S. cerevisiae

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 the proteasome/bivalent inhibitor complexes confirmed independent recognition and binding of the inhibitory head groups . Their inhibitory potencies, which are by 2 orders of magnitude enhanced, compared with pegylated monovalent inhibitors, result from the bivalent binding . The principle of multivalency, ubiquitous in nature, has been successfully applied in the past to enhance affinity and avidity of ligands in molecular recognition processes . The present study confirms its utility also for inhibition of multicatalytic protease complexes.

Proc Natl Acad Sci U S A, 1999 May 11, 96(10), 5406 - 11
Snt309p modulates interactions of Prp19p with its associated components to stabilize the Prp19p-associated complex essential for pre-mRNA splicing; Chen HR et al.; The SNT309 gene was identified via a mutation that causes lethality of cells in combination with a prp19 mutation . We showed previously that Snt309p is a component of the Prp19p-associated complex and that Snt309p, like Prp19p, is associated with the spliceosome immediately after or concomitantly with dissociation of U4 from the spliceosome . We show here that extracts prepared from the SNT309-deleted strain (DeltaSNT309) were defective in splicing but could be complemented by addition of the purified Prp19p-associated complex . Isolation of the Prp19p-associated complex from DeltaSNT309 extracts indicated that the complex was destabilized in the absence of Snt309p and dissociated on affinity chromatography, suggesting a role of Snt309p in stabilization of the Prp19p-associated complex . Addition of the affinity-purified Prp19p-Snt309p binary complex to DeltaSNT309 extracts could reconstitute the Prp19p-associated complex . Genetic analysis further suggests that Snt309p plays a role in modulating interactions of Prp19p with other associated components to facilitate formation of the Prp19p-associated complex . A model for how Snt309p modulates such interactions is proposed.

Proc Natl Acad Sci U S A, 1999 May 11, 96(10), 5382 - 7
P/CAF associates with cyclin D1 and potentiates its activation of the estrogen receptor; McMahon C et al.; Cyclin D1 is overexpressed in a significant percentage of human breast cancers, particularly in those that also express the estrogen receptor (ER) . We and others have demonstrated previously that experimentally overexpressed cyclin D1 can associate with the ER and stimulate its transcriptional functions in the absence of estrogen . This effect is separable from the established function of cyclin D1 as a regulator of cyclin-dependent kinases . Here, we demonstrate that cyclin D1 can also interact with the histone acetyltransferase, p300/CREB-binding protein-associated protein (P/CAF), thereby facilitating an association between P/CAF and the ER . Ectopic expression of P/CAF potentiates cyclin D1-stimulated ER activity in a dose-dependent manner . This effect is largely dependent on the acetyltransferase activity of P/CAF . These results suggest that cyclin D1 may trigger the activation of the ER through the recruitment of P/CAF, by providing histone acetyltransferase activity and, potentially, links to additional P/CAF-associated transcriptional coactivators.

Nature, 1999 Apr 29, 398(6730), 818 - 23
Cfi1 prevents premature exit from mitosis by anchoring Cdc14 phosphatase in the nucleolus; Visintin R et al.; In eukaryotes, the activation of mitotic cyclin-dependent kinases (CDKs) induces mitosis, and their inactivation causes cells to leave mitosis . In budding yeast, two redundant mechanisms induce the inactivation of mitotic CDKs . In one mechanism, a specialized ubiquitin-dependent proteolytic system (called the APC-dependent proteolysis machinery) degrades the mitotic (Clb) cyclin subunit . In the other, the kinase-inhibitor Sic1 binds to mitotic CDKs and inhibits their kinase activity . The highly conserved protein phosphatase Cdc14 promotes both Clb degradation and Sic1 accumulation . Cdc14 promotes SIC1 transcription and the stabilization of Sic1 protein by dephosphorylating Sicl and its transcription factor Swi5 . Cdc14 activates the degradation of Clb cyclins by dephosphorylating the APC-specificity factor Cdh1 . So how is Cdc14 regulated? Here we show that Cdc14 is sequestered in the nucleolus for most of the cell cycle . During nuclear division, Cdc14 is released from the nucleolus, allowing it to reach its targets . A highly conserved signalling cascade, critical for the exit from mitosis, is required for this movement of Cdc14 during anaphase . Furthermore, we have identified a negative regulator of Cdc14, Cfi1, that anchors Cdc14 in the nucleolus.

Yeast, 1999 Apr, 15(6), 513 - 26
How to bring orphan genes into functional families; Bianchi MM et al.; In the framework of the B1 Consortium of the EUROFAN-1 project, we set up a series of simple phenotypic tests that can be performed on a large number of strains at a time . This methodological approach was intended to help assign functions of putative genes coding for unknown proteins to several specific aspects of cell biology . The tests were chosen to study phenotypes which should be affected by numerous genes . In this report, we examined the sensitivity/resistance or the adaptation of the cell to physical or chemical stresses (thermotolerance, osmotolerance and ethanol sensitivity), the effects of the alteration of the level of protein phosphorylation (sensitivity or resistance to compounds affecting the activity of protein kinases or phosphatases) and the effects of compounds interfering with synthesis of nucleic acids or proteins . Deletions in 66 genes of unknown function have been tested in 21 different conditions . In many deletant strains, phenotypes were observed and, for the most promising candidates, tetrad analysis was performed in order to verify co-segregation of the deletion marker with the phenotype.

J Virol, 1999 Jun, 73(6), 5231 - 9
An orphan G protein-coupled receptor, GPR1, acts as a coreceptor to allow replication of human immunodeficiency virus types 1 and 2 in brain-derived cells; Shimizu N et al.; Twelve G protein-coupled receptors, including chemokine receptors, act as coreceptors and determinants for the cell tropisms of human immunodeficiency virus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus (SIV) . We isolated HIV-1 variants from T-cell-line (T)- and macrophage (M)-tropic (i.e., dualtropic) (R5-R3-X4) HIV-1 strains and also produced six HIV-1 mutants carrying single-point amino acid substitutions at the tip of the V3 region of the Env protein of HIV-1 . These variants and three mutants infected brain-derived CD4-positive cells that are resistant to M-, T-, or dualtropic (R5, X4, or R5-X4) HIV-1 strains . However, a factor that determines this cell tropism has not been identified . This study shows that primary brain-derived fibroblast-like cell strains, BT-3 and BT-20/N, as well as a CD4-transduced glioma cell line, U87/CD4, which were susceptible to these HIV-1 variants and mutants and the HIV-2ROD strain, expressed mRNA of an orphan G protein-coupled receptor (GPCR), GPR1 . When a CD4-positive cell line which was strictly resistant to infection with diverse HIV-1 and HIV-2 strains was transduced with GPR1, the cell line became susceptible to these HIV-1 variants and mutants and to an HIV-2 strain but not to T- or dualtropic HIV-1 strains, and numerous syncytia formed after infection . These results indicate that GPR1 functions as a coreceptor for the HIV-1 variants and mutants and for the HIV-2ROD strain in vitro.

Mol Biol Cell, 1999 May, 10(5), 1553 - 68
Characterization of Fus3 localization: active Fus3 localizes in complexes of varying size and specific activity; Choi KY et al.; The MAP kinase Fus3 regulates many different signal transduction outputs that govern the ability of Saccharomyces cerevisiae haploid cells to mate . Here we characterize Fus3 localization and association with other proteins . By indirect immunofluorescence, Fus3 localizes in punctate spots throughout the cytoplasm and nucleus, with slightly enhanced nuclear localization after pheromone stimulation . This broad distribution is consistent with the critical role Fus3 plays in mating and contrasts that of Kss1, which concentrates in the nucleus and is not required for mating . The majority of Fus3 is soluble and not bound to any one protein; however, a fraction is stably bound to two proteins of approximately 60 and approximately 70 kDa . Based on fractionation and gradient density centrifugation properties, Fus3 exists in a number of complexes, with its activity critically dependent upon association with other proteins . In the presence of alpha factor, nearly all of the active Fus3 localizes in complexes of varying size and specific activity, whereas monomeric Fus3 has little activity . Fus3 has highest specific activity within a 350- to 500-kDa complex previously shown to contain Ste5, Ste11, and Ste7 . Ste5 is required for Fus3 to exist in this complex . Upon alpha factor withdrawal, a pool of Fus3 retains activity for more than one cell cycle . Collectively, these results support Ste5's role as a tether and suggest that association of Fus3 in complexes in the presence of pheromone may prevent inactivation in addition to enhancing activation.

Mol Biol Cell, 1999 May, 10(5), 1367 - 79
Apg7p/Cvt2p: A novel protein-activating enzyme essential for autophagy; Tanida I et al.; In the yeast Saccharomyces cerevisiae, the Apg12p-Apg5p conjugating system is essential for autophagy . Apg7p is required for the conjugation reaction, because Apg12p is unable to form a conjugate with Apg5p in the apg7/cvt2 mutant . Apg7p shows a significant similarity to a ubiquitin-activating enzyme, Uba1p . In this article, we investigated the function of Apg7p as an Apg12p-activating enzyme . Hemagglutinin-tagged Apg12p was coimmunoprecipitated with c-myc-tagged Apg7p . A two-hybrid experiment confirmed the interaction . The coimmunoprecipitation was sensitive to a thiol-reducing reagent . Furthermore, a thioester conjugate of Apg7p was detected in a lysate of cells overexpressing both Apg7p and Apg12p . These results indicated that Apg12p interacts with Apg7p via a thioester bond . Mutational analyses of Apg7p suggested that Cys507 of Apg7p is an active site cysteine and that both the ATP-binding domain and the cysteine residue are essential for the conjugation of Apg7p with Apg12p to form the Apg12p-Apg5p conjugate . Cells expressing mutant Apg7ps, Apg7pG333A, or Apg7pC507A showed defects in autophagy and cytoplasm-to-vacuole targeting of aminopeptidase I . These results indicated that Apg7p functions as a novel protein-activating enzyme necessary for Apg12p-Apg5p conjugation.

Biophys J, 1999 May, 76(5), 2752 - 9
Thermodynamics and kinetics of a folded-folded' transition at valine-9 of a GCN4-like leucine zipper; d'Avignon DA et al.; Spin inversion transfer (SIT) NMR experiments are reported probing the thermodynamics and kinetics of interconversion of two folded forms of a GCN4-like leucine zipper near room temperature . The peptide is 13Calpha-labeled at position V9(a) and results are compared with prior findings for position L13(e) . The SIT data are interpreted via a Bayesian analysis, yielding local values of T1a, T1b, kab, kba, and Keq as functions of temperature for the transition FaV9 right arrow over left arrow FbV9 between locally folded dimeric forms . Equilibrium constants, determined from relative spin counts at spin equilibrium, agree well with the ratios kab/kba from the dynamic SIT experiments . Thermodynamic and kinetic parameters are similar for V9(a) and L13(e), but not the same, confirming that the molecular conformational population is not two-state . The energetic parameters determined for both sites are examined, yielding conclusions that apply to both and are robust to uncertainties in the preexponential factor (kT/h) of the Eyring equation . These conclusions are 1) the activation free energy is substantial, requiring a sparsely populated transition state; 2) the transition state's enthalpy far exceeds that of either Fa or Fb; 3) the transition state's entropy far exceeds that of Fa, but is comparable to that of Fb; 4) "Arrhenius kinetics" characterize the temperature dependence of both kab and kba, indicating that the temperatures of slow interconversion are not below that of the glass transition . Any postulated free energy surface for these coiled coils must satisfy these constraints.

Mol Cell, 1999 Apr, 3(4), 535 - 41
ROC1, a homolog of APC11, represents a family of cullin partners with an associated ubiquitin ligase activity; Ohta T et al.; We have identified two highly conserved RING finger proteins, ROC1 and ROC2, that are homologous to APC11, a subunit of the anaphase-promoting complex . ROC1 and ROC2 commonly interact with all cullins while APC11 specifically interacts with APC2, a cullin-related APC subunit . YeastROC1 encodes an essential gene whose reduced expression resulted in multiple, elongated buds and accumulation of Sic1p and Cln2p . ROC1 and APC11 immunocomplexes can catalyze isopeptide ligations to form polyubiquitin chains in an E1- and E2-dependent manner . ROC1 mutations completely abolished their ligase activity without noticeable changes in associated proteins . Ubiquitination of phosphorylated I kappa B alpha can be catalyzed by the ROC1 immunocomplex in vitro . Hence, combinations of ROC/APC11 and cullin proteins proteins potentially constitute a wide variety of ubiquitin ligases.

Nutr Rev, 1999 Apr, 57(4), 114 - 23
Iron transport across biologic membranes; Andrews NC et al.; Iron is essential for life, but is toxic in excess . Nearly all organisms have therefore developed regulated mechanisms for efficient transport of iron into cells . This paper reviews the current understanding of iron transport, focusing on valuable lessons from studies of yeast iron transport and the discovery of the first mammalian transmembrane iron transporter.

EMBO J, 1999 May 4, 18(9), 2621 - 30
Molecular cloning of the maize gene crp1 reveals similarity between regulators of mitochondrial and chloroplast gene expression; Fisk DG et al.; The maize nuclear gene crp1 is required for the translation of the chloroplast petA and petD mRNAs and for the processing of the petD mRNA from a polycistronic precursor . In order to understand the biochemical role of the crp1 gene product and the interconnections between chloroplast translation and RNA metabolism, the crp1 gene and cDNA were cloned . The predicted crp1 gene product (CRP1) is related to nuclear genes in fungi that play an analogous role in mitochondrial gene expression, suggesting an underlying mechanistic similarity . Analysis of double mutants that lack both chloroplast ribosomes and crp1 function indicated that CRP1 activates a site-specific endoribonuclease independently of any role it plays in translation . Antibodies prepared to recombinant CRP1 were used to demonstrate that CRP1 is localized to the chloroplast stroma and that it is a component of a multisubunit complex . The CRP1 complex is not associated detectably with either chloroplast membranes or chloroplast ribosomes . Models for CRP1 function and its relationship to other activators of organellar translation are discussed.

EMBO J, 1999 May 4, 18(9), 2563 - 79
Solution structure of the HMG protein NHP6A and its interaction with DNA reveals the structural determinants for non-sequence-specific binding; Allain FH et al.; NHP6A is a chromatin-associated protein from Saccharomyces cerevisiae belonging to the HMG1/2 family of non-specific DNA binding proteins . NHP6A has only one HMG DNA binding domain and forms relatively stable complexes with DNA . We have determined the solution structure of NHP6A and constructed an NMR-based model structure of the DNA complex . The free NHP6A folds into an L-shaped three alpha-helix structure, and contains an unstructured 17 amino acid basic tail N-terminal to the HMG box . Intermolecular NOEs assigned between NHP6A and a 15 bp 13C,15N-labeled DNA duplex containing the SRY recognition sequence have positioned the NHP6A HMG domain onto the minor groove of the DNA at a site that is shifted by 1 bp and in reverse orientation from that found in the SRY-DNA complex . In the model structure of the NHP6A-DNA complex, the N-terminal basic tail is wrapped around the major groove in a manner mimicking the C-terminal tail of LEF1 . The DNA in the complex is severely distorted and contains two adjacent kinks where side chains of methionine and phenylalanine that are important for bending are inserted . The NHP6A-DNA model structure provides insight into how this class of architectural DNA binding proteins may select preferential binding sites.

EMBO J, 1999 May 4, 18(9), 2551 - 62
A WW domain-containing yes-associated protein (YAP) is a novel transcriptional co-activator; Yagi R et al.; A protein module called the WW domain recognizes and binds to a short oligopeptide called the PY motif, PPxY, to mediate protein-protein interactions . The PY motif is present in the transcription activation domains of a wide range of transcription factors including c-Jun, AP-2, NF-E2, C/EBPalpha and PEBP2/CBF, suggesting that it plays an important role in transcriptional activation . We show here that mutation of the PY motif in the subregion of the activation domain of the DNA-binding subunit of PEBP2, PEBP2alpha, abolishes its transactivation function . Using yeast two-hybrid screening, we demonstrate that Yes-associated protein (YAP) binds to the PY motif of PEBP2alpha through its WW domain . The C-terminal region of YAP fused to the DNA-binding domain of GAL4 showed transactivation as strong as that of GAL4-VP16 . Exogenously expressed YAP conferred transcription-stimulating activity on the PY motif fused to the GAL4 DNA-binding domain as well as to native PEBP2alpha . The osteocalcin promoter was stimulated by exogenous PEBP2alphaA and a dominant negative form of YAP strongly inhibited this activity, suggesting YAP involvement in this promoter activity in vivo . These results indicate that the PY motif is a novel transcription activation domain that functions by recruiting YAP as a strong transcription activator to target genes.

EMBO J, 1999 May 4, 18(9), 2435 - 48
E2F mediates developmental and cell cycle regulation of ORC1 in Drosophila; Asano M et al.; Throughout the cell cycle of Saccharomyces cerevisiae, the level of origin recognition complex (ORC) is constant and ORCs are bound constitutively to replication origins . Replication is regulated by the recruitment of additional factors such as CDC6 . ORC components are widely conserved, and it generally has been assumed that they are also stable factors bound to origins throughout the cell cycle . In this report, we show that the level of the ORC1 subunit changes dramatically throughout Drosophila development . The accumulation of ORC1 is regulated by E2F-dependent transcription . In embryos, ORC1 accumulates preferentially in proliferating cells . In the eye imaginal disc, ORC1 accumulation is cell cycle regulated, with high levels in late G1 and S phase . In the ovary, the sub-nuclear distribution of ORC1 shifts during a developmentally regulated switch from endoreplication of the entire genome to amplification of the chorion gene clusters . Furthermore, we find that overexpression of ORC1 alters the pattern of DNA synthesis in the eye disc and the ovary . Thus, replication origin activity appears to be governed in part by the level of ORC1 in Drosophila.

Nature, 1999 Apr 22, 398(6729), 728 - 31
Binding of double-strand breaks in DNA by human Rad52 protein; Van Dyck E et al.; Double-strand breaks (DSBs) in DNA are caused by ionizing radiation . These chromosomal breaks can kill the cell unless repaired efficiently, and inefficient or inappropriate repair can lead to mutation, gene translocation and cancer . Two proteins that participate in the repair of DSBs are Rad52 and Ku: in lower eukaryotes such as yeast, DSBs are repaired by Rad52-dependent homologous recombination, whereas vertebrates repair DSBs primarily by Ku-dependent non-homologous end-joining . The contribution of homologous recombination to vertebrate DSB repair, however, is important . Biochemical studies indicate that Ku binds to DNA ends and facilitates end-joining . Here we show that human Rad52, like Ku, binds directly to DSBs, protects them from exonuclease attack and facilitates end-to-end interactions . A model for repair is proposed in which either Ku or Rad52 binds the DSB . Ku directs DSBs into the non-homologous end-joining repair pathway, whereas Rad52 initiates repair by homologous recombination . Ku and Rad52, therefore, direct entry into alternative pathways for the repair of DNA breaks.

Neurochem Res, 1999 Apr, 24(4), 581 - 6
Rhizomelic chondrodysplasia punctata, a peroxisomal biogenesis disorder caused by defects in Pex7p, a peroxisomal protein import receptor: a minireview; Purdue PE et al.; Rhizomelic chondrodysplasia punctata (RCDP) is a lethal autosomal recessive disease corresponding to complementation group 11 (CG11), the second most common of the thirteen CGs of peroxisomal biogenesis disorders (PBDs) . RCDP is characterized by proximal limb shortening, severely disturbed endochondrial bone formation, and mental retardation, but there is an absence of the neuronal migration defect found in the other PBDs . Plasmalogen biosynthesis and phytanic acid oxidation are deficient, but very long chain fatty acid (VLCFA) oxidation is normal . At the cellular level, RCDP is unique in that the biogenesis of most peroxisomal proteins is normal, but a specific subset of at least four, and maybe more, peroxisomal matrix proteins fail to be imported from the cytosol . In this review, we discuss recent advances in understanding RCDP, most prominently the cloning of the affected gene, PEX7, and identification of PEX7 mutations in RCDP patients . Human PEX7 was identified by virtue of its sequence similarity to its Saccharomyces cerevisiae ortholog, which had previously been shown to encode Pex7p, an import receptor for type 2 peroxisomal targeting sequences (PTS2) . Normal human PEX7 expression rescues the cellular defects in cultured RCDP cells, and cDNA sequence analysis has identified a variety of PEX7 mutations in RCDP patients, including a deletion of 100 nucleotides, probably due to a splice site mutation, and a prevalent nonsense mutation which results in loss of the carboxyterminal 32 amino acids . Identification of RCDP as a PTS2 import disorder explains the observation that several, but not all, peroxisomal matrix proteins are mistargeted in this disease; three of the four proteins deficient in RCDP have now been shown to be PTS2-targeted.

Curr Biol, 1999 Apr 22, 9(8), 393 - 404
PDK1 acquires PDK2 activity in the presence of a synthetic peptide derived from the carboxyl terminus of PRK2; Balendran A et al.; BACKGROUND: Protein kinase B (PKB) is activated by phosphorylation of Thr308 and of Ser473 . Thr308 is phosphorylated by the 3-phosphoinositide-dependent protein kinase-1 (PDK1) but the identity of the kinase that phosphorylates Ser473 (provisionally termed PDK2) is unknown . RESULTS: The kinase domain of PDK1 interacts with a region of protein kinase C-related kinase-2 (PRK2), termed the PDK1-interacting fragment (PIF) . PIF is situated carboxy-terminal to the kinase domain of PRK2, and contains a consensus motif for phosphorylation by PDK2 similar to that found in PKBalpha, except that the residue equivalent to Ser473 is aspartic acid . Mutation of any of the conserved residues in the PDK2 motif of PIF prevented interaction of PIF with PDK1 . Remarkably, interaction of PDK1 with PIF, or with a synthetic peptide encompassing the PDK2 consensus sequence of PIF, converted PDK1 from an enzyme that could phosphorylate only Thr308 of PKBalpha to one that phosphorylates both Thr308 and Ser473 of PKBalpha in a manner dependent on phosphatidylinositol (3,4,5) trisphosphate (PtdIns(3,4,5)P3) . Furthermore, the interaction of PIF with PDK1 converted the PDK1 from a form that is not directly activated by PtdIns(3,4,5)P3 to a form that is activated threefold by PtdIns(3,4,5)P3 . We have partially purified a kinase from brain extract that phosphorylates Ser473 of PKBalpha in a PtdIns(3,4,5)P3-dependent manner and that is immunoprecipitated with PDK1 antibodies . CONCLUSIONS: PDK1 and PDK2 might be the same enzyme, the substrate specificity and activity of PDK1 being regulated through its interaction with another protein(s) . PRK2 is a probable substrate for PDK1.

J Cell Biol, 1999 May 3, 145(3), 447 - 55
Phosphorylation regulates in vivo interaction and molecular targeting of serine/arginine-rich pre-mRNA splicing factors; Yeakley JM et al.; The SR superfamily of splicing factors and regulators is characterized by arginine/serine (RS)-rich domains, which are extensively modified by phosphorylation in cells . In vitro binding studies revealed that RS domain-mediated protein interactions can be differentially affected by phosphorylation . Taking advantage of the single nonessential SR protein-specific kinase Sky1p in Saccharomyces cerevisiae, we investigated RS domain interactions in vivo using the two-hybrid assay . Strikingly, all RS domain-mediated interactions were abolished by SKY1 deletion and were rescuable by yeast or mammalian SR protein-specific kinases, indicating that phosphorylation has a far greater impact on RS domain interactions in vivo than in vitro . To understand this dramatic effect, we examined the localization of SR proteins and found that SC35 was shifted to the cytoplasm in sky1Delta yeast, although this phenomenon was not obvious with ASF/SF2, indicating that nuclear import of SR proteins may be differentially regulated by phosphorylation . Using a transcriptional repression assay, we further showed that most LexA-SR fusion proteins depend on Sky1p to efficiently recognize the LexA binding site in a reporter, suggesting that molecular targeting of RS domain-containing proteins within the nucleus was also affected . Together, these results reveal multiple phosphorylation-dependent steps for SR proteins to interact with one another efficiently and specifically, which may ultimately determine the splicing activity and specificity of these factors in mammalian cells.

Genetics, 1999 May, 152(1), 153 - 66
Genetic and biochemical interactions involving tricarboxylic acid cycle (TCA) function using a collection of mutants defective in all TCA cycle genes; Przybyla-Zawislak B et al.; The eight enzymes of the tricarboxylic acid (TCA) cycle are encoded by at least 15 different nuclear genes in Saccharomyces cerevisiae . We have constructed a set of yeast strains defective in these genes as part of a comprehensive analysis of the interactions among the TCA cycle proteins . The 15 major TCA cycle genes can be sorted into five phenotypic categories on the basis of their growth on nonfermentable carbon sources . We have previously reported a novel phenotype associated with mutants defective in the IDH2 gene encoding the Idh2p subunit of the NAD+-dependent isocitrate dehydrogenase (NAD-IDH) . Null and nonsense idh2 mutants grow poorly on glycerol, but growth can be enhanced by extragenic mutations, termed glycerol suppressors, in the CIT1 gene encoding the TCA cycle citrate synthase and in other genes of oxidative metabolism . The TCA cycle mutant collection was utilized to search for other genes that can suppress idh2 mutants and to identify TCA cycle genes that display a similar suppressible growth phenotype on glycerol . Mutations in 7 TCA cycle genes were capable of functioning as suppressors for growth of idh2 mutants on glycerol . The only other TCA cycle gene to display the glycerol-suppressor-accumulation phenotype was IDH1, which encodes the companion Idh1p subunit of NAD-IDH . These results provide genetic evidence that NAD-IDH plays a unique role in TCA cycle function.

Genetics, 1999 May, 152(1), 143 - 52
RAD50 and RAD51 define two pathways that collaborate to maintain telomeres in the absence of telomerase; Le S et al.; Telomere length is maintained by the de novo addition of telomere repeats by telomerase, yet recombination can elongate telomeres in the absence of telomerase . When the yeast telomerase RNA component, TLC1, is deleted, telomeres shorten and most cells die . However, gene conversion mediated by the RAD52 pathway allows telomere lengthening in rare survivor cells . To further investigate the role of recombination in telomere maintenance, we assayed telomere length and the ability to generate survivors in several isogenic DNA recombination mutants, including rad50, rad51, rad52, rad54, rad57, xrs2, and mre11 . The rad51, rad52, rad54, and rad57 mutations increased the rate of cell death in the absence of TLC1 . In contrast, although the rad50, xrs2, and mre11 strains initially had short telomeres, double mutants with tlc1 did not affect the rate of cell death, and survivors were generated at later times than tlc1 alone . While none of the double mutants of recombination genes and tlc1 (except rad52 tlc1) blocked the ability to generate survivors, a rad50 rad51 tlc1 triple mutant did not allow the generation of survivors . Thus RAD50 and RAD51 define two separate pathways that collaborate to allow cells to survive in the absence of telomerase.

Trends Cell Biol, 1999 Apr, 9(4), 150 - 3
Targeting vesicles to specific sites on the plasma membrane: the role of the sec6/8 complex; Hsu SC et al.; The delivery of secretory vesicles to appropriate docking and fusion sites on the plasma membrane is crucial for many cellular functions, including formation of synapses, exocytosis of neurotransmitter, establishment and maintenance of cell polarity, cell growth and plasma membrane wound healing . Cell-biological, genetic and biochemical approaches have identified crucial proteins and protein interactions important for vesicle docking and fusion . However, a description of the molecular mechanisms underlying vesicle targeting to specific membrane-fusion sites remains elusive . This review discusses a set of proteins that might direct vesicles to specific domains of the plasma membrane.

Trends Cell Biol, 1999 Apr, 9(4), 125 - 8
PTEN: a tumour suppressor that functions as a phospholipid phosphatase; Maehama T et al.; The tumour suppressor PTEN has been implicated in a large number of human tumours and is conserved from humans to worms . Characterization of PTEN protein showed that it is a phosphatase that acts on proteins and on 3-phosphorylated phosphoinositides, including phosphatidylinositol (3,4,5)-trisphosphate, and can therefore modulate signal-transduction pathways that involve lipid second messengers . Recent results indicate that at least part of its role is to regulate the activity of the serine/threonine kinase AKT/PKB, and thus influence cell survival signalling . This article discusses the function of PTEN and how this could be linked to its activity as a tumour suppressor.

Mol Phylogenet Evol, 1999 Jun, 12(1), 57 - 66
Molecular evolution of type 1 serine/threonine protein phosphatases; Lin Q et al.; Type 1 serine/threonine protein phosphatases (PP1s) play key roles in many cellular processes . To understand the evolutionary relationships among PP1s from various kingdoms and to provide a valid basis to evaluate the structure-function relationships of these phosphatases, 44 PP1 sequences were aligned, revealing a high sequence similarity among PP1 homologs . About one-third of the total amino acids are conserved in all the sequences studied . Most of these conserved amino acids are located within a 270-amino-acid core region . They include most sites critical to the activity and regulation of PP1s based on three-dimensional structural studies of mammalian PP1s . Positional variation analysis using a sliding window approach revealed two variable blocks in the 270-amino-acid core region . The major variable block corresponds to a subdomain composed of three alpha-helices (alphaG, alphaH, and alphaI) and three beta-sheets (beta7, beta8, and beta9) . Phylogenetic analyses suggested that plant and animal PP1s form distinct monophyletic groups . The plant PP1 family contains several subgroups that may be older than the monocot-dicot divergence . In the animal PP1 family, different vertebrate isoforms appear to form distinct subgroups . Relative substitution rate studies indicated that plant PP1s are more diverse than animal PP1s, with an average substitution rate 1.5 times as large as that of animal PP1s . The possible involvement of PP1s in the establishment of multicellularity is discussed .

J Biol Chem, 1999 May 7, 274(19), 13619 - 28
Cloning and characterization of a mammalian lithium-sensitive bisphosphate 3'-nucleotidase inhibited by inositol 1,4-bisphosphate; Spiegelberg BD et al.; Discovery of a structurally conserved metal-dependent lithium-inhibited phosphomonoesterase protein family has identified several potential cellular targets of lithium as used to treat manic depression . Here we describe identification of a novel family member using a "computer cloning" strategy . Human and murine cDNA clones encoded proteins sharing 92% identity and were highly expressed in kidney . Native and recombinant protein harbored intrinsic magnesium-dependent bisphosphate nucleotidase activity (BPntase), which removed the 3'-phosphate from 3'-5' bisphosphate nucleosides and 3'-phosphoadenosine 5'-phosphosulfate with Km and Vmax values of 0.5 microM and 40 micromol/min/mg . Lithium uncompetitively inhibited activity with a Ki of 157 microM . Interestingly, BPntase was competitively inhibited by inositol 1,4-bisphosphate with a Ki of 15 microM . Expression of mammalian BPntase complemented defects in hal2/met22 mutant yeast . These data suggest that BPntase's physiologic role in nucleotide metabolism may be regulated by inositol signaling pathways . The presence of high levels of BPntase in the kidney are provocative in light of the roles of bisphosphorylated nucleotides in regulating salt tolerance, sulfur assimilation, detoxification, and lithium toxicity . We propose that inhibition of human BPntase may account for lithium-induced nephrotoxicity, which may be overcome by supplementation of current therapeutic regimes with inhibitors of nucleotide biosynthesis, such as methionine.

Science, 1999 Apr 30, 284(5415), 805 - 8
Undetectable intracellular free copper: the requirement of a copper chaperone for superoxide dismutase; Rae TD et al.; The copper chaperone for the superoxide dismutase (CCS) gene is necessary for expression of an active, copper-bound form of superoxide dismutase (SOD1) in vivo in spite of the high affinity of SOD1 for copper (dissociation constant = 6 fM) and the high intracellular concentrations of both SOD1 (10 microM in yeast) and copper (70 microM in yeast) . In vitro studies demonstrated that purified Cu(I)-yCCS protein is sufficient for direct copper activation of apo-ySOD1 but is necessary only when the concentration of free copper ions ({Cu}free) is strictly limited . Moreover, the physiological requirement for yCCS in vivo was readily bypassed by elevated copper concentrations and abrogation of intracellular copper-scavenging systems such as the metallothioneins . This metallochaperone protein activates the target enzyme through direct insertion of the copper cofactor and apparently functions to protect the metal ion from binding to intracellular copper scavengers . These results indicate that intracellular {Cu}free is limited to less than one free copper ion per cell and suggest that a pool of free copper ions is not used in physiological activation of metalloenzymes.

Biochemistry, 1999 Apr 27, 38(17), 5430 - 7
A potent oligosaccharyl transferase inhibitor that crosses the intracellular endoplasmic reticulum membrane; Eason PD et al.; Recent work has resulted in the development of potent inhibitors of oligosaccharyl transferase (OT), the enzyme that catalyzes the cotranslational glycosylation of asparagine {Hendrickson, T . L., Spencer, J . R., Kato, M., and Imperiali, B . (1996) J . Am . Chem . Soc . 118, 7636-7637; Kellenberger, C., Hendrickson, T . L., and Imperiali, B . (1997) Biochemistry 36, 12554-12559} . However, no specific OT inhibitors that function in the cellular environment have yet been reported . The peptide cyclo(hex-Amb-Cys)-Thr-Val-Thr-Nph-NH2 was previously shown to exhibit nanomolar inhibition (Ki = 37 nM) through slow tight binding kinetics {Hendrickson, T . L., Spencer, J . R., Kato, M., and Imperiali, B . (1996) J . Am . Chem . Soc . 118, 7636-7637} . Included herein is the redesign of this prototype inhibitor for achieving both passive and active translocation into model membrane systems representing the endoplasmic reticulum (ER) . The strategy for passive transport involved the incorporation of a membrane permeable import function previously shown to carry various peptides across the outer as well as the interior cellular membranes {Rojas, M., Donahue, J . P., Tan, Z., and Lin, Y.-Z . (1998) Nat . Biotechnol . 16, 370-375} . Assessment of function in intact ER membranes revealed that the inhibitor targeted toward passive diffusion demonstrated concentration-dependent inhibition of two different glycosylation substrates . Thus, this modified inhibitor achieved potent inhibition of glycosylation after being successfully transported through the ER membrane . In the active translocation approach, the lead OT inhibitor and a corresponding substrate were redesigned to include features recognized by the transporter associated with antigen processing (TAP) . This protein translocates peptides into the lumen of the ER {Heemels, M.-T., Schumacher, T . N . M., Wonigeit, K., and Ploegh, H . L . (1993) Science 262, 2059-2063} . However, although acceptance of the cyclized substrate by the TAP receptor was demonstrated via efficient transport and glycosylation, the modified inhibitor was not translocated by TAP machinery, and therefore, active translocation was achieved for the modified substrate only . Both of these ER transport methods afforded redesigned OT inhibitors that retained their inhibitor properties in vitro, regardless of the extensions to the carboxy-terminus of the root inhibitor . The above family of redesigned inhibitors provides a template for generating a transcellular pathway and represents the first step toward OT inhibition in intact cells.

Biochemistry, 1999 Apr 27, 38(17), 5378 - 85
Trimerization specificity in HIV-1 gp41: analysis with a GCN4 leucine zipper model; Shu W et al.; The envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1) consists of a complex of two noncovalently associated subunits, gp120 and gp41 . Formation of gp120/gp41 oligomers is thought to be dependent on a 4-3 hydrophobic (heptad) repeat located in the amino-terminal region of the gp41 molecule . We have investigated the role of this heptad repeat in determining the oligomeric structure of gp41 by introducing its buried core residues into the first (a) and fourth (d) positions of the GCN4 leucine-zipper dimerization domain . The mutant peptides fold into trimeric, helical structures, as shown by circular dichroism and equilibrium sedimentation centrifugation . The 2.4 A resolution crystal structure of one such trimer reveals a parallel three-stranded, alpha-helical coiled coil . Thus, the buried core residues from the gp41 heptad repeat direct trimer formation . We suggest that the conserved amino-terminal heptad repeat within the gp41 ectodomain possesses trimerization specificity.

Cell, 1999 Apr 16, 97(2), 245 - 56
Net1, a Sir2-associated nucleolar protein required for rDNA silencing and nucleolar integrity; Straight AF et al.; The Sir2 protein mediates gene silencing and repression of recombination at the rDNA repeats in budding yeast . Here we show that Sir2 executes these functions as a component of a nucleolar complex designated RENT (regulator of nucleolar silencing and telophase exit) . Net1, a core subunit of this complex, preferentially cross-links to the rDNA repeats, but not to silent DNA regions near telomeres or to active genes, and tethers the RENT complex to rDNA . Net1 is furthermore required for rDNA silencing and nucleolar integrity . During interphase, Net1 and Sir2 colocalize to a subdomain within the nucleous, but at the end of mitosis a fraction of Sir2 leaves the nucleolus and disperses as foci throughout the nucleus, suggesting that the structure of rDNA silent chromatin changes during the cell cycle . Our findings suggest that a protein complex shown to regulate exit from mitosis is also involved in gene silencing.

Cell, 1999 Apr 16, 97(2), 233 - 44
Exit from mitosis is triggered by Tem1-dependent release of the protein phosphatase Cdc14 from nucleolar RENT complex; Shou W et al.; Exit from mitosis in budding yeast requires a group of essential proteins--including the GTPase Tem1 and the protein phosphatase Cdc14--that downregulate cyclin-dependent kinase activity . We identified a mutation, net1-1, that bypasses the lethality of tem1 delta . NET1 encodes a novel protein, and mass spectrometric analysis reveals that it is a key component of a multifunctional complex, denoted RENT (for regulator of nucleolar silencing and telophase), that also contains Cdc14 and the silencing regulator Sir2 . From G1 through anaphase, RENT localizes to the nucleolus, and Cdc14 activity is inhibited by Net1 . In late anaphase, Cdc14 dissociates from RENT, disperses throughout the cell in a Tem1-dependent manner, and ultimately triggers mitotic exit . Nucleolar sequestration may be a general mechanism for the regulation of diverse biological processes.

Nucleic Acids Res, 1999 May 15, 27(10), 2165 - 74
Isolation of Ku70-binding proteins (KUBs); Yang CR et al.; DNA-dependent protein kinase (DNA-PK) plays a critical role in resealing DNA double-stand breaks by non-homologous end joining . Aside from DNA-PK, XRCC4 and DNA ligase IV, other proteins which play a role(s) in this repair pathway remain unknown; DNA-PK contains a catalytic subunit (DNA-PKcs) and a DNA binding subunit (Ku70 and Ku80) . We isolated Ku70-binding proteins (KUB1-KUB4) using yeast two-hybrid analyses . Sequence analyses revealed KUB1 to be apolipoprotein J (apoJ), also known as X-ray-inducible transcript 8 (XIP8), testosterone-repressed prostate message-2 (TRPM-2) and clusterin . KUB2 is Ku80 . KUB3 and KUB4 are unknown, >10 kb trans-cripts . Interactions of apoJ/XIP8 or KUB3 with Ku70 were confirmed by co-immunoprecipitation analyses in MCF-7:WS8 breast cancer or IMR-90 normal lung fibroblast cells, respectively . The interaction of apoJ/XIP8 with Ku70 was confirmed by far-western analyses . Stable over-expression of full-length apoJ/XIP8 in MCF-7:WS8 caused decreased Ku70/Ku80 DNA end binding that was restored by apoJ/XIP8 monoclonal antibodies . The role of apoJ/XIP8 in ionizing radiation resistance/sensitivity is under investigation.

Nucleic Acids Res, 1999 May 15, 27(10), 2091 - 8
Early activated replication origins within the cell cycle-regulated histone H4 genes in Physarum; Benard M et al.; It was previously shown that the two members of the cell cycle-regulated histone H4 gene family, H4-1 and H4-2, are replicated at the onset of S phase in the naturally synchronous plasmodium of Physarum polycephalum, suggesting that they are flanked by replication origins . It was further shown that a DNA fragment upstream of the H4-1 gene is able to confer autonomous replication of a plasmid in the budding yeast . In this paper, we re-investigated replication of the unlinked Physarum histone H4 genes by mapping the replication origin of these two loci using alkaline agarose gel and neutral/neutral 2-dimensional agarose gel electrophoreses . We showed that the two replicons containing the H4 genes are simultaneously activated at the onset of S phase and we mapped an efficient, bidirectional replication origin in the vicinity of each gene . Our data demonstrated that the Physarum sequence that functions as an ARS in yeast is not the site of replication initiation at the H4-1 locus . We also observed a stalling of the rightward moving replication fork downstream of the H4-1 gene, in a region where transient topoisomerase II sites were previously mapped . Our results further extend the concept of replication/transcription coupling in Physarum to cell cycle-regulated genes.

FEBS Lett, 1999 Mar 19, 447(1), 5 - 9
MDM2 interacts with MDMX through their RING finger domains; Tanimura S et al.; The N-terminus of MDM2 proto-oncoprotein interacts with p53 and down modulates p53 activity by inhibiting transcriptional activity and promoting p53 degradation . MDMX is structurally related to MDM2 and also binds to p53 . However, the function of MDMX has not been clarified yet . We found that MDM2 hetero-oligomerized with MDMX through their C-terminal RING finger domains . Yeast two-hybrid analysis revealed that the hetero-oligomerization between MDMX and MDM2 was more stable than the homo-oligomerization of each protein . MDM2 has been shown to be degraded by the ubiquitin-proteasome pathway, while MDMX was a stable protein . Interaction of MDMX with MDM2 through the C-terminal RING finger domains resulted in inhibiting degradation of MDM2 . These data indicate that MDMX functions as a regulator of MDM2.

FEBS Lett, 1999 Apr 9, 448(2-3), 297 - 300
Clusterin (SGP-2) gene expression is cell cycle dependent in normal human dermal fibroblasts; Bettuzzi S et al.; In confluent human dermal fibroblasts brought to quiescence (G0) by serum starvation, the S phase peaked at 24 h after serum re-addiction and G2/M phase peaked at 36 h . This was confirmed by titration of h-gas1 mRNA (a marker of G0 phase) and histone H3 (a marker of S phase) . Clusterin mRNA accumulation progressively increased in cells proceeding to confluence after seeding and to quiescence upon serum starvation, and peaked at around G0, in parallel with h-gas1 mRNA . At 6 h (roughly G1 phase) clusterin transcript formed a second peak, followed by a gradual decrease until 36 h . Correspondence of clusterin protein accumulation to its mRNA occurred solely with regard to the G0 peak but not to the second one . The possible meaning of the cell cycle related clusterin gene expression is discussed.

Adv Enzymol Relat Areas Mol Biol, 1999, 73, 209 - 67, xii
The enzymes of glutathione synthesis: gamma-glutamylcysteine synthetase; Griffith OW et al.; The metabolite glutathione fulfills many important and chemically complex roles in protecting cellular components from the deleterious effects of toxic species . GSH combines with hydroxyl radical, peroxynitrite, and hydroperoxides, as well as reactive electrophiles, including activated phosphoramide mustard . This thiol-containing reductant also maintains so-called thiol-enzymes in their catalytically active form, and maintains vitamins C and E in their biologically active forms . The key step in glutathione synthesis, namely the ATP-dependent synthesis of gamma-glutamylcysteine, is the topic of this review . Details are presented on (a) the enzyme's purification and protein chemistry, (b) the successful cDNA cloning, and characterization of the genes responsible for the biosynthesis of this enzyme . After considering aspects of the role of overexpression of this synthetase in terms of cancer chemotherapy, attention is focused on post-translational regulation . The remainder of the review deals with the catalytic mechanism (including substrate specificity, reactions catalyzed, steady-state kinetics, and chemical mechanism) as well as the inhibition of the enzyme (via feedback inhibition, reaction with S-alkyl homocysteine sulfoximine inhibitors, the clinical use of buthionine sulfoximine with cancer patients, and inactivation by cystamine, chloroketones, and various nitric oxide donors).

J Biomol Struct Dyn, 1999 Feb, 16(4), 757 - 74
The effect of queuosine on tRNA structure and function; Morris RC et al.; Computational modeling was performed to determine the potential function of the queuosine modification of tRNA found in wobble position 34 of tRNAasp, tRNAasn, tRNAhis, and tRNAtyr . Using the crystal structure of tRNAasp and a tRNA-tRNA-mRNA complex model, we show that the queuosine modification serves as a structurally restrictive base for tRNA anticodon loop flexibility . An extended intraresidue and intramolecular hydrogen bonding network is established by queuosine . The quaternary amine of the 7-aminomethyl side chain hydrogen bonds with the base's carbonyl oxygen . This positions the dihydroxycyclopentenediol ring of queuosine in proper orientation for hydrogen bonding with the backbone of the neighboring uridine 33 residue . The interresidue association stabilizes the formation of a cross-loop hydrogen bond between the uridine 33 base and the phosphoribosyl backbone of the cytosine at position 36 . Additional interactions between RNAs in the translation complex were studied with regard to potential codon context and codon bias effects . Neither steric nor electrostatic interaction occurs between aminoacyl- and peptidyl-site tRNA anticodon loops that are modified with queuosine . However, there is a difference in the strength of anticodon/codon associations (codon bias) based on the presence or lack of queuosine in the wobble position of the tRNA . Unmodified (guanosine-containing) tRNAasp forms a very stable association with cytosine (GAC), but is much less stable in complex with a uridine-containing codon (GAU) . Queuosine-modified tRNAasp exhibits no bias for either of cognate codons GAC or GAU and demonstrates a lower binding energy similar to the wobble pairing of guanosine-containing tRNA with a GAU codon . This is proposed to be due to the inflexibility of the queuosine-modified anticodon loop to accommodate proper positioning for optimal Watson-Crick type associations . A preliminary survey of codon usage patterns in oncodevelopmental versus housekeeping gene transcripts suggests a significant difference in bias for the queuosine-associated codons . Therefore, the queuosine modification may have the potential to influence cellular growth and differentiation by codon bias-based regulation of protein synthesis for discrete mRNA transcripts.

Science, 1999 Apr 23, 284(5414), 657 - 61
Rbx1, a component of the VHL tumor suppressor complex and SCF ubiquitin ligase; Kamura T et al.; The von Hippel-Lindau (VHL) tumor suppressor gene is mutated in most human kidney cancers . The VHL protein is part of a complex that includes Elongin B, Elongin C, and Cullin-2, proteins associated with transcriptional elongation and ubiquitination . Here it is shown that the endogenous VHL complex in rat liver also includes Rbx1, an evolutionarily conserved protein that contains a RING-H2 fingerlike motif and that interacts with Cullins . The yeast homolog of Rbx1 is a subunit and potent activator of the Cdc53-containing SCFCdc4 ubiquitin ligase required for ubiquitination of the cyclin-dependent kinase inhibitor Sic1 and for the G1 to S cell cycle transition . These findings provide a further link between VHL and the cellular ubiquitination machinery.

Eur J Biochem, 1999 Apr, 261(2), 517 - 23
Nonselective coupling of the human mu-opioid receptor to multiple inhibitory G-protein isoforms; Gaibelet G et al.; The human mu-opioid receptor was expressed in Saccharomyces cerevisiae . Binding of {3H}diprenorphine to yeast spheroplasts was specific and saturable (Kd = 1 nm, Bmax = 0.2-1 pmol x mg-1 of membrane proteins) . Inhibition of {3H}diprenorphine binding by antagonists and agonists with varying opioid selectivities (mu, delta and kappa) occurred with the same order of potency as in mammalian tissues . Affinities of antagonists were the same with yeast spheroplasts as in reference tissues whereas those of agonists, except etorphine and buprenorphine, were 10-fold to 100-fold lower . Addition of heterotrimeric Gi,o-proteins purified from bovine brain shifted the mu-opioid receptor into a high-affinity state for agonists . Using individually purified Galpha-subunits re-associated with betagamma-dimers, we showed that alphao1, alphao2, alphai1, alphai2 and alphai3 reconstituted high-affinity agonist binding with equal efficiency . This suggests that the structural determinants of the mu-opioid receptor responsible for G-protein coupling are not able to confer a high degree of specificity towards any member of the Gi,o family . The selective effects of opioid observed in specialized tissues upon opioid stimulation may be a result of regulation of G-protein activity by cell-specific factors which should conveniently be analysed using the reconstitution assay described here.

FEBS Lett, 1999 Mar 26, 447(2-3), 325 - 8
The NH2-terminal region of the active domain of sonic hedgehog is necessary for its signal transduction; Katsuura M et al.; The NH2-terminal domain of sonic hedgehog (residue 25-198) was expressed in both yeast and animal cells . The yeast-derived NH2-terminal domain of sonic hedgehog was less active by far than the animal cell-derived counterpart . The yeast-derived NH2-terminal domain of sonic hedgehog lacked 10 amino acids from the NH2-terminus . This cleavage of the yeast-derived NH2-terminal domain of sonic hedgehog might due to Kex 2 . In contrast, a mutant yeast-derived NH2-terminal domain of sonic hedgehog (Lys-33 to Thr) retained its NH2-terminus and its activity was comparable to that of the animal cell-derived NH2-terminal domain of sonic hedgehog . The NH2-terminal deleted NH2-terminal domain of sonic hedgehog completely lost its activity, nevertheless it inhibited the alkaline phosphatase activity induced by the animal cell-derived NH2-terminal domain of sonic hedgehog in a dose-dependent manner . These data suggest that the NH2-terminal deleted NH2-terminal domain of sonic hedgehog retains a receptor-binding ability and that the NH2-terminal peptide of the NH2-terminal domain of sonic hedgehog is necessary for its signal transduction.

FEBS Lett, 1999 Mar 26, 447(2-3), 311 - 4
A potential NES of the Epstein-Barr virus nuclear antigen 1 (EBNA1) does not confer shuttling; Fischer N et al.; The Epstein-Barr virus nuclear antigen 1 (EBNA1) is a multifunctional protein involved in the replication and maintenance of the viral episome . We identified a potential Rev-like nuclear export signal (NES) which, however, does not confer the export of EBNA1 . In the yeast two-hybrid system EBNA1 does not bind to the nuclear exporter Crm1p . In spite of the RNA-binding ability of EBNA1 and its structural homologies to RNA binding proteins like hnRNP U and/or A1, EBNA1 does not shuttle to the cytoplasm in heterokaryon analysis . We propose the function of the RNA binding of EBNA1 in retaining RNAs to the nucleus.

FEBS Lett, 1999 Mar 26, 447(2-3), 292 - 6
Identification of the sequence responsible for the nuclear localization of human Cdc6; Takei Y et al.; The Cdc6 is the essential protein for the initiation of DNA replication . Cdc6 is localized in the G1 nucleus, and abnormal nuclear localization of this protein induces irregular initiation of DNA replication . We identified here that amino acids K57 and R58 in the human Cdc6 protein play an important role in the nuclear localization of the protein . The fundamental features of the mechanism regulating the localization of Cdc6 seem to be maintained in yeast, Xenopus, and human, since the amino acid sequence surrounding K57 and R58, (S/T)PXKR(L/I), is conserved in these species . Substitution of amino acid residue S54 with E and not Q blocked partially the nuclear localization of the protein, implying that the phosphorylation at S54 is involved in the regulating mechanism of the cell cycle-dependent localization of Cdc6.

FEBS Lett, 1999 Mar 26, 447(2-3), 213 - 6
Vacuolar processing enzyme is self-catalytically activated by sequential removal of the C-terminal and N-terminal propeptides; Hiraiwa N et al.; A vacuolar processing enzyme (VPE) responsible for maturation of various vacuolar proteins is synthesized as an inactive precursor . To clarify how to convert the VPE precursor into the active enzyme, we expressed point mutated VPE precursors of castor bean in the pep4 strain of Saccharomyces cerevisiae . A VPE with a substitution of the active site Cys with Gly showed no ability to convert itself into the mature form, although a wild VPE had the ability . The mutated VPE was converted by the action of the VPE that had been purified from castor bean . Substitution of the conserved Asp-Asp at the putative cleavage site of the C-terminal propeptide with Gly-Gly abolished both the conversion into the mature form and the activation of the mutated VPE . In vitro assay with synthetic peptides demonstrated that a VPE exhibited activity towards Asp residues and that a VPE cleaved an Asp-Gln bond to remove the N-terminal propeptide . Taken together, the results indicate that the VPE is self-catalytically maturated to be converted into the active enzyme by removal of the C-terminal propeptide and subsequent removal of the N-terminal one.

Biochimie, 1999 Jan-Feb, 81(1-2), 161 - 71
Radiation inducible DNA repair processes in eukaryotes; Eckardt-Schupp F et al.; Eukaryotic cells respond to radiation-induced damage in DNA and other cellular components by turning on cascades of regulatory events which constitute a complex network of pathways of cell cycle checkpoints, DNA repair and damage tolerance mechanisms, recombination and delayed cell death (apoptosis) . By virtue of the high homology in structure and function of yeast and mammalian proteins several DNA repair pathways that may be upregulated in response to radiation, and some of their regulatory factors involved in sensing of damage, signal transduction by protein kinase cascades and transcription have been identified . In yeast, genes for DNA synthesis and replicative damage bypass, for base and nucleotide excision repair, in particular global genome repair, and for crucial steps in DNA double strand break repair by homologous recombination show enhanced expression in response to radiation . In mammalian cells, the identification of homologous genes and upregulated homologous DNA repair pathways makes fast progress . It is, however, evident that the regulatory network is considerably more complex than in yeast . The improved understanding on the molecular level of the radiation-inducible cellular responses to radiation is of high public interest . Especially, the response to very low doses may have relevance for the risk estimation for ionising radiation and, possibly as well, ultraviolet light (UV-B), and for the design of suitable dose fractionation schemes for radiotherapy.

Biochimie, 1999 Jan-Feb, 81(1-2), 117 - 25
Regulation of the DNA-dependent protein kinase (DNA-PK) activity in eukaryotic cells; Muller C et al.; The DNA-dependent protein kinase (DNA-PK) is a trimeric nuclear serine/threonine protein kinase consisting of a large catalytic sub-unit and the Ku heterodimer that regulates kinase activity by its association with DNA . DNA-PK is a major component of the DNA double strand break repair apparatus, and cells deficient in one of its component are hypersensitive to ionizing radiation . DNA-PK is also required to lymphoid V(D)J recombination and its absence confers in mice a severe combined immunodeficiency phenotype . The purpose of this review is to summarize the current knowledge on the mechanisms that contribute to regulate DNA-PK activity in vivo or in vitro and relates them to the role of DNA-PK in cellular functions . Finally, the studies devoted to drug-inhibition of DNA-PK in order to enhance cancer therapy by DNA-damaging agents are presented.

Biochimie, 1999 Jan-Feb, 81(1-2), 107 - 16
Mammalian X-ray-sensitive mutants which are defective in non-homologous (illegitimate) DNA double-strand break repair; Zdzienicka MZ; In all organisms multiple pathways to repair DNA double-strand breaks (DSB) have been identified . In mammalian cells DSB are repaired by two distinct pathways, homologous and non-homologous (illegitimate) recombination . X-ray-sensitive mutants have provided a tool for the identification and understanding of the illegitimate recombination pathway in mammalian cells . Two (sub-)pathways can be distinguished, the first mediated by DNA-PK-dependent protein kinase (DNA-PK), and the second directed by the hMre11/hRad50 complex . A variety of mutants impaired in DSB repair by illegitimate recombination, with mutations in Ku, DNA-PKcs, XRCC4 or nibrin, have been described . Herein, the characterization of these mutants with respect to the impaired cellular function and the molecular defect is provided . Further studies on these mutants, as well as on new mutants impaired in as-of-yet unidentified pathways, should be helpful to a better understanding of DSB repair and of the processes leading to genome instability and cancer.

Biochimie, 1999 Jan-Feb, 81(1-2), 87 - 105
The contribution of homologous recombination in preserving genome integrity in mammalian cells; Thompson LH et al.; Although it is clear that mammalian somatic cells possess the enzymatic machinery to perform homologous recombination of DNA molecules, the importance of this process in mitigating DNA damage has been uncertain . An initial genetic framework for studying homologous recombinational repair (HRR) has come from identifying relevant genes by homology or by their ability to correct mutants whose phenotypes are suggestive of recombinational defects . While yeast has been an invaluable guide, higher eukaryotes diverge in the details and complexity of HRR . For eliminating DSBs, HRR and end-joining pathways share the burden, with HRR contributing critically during S and G2 phases . It is likely that the removal of interstrand cross-links is absolutely dependent on efficient HRR, as suggested by the extraordinary sensitivity of the ercc1, xpf/ercc4, xrcc2, and xrcc3 mutants to cross-linking chemicals . Similarly, chromosome stability in untreated cells requires intact HRR, which may eliminate DSBs arising during DNA replication and thereby prevent chromosome aberrations . Complex regulation of HRR by cell cycle checkpoint and surveillance functions is suggested not only by direct interactions between human Rad51 and p53, c-Abl, and BRCA2, but also by very high recombination rates in p53-deficient cells.

Biochimie, 1999 Jan-Feb, 81(1-2), 77 - 85
The role of homologous recombination processes in the repair of severe forms of DNA damage in mammalian cells; Thacker J; The role of homologous recombination processes in the repair of severe forms of DNA damage is reviewed, with particular attention to the functions of members of the recA/RAD51 family of genes . In the yeast Saccharomyces cerevisiae, several of the gene products involved in homologous recombination repair (HRR) have been studied in detail, and a picture is beginning to emerge of the repair mechanism for DNA double-strand breaks . Knowledge is fragmentary for other eukaryotic organisms and for other types of DNA damage . In mammalian cells, while it has been known for some years that HRR occurs, the relative importance of the process in repairing DNA damage is unknown and very few of the gene products involved have been identified . Very recently, a number of RAD51-like genes have been identified in mammals, either through cloning genes complementing cell lines sensitive to DNA-damaging agents (XRCC2, XRCC3), or through homology searches (RAD51L1, RAD51L2, RAD51L3) . As yet the role of these genes and their possible functions are speculative, although the combination of sequence conservation and gene expression patterns suggest that they function in HRR pathways.

Leukemia, 1999 Apr, 13(4), 595 - 600
Expressions of four major protein Ser/Thr phosphatases in human primary leukemic cells; Yamamoto M et al.; Activity and expression of four major protein serine/threonine (Ser/Thr) phosphatases, protein phosphatase type 1 (PP1), protein phosphatase type 2A (PP2A), protein phosphatase type 2B (PP2B) and protein phosphatase type 2C (PP2C) were evaluated in normal peripheral leukocytes, and in various leukemic cells from patients with acute myelogenous leukemia (AML), common acute lymphocytic leukemia (cALL), or chronic lymphocytic leukemia (CLL) . PP1 was the most abundant phosphatase in blood cells, and relative abundance of each phosphatase was: PP1 > PP2A > PP2B approximately = PP2C . PP1 activity and its expressions were higher in blasts of AML-M4 and -M5 than in cells of AML-M1, cALL and CLL . PP2A activity and its expression were higher in blasts of AML-M3, -M4 and -M5 than in cells of AML-M1, cALL and CLL . Activity and expression of both PP1 and PP2A in normal monocytes were highest, and PP2A activity in normal neutrophils was lowest among normal leukocytes . PP2B activity and its expression were higher in blasts of AML-M2, -M3 and normal lymphocytes . PP2C activity and its expression were relatively constant in various leukemic cell types . Activities of PP1 and PP2A of AML blasts correlated positively with the expression of CD11b, whereas activities of PP1 and PP2B correlated negatively with the expression of CD7 . Thus, each phosphatase was ubiquitously but differently expressed in various leukemic cell types and in normal leukocytes . These data also suggest that expressions of PP1, PP2A and PP2B are relatively low in leukemic blasts arresting at the stage of early pluripotent stem cells, and are differently modulated during the course of myelomonocytic commitment and maturation.

Science, 1999 Apr 23, 284(5414), 662 - 5
Reconstitution of G1 cyclin ubiquitination with complexes containing SCFGrr1 and Rbx1; Skowyra D et al.; Control of cyclin levels is critical for proper cell cycle regulation . In yeast, the stability of the G1 cyclin Cln1 is controlled by phosphorylation-dependent ubiquitination . Here it is shown that this reaction can be reconstituted in vitro with an SCF E3 ubiquitin ligase complex . Phosphorylated Cln1 was ubiquitinated by SCF (Skp1-Cdc53-F-box protein) complexes containing the F-box protein Grr1, Rbx1, and the E2 Cdc34 . Rbx1 promotes association of Cdc34 with Cdc53 and stimulates Cdc34 auto-ubiquitination in the context of Cdc53 or SCF complexes . Rbx1, which is also a component of the von Hippel-Lindau tumor suppressor complex, may define a previously unrecognized class of E3-associated proteins.

Biochemistry, 1999 Apr 20, 38(16), 5155 - 63
Site-specific inhibition of transcription factor binding to DNA by a metallointercalator; Odom DT et al.; The metallointercalator Lambda-1-Rh(MGP)2phi5+ binds tightly and specifically to the site 5'-CATATG-3' in the major groove of double helical DNA by a combination of direct readout and shape selection . To examine competitive interactions between this small metal complex and a DNA-binding transcription factor, the preferred binding site for Lambda-1-Rh(MGP)2phi5+ was engineered into the AP-1 recognition element (ARE) of the major-groove binding bZIP transcription factor yAP-1, the yeast analogue of mammalian AP-1 . Binding experiments confirmed that the modified ARE retained normal yAP-1 binding affinity . Photocleavage experiments demonstrated that the modified ARE contained a high-affinity binding site for Lambda-1-Rh(MGP)2phi5+, whereas the native ARE showed no interaction . Competition experiments using gel shift mobility assays demonstrated that Lambda-1-Rh(MGP)2phi5+ at 120 nM competes 50% of yAP-1 binding to the 5'-CATATG-3' containing oligonucleotide . In contrast, competitive disruption of protein binding to the native ARE requires 3 microM Lambda-1-Rh(MGP)2phi5+ . Metallointercalator derivatives, including geometric isomers of Lambda-1-Rh(MGP)2phi5+, show no specific binding to the target site and show no inhibition of yAP-1/DNA complexes at concentrations as high as 20 microM . Thus, metallointercalators can be tuned to show selectivity for major groove sites on DNA comparable to transcription factors and indeed can inhibit transcription factor binding site selectively.

Neurochem Int, 1999 Feb, 34(2), 131 - 6
Changes in proteasome expression and activity during differentiation of neuronal precursor NTera 2 clone D1 cells; Wojcik C et al.; The effect of differentiation of the human neuronal progenitor cell line NTera 2 clone D1 (NT2/D1) by retinoic acid on components of the proteasome system was studied . The chymotrypsin-like and peptidylglutamyl peptide bond hydrolyzing activities of the proteasome increased five weeks after retinoic acid, and following treatment with mitotic inhibitors returned to levels detected in non-differentiated cells . A selective induction of the MHC class II region encoded LMP7 and LMP2 proteasome subunits occurred during differentiation, whereas there were no changes in the expression of the constitutive LMP2 counterpart (delta-subunit) or the constitutive C2 subunit . Immunofluorescence revealed marked LMP7 accumulation in fully differentiated cells, with no changes in the labeling pattern of the constitutive proteasome antigens . The expression of the alpha-subunit of the PA28 proteasome activator was down-regulated in fully differentiated neurons, but was not correlated with changes in enzymatic activity . Changes in proteasome activity and composition may contribute to the processes leading to differentiation of human neurons in vitro and to the properties of fully differentiated neurons.

J Biol Chem, 1999 Apr 30, 274(18), 12748 - 52
Radiation-induced assembly of Rad51 and Rad52 recombination complex requires ATM and c-Abl; Chen G et al.; Cells from individuals with the recessive cancer-prone disorder ataxia telangiectasia (A-T) are hypersensitive to ionizing radiation (I-R) . ATM (mutated in A-T) is a protein kinase whose activity is stimulated by I-R . c-Abl, a nonreceptor tyrosine kinase, interacts with ATM and is activated by ATM following I-R . Rad51 is a homologue of bacterial RecA protein required for DNA recombination and repair . Here we demonstrate that there is an I-R-induced Rad51 tyrosine phosphorylation, and this induction is dependent on both ATM and c-Abl . ATM, c-Abl, and Rad51 can be co-immunoprecipitated from cell extracts . Consistent with the physical interaction, c-Abl phosphorylates Rad51 in vitro and in vivo . In assays using purified components, phosphorylation of Rad51 by c-Abl enhances complex formation between Rad51 and Rad52, which cooperates with Rad51 in recombination and repair . After I-R, an increase in association between Rad51 and Rad52 occurs in wild-type cells but not in cells with mutations that compromise ATM or c-Abl . Our data suggest signaling mediated through ATM, and c-Abl is required for the correct post-translational modification of Rad51, which is critical for the assembly of Rad51 repair protein complex following I-R.

J Biol Chem, 1999 Apr 30, 274(18), 12642 - 9
Characterization of a novel type of serine/threonine kinase that specifically phosphorylates the human goodpasture antigen; Raya A et al.; Goodpasture disease is an autoimmune disorder that occurs naturally only in humans . Also exclusive to humans is the phosphorylation process that targets the unique N-terminal region of the Goodpasture antigen . Here we report the molecular cloning of GPBP (Goodpasture antigen-binding protein), a previously unknown 624-residue polypeptide . Although the predicted sequence does not meet the conventional structural requirements for a protein kinase, its recombinant counterpart specifically binds to and phosphorylates the exclusive N-terminal region of the human Goodpasture antigen in vitro . This novel kinase is widely expressed in human tissues but shows preferential expression in the histological structures that are targets of common autoimmune responses . The work presented in this report highlights a novel gene to be explored in human autoimmunity.

J Biol Chem, 1999 Apr 30, 274(18), 12593 - 604
The peroxin Pex14p . cDNA cloning by functional complementation on a Chinese hamster ovary cell mutant, characterization, and functional analysis; Shimizu N et al.; Rat cDNA encoding a 376-amino acid peroxin was isolated by functional complementation of a peroxisome-deficient Chinese hamster ovary cell mutant, ZP110, of complementation group 14 (CG14) . The primary sequence showed 28 and 24% amino acid identity with the yeast Pex14p from Hansenula polymorpha and Saccharomyces cerevisiae, respectively; therefore, we termed this cDNA rat PEX14 (RnPEX14) . Human and Chinese hamster Pex14p showed 96 and 94% identity to rat Pex14p, except that both Pex14p comprised 377 amino acids . Pex14p was characterized as an integral membrane protein of peroxisomes, exposing its N- and C-terminal parts to the cytosol . Pex14p interacts with both Pex5p and Pex7p, the receptors for peroxisome targeting signal type 1 (PTS1) and PTS2, respectively, together with the receptors' cargoes, PTS1 and PTS2 proteins . Mutation in PEX14 from ZP161, the same CG as ZP110, was determined by reverse transcription-PCR as follows . A 133-base pair deletion at nucleotide residues 37-169 in one allele created a termination codon at 40-42; in addition to this mutation, 103 base pairs were deleted at positions 385-487, resulting in the second termination immediately downstream the second deletion site in the other allele . Neither of these two mutant forms of Pex14p restored peroxisome biogenesis in ZP110 and ZP161, thereby demonstrating PEX14 to be responsible for peroxisome deficiency in CG14.

J Biol Chem, 1999 Apr 30, 274(18), 12408 - 13
Role of the copper-binding domain in the copper transport function of ATP7B, the P-type ATPase defective in Wilson disease; Forbes JR et al.; We have analyzed the functional effect of site-directed mutations and deletions in the copper-binding domain of ATP7B (the copper transporting P-type ATPase defective in Wilson disease) using a yeast complementation assay . We have shown that the sixth copper-binding motif alone is sufficient, but not essential, for normal ATP7B function . The N-terminal two or three copper-binding motifs alone are not sufficient for ATP7B function . The first two or three N-terminal motifs of the copper-binding domain are not equivalent to, and cannot replace, the C-terminal motifs when placed in the same sequence position with respect to the transmembrane channel . From our data, we propose that the copper-binding motifs closest to the channel are required for the copper-transport function of ATP7B . We propose that cooperative copper binding to the copper-binding domain of ATP7B is not critical for copper transport function, but that cooperative copper binding involving the N-terminal two or three copper-binding motifs may be involved in initiating copper-dependent intracellular trafficking . Our data also suggest a functional difference between the copper-binding domains of ATP7A and ATP7B.

Int J Cancer, 1999 May 5, 81(3), 459 - 66
Identification of a SART-1-derived peptide capable of inducing HLA-A24-restricted and tumor-specific cytotoxic T lymphocytes; Kikuchi M et al.; We have described the SART-1 gene-encoding peptides recognized by HLA-A2601-restricted and tumor-specific cytotoxic T lymphocytes (CTLs) . We now have investigated whether SART-1 encodes peptides capable of inducing the HLA-A24-restricted CTLs . Among the 18 different peptides with HLA-A24-binding motifs, the SART-1(690-698) peptide (EYRGFTQDF) was most strongly recognized by the HLA-A24-restricted and tumor-specific CTLs established from an esophageal cancer patient . After a third stimulation in vitro, this peptide induced HLA-A24-restricted CTLs recognizing the SART-1(259)+ tumor cells in PBMCs of all HLA-A24 homozygous and the majority of HLA-A24 heterozygous cancer patients and healthy donors tested . A similar activity, induction of CTLs from PBMCs, was observed in the Saccharomyces cerevisiae-derived nonapeptide (EYRGFTPMF) that shares 7 amino acids with the SART-1(690-698) peptide . The SART-1(690-698) peptide-induced CTL activity was significantly higher in PBMCs of HLA-A24 homozygotes than in HLA-A24 heterozygotes . The CTL precursor frequency in PBMCs after a third stimulation in vitro with the SART-1(690-698) peptide was high (>1/200) in both cancer patients and healthy donors . The SART-1(690-698) peptide could thus be useful for specific immunotherapy of HLA-A24+ cancer patients.

Exp Hematol, 1999 Apr, 27(4), 751 - 61
Generation of phagocytic MAK and MAC-DC for therapeutic use: characterization and in vitro functional properties; Boyer A et al.; Phagocytic cells with macrophage or dendritic cell phenotype, able to capture and ingest tumor cells, were derived in large numbers from peripheral blood mononuclear cells using two different activation procedures . Peripheral blood mononuclear cells were stimulated in nonadherent conditions in the presence of human AB serum with either granulocyte-macrophage colony-stimulating factor and dihydroxy-vitamin D3 for 7 days and with interferon-gamma for the last 18 hours to obtain activated macrophages (MAK) or with granulocyte-macrophage colony-stimulating factor and interleukin-13 for 7 days (with fresh interleukin-13 added on day 4) to obtain macrophage-dendritic cells (MAC-DC) . A strong ability of MAC-DC to phagocytose yeasts was observed, in contrast to a low-intermediate phagocytosis capacity by MAK . Both CD14+ FCgammaR+ (FcgammaRI/CD64, FcgammaRII/CD32, FcgammaRIII/CD16) MAK and CD1a+/CD86+, CD14- MAC-DC were able to phagocytose whole tumor cells . However, only MAK phagocytosis was enhanced by FcgammaR engagement . MAK but not MAC-DC could lyse tumor cell in antibody-dependent cell cytotoxicity assays, via FcgammaRI . Thus, MAK as well as MAC-DC may represent valuable tools for different in vivo therapy strategies that do or do not include the use of monoclonal antibodies.

Biochim Biophys Acta, 1999 Apr 12, 1431(1), 37 - 46
Comparison of physical chemical properties of llama VHH antibody fragments and mouse monoclonal antibodies; van der Linden RH et al.; Antigen specific llama VHH antibody fragments were compared to antigen specific mouse monoclonal antibodies with respect to specificity, affinity and stability . The llama VHH antibody fragments and the mouse monoclonal antibodies investigated were shown to be highly specific for the protein antigen hCG or the hapten antigen RR-6 . The affinity of the interaction between monovalent llama VHH antibody fragments and their antigen is close to the nanomolar range, similar to the bivalent mouse monoclonal antibodies studied . Llama VHH antibody fragments are similar to mouse monoclonal antibodies with respect to antigen binding in the presence of ammonium thiocyanate and ethanol . The results show that relative to antigen specific mouse monoclonal antibodies, antigen specific llama VHH fragments are extremely temperature stable . Two out of six llama VHHs are able to bind antigen specifically at temperatures as high as 90 degrees C, whereas four out of four mouse monoclonal antibodies are not functional at this temperature . Together with the finding that llama VHH fragments can be produced at high yield in Saccharomyces cerevisiae, these findings indicate that in the near future antigen specific llama VHH fragments can be used in for antibodies unexpected products and processes.

Biochim Biophys Acta, 1999 Apr 14, 1445(1), 160 - 4
Molecular cloning and sequencing of cDNAs encoding homologues of human Ku70 and Ku80 autoantigen from Xenopus and their expression in various Xenopus tissues; Yagura T et al.; We isolated cDNA clones encoding Ku70 and Ku80 homologues of Xenopus laevis from a cDNA library prepared from Xenopus oocytes . The nucleotide sequences of these Ku70 and Ku80 homologues have coding sequences of 1833 bp and a 611 aa protein, and 2178 bp and a 726 aa protein, respectively . The amino acid sequences deduced from the open reading frame of the Ku70 and Ku80 cDNA clones were highly homologous to those from Ku genes previously isolated, such as human (ca . 65% and ca . 62% identity, respectively) and mouse (ca . 65% and ca . 60%), and show a certain degree of homology to Drosophila (ca . 27% with Ku70), Caenorhabditis elegans (ca . 20% with Ku80) and Saccharomyces cerevisiae (ca . 23% and ca . 19%) . Our detailed comparison of the predicted amino acid sequences among these species revealed the highly conserved octa-peptide LPFXXDIR common to both Xenopus Ku70 and Ku80 homologues in the region showing the high homology throughout the species tested . A Northern analysis using specific cDNA probes showed that Ku poly(A)+ mRNAs are expressed at high levels in Xenopus adult oocyte and testis.

Curr Opin Cell Biol, 1999 Apr, 11(2), 211 - 8
Organization and regulation of mitogen-activated protein kinase signaling pathways; Garrington TP et al.; Mitogen-activated protein kinases (MAPKs) are components of a three kinase regulatory cascade . There are multiple members of each component family of kinases in the MAPK module . Specificity of regulation is achieved by organization of MAPK modules, in part, by use of scaffolding and anchoring proteins . Scaffold proteins bring together specific kinases for selective activation, sequestration and localization of signaling complexes . The recent elucidation of scaffolding mechanisms for MAPK pathways has begun to solve the puzzle of how specificity in signaling can be achieved for each MAPK pathway in different cell types and in response to different stimuli . As new MAPK members are defined, determining their organization in kinase modules will be critical in understanding their select role in cellular regulation.

Curr Opin Cell Biol, 1999 Apr, 11(2), 241 - 7
In or out? Regulating nuclear transport; Hood JK et al.; The compartmentalization of proteins within the nucleus or cytoplasm of a eukaryotic cell offers opportunity for regulation of cell cycle progression and signalling pathways . Nuclear localization of proteins is determined by their ability to interact with specific nuclear import and export factors . In the past year, substrate phosphorylation has emerged as a common mechanism for controlling this interaction.

Curr Biol, 1999 Apr 8, 9(7), 385 - 8
A novel Golgi-localisation domain shared by a class of coiled-coil peripheral membrane proteins; Kjer-Nielsen L et al.; The mechanism by which peripheral membrane proteins are targeted to the cytoplasmic face of the Golgi apparatus is poorly understood . Previously, we have identified a carboxy-terminal domain of the trans-Golgi-network (TGN) protein p230 that is responsible for Golgi localisation {1} . Here, we report the identification of a similar Golgi-localisation domain (GLD, also termed the 'GRIP' domain - see the paper by Munro and Nichols elsewhere in this issue) in a family of putative peripheral membrane proteins from lower and higher eucaryotes . The majority of family members have a domain structure similar to that of p230, with extensive coiled-coil regions (>80%) and the potential GLD located in a non-coiled-coil domain at the carboxyl terminus . Previously reported proteins in this family include human golgin-97 and Saccharomyces cerevisiae Imh1p . By constructing chimeric cDNAs encoding carboxy-terminal regions of these family members fused to green fluorescent protein (GFP), we have directly demonstrated that the GLD of p230, golgin-97, the newly identified human protein GCC1p and yeast Imh1p functions as a Golgi-targeting domain in transfected mammalian cells . Site-directed mutagenesis of the GLDs identified two conserved aromatic residues that are critical for the function of this targeting domain . Endogenous p230 was displaced from the Golgi membranes in transfected cells expressing high levels of GFP fused to the GLD of either p230 or golgin-97, indicating that different GLDs interact with similar membrane determinants . Thus, we have identified a family of coiled-coil proteins that share a domain shown to be sufficient for the localisation of peripheral membrane proteins to the Golgi apparatus.

Curr Biol, 1999 Apr 8, 9(7), 373 - 6
F-box/WD-repeat proteins pop1p and Sud1p/Pop2p form complexes that bind and direct the proteolysis of cdc18p; Wolf DA et al.; Ubiquitin-dependent proteolysis plays an important role in cell-cycle control {1} {2} . In budding yeast, the protein Skp1p, the cullin-family member Cdc53p, and the F-box/WD-repeat protein Cdc4p form the SCFCdc4p ubiquitin ligase complex, which targets the cyclin-dependent kinase (Cdk) inhibitor Sic1p for proteolysis {3} {4} {5} {6} {7} {8} . Sic1p is recruited to the SCFCdc4p complex by binding to the WD-repeat region of Cdc4p {5} {6}, while Skp1p binds to the F-box of Cdc4p {9} . In fission yeast, two distinct Cdc4p-related proteins, Pop1p/Ste16p {10} {11} and the recently identified Sud1p/Pop2p {12}, regulate the stability of the replication initiator Cdc18p and the Cdk inhibitor Rum1p . We show here that, despite their structural and functional similarities, the pop1 and pop2 genes fail to complement each other's deletion phenotypes, indicating that they perform non-redundant, but potentially interdependent, functions in proteolysis . Consistent with this hypothesis, Pop1p and Pop2p formed heterooligomeric complexes when overexpressed, and binding of Cdc18p to Pop2p was dependent on Pop1p . The Pop1p-Pop2p interaction was mediated by the amino-terminal domain of Pop2p which, when fused to full-length Pop1p, rescued the phenotype of a Deltapop1Deltapop2 double mutant . Thus, close physical proximity of two distinct F-box/WD-repeat proteins directs proteolysis mediated by the SCFPop ubiquitin ligase complex.

Curr Biol, 1999 Apr 8, 9(7), 351 - 60
Inducible recruitment of Cdc42 or WASP to a cell-surface receptor triggers actin polymerization and filopodium formation; Castellano F et al.; BACKGROUND: Cdc42, a GTP-binding protein of the Rho family, controls actin cytoskeletal organization and helps to generate actin-based protruding structures, such as filopodia . In vitro, Cdc42 regulates actin polymerization by facilitating the creation of free barbed ends - the more rapidly growing ends of actin filaments - and subsequent elongation at these ends . The Wiskott- Aldrich syndrome protein, WASP, which has a pleckstrin-homology domain and a Cdc42/Rac-binding motif, has been implicated in cell signaling and cytoskeleton reorganization . We have investigated the consequences of local recruitment of activated Cdc42 or WASP to the plasma membrane . RESULTS: We used an activated Cdc42 protein that could be recruited to an engineered membrane receptor by adding rapamycin as a bridge, and added antibody-coupled beads to aggregate these receptors . Inducible recruitment of Cdc42 to clusters of receptors stimulated actin polymerization, resulting in the formation of membrane protrusions . Cdc42-induced protrusions were enriched in the vasodilator-stimulated phosphoprotein VASP and the focal-adhesion-associated proteins zyxin and ezrin . The Cdc42 effector WASP could also induce the formation of protrusions, albeit of different morphology . CONCLUSIONS: This is the first demonstration that the local recruitment of activated Cdc42 or its downstream effector, WASP, to a membrane receptor in whole cells is sufficient to trigger actin polymerization that results in the formation of membrane protrusions . Our data suggest that Cdc42-induced actin-based protrusions result from the local and serial recruitment of cytoskeletal proteins including zyxin, VASP, and ezrin.

Curr Biol, 1999 Apr 8, 9(7), R258 - 60
Membrane transport: Take your fusion partners; Clague MJ; Recent studies of how vesicles are targeted to fuse with specific membranes inside cells highlight a role for extended coiled-coil proteins in tethering partner membranes prior to formation of the 'SNARE complex' that mediates the fusion reaction . The tethering protein is recruited to membranes by a Rab family GTPase

Curr Biol, 1999 Mar 25, 9(6), 325 - 8
Mouse Rad54 affects DNA conformation and DNA-damage-induced Rad51 foci formation; Tan TL et al.; Error-free repair by homologous recombination of DNA double-strand breaks induced by ionizing radiation (IR) requires the Rad52 group proteins, including Rad51 and Rad54, in the yeast Saccharomyces cerevisiae {1} . The formation of a 'joint' molecule between the damaged DNA and the homologous repair template is a key step in recombination mediated by Rad51 and stimulated by Rad54 {2} {3} {4} {5} . Mammalian homologs of Rad51 and Rad54 have been identified {2} {3} {6} . Here, we demonstrate that mouse Rad54 (mRad54) formed IR-induced nuclear foci that colocalized with mRad51 . Interaction between mRad51 and mRad54 was induced by genotoxic stress, but only when lesions that required mRad54 for their repair were formed . Interestingly, mRad54 was essential for the formation of IR-induced mRad51 foci . Rad54 belongs to the SWI2/SNF2 protein family, members of which modulate protein-DNA interactions in an ATP-driven manner {7} . Results of a topological assay suggested that purified human Rad54 (hRad54) protein can unwind double-stranded (ds) DNA at the expense of ATP hydrolysis . Unwinding of the homologous repair template could promote the formation or stabilization of hRad51-mediated joint molecules . Rad54 appears to be required downstream of other Rad52 group proteins, such as Rad52 and the Rad55-Rad57 heterodimer, that assist Rad51 in interacting with the broken DNA {2} {3} {4}.

Curr Biol, 1999 Mar 25, 9(6), 333 - 6
Structural elements required for the localization of ASH1 mRNA and of a green fluorescent protein reporter particle in vivo; Chartrand P et al.; The sorting of the Ash1 protein to the daughter nucleus of Saccharomyces cerevisiae in late anaphase of the budding cycle correlates with the localization of ASH1 mRNA at the bud tip {1} {2} . Although the 3' untranslated region (3' UTR) of ASH1 is sufficient to localize a reporter mRNA, it is not necessary, a result which indicates that other sequences are involved {1} . We report the identification of three additional cis-acting elements in the coding region . Each element alone, when fused to a lacZ reporter gene, was sufficient for the localization of the lacZ mRNA reporter to the bud . A fine-structure analysis of the 3' UTR element showed that its function in mRNA localization did not depend on a specific sequence but on the secondary and tertiary structure of a minimal 118 nucleotide stem-loop . Mutations in the stem-loop that affect the localization of the lacZ mRNA reporter also affected the formation of the localization particles, in living cells, composed of a green fluorescent protein (GFP) complexed with lacZ-ASH1-3' UTR mRNA {3} . A specific stem-loop in the 3' UTR of the ASH1 mRNA is therefore required for both localization and particle formation, suggesting that complex formation is part of the localization mechanism . An analysis on one of the coding-region elements revealed a comparable stem-loop structure with similar functional requirements.

Curr Biol, 1999 Mar 25, 9(6), 292 - 301
Caenorhabditis elegans inhibitor of apoptosis protein (IAP) homologue BIR-1 plays a conserved role in cytokinesis; Fraser AG et al.; BACKGROUND: Inhibitor of apoptosis proteins (IAPs) suppress apoptotic cell death in several model systems and are highly conserved between insects and mammals . All IAPs contain at least one copy of the approximately 70 amino-acid baculovirus IAP repeat (BIR), and this domain is essential for the anti-apoptotic activity of the IAPs . Both the marked structural diversity of IAPs and the identification of BIR-containing proteins (BIRPs) in yeast, however, have led to the suggestion that BIRPs might play roles in other, as yet unidentified, cellular processes besides apoptosis . Survivin, a human BIRP, is upregulated 40-fold at G2-M phase and binds to mitotic spindles, although its role at the spindle is still unclear . RESULTS: We have identified and characterised two Caenorhabditis elegans BIRPs,BIR-1 and BIR-2; these proteins are the only BIRPs in C . elegans . The bir-1 gene is highly expressed during embryogenesis with detectable expression throughout other stages of development; bir-2 expression is detectable only in adults and embryos . Overexpression of bir-1 was unable to inhibit developmentally occurring cell death in C . elegans and inhibition of bir-1 expression did not increase cell death . Instead, embryos lacking bir-1 were unable to complete cytokinesis and they became multinucleate . This cytokinesis defect could be partially suppressed by transgenic expression of survivin, the mammalian BIRP most structurally related to BIR-1, suggesting a conserved role for BIRPs in the regulation of cytokinesis . CONCLUSIONS: BIR-1, a C . elegans BIRP, is probably not involved in the general regulation of apoptosis but is required for embryonic cytokinesis . We suggest that BIRPs may regulate cytoskeletal changes in diverse biological processes including cytokinesis and apoptosis.

Curr Biol, 1999 Mar 25, 9(6), R221 - 4
Transcriptional regulation: SWItching circuitry; Wade PA et al.; Proteins of the SWI/SNF family disrupt chromatin, hydrolysing ATP in the process . How they do so is still mysterious, but recent studies indicate that they can be targeted to the nuclear infrastructure and to particular genes, where they cooperate with other enzymes to activate or repress transcription.

Nat Biotechnol, 1999 Apr, 17(4), 379 - 84
Directed evolution of a fungal peroxidase; Cherry JR et al.; The Coprinus cinereus (CiP) heme peroxidase was subjected to multiple rounds of directed evolution in an effort to produce a mutant suitable for use as a dye-transfer inhibitor in laundry detergent . The wild-type peroxidase is rapidly inactivated under laundry conditions due to the high pH (10.5), high temperature (50 degrees C), and high peroxide concentration (5-10 mM) . Peroxidase mutants were initially generated using two parallel approaches: site-directed mutagenesis based on structure-function considerations, and error-prone PCR to create random mutations . Mutants were expressed in Saccharomyces cerevisiae and screened for improved stability by measuring residual activity after incubation under conditions mimicking those in a washing machine . Manually combining mutations from the site-directed and random approaches led to a mutant with 110 times the thermal stability and 2.8 times the oxidative stability of wild-type CiP . In the final two rounds, mutants were randomly recombined by using the efficient yeast homologous recombination system to shuffle point mutations among a large number of parents . This in vivo shuffling led to the most dramatic improvements in oxidative stability, yielding a mutant with 174 times the thermal stability and 100 times the oxidative stability of wild-type CiP.

J Cell Biol, 1999 Apr 19, 145(2), 237 - 54
RAE1 is a shuttling mRNA export factor that binds to a GLEBS-like NUP98 motif at the nuclear pore complex through multiple domains; Pritchard CE et al.; Gle2p is implicated in nuclear export of poly(A)+ RNA and nuclear pore complex (NPC) structure and distribution in Saccharomyces cerevisiae . Gle2p is anchored at the nuclear envelope (NE) via a short Gle2p-binding motif within Nup116p called GLEBS . The molecular mechanism by which Gle2p and the Gle2p-Nup116p interaction function in mRNA export is unknown . Here we show that RAE1, the mammalian homologue of Gle2p, binds to a GLEBS-like NUP98 motif at the NPC through multiple domains that include WD-repeats and a COOH-terminal non-WD-repeat extension . This interaction is direct, as evidenced by in vitro binding studies and chemical cross-linking . Microinjection experiments performed in Xenopus laevis oocytes demonstrate that RAE1 shuttles between the nucleus and the cytoplasm and is exported from the nucleus in a temperature-dependent and RanGTP-independent manner . Docking of RAE1 to the NE is highly dependent on new mRNA synthesis . Overexpression of the GLEBS-like motif also inhibits NE binding of RAE1 and induces nuclear accumulation of poly(A)+ RNA . Both effects are abrogated either by the introduction of point mutations in the GLEBS-like motif or by overexpression of RAE1, indicating a direct role for RAE1 and the NUP98-RAE1 interaction in mRNA export . Together, our data suggest that RAE1 is a shuttling transport factor that directly contributes to nuclear export of mRNAs through its ability to anchor to a specific NUP98 motif at the NPC.

Pharmacogenetics, 1999 Feb, 9(1), 61 - 70
High yield purification and characterization of engineered human P450 1A2 and generation of immuno-inhibitor antibodies; Louerat-Oriou B et al.; P450 S12, an engineered human P450 1A2 containing the 88-first amino-acids of the P450 1A1, demonstrates particularly high expression level in yeast while exhibiting catalytic properties very similar to the moderately expressed natural human P450 1A2 . To facilitate P450 purification by nickel chelate chromatography, C-terminal extensions including histidine tags were tested . The -G(H)4 extension was found to be particularly efficient for permitting high expression levels without any catalytic alteration . This engineered P450 was purified to electrophoretic homogeneity (18 nmol/mg of protein) at a very high yield (87%) without any detectable formation of P420 . P450 S12 activities were reconstituted in the presence of yeast and Arabidopsis thaliana (ATR1) NADPH-P450 reductases . The plant reductase supported better ethoxyresorufin-, methoxyresorufin- and phenacetin-O-dealkylase activities than the yeast reductase in reconstituted systems . Interestingly, polyclonal antibodies raised against purified P450 S12 selectively recognized in Western blot and fully immuno-inhibited the natural or recombinant P450 1A2 with very limited or no cross-reaction with P450 1A1 and other isoenzymes.

Oncogene, 1999 Mar 4, 18(9), 1677 - 87
The translation initiation factor, hu-Sui1 may be a target of hepatitis B X antigen in hepatocarcinogenesis; Lian Z et al.; The role of hepatitis B virus X antigen in the development of hepatocellular carcinoma was explored by stably transfecting HepG2 cells with an X antigen expression vector, and identifying the differences in gene expression that distinguish X positive from X negative cells by subtractive PCR . One differentially expressed gene, the human homolog of sui1 (hu-sui1), encodes a translation initiation factor whose expression was suppressed by X antigen in HepG2 cells . Hu-Sui1 was also expressed in nontumor liver but not in tumor cells from patients with hepatocellular carcinoma . Introduction of hu-sui1 into HepG2 cells inhibited cell growth in culture, in soft agar, and partially inhibited tumor formation in nude mice . Hence, the suppression of hu-sui1 by X antigen may result in the abrogation of negative growth regulation and contribute to the development of hepatocellular carcinoma.

Genome Res, 1999 Apr, 9(4), 383 - 92
Genome-scale cloning and expression of individual open reading frames using topoisomerase I-mediated ligation; Heyman JA et al.; The in vitro cloning of DNA molecules traditionally uses PCR amplification or site-specific restriction endonucleases to generate linear DNA inserts with defined termini and requires DNA ligase to covalently join those inserts to vectors with the corresponding ends . We have used the properties of Vaccinia DNA topoisomerase I to develop a ligase-free technology for the covalent joining of DNA fragments to suitable plasmid vectors . This system is much more efficient than cloning methods that require ligase because the rapid DNA rejoining activity of Vaccinia topoisomerase I allows ligation in only 5 min at room temperature, whereas the enzyme's high substrate specificity ensures a low rate of vector-alone transformants . We have used this topoisomerase I-mediated cloning technology to develop a process for accelerated cloning and expression of individual ORFs . Its suitability for genome-scale molecular cloning and expression is demonstrated in this report.

Development, 1999 May, 126(10), 2253 - 60
Direct regulatory interaction of the eyeless protein with an eye-specific enhancer in the sine oculis gene during eye induction in Drosophila; Niimi T et al.; The Pax-6 gene encodes a transcription factor with two DNA-binding domains, a paired and a homeodomain, and is expressed during eye morphogenesis and development of the nervous system . Pax-6 homologs have been isolated from a wide variety of organisms ranging from flatworms to humans . Since loss-of-function mutants in insects and mammals lead to an eyeless phenotype and Pax-6 orthologs from distantly related species are capable of inducing ectopic eyes in Drosophila, we have proposed that Pax-6 is a universal master control gene for eye morphogenesis . To determine the extent of evolutionary conservation of the eye morphogenetic pathway, we have begun to identify subordinate target genes of Pax-6 . Previously we have shown that expression of two genes, sine oculis (so) and eyes absent (eya), is induced by eyeless (ey), the Pax-6 homolog of Drosophila . Here we present evidence from ectopic expression studies in transgenic flies, from transcription activation studies in yeast, and from gel shift assays in vitro that the EY protein activates transcription of sine oculis by direct interaction with an eye-specific enhancer in the long intron of the so gene.

Mol Cell Biol, 1999 May, 19(5), 3748 - 59
Role for Hsp90-associated cochaperone p23 in estrogen receptor signal transduction; Knoblauch R et al.; The mechanism of signal transduction by the estrogen receptor (ER) is complex and not fully understood . In addition to the ER, a number of accessory proteins are apparently required to efficiently transduce the steroid hormone signal . In the absence of estradiol, the ER, like other steroid receptors, is complexed with Hsp90 and other molecular chaperone components, including an immunophilin, and p23 . This Hsp90-based chaperone complex is thought to repress the ER's transcriptional regulatory activities while maintaining the receptor in a conformation that is competent for high-affinity steroid binding . However, a role for p23 in ER signal transduction has not been demonstrated . Using a mutant ER (G400V) with decreased hormone binding capacity as a substrate in a dosage suppression screen in yeast cells (Saccharomyces cerevisiae), we identified the yeast homologue of the human p23 protein (yhp23) as a positive regulator of ER function . Overexpression of yhp23 in yeast cells increases ER transcriptional activation by increasing estradiol binding in vivo . Importantly, the magnitude of the effect of yhp23 on ER transcriptional activation is inversely proportional to the concentration of both ER and estradiol in the cell . Under conditions of high ER expression, ER transcriptional activity is largely independent of yhp23, whereas at low levels of ER expression, ER transcriptional activation is primarily dependent on yhp23 . The same relationship holds for estradiol levels . We further demonstrate that yhp23 colocalizes with the ER in vivo . Using a yhp23-green fluorescent protein fusion protein, we observed a redistribution of yhp23 from the cytoplasm to the nucleus upon coexpression with ER . This nuclear localization of yhp23 was reversed by the addition of estradiol, a finding consistent with yhp23's proposed role as part of the aporeceptor complex . Expression of human p23 in yeast partially complements the loss of yhp23 function with respect to ER signaling . Finally, ectopic expression of human p23 in MCF-7 breast cancer cells increases both hormone-dependent and hormone-independent transcriptional activation by the ER . Together, these results strongly suggest that p23 plays an important role in ER signal transduction.

Mol Cell Biol, 1999 May, 19(5), 3466 - 73
Specific acetylation of chromosomal protein HMG-17 by PCAF alters its interaction with nucleosomes; Herrera JE et al.; Nonhistone chromosomal proteins HMG-14 and HMG-17 are closely related nucleosomal binding proteins that unfold the higher-order chromatin structure, thereby enhancing the transcription and replication potential of chromatin . Here we report that PCAF, a transcription coactivator with intrinsic histone acetyltransferase activity, specifically acetylates HMG-17 but not HMG-14 . Using mass spectrum sequence analysis, we identified the lysine at position 2 as the predominant site acetylated by PCAF . Lysine 2 is a prominent acetylation site in vivo, suggesting that this PCAF-mediated acetylation is physiologically relevant . Experiments with HMG-17 deletion mutants and competition studies with various protein fragments indicate that the specific acetylation of HMG-17 is not determined solely by the primary sequence near the acetylation site . By equilibrium dialysis we demonstrated that acetylation reduces the affinity of HMG-17 to nucleosome cores . In addition, we found that the binding of HMG-14 and HMG-17 to nucleosome cores inhibits the PCAF-mediated acetylation of histone H3 . Thus, the presence of HMG-14 and HMG-17 affects the ability of PCAF to acetylate chromatin, while the acetylation of HMG-17 reduces its binding affinity to chromatin . Conceivably, in HMG-17-containing chromatin, acetylation of HMG-17 precedes the acetylation of histones.

Mol Cell Biol, 1999 May, 19(5), 3435 - 42
Prohibitins regulate membrane protein degradation by the m-AAA protease in mitochondria; Steglich G et al.; Prohibitins comprise a protein family in eukaryotic cells with potential roles in senescence and tumor suppression . Phb1p and Phb2p, members of the prohibitin family in Saccharomyces cerevisiae, have been implicated in the regulation of the replicative life span of the cells and in the maintenance of mitochondrial morphology . The functional activities of these proteins, however, have not been elucidated . We demonstrate here that prohibitins regulate the turnover of membrane proteins by the m-AAA protease, a conserved ATP-dependent protease in the inner membrane of mitochondria . The m-AAA protease is composed of the homologous subunits Yta10p (Afg3p) and Yta12p (Rca1p) . Deletion of PHB1 or PHB2 impairs growth of Deltayta10 or Deltayta12 cells but does not affect cell growth in the presence of the m-AAA protease . A prohibitin complex with a native molecular mass of approximately 2 MDa containing Phb1p and Phb2p forms a supercomplex with the m-AAA protease . Proteolysis of nonassembled inner membrane proteins by the m-AAA protease is accelerated in mitochondria lacking Phb1p or Phb2p, indicating a negative regulatory effect of prohibitins on m-AAA protease activity . These results functionally link members of two conserved protein families in eukaryotes to the degradation of membrane proteins in mitochondria.

Mol Cell Biol, 1999 May, 19(5), 3267 - 77
The C terminus of Ku80 activates the DNA-dependent protein kinase catalytic subunit; Singleton BK et al.; Ku is a heterodimeric protein with double-stranded DNA end-binding activity that operates in the process of nonhomologous end joining . Ku is thought to target the DNA-dependent protein kinase (DNA-PK) complex to the DNA and, when DNA bound, can interact and activate the DNA-PK catalytic subunit (DNA-PKcs) . We have carried out a 3' deletion analysis of Ku80, the larger subunit of Ku, and shown that the C-terminal 178 amino acid residues are dispensable for DNA end-binding activity but are required for efficient interaction of Ku with DNA-PKcs . Cells expressing Ku80 proteins that lack the terminal 178 residues have low DNA-PK activity, are radiation sensitive, and can recombine the signal junctions but not the coding junctions during V(D)J recombination . These cells have therefore acquired the phenotype of mouse SCID cells despite expressing DNA-PKcs protein, suggesting that an interaction between DNA-PKcs and Ku, involving the C-terminal region of Ku80, is required for DNA double-strand break rejoining and coding but not signal joint formation . To gain further insight into important domains in Ku80, we report a point mutational change in Ku80 in the defective xrs-2 cell line . This residue is conserved among species and lies outside of the previously reported Ku70-Ku80 interaction domain . The mutational change nonetheless abrogates the Ku70-Ku80 interaction and DNA end-binding activity.

J Biol Chem, 1999 Apr 23, 274(17), 12036 - 42
Identification of the activating and conjugating enzymes of the NEDD8 conjugation pathway; Gong L et al.; NEDD8 is a ubiquitin-like molecule that can be covalently conjugated to a limited number of cellular proteins, such as Cdc53/cullin . We have previously reported that the C terminus of NEDD8 is efficiently processed to expose Gly-76, which is required for conjugation to target proteins . A combination of data base searches and polymerase chain reaction cloning was used to identify a cDNA encoding human UBA3, which is 38% identical to the yeast homologue, 22% identical to human UBA2, and 19% identical to the C-terminal region of human UBE1 . The human UBA3 gene is located on chromosome 3p13 and gave rise to a 2.2-kilobase pair transcript that was detected in all tissues . Human UBA3 could be precipitated with glutathione S-transferase (GST)-NEDD8, but not with GST-ubiquitin or GST-sentrin-1 . Moreover, human UBA3 could form a beta-mercaptoethanol-sensitive conjugate with NEDD8 in the presence of APP-BP1, a protein with sequence homology to the N-terminal half of ubiquitin-activating enzyme . We have also cloned human UBC12 and demonstrated that it could form a thiol ester linkage with NEDD8 in the presence of the activating enzyme complex . Identification of the activating and conjugating enzymes of the NEDD8 conjugation pathway should allow for a more detailed study of the role of NEDD8 modification in health and disease.

J Biol Chem, 1999 Apr 23, 274(17), 11672 - 8
The CREB constitutive activation domain interacts with TATA-binding protein-associated factor 110 (TAF110) through specific hydrophobic residues in one of the three subdomains required for both activation and TAF110 binding; Felinski EA et al.; The cAMP response element-binding protein (CREB) mediates both basal and PKA-inducible transcription through two separate and independently active domains, the constitutive activation domain (CAD) and the kinase-inducible domain, respectively . The CREB CAD interacts with the general transcription factor TFIID through one or more of the TATA-binding protein-associated factors (TAFs), one of which is TAF110 . The CAD is composed of three subdomains, rich in either serine, hydrophobic amino acids, or glutamine . In the present study, analysis of deletion mutants of the CAD showed that all three CAD subdomains were required for effective interaction with TAF110 in a yeast two-hybrid assay . Therefore, a library of random point mutations within the CAD was analyzed in a reverse two-hybrid screen to identify amino acids that are essential for interaction with the TAF . Interaction defects resulted solely from mutations of hydrophobic amino acid residues within the hydrophobic cluster to charged amino acid residues . Together, the deletion and mutation analyses suggest that the entire CAD provides an environment for a specific hydrophobic interaction with TAF110 that is crucial for interaction . Our results provide further evidence for a model of basal activation by CREB involving interaction with TAF110 that promotes recruitment or stabilization of TFIID binding to the promoter, which facilitates pre-initiation complex assembly.

Microbiology, 1999 Jan, 145 ( Pt 1), 75 - 87
Functional analysis of upstream regulating regions from the Yarrowia lipolytica XPR2 promoter; Madzak C et al.; The XPR2 gene from Yarrowia lipolytica encodes an inducible alkaline extracellular protease . Its complex regulation involves pH, carbon, nitrogen and peptones . Two previously identified upstream activating sequence (UAS) regions were analysed in a reporter system, outside the XPR2 context . Fragments from the UAS regions were inserted upstream of a minimal LEU2 promoter directing the expression of a reporter gene . The activity of the hybrid promoters was assessed following integration into the Y . lipolytica genome . This study confirmed the presence of two UASs composed of several interacting elements . Within the distal UAS (UAS1), a TUF/RAP1 binding site exhibited a UAS activity, which was enhanced by the presence of two adjacent repeats, overlapping sites similar to the CAR1 upstream repressing sequence from Saccharomyces cerevisiae . Within the proximal UAS (UAS2), the UAS activity required the interaction of both an ABF1-like binding site and a decameric repeat, containing Aspergillus nidulans PacC site consensus sequences . This decameric repeat was able to mediate repression due to carbon and/or nitrogen sources as well as pH-dependent activation . A study in the context of trans-regulatory mutations in the Y . lipolytica RIM101 gene showed that the PacC-like sites, potential binding sites for YlRim101p, were implicated in the derepression of UAS2-driven expression at neutral-alkaline pH . The in vivo response of the PacC-like decamers to external pH was dependent on the status of the pH-regulated activator YlRim101p, which is homologous to the A . nidulans PacC regulator . The carbon/nitrogen regulation imposed on the decamers was shown to be independent of YlRim101p and to override its effects.

Br J Cancer, 1999 Apr, 79(11-12), 1657 - 64
Radiation-induced G1 arrest is not defective in fibroblasts from Li-Fraumeni families without TP53 mutations; Boyle JM et al.; Radiation-induced G1 arrest was studied in four classes of early passage skin fibroblasts comprising 12 normals, 12 heterozygous (mut/wt) TP53 mutation-carriers, two homozygous (mut/-) TP53 mutation-carriers and 16 strains from nine Li-Fraumeni syndrome or Li-Fraumeni-like families in which no TP53 mutation has been found, despite sequencing of all exons, exon-intron boundaries, 3' and 5' untranslated regions and promoter regions . In an assay of p53 allelic expression in yeast, cDNAs from these non-mutation strains behaved as wild-type p53 . Using two different assays, we found G1 arrest was reduced in heterozygous strains with mis-sense mutations and one truncation mutation, when compared to the range established for the normal cells . Heterozygous strains with mutations at splice sites behaved like normal cells, whilst homozygous (mut/-) strains showed either extremely reduced, or no, arrest . Strains from all nine non-mutation families gave responses within the normal range . Exceptions to the previously reported inverse correlation between G1 arrest and clonogenic radiation resistance were observed, indicating that these phenotypes are not strictly interdependent.

Plant J, 1999 Feb, 17(4), 373 - 83
The Arabidopsis HAL2-like gene family includes a novel sodium-sensitive phosphatase; Gil-Mascarell R et al.; The yeast HAL2 gene encodes a lithium- and sodium-sensitive phosphatase that hydrolyses 3'-phosphoadenosine-5'-phosphate (PAP) . Salt toxicity in yeast results from Hal2 inhibition and accumulation of PAP, which inhibits sulphate assimilation and RNA processing . We have investigated whether the model plant Arabidopsis thaliana contains sodium-sensitive PAP phosphatases . The Arabidopsis HAL2-like gene family is composed of three members: AtAHL and AtSAL2, characterized in the present work, and the previously identified AtSAL1 . The AtAHL and AtSAL2 cDNAs complement the auxotrophy for methionine of the yeast hal2 mutant and the recombinant proteins catalyse the conversion of PAP to AMP in a Mg(2+)-dependent reaction sensitive to inhibition by Ca2+ and Li+ . The PAP phosphatase activity of AtAHL is sensitive to physiological concentrations of Na+, whereas the activities of AtSAL1 and AtSAL2 are not . Another important difference is that AtAHL is very specific for PAP while AtSAL1 and AtSAL2 also act as inositol polyphosphate 1-phosphatases . AtAHL constitutes a novel type of sodium-sensitive PAP phosphatase which could act co-ordinately with plant sulphotransferases and serve as target of salt toxicity in plants.

EMBO J, 1999 Apr 15, 18(8), 2254 - 64
Continuous and widespread roles for the Swi-Snf complex in transcription; Biggar SR et al.; Chromatin presents a significant obstacle to transcription, but two means of overcoming its repressive effects, histone acetylation and the activities of the Swi-Snf complex, have been proposed . Histone acetylation and Swi-Snf activity have been shown to be crucial for transcriptional induction and to facilitate binding of transcription factors to DNA . By regulating the activity of the Swi-Snf complex in vivo, we found that active transcription requires continuous Swi-Snf function, demonstrating a role for this complex beyond the induction of transcription . Despite the presumably generalized packaging of genes into chromatin, previous studies have indicated that the transcriptional requirements for the histone acetyltransferase, Gcn5, and the Swi-Snf complex are limited to a handful of genes . However, inactivating Swi-Snf function in cells also lacking GCN5 revealed defects in transcription of several genes previously thought to be SWI-SNF- and GCN5-independent . These findings suggest that chromatin remodeling plays a widespread role in gene expression and that these two chromatin remodeling activities perform independent and overlapping functions during transcriptional activation.

Nat Struct Biol, 1999 Apr, 6(4), 322 - 6
Quaternary changes in topoisomerase II may direct orthogonal movement of two DNA strands; Fass D et al.; Type II DNA topoisomerases mediate the passage of one DNA duplex through a transient break in another, an event essential for chromosome segregation and cell viability . The active sites of the type II topoisomerase dimer associate covalently with the DNA break-points and must separate by at least the width of the second DNA duplex to accommodate transport . A new structure of the Saccharomyces cerevisiae topoisomerase II DNA-binding and cleavage core suggests that in addition to conformational changes in the DNA-opening platform, a dramatic reorganization of accessory domains may occur during catalysis . These conformational differences have implications for both the DNA-breaking and duplex-transport events in the topo II reaction mechanism, suggest a mechanism by which two distinct drug-resistance loci interact, and illustrate the scope of structural changes in the cycling of molecular machines.

J Immunol, 1999 Apr 15, 162(8), 5019 - 24
Mutations that cause the Wiskott-Aldrich syndrome impair the interaction of Wiskott-Aldrich syndrome protein (WASP) with WASP interacting protein; Stewart DM et al.; Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disorder characterized by thrombocytopenia, eczema, immune deficiency, and a proclivity toward lymphoid malignancy . Lymphocytes of affected individuals show defects of activation, motility, and cytoskeletal structure . The disease gene encodes a 502-amino acid protein named the WAS protein (WASP) . Studies have identified a number of important interactions that place WASP in a role of integrating signaling pathways with cytoskeletal function . We performed a two-hybrid screen to identify proteins interacting with WASP and cloned a proline-rich protein as a specific WASP interactor . Our clone of this protein, termed WASP interacting protein (WIP) by others, shows a difference in seven amino acid residues, compared with the previously published sequence revealing an additional profilin binding motif . Deletion mutant analysis reveals that WASP residues 101-151 are necessary for WASP-WIP interaction . Point mutant analyses in the two-hybrid system and in vitro show impairment of WASP-WIP interaction with three WASP missense mutants known to cause WAS . We conclude that impaired WASP-WIP interaction may contribute to WAS.

J Immunol, 1999 Apr 15, 162(8), 4697 - 704
Binding motifs of copolymer 1 to multiple sclerosis- and rheumatoid arthritis-associated HLA-DR molecules; Fridkis-Hareli M et al.; Copolymer 1 (Cop 1, poly (Y, E, A, K)) is a random synthetic amino acid copolymer effective in the treatment of relapsing forms of multiple sclerosis (MS) . Cop 1 binds promiscuously, with high affinity and in a peptide-specific manner to purified MS-associated HLA-DR2 (DRB1*1501) and rheumatoid arthritis-associated HLA-DR1 (DRB1*0101) or HLA-DR4 (DRB1*0401) molecules . In the present work at least 95% of added Cop 1 could be bound to recombinant "empty" HLA-DR1 and -DR4, and 80% could be bound to HLA-DR2 proteins . Amino acid composition, HPLC profiles, and sequencing patterns of Cop 1 eluted by acid extraction from HLA-DR molecules were similar to those of the unseparated Cop 1 . Protruding N-terminal ends of Cop 1 bound to HLA-DR1, -DR2, or -DR4 molecules were then treated with aminopeptidase I, followed by elution, HPLC, and pool sequencing . In contrast to untreated or unbound Cop 1, this material exhibited distinct motifs at some positions with increases in levels of E at the first and second cycles, of K at the second and third cycles, and of Y (presumably at P1 of the bound peptide) at the third to fifth cycles, regardless of the HLA-DR molecule employed . No preference was seen at the following cycles that were mainly A . These first pooled HLA-DR binding epitopes provide clues to the components of Cop 1 that are biologically active in suppressing MS and possibly rheumatoid arthritis.

Trends Cell Biol, 1999 Mar, 9(3), 107 - 12
Gettin' down with ubiquitin: turning off cell-surface receptors, transporters and channels; Hicke L; G-protein-coupled receptors and transporters in Saccharomyces cerevisiae are modified with ubiquitin in response to ligand biding . In most cases, the proteasome does not recognize these ubiquitinated proteins . Instead, ubiquitination serves to trigger internalization and degradation of plasma membrane proteins in the lysosome-like vacuole . A number of mammalian receptors and at least one ion channel undergo ubiquitination at the plasma membrane, and this modification is required for their downregulation . Some of these cell-surface proteins appear to be degraded by both the proteasome and lysosomal proteases . Recent evidence indicates that other proteins required for receptor internalization might also be regulated by ubiquitination, suggesting that ubiquitin plays diverse roles in regulating plasma membrane protein activity.

Mol Cell, 1999 Mar, 3(3), 361 - 70
Identity between TRAP and SMCC complexes indicates novel pathways for the function of nuclear receptors and diverse mammalian activators; Ito M et al.; The human thyroid hormone receptor-associated protein (TRAP) complex, an earlier described coactivator for nuclear receptors, and an SRB- and MED-containing cofactor complex (SMCC) that mediates activation by Gal4-p53 are shown to be virtually the same with respect to specific polypeptide subunits, coactivator functions, and mechanisms of action (activator interactions) . In parallel with ligand-dependent interactions of nuclear receptors with the TRAP220 subunit, p53 and VP16 activation domains interact directly with a newly cloned TRAP80 subunit . These results indicate novel pathways for the function of nuclear receptors and other activators (p53 and VP16) through a common coactivator complex that is likely to target RNA polymerase II . Identification of the TRAP230 subunit as a previously predicted gene product also suggests a coactivator-related transcription defect in certain disease states.

Mol Cell, 1999 Mar, 3(3), 287 - 96
The proapoptotic activity of the Bcl-2 family member Bim is regulated by interaction with the dynein motor complex; Puthalakath H et al.; Bcl-2 family members that have only a single Bcl-2 homology domain, BH3, are potent inducers of apoptosis, and some appear to play a critical role in developmentally programmed cell death . We examined the regulation of the proapoptotic activity of the BH3-only protein Bim . In healthy cells, most Bim molecules were bound to LC8 cytoplasmic dynein light chain and thereby sequestered to the microtubule-associated dynein motor complex . Certain apoptotic stimuli disrupted the interaction between LC8 and the dynein motor complex . This freed Bim to translocate together with LC8 to Bcl-2 and to neutralize its antiapoptotic activity . This process did not require caspase activity and therefore constitutes an initiating event in apoptosis signaling.

Mol Cell, 1999 Mar, 3(3), 275 - 85
Brefeldin A acts to stabilize an abortive ARF-GDP-Sec7 domain protein complex: involvement of specific residues of the Sec7 domain; Peyroche A et al.; We demonstrate that the major in vivo targets of brefeldin A (BFA) in the secretory pathway of budding yeast are the three members of the Sec7 domain family of ARF exchange factors: Gea1p and Gea2p (functionally interchangeable) and Sec7p . Specific residues within the Sec7 domain are important for BFA inhibition of ARF exchange activity, since mutations in these residues of Gea1p (sensitive to BFA) and of ARNO (resistant to BFA) reverse the sensitivity of each to BFA in vivo and in vitro . We show that the target of BFA inhibition of ARF exchange activity is an ARF-GDP-Sec7 domain protein complex, and that BFA acts to stabilize this complex to a greater extent for a BFA-sensitive Sec7 domain than for a resistant one.

Biochem Biophys Res Commun, 1999 Apr 13, 257(2), 333 - 9
Cloning, expression, and chromosomal mapping of a human ATPase II gene, member of the third subfamily of P-type ATPases and orthologous to the presumed bovine and murine aminophospholipid translocase; Mouro I et al.; Recently, a P-type ATPase was cloned from bovine chromaffin granules (b-ATPase II) and a mouse teratocarcinoma cell line (m-ATPase II) and was shown to be homologous to the Saccharomyces cerevisiae DRS2 gene, the inactivation of which resulted in defective transport of phosphatidylserine . Here, we report the cloning from a human skeletal muscle cDNA library of a human ATPase II (h-ATPase II), orthologous to the presumed bovine and mouse aminophospholipid translocase (95.3 and 95.9% amino acid identity, respectively) . Compared with the bovine and mouse counterparts, the cloned h-ATPase II polypeptide exhibits a similar membrane topology, but contains 15 additional amino acids (1163 vs 1148) located in the second intracytoplasmic loop, near the DKTGTLT-phosphorylation site . However, RT-PCR analysis performed with RNA from different human tissues and cell lines revealed that the coding sequence for these 15 residues is sometimes present and sometimes absent, most likely as a result of a tissue-specific alternative splicing event . The h-ATPase II gene, which was mapped to chromosome 4p14-p12, is expressed as a 9.5-kb RNA species in a large variety of tissues, but was not detected in liver, testis, and placenta, nor in the erythroleukemic cell line K562 .

Mol Biol Cell, 1999 Apr, 10(4), 1191 - 203
The Drosophila melanogaster homologue of the Xeroderma pigmentosum D gene product is located in euchromatic regions and has a dynamic response to UV light-induced lesions in polytene chromosomes; Reynaud E et al.; The XPD/ERCC2/Rad3 gene is required for excision repair of UV-damaged DNA and is an important component of nucleotide excision repair . Mutations in the XPD gene generate the cancer-prone syndrome, xeroderma pigmentosum, Cockayne's syndrome, and trichothiodystrophy . XPD has a 5'- to 3'-helicase activity and is a component of the TFIIH transcription factor, which is essential for RNA polymerase II elongation . We present here the characterization of the Drosophila melanogaster XPD gene (DmXPD) . DmXPD encodes a product that is highly related to its human homologue . The DmXPD protein is ubiquitous during development . In embryos at the syncytial blastoderm stage, DmXPD is cytoplasmic . At the onset of transcription in somatic cells and during gastrulation in germ cells, DmXPD moves to the nuclei . Distribution analysis in polytene chromosomes shows that DmXPD is highly concentrated in the interbands, especially in the highly transcribed regions known as puffs . UV-light irradiation of third-instar larvae induces an increase in the signal intensity and in the number of sites where the DmXPD protein is located in polytene chromosomes, indicating that the DmXPD protein is recruited intensively in the chromosomes as a response to DNA damage . This is the first time that the response to DNA damage by UV-light irradiation can be visualized directly on the chromosomes using one of the TFIIH components.

Mol Biol Cell, 1999 Apr, 10(4), 1043 - 59
Two endoplasmic reticulum (ER) membrane proteins that facilitate ER-to-Golgi transport of glycosylphosphatidylinositol-anchored proteins; Barz WP et al.; Many eukaryotic cell surface proteins are anchored in the lipid bilayer through glycosylphosphatidylinositol (GPI) . GPI anchors are covalently attached in the endoplasmic reticulum (ER) . The modified proteins are then transported through the secretory pathway to the cell surface . We have identified two genes in Saccharomyces cerevisiae, LAG1 and a novel gene termed DGT1 (for "delayed GPI-anchored protein transport"), encoding structurally related proteins with multiple membrane-spanning domains . Both proteins are localized to the ER, as demonstrated by immunofluorescence microscopy . Deletion of either gene caused no detectable phenotype, whereas lag1Delta dgt1Delta cells displayed growth defects and a significant delay in ER-to-Golgi transport of GPI-anchored proteins, suggesting that LAG1 and DGT1 encode functionally redundant or overlapping proteins . The rate of GPI anchor attachment was not affected, nor was the transport rate of several non-GPI-anchored proteins . Consistent with a role of Lag1p and Dgt1p in GPI-anchored protein transport, lag1Delta dgt1Delta cells deposit abnormal, multilayered cell walls . Both proteins have significant sequence similarity to TRAM, a mammalian membrane protein thought to be involved in protein translocation across the ER membrane . In vivo translocation studies, however, did not detect any defects in protein translocation in lag1Delta dgt1Delta cells, suggesting that neither yeast gene plays a role in this process . Instead, we propose that Lag1p and Dgt1p facilitate efficient ER-to-Golgi transport of GPI-anchored proteins.

Exp Gerontol, 1999 Jan, 34(1), 1 - 6
Longevity, genes, and aging: a view provided by a genetic model system; Jazwinski SM; The genetic analysis of aging in the yeast Saccharomyces cerevisiae has revealed the importance of metabolic capacity, resistance to stress, integrity of gene regulation, and genetic stability for longevity . A balance between these life maintenance processes is sustained by the RAS2 gene, which channels cellular resources among them . This gene cooperates with mitochondria and PHB1 in metabolic adjustments important for longevity . It also modulates stress responses . Transcriptional silencing of heterochromatic regions of the genome is lost during aging, suggesting that gene dysregulation accompanies the aging process . There is evidence that this age change plays a causal role . Aging possesses features of a nonlinear process, and it is likely that application of nonlinear system methodology to aging will be productive.

Hum Mol Genet, 1999 May, 8(5), 743 - 9
Mutation of a putative mitochondrial iron transporter gene (ABC7) in X-linked sideroblastic anemia and ataxia (XLSA/A); Allikmets R et al.; X-linked sideroblastic anemia and ataxia (XLSA/A) is a recessive disorder characterized by an infantile to early childhood onset of non-progressive cerebellar ataxia and mild anemia with hypochromia and microcytosis . A gene encoding an ATP-binding cassette (ABC) transporter was mapped to Xq13, a region previously shown by linkage analysis to harbor the XLSA/A gene . This gene, ABC7, is an ortholog of the yeast ATM1 gene whose product localizes to the mitochondrial inner membrane and is involved in iron homeostasis . The full-length ABC7 cDNA was cloned and the entire coding region screened for mutations in a kindred in which five male members manifested XLSA/A . An I400M variant was identified in a predicted transmembrane segment of the ABC7 gene in patients with XLSA/A . The mutation was shown to segregate with the disease in the family and was not detected in at least 600 chromosomes of general population controls . Introduction of the corresponding mutation into the Saccharomyces cerevisiae ATM1 gene resulted in a partial loss of function of the yeast Atm1 protein . In addition, the human wild-type ABC7 protein was able to complement ATM1 deletion in yeast . These data indicate that ABC7 is the causal gene of XLSA/A and that XLSA/A is a mitochondrial disease caused by a mutation in the nuclear genome.

J Biol Chem, 1999 Apr 16, 274(16), 11245 - 52
The role of antiapoptotic Bcl-2 family members in endothelial apoptosis elucidated with antisense oligonucleotides; Ackermann EJ et al.; In this study, we utilized potent antisense oligonucleotides to examine the role of two Bcl-2 family members found in human umbilical vein endothelial cells (HUVEC) . The first, A1, is thought to be a TNF-alpha-inducible cytoprotective gene, and the second, Bcl-XL, is constitutively expressed . Inhibition of the constitutive levels of Bcl-XL caused 10-25% of the cell population to undergo apoptosis and increased the susceptibility of cells to treatment with low concentrations of staurosporin or ceramide . The caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-CH2 prevented DNA fragmentation and DeltaYm loss caused by Bcl-XL inhibition or Bcl-XL inhibition combined with staurosporin . However, disruption of DeltaYm caused by Bcl-XL inhibition combined with ceramide treatment was not inhibited by benzyloxycarbonyl-Val-Ala-Asp(OMe)-CH2, although DNA fragmentation was completely prevented . Taken together, these results demonstrate a direct protective role for Bcl-XL under normal resting conditions and under low level apoptotic challenges to HUVEC . Furthermore, Bcl-XL protects cells from caspase-dependent and -independent mechanisms of DeltaYm disruption . In contrast to Bcl-XL, A1 inhibition did not show a marked effect on the susceptibility of HUVEC to undergo apoptosis in response to TNF-alpha, ceramide, or staurosporin . These results demonstrate that although A1 may be a cytoprotective gene induced by TNF-alpha, it is not primarily responsible for HUVEC resistance to this cytokine.

J Struct Biol, 1999 Mar, 125(1), 1 - 10
Side-chains configurations in coiled coils revealed by the 5.15-A meridional reflection on hard alpha-keratin X-ray diffraction patterns; Busson B et al.; The origin of the 5.15-A meridional reflection on hard alpha-keratin X-ray diffraction patterns is discussed in terms of side-chains conformations . We show it to reveal specific configurations of the side chains which are common to all two-stranded alpha-helical coiled coils . Combining literature data on crystallised coiled coil pieces and molecular dynamics results with our X-ray diffraction pattern simulations, we propose rules for the attribution of chi1 torsion angles for coiled coils involved in fibres whose structure cannot be resolved at atomic resolution: in a (a b c d e f g) heptad repeat, a and d residues, respectively, adopt mean t and g+ configurations, whereas statistical rules are given for the other residues .

J Cell Sci, 1999 May, 112 ( Pt 9), 1325 - 36
New actin mutants allow further characterization of the nucleotide binding cleft and drug binding sites; Belmont LD et al.; We have generated 9 site-specific mutations in Saccharomyces cerevisiae actin . These mutants display a variety of phenotypes when expressed in vivo, including slow actin filament turnover, slow fluid-phase endocytosis, and defects in actin organization . Actin mutation D157E confers resistance to the actin-sequestering drug, latrunculin A . Latrunculin A inhibits nucleotide exchange on wild-type yeast actin but not on D157E actin, suggesting that this residue is part of the latrunculin A binding site . We have refined our earlier map of the phalloidin binding site on actin, demonstrating a requirement for residue G158 in addition to D179 and R177 . The nine new actin mutants as well as a large collection of existing actin mutants were also used to identify the putative binding site of another actin binding drug, tolytoxin, on actin . The actin alleles that result in decreased sensitivity to this drug cluster at a site near the nucleotide-binding pocket . Actin purified from one of these mutants has a reduced affinity for tolytoxin . In addition, tolytoxin causes a 2.4-fold increase in the t1/2 of ATP exchange, further suggesting that this drug binds near the nucleotide-binding pocket of actin . We note that the binding sites for latrunculin A, phalloidin, and tolytoxin all map close to the actin nucleotide binding pocket.

Biochemistry, 1999 Apr 6, 38(14), 4572 - 85
Plasmin desensitization of the PAR1 thrombin receptor: kinetics, sites of truncation, and implications for thrombolytic therapy; Kuliopulos A et al.; It has been hypothesized that protease-activated receptors may be activated and attenuated by more than one protease . Here, we explore a desensitization mechanism of the PAR1 thrombin receptor by anticoagulant proteases and provide an explanation to the enigma of why plasmin/tissue plasminogen activator (t-PA) can both activate and deactivate platelets prior to thrombin treatment . By using a soluble N-terminal exodomain (TR78) as a model for the full-length receptor, we were able to unambiguously compare cleavage rates and specificities among the serum proteases . Thrombin cleaves TR78 at the R41-S42 peptide bond with a kcat of 120 s-1 and a KM of 16 microM to produce TR62 (residues 42-103) . We found that, of the anticoagulant proteases, only plasmin can rapidly truncate the soluble exodomain at the R70/K76/K82 sites located on a linker region that tethers the ligand to the body of the receptor . Plasmin cleavage of the TR78 exodomain is nearly equivalent to that of thrombin cleavage at R41 with similar rates (kcat = 30 s-1) and affinity (KM = 18 microM) . Specificity was demonstrated since there is no observed cleavage at the five other potential plasmin-cleavage sites . Plasmin also cleaves the TR78 exodomain at the R41 thrombin-cleavage site generating transiently activated exodomain . We directly demonstrated that plasmin cleaves these same sites in full-length membrane-embedded receptor expressed in yeast and COS7 fibroblasts . The rate of plasmin truncation is similar between the extensively glycosylated COS7-expressed receptor and the nonglycosylated yeast-produced receptor . Mutation of the R70/K76/K82 sites to A70/A76/A82 eliminates plasmin truncation and desensitization of thrombin-dependent Ca2+ signaling and converts PAR1 into a plasmin-activated receptor with full agonist activity for plasmin . Plasmin does not desensitize the Ca2+ response of platelets or COS7 cells to SFLLRN consistent with intermolecular ligand-binding sites being located to the C-terminal side of K82 . Truncation of the wild-type receptor at the C-terminal plasmin-cleavage sites removes the N-terminal tethered ligand or preligand, thereby providing an effective pathway for PAR1 desensitization in vivo.

Biochemistry, 1999 Apr 6, 38(14), 4493 - 503
Hydrogen exchange behavior of {U-15N}-labeled oxidized and reduced iso-1-cytochrome c; Baxter SM et al.; Heteronuclear NMR spectroscopy was used to measure the hydrogen-deuterium exchange rates of backbone amide hydrogens in both oxidized and reduced {U-15N}iso-1-cytochrome c from the yeast Saccharomyces cerevisiae . The exchange data confirm previously reported data {Marmorino et al . (1993) Protein Sci . 2, 1966-1974}, resolve several inconsistencies, and provide more thorough coverage of exchange rates throughout the cytochrome c protein in both oxidation states . Combining the data previously collected on unlabeled C102T with the current data collected on {U-15N}C102T, exchange rates for 53 protons in the oxidized state and 52 protons in the reduced state can now be reported . Most significantly, hydrogen exchange measurements on {U-15N}iso-1-cytochrome c allowed the observation of exchange behavior of the secondary structures, such as large loops, that are not extensively hydrogen-bonded . For the helices, the most slowly exchanging protons are found in the middle of the helix, with more rapidly exchanging protons at the helix ends . The observation for the Omega-loops in cytochrome c is just the opposite . In the loops, the ends contain the most slowly exchanging protons and the loop middles allow more rapid exchange . This is found to be true in cytochrome c loops, even though the loop ends are not attached to any regular secondary structures . Some of the exchange data are strikingly inconsistent with data collected on the C102S variant at a different pH, which suggests pH-dependent dynamic differences in the protein structure . This new hydrogen exchange data for loop residues could have implications for the substructure model of eukaryotic cytochrome c folding . Isotopic labeling of variant forms of cytochrome c can now be used to answer many questions about the structure and folding of this model protein.

Biochemistry, 1999 Apr 6, 38(14), 4480 - 92
Assignment of 15N chemical shifts and 15N relaxation measurements for oxidized and reduced iso-1-cytochrome c; Fetrow JS et al.; A protocol for complete isotopic labeling of iso-1-cytochrome c from the eukaryote Saccharomyces cerevisiae is reported . Assignments are reported for the vast majority of the 15N amide resonances in both oxidized and reduced states . 15N heteronuclear relaxation experiments were collected to study the picosecond-nanosecond backbone dynamics of this protein . Relaxation rates were computed and fit to spectral density functions by a model-free analysis . Backbone amides in the overlapping loop B/C region are the most flexible on the picosecond-nanosecond time scale in both forms of the protein . The results show that, on average, the protein backbone is slightly more dynamic in the oxidized than the reduced state, though not significantly so . Exchange terms, which suggest significant motion on a time scale at least an order of magnitude slower than the overall correlation time of 5.2 ns, were required for only two residues in the reduced state and 27 residues in the oxidized state . When analyzed on a per-residue basis, the lower order parameters found in the oxidized state were scattered throughout the protein, with a few continuous segments found in loop C and the C-terminal helix, suggesting greater flexibility of these regions in the oxidized state . The results provide dynamic interpretations for previously presented structural and functional data, including redox-dependent changes that occur in the protein . The way is now paved for extensive dynamic analysis of variant cytochromes c.

Biochemistry, 1999 Apr 6, 38(14), 4374 - 81
Role of the 20-hydroxyl group in camptothecin binding by the topoisomerase I-DNA binary complex; Wang X et al.; Recent findings concerning the structure of the covalent binary complex formed by DNA topisomerase I and its DNA substrate, as well as the nature of interactions with inhibitors that bind reversibly to this binary complex, have led to two proposed models for the binding of the prototype inhibitor camptothecin to the DNA-topisomerase I binary complex . While these models differ in many regards, they both suggest the involvement of the 20-OH group of camptothecin in a donor hydrogen bond with an enzyme side chain functional group . Presently, five analogues of camptothecin that differ only at C-20 have been evaluated for their ability to bind to the topoisomerase I-DNA binary complex and thereby inhibit enzyme function . Both 20-chloro- and 20-bromocamptothecin bound as well to the enzyme-DNA binary complex as 20-aminoCPT despite the absence of a substituent at C-20 capable of contributing a donor hydrogen bond.

Biochemistry, 1999 Mar 30, 38(13), 4008 - 17
Stability of the dimerization domain effects the cooperative DNA binding of short peptides; Aizawa Y et al.; The basic region peptide derived from the basic leucine zipper protein GCN4 bound specifically to the native GCN4 binding sequences in a dimeric form when the beta-cyclodextrin/adamantane dimerization domain was introduced at the C-terminus of the GCN4 basic region peptide . We describe here how the structure and stability of the dimerization domain affect the cooperative formation of the peptide dimer-DNA complex . The basic region peptides with five different guest molecules were synthesized, and their equilibrium dissociation constants with a peptide possessing beta-cyclodextrin were determined . These values, ranging from 1.3 to 15 microM, were used to estimate the stability of the complexes between the dimers with various guest/cyclodextrin dimerization domains and GCN4 target sequences . An efficient cooperative formation of the dimer complexes at the GCN4 binding sequence was observed when the adamantyl group was replaced with the norbornyl or noradamantyl group, but not with the cyclohexyl group that formed a beta-cyclodextrin complex with a stability that was 1 order of magnitude lower than that of the adamantyl group . Thus, cooperative formation of the stable dimer-DNA complex appeared to be effected by the stability of the dimerization domain . For the peptides that cooperatively formed dimer-DNA complexes, there was no linear correlation between the stability of the inclusion complex and that of the dimer-DNA complex . With the beta-cyclodextrin/adamantane dimerization domain, the basic region peptide dimer preferred to bind to a palindromic 5'-ATGACGTCAT-3' sequence over the sequence lacking the central G.C base pair and that with an additional G.C base pair in the middle . Changing the adamantyl group into a norbornyl group did not alter the preferential binding of the peptide dimers to the palindromic sequence, but slightly affected the selectivity of the dimer for other nonpalindromic sequences . The helical contents of the peptides in the DNA-bound dimer with the adamantyl group were decreased by reducing the stability of the dimer-DNA complex, which was possibly caused by deformation of the helical structure proximal to the dimerization domain.

Bioessays, 1999 Feb, 21(2), 121 - 30
Comparative genomics: the key to understanding the Human Genome Project; Clark MS; The sequencing of the human genome is well underway . Technology has advanced, such that the total genomic sequence is possible, along with an extensive catalogue of genes via comprehensive cDNA libraries . With the recent completion of the Saccharomyces cerevisiae sequencing project and the imminent completion of that of Caenorhabditis elegans, the most frequently asked question is how much can sequence data alone tell us? The answer is that that a DNA sequence taken in isolation from a single organism reveals very little . The vast majority of DNA in most organisms is noncoding . Protein coding sequences or genes cannot function as isolated units without interaction with noncoding DNA and neighboring genes . This genomic environment is specific to each organism . In order to understand this we need to look at similar genes in different organisms, to determine how function and position has changed over the course of evolution . By understanding evolutionary processes we can gain a greater insight into what makes a gene and the wider processes of genetics and inheritance . Comparative genomics (with model organisms), once the poor relation of the human genome project, is starting to provide the key to unlock the DNA code.

Biotechnol Bioeng, 1998 Apr 20-May 5, 58(2-3), 139 - 48
On-line metabolic pathway analysis based on metabolic signal flow diagram; Shi H et al.; In this work, an integrated modeling approach based on a metabolic signal flow diagram and cellular energetics was used to model the metabolic pathway analysis for the cultivation of yeast on glucose . This approach enables us to make a clear analysis of the flow direction of the carbon fluxes in the metabolic pathways as well as of the degree of activation of a particular pathway for the synthesis of biomaterials for cell growth . The analyses demonstrate that the main metabolic pathways of Saccharomyces cerevisiae change significantly during batch culture . Carbon flow direction is toward glycolysis to satisfy the increase of requirement for precursors and energy . The enzymatic activation of TCA cycle seems to always be at normal level, which may result in the overflow of ethanol due to its limited capacity . The advantage of this approach is that it adopts both virtues of the metabolic signal flow diagram and the simple network analysis method, focusing on the investigation of the flow directions of carbon fluxes and the degree of activation of a particular pathway or reaction loop . All of the variables used in the model equations were determined on-line; the information obtained from the calculated metabolic coefficients may result in a better understanding of cell physiology and help to evaluate the state of the cell culture process .

Mol Genet Metab, 1999 Apr, 66(4), 309 - 13
A murine model for juvenile NCL: gene targeting of mouse Cln3; Greene ND et al.; JNCL is a neurodegenerative disease of childhood caused by mutations in the CLN3 gene . A mouse model for JNCL was created by disrupting exons 1-6 of Cln3, resulting in a null allele . Cln3 null mice appear clinically normal at 5 months of age; however, like JNCL patients, they exhibit intracellular accumulation of autofluorescent material . A second approach will generate mice in which exons 7 and 8 of Cln3 are deleted, mimicking the common mutation in JNCL patients .

Mol Genet Metab, 1999 Apr, 66(4), 294 - 308
CLN3 defines a novel antiapoptotic pathway operative in neurodegeneration and mediated by ceramide; Puranam KL et al.; Juvenile neuronal ceroid lipofuscinosis or Batten disease (JNCL) is a neurodegenerative disorder characterized by blindness, seizures, cognitive decline and early death . Brain atrophy and retinitis pigmentosa ensue because of neuronal and photoreceptor apoptosis . The CLN3 gene defective in JNCL encodes a novel 438 amino acid protein . Most affected genes harbor a deletion resulting in a truncated protein . CLN3 overexpression in NT2 cells enhances growth, reverses growth inhibition induced by serum starvation and protects from apoptosis induced by vincristine, staurosporine, and etoposide but not from death caused by ceramide . CLN3 modulates endogenous and vincristine-activated ceramide, and therefore suppresses apoptosis by impacting generation of ceramide .

Mol Genet Metab, 1999 Apr, 66(4), 283 - 9
Tissue expression and subcellular localization of CLN3, the Batten disease protein; Margraf LR et al.; Juvenile neuronal ceroid lipofuscinosis (Batten disease) is a progressive neurologic disorder which results from mutations in the CLN3 gene, which normally produces a 48-kDa polypeptide of unknown function . To help characterize the CLN3 protein, we have studied its tissue distribution and subcellular localization in human tissues using three epitope-specific polyclonal antibodies to human CLN3 by immunoblot, immunocytochemical, and immunoelectron microscopic analysis . The most abundant CLN3 protein expression was in the gray matter of the brain, where it was localized to astrocytes, capillary endothelium, and neurons . CLN3 was also evident in peripheral nerve, in pancreatic islet cells, and within the seminiferous tubules in the testis . Staining was generally diffuse within the cytoplasm with some nuclear reactivity . Subcellular localization identified the CLN3 protein within the nucleus and along cell membranes . These results were contrasted with the cellular distribution of palmitoyl-protein thioesterase (PPT), the enzyme whose deficiency is responsible for infantile neuronal ceroid lipofuscinosis (CLN1) . PPT was most abundant in brain and visceral macrophages where it displayed a coarse granular staining pattern typical of lysosomal distribution . Immunoelectron microscopy confirmed that PPT immunoreactivity was limited to lysosomes .

Mol Genet Metab, 1999 Apr, 66(4), 277 - 82
Expression studies of CLN3 protein (battenin) in fusion with the green fluorescent protein in mammalian cells in vitro; Golabek AA et al.; The gene for Batten disease, the CLN3 gene, encodes a novel, highly hydrophobic, multitransmembrane protein, predicted to consist of 438 amino acid residues . We have expressed a full-length CLN3 protein in fusion with green fluorescent protein in various cell lines to provide its initial biochemical characterization and subcellular localization . By using Western blotting, Percoll density gradient fractionation, and Triton X-114 extraction, we demonstrate that the product of the CLN3 gene, which we call battenin, in mammalian expression system studied is a highly glycosylated protein of lysosomal membrane . In addition our data suggest that CLN3 protein is processed proteolytically in acidic compartments of the cell . Thus, battenin represents the novel constituent of a growing family of lysosomal membrane proteins .

Mol Genet Metab, 1999 Apr, 66(4), 272 - 6
Posttranslational modification of CLN3 protein and its possible functional implication; Michalewski MP et al.; The CLN3 gene associated with Batten disease and encoding a novel protein of a predicted 438 amino acids was cloned in 1995 by the International Batten Disease Consortium . The function of CLN3 protein remains unknown . Computer-based analysis predicted that CLN3 may contain several posttranslational modifications . Thus, to study the posttranslational modification of CLN3 protein, we have expressed a full-length CLN3 protein as a C-terminal fusion with green fluorescent protein of the jellyfish Aequerea victoria in a Chinese hamster ovary cell line . Previously, we have shown that CLN3 is a glycosylated protein from lysosomal compartment, and now, by using in vivo labeling with 32P, detection with anti-phosphoamino acid antibodies, and phosphoamino acid analysis, we demonstrate that CLN3 is a phosphorylated protein . We demonstrate that CLN3 protein does not undergo mannose 6-phosphate modification and that it is a membrane protein . Furthermore, we show that the level of CLN3 protein phosphorylation may be modulated by several protein kinases and phosphatases activators or inhibitors .

Mol Genet Metab, 1999 Apr, 66(4), 265 - 71
Analysis of intracellular distribution and trafficking of the CLN3 protein in fusion with the green fluorescent protein in vitro; Kida E et al.; CLN3 gene, associated with juvenile neuronal ceroid lipofuscinosis, encodes a novel protein of a predicted 438 amino acid residues . We have expressed a full-length CLN3 protein and fragments thereof in fusion with green fluorescent protein in Chinese hamster ovary and human neuroblastoma cell lines to study its subcellular localization and intracellular trafficking pattern . By using laser scanning confocal microscopy, we demonstrate that the full-length CLN3 fusion protein is targeted to lysosomal compartments . Tunicamycin treatment did not alter the lysosomal targeting of the CLN3 protein, which indicates that extensive N-glycosylation of the full-length CLN3 fusion protein is not engaged in its lysosomal sorting . Monensin produced retention of CLN3 fusion protein in vesicular structure of the Golgi apparatus in the perinuclear space, suggesting that CLN3 fusion protein is transported to the lysosomal compartments through the trans-Golgi cisternae . Neither of the truncated CLN3 fusion proteins encompassing its 1-138, 1-322, and 138-438 amino acid residues was disclosed in lysosomal compartments . However, CLN3 fusion protein showing double-point mutations at amino acid residues 425 and 426, thus at its putative dileucine lysosomal signaling motif, was still targeted to lysosomes, suggesting that a dileucine motif alone is not sufficient for lysosomal sorting of the CLN3 fusion protein .

Mol Genet Metab, 1999 Apr, 66(4), 261 - 4
Studies of membrane association of CLN3 protein; Kaczmarski W et al.; The product of the CLN3 gene is a novel protein of unknown function . Simulations using amphiphacy algorithms have shown that structurally CLN3 may be another candidate for the family of membranous proteins . Signals controlling intracellular targeting of many membrane proteins are present as short sequences within their cytoplasmic domains . In fact, the sequence of CLN3 protein contains several such signaling sequences, which are conserved among mammals . First, at the N-terminus, potential N-myristoylation motif is present . Second, the C-terminal part of CLN3 protein contains both the dileucine motif, which is a potential lysosomal targeting signal, and the prenylation motif . There is scanty evidence of lysosomal and/or mitochondrial localization of CLN3 protein . However, the question of where the functional site of the cln3 protein exists in vivo remains unanswered . From theoretical calculations, we hypothesized that CLN3 should be an integral part of the membranous micro-environment . First, to test this hypothesis, we initiated detergent-partitioning experiments, localizing CLN3 predominantly in a pool of membranous protein . Further studies have shown that CLN3 protein integrates spontaneously with cellular membranes . Second, based on the prenylation results of CLN3 protein in vitro, we discussed the possible topological consequences of C-terminal fragment of CLN3 protein .

Mol Genet Metab, 1999 Apr, 66(4), 253 - 60
Intracellular trafficking of the JNCL protein CLN3; Haskell RE et al.; Juvenile neuronal ceroid lipofuscinosis is a lysosomal storage disease that causes visual impairment, progressive mental deterioration, and eventually death . A predominant 1.02-kb deletion as well as other mutations have been described in the CLN3 gene . Lacking significant identity with proteins of known function and no overt targeting signals within the primary amino acid sequence, accurate predictions of the intracellular location and function could not be made . Further, recent conflicting reports identified CLN3 as either a lysosomal or a mitochondrial protein . Transfection experiments using native and epitope-tagged fusion proteins were evaluated to help delineate CLN3 localization . We confirmed by immunohistochemistry and brefeldin A treatment that NH2-terminal green fluorescence protein (GFP)-CLN3 fusion proteins were retained in the Golgi apparatus, with no colocalization with mitochondrial markers . Anti-CLN3 antibodies directed against amino acids 67-90 of CLN3 were generated and shown to be specific for a 50-kDa protein in HEK 293 cells and GFP-CLN3 in transfected cells . However, cells transfected with nontagged CLN3 or carboxyl-terminal-tagged CLN3 were not immunoreactive with anti-CLN3 antibodies, suggesting that normally, the amino terminus interacts with other molecules . Thus, tags on the NH2-terminus probably inhibited these interactions and movement of CLN3 from the Golgi to more distal compartments . Also, CLN3 tagged at the COOH-terminus with either GFP or FLAG epitopes were retained in the ER, indicating a role for the COOH-terminus in trafficking . Taken together, these data confirm that CLN3 traffics through the ER and Golgi .

Mol Genet Metab, 1999 Apr, 66(4), 248 - 52
Reevaluation of neuronal ceroid lipofuscinoses: atypical juvenile onset may be the result of CLN2 mutations; Wisniewski KE et al.; This study describes the phenotype/genotype analyses of 56 probands with a juvenile onset, some of which had atypical features of neuronal ceroid lipofuscinosis, collected at the New York State Institute for Basic Research (IBR) . In this group, we found probands with abundant curvilinear profiles in lysosomal storage material, deficiency of pepstatin-insensitive peptidase, and mutations in the CLN2 gene, as well as patients with a predominance of granular osmiophilic deposits in the lysosomal storage material, deficiency of palmitoyl-protein thioesterase, and mutations in the CLN1 gene . We have divided the probands into two categories: typical (or classic) and atypical . Most of the typical and atypical probands had onset of symptoms about or after 4 years of age . Interfamiliar and intrafamiliar variations were found, especially in the speed of becoming practically blind . Thus, our study indicates that some mutations in the CLN1, CLN2, and CLN3 genes may be associated with late onset of the disease process, may have a more benign clinical course, and clinic overlap with other forms of neuronal ceroid lipofuscinosis .

J Pediatr, 1999 Apr, 134(4), 447 - 52
Serologic testing for inflammatory bowel disease; Hoffenberg EJ et al.; OBJECTIVES: To determine the accuracy of anti-neutrophil cytoplasmic antibodies (ANCAs) and anti-Saccharomyces cerevisiae antibodies (ASCA) in distinguishing patients with inflammatory bowel disease from patients with other disorders, seen in a pediatric gastroenterology clinic setting, and in distinguishing ulcerative colitis (UC) from Crohn's disease (CD) . STUDY DESIGN: Serum samples from 120 children with new or established diagnoses of UC (n = 25) or CD (n = 20) and control children (n = 74) were analyzed in blinded fashion for the presence of IgG ANCAs and IgA and IgG ASCA . RESULTS: The highest sensitivity for detecting inflammatory bowel disease, 71%, was achieved by using ANCAs and ASCA together . The best test for UC was ANCAs, which had a sensitivity of 80% . However, the ANCA pattern characteristic of UC, perinuclear ANCAs eliminated by DNAse, had a sensitivity of 60% . High-titer ANCAs were specific for UC, whereas ASCA were specific for CD . CONCLUSIONS: Testing for ANCAs and ASCA together did not achieve sensitivity necessary for population screening . However, ANCAs and ASCA may be helpful in evaluating children suspected of having inflammatory bowel disease and in distinguishing UC from CD.

Radiat Res, 1999 Apr, 151(4), 414 - 22
Protein-DNA complexes containing DNA-dependent protein kinase in crude extracts from human and rodent cells; Ting NS et al.; The DNA-dependent protein kinase (DNA-PK) is composed of a large catalytic subunit (DNA-PKcs) and a DNA-binding protein, Ku . Cells lacking DNA-PK activity are radiosensitive and are defective in DNA double-strand break repair and V(D)J recombination . Although much information regarding the interactions of Ku with DNA ends is available, relatively little is known about the interaction of DNA-PKcs with DNA-bound Ku . Here we show, using electrophoretic mobility shift assays, that chemical crosslinkers enhance the formation of protein-DNA complexes containing DNA-PKcs, Ku and other proteins in extracts from cells of normal human cell lines . Extracts from cells of the radiosensitive human cell line M059J, which lacks DNA-PKcs, are not competent to form these protein-DNA complexes, while addition of purified DNA-PKcs protein restores complex formation . This assay may be useful for screening for DNA-PK function in cells of human cell lines and for identifying proteins that interact with the DNA-PK-DNA complex . We also show that Ku protein in rodent cells can interact with human DNA-PKcs; however, this assay may be less useful for studying Ku/DNA-PKcs interactions in cells of rodent cell lines due to the low abundance of DNA-PKcs in these cells.

Radiat Res, 1999 Apr, 151(4), 408 - 13
Radiation-induced recombination is dependent on Ku80; Stevens CW et al.; We have recently shown that irradiating cells prior to transfection induces recombination, as manifested by increased stable transduction of both plasmid and adenoviral vectors . We hypothesized that Ku proteins, which have previously been shown to be involved in both recombination and the repair of DNA damage after irradiation, would likely be important mediators of radiation-induced recombination . The present work demonstrates that Ku80 is essential for radiation-induced recombination . While human and hamster Ku80 are equally effective at restoring the transfection efficiency and radiation resistance of xrs-5 cells, human Ku80 is much more effective at radiation-induced recombination than hamster Ku80 . This difference is not due to differences in Ku80 expression or DNA end-binding activity, but it may be due to structural differences between human and hamster Ku80.

Radiat Res, 1999 Apr, 151(4), 398 - 407
Rejoining of DNA double-strand breaks in Ku80-deficient mouse fibroblasts; Wachsberger PR et al.; The role of Ku80 in the repair of DNA double-strand breaks (DSBs) was examined in fibroblasts derived from a Ku80 knockout mouse model described by Nussenzweig et al . (Nature 382, 551-555, 1996) . Primary fibroblasts from Ku80+/+ and Ku80-/- mice were immortalized by transfection with plasmids containing either the human MYC proto-oncogene or the Simian virus 40 (SV40) T antigen and were used to measure induction and rejoining of DSBs after exposure to ionizing radiation . The number of DSBs in the cells was quantified by either asymmetric field-inversion gel electrophoresis (AFIGE) or clamped homogeneous electrical-field gel electrophoresis (CHEF) . The latter method was introduced for a more reliable quantification of repair even when DNA degradation occurs in a fraction of the irradiated cell population during the postirradiation incubation time . The results confirm that Ku80-deficient mouse fibroblasts are sensitive to ionizing radiation and demonstrate that the increased radiosensitivity may result from a deficiency in DSB rejoining . The results further indicate that unless techniques are employed that allow for distinction between DNA degradation and DNA repair, erroneous conclusions may be drawn regarding the potential of cells to repair DSBs.

J Cell Biol, 1999 Apr 5, 145(1), 29 - 43
A nonerythroid isoform of protein 4.1R interacts with the nuclear mitotic apparatus (NuMA) protein; Mattagajasingh SN et al.; Red blood cell protein 4.1 (4.1R) is an 80- kD erythrocyte phosphoprotein that stabilizes the spectrin/actin cytoskeleton . In nonerythroid cells, multiple 4.1R isoforms arise from a single gene by alternative splicing and predominantly code for a 135-kD isoform . This isoform contains a 209 amino acid extension at its NH2 terminus (head piece; HP) . Immunoreactive epitopes specific for HP have been detected within the cell nucleus, nuclear matrix, centrosomes, and parts of the mitotic apparatus in dividing cells . Using a yeast two-hybrid system, in vitro binding assays, coimmunolocalization, and coimmunoprecipitation studies, we show that a 135-kD 4.1R isoform specifically interacts with the nuclear mitotic apparatus (NuMA) protein . NuMA and 4.1R partially colocalize in the interphase nucleus of MDCK cells and redistribute to the spindle poles early in mitosis . Protein 4.1R associates with NuMA in the interphase nucleus and forms a complex with spindle pole organizing proteins, NuMA, dynein, and dynactin during cell division . Overexpression of a 135-kD isoform of 4.1R alters the normal distribution of NuMA in the interphase nucleus . The minimal sequence sufficient for this interaction has been mapped to the amino acids encoded by exons 20 and 21 of 4.1R and residues 1788-1810 of NuMA . Our results not only suggest that 4.1R could, possibly, play an important role in organizing the nuclear architecture, mitotic spindle, and spindle poles, but also could define a novel role for its 22-24-kD domain.

Transfus Clin Biol, 1999 Feb, 6(1), 7 - 16
Role of the 37 kDa laminin receptor precursor in the life cycle of prions; Rieger R et al.; Prions are thought to consist of infectious proteins that cause, in the absence of detectable nucleic acid, a group of fatal neurodegenerative diseases, called transmissible spongiform encephalopathies (TSE) . Among these diseases are bovine spongiform encephalopathy (BSE), scrapie of sheep and Creutzfeldt-Jakob disease (CJD) in humans . They occur as sporadic, infectious or genetic disorders and have in common the accumulation of an abnormal, pathogenic isoform of the cellular prion protein PrPc which is converted in a post-translational process into PrPSc concomitant with conformational changes of the protein . During this process PrPc acquires a high beta-sheet content and becomes partially resistant to proteases . The mechanism of this conversion as well as the physiological function of the cellular prion protein PrPc are poorly understood, but studies employing PrP knock-out mice demonstrated that PrPc is required for the development of prion diseases . The involvement of co-factors such as chaperones, receptors or an unknown protein, designated "protein X" in the conversion process are discussed . In a yeast two-hybrid screen we have identified the 37 kDa laminin receptor precursor (LRP) as an interactor of the cellular prion protein and this interaction could be confirmed by co-infection and co-transfection studies in mammalian and insect cells . LRP evolved from the ribosomal protein p40 essential for protein synthesis lacking any laminin binding activity to a cell surface receptor binding laminin, elastin and carbohydrates . The gene encoding 37 kDa LRP/p40 has been identified in a variety of species including the sea urchin Urechis caupo, Chlorohydra viridissima, the archaebacterium Haloarcula marismortui, the yeast Saccharomyces cerevisiae as well as in mammals where it is highly conserved . LRP works as a receptor for alphaviruses and is associated with the metastatic potential of solid tumors where it was first identified . The 37 kDa LRP forms its mature 67 kDa isoform with high laminin binding capacity by an unknown mechanism involving acylation . The multifunctionality of LRP as a ribosomal protein and a cell surface receptor for infectious agents such as viruses and prions might be extended by additional properties.

J Biol Chem, 1999 Apr 9, 274(15), 10618 - 24
Identification of the enzyme required for activation of the small ubiquitin-like protein SUMO-1; Desterro JM et al.; The ubiquitin-like protein SUMO-1 is conjugated to a variety of proteins including Ran GTPase-activating protein 1 (RanGAP1), IkappaBalpha, and PML . SUMO-1-modified proteins display altered subcellular targeting and/or stability . We have purified the SUMO-1-activating enzyme from human cells and shown that it contains two subunits of 38 and 72 kDa . Isolation of cDNAs for each subunit indicates that they are homologous to ubiquitin-activating enzymes and to the Saccharomyces cerevisiae enzymes responsible for conjugation of Smt3p and Rub-1p . In vitro, recombinant SAE1/SAE2 (SUMO-1-activating enzyme) was capable of catalyzing the ATP-dependent formation of a thioester linkage between SUMO-1 and SAE2 . The addition of the SUMO-1-conjugating enzyme Ubch9 resulted in efficient transfer of the thioester-linked SUMO-1 from SAE2 to Ubch9 . In the presence of SAE1/SAE2, Ubch9, and ATP, SUMO-1 was efficiently conjugated to the protein substrate IkappaBalpha . As SAE1/SAE2, Ubch9, SUMO-1, and IkappaBalpha are all homogeneous, recombinant proteins, it appears that SUMO-1 conjugation of IkappaBalpha in vitro does not require the equivalent of an E3 ubiquitin protein ligase activity.

Eur J Biochem, 1999 Apr, 261(1), 236 - 43
Acn9 is a novel protein of gluconeogenesis that is located in the mitochondrial intermembrane space; Dennis RA et al.; Previous studies have indicated that the Acn9 protein is involved in gluconeogenesis . Yeast mutants defective in the ACN9 gene display phenotypes identical with mutants defective in metabolic enzymes required for carbon assimilation . These phenotypes include the inability to utilize acetate as a carbon and energy source, elevated levels of enzymes of the glyoxylate cycle, gluconeogenesis and acetyl-CoA mobilization, and a deficiency in de novo synthesis of glucose from ethanol . The ACN9 gene was isolated by functional complementation of the acetate growth defect of an acn9 mutant . The open reading frame corresponds to YDR511w, and encodes a protein of unknown function . Homologs have been identified in human, mouse, and nematode databases . Two mutant alleles were sequenced . The mutations altered amino acid residues that are conserved among members of the new gene family . ACN9 gene expression was slightly repressed by glucose, and the level of the transcript was approximately 100-fold lower than that of glyoxylate or tricarboxylic acid cycle enzymes . A functional epitope-tagged form of Acn9 was expressed to study expression and the subcellular localization of the protein . The tagged protein was localized to the mitochondrial intermembrane space.

J Korean Med Sci, 1999 Feb, 14(1), 27 - 33
Morphological and biochemical analysis of anti-nuclear matrix protein antibodies in human sera; Yu E et al.; Autoimmune sera have been used in the diagnosis of autoimmune diseases as well as the analysis of nuclear substructures . In an attempt to study the biological characteristics of the nuclear matrix, we screened human sera using immunofluorescent staining and immunoblot . We detected antibodies against nuclear matrix (NM), a remnant nonchromatin protein compartment after the treatment of detergent, salt and nuclease, in 212 out of 284 tested sera (74.6%) by immunoblot . Peptides with molecular weights of 70 kDa, 50 kDa and 25 kDa were detected in the order of frequency . Clinical informations of 198 out of 212 cases were available and went as follows: 38 cases were autoimmune diseases, such as systemic lupus erythematosus and rheumatoid arthritis; 132 non-autoimmune and non-neoplastic diseases; 16 neoplastic diseases and 12 cases unclassified . The immunofluorescent staining intensity by anti-nuclear matrix protein (NMP) antibodies decreased variably, but fibrillogranular, speckled and nucleolar immunolocalization patterns were retained after in situ fractionation . Ku70 and La protein were detected by anti-NMP antibodies . Immunolocalization by anti-NMP antibodies indicates that the NMPs constitute a variety of characteristic nuclear substructures and may serve as autoantigens in diverse human diseases . In addition, the presence of Ku70 and La protein as NMPs suggests that the NM can be functionally active in association with DNA or RNA.

Mol Gen Genet, 1999 Mar, 261(2), 251 - 8
Cloning and characterisation of the sagA gene of Aspergillus nidulans: a gene which affects sensitivity to DNA-damaging agents; Jones GW et al.; Mutations within the sagA gene of Aspergillus nidulans cause sensitisation to DNA-damaging chemicals but have no effect upon spontaneous or damage-induced mutation frequency . The sagA gene was cloned on a 19-kb cosmid-derived fragment by functional complementation of a sagA1 sagC3 double mutant; subsequently, a fragment of the gene was also isolated on a 3.9-kb genomic subclone . Initial sequencing of a small section of the 19-kb fragment allowed the design of primers that were subsequently used in RTPCR experiments to show that this DNA is transcribed . A 277-bp fragment derived from the transcribed region was used to screen an A . nidulans cDNA library, resulting in the isolation of a 1.4-kb partial cDNA clone which had sequence overlap with the genomic sagA fragment . This partial cDNA was incomplete but appeared to contain the whole coding region of sagA . The sagA1 mutant was shown to possess two mutations; a G-T transversion and a+ 1 frameshift due to insertion of a T . causing disruption to the C-terminal region of the SagA protein . Translation of the sagA cDNA predicts a protein of 378 amino acids, which has homology to the Saccharomyces cerevisiae End3 protein and also to certain mammalian proteins capable of causing cell transformation.

Mutat Res, 1999 Mar 10, 433(2), 109 - 16
Molecular cloning, expression and chromosomal localisation of the mouse Rev3l gene, encoding the catalytic subunit of polymerase zeta; Van Sloun PP et al.; The REV3 gene of Saccharomyces cerevisiae encodes the catalytic subunit of DNA polymerase zeta which is involved in translesion synthesis . The mouse homolog of this gene, Rev3l, was cloned and sequenced . The gene encodes a putative protein of 3122 amino acids . The sequence conservation to its yeast counterpart is restricted to several regions . In the carboxy-terminal part of the protein all six domains are present that are characteristic for alpha-type DNA polymerases . In the amino-terminal part of the protein two regions can be identified with considerable similarity to the NT boxes of mouse polymerase delta . In addition, a region of 60 residues unique for the REV3 homologs can be found in the middle part of the protein . The mouse REV3L protein shows strong sequence conservation with the recently cloned human REV3L protein (86% identity overall) . Northern blot analysis of various tissues of the mouse revealed that transcription of the Rev3l gene was highest in brain, ovaries and testis . The human REV3L gene was localised to the long arm of chromosome 6, region 21-22 . The mouse equivalent maps to chromosome 10, distal to the c-myb gene, close to the Macs gene.

Mutat Res, 1999 Mar 10, 433(2), 89 - 98
A full-length cDNA of hREV3 is predicted to encode DNA polymerase zeta for damage-induced mutagenesis in humans; Lin W et al.; DNA damage can cause mutations which in turn may lead to carcinogenesis . In the yeast Saccharomyces cerevisiae, DNA damage-induced mutagenesis pathway requires the REV3 gene . It encodes the catalytic subunit of DNA polymerase zeta that specifically functions in translesion DNA synthesis . We have cloned a cDNA of the human homologue of REV3 (hREV3), which consists of 10,716 bp and codes for a protein of 3130 amino acid residues (352,737 Da) . Its C-terminal 755 amino acids show extensive homology with the yeast protein at the C-terminus: 43% identity and 74% similarity . This region contains the six highly conserved DNA polymerase motifs . Furthermore, we have identified four sequence motifs in the N-terminal region outside the polymerase domain that are conserved in DNA polymerase delta from various sources . Three of which are present in DNA polymerase zeta encoded by human, yeast, and plant REV3 genes, indicating that this protein is a member of the DNA polymerase delta family . DNA polymerases delta and zeta are structurally distinguished by the presence of a specific delta IV motif in the former and motifs zeta I and zeta II in the latter, respectively . Human DNA polymerase zeta is ubiquitously expressed in various tissues, consistent with the notion that the hREV3 pathway may be a fundamental mechanism of damage-induced mutagenesis in humans.

J Biochem (Tokyo), 1999 Apr, 125(4), 818 - 25
The human PMS2L proteins do not interact with hMLH1, a major DNA mismatch repair protein; Kondo E et al.; The human PMS2 gene encodes one of the bacterial mutL homologs that is associated with hereditary nonpolyposis colorectal cancer (HNPCC) . One of the interesting features of the hPMS2 gene is that it is part of a multiple gene family which is localized on chromosome bands 7p22, 7p12-p13, 7q11, and 7q22 . Here we report four newly identified hPMS2-like (PMS2L) genes . All four novel members of the PMS2L gene family encode relatively short polypeptides composed of the amino-terminal portion of hPMS2 and are expressed ubiquitously except in the heart . To clarify whether the PMS2L polypeptides contribute to the DNA mismatch repair (MMR) pathway through an interaction with hMLH1, we have performed a yeast two-hybrid assay and an immunoprecipitation study using an hPMS2 mutant cell line, HEC-1-A . Our results clearly indicate that hMLH1 does not interact with two representative PMS2Ls, whereas the carboxyl-terminal portion of hPMS2, not the amino-terminal portion, does interact with hMLH1 . Thus, PMS2Ls are not likely to participate in the MMR pathway through association with hMLH1; they must play some other roles in the living cells.

Genetics, 1999 Apr, 151(4), 1459 - 70
Dna2 mutants reveal interactions with Dna polymerase alpha and Ctf4, a Pol alpha accessory factor, and show that full Dna2 helicase activity is not essential for growth; Formosa T et al.; Mutations in the gene for the conserved, essential nuclease-helicase Dna2 from the yeast Saccharomyces cerevisiae were found to interact genetically with POL1 and CTF4, which encode a DNA Polymerase alpha subunit and an associated protein, suggesting that Dna2 acts in a process that involves Pol alpha . DNA2 alleles were isolated that cause either temperature sensitivity, sensitivity to alkylation damage, or both . The alkylation-sensitive alleles clustered in the helicase domain, including changes in residues required for helicase activity in related proteins . Additional mutations known or expected to destroy the ATPase and helicase activities of Dna2 were constructed and found to support growth on some media but to cause alkylation sensitivity . Only damage-sensitive alleles were lethal in combination with a ctf4 deletion . Full activity of the Dna2 helicase function is therefore not needed for viability, but is required for repairing damage and for tolerating loss of Ctf4 . Arrest of dna2 mutants was RAD9 dependent, but deleting this checkpoint resulted in either no effect or suppression of defects, including the synthetic lethality with ctf4 . Dna2 therefore appears to act in repair or lagging strand synthesis together with Pol alpha and Ctf4, in a role that is optimal with, but does not require, full helicase activity.

Genetics, 1999 Apr, 151(4), 1409 - 23
Removal of one nonhomologous DNA end during gene conversion by a RAD1- and MSH2-independent pathway; Colaiacovo MP et al.; Repair of a double-strand break (DSB) by homologous recombination depends on the invasion of a 3'-ended strand into an intact template sequence to initiate new DNA synthesis . When the end of the invading DNA is not homologous to the donor, the nonhomologous sequences must be removed before new synthesis can begin . In Saccharomyces cerevisiae, the removal of these ends depends on both the nucleotide excision repair endonuclease Rad1p/Rad10p and the mismatch repair proteins Msh2p/Msh3p . In rad1 or msh2 mutants, when both ends of the DSB have nonhomologous ends, repair is reduced approximately 90-fold compared to a plasmid with perfect ends; however, with only one nonhomologous end, repair is reduced on average only 5-fold . These results suggest that yeast has an alternative, but less efficient, way to remove a nonhomologous tail from the second end participating in gene conversion . When the removal of one nonhomologous end is impaired in rad1 and msh2 mutants, there is also a 1-hr delay in the appearance of crossover products of gene conversion, compared to noncrossovers . We interpret these results in terms of the formation and resolution of alternative intermediates of a synthesis-dependent strand annealing mechanism.

Development, 1999 May, 126(9), 2021 - 31
C . elegans MAC-1, an essential member of the AAA family of ATPases, can bind CED-4 and prevent cell death; Wu D et al.; In the nematode Caenorhabditis elegans, CED-4 plays a central role in the regulation of programmed cell death . To identify proteins with essential or pleiotropic activities that might also regulate cell death, we used the yeast two-hybrid system to screen for CED-4-binding proteins . We identified MAC-1, a member of the AAA family of ATPases that is similar to Smallminded of Drosophila . Immunoprecipitation studies confirm that MAC-1 interacts with CED-4, and also with Apaf-1, the mammalian homologue of CED-4 . Furthermore, MAC-1 can form a multi-protein complex that also includes CED-3 or CED-9 . A MAC-1 transgene under the control of a heat shock promoter prevents some natural cell deaths in C . elegans, and this protection is enhanced in a ced-9(n1950sd)/+ genetic background . We observe a similar effect in mammalian cells, where expression of MAC-1 can prevent CED-4 and CED-3 from inducing apoptosis . Finally, mac-1 is an essential gene, since inactivation by RNA-mediated interference causes worms to arrest early in larval development . This arrest is similar to that observed in Smallminded mutants, but is not related to the ability of MAC-1 to bind CED-4, since it still occurs in ced-3 or ced-4 null mutants . These results suggest that MAC-1 identifies a new class of proteins that are essential for development, and which might regulate cell death in specific circumstances.

Biotechnol Bioeng, 1998 Dec 5, 60(5), 568 - 79
Preparation of a new thermo-responsive adsorbent with maltose as a ligand and its application to affinity precipitation; Hoshino K et al.; A thermo-responsive polymer on which maltose was covalently immobilized as an affinity ligand was newly synthesized for purification of thermolabile proteins from the crude solution by affinity precipitation . Among the thermo-responsive polymers synthesized as carriers for adsorbent, poly(N-acryloylpiperidine)-cysteamine (pAP) has a lower critical solution temperature (LCST) of around 4 degrees C, at which its solubility exhibits a sharp change . Adsorbent for affinity precipitation was prepared by combining pAP with maltose using trimethylamine-borane as a reducing reagent . This adsorbent (pAPM) obtained showed a good solubility response: pAPM in the basal buffer (pH 7.0) became soluble below 4 degrees C and was completely insoluble above 8 degrees C . The affinity precipitation method using pAPM consisted of the following four steps: adsorption at 4 degrees C, precipitation of the complex at 10 degrees C, desorption by adding the desorption reagent at 4 degrees C, and recovery of a target protein at 10 degrees C . In the affinity precipitation of Con A from the crude extract of jack bean meal, 82% of Con A added was recovered with 80% purity by addition of 0.2 M methyl-alpha-D-mannopyranoside as a desorption reagent . In the repeated purification of Con A from the crude extract, pAPM could be satisfactorily reused without decrease in the affinity performance . Moreover, when pAPM was used for the purification of thermolabile alpha-glucosidase from the cell-free extract of Saccharomyces cerevisiae, 68% of total activity added was recovered and the specific activity per amount of protein of the purified solution was enhanced 206-fold higher than that of the cell-free extract without thermal deactivation of the enzyme .

Biotechnol Bioeng, 1998 Oct 20, 60(2), 190 - 6
Enzymatic transformations in supersaturated substrate solutions: I . A general study with glycosidases; Millqvist-Fureby A et al.; The results of an initial study of enzymatic catalysis in metastable supersaturated solutions of carbohydrates are presented . It has been shown that such solutions, formed in the presence of small amounts of water and alcohol as plasticizers, are sufficiently stable under ambient conditions to enable enzymatic transformations of substrates . A partial phase diagram for a system consisting of glucose, water, and (poly)ethylene glycol was constructed to identify the regions which are most suitable for biotransformations . It was confirmed that the glass transition in this system occurred below the reaction temperature at any given composition of the constituent components . Several glycosidases were found to be catalytically active in this medium and the activity of beta-glucosidase from almond was determined at several compositions of the reaction mixture and related to the corresponding regions of the phase diagram . The synthetic utility of the system was illustrated by glucosylation of several alpha,omega-alkyldiols, short-chain polyethylene glycols, and hydroxyalkyl and glyceryl monoacrylates .

Biotechnol Bioeng, 1998 Mar 5, 57(5), 610 - 9
Identification and control of oxidative metabolism in Ssaccharomyces cerevisiae during transient growth using calorimetric measurements; Duboc P et al.; The objective of this study was to characterize the dynamic adaptation of the oxidative capacity of Saccharomyces cerevisiae to an increase in the glucose supply rate and its implications for the control of a continuous culture designed to produce biomass without allowing glucose to be diverted into the reductive metabolism . Continuous cultures subjected to a sudden shift-up in the dilution rate showed that the glucose uptake rate increased immediately to the new feeding rate but that the oxygen consumption could not follow fast enough to ensure a completely oxidative metabolism . Thus, part of the glucose assimilated was degraded by the reductive metabolism, resulting in a temporary decrease of biomass concentration, even if the final dilution rate was below Dcrit . The dynamic increase of the specific oxygen consumption rate, qO2, was characterized by an initial immediate jump followed by a first-order increase to the maximum value . It could be modeled using three parameters denoted qjumpO2, qmaxO2, and a time constant tau . The values for the first two of the parameters varied considerably from one shift to another, even when they were performed under identical conditions . On the basis of this model, a time-dependent feed flow rate function was derived that should permit an increase in the dilution rate from one value to another without provoking the appearance of reductive metabolism . The idea was to increase the glucose supply in parallel with the dynamic increase of the oxidative capacity of the culture, so that all of the assimilated glucose could always be oxidized . Nevertheless, corresponding feed-profile experiments showed that deviations in the reductive metabolism could not be completely suppressed due to variability in the model parameters . Therefore, a proportional feedback controller using heat evolution rate measurements was implemented . Calorimetry provides an excellent and rapid estimate of the metabolic activity . Satisfactory control was achieved and led to constant biomass yields . Ethanol accumulated only up to 0.49 g L-1 as compared to an accumulation of 1.82 g L-1 without on-line control in the shift-up experiment to the same final dilution rate .

FEBS Lett, 1999 Mar 5, 446(1), 108 - 12
Alpha4 protein as a common regulator of type 2A-related serine/threonine protein phosphatases; Nanahoshi M et al.; The catalytic activity of the C subunit of serine/threonine phosphatase 2A is regulated by the association with A (PR65) and B subunits . It has been reported that the alpha4 protein, a yeast homolog of the Tap42 protein, binds the C subunit of serine/threonine phosphatase 2A and protein phosphatase 2A-related protein phosphatases such as protein phosphatase 4 and protein phosphatase 6 . In the present study, we showed that alpha4 binds these three phosphatases and the association of alpha4 reduces the activities of these phosphatases in vitro . In contrast, PR65 binds to the C subunit of serine/threonine phosphatase 2A but not to protein phosphatase 4 and protein phosphatase 6 . These results suggest that the alpha4 protein is a common regulator of the C subunit of serine/threonine phosphatase 2A and protein phosphatase 2A-related protein phosphatases.

Chem Biol, 1999 Apr, 6(4), 197 - 204
Bifunctional inhibitors of the trypsin-like activity of eukaryotic proteasomes; Loidl G et al.; BACKGROUND: The 20S proteasome is a multicatalytic protease complex that exhibits trypsin-like, chymotrypsin-like and post-glutamyl-peptide hydrolytic activities associated with the active sites of the beta2, beta5 and beta1 subunits, respectively . Modulation of these activities using inhibitors is essential for a better understanding of the proteasome's mechanism of action . Although there are highly selective inhibitors of the proteasome's chymotryptic activity, inhibitors of similar specificity have not yet been identified for the other activities . RESULTS: The X-ray structure of the yeast proteasome reveals that the sidechain of Cys118 of the beta3 subunit protrudes into the S3 subsite of the beta2 active site . The location of this residue was exploited for the rational design of bidentated inhibitors containing a maleinimide moiety at the P3 position for covalent linkage to the thiol group and a carboxy-terminal aldehyde group for hemiacetal formation with the Thr1 hydroxyl group of the active site . Structure-based modelling was used to determine the optimal spacing of the maleinimide group from the P2-P1 dipeptide aldehydes and the specificity of the S1 subsite was exploited to limit the inhibitory activity to the beta2 active site . X-ray crystallographic analysis of a yeast proteasome-inhibitor adduct confirmed the expected irreversible binding of the inhibitor to the P3 subsite . CONCLUSIONS: Maleoyl-beta-alanyl-valyl-arginal is a new type of inhibitor that is highly selective for the trypsin-like activity of eukaryotic proteasomes . Despite the reactivity of the maleinimide group towards thiols, and therefore the limited use of this inhibitor for in vitro studies, it might represent an interesting new biochemical tool.

Br J Cancer, 1999 Mar, 79(7-8), 1037 - 41
Ku70/80 gene expression and DNA-dependent protein kinase (DNA-PK) activity do not correlate with double-strand break (dsb) repair capacity and cellular radiosensitivity in normal human fibroblasts; Kasten U et al.; The expression of the Ku70 and Ku80 genes as well as the activity of the DNA-dependent protein kinase (DNA-PK) were studied in 11 normal human fibroblast lines . The proteins studied are known to be part of a double-strand break (dsb) repair complex involved in non-homologous recombination, as was demonstrated for the radiosensitive rodent mutant cell lines of the complementation groups 5-7 . The 11 fibroblast lines used in this study represent a typical spectrum of normal human radiosensitivity with the surviving fraction measured for a dose of 3.5 Gy, SF3.5 GY, ranging from 0.03 to 0.28 . These differences in cell survival were previously shown to correlate with the number of non-repaired dsbs . We found that the mRNA signal intensities of both Ku70 and Ku80 genes were fairly similar for the 11 cell lines investigated . In addition, the DNA-PK activity determined by the pulldown assay was fairly constant in these fibroblast lines . Despite the correlation between cell survival and dsb repair capacity, there was no correlation between dsb repair capacity and DNA-PK activity in the tested normal human fibroblast lines . Obviously, in this respect, other proteins/pathways appear to be more relevant.

DNA Cell Biol, 1999 Mar, 18(3), 197 - 208
Ku autoimmune antigen is involved in placental regulation of rat P450c17 gene transcription; Zhang P et al.; The steroidogenic enzyme P450c17 (17alpha hydroxylase/C17,20 lyase) regulates a key branchpoint in steroidogenesis, as its activity directs the steroid biosynthetic pathways toward glucocorticoid or sex hormone synthesis . Expression of the P450c17 gene is transcriptionally regulated in steroidogenic tissues by cAMP . We showed that DNA between -84 and -55 in the rat P450c17 gene was bound uniquely by steroidogenic factor-1 (SF-1), which regulated both basal and cAMP-stimulated transcription in mouse adrenocortical and Leydig cells . SF-1 gene ablation experiments in mice indicate that SF-1 is not mandatory for placental steroidogenesis . We studied P450c17 gene regulation in the placenta using human placental JEG-3 trophoblast cells . Transfection of reporter luciferase gene constructs containing serial deletions of the 5' flanking region of the rat P450c17 gene showed that DNA between -98 and +13 mediated basal and cAMP-regulated transcription in placental JEG-3 cells, as it did in adrenal and Leydig cells . DNase footprints further identified a region between -88 and the TATA box that was bound by protein . Transfection of luciferase reporter constructs containing -84 to -55 of the rat P450c17 DNA ligated to the minimal promoter of the thymidine kinase gene showed that this DNA increased both basal and cAMP-simulated luciferase activity . Gel mobility shift assays identified two DNA-protein complexes with JEG-3 cell nuclear extracts that were different from complexes formed with MA-10 cell extracts and did not involve SF-1 . Mutational analysis of the -84/-55 DNA showed that JEG-3 nuclear proteins bound to a site containing, but not identical to, the SF-1 sequence . One complex involved Ku autoimmune antigen, which bound to DNA sequence specifically . Overexpression of Ku antigen in MA-10 cells stimulated rat P450c17 gene transcription, thus demonstrating a biologic effect of Ku . Ku also bound to a similar region of the human P450c17 gene, and the DNA region to which Ku bound was transcriptionally active in JEG-3 cells . Ku was also found in extracts from rat placenta and bound to the -84/-55 rat P450c17 DNA . These data demonstrate a role of Ku in regulating P450c17 gene expression . These data further indicate that although human P450c17 is not normally expressed in the placenta, factors that could activate this gene are indeed present.

Trends Genet, 1999 Feb, 15(2), 74 - 80
RNA surveillance . Unforeseen consequences for gene expression, inherited genetic disorders and cancer; Culbertson MR; Messenger RNAs are monitored for errors that arise during gene expression by a mechanism called RNA surveillance, with the result that most mRNAs that cannot be translated along their full length are rapidly degraded . This ensures that truncated proteins are seldom made, reducing the accumulation of rogue proteins that might be deleterious . The pathway leading to accelerated mRNA decay is referred to as nonsense-mediated mRNA decay (NMD) . The proteins that catalyze steps in NMD in yeast serve two roles, one to monitor errors in gene expression and the other to control the abundance of endogenous wild-type mRNAs as part of the normal repertoire of gene expression . The NMD pathway has a direct impact on hundreds of genetic disorders in the human population, where about a quarter of all known mutations are predicted to trigger NMD.

Trends Genet, 1999 Feb, 15(2), 43 - 5
New lines of host defense: inhibition of Ty1 retrotransposition by Fus3p and NER/TFIIH; Curcio MJ et al.; The genomes of all organisms examined contain transposons whose uncontrolled movement threatens genome function . Fortunately, host cells have evolved defense mechanisms to minimize the level of transposition . In this review we discuss recent work showing that proteins involved in signal transduction and RNA transcription/DNA repair inhibit Ty1 retrotransposition in the yeast Saccharomyces cerevisiae . On the basis of these examples, we hypothesize that the level of Ty1 retrotransposition may be modulated in response to environmental stress signals that affect cellular differentiation and DNA repair.

Proc Natl Acad Sci U S A, 1999 Mar 30, 96(7), 3712 - 7
The beta2-adrenergic receptor/betaarrestin complex recruits the clathrin adaptor AP-2 during endocytosis; Laporte SA et al.; betaarrestins mediate the desensitization of the beta2-adrenergic receptor (beta2AR) and many other G protein-coupled receptors (GPCRs) . Additionally, betaarrestins initiate the endocytosis of these receptors via clathrin coated-pits and interact directly with clathrin . Consequently, it has been proposed that betaarrestins serve as clathrin adaptors for the GPCR family by linking these receptors to clathrin lattices . AP-2, the heterotetrameric clathrin adaptor protein, has been demonstrated to mediate the internalization of many types of plasma membrane proteins other than GPCRs . AP-2 interacts with the clathrin heavy chain and cytoplasmic domains of receptors such as those for epidermal growth factor and transferrin . In the present study we demonstrate the formation of an agonist-induced multimeric complex containing a GPCR, betaarrestin 2, and the beta2-adaptin subunit of AP-2 . beta2-Adaptin binds betaarrestin 2 in a yeast two-hybrid assay and coimmunoprecipitates with betaarrestins and beta2AR in an agonist-dependent manner in HEK-293 cells . Moreover, beta2-adaptin translocates from the cytosol to the plasma membrane in response to the beta2AR agonist isoproterenol and colocalizes with beta2AR in clathrin-coated pits . Finally, expression of betaarrestin 2 minigene constructs containing the beta2-adaptin interacting region inhibits beta2AR endocytosis . These findings point to a role for AP-2 in GPCR endocytosis, and they suggest that AP-2 functions as a clathrin adaptor for the endocytosis of diverse classes of membrane receptors.

Proc Natl Acad Sci U S A, 1999 Mar 30, 96(7), 3634 - 9
Uncoupling of transfer of the presequence and unfolding of the mature domain in precursor translocation across the mitochondrial outer membrane; Kanamori T et al.; Translocation of mitochondrial precursor proteins across the mitochondrial outer membrane is facilitated by the translocase of the outer membrane (TOM) complex . By using site-specific photocrosslinking, we have mapped interactions between TOM proteins and a mitochondrial precursor protein arrested at two distinct stages, stage A (accumulated at 0 degrees C) and stage B (accumulated at 30 degrees C), in the translocation across the outer membrane at high resolution not achieved previously . Although the stage A and stage B intermediates were assigned previously to the forms bound to the cis site and the trans site of the TOM complex, respectively, the results of crosslinking indicate that the presequence of the intermediates at both stage A and stage B is already on the trans side of the outer membrane . The mature domain is unfolded and bound to Tom40 at stage B whereas it remains folded at stage A . After dissociation from the TOM complex, translocation of the stage B intermediate, but not of the stage A intermediate, across the inner membrane was promoted by the intermembrane-space domain of Tom22 . We propose a new model for protein translocation across the outer membrane, where translocation of the presequence and unfolding of the mature domain are not necessarily coupled.

Proc Natl Acad Sci U S A, 1999 Mar 30, 96(7), 3572 - 7
Crystal structure of human p32, a doughnut-shaped acidic mitochondrial matrix protein; Jiang J et al.; Human p32 (also known as SF2-associated p32, p32/TAP, and gC1qR) is a conserved eukaryotic protein that localizes predominantly in the mitochondrial matrix . It is thought to be involved in mitochondrial oxidative phosphorylation and in nucleus-mitochondrion interactions . We report the crystal structure of p32 determined at 2.25 A resolution . The structure reveals that p32 adopts a novel fold with seven consecutive antiparallel beta-strands flanked by one N-terminal and two C-terminal alpha-helices . Three monomers form a doughnut-shaped quaternary structure with an unusually asymmetric charge distribution on the surface . The implications of the structure on previously proposed functions of p32 are discussed and new specific functional properties are suggested.

Proc Natl Acad Sci U S A, 1999 Mar 30, 96(7), 3507 - 12
Design of highly specific cytotoxins by using trans-splicing ribozymes; Ayre BG et al.; We have designed ribozymes based on a self-splicing group I intron that can trans-splice exon sequences into a chosen RNA target to create a functional chimeric mRNA and provide a highly specific trigger for gene expression . We have targeted ribozymes against the coat protein mRNA of a widespread plant pathogen, cucumber mosaic virus . The ribozymes were designed to trans-splice the coding sequence of the diphtheria toxin A chain in frame with the viral initiation codon of the target sequence . Diphtheria toxin A chain catalyzes the ADP ribosylation of elongation factor 2 and can cause the cessation of protein translation . In a Saccharomyces cerevisiae model system, ribozyme expression was shown to specifically inhibit the growth of cells expressing the virus mRNA . A point mutation at the target splice site alleviated this ribozyme-mediated toxicity . Increasing the extent of base pairing between the ribozyme and target dramatically increased specific expression of the cytotoxin and reduced illegitimate toxicity in vivo . Trans-splicing ribozymes may provide a new class of agents for engineering virus resistance and therapeutic cytotoxins.

Anal Biochem, 1999 Apr 10, 269(1), 133 - 8
Viral protease assay based on GAL4 inactivation is applicable to high-throughput screening in mammalian cells; Lawler JF Jr et al.; We present an assay for viral proteases that relies on the proteolytic cleavage of substrate leading to the dissociation of the yeast transcription factor GAL4 . A consensus substrate for the cytomegalovirus protease is fused between the DNA binding and transactivating domains of GAL4 . Proteolysis inactivates the transcription factor which drives a luciferase reporter system . The assay is performed in mammalian cells, has a robust signal-to-noise ratio, and assesses proteolysis in a physiologic context . A unique feature of the assay is its ability to detect inhibitors of viral replication that act on viral targets other than the protease .

Eur J Biochem, 1999 Mar, 260(2), 336 - 46
A novel human DNA-binding protein with sequence similarity to a subfamily of redox proteins which is able to repress RNA-polymerase-III-driven transcription of the Alu-family retroposons in vitro; Kropotov A et al.; In this study we identified a novel protein which may contribute to the transcriptional inactivity of Alu retroposons in vivo . A human cDNA clone encoding this protein (ACR1) was isolated from a human expression library using South-western screening with an Alu subfragment, implicated in the regulation of Alu in vitro transcription and interacting with a HeLa nuclear protein down-regulated in adenovirus-infected cells . Bacterially expressed ACR1 is demonstrated to inhibit RNA polymerase III (Pol III)-dependent Alu transcription in vitro but showed no repression of transcription of a tRNA gene or of a reporter gene under control of a Pol II promoter . ACR1 mRNA is also found to be down-regulated in adenovirus-infected HeLa cells, consistent with a possible repressor function of the protein in vivo . ACR1 is mainly (but not exclusively) located in cytoplasm and appears to be a member of a weakly characterized redox protein family having a central, highly conserved sequence motif, PGAFTPXCXXXXLP . One member of the family identified earlier as peroxisomal membrane protein (PMP)20 is known to interact in a sequence-specific manner with a yeast homolog of mammalian cyclosporin-A-binding protein cyclophilin, and mammalian cyclophilin A (an abundant ubiquitously expressed protein) is known to interact with human transcriptional repressor YY1, which is a major sequence-specific Alu-binding protein in human cells . It appears, therefore, that transcriptional silencing of Alu in vivo is a result of complex interactions of many proteins which bind to its Pol III promoter.

Mutat Res, 1999 Mar, 436(2), 157 - 78
Somatic hypermutation and the three R's: repair, replication and recombination; Harris RS et al.; Somatic hypermutation introduces single base changes into the rearranged variable (V) regions of antigen activated B cells at a rate of approximately 1 mutation per kilobase per generation . This is nearly a million-fold higher than the typical mutation rate in a mammalian somatic cell . Rampant mutation at this level could have a devastating effect, but somatic hypermutation is accurately targeted and tightly regulated . Here, we provide an overview of immunoglobulin gene somatic hypermutation; discuss mechanisms of mutation in model organisms that may be relevant to the hypermutation mechanism; and review recent advances toward understanding the possible role(s) of DNA repair, replication, and recombination in this fascinating process .

Hum Mutat, 1999, 13(2), 170 - 1
Detection of protein truncating mutations in exons 1-14 of the APC gene using an in vivo fusion protein assay . Mutations in brief no . 214 . Online; Andreutti-Zaugg C et al.; About 80% of the mutations identified to date in the Adenomatous Polyposis Coli (APC) gene have been found in the 5' half of the coding sequence, the vast majority of which (>95%) are nonsense or frameshift mutations that result in the loss of the carboxyl terminus of APC protein . Using a stop codon assay in yeast recently developed by others (Ishioka et al., 1997), we have screened the 5' half of the APC gene for mutations in 7 unrelated families affected with Familial Adenomatous Polyposis . The assay relies on the expression of a yeast reporter gene fused in frame to one of 3 contiguous segments of the APC open reading frame . Here we report on the detection by this assay of 5 germline mutations, 4 of which lie upstream of exon 15, where lesions appear to be sometimes difficult to detect by standard methods.

FEBS Lett, 1999 Feb 26, 445(2-3), 315 - 20
Pax6 and Pdx1 form a functional complex on the rat somatostatin gene upstream enhancer; Andersen FG et al.; The somatostatin upstream enhancer (SMS-UE) is a highly complex enhancer element . The distal A-element contains overlapping Pdx1 and Pbx binding sites . However, a point mutation in the A-element that abolishes both Pdxl and Pbx binding does not impair promoter activity . In contrast, a point mutation that selectively eliminates Pdx1 binding to a proximal B-element reduces the promoter activity . The B-element completely overlaps with a Pax6 binding site, the C-element . A point mutation in the C-element demonstrates that Pax6 binding is essential for promoter activity . Interestingly, a block mutation in the A-element reduces both Pax6 binding and promoter activity . In heterologous cells, Pdx1 potentiated Pax6 mediated activation of a somatostatin reporter . We conclude that the beta/delta-cell-specific activity of the SMS-UE is achieved through simultaneous binding of Pdx1 and Pax6 to the B- and C-elements, respectively . Furthermore, the A-element appears to stabilise Pax6 binding.

Mol Cell Biochem, 1999 Jan, 191(1-2), 143 - 8
A review of progress towards elucidating the role of protein kinase CK2 in polymerase III transcription: regulation of the TATA binding protein; Ghavidel A et al.; We have investigated the molecular basis of the requirement for protein kinase CK2 in nuclear transcription in Saccharomyces cerevisiae . In vivo and in vitro analysis has demonstrated that CK2 is required for efficient transcription of the tRNA and 55 rRNA genes by RNA polymerase III . This suggests that a component of the pol III transcription machinery is regulated by CK2 . We tested this possibility by a biochemical complementation approach in which components of the pol III transcription machinery from wild type cells were tested for their ability to rescue transcription in extract from a conditionally CK2-deficient mutant . We found that pol III transcription initiation factor IIIB (TFIIIB) fully restores transcription in CK2-deficient extract . Further in vitro studies revealed that TFIIIB must be phosphorylated to be active, that a single subunit of wild type TFIIIB, the TATA binding protein (TBP), is efficiently phosphorylated by CK2, and that recombinant TBP and a limiting amount of CK2 rescues transcription in CK2-deficient extract . We conclude that TBP is the physiological target of CK2 among the components of the pol III transcription machinery . The implications of this result are discussed in the context of previous data concerning the regulation of TFIIIB.

Mol Cell Biochem, 1999 Jan, 191(1-2), 121 - 8
BTF3 is a potential new substrate of protein kinase CK2; Grein S et al.; BTF3, initially discovered as a factor required for transcription inititation of RNA polymerase II, is expressed in two isoforms, termed a and b . BTF3b, the transcriptionally inactive isoform, was identified as an interaction partner of protein kinase CK2 subunit beta employing the interaction trap system for screening ofa HeLa cDNA fusion library . We report here on the interaction between the other isoform, BTF3a, and protein kinase CK2 . The complete cDNA of BTF3a was cloned by RT-PCR and used for analysis in the two-hybrid system with a three-reporter yeast strain . Interaction of BTF3a with CK2 subunits alpha, alpha' or beta was detectable by one of three reporters, whereas the CK2beta - BTF3a interaction was activating two reporters . It was also shown that BTF3a is phosphorylated in vitro by the alpha2beta2 holoenzyme, but not by alpha or alpha' alone, indicating the requirement of beta for substrate recognition . Immunoprecipitations of GST-fused BTF3a carried out in vitro resulted in co-precipitation of beta . Similarly, GST-BTF3a, but not GST alone isolated with glutathione agarose beads from buffer containing recombinant CK2 subunits was found complexed with alpha and beta, likely representing alpha2beta2 holoenzyme . The data show a weak, nevertheless specific interaction of protein kinase CK2 via subunit beta with the putative transcription factor BTF3a in vitro and in vivo, and a role of BTF3a as a potential new substrate for CK2.

Mol Cell Biochem, 1999 Jan, 191(1-2), 105 - 9
Searching interaction partners of protein kinase CK2beta subunit by two-hybrid screening; Grein S et al.; To date, the intracellular regulation of protein kinase CK2 is unknown . However it was observed that the enzyme associates with several intracellular proteins and the formation of such molecular complexes may represent a mechanism for the control of CK2 activity . Using the Interaction Trap system in yeast, with the CK2beta as a bait, we looked for CK2 partners . We present the identification of new potential partners of CK2beta and it is hoped that their classification will help in understanding the physiological roles and the regulation of CK2 in the cell.

Mol Cell Biochem, 1999 Jan, 191(1-2), 85 - 95
Mutations in the C-terminal domain of topoisomerase II affect meiotic function and interaction with the casein kinase 2 beta subunit; Leroy D et al.; Topoisomerase II is a major target of the protein kinase casein kinase 2 (PK CK2) in vivo . All major phosphorylation acceptor sites in the yeast enzyme are found in the C-terminal 350aa . The acceptor sites are generally clustered such that there is more than one modified Ser or Thr within a short peptide . Mutagenesis of the predicted acceptor sites have confirmed that five of the eight predicted sites are targeted in vitro and in vivo by PK CK2 . Mutation to nonphosphorylatable, neutral residues provokes at most a 10% increase in mitotic doubling time . Truncation of the enzyme leaves the enzyme catalytically active, but slightly lengthens the doubling time during mitotic growth and impedes progress through meiosis . Since this could reflect the loss of interaction with an important ligand, we have examined whether the C-terminal domain of the yeast enzyme mediates interaction with the regulatory beta subunit of PK CK2, which was previously reported to bind topoisomerase II . We find that point mutation of the phospho-acceptor sites does not abrogate the interaction with a small region of PK CK2beta, while truncation at aa1276 or aa1236 does . The site of interaction within PK CK2beta does not coincide with the highly negatively charged spermine binding site.

RNA, 1999 Mar, 5(3), 455 - 67
Nop58p is a common component of the box C+D snoRNPs that is required for snoRNA stability; Lafontaine DL et al.; Eukaryotic nucleoli contain a large family of box C+D small nucleolar RNA (snoRNA) species, all of which are associated with a common protein Nop1p/fibrillarin . Nop58p was identified in a screen for synthetic lethality with Nop1p and shown to be an essential nucleolar protein . Here we report that a Protein A-tagged version of Nop58p coprecipitates all tested box C+D snoRNAs and that genetic depletion of Nop58p leads to the loss of all tested box C+D snoRNAs . The box H+ACA class of snoRNAs are not coprecipitated with Nop58p, and are not codepleted . The yeast box C+D snoRNAs include two species, U3 and U14, that are required for the early cleavages in pre-rRNA processing . Consistent with this, Nop58p depletion leads to a strong inhibition of pre-rRNA processing and 18S rRNA synthesis . Unexpectedly, depletion of Nop58p leads to the accumulation of 3' extended forms of U3 and U24, showing that the protein is also involved in snoRNA synthesis . Nop58p is the second common component of the box C+D snoRNPs to be identified and the first to be shown to be required for the stability and for the synthesis of these snoRNAs.

J Photochem Photobiol B, 1998 Dec, 47(2-3), 173 - 80
Visible radiation effects on flavocytochrome b2 in dilute aqueous solution: a steady-state and laser flash photolysis study; Bhattacharya D et al.; Irradiation of flavocytochrome b2 by visible radiation at 450 nm in dilute aqueous solution is found to have a devastating effect not only on its activity but also on the important flavin mononucleotide (FMN) constituents . The active site and the substrate binding site are also found to be largely modified on exposure to visible radiation . This has a telling effect on the constituent aromatic amino acids, tryptophan and tyrosine, and therefore justifies the role of FMN as a very potent photosensitizer . Partial unfolding of the irradiated enzyme molecule is also observed . Damage is much greater in deaerated conditions, which indicates that molecular oxygen plays a protecting role in this particular system . The inactivation is mediated through rapid electron transfer from tryptophan and tyrosine to excited flavin, forming flavin semiquinone and tryptophanyl and tyrosinyl radicals, which in turn cause permanent damage at the molecular level.

Eur J Biochem, 1999 Feb, 259(3), 877 - 86
An intrinsic curvature towards the minor groove in the cAMP-responsive element DNA found by combined NMR and molecular modelling studies; Chaoui M et al.; The cAMP-responsive element (CRE, 5'-TGACGTCA-3') is essential to the transcriptional function of numerous gene promoters in eukaryotic cells . We carried out NMR restrained molecular mechanics studies using two different force fields (Flex and "AMBER94") on a hexadecanucleotide d(GAGATGACGTCATCTC) containing CRE . Results indicated that free CRE is a B-DNA that is intrinsically curved towards the minor groove . To our knowledge, NMR restraints have not previously been useful in accounting for a global DNA curvature . In order to validate the bend in CRE, we applied a new strategy in which DNA structures displaying different curvatures were generated and then compared with NMR data . Conformations of CRE curved towards the minor groove provided the best agreement with NMR data . Our results contrast with previous results obtained from NMR restrained modelling and gel methods; these suggested conformations that were straight or curved towards the major groove, respectively . The curve in free CRE is spread along the DNA helix: several kinks are repeated in phase within the helical turn, although they are centred mainly on CpG in between the TGA half-sites, thus slightly increasing their spacing within the major groove . Comparison with the crystal structure of CRE complexed to general control protein 4 showed that the curve orientation is reversed from the minor to the major groove upon protein binding, due to a helix distortion concentrated mainly on CpG.

Eur J Biochem, 1999 Feb, 259(3), 719 - 25
Cloning, sequencing and functional expression of a cDNA encoding porcine pancreatic preprocarboxypeptidase A1; Darnis S et al.; A full-length cDNA clone coding for porcine pancreatic preprocarboxypeptidase A1 (prePCPA1) was isolated from a cDNA library . The open reading frame (ORF) of the nucleotide sequence was 1260 nt in length and encoded a protein of 419 amino acids (aa) . The cDNA included a short signal peptide of 16 aa and a 94 aa-long activation segment . The calculated molecular mass of the mature proenzyme was 45561 Da, in accordance with that of the purified porcine pancreatic PCPA1 . The deduced aa sequence of the corresponding enzyme differed from that predicted by the three-dimensional structure by 40 aa, and showed 85% identity and 55% identity to that of procarboxypeptidases A1 and A2, respectively . Moreover the sequence was identical to that of several independent cDNA clones, suggesting that it is the major transcribed gene . No evidence for a second variant was observed in the cDNA library and PCPA2 is apparently absent from the porcine pancreas . The cDNA was expressed in Saccharomyces cerevisiae under the control of the yeast triose phosphate isomerase promoter . The signal peptide of the PCPA protein efficiently directed its secretion into the culture medium (1.5 mg.L-1) as a protein of the predicted size . The recombinant proenzyme was analyzed by immunological and enzymological methods . Its activation behavior was comparable with that of the native form and led to a 35-kDa active enzyme.

J Biol Chem, 1999 Apr 2, 274(14), 9455 - 62
Cef1p is a component of the Prp19p-associated complex and essential for pre-mRNA splicing; Tsai WY et al.; The Prp19p protein of the budding yeast Saccharomyces cerevisiae is an essential splicing factor and is associated with the spliceosome during the splicing reaction . We have previously shown that Prp19p is not tightly associated with small nuclear ribonucleoprotein particles but is associated with a protein complex consisting of at least eight protein components . By sequencing components of the affinity-purified complex, we have identified Cef1p as a component of the Prp19p-associated complex, Ntc85p . Cef1p could directly interact with Prp19p and was required for pre-mRNA splicing both in vivo and in vitro . The c-Myb DNA binding motif at the amino terminus of Cef1p was required for cellular growth but not for interaction of Cef1p with Prp19p or Cef1p self-interaction . We have identified a small region of 30 amino acid residues near the carboxyl terminus required for both cell viability and protein-protein interactions . Cef1p was associated with the spliceosome in the same manner as Prp19p, i.e . concomitant with or immediately after dissociation of U4 . The anti-Cef1p antibody inhibited binding to the spliceosome of Cef1p, Prp19p, and at least three other components of the Prp19p-associated complex, suggesting that the Prp19p-associated complex is likely associated with the spliceosome and functions as an integral complex.

J Biol Chem, 1999 Apr 2, 274(14), 9169 - 74
Thrombin, a survival factor for cultured myoblasts; Chinni C et al.; Three members of the family of protease-activated receptors (PARs), PARs-1, -3 and -4, have been identified as thrombin receptors . PAR-1 is expressed by primary myoblast cultures, and expression is repressed once myoblasts fuse to form myotubes . The current study was undertaken to investigate the hypothesis that thrombin inhibits myoblast fusion . Primary rodent myoblast cultures were deprived of serum to promote myoblast fusion and then cultured in the presence or absence of thrombin . Thrombin inhibited myoblast fusion, but another notable effect was observed; 50% of control cells were apoptotic within 24 h of serum deprivation, whereas less than 15% of thrombin-treated cells showed signs of apoptosis . Proteolysis was required for the effect of thrombin, but no other serine protease tested mimicked the action of thrombin . Neither a PAR-1- nor a PAR-4-activating peptide inhibited apoptosis or fusion, and myoblast cultures were negative for PAR-3 expression . Myoblasts exposed to thrombin for 1 h and then changed to medium without thrombin accumulated apoptosis inhibitory activity in their medium over the subsequent 20 h . Thus the protective action of thrombin appears to be effected through cleavage of an unidentified thrombin receptor, leading to secretion of a downstream apoptosis inhibitory factor . These results demonstrate that thrombin functions as a survival factor for myoblasts and is likely to play an important role in muscle development and repair.

Biochem Biophys Res Commun, 1999 Apr 2, 257(1), 111 - 6
Characterization of a novel Ras-binding protein Ce-FLI-1 comprising leucine-rich repeats and gelsolin-like domains; Goshima M et al.; Ras proteins are conserved from yeasts to mammals and implicated in regulation of the actin cytoskeleton . The flightless-1 (fli-1) gene of Drosophila melanogaster and its homologs in Caenorhabditis elegans and humans encode proteins (FLI-1) comprising a fusion of a leucine-rich repeats (LRRs) domain and a gelsolin-like domain . This LRRs domain is highly homologous to those of three proteins involved in Ras-mediated signaling; Saccharomyces cerevisiae adenylyl cyclase, C . elegans SUR-8, and mammalian RSP-1 . Here we report that the LRRs domain of C . elegans FLI-1 (Ce-FLI-1) associates directly with Ras (Kd = 11 nM) and, when overexpressed, suppresses the heat shock sensitive phenotype of yeast cells bearing the activated RAS2 gene (RAS2(Val-19)) . Further, the gelsolin-like domain of Ce-FLI-1 is shown to possess a Ca2+-independent G-actin-binding activity as well as F-actin-binding and -severing activities . FLI-1 may be involved in regulation of the actin cytoskeleton through Ras .

Biochem Biophys Res Commun, 1999 Apr 2, 257(1), 100 - 5
Identification of NEDD8-conjugation site in human cullin-2; Wada H et al.; NEDD8 is a novel ubiquitin-like protein that has been shown to conjugate to nuclear proteins in a manner analogous to ubiquitination and sentrinization . Recently, human cullin-4A was reported to be conjugated by a single molecule of NEDD8 . Here, we show that human cullin-2 is also conjugated by a single molecule of the NEDD8 . The C-terminal 171-amino-acid residues in human cullin-2 are sufficient for NEDD8-conjugation . In addition, the equivalent C-terminal fragments of other cullins have been shown to be conjugated by NEDD8 . Mapping of the NEDD8-conjugation site revealed that Lys-689 in human cullin-2 is conjugated by NEDD8 . Interestingly, the Lys residue at position 689 in cullin-2 is conserved in all cullin family members, including human cullin-1, -2, -3, -4A, -4B, and -5 and yeast cullin (Cdc53), suggesting the possibility that other cullin family members are conjugated by NEDD8/Rub1 at a Lys residue of equivalent position .

Biochem Biophys Res Commun, 1999 Apr 2, 257(1), 12 - 8
Role of Suc1 in the activation of the cyclosome by protein kinase Cdk1/cyclin B; Shteinberg M et al.; A large complex, called the cyclosome or anaphase-promoting complex, has specific and regulated protein-ubiquitin ligase activity that targets mitotic regulators (such as cyclin B) for degradation at the end of mitosis . In early embryonic cell cycles the cyclosome is inactive in the interphase, but is subsequently converted by protein kinase Cdk1/cyclin B to an active, phosphorylated form, in a process that includes an initial lag period . This time lag may be important to prevent premature self-inactivation of Cdk1/cyclin B before the end of mitosis . We have previously observed that the phosphorylated form of the cyclosome binds to Suc1, a protein that associates with Cdk1 and with phosphate-containing compounds . We now report that low, physiological concentrations of Suc1 stimulate the activation of the interphase form of the cyclosome by the protein kinase . When Suc1 was present from the beginning of the incubation together with protein kinase Cdk1/cyclin B, activation of the cyclosome took place with the normal lag kinetics . However, when interphase cyclosome was first incubated with protein kinase Cdk1/cyclin B without Suc1, the subsequent addition of Suc1 caused a rapid burst of cyclosome activation and the lag was completely abolished . These findings are consistent with the interpretation that following initial slow phosphorylations of the cyclosome by the protein kinase, Suc1 accelerates multiple phosphorylations that culminate in the full activation of the cyclosome . In support of this interpretation, we find that Suc1 stimulates the phosphorylation of several proteins in the preparation of interphase cyclosome and that the effect of Suc1 on phosphorylation was augmented by prior incubation of interphase cyclosome with protein kinase Cdk1/cyclin B .

Plant Mol Biol, 1999 Feb, 39(3), 515 - 25
Characterization and functional analysis of Arabidopsis TFIIA reveal that the evolutionarily unconserved region of the large subunit has a transcription activation domain; Li YF et al.; TFIIA has initially been identified as a component of transcription initiation complex of RNA polymerase II . Its role in transcription has been controversial . In this paper, we report the characterization and functional analysis of both the Arabidopsis TFIIA large and small subunits . Sequence analysis revealed that Arabidopsis TFIIA is structurally more related to animal than to yeast counterparts . Arabidopsis has at least two genes for the large subunit and one for the small subunit . Both types of genes are constitutively transcribed in various plant organs . The proteins encoded by the cDNA interact each other in yeast 2-hybrid system . Only the N-terminal part of the large subunit is necessary for the interaction with the small subunit . Recombinant Arabidopsis TFIIA polypeptides bind to TBP-DNA complex in gel shift assays . The large subunit of TFIIA can stimulate transcription in yeast and in plant cells when fused to a DNA-binding domain binding to cis sequences upstream of a minimal promoter . This trans-activating activity is localized to a 35 amino acid segment within the evolutionarily unconserved central region.

Mol Reprod Dev, 1999 Apr, 52(4), 392 - 405
Tlk, a novel evolutionarily conserved murine serine threonine kinase, encodes multiple testis transcripts; Shalom S et al.; Hypothesizing that genes important in meiotic processes in mammals might have evolutionarily conserved counterparts in lower organisms, we used the yeast IME2 meiotic gene (serine threonine kinase) as a probe for screening a mouse testis cDNA library . This screening resulted in identification of a novel putative serine threonine kinase . Although it did not exhibit significant homology to IME2, it did show significant sequence homology to the Tousled kinase in Arabidopsis . Tousled is associated with various differentiative processes including differentiation of the reproductive organs . The new murine gene was designated accordingly Tlk (Tousled like kinase) . Tousled like kinase sequences have been reported to occur in C . elegans and in the human . Positive hybridization signals obtained in zooblot analysis suggest evolutionary conservation of Tlk throughout the phylogenetic ladder . Four distinct Tlk transcripts were detected in mouse testis, at least one of which is testis-specific . Northern and in situ hybridization analyses revealed that in normal testis, Tlk is expressed predominantly in pachytene spermatocytes and in round spermatids . Transcripts differ from one another in their 3' untranslated region, resulting from use of different polyadenylation sites, and in the length of their 5' region . Within the coding region, three of the putative peptides share the kinase and C-terminal domains but differ in their N-terminal domain, suggesting that the latter may be involved in the regulation of Tlk's function . We conclude that although Tlk might have an essential role in all tissues, these kinases are likely to take part in the complex array of phosphorylations involved in regulating spermatogenesis.

J Gen Virol, 1999 Mar, 80 ( Pt 3), 607 - 15
A cellular protein which binds hepatitis B virus but not hepatitis B surface antigen; Harvey TJ et al.; The envelope of hepatitis B virus (HBV) consists of three related proteins known as the large (L), middle (M) and small (S) hepatitis B surface antigens (HBsAg) . L-HBsAg has a 108-119 amino acid extension at the N terminus compared with M-HBsAg and contains the preS1 sequence of the HBV envelope . Previous research has identified this region as the likely virus attachment protein which is thought to interact with the cellular receptor for the virus . However, as the receptor has still not been identified unequivocally, we used the preS1 region of L-HBsAg to screen a human liver cDNA library by the yeast two-hybrid system . Several positive clones were isolated which encoded cellular proteins that interacted with the HBV preS1 protein . The specificity was examined in an independent manner in experiments in which baculovirus-derived glutathione S-transferase (GST)-preS1 was incubated with 35S-labelled protein expressed by in vitro translation from the positive clones . The intensity of the interactions using this alternative approach mirrored those observed in the yeast two-hybrid system and two proteins (an unidentified protein and a mitochondrial protein) were selected for further study . The specificity of the binding reaction between the preS1 protein and these two proteins was further confirmed in a competition assay; HBV purified from serum, but not purified HBsAg, was able to compete with preS1 and thus block GST-preS1 binding to the unidentified protein but not to the mitochondrial protein . The unidentified protein was then expressed as a fusion protein with GST and this was able to bind HBV virions in a direct manner.

Appl Microbiol Biotechnol, 1999 Feb, 51(2), 215 - 22
Cloning and characterization of an endo-beta-1,3(4)glucanase and an aspartic protease from Phaffia rhodozyma CBS 6938; Bang ML et al.; We describe the identification and expression cloning of two novel enzymes, a beta-glucanase and an aspartic protease, secreted from the basidiomycetous yeast Phaffia rhodozyma . A cDNA library from P . rhodozyma CBS 6938 was constructed, and full-length cDNA encoding an endo-1,3(4)-beta-glucanase (bg1) and an aspartic protease (pr1) were cloned by expression cloning in Saccharomyces cerevisiae W3124 . The bg1 cDNA encodes a 424-residue precursor protein with a putative signal peptide . The pr1 cDNA encodes a 405-residue prepropolypeptide with an 81-residue leader peptide . The aspartic protease was purified and characterized . It has a molecular mass of 36 kDa, an isoelectric point of pH 7.5, a pH activity optimum at 4.0-6.0, and a temperature activity optimum around 40 degrees C . Both enzymes show only low sequence identity to other known enzymes.

Biochemistry, 1999 Mar 23, 38(12), 3683 - 93
Mutation of a strictly conserved, active-site residue alters substrate specificity and cofactor biogenesis in a copper amine oxidase; Hevel JM et al.; The copper amine oxidases (CAOs) catalyze both the single-turnover modification of a peptidyl tyrosine to form the active-site cofactor 2,4,5-trihydroxyphenylalanine quinone (TPQ) and the oxidative deamination of primary amines using TPQ . The function of a strictly conserved tyrosine located within hydrogen-bonding distance to TPQ has been explored by employing site-directed mutagenesis on the enzyme from H . polymorpha to form the mutants Y305A, Y305C, and Y305F . Both Y305A and Y305C behave similarly with regard to aliphatic amine oxidase activity, showing 3-7-fold decreases in kinetic parameters relative to WT, while the more conservative substitution of Y305F results in a >100-fold decrease in kcat and >500-fold decrease in kcat/Km relative to WT for the reductive half-reaction . The oxidation of benzylamine by all three mutants is severely impaired, with very significant effects seen in the oxidative half-reaction . CAO activity was studied as a function of pH for WT and Y305A proteins . Profiles for WT-catalyzed methylamine oxidation and Y305A-catalyzed ethylamine oxidation are comparable, while profiles of Y305A-catalyzed methylamine oxidation suggest the pH-dependent build-up of an inhibitory intermediate, which was subsequently observed spectrophotometrically and is attributed to the product Schiff base . The relative effects of mutations at Y305 on catalytic turnover are, thus, concluded to be dependent on the nature of the amino acid which substitutes for tyrosine and the substrate used in amine oxidase assays . TPQ biogenesis experiments demonstrate a approximately 800-fold decrease in kobs for apo-Y305A compared to WT . Despite the strict conservation of Tyr305 in all CAOs, neither biogenesis nor catalytic turnover is abolished upon mutation of this residue . We propose an important, but nonessential, role for Tyr305 in the positioning of the TPQ precursor for biogenesis, and in the maintenance of the correct conformation for TPQ-derived intermediates during catalytic turnover.

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