtained for yeast cells shows similar RBEs of electrons for yeast and mammalian cells; however, for 4He and light ions in the LET range of 100 to 1000 keV/microm, the RBEs for yeast are significantly higher compared with mammalian cells . These characteristics of the RBE-LET relationships for yeast and mammalian cells are attributed to the fraction of small DNA fragments induced by particles when traversing the higher-order chromatin structures which are different to some extent in these two cell types. Radiat Res, 1999 May, 151(5), 532 - 9 Overexpression of human Ku70/Ku80 in rat cells resulting in reduced DSB repair capacity with appropriate increase in cell radiosensitivity but with no effect on cell recovery; Kasten U et al.; The effect of an overexpression of human Ku70/80 was studied using cells of the rat cell lines Rat-1 and R7080, the latter being transfected with the human cDNAs for Ku70 and Ku80 . The overexpression was found to result in a 20% reduction of the DNA-PK activity . The kinetics of DSB repair, which was studied after exposure of the cells to 30 Gy of X rays, was biphasic and had identical half-times for Rat-1 and R7080 cells (tfast = 7 min and tslow = 135 min) . However, there was a significant difference between the cell lines in the fractions of DSBs repaired with slow and fast kinetics . In R7080 cells, about twice as many DSBs were repaired with slow kinetics compared to Rat-1 cells (34% compared to 16%) . A similar difference was found in the number of residual DSBs (3.6% compared to 2.0%) . R7080 cells also showed a reduced capacity to repair chromosome damage as detected by the PCC technique . Concerning cell killing, R7080 cells were clearly more radiosensitive than Rat-1 cells (D0.1 = 6.4 compared to 10.5 Gy), and this increase in sensitivity correlated well with the increase in residual DSBs . The two cell lines, however, did not vary in cell recovery . For sublethal as well as potentially lethal damage, Rat-1 and R7080 cells showed identical recovery ratios . These data demonstrate that the overexpression of human Ku70/Ku80 led to a reduced capacity for DSB repair with an associated increase in cell sensitivity but with no effect on cell recovery. Proc Natl Acad Sci U S A, 1999 May 11, 96(10), 5510 - 5 Conjugation of the ubiquitin-like protein NEDD8 to cullin-2 is linked to von Hippel-Lindau tumor suppressor function; Liakopoulos D et al.; The von Hippel-Lindau tumor suppressor protein pVHL assembles with cullin-2 (hCUL-2) and elongin B/C forming a protein complex, CBCVHL, that resembles SKP1-CDC53-F-box protein ubiquitin ligases . Here, we show that hCUL-2 is modified by the conserved ubiquitin-like protein NEDD8 and that NEDD8-hCUL-2 conjugates are part of CBCVHL complexes in vivo . Remarkably, the formation of these conjugates is stimulated by the pVHL tumor suppressor . A tumorigenic pVHL variant, however, is essentially deficient in this activity . Thus, ligation of NEDD8 to hCUL-2 is linked to pVHL activity and may be important for pVHL tumor suppressor function. Proc Natl Acad Sci U S A, 1999 May 11, 96(10), 5418 - 22 Bivalency as a principle for proteasome inhibition; Loidl G et al.; The proteasome, a multicatalytic protease, is known to degrade unfolded polypeptides with low specificity in substrate selection and cleavage pattern . This lack of well-defined substrate specificities makes the design of peptide-based highly selective inhibitors extremely difficult . However, the x-ray structure of the proteasome from Saccharomyces cerevisiae reveals a unique topography of the six active sites in the inner chamber of the protease, which lends itself to strategies of specific multivalent inhibition . Structure-derived active site separation distances were exploited for the design of homo- and heterobivalent inhibitors based on peptide aldehyde head groups and polyoxyethylene as spacer element . Polyoxyethylene was chosen as a flexible, linear, and proteasome-resistant polymer to mimic unfolded polypeptide chains and thus to allow access to the proteolytic chamber . Spacer lengths were selected that satisfy the inter- and intra-ring distances for occupation of the active sites from the S subsites . X-ray analysis of