S. cerevisiae

J Mol Biol, 2000 Mar 3, 296(4), 1105 - 16
Preformed secondary structure drives the association reaction of GCN4-p1, a model coiled-coil system; Zitzewitz JA et al.; The structure of the transition state for the rate-limiting step in the folding and association of the homodimeric coiled-coil peptide GCN4-p1, was probed by mutational analysis . A series of quadruple amino acid replacements that spanned the helix propensity scale were made at the four external f positions in the heptad repeat . Equilibrium and kinetic circular dichroism studies demonstrate that both the stability and the unfolding and refolding rate constants vary with helix propensity but also reflect interactions of the altered side-chains with their local environments . Pairwise replacements and fragment studies show that the two C-terminal heptads are the likely source of the nucleating helices . Helix-helix recognition between preformed elements of secondary structure plays an important role in this fundamental folding reaction .

Z Naturforsch {C}, 1999 Dec, 54(12), 1084 - 8
Carboxymethylated glucan inhibits lipid peroxidation in liposomes; Babincova M et al.; Protective capabilities were studied of carboxymethylated (1-->3)-beta-D-glucan from Saccharomyces cerevisiae cell wall against lipid peroxidation in phosphatidylcholine liposomes induced by OH radicals produced with Fenton's reagent (H2O2/Fe2+) and also by microwave radiation using absorption UV-VIS spectrophotometry . A significant decrease in the conjugated diene production, quantified as Klein oxidation index, was observed in the presence of a moderate amount of added glucan . Increase of the oxidation index was accompanied with enhanced carboxyfluorescein leakage as a result of liposome membrane destabilization . This process was markedly suppressed with glucan present in the liposome suspension . Therefore, glucan may be considered as a potent protector against microwave radiation-induced cell damage.

Biochemistry, 2000 Feb 15, 39(6), 1234 - 42
Structural and mechanistic basis for the activation of a low-molecular weight protein tyrosine phosphatase by adenine; Wang S et al.; Although the activation of low-molecular weight protein tyrosine phosphatases by certain purines and purine derivatives was first described three decades ago, the mechanism of this rate enhancement was unknown . As an example, adenine activates the yeast low-molecular weight protein tyrosine phosphatase LTP1 more than 30-fold . To examine the structural and mechanistic basis of this phenomenon, we have determined the crystal structure of yeast LTP1 complexed with adenine . In the crystal structure, an adenine molecule is found bound in the active site cavity, sandwiched between the side chains of two large hydrophobic residues at the active site . Hydrogen bonding to the side chains of other active site residues, as well as some water-mediated hydrogen bonds, also helps to fix the position of the bound adenine molecule . An ordered water was found in proximity to the bound phosphate ion present in the active site, held by hydrogen bonding to N3 of adenine and Odelta1 of Asp-132 . On the basis of the crystal structure, we propose that this water molecule is the nucleophile that participates in the dephosphorylation of the phosphoenzyme intermediate . Solvent isotope effect studies show that there is no rate-determining transfer of a solvent-derived proton in the transition state for the dephosphorylation of the phosphoenzyme intermediate . Such an absence of general base catalysis of water attack is consistent with the stability of the leaving group, namely, the thiolate anion of Cys-13 . Consequently, adenine activates the enzyme by binding and orienting a water nucleophile in proximity to the phosphoryl group of the phosphoenzyme intermediate, thus increasing the rate of the dephosphorylation step, a step that is normally the rate-limiting step of this enzymatic reaction.

Biochemistry, 2000 Feb 15, 39(6), 1223 - 33
Mutagenesis of E477 or K505 in the B' domain of human topoisomerase II beta increases the requirement for magnesium ions during strand passage; West KL et al.; A type II topoisomerase is essential for decatenating DNA replication products, and it accomplishes this task by passing one DNA duplex through a transient break in a second duplex . The B' domain of topoisomerase II contains three highly conserved motifs, EGDSA, PL(R/K)GK(I/L/M)LNVR, and IMTD(Q/A)DXD . We have investigated these motifs in topoisomerase II beta by mutagenesis, and report that they play a critical role in establishing the DNA cleavage-religation equilibrium . In addition, the mutations E477Q (EGDSA) and K505E (PLRGKILNVR) increase the optimal magnesium ion concentration for strand passage, without affecting the Mg(2+) dependence of ATP hydrolysis . It is likely that the binding affinity of the magnesium ion(s) specifically required for DNA cleavage has been reduced by these mutations . The crystal structure of yeast topo II indicates that residues E477 and K505 may help to position the three aspartate residues of the IMTD(Q/A)DXD motif for magnesium ion coordination, and we propose two possible locations for the magnesium ion binding site(s) . These observations are consistent with a previous model in which the B' domain is positioned such that these acidic residues lie next to the active site tyrosine residue . A magnesium ion bound by these aspartate residues could therefore mediate the DNA cleavage-religation reaction.

Biopolymers, 2000 Apr 5, 53(4), 293 - 307
Osmolyte-induced changes in protein conformational equilibria; Saunders AJ et al.; Examining solute-induced changes in protein conformational equilibria is a long-standing method for probing the role of water in maintaining protein stability . Interpreting the molecular details governing the solute-induced effects, however, remains controversial . We present experimental and theoretical data for osmolyte-induced changes in the stabilities of the A and N states of yeast iso-1-ferricytochrome c . Using polyol osmolytes of increasing size, we observe that osmolytes alone induce A-state formation from acid-denatured cytochrome c and N state formation from the thermally denatured protein . The stabilities of the A and N states increase linearly with osmolyte concentration . Interestingly, osmolytes stabilize the A state to a greater degree than the N state . To interpret the data, we divide the free energy for the reaction into contributions from nonspecific steric repulsions (excluded volume effects) and from binding interactions . We use scaled particle theory (SPT) to estimate the free energy contributions from steric repulsions, and we estimate the contributions from water-protein and osmolyte-protein binding interactions by comparing the SPT calculations to experimental data . We conclude that excluded volume effects are the primary stabilizing force, with changes in water-protein and solute-protein binding interactions making favorable contributions to stability of the A state and unfavorable contributions to the stability of the N state . The validity of our interpretation is strengthened by analysis of data on osmolyte-induced protein stabilization from the literature, and by comparison with other analyses of solute-induced changes in conformational equilibria .

Nucleic Acids Res, 2000 Mar 15, 28(6), 1407 - 17
A new double-stranded RNA-binding protein that interacts with PKR; Coolidge CJ et al.; We have identified a 74 kDa double-stranded (ds)RNA-binding protein that shares extensive homology with the mouse spermatid perinuclear RNA-binding (Spnr) protein . p74 contains two dsRNA-binding motifs (dsRBMs) that are essential for preferential binding to dsRNA . Previously, dsRNA-binding proteins were shown to undergo homo- and heterodimerization, raising the possibility that regulation of activity could be controlled by interactions between different family members . Homodimerization is required to activate the dsRNA-dependent protein kinase PKR, whereas hetero-dimerization between PKR and other dsRNA-binding proteins can inhibit kinase activity . We have found that p74 also interacts with PKR, both the wild-type enzyme and a catalytically defective mutant (K296R) . While co-expression of p74 and wild-type PKR in the yeast Saccharomyces cerevisiae did not alter PKR activity, co-expression of p74 and the catalytically defective K296R mutant surprisingly resulted in abnormal morphology and cell death in transformants that maintained a high level of p74 expression . These transformants could be rescued by overexpression of the alpha-subunit of wild-type eukaryotic translation initiation factor 2 (eIF2alpha), one of the known substrates for PKR . We hypothesize that competing heterodimers between p74-K296R PKR and eIF2alpha-K296R PKR may control cell growth such that stabilization of the p74-K296R PKR heterodimer induces abnormal morphology and cell death.

Nucleic Acids Res, 2000 Mar 15, 28(6), 1365 - 73
Assembly of archaeal signal recognition particle from recombinant components; Bhuiyan SH et al.; Signal recognition particle (SRP) takes part in protein targeting and secretion in all organisms . Searches for components of archaeal SRP in primary databases and completed genomes indicated that archaea possess only homologs of SRP RNA, and proteins SRP19 and SRP54 . A recombinant SRP was assembled from cloned, expressed and purified components of the hyperthermophilic archaeon Archaeoglobus fulgidus . Recombinant Af-SRP54 associated with the signal peptide of bovine pre-prolactin translated in vitro . As in mammalian SRP, Af-SRP54 binding to Af-SRP RNA required protein Af-SRP19, although notable amounts bound in absence of Af-SRP19 . Archaeoglobus fulgidus SRP proteins also bound to full-length SRP RNA of the archaeon Methanococcus jannaschii, to eukaryotic human SRP RNA, and to truncated versions which corresponded to the large domain of SRP . Dependence on SRP19 was most pronounced with components from the same species . Reconstitutions with heterologous components revealed a significant potential of human SRP proteins to bind to archaeal SRP RNAs . Surprisingly, M.jannaschii SRP RNA bound to human SRP54M quantitatively in the absence of SRP19 . This is the first report of reconstitution of an archaeal SRP from recombinantly expressed purified components . The results highlight structural and functional conservation of SRP assembly between archaea and eucarya.

IUBMB Life, 1999 Dec, 48(6), 593 - 9
5' to 3' single strand DNA exonuclease activity in a preparation of human Ku protein; Morozov VE et al.; We describe a novel 5' to 3' single-strand exonuclease activity exhibited by a Ku preparation purified from a human cell line . The enzyme removes 5' single-strand extensions from duplex DNA molecules . The exonuclease and helicase activities respond reciprocally to changes in ATP concentrations: Nuclease activity is inhibited at the ATP concentrations that are optimal for the helicase . The exonuclease activity does not require divalent cations . The potential implications of the exonuclease activity findings for repair of double-strand breaks and recombination processes are discussed.

FEBS Lett, 2000 Feb 18, 468(1), 101 - 4
Structure of the cytosolic domain of TOM5, a mitochondrial import protein; Hammen PK et al.; TOM5 is a small outer mitochondrial membrane protein in Saccharomyces cerevisiae and is part of a multi-protein translocator complex, which mediates protein import into mitochondria . Presently, nothing is known about the conformational preferences of TOM5 or other mitochondrial import proteins . In this report, circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy are used to determine the conformational preferences of the cytosolic domain of TOM5 . The CD spectra show evidence of a helical structure that is invariant with pH . NOESY data revealed that TOM5 forms a stable helical core between E11 and R15 with a less structurally rigid helix extending to the C-terminus.

Arch Biochem Biophys, 2000 Mar 1, 375(1), 138 - 44
Reactivity of the cysteine residues in the protein splicing active center of the Mycobacterium tuberculosis RecA intein; Shingledecker K et al.; Protein splicing involves the self-catalyzed excision of an intervening polypeptide segment, an intein, from a precursor protein . The first two steps in the protein splicing process lead to the formation of ester intermediates through nucleophilic attacks by the side chains of cysteine, serine, or threonine residues adjacent to the splice junctions . Since both nucleophilic residues in the Mycobacterium tuberculosis RecA intein are cysteine, their reactivities could be compared by sulfhydryl group titration . This was accomplished by using fusion proteins containing a truncated RecA intein modified by mutation to prevent protein splicing, in which the cysteines at the splice junctions were the only sulfhydryl groups . The ability to undergo hydroxylamine-induced cleavage at the upstream splice junction showed that the modified intein was not impaired in the ability to form ester intermediates . Sulfhydryl titration with iodoacetamide, monitored by quantitating the residual thiols after reaction with a maleimide derivative of biotin, revealed a striking difference in the apparent pK(a) values of the cysteines at the two splice junctions . The apparent pK(a) of the cysteine at the upstream splice junction, which initiates the N-S acyl rearrangement leading to the linear ester intermediate, was approximately 8.2, whereas that of the cysteine residue at the downstream splice junction, which initiates the transesterification reaction converting the linear ester to the branched ester intermediate, was about 5.8 . This suggests that the transesterification step is facilitated by an unusually low pK(a) of the attacking thiol group . Comparison of the rates of cleavage of the linear ester intermediates derived from the M . tuberculosis RecA and the Saccharomyces cerevisiae VMA inteins by dithiothreitol and hydroxylamine revealed that the former reacted relatively more slowly with dithiothreitol, suggesting that the RecA intein has diverged in the course of evolution to react preferentially with thiolate anions and thus lacks the basic groups that may facilitate nucleophilic attack by thiols in other inteins .

FEBS Lett, 2000 Jan 28, 466(2-3), 239 - 43
cDNA-derived amino acid sequence of acetoacetyl-CoA synthetase from rat liver; Iwahori A et al.; In order to examine the primary structure of acetoacetyl-CoA synthetase (acetoacetate-CoA ligase, EC 6.2.1.16; AA-CoA synthetase), the cDNA clone encoding this enzyme has been isolated from the cDNA library which was prepared from the liver of rat fed a diet supplemented with 4% cholestyramine and 0.4% pravastatin for 4 days . Nucleotide sequence analysis of cloned cDNA revealed that AA-CoA synthetase of rat liver contains an open reading frame of 2019 nucleotides, and the deduced amino acid sequence (672 amino acid residues) bears 25.0 and 38.9% homologies with acetyl-CoA synthetases of Saccharomyces cerevisiae and Archaeoglobus fulgidus, respectively.

J Biol Chem, 2000 Feb 25, 275(8), 6038 - 44
Metalloproteolytic release of endothelial cell protein C receptor; Xu J et al.; Previous studies observed that there is about 100 ng/ml soluble endothelial cell protein C receptor (EPCR) in human plasma and that the levels increase in inflammatory diseases . In this study we examine the potential mechanisms involved in release of EPCR from cells . We find that EPCR is released from the surface of endothelium and transfected 293 cells by a metalloprotease in a constitutive fashion . The mass of soluble EPCR is 4 kDa less than intact EPCR . Release is blocked by either the hydroxamic acid based inhibitor, KD-IX-73-4 or by 1,10-phenanthroline, but not by matrix metalloprotease inhibitors . Release is stimulated by phorbol 12-myristate 13-acetate, thrombin, interleukin-1beta, and hydrogen peroxide . Stimulation with these agents reduces EPCR expression levels sufficiently to decrease the rate of protein C activation to a limited extent . The influence of phorbol 12-myristate 13-acetate on both EPCR release and inhibition of protein C activation are enhanced by microtubule disruption with nocodazole . EPCR release is augmented by transfection of EPCR expressing 293 cells with caveolin, suggesting that release is caveolae dependent . These studies indicate that metalloproteolytic release of EPCR is a highly regulated process that is sensitive to both coagulation factors and inflammatory mediators.

J Biol Chem, 2000 Feb 25, 275(8), 5904 - 10
Association of human origin recognition complex 1 with chromatin DNA and nuclease-resistant nuclear structures; Tatsumi Y et al.; An origin recognition complex (ORC) consisting of six polypeptides has been identified as a DNA replication origin-binding factor in Saccharomyces cerevisiae . Homologues of ORC subunits have been discovered among eukaryotes, and we have prepared monoclonal antibodies against a human homologue of ORC1 (hORC1) to study its localization in human cells . It was thus found to associate with nuclei throughout the cell cycle and to be resistant to nonionic detergent treatment, in contrast to MCM proteins, which are other replication factors, the association of which with nuclei is clearly dependent on the phase of the cell cycle . A characteristic feature of hORC1 is dissociation by NaCl in a narrow concentration range around 0.25 M, suggesting interaction with some specific partner(s) in nuclei . Nuclease treatment experiments and UV cross-linking experiments further indicated interaction with both nuclease-resistant nuclear structures and chromatin DNA . Although its DNA binding was unaffected, some variation in the cell cycle was apparent, the association with nuclear structures being less stable in the M phase . Interestingly, the less stable association occurred concomitantly with hyperphosphorylation of hORC1, suggesting that this hyperphosphorylation may be involved in M phase changes.

J Biol Chem, 2000 Feb 25, 275(8), 5874 - 9
Dissecting the interactions between NTF2, RanGDP, and the nucleoporin XFXFG repeats; Chaillan-Huntington C et al.; We have used a range of complementary biochemical and biophysical methods to investigate the interactions between nuclear transport factor 2 (NTF2), the Ras family GTPase Ran, and XFXFG nucleoporin repeats that are crucial for nuclear trafficking . Microcalorimetry, microtiter plate binding, and fluorescence quenching in solution are all consistent with the binding constant for the NTF2-RanGDP interaction being in the 100 nM range, whereas the interaction between NTF2 and XFXFG repeat-containing nucleoporins such as Nsp1p is in the 1 microM range . Although the accumulation of NTF2 at the nuclear envelope is enhanced by RanGDP, we show that Ran binding does not alter the affinity of NTF2 for nucleoporins nor does the binding of nucleoporins alter the affinity of NTF2 for RanGDP . These results indicate that, instead, Ran increases the binding of NTF2 to nucleoporins by another mechanism, most probably by Ran itself binding to nucleoporins and NTF2 binding to this nuclear pore-associated Ran.

J Biol Chem, 2000 Feb 25, 275(8), 5767 - 72
The assembly factor Atp11p binds to the beta-subunit of the mitochondrial F(1)-ATPase; Wang ZG et al.; Atp11p is a protein of Saccharomyces cerevisiae required for the assembly of the F(1) component of the mitochondrial F(1)F(0)-ATP synthase . This study presents evidence that Atp11p binds selectively to the beta-subunit of F(1) . Under conditions in which avidin-Sepharose beads specifically adsorbed biotinylated Atp11p from yeast mitochondrial extracts, the F(1) beta-subunit coprecipitated with the tagged Atp11p protein . Binding interactions between Atp11p and the entire beta-subunit of F(1) or fragments of the beta-subunit were also revealed by a yeast two-hybrid screen: Atp11p bound to a region of the nucleotide-binding domain of the beta-subunit located between Gly(114) and Leu(318) . Certain elements of this sequence that would be accessible to Atp11p in the free beta-subunit make contact with adjacent alpha-subunits in the assembled enzyme . This observation suggests that the alpha-subunits may exchange for bound Atp11p during the process of F(1) assembly.

Proc Natl Acad Sci U S A, 2000 Feb 29, 97(5), 1988 - 92
An artificial transcriptional activating region with unusual properties; Lu X et al.; We describe a series of transcriptional activators generated by adding amino acids (eight in one case, six in another) to fragments of the yeast Saccharomyces cerevisiae activator Gal4 that dimerize and bind DNA . One of the novel activating regions identified by this procedure is unusual, compared with previously characterized yeast activating regions, in the following ways: it works more strongly than does Gal4's natural activating region as assayed in yeast; it is devoid of acidic residues; and several lines of evidence suggest that it sees targets in the yeast transcriptional machinery at least partially distinct from those seen by Gal4's activating region.

Proc Natl Acad Sci U S A, 2000 Feb 29, 97(5), 2011 - 6
Anatomy of a proficient enzyme: the structure of orotidine 5'-monophosphate decarboxylase in the presence and absence of a potential transition state analog; Miller BG et al.; Orotidine 5'-phosphate decarboxylase produces the largest rate enhancement that has been reported for any enzyme . The crystal structure of the recombinant Saccharomyces cerevisiae enzyme has been determined in the absence and presence of the proposed transition state analog 6-hydroxyuridine 5'-phosphate, at a resolution of 2.1 A and 2.4 A, respectively . Orotidine 5'-phosphate decarboxylase folds as a TIM-barrel with the ligand binding site near the open end of the barrel . The binding of 6-hydroxyuridine 5'-phosphate is accompanied by protein loop movements that envelop the ligand almost completely, forming numerous favorable interactions with the phosphoryl group, the ribofuranosyl group, and the pyrimidine ring . Lysine-93 appears to be anchored in such a way as to optimize electrostatic interactions with developing negative charge at C-6 of the pyrimidine ring, and to donate the proton that replaces the carboxylate group at C-6 of the product . In addition, H-bonds from the active site to O-2 and O-4 help to delocalize negative charge in the transition state . Interactions between the enzyme and the phosphoribosyl group anchor the pyrimidine within the active site, helping to explain the phosphoribosyl group's remarkably large contribution to catalysis despite its distance from the site of decarboxylation.

J Mass Spectrom, 2000 Feb, 35(2), 258 - 64
Mapping of surrogate markers of cellular components and structures using laser desorption/ionization mass spectrometry; Koomen JM et al.; Laser desorption/ionization mass spectrometry (LDI-MS) has been used to assess the potential of using surrogate markers, bound to cellular structures containing nucleic acids, to image or map the position of these structures within biological samples . In this study, organic dyes were used as markers because of their established use in the histochemical marking of nucleic acids, and also because they are amenable to LDI-MS . Eight cationic dyes were tested and all could be desorbed from nucleic acid samples without additional matrix after specifically binding to these molecules . Methylene Blue was the best of these based on its sensitivity to detection by LDI-MS and the fact that it can be washed from the tissue in areas where it was not specifically bound to provide low-intensity background signals . Experiments are reported which characterize the M(+) ion signal obtained from Methylene Blue with regard to sensitivity, reproducibility and possible use for quantitation . This dye was used to map (with a lateral resolution of 25 microm) several nucleic acid-containing samples spotted on prepared surfaces, and to image the location of nucleic acids in two model tissues, retinal vertical sections and thyroid whole mount sections .

Curr Biol, 2000 Feb 10, 10(3), 165 - 8
Lif1p targets the DNA ligase Lig4p to sites of DNA double-strand breaks; Teo SH et al.; DNA ligases catalyse the joining of DNA single- and double-strand breaks . Saccharomyces cerevisiae Cdc9p is a homologue of mammalian DNA ligase I and is required for DNA replication, recombination and single-strand break repair . The other yeast ligase, Lig4p/Dnl4p, is a homologue of mammalian DNA ligase IV, and functions in the non-homologous end-joining (NHEJ) pathway of DNA double-strand break repair {1} {2} {3} {4} . Lig4p interacts with Lif1p, the yeast homologue of the human ligase IV-associated protein, XRCC4 {5} . This interaction takes place through the carboxy-terminal domain of Lig4p and is required for Lig4p stability . We show that the carboxy-terminal interaction region of Lig4p is necessary for NHEJ but, when fused to Cdc9p, is insufficient to confer NHEJ function to Cdc9p . Also, Lif1p stimulates the in vitro catalytic activity of Lig4p in adenylation and DNA ligation . Nevertheless, Lig4p is inactive in NHEJ in the absence of Lif1p in vivo, even when Lig4p is stably expressed . We show that Lif1p binds DNA in vitro and, through in vivo cross-linking and chromatin immuno precipitation assays, demonstrate that it targets Lig4p to chromosomal DNA double-strand breaks . Furthermore, this targeting requires another key NHEJ protein, Ku.

Biochem Biophys Res Commun, 2000 Feb 24, 268(3), 704 - 10
The human PEX3 gene encoding a peroxisomal assembly protein: genomic organization, positional mapping, and mutation analysis in candidate phenotypes; Muntau AC et al.; In yeasts, the peroxin Pex3p was identified as a peroxisomal integral membrane protein that presumably plays a role in the early steps of peroxisomal assembly . In humans, defects of peroxins cause peroxisomal biogenesis disorders such as Zellweger syndrome . We previously reported data on the human PEX3 cDNA and its protein, which in addition to the peroxisomal targeting sequence contains a putative endoplasmic reticulum targeting signal . Here we report the genomic organization, sequencing of the putative promoter region, chromosomal localization, and physical mapping of the human PEX3 gene . The gene is composed of 12 exons and 11 introns spanning a region of approximately 40 kb . The highly conserved putative promoter region is very GC rich, lacks typical TATA and CCAAT boxes, and contains potential Sp1, AP1, and AP2 binding sites . The gene was localized to chromosome 6q23-24 and D6S279 was identified to be the closest positional marker . As yeast mutants deficient in PEX3 have been shown to lack peroxisomes as well as any peroxisomal remnant structures, human PEX3 is a candidate gene for peroxisomal assembly disorders . Mutation analysis of the human PEX3 gene was therefore performed in fibroblasts from patients suffering from peroxisome biogenesis disorders . Complementation groups 1, 4, 7, 8, and 9 according to the numbering system of Kennedy Krieger Institute were analyzed but no difference to the wild-type sequence was detected . PEX3 mutations were therefore excluded as the molecular basis of the peroxisomal defect in these complementation groups .

Biochem Biophys Res Commun, 2000 Feb 16, 268(2), 530 - 4
Human p55(CDC)/Cdc20 associates with cyclin A and is phosphorylated by the cyclin A-Cdk2 complex; Ohtoshi A et al.; The initiation of anaphase and exit from mitosis depend on the activation of the anaphase-promoting complex/cyclosome (APC/C), a multicomponent, ubiquitin-protein ligase . The WD-repeat protein called p55(CDC)(Cdc20) directly binds to and activates APC/C . By using yeast two-hybrid screening, we found that cyclin A, a critical cell cycle regulator in the S and G2/M phases, specifically interacts with p55(CDC) . Ectopically expressed p55(CDC) and cyclin A form a stable protein complex in mammalian cells . The p55(CDC)-cyclin A interaction occurs through the region containing the WD repeats of p55(CDC) and the region between the destruction box and the cyclin box of cyclin A . In addition to the physical interaction, p55(CDC) is phosphorylated by cyclin A-associated kinase . These findings suggest that the function of p55(CDC) is mediated or regulated by its complex formation with cyclin A .

Curr Opin Genet Dev, 2000 Feb, 10(1), 26 - 31
Complexity in the spindle checkpoint; Burke DJ; Cell viability requires accurate chromosome segregation at mitosis . The spindle checkpoint ensures that anaphase is not attempted until the sister chromatids of each chromosome are attached to spindle microtubules from opposite poles . The checkpoint mechanism involves a signal transduction cascade that is more complex than was originally envisioned.

Cell, 2000 Feb 4, 100(3), 333 - 43
Role of Sec61alpha in the regulated transfer of the ribosome-nascent chain complex from the signal recognition particle to the translocation channel; Song W et al.; Targeting of ribosome-nascent chain complexes to the translocon in the endoplasmic reticulum is mediated by the concerted action of the signal recognition particle (SRP) and the SRP receptor (SR) . Ribosome-stripped microsomes were digested with proteases to sever cytoplasmic domains of SRalpha, SRbeta, TRAM, and the Sec61 complex . We characterized protein translocation intermediates that accumulate when Sec61alpha or SRbeta is inactivated by proteolysis . In the absence of a functional Sec61 complex, dissociation of SRP54 from the signal sequence is blocked . Experiments using SR proteoliposomes confirmed the assembly of a membrane-bound posttargeting intermediate . These results strongly suggest that the Sec61 complex regulates the GTP hydrolysis cycle of the SRP-SR complex at the stage of signal sequence dissociation from SRP54.

EMBO J, 2000 Feb 15, 19(4), 683 - 90
The novel coactivator C1 (HCF) coordinates multiprotein enhancer formation and mediates transcription activation by GABP; Vogel JL et al.; Transcription of the herpes simplex virus 1 (HSV-1) immediate early (IE) genes is determined by multiprotein enhancer complexes . The core enhancer assembly requires the interactions of the POU-homeodomain protein Oct-1, the viral transactivator alphaTIF and the cellular factor C1 (HCF) . In this context, the C1 factor interacts with each protein to assemble the stable enhancer complex . In addition, the IE enhancer cores contain adjacent binding sites for other cellular transcription factors such as Sp1 and GA-binding protein (GABP) . In this study, a direct interaction of the C1 factor with GABP is demonstrated, defining the C1 factor as the critical coordinator of the enhancer complex assembly . In addition, mutations that reduce the GABP transactivation potential also impair the C1-GABP interaction, indicating that the C1 factor functions as a novel coactivator of GABP-mediated transcription . The interaction and coordinated assembly of the enhancer proteins by the C1 factor may be critical for the regulation of the HSV lytic-latent cycle.

EMBO J, 2000 Feb 15, 19(4), 672 - 82
A single point mutation in TFIIA suppresses NC2 requirement in vivo; Xie J et al.; Negative cofactor 2 (NC2) is a dimeric histone-fold complex that represses RNA polymerase II transcription through binding to TATA-box-binding protein (TBP) and inhibition of the general transcription factors TFIIA and TFIIB . Here we study molecular mechanisms of repression by human NC2 in vivo in yeast . Yeast NC2 genes are essential and can be exchanged with human NC2 . The physiologically relevant regions of NC2 have been determined and shown to match the histone-fold dimerization motif . A suppressor screen based upon limiting concentrations of NC2beta yielded a cold-sensitive mutant in the yeast TFIIA subunit Toa1 . The single point mutation in Toa1 alleviates the requirement for both subunits of NC2 . Biochemical characterization indicated that mutant (mt)-Toa1 dimerizes well with Toa2; it supports specific recognition of the TATA box by TBP but forms less stable TBP-TFIIA-DNA complexes . Wild-type but not the mt-Toa1 can relieve NC2 effects in purified transcription systems . These data provide evidence for a dimeric NC2 complex that is in an equilibrium with TFIIA after the initial binding of TBP to promoter TATA boxes.

EMBO J, 2000 Feb 15, 19(4), 581 - 8
Crystal structure of a class I alpha1,2-mannosidase involved in N-glycan processing and endoplasmic reticulum quality control; Vallee F et al.; Mannose trimming is not only essential for N-glycan maturation in mammalian cells but also triggers degradation of misfolded glycoproteins . The crystal structure of the class I alpha1, 2-mannosidase that trims Man(9)GlcNAc(2) to Man(8)GlcNAc(2 )isomer B in the endoplasmic reticulum of Saccharomyces cerevisiae reveals a novel (alphaalpha)(7)-barrel in which an N-glycan from one molecule extends into the barrel of an adjacent molecule, interacting with the essential acidic residues and calcium ion . The observed protein-carbohydrate interactions provide the first insight into the catalytic mechanism and specificity of this eukaryotic enzyme family and may be used to design inhibitors that prevent degradation of misfolded glycoproteins in genetic diseases.

FEBS Lett, 2000 Feb 11, 467(2-3), 359 - 64
The polyomavirus major capsid protein VP1 interacts with the nuclear matrix regulatory protein YY1; Palkova Z et al.; Polyomavirus reaches the nucleus in a still encapsidated form, and the viral genome is readily found in association with the nuclear matrix . This association is thought to be essential for viral replication . In order to identify the protein(s) involved in the virus-nuclear matrix interaction, we focused on the possible roles exerted by the multifunctional cellular nuclear matrix protein Yin Yang 1 (YY1) and by the viral major capsid protein VP1 . In the present work we report on the in vivo association between YY1 and VP1 . Using the yeast two-hybrid system we demonstrate that the VP1 and YY1 proteins physically interact through the D-E region of VP1 and the activation domain of YY1.

FEBS Lett, 2000 Feb 11, 467(2-3), 348 - 55
A family of ubiquitin-like proteins binds the ATPase domain of Hsp70-like Stch; Kaye FJ et al.; We have isolated two human ubiquitin-like (UbL) proteins that bind to a short peptide within the ATPase domain of the Hsp70-like Stch protein . Chap1 is a duplicated homologue of the yeast Dsk2 gene that is required for transit through the G2/M phase of the cell cycle and expression of the human full-length cDNA restored viability and suppressed the G2/M arrest phenotype of dsk2Delta rad23Delta Saccharomyces cerevisiae mutants . Chap2 is a homologue for Xenopus scythe which is an essential component of reaper-induced apoptosis in egg extracts . While the N-terminal UbL domains were not essential for Stch binding, Chap1/Dsk2 contains a Sti1-like repeat sequence that is required for binding to Stch and is also conserved in the Hsp70 binding proteins, Hip and p60/Sti1/Hop . These findings extend the association between Hsp70 members and genes encoding UbL sequences and suggest a broader role for the Hsp70-like ATPase family in regulating cell cycle and cell death events.

FEBS Lett, 2000 Feb 11, 467(2-3), 263 - 7
Expression of fatty acid-CoA ligase 4 during development and in brain; Cao Y et al.; Fatty acid utilization is initiated by fatty acid-CoA ligase, which converts free fatty acids into fatty acyl-CoA esters . We have cloned previously the human long-chain fatty acid-CoA ligase 4 (FACL4), which is a central enzyme in controlling the free arachidonic acid level in cells and thereby regulating eicosanoid production . We report here the expression of this gene in tissues, particularly in different parts of the brain . We found that FACL4 encoded a 75 kDa enzyme and that there was a modified translation product expressed in the brain . FACL4 was expressed in early stages of development with a significant amount of FACL4 mRNA detected in an E7 mouse embryo . In addition, FACL4 was highly expressed in both adult and newborn mouse brain especially in the granule cells of the dentate gyrus and the pyramidal cell layer of CA1 in hippocampus, and the granular cell layer and Purkinje cells of the cerebellum.

J Pept Sci, 2000 Jan, 6(1), 36 - 46
Synthesis of bivalent inhibitors of eucaryotic proteasomes; Loidl G et al.; Based on the peculiar spatial array of the active sites in the internal chamber of the multicatalytic proteasome, as derived from the X-ray structure of yeast proteasome, homo- and heterobivalent inhibitors were designed and synthesized to exploit the principle of multivalency for enhancing inhibition potency . Peptidic bis-aldehyde compounds of the octapeptide size were synthesized to address adjacent active sites, whilst a PEG spacer with a statistical length distribution of 19-25 monomers was used to link two identical or different tripeptide aldehydes as binding heads . These bis-aldehyde compounds were synthesized applying both methods in solution and solid phase peptide synthesis . Bivalent binding was observed only for the PEG-spaced inhibitors suggesting that binding from the primed side prevents hemiacetal formation with the active site threonine residue.

Biochem Biophys Res Commun, 2000 Jan 27, 267(3), 692 - 6
Interferon-alpha signaling promotes nucleus-to-cytoplasmic redistribution of p95Vav, and formation of a multisubunit complex involving Vav, Ku80, and Tyk2; Adam L et al.; Interferons (IFNs) are a family of hormone-like secretory proteins with multiple phenotypical changes, including gene expression and morphological alterations . Earlier studies have shown that IFN-activated Tyk2 kinase physical associates with p95Vav (Vav), a proto-oncogene gene product expressed in hematopoietic cells . Since Tyk2 is a cytoplasmic kinase and Vav is believed to be localized in the nuclear compartment, here we explored the possibility of Vav redistribution in IFN-alpha-activated cells, using the U266 human myeloma cell line as a model system . Using biochemical assays and in situ confocal microscopy, we demonstrate that IFN-alpha treatment triggers a rapid (10 min) translocation of Vav from the nuclear compartment to the cytoplasm . In addition, we also show the existence of IFN-alpha-induced physical interaction between Vav and Ku80, Ku80, and Tyk2, and among Vav, Ku80, and Tyk2 in the cytoplasmic compartment of IFN-stimulated cells . The observed IFN-alpha-induced association among Vav, Ku80, and Tyk2 was dependent on cellular tyrosine kinase activity . Since recently Vav has been shown to promote the GDP/GTP exchange activity of the cytoskeleton signaling molecule small GTPase Rac1 and activates its downstream signaling, our present findings raise the possibility of involvement of the small GTPase in IFN signaling leading to its biological effects, including cytoskeleton reorganization .

Exp Parasitol, 2000 Feb, 94(2), 75 - 83
Schistosoma mansoni: differential expression of cathepsins L1 and L2 suggests discrete biological functions for each enzyme; Brady CP et al.; Schistosoma mansoni cathepsins L1 (SmCL1) and L2 (SmCL2) were expressed as active recombinant proteinases in Saccharomyces cerevisiae . The recombinant enzymes exhibited substrate preferences characteristic of cathepsin-L-like cysteine proteinases . However, the enzymes differed in their substrate specificities; SmCL1 cleaved Boc-Val-Leu-Lys-NHMec with a higher efficiency than it cleaved Z-Phe-Arg-NHMec, whereas the opposite was true for SmCL2 . The enzymes also differed in their pH profiles of activity; SmCL1 exhibited a broad pH profile with an optimum of pH 6 . 5, while SmCL2 was active only in the acidic pH range with an optimum of 5.35 . Immunoblot and RT-PCR analyses revealed that the native forms of both SmCL1 and SmCL2 are expressed in male and female worms, but at higher levels in adult female compared to male schistosomes . Additionally, both enzymes were observed in the excretory/secretory products of adult worms . The RT-PCR analysis indicated that neither enzyme is expressed in S . mansoni eggs or in miracidia, suggesting that the cathepsin-L-like activity that has been previously reported to be expressed in these stages may be the product of another gene(s) . Cercariae do not express SmCL2, but appear to express SmCL1 in its inactive precursor form . Together with the findings of previous immunolocalization and phylogenetic analyses, the results reported here demonstrate that SmCL1 and SmCL2 are distinct cathepsin cysteine proteinases and strongly suggest that they play discrete biological roles .

Int Rev Immunol, 1999, 18(5-6), 429 - 48
Ribosomal proteins in cell proliferation and apoptosis; Chen FW et al.; Ribosomal proteins have the complex task of coordinating protein biosynthesis to maintain cell homeostasis and survival . Recent evidence suggests that a number of ribosomal proteins have secondary functions independent of their involvement in protein biosynthesis . A number of these proteins function as cell proliferation regulators and in some instances as inducers of cell death . Specifically, expression of human ribosomal protein L13a has been shown to induce apoptosis, presumably by arresting cell growth in the G2/M phase of the cell cycle . In addition, inhibition of expression of L13a induces apoptosis in target cells, suggesting that this protein is necessary for cell survival . Similar results have been obtained in the yeast Saccharomyces cerevisiae, where inactivation of the yeast homologues of L13a, rp22 and rp23, by homologous recombination results in severe growth retardation and death . In addition, a closely related ribosomal protein, L7, arrests cells in G1 and also induces apoptosis . Thus, it appears that a group of ribosomal proteins may function as cell cycle checkpoints and compose a new family of cell proliferation regulators.

Acta Virol, 1999 Feb, 43(1), 5 - 18
The "small" polydisperse cytoplasmic extrachromosomal DNA of chicken leukaemic myeloblasts and the avian myeloblastosis virus core-bound DNA seem to descend from origin regions of chromosomal DNA replication; Pajer P et al.; Nucleotide sequences are presented for 12, 7 and 12 cloned extrachromosomal DNAs by nature harbored in nucleoprotein (NP) complexes forming chicken leukaemic myeloblast (CHLM) post microsomal sediment (POMS) components A, B and C, respectively, and for 11 cloned avian myeloblastosis virus (AMV) DNAs . Analysis of the abundance of sequence motifs significant for eukaryotic chromosomal DNA replication origin (ori) regions (and their initiation zones) has shown that these DNAs are reminiscent of cell DNA fragments enriched in ori sequences (Rao et al., 1990) and/or sequence features of several eukaryotic chromosomal oris containing clusters of modular sequence elements (Dobbs et al., 1994) . Accordingly, these DNAs, with an (A + T) content prevalently higher than that of the total cell DNA, revealed the presence of asymmetrically distributed (A + T)-rich stretches, scaffold attachment region (SAR) T consensuses, polypyrimidine nucleotide (poly(Py)) tracts and minimal Saccharomyces cerevisiae autonomously replicating sequence (ARS) consensus, in abundance comparable with that of these sequences of DNA fragments enriched in oris . All these DNAs were found to be enriched also in sequence elements held as primase (Pr) attachment sites . Moreover, DNAs of POMS component B and those of AMV DNA were found to be enriched in the asymmetric pyrimidine (Py) heptanucleotide motif of Waltz et al . (1996) occurring in the initiation zones of ori region . Consequently, these extrachromosomal DNAs, portion of which represents a precursor of AMV DNA, seem to descent from initiation zones of various ori regions of an early replicating chromosomal myeloblast DNA . In addition, a possible explanation of the inclination of these DNAs to form multimers is presented.

Eur J Biochem, 2000 Feb, 267(4), 1008 - 18
High resolution solution structure of the protein part of Cu7 metallothionein; Bertini I et al.; The three-dimensional solution structure of the protein part of Cu7 metallothionein (Cu7MT) of Saccharomyces cerevisiae has been attempted by 1H two-dimensional NMR spectroscopy at 800 MHz . The protein part constitutes 53 amino acids . A total of 1192 NOEs, of which 1048 are meaningful, were used to determine the solution structure of the first 40 residues, the last 13 residues being disordered . A family of 30 structures was generated . Root-mean-square deviation (rmsd) values from the average structure of 0.32 +/- 0.13 A and 0.61 +/- 0.15 A for backbone and all heavy atoms, respectively, were obtained for the residues 2-40 . The ten copper-coordinating cysteine sulfurs and the empty spaces around them are well defined . The structure of the protein part is similar but not identical to the available ones of the same holoprotein and of the Ag7 metallothionein, and is qualitatively superior . If the same metal-sulfur connectivities reported in the literature from 1H-109Ag heteronuclear multiple quantum coherence spectroscopy are assumed to hold for the present copper derivative, a peptide structure is obtained which is again similar, but still not identical, within indetermination, to that available . The structure of the copper polymetallic center may well be different from that proposed for the silver derivative, and indeed a number of different arrangements of the seven copper ions are consistent with the present highly refined structure of the protein part.

J Biol Chem, 2000 Feb 18, 275(7), 4897 - 905
Binding and phosphorylation of a novel male germ cell-specific cGMP-dependent protein kinase-anchoring protein by cGMP-dependent protein kinase Ialpha; Yuasa K et al.; cGMP-dependent protein kinase (cGK) is a major cellular receptor of cGMP and plays important roles in cGMP-dependent signal transduction pathways . To isolate the components of the cGMP/cGK signaling pathway such as substrates and regulatory proteins of cGK, we employed the yeast two-hybrid system using cGK-Ialpha as a bait and isolated a novel male germ cell-specific 42-kDa protein, GKAP42 (42-kDa cGMP-dependent protein kinase anchoring protein) . Although the N-terminal region (amino acids 1-66) of cGK-Ialpha is sufficient for the association with GKAP42, GKAP42 could not interact with cGK-Ibeta, cGK-II, or cAMP-dependent protein kinase . GKAP42 mRNA is specifically expressed in testis, where it is restricted to the spermatocytes and early round spermatids . Endogenous cGK-I is co-immunoprecipitated with anti-GKAP42 antibody from mouse testis tissue, suggesting that cGK-I physiologically interacts with GKAP42 . Immunocytochemical observations revealed that GKAP42 is localized to the Golgi complex and that cGK-Ialpha is co-localized to the Golgi complex when coexpressed with GKAP42 . Although both cGK-Ialpha and -Ibeta, but not cAMP-dependent protein kinase, phosphorylated GKAP42 in vitro, GKAP42 was a good substrate only for cGK-Ialpha in intact cells, suggesting that the association with kinase protein is required for the phosphorylation in vivo . Finally, we demonstrated that the kinase-deficient mutant of cGK-Ialpha stably associates with GKAP42 and that binding of cGMP to cGK-Ialpha facilitates their release from GKAP42 . These findings suggest that GKAP42 functions as an anchoring protein for cGK-Ialpha and that cGK-Ialpha may participate in germ cell development through phosphorylation of Golgi-associated proteins such as GKAP42.

J Biol Chem, 2000 Feb 18, 275(7), 4827 - 33
ERO1-L, a human protein that favors disulfide bond formation in the endoplasmic reticulum; Cabibbo A et al.; Oxidizing conditions must be maintained in the endoplasmic reticulum (ER) to allow the formation of disulfide bonds in secretory proteins . Here we report the cloning and characterization of a mammalian gene (ERO1-L) that shares extensive homology with the Saccharomyces cerevisiae ERO1 gene, required in yeast for oxidative protein folding . When expressed in mammalian cells, the product of the human ERO1-L gene co-localizes with ER markers and displays Endo-H-sensitive glycans . In isolated microsomes, ERO1-L behaves as a type II integral membrane protein . ERO1-L is able to complement several phenotypic traits of the yeast thermosensitive mutant ero1-1, including temperature and dithiothreitol sensitivity, and intrachain disulfide bond formation in carboxypeptidase Y . ERO1-L is no longer functional when either one of the highly conserved Cys-394 or Cys-397 is mutated . These results strongly suggest that ERO1-L is involved in oxidative ER protein folding in mammalian cells.

J Biol Chem, 2000 Feb 18, 275(7), 4571 - 8
Identification of Cox20p, a novel protein involved in the maturation and assembly of cytochrome oxidase subunit 2; Hell K et al.; We have identified Cox20p, a 23.8-kDa protein of the mitochondrial inner membrane that is involved in the biogenesis of the yeast cytochrome oxidase complex . Cytochrome oxidase subunit 2 (Cox2p) accumulates as a precursor in cox20 mutants, suggesting a defect in biogenesis of this mitochondrially encoded protein . The inability of cox20 mutants to process the subunit 2 precursor (pCox2p) is not due to impaired export of the protein across the inner membrane or to an inactive Imp1p/Imp2p peptidase . Rather, Cox20p specifically binds the newly synthesized pCox2p, a step required to present the exported pCox2p as a substrate to the Imp1p peptidase . All of the endogenous pCox2p accumulated in an Deltaimp1 mutant, and a small fraction of Cox2p in wild type yeast, is detected in a complex with Cox20p . Following maturation Cox2p remained associated with Cox20p, prior to assembling into the cytochrome oxidase complex . We propose that Cox20p acts as a membrane-bound chaperone necessary for cleavage of pCox2p and for interaction of the mature protein with other subunits of cytochrome oxidase in a later step of the assembly process.

Mol Cell Biol, 2000 Mar, 20(5), 1772 - 83
Mouse A6/twinfilin is an actin monomer-binding protein that localizes to the regions of rapid actin dynamics; Vartiainen M et al.; In our database searches, we have identified mammalian homologues of yeast actin-binding protein, twinfilin . Previous studies suggested that these mammalian proteins were tyrosine kinases, and therefore they were named A6 protein tyrosine kinase . In contrast to these earlier studies, we did not find any tyrosine kinase activity in our recombinant protein . However, biochemical analysis showed that mouse A6/twinfilin forms a complex with actin monomer and prevents actin filament assembly in vitro . A6/twinfilin mRNA is expressed in most adult tissues but not in skeletal muscle and spleen . In mouse cells, A6/twinfilin protein is concentrated to the areas at the cell cortex which overlap with G-actin-rich actin structures . A6/twinfilin also colocalizes with the activated forms of small GTPases Rac1 and Cdc42 to membrane ruffles and to cell-cell contacts, respectively . Furthermore, expression of the activated Rac1(V12) in NIH 3T3 cells leads to an increased A6/twinfilin localization to nucleus and cell cortex, whereas a dominant negative form of Rac1(V12,N17) induces A6/twinfilin localization to cytoplasm . Taken together, these studies show that mouse A6/twinfilin is an actin monomer-binding protein whose localization to cortical G-actin-rich structures may be regulated by the small GTPase Rac1.

Mol Cell Biol, 2000 Mar, 20(5), 1537 - 45
Nck-interacting Ste20 kinase couples Eph receptors to c-Jun N-terminal kinase and integrin activation; Becker E et al.; The mammalian Ste20 kinase Nck-interacting kinase (NIK) specifically activates the c-Jun amino-terminal kinase (JNK) mitogen-activated protein kinase module . NIK also binds the SH3 domains of the SH2/SH3 adapter protein Nck . To determine whether Nck functions as an adapter to couple NIK to a receptor tyrosine kinase signaling pathway, we determined whether NIK is activated by Eph receptors (EphR) . EphRs constitute the largest family of receptor tyrosine kinases (RTK), and members of this family play important roles in patterning of the nervous and vascular systems . In this report, we show that NIK kinase activity is specifically increased in cells stimulated by two EphRs, EphB1 and EphB2 . EphB1 kinase activity and phosphorylation of a juxtamembrane tyrosine (Y594), conserved in all Eph receptors, are both critical for NIK activation by EphB1 . Although pY594 in the EphB1R has previously been shown to bind the SH2 domain of Nck, we found that stimulation of EphB1 and EphB2 led predominantly to a complex between NIK/Nck, p62(dok), RasGAP, and an unidentified 145-kDa tyrosine-phosphorylated protein . Tyrosine-phosphorylated p62(dok) most probably binds directly to the SH2 domain of Nck and RasGAP and indirectly to NIK bound to the SH3 domain of Nck . We found that NIK activation is also critical for coupling EphB1R to biological responses that include the activation of integrins and JNK by EphB1 . Taken together, these findings support a model in which the recruitment of the Ste20 kinase NIK to phosphotyrosine-containing proteins by Nck is an important proximal step in the signaling cascade downstream of EphRs.

J Mol Biol, 2000 Feb 18, 296(2), 421 - 33
Extensive central disruption of a four-way junction on binding CCE1 resolving enzyme; Declais AC et al.; Junction-resolving enzymes are nucleases that are selective for the structure of the four-way DNA junction that is important in genetic recombination . They exhibit selectivity for the structure of the junction, but they also manipulate the structure . Local disruption of DNA structure around the centre of the junction by CCE1 of Saccharomyces cerevisiae has been investigated using 2-aminopurine fluorescence . On binding CCE1, 2-aminopurine bases located at the point of strand exchange exhibit a large increase in fluorescence intensity (up to 39-fold enhancement), consistent with complete unstacking . This was observed for all positions around the centre of the junction, both 5' and 3' to the point of strand exchange . Thymine bases complementary to the modified adenine bases adjacent to the junction centre were strongly reactive to potassium permanganate . The results indicate that binding of CCE1 results in a complete unpairing of the four central base-pairs of the junction, with a lesser disruption of the next base-pairs .

DNA Cell Biol, 2000 Jan, 19(1), 9 - 18
C/EBPalpha is required to maintain postmitotic growth arrest in adipocytes; Tao H et al.; Terminal differentiation is often coupled with irreversible loss of proliferative potential . The CCAAT enhancer binding protein alpha (C/EBPalpha) preferentially accumulates in postmitotic, differentiated 3T3-L1 adipocytes but declines during tumor necrosis factor alpha (TNFalpha)-induced dedifferentiation . We have discovered that this decline in C/EBPalpha correlates with an increased mitotic growth potential . In order to further investigate the antimitotic activity of C/EBPalpha, we introduced antisense C/EBPalpha RNA into 3T3-L1 cells to block endogenous C/EBPalpha expression . When treated according to the standard differentiation protocol, stable cells lines harboring antisense C/EBPalpha RNA did not differentiate into fat-laden adipocytes, consistent with previous findings (Lin F, Lane MD, Genes Dev 1992;6:533-544) . We found that these undifferentiated cells expressing antisense-C/EBPalpha can reenter the cell cycle after mitogenic stimulation at a time in development when parental 3T3-L1 cells cannot . Moreover, the expression profiles of the growth-arrest-associated genes gas1 and gas2 revealed that the antisense C/EBPalpha-expressing cells withdrew from the cell cycle after the period of clonal expansion but failed to progress to the state of least proliferative potential characteristic of terminally differentiated adipocytes.

Nature, 2000 Jan 27, 403(6768), 451 - 6
DNA-bound structures and mutants reveal abasic DNA binding by APE1 and DNA repair coordination {corrected}; Mol CD et al.; Non-coding apurinic/apyrimidinic (AP) sites in DNA are continually created in cells both spontaneously and by damage-specific DNA glycosylases . The biologically critical human base excision repair enzyme APE1 cleaves the DNA sugar-phosphate backbone at a position 5' of AP sites to prime DNA repair synthesis . Here we report three co-crystal structures of human APE1 bound to abasic DNA which show that APE1 uses a rigid, pre-formed, positively charged surface to kink the DNA helix and engulf the AP-DNA strand . APE1 inserts loops into both the DNA major and minor grooves and binds a flipped-out AP site in a pocket that excludes DNA bases and racemized beta-anomer AP sites . Both the APE1 active-site geometry and a complex with cleaved AP-DNA and Mn2+ support a testable structure-based catalytic mechanism . Alanine substitutions of the residues that penetrate the DNA helix unexpectedly show that human APE1 is structurally optimized to retain the cleaved DNA product . These structural and mutational results show how APE1 probably displaces bound glycosylases and retains the nicked DNA product, suggesting that APE1 acts in vivo to coordinate the orderly transfer of unstable DNA damage intermediates between the excision and synthesis steps of DNA repair.

Cancer Res, 2000 Jan 15, 60(2), 238 - 41
Nickel compounds are novel inhibitors of histone H4 acetylation; Broday L et al.; Environmental factors influence carcinogenesis by interfering with a variety of cellular targets . Carcinogenic nickel compounds, although generally inactive in most gene mutation assays, induce chromosomal damage in heterochromatic regions and cause silencing of reporter genes when they are located near telomere or heterochromatin in either yeast or mammalian cells . We studied the effects of nickel on the lysine acetylation status of the NH2-terminal region of histone H4 . At nontoxic levels, nickel decreased the levels of histone H4 acetylation in vivo in both yeast and mammalian cells, affecting only lysine 12 in mammalian cells and all of the four lysine residues in yeast . In yeast, lysine 12 and 16 were more greatly affected than lysine 5 and 8 . Interestingly, a histidine Ni2+ anchoring site is found at position 18 from the NH2-terminal tail of H4 . Nickel was also found to inhibit the acetylation of H4 in vitro using purified recombinant histone acetyltransferase . To our knowledge, this is the first agent shown to decrease histone H4 acetylation at nontoxic levels.

J Theor Biol, 2000 Feb 21, 202(4), 273 - 82
Distributions of dimeric tandem repeats in non-coding and coding DNA sequences; Dokholyan NV et al.; We study the length distribution functions for the 16 possible distinct dimeric tandem repeats in DNA sequences of diverse taxonomic partitions of GenBank (known human and mouse genomes, and complete genomes of Caenorhabditis elegans and yeast) . For coding DNA, we find that all 16 distribution functions are exponential . For non-coding DNA, the distribution functions for most of the dimeric repeats have surprisingly long tails, that fit a power-law function . We hypothesize that: (i) the exponential distributions of dimeric repeats in protein coding sequences indicate strong evolutionary pressure against tandem repeat expansion in coding DNA sequences; and (ii) long tails in the distributions of dimers in non-coding DNA may be a result of various mutational mechanisms . These long, non-exponential tails in the distribution of dimeric repeats in non-coding DNA are hypothesized to be due to the higher tolerance of non-coding DNA to mutations . By comparing genomes of various phylogenetic types of organisms, we find that the shapes of the distributions are not universal, but rather depend on the specific class of species and the type of a dimer .

Arch Biochem Biophys, 2000 Feb 15, 374(2), 181 - 8
Isolation and characterization of a novel 530-kDa protein complex (PC530) capable of associating with the 20S proteasome from starfish oocytes; Tanaka E et al.; A novel protein complex called PC530 was purified concomitantly with proteasomes from oocytes of the starfish, Asterina pectinifera, by chromatography with DEAE-cellulose, phosphocellulose, Mono Q, and Superose 6 columns . The molecular mass of this complex was estimated to be 530 kDa by Ferguson plot analysis and about 500 kDa by Superose 6 gel filtration . Since the 1500-kDa proteasome fractions contain the PC530 subunits as well as the 20S proteasomal subunits, and also since the purified PC530 and the 20S proteasome were cross-linked with a bifunctional cross-linking reagent, it is thought that PC530 is able to associate with the 20S proteasome . The PC530 comprises six main subunits with molecular masses of 105, 70, 50, 34, 30, and 23 kDa . The 70-kDa subunit showed a sequence similarity to the S3/p58/Sun2/Rpn3p subunit of the 26S proteasome, whereas the other subunits showed little or no appreciable similarity to the mammalian and yeast regulatory subunits . These results indicate that starfish oocytes contain a novel 530-kDa protein complex capable of associating with the 20S proteasome, which is distinctly different from PA700 or the 19S regulatory complex in molecular size and subunit composition .

Nucleic Acids Res, 2000 Mar 1, 28(5), 1211 - 20
Poly(A)-binding protein I of Leishmania: functional analysis and localisation in trypanosomatid parasites; Bates EJ et al.; Regulation of gene expression in trypanosomatid parasites is predominantly post-transcriptional . Primary transcripts are trans-spliced and polyadenylated to generate mature mRNAs and transcript stability is a major factor controlling stage-specific gene expression . Degenerate PCR has been used to clone the gene encoding the Leishmania homologue of poly(A)-binding protein (Lm PAB1), as an approach to the identification of trans-acting factors involved in this atypical mode of eukaryotic gene expression . lmpab1 is a single copy gene encoding a 63 kDa protein which shares major structural features but only 35-40% amino acid identity with other PAB1 sequences, including those of other trypanosomatids . Lm PAB1 is expressed at constant levels during parasite differentiation and is phosphorylated in vivo . It is localised predominantly in the cytoplasm but inhibition of transcription with actinomycin D also reveals diffuse localisation in the nucleus . Lm PAB1 binds poly(A) with high specificity and affinity but fails to complement a null mutation in Saccharomyces cerevisiae . These properties are indicative of functional divergence in vivo.

J Virol, 2000 Mar, 74(5), 2372 - 82
A chimeric protein containing the N terminus of the adeno-associated virus Rep protein recognizes its target site in an in vivo assay; Cathomen T et al.; The Rep78 and Rep68 proteins of adeno-associated virus (AAV) type 2 are involved in DNA replication, regulation of gene expression, and targeting site-specific integration . They bind to a specific Rep recognition sequence (RRS) found in both the viral inverted terminal repeats and the AAVS1 integration locus on human chromosome 19 . Previous in vitro studies implied that an N-terminal segment of Rep is involved in DNA recognition, while additional domains might stabilize binding and mediate multimerization . In order to define the minimal requirements for Rep to recognize its target site in the human genome, we developed one-hybrid assays in which DNA-protein interactions are detected in vivo . Chimeric proteins consisting of the N terminus of Rep fused to different oligomerization motifs and a transcriptional activation domain were analyzed for oligomerization, DNA binding, and activation of reporter gene expression . Expression of reporter genes was driven from RRS motifs cloned upstream of minimal promoters and examined in mammalian cells from transfected plasmids and in Saccharomyces cerevisiae from a reporter cassette integrated into the yeast genome . Our results show for the first time that chimeric proteins containing the amino-terminal 244 residues of Rep are able to target the RRS in vitro and in vivo when incorporated into artificial multimers . These studies suggest that chimeric proteins may be used to harness the unique targeting feature of AAV for gene therapy applications.

Biosci Biotechnol Biochem, 1999 Dec, 63(12), 2082 - 90
Encoding of a cytochrome P450-dependent lauric acid monooxygenase by CYP703A1 specifically expressed in the floral buds of petunia hybrida; Imaishi H et al.; The cDNA clone of novel cytochrome P450 CYP703A1 from petunia floral buds was isolated by RT-PCR . The nucleotide sequences of this cDNA clone contained the open reading frame that has been predicted to encode polypeptides consisting of 539 amino acid residues . A significantly high level of the transcript of the cyp703A1 gene was found in the early stage of petunia flower buds, but not in the leaves, stems and roots . The 1041bp 5'-flanking sequences of the cyp703A1 gene contained the conserved motifs of ATHB-1, AGAMOUS, MYB.Ph3, P and SBF-1 binding boxes . CYP703A1 cDNA was expressed in yeast Saccharomyces cerevisiae AH22 cells under the control of an alcohol dehydrogenase I promoter and terminator . The recombinant yeast microsomes containing the CYP703A1 hemoprotein were found to metabolize lauric acid . Based on these results, CYP703A1 was specifically expressed in the early stage of flower development and appeared to participate in the monooxygenation of fatty acids.

FEBS Lett, 2000 Feb 4, 467(1), 111 - 6
The molecular chaperone Cdc37 is required for Ste11 function and pheromone-induced cell cycle arrest; Abbas-Terki T et al.; The molecular chaperone Cdc37 is thought to act in part as a targeting subunit of the heat-shock protein 90 (Hsp90) chaperone complex . We demonstrate here that Cdc37 is required for activity of the kinase Ste11 in budding yeast . A cdc37 mutant strain is defective in Ste11-mediated pheromone signaling and in accumulation and functional maturation of the constitutively active Ste11 version Ste11DeltaN . Moreover, Cdc37, Ste11DeltaN and Hsp90 coprecipitate pairwise . Thus, Hsp90 and Cdc37 may transiently associate with Ste11 to promote proper folding and/or association with additional regulatory factors . Our results establish Ste11 as the first endogenous Cdc37 client protein in yeast.

Plant Cell, 2000 Feb, 12(2), 279 - 90
The Arabidopsis NPR1/NIM1 protein enhances the DNA binding activity of a subgroup of the TGA family of bZIP transcription factors; Despres C et al.; The Arabidopsis NPR1 gene is essential in activating systemic, inducible plant defense responses . To gain a better understanding of NPR1 function, we conducted a yeast two-hybrid screening procedure and identified a differential interaction between NPR1 and all known members of the Arabidopsis TGA family of basic leucine zipper transcription factors . In the electrophoretic mobility shift assay, NPR1 substantially increased the binding of TGA2 to its cognate promoter element (as-1) as well as to a positive salicylic acid-inducible element (LS7) and a negative element (LS5) in the promoter of the pathogenesis-related PR-1 gene . Proteins encoded by npr1 mutants interacted poorly with TGA2 and did not substantially increase TGA2 binding to the as-1, LS5, or LS7 elements, thus establishing a link between the loss of disease resistance and the loss of TGA2 interaction and NPR1-enhanced DNA binding . Coupled with observations that the DNA binding activity of TGA factors is deregulated in npr1 plants, the results suggest that NPR1-mediated DNA binding of TGA2 is critical for activation of defense genes.

Plant Cell, 2000 Feb, 12(2), 237 - 48
The Cochliobolus carbonum SNF1 gene is required for cell wall-degrading enzyme expression and virulence on maize; Tonukari NJ et al.; The production of cell wall-degrading enzymes (wall depolymerases) by plant pathogenic fungi is under catabolite (glucose) repression . In Saccharomyces cerevisiae, the SNF1 gene is required for expression of catabolite-repressed genes when glucose is limiting . An ortholog of SNF1, ccSNF1, was isolated from the maize pathogen Cochliobolus carbonum, and ccsnf1 mutants of HC toxin-producing (Tox2(+)) and HC toxin-nonproducing (Tox2(-)) strains were created by targeted gene replacement . Growth in vitro of the ccsnf1 mutants was reduced by 50 to 95% on complex carbon sources such as xylan, pectin, or purified maize cell walls . Growth on simple sugars was affected, depending on the sugar . Whereas growth on glucose, fructose, or sucrose was normal, growth on galactose, galacturonic acid, maltose, or xylose was somewhat reduced, and growth on arabinose was strongly reduced . Production of HC toxin was normal in the Tox2(+) ccsnf1 mutant, as were conidiation, conidial morphology, conidial germination, and in vitro appressorium formation . Activities of secreted beta-1,3-glucanase, pectinase, and xylanase in culture filtrates of the Tox2(+) ccsnf1 mutant were reduced by 53, 24, and 65%, respectively . mRNA expression was downregulated under conditions that induced the following genes encoding secreted wall-degrading enzymes: XYL1, XYL2, XYL3, XYL4, XYP1, ARF1, MLG1, EXG1, PGN1, and PGX1 . The Tox2(+) ccsnf1 mutant was much less virulent on susceptible maize, forming fewer spreading lesions; however, the morphology of the lesions was unchanged . The Tox2(-) ccsnf1 mutant also formed fewer nonspreading lesions, which also retained their normal morphology . The results indicate that ccSNF1 is required for biochemical processes important in pathogenesis by C . carbonum and suggest that penetration is the single most important step at which ccSNF1 is required . The specific biochemical processes controlled by ccSNF1 probably include, but are not necessarily restricted to, the ability to degrade polymers of the plant cell wall and to take up and metabolize the sugars produced.

Virology, 2000 Feb 15, 267(2), 185 - 98
Multiple interactions among proteins encoded by the mite-transmitted wheat streak mosaic tritimovirus; Choi IR et al.; The genome organization of the mite-transmitted wheat streak mosaic virus (WSMV) appears to parallel that of members of the Potyviridae with monopartite genomes, but there are substantial amino acid dissimilarities with other potyviral polyproteins . To initiate studies on the functions of WSMV-encoded proteins, a protein interaction map was generated using a yeast two-hybrid system . Because the pathway of proteolytic maturation of the WSMV polyprotein has not been experimentally determined, random libraries of WSMV cDNA were made both in DNA-binding domain and activation domain plasmid vectors and introduced into yeast . Sequence analysis of multiple interacting pairs revealed that interactions largely occurred between domains within two groups of proteins . The first involved interactions among nuclear inclusion protein a, nuclear inclusion protein b, and coat protein (CP), and the second involved helper component-proteinase (HC-Pro) and cylindrical inclusion protein (CI) . Further immunoblot and deletion mapping analyses of the interactions suggest that subdomains of CI, HC-Pro, and P1 interact with one another . The two-hybrid assay was then performed using full-length genes of CI, HC-Pro, P1, P3, and CP, but no heterologous interactions were detected . In vitro binding assay using glutathione-S-transferase fusion proteins and in vitro translation products, however, revealed mutual interactions among CI, HC-Pro, P1, and P3 . The failure to detect interactions between full-length proteins by the two-hybrid assay might be due to adverse effects of expression of viral proteins in yeast cells . The capacity to participate in multiple homomeric and heteromeric molecular interactions is consistent with the pleiotropic nature of many potyviral gene mutants and suggests mechanisms for regulation of various viral processes via a network of viral protein complexes .

Biotechnol Prog, 2000 Jan-Feb, 16(1), 44 - 51
Macro approach and fuzzy modeling of entrapped biocatalyst; Ruggeri B et al.; The interactions between a strain of Saccharomyces cerevisiae and an alginate matrix are investigated to ascertain the main factors affecting the bioreaction evolution . During the tests several parameters (glucose, ethanol, calcium ion and biomass concentration, pH, and alginate bed diameter) were evaluated, coupled with microscopic investigation inside the beads to determine the spatial biomass distribution . A detailed analysis of macro parameters and a correlation among them are proposed using a fuzzy algorithm . A global two-step fuzzy model results in which biomass distribution inside the beads is represented as a hidden parameter.

J Cell Biol, 2000 Feb 7, 148(3), 465 - 80
Apg9p/Cvt7p is an integral membrane protein required for transport vesicle formation in the Cvt and autophagy pathways; Noda T et al.; In nutrient-rich, vegetative conditions, the yeast Saccharomyces cerevisiae transports a resident protease, aminopeptidase I (API), to the vacuole by the cytoplasm to vacuole targeting (Cvt) pathway, thus contributing to the degradative capacity of this organelle . When cells subsequently encounter starvation conditions, the machinery that recruited precursor API (prAPI) also sequesters bulk cytosol for delivery, breakdown, and recycling in the vacuole by the autophagy pathway . Each of these overlapping alternative transport pathways is specifically mobilized depending on environmental cues . The basic mechanism of cargo packaging and delivery involves the formation of a double-membrane transport vesicle around prAPI and/or bulk cytosol . Upon completion, these Cvt and autophagic vesicles are targeted to the vacuole to allow delivery of their lumenal contents . Key questions remain regarding the origin and formation of the transport vesicle . In this study, we have cloned the APG9/CVT7 gene and characterized the gene product . Apg9p/Cvt7p is the first characterized integral membrane protein required for Cvt and autophagy transport . Biochemical and morphological analyses indicate that Apg9p/Cvt7p is localized to large perivacuolar punctate structures, but does not colocalize with typical endomembrane marker proteins . Finally, we have isolated a temperature conditional allele of APG9/CVT7 and demonstrate the direct role of Apg9p/Cvt7p in the formation of the Cvt and autophagic vesicles . From these results, we propose that Apg9p/Cvt7p may serve as a marker for a specialized compartment essential for these vesicle-mediated alternative targeting pathways.

J Biol Chem, 2000 Feb 11, 275(6), 4311 - 22
PSK, a novel STE20-like kinase derived from prostatic carcinoma that activates the c-Jun N-terminal kinase mitogen-activated protein kinase pathway and regulates actin cytoskeletal organization; Moore TM et al.; Degenerate polymerase chain reaction against conserved kinase catalytic subdomains identified 15 tyrosine and serine-threonine kinases expressed in surgically removed prostatic carcinoma tissues, including six receptor kinases (PDGFBR, IGF1-R, VEGFR2, MET, RYK, and EPH-A1), six non-receptor kinases (ABL, JAK1, JAK2, TYK2, PLK-1, and EMK), and three novel kinases . Several of these kinases are oncogenic, and may function in the development of prostate cancer . One of the novel kinases is a new member of the sterile 20 (STE20) family of serine-threonine kinases which we have called prostate-derived STE20-like kinase (PSK) and characterized functionally . PSK encodes an open reading frame of 3705 nucleotides and contains an N-terminal kinase domain . Immunoprecipitated PSK phosphorylates myelin basic protein and transfected PSK stimulates MKK4 and MKK7 and activates the c-Jun N-terminal kinase mitogen-activated protein kinase pathway . Microinjection of PSK into cells results in localization of PSK to a vesicular compartment and causes a marked reduction in actin stress fibers . In contrast, C-terminally truncated PSK (1-349) did not localize to this compartment or induce a decrease in stress fibers demonstrating a requirement for the C terminus . Kinase-defective PSK (K57A) was unable to reduce stress fibers . PSK is the first member of the STE20 family lacking a Cdc42/Rac binding domain that has been shown to regulate both the c-Jun N-terminal kinase mitogen-activated protein kinase pathway and the actin cytoskeleton.

J Biol Chem, 2000 Feb 11, 275(6), 4081 - 91
Random mutagenesis and functional analysis of the Ran-binding protein, RanBP1; Petersen C et al.; Ran GTPase is required for nucleocytoplasmic transport of many types of cargo . Several proteins that recognize Ran in its GTP-bound state (Ran x GTP) possess a conserved Ran-binding domain (RanBD) . Ran-binding protein-1 (RanBP1) has a single RanBD and is required for RanGAP-mediated GTP hydrolysis and release of Ran from nuclear transport receptors (karyopherins) . In budding yeast (Saccharomyces cerevisiae), RanBP1 is encoded by the essential YRB1 gene; expression of mouse RanBP1 cDNA rescues the lethality of Yrb1-deficient cells . We generated libraries of mouse RanBP1 mutants and examined 11 mutants in vitro and for their ability to complement a temperature-sensitive yrb1 mutant (yrb1-51(ts)) in vivo . In 9 of the mutants, the alteration was a change in a residue (or 2 residues) that is conserved in all known RanBDs . However, 4 of these 9 mutants displayed biochemical properties indistinguishable from that of wild-type RanBP1 . These mutants bound to Ran x GTP, stimulated RanGAP, inhibited the exchange activity of RCC1, and rescued growth of the yrb1-51(ts) yeast cells . Two of the 9 mutants altered in residues thought to be essential for interaction with Ran were unable to rescue growth of the yrb1(ts) mutant and did not bind detectably to Ran in vitro . However, one of these 2 mutants (and 2 others that were crippled in other RanBP1 functions) retained some ability to co-activate RanGAP . A truncated form of RanBP1 (lacking its nuclear export signal) was able to complement the yrb1(ts) mutation . When driven from the YRB1 promoter, 4 of the 5 mutants most impaired for Ran binding were unable to rescue growth of the yrb1(ts) cells; remarkably, these mutants could nevertheless form ternary complexes with importin-5 or importin-beta and Ran-GTP . The same mutants stimulated only inefficiently RanGAP-mediated GTP hydrolysis of the Ran x GTP x importin-5 complex . Thus, the essential biological activity of RanBP1 in budding yeast correlates not with Ran x GTP binding per se or with the ability to form ternary complexes with karyopherins, but with the capacity to potentiate RanGAP activity toward GTP-bound Ran in these complexes.

J Biol Chem, 2000 Feb 11, 275(6), 3810 - 8
Identification of the transactivation domain of the transcription factor Sox-2 and an associated co-activator; Nowling TK et al.; The importance of interactions between Sox and POU transcription factors in the regulation of gene expression is becoming increasingly apparent . Recently, many examples of the involvement of Sox-POU partnerships in transcription have been discovered, including a partnership between Sox-2 and Oct-3 . Little is known about the mechanisms by which these factors modulate transcription . To better understand the molecular interactions involved, we mapped the location of the transactivation domain of Sox-2 . This was done in the context of its interaction with Oct-3, as well as its ability to transactivate as a fusion protein linked to the DNA-binding domain of Gal4 . Both approaches demonstrated that Sox-2 contains a transactivation domain in its C-terminal half, containing a serine-rich region and the C terminus . We also determined that the viral oncoprotein E1a inhibits the ability of the Gal4/Sox-2 fusion protein to transactivate, as well as the transcriptional activation mediated by the combined action of Sox-2 and Oct-3 . In contrast, a mutant form of E1a, unable to bind p300, lacks both of these effects . Importantly, we determined that p300 overcomes the inhibitory effects of E1a in both assays . Together, these findings suggest that Sox-2 mediates its effects, at least in part, through the co-activator p300.

Curr Biol, 2000 Jan 13, 10(1), 39 - 42
A novel Rad24 checkpoint protein complex closely related to replication factor C; Green CM et al.; Rad24 functions in the DNA damage checkpoint pathway of Saccharomyces cerevisiae . Here, analysis of Rad24 in whole cell extracts demonstrated that its mass was considerably greater than its predicted molecular weight, suggesting that Rad24 is a component of a protein complex . The Rad24 complex was purified to homogeneity . In addition to Rad24, the complex included polypeptides of 40 kDa and 35 kDa . The 40 kDa species was found by mass spectrometry to contain Rfc2 and Rfc3, subunits of replication factor C (RFC), a five subunit protein that is required for the loading of polymerases onto DNA during replication and repair {3} . We hypothesised that other RFC subunits, all of which share sequence homologles with Rad24, might also be components of the Rad24 complex . Reciprocal co-immunoprecipitation studies were performed using extracts prepared from strains containing epitope-tagged RFC proteins . These experiments showed that the small RFC proteins, Rfc2, Rfc3, Rfc4 and Rfc5, interacted with Rad24, whereas the Rfc1 subunit did not . We suggest that this RFC-like Rad24 complex may function as a structure-specific sensor in the DNA damage checkpoint pathway.

Mol Gen Genet, 2000 Jan, 262(6), 1147 - 56
SLG1 plays a role during G1 in the decision to enter or exit the cell cycle; Ivanovska I et al.; Saccharomyces cerevisiae cells decide to divide during G1 . If nutrients are abundant, cells pass through START and coordinately undergo DNA replication, bud emergence, and spindle pole body duplication . Phenotypic analysis of the slg1delta mutant revealed that this mutation uncouples post-START events . At the nonpermissive temperature, slg1delta cells that have undergone bud emergence but not DNA replication or SPB duplication accumulate . Furthermore, while wild-type cells arrest in GO when starved, the slg1delta mutant fails to arrest at this point; instead, cells with small buds accumulate . The slg1delta mutation displayed genetic interactions with cdc34, which encodes a regulator of exit from G1 . This is consistent with a role of SLG1 in G1 regulation . Epitope-tagged Slg1p cofractionated with the plasma membrane, suggesting that Slglp may function by integrating external cues and relaying them to the interior of the cell . We propose that SLG1 plays a regulatory role in bud emergence or stationary phase.

Mol Gen Genet, 2000 Jan, 262(6), 1001 - 11
Developmental and metabolic regulation of the phosphoglucomutase-encoding gene, pgmB, of Aspergillus nidulans; Hoffmann B et al.; We have isolated the pgmB gene from Aspergillus nidulans, which encodes a phosphoglucomutase, one of the key enzymes in carbohydrate metabolism . The pgmB gene is located on chromosome VII and its ORF encodes 557 amino acids . Mutant phenotypes were analysed by expression of high levels of pgmB antisense RNA, which lead to a loss of detectable levels of sense RNA . Under conditions of antisense RNA expression, a 30% reduction in the growth rate was observed in comparison to wild-type . On the enzyme level, expression of pgmB antisense RNA resulted in a 35% reduction in total phosphoglucomutase activity . Two pgmB mRNAs were observed under all conditions tested and differ with respect to the location of the poly(A) site . Expression of pgmB driven by the GAL1 promoter in Saccharomyces cerevisiae complemented the growth phenotype of a pgm2delta mutant strain and suppressed the sensitivity of a gcn4delta mutant strain to amino acid starvation in the presence of galactose . Cultivation of A . nidulans in the presence of glucose or galactose as carbon source did not affect transcription of pgmB . However, amino acid starvation conditions resulted in a six-fold reduction in the level of pgmB mRNA, while genes for amino acid biosynthesis showed increased transcription . Transcription of pgmB was low during hyphal growth and in the sexual phase of development, but was significantly increased during the asexual stage of the A . nidulans life cycle.

Genome, 1999 Dec, 42(6), 1077 - 87
Sequence and phylogenetic analysis of the SNF4/AMPK gamma subunit gene from Drosophila melanogaster; Yoshida EN et al.; To optimize gene expression under different environmental conditions, many organisms have evolved systems which can quickly up- and down-regulate the activity of other genes . Recently, the SNF1 kinase complex from yeast and the AMP-activated protein kinase complex from mammals have been shown to represent homologous metabolic sensors that are key to regulating energy levels under times of metabolic stress . Using heterologous probing, we have cloned the Drosophila melanogaster homologue of SNF4, the noncatalytic effector subunit from this kinase complex . A sequence corresponding to the partial genomic sequence as well as the full-length cDNA was obtained, and shows that the D . melanogaster SNF4 is encoded in a 1944-bp cDNA representing a protein of 648 amino acids (aa) . Southern analysis of Drosophila genomic DNA in concert with a survey of mammalian SNF4 ESTs indicates that in metazoans, SNF4 is a duplicated gene, and possibly even a larger gene family . We propose that one gene copy codes for a short (330 aa) protein, whereas the second locus codes for a longer version (<410 aa) that is extended at the carboxy terminus, as typified by the Drosophila homologue presented here . Phylogenetic analysis of yeast, invertebrate, and multiple mammalian isoforms of SNF4 shows that the gene duplication likely occurred early in the metazoan lineage, as the protein products of the different loci are relatively divergent . When the phylogeny was extended beyond the SNF4 gene family, SNF4 shares sequence similarity with other cystathionine-beta-synthase domain-containing proteins, including IMP dehydrogenase and a variety of uncharacterized Methanococcus proteins.

J Steroid Biochem Mol Biol, 1999 Dec 15, 71(3-4), 93 - 102
The nuclear-receptor interacting protein (RIP) 140 binds to the human glucocorticoid receptor and modulates hormone-dependent transactivation; Windahl SH et al.; The glucocorticoid receptor (GR) regulates target gene expression in response to corticosteroid hormones . We have investigated the mechanism of transcriptional activation by the GR by studying the role of the receptor interacting protein RIP140 . Both in vivo and in vitro protein-protein interaction assays revealed a ligand-dependent interaction between the GR and RIP140 . The ligand binding domain of the GR was sufficient for this interaction, while both the N- and C-terminal regions of RIP140 bound to the receptor . In a yeast transactivation assay RIP140 and SRC-1, a member of the steroid receptor coactivator family of proteins, both enhanced the transactivation activity of a GR protein (GRA-1) in which the potent N-terminal tau1 transactivation domain has been deleted . In contrast, in COS-7 cells increasing amounts of RIP140 significantly inhibited GRdeltatau1 function . In cotransfection studies in COS-7 cells, RIP140 also inhibited receptor activity in presence of both SRC-1 and the coactivator protein CBP together . Thus, in yeast cells a stimulation of receptor activity was observed, while in mammalian cells RIP140 repressed GR function . Taken together, these data suggest that, (1) RIP140 is a target protein for the GR and (2) RIP140 can modulate the transactivation activity of the receptor.

Anal Chem, 2000 Jan 15, 72(2), 267 - 75
A generic strategy to analyze the spatial organization of multi-protein complexes by cross-linking and mass spectrometry; Rappsilber J et al.; Most cellular functions are performed by multi-protein complexes . The identity of the members of such complexes can now be determined by mass spectrometry . Here we show that mass spectrometry can also be used in order to define the spatial organization of these complexes . In this approach, components of a protein complex are purified via molecular interactions using an affinity tagged member and the purified complex is then partially cross-linked . The products are separated by gel electrophoresis and their constituent components identified by mass spectrometry yielding nearest-neighbor relationships . In this study, a member of the yeast nuclear pore complex (Nup85p) was tagged and a six-member sub-complex of the pore was cross-linked and analyzed by 1D SDS-PAGE . Cross-linking reactions were optimized for yield and number of products . Analysis by MALDI mass spectrometry resulted in the identification of protein constituents in the cross-linked bands even at a level of a few hundred femtomoles . Based on these results, a model of the spatial organization of the complex was derived that was later supported by biological experiments . This work demonstrates, that the use of mass spectrometry is the method of choice for analyzing cross-linking experiments aiming on nearest neighbor relationships.

J Neurosci Res, 2000 Jan 1, 59(1), 94 - 9
GAL4/UAS-WGA system as a powerful tool for tracing Drosophila transsynaptic neural pathways; Tabuchi K et al.; Visualization of specific transsynaptic neural pathways is an indispensable technique for understanding the relationship between structure and function in the nervous system . Here, we demonstrate the application of the wheat germ agglutinin (WGA) transgene technique for tracing transsynaptic neural pathways in Drosophila . The intracellular localization of WGA was examined by immunoelectron microscopy . WGA signals were detected in granule-like structures in both the outer photoreceptor cells expressing WGA and the second-order laminar neurons . Misexpression of tetanus toxin (TNT), which inactivates N-synaptobrevin, in the outer photoreceptor cells resulted in the elimination of on/off transients in electroretinogram (ERG) recordings and in a great reduction in WGA transfer into laminar neurons, suggesting that anterograde WGA transsynaptic transfer is dependent mainly on synaptic transmission . Retrograde WGA transfer was also detected upon its forced expression in muscle cells . WGA primarily expressed in muscle cells was taken up by motoneuron axons and transported to their cell bodies in the ventral nerve cord, suggesting that WGA can trace motoneuronal pathways in combination with the muscle-specific GAL4 driver . Thus, the GAL4/UAS-WGA system should facilitate the dissection of the Drosophila neural circuit formation and/or synaptic activity in various regions and at various developmental stages.

J Neurosci Res, 2000 Jan 1, 59(1), 19 - 23
Localization and processing of CLN3, the protein associated to Batten disease: where is it and what does it do?
Pearce DA.
Although the CLN3 gene for Batten disease, the most common inherited neurovisceral storage disease of childhood, was identified in 1995, the function of the corresponding protein still remains elusive . A key to understanding the pathology of this devastating disease will be to elucidate the function of CLN3 at the molecular level . CLN3 has proven difficult to study, as it is predicted to be a membrane protein, and is of apparently low abundance in cells . Different groups have reported differing subcellular localization of CLN3 . The purpose of this review is to critically examine the various cell biological approaches undertaken to localize CLN3 and to piece together a potential function for CLN3 in neuronal cells . The most likely conclusion of this is that CLN3 is a lysosomal/endosomal protein that is trafficked through the endoplasmic reticulum (ER) and Golgi . Furthermore, studies are required to confirm whether CLN3 has a potential role in the recycling of synaptic vesicles through the endosome/lysosome.

Science, 2000 Feb 4, 287(5454), 873 - 80
Signaling and circuitry of multiple MAPK pathways revealed by a matrix of global gene expression profiles; Roberts CJ et al.; Genome-wide transcript profiling was used to monitor signal transduction during yeast pheromone response . Genetic manipulations allowed analysis of changes in gene expression underlying pheromone signaling, cell cycle control, and polarized morphogenesis . A two-dimensional hierarchical clustered matrix, covering 383 of the most highly regulated genes, was constructed from 46 diverse experimental conditions . Diagnostic subsets of coexpressed genes reflected signaling activity, cross talk, and overlap of multiple mitogen-activated protein kinase (MAPK) pathways . Analysis of the profiles specified by two different MAPKs-Fus3p and Kss1p-revealed functional overlap of the filamentous growth and mating responses . Global transcript analysis reflects biological responses associated with the activation and perturbation of signal transduction pathways.

Biochem J, 2000 Feb 15, 346 Pt 1, 63 - 70
Activation of exocytosis by cross-linking of the IgE receptor is dependent on ADP-ribosylation factor 1-regulated phospholipase D in RBL-2H3 mast cells: evidence that the mechanism of activation is via regulation of phosphatidylinositol 4,5-bisphosphate synthesis; Way G et al.; The physiological stimulus to exocytosis in mast cells is the cross-linking of the high-affinity IgE receptor, FcepsilonR1, with antigen . We demonstrate a novel function for ADP-ribosylation factor 1 (ARF1) in the regulation of antigen-stimulated secretion using cytosol-depleted RBL-2H3 mast cells for reconstitution of secretory responses . When antigen is used as the stimulus, ARF1 also reconstitutes phospholipase D activation . Using ethanol to divert the phosphatidic acid (the product of phospholipase D activity) to phosphatidylethanol causes inhibition of ARF1-reconstituted secretion . In addition . ARF1 causes an increase in phosphatidylinositol 4,5-bisphosphate (PIP(2)) levels at the expense of phosphatidylinositol 4-monophosphate . The requirement for PIP(2) in exocytosis was confirmed by using phosphatidylinositol transfer protein (PITPalpha) to increase PIP(2) levels . Exocytosis, restored by either ARF1 or PITPalpha, was inhibited when PIP(2) levels were depleted by phospholipase Cdelta1 . We conclude that the function of ARF1 and PITPalpha is to increase the local synthesis of PIP(2), the function of which in exocytosis is likely to be linked to lipid-protein interactions, whereby recruitment of key components of the exocytotic machinery are targeted to the appropriate membrane compartment.

Biochim Biophys Acta, 2000 Feb 14, 1470(1), R21 - 8
Keystone Symposia on Molecular and Cellular Biology: 'The molecular basis of cancer' . Organizers: Carol L . Prives and George F . Vande Woude, Taos, NM, 15-21 March 1999; Wang JY et al.; The Keystone Symposium on the Molecular Basis of Cancer was an excellent meeting, which stimulated the exchange of a great deal of information . This report was prepared to organize some of the results that provided new insights into the regulation of cell proliferation and apoptosis . We were unable to report on all of the talks and posters due mostly to our limited capacity to absorb and digest the large amount of results presented at the meeting . We apologize to those whose results were not covered in this report.

J Mol Biol, 2000 Feb 11, 296(1), 217 - 28
Measuring denatured state energetics: deviations from random coil behavior and implications for the folding of iso-1-cytochrome c; Godbole S et al.; The changes in the free energy of the denatured state of a set of yeast iso-1-cytochrome c variants with single surface histidine residues have been measured in 3 M guanidine hydrochloride . The thermodynamics of unfolding by guanidine hydrochloride is also reported . All variants have decreased stability relative to the wild-type protein . The free energy of the denatured state was determined in 3 M guanidine hydrochloride by evaluating the strength of heme-histidine ligation through determination of the pK(a) for loss of histidine binding to the heme . The data are corrected for the presence of the N-terminal amino group which also ligates to the heme under similar solution conditions . Significant deviations from random coil behavior are observed . Relative to a variant with a single histidine at position 26, residual structure of the order of -1.0 to -2.5 kcal/mol is seen for the other variants studied . The data explain the slower folding of yeast iso-1-cytochrome c relative to the horse protein . The greater number of histidines and the greater strength of ligation are expected to slow conversion of the histidine-misligated forms to the obligatory aquo-heme intermediate during the ligand exchange phase of folding . The particularly strong association of histidine residues at positions 54 and 89 may indicate regions of the protein with strong energetic propensities to collapse against the heme during early folding events, consistent with available data in the literature on early folding events for cytochrome c .

J Mol Biol, 2000 Jan 28, 295(4), 791 - 801
DNA polymerase switching: I . Replication factor C displaces DNA polymerase alpha prior to PCNA loading; Maga G et al.; An important not yet fully understood event in DNA replication is the DNA polymerase (pol) switch from pol alpha to pol delta . Indirect evidence suggested that the clamp loader replication factor C (RF-C) plays an important role, since a replication competent protein complex containing pol alpha, pol delta and RF-C could perform pol switching in the presence of proliferating cell nuclear antigen (PCNA) . By using purified pol alpha/primase, pol delta, RF-C, PCNA and RP-A we show that: (i) RF-C can inhibit pol alpha in the presence of ATP prior to PCNA loading, (ii) RF-C decreases the affinity of pol alpha for the 3'OH primer ends, (iii) the inhibition of pol alpha by RF-C is released upon PCNA loading, (iv) ATP hydrolysis is required for PCNA loading and subsequent release of inhibition of pol alpha, (v) under these conditions a switching from pol alpha/primase to pol delta is evident . Thus, RF-C appears to be critical for the pol alpha to pol delta switching . Based on these results, a model is proposed in which RF-C induces the pol switching by sequestering the 3'-OH end from pol alpha and subsequently recruiting PCNA to DNA .

Nat Struct Biol, 2000 Feb, 7(2), 161 - 6
Asparagine-mediated self-association of a model transmembrane helix; Choma C et al.; In membrane proteins, the extent to which polarity, hydrogen bonding, and van der Waals packing interactions of the buried, internal residues direct protein folding and association of transmembrane segments is poorly understood . The energetics associated with these various interactions should differ substantially between membrane versus water-soluble proteins . To help evaluate these energetics, we have altered a water-soluble, two-stranded coiled-coil peptide to render its sequence soluble in membranes . The membrane-soluble peptide associates in a monomer-dimer-trimer equilibrium, in which the trimer predominates at the highest peptide/detergent ratios . The oligomers are stabilized by a buried Asn side chain . Mutation of this Asn to Val essentially eliminates oligomerization of the membrane-soluble peptide . Thus, within a membrane-like environment, interactions involving a polar Asn side chain provide a strong thermodynamic driving force for membrane helix association.

Nat Struct Biol, 2000 Feb, 7(2), 154 - 60
Interhelical hydrogen bonding drives strong interactions in membrane proteins; Zhou FX et al.; Polar residues in transmembrane alpha-helices may strongly influence the folding or association of integral membrane proteins . To test whether a motif that promotes helix association in a soluble protein could do the same within a membrane, we designed a model transmembrane helix based on the GCN4 leucine zipper . We found in both detergent micelles and biological membranes that helix association is driven strongly by asparagine, independent of the rest of the hydrophobic leucine and/or valine sequence . Hydrogen bonding between membrane helices gives stronger associations than the packing of surfaces in glycophorin A helices, creating an opportunity to stabilize structures, but also implying a danger that non-specific interactions might occur . Thus, membrane proteins may fold to avoid exposure of strongly hydrogen bonding groups at their lipid exposed surfaces.

Nat Struct Biol, 2000 Feb, 7(2), 97 - 9
Are DEAD-box proteins becoming respectable helicases?
Linder P, Daugeron MC.
The vaccinia NPH-II RNA helicase, a member of the DEAD/DExH-box protein family, has been shown to be a processive, unidirectional RNA helicase with a step size of about one half turn of a helix . This finding demonstrates that RNA helicases can function as molecular motors.

Nat Struct Biol, 2000 Feb, 7(2), 91 - 4
Understanding membrane protein structure by design; Bowie JU; In contrast to soluble proteins, the primary interactions that specify and stabilize membrane protein structures are still largely a matter of speculation . Although van der Waals interactions have been gaining increasing favor as the dominant player, new results demonstrate the strength of hydrogen bonding in a membrane environment.

Proc Natl Acad Sci U S A, 2000 Feb 1, 97(3), 978 - 83
A relation between the principal axes of inertia and ligand binding; Foote J et al.; The principal axes of inertia are eigenvectors that can be calculated for any rigid body . We report studies of the position of the principal axes in crystallographically solved protein molecules . We find with high frequency that at least one principal axis penetrates the surface of the respective protein in a region used for ligand binding . In antibody variable regions, an axis goes through the third hypervariable loop of the heavy chain . In major histocompatibility complex proteins, an axis goes through the peptide-binding groove . In protein-protein heterodimers, a principal axis of one subunit will often penetrate the interface formed with the other subunit . In many of these protein-protein complexes, the axis specifically intersects residues known to be critical for molecular recognition.

Oncogene, 2000 Jan 20, 19(3), 444 - 51
c-Myb acetylation at the carboxyl-terminal conserved domain by transcriptional co-activator p300; Tomita A et al.; Transcription factor c-Myb plays important roles in cell survival and differentiation in immature hematopoietic cells . Here we demonstrate that c-Myb is acetylated at the carboxyl-terminal conserved domain by histone acetyltransferase p300 both in vitro and in vivo . The acetylation sites in vivo have been located at the lysine residues of the conserved domain (K471, K480, K485) by the use of the mutant Myb (Myb-KAmut), in which all three lysine residues are substituted into alanine . Electrophoretic mobility shift assay reveals that Myb-KAmut shows higher DNA binding activity than wild type c-Myb and that acetylation of c-Myb in vitro by p300 causes dramatic increase in DNA binding activity . Accordingly, transactivation activity of both mim-1 and CD34 promoters by Myb-KAmut is higher than that driven by wild type c-Myb . Furthermore, the bromodomain of p300, in addition to the histone acetyltransferase (HAT) domain, is required for effective acetylation of c-Myb, and hGCN5 is revealed to be a factor acetyl-transferase for c-Myb in vitro . We present a new manner of post-translational modification of the c-Myb protein and the potential significance of the acetylation in c-Myb.

EMBO J, 2000 Feb 1, 19(3), 463 - 71
The controlling role of ATM in homologous recombinational repair of DNA damage; Morrison C et al.; The human genetic disorder ataxia telangiectasia (A-T), caused by mutation in the ATM gene, is characterized by chromosomal instability, radiosensitivity and defective cell cycle checkpoint activation . DNA double-strand breaks (dsbs) persist in A-T cells after irradiation, but the underlying defect is unclear . To investigate ATM's interactions with dsb repair pathways, we disrupted ATM along with other genes involved in the principal, complementary dsb repair pathways of homologous recombination (HR) or non-homologous end-joining (NHEJ) in chicken DT40 cells . ATM(-/-) cells show altered kinetics of radiation-induced Rad51 and Rad54 focus formation . Ku70-deficient (NHEJ(-)) ATM(-/-) chicken DT40 cells show radiosensitivity and high radiation-induced chromosomal aberration frequencies, while Rad54-defective (HR(-)) ATM(-/-) cells show only slightly elevated aberration levels after irradiation, placing ATM and HR on the same pathway . These results reveal that ATM defects impair HR-mediated dsb repair and may link cell cycle checkpoints to HR activation.

EMBO J, 2000 Feb 1, 19(3), 421 - 33
The Bbp1p-Mps2p complex connects the SPB to the nuclear envelope and is essential for SPB duplication; Schramm C et al.; In budding yeast, microtubules are organized by the spindle pole body (SPB), which is embedded in the nuclear envelope via its central plaque structure . Here, we describe the identification of BBP1 in a suppressor screen with a conditional lethal allele of SPC29 . Bbp1p was detected at the central plaque periphery of the SPB and bbp1-1 cells were found to be defective in SPB duplication . bbp1-1 cells extend their satellite into a duplication plaque like wild-type cells; however, this duplication plaque then fails to insert properly into the nuclear envelope and does not assemble a functional inner plaque . This function in SPB duplication is probably fulfilled by a stable complex of Bbp1p and Mps2p, a nuclear envelope protein that is also essential for duplication plaque insertion . In addition, we found that Bbp1p interacts with Spc29p and the half-bridge component Kar1p . These interactions are likely to play a role in connecting the SPB with the nuclear envelope and the central plaque with the half-bridge.

EMBO J, 2000 Feb 1, 19(3), 317 - 23
Crystal structure of a fibrillarin homologue from Methanococcus jannaschii, a hyperthermophile, at 1.6 A resolution; Wang H et al.; Fibrillarin is a phylogenetically conserved protein essential for efficient processing of pre-rRNA through its association with a class of small nucleolar RNAs during ribosomal biogenesis . The protein is the antigen for the autoimmune disease scleroderma . Here we report the crystal structure of the fibrillarin homologue from Methanococcus jannaschii, a hyperthermophile, at 1.6 A resolution . The structure consists of two domains, with a novel fold in the N-terminal region and a methyltransferase-like domain in the C-terminal region . Mapping temperature-sensitive mutations found in yeast fibrillarin Nop1 to the Methanococcus homologue structure reveals that many of the mutations cluster in the core of the methyltransferase-like domain.

J Exp Zool, 2000 Feb 15, 286(3), 231 - 7
Enzyme activities involved in lipid metabolism during embryonic development of Macrobrachium borellii; Gonzalez-Baro MR et al.; The activities of the enzymatic systems involved in the activation and degradation of fatty acids, and in the synthesis of triacylglycerols and phospholipids were studied in vitro using total cellular homogenate and subcellular fractions of eggs of the shrimp Macrobrachium borellii at different developing stages . Egg development was divided into seven stages based on morphological features of the embryo . Palmitoyl-CoA ligase activity increased as the embryo developed and showed its maximum at stage V . An increase in the synthesis of triacylglycerols and diacylglycerols was also observed at this stage . Diacylglycerylethers were synthesized more actively during the first stages of development . The higher specific activity observed in total homogenate than in microsomal fraction suggested that their synthesis was not exclusively microsomal . Phospholipid synthesis was very active all along development, reflecting active membrane biosynthesis . The highest activity of the cytosolic triacylglycerol lipase was observed at stage V . Fatty acid degradation, measured as mitochondrial beta-oxidation activity, did not vary significantly during development . We conclude that both the anabolic and catabolic processes concerning lipid metabolism are very active, with values similar to those described for adult hepatopancreas, revealing the major role of lipids during shrimp embryogenesis energetics, and that the highest activities of lipid synthesis-hydrolysis take place at stage V when embryos are under active organogenesis . J . Exp . Zool . 286:231-237, 2000 .

J Mol Biol, 2000 Feb 4, 295(5), 1139 - 62
Reconstitution of the KRAB-KAP-1 repressor complex: a model system for defining the molecular anatomy of RING-B box-coiled-coil domain-mediated protein-protein interactions; Peng H et al.; The KRAB domain is a 75 amino acid residue transcriptional repression module commonly found in eukaryotic zinc-finger proteins . KRAB-mediated gene silencing requires binding to the corepressor KAP-1 . The KRAB:KAP-1 interaction requires the RING-B box-coiled coil (RBCC) domain of KAP-1, which is a widely distributed motif, hypothesized to be a protein-protein interface . Little is known about RBCC-mediated ligand binding and the role of the individual sub-domains in recognition and specificity . We have addressed these issues by reconstituting and characterizing the KRAB:KAP-1-RBCC interaction using purified components . Our results show that KRAB binding to KAP-1 is direct and specific, as the related RBCC domains from TIF1alpha and MID1 do not bind the KRAB domain . A combination of gel filtration, analytical ultracentrifugation, chemical cross-linking, non-denaturing gel electrophoresis, and site-directed mutagenesis techniques has revealed that the KAP-1-RBCC must oligomerize likely as a homo-trimer in order to bind the KRAB domain . The RING finger, B2 box, and coiled-coil region are required for oligomerization of KAP-1-RBCC and KRAB binding, as mutations in these domains concomitantly abolished these functions . KRAB domain binding stabilized the homo-oligomeric state of the KAP-1-RBCC as detected by chemical cross-linking and velocity sedimentation studies . Mutant KAP-1-RBCC molecules hetero-oligomerize with the wild-type KAP-1, but these complexes were inactive for KRAB binding, suggesting a potential dominant negative activity . Substitution of the coiled-coil region with heterologous dimerization, trimerization, or tetramerization domains failed to recapitulate KRAB domain binding . Chimeric KAP-1-RBCC proteins containing either the RING, RING-B box, or coiled-coil regions from MID1 also failed to bind the KRAB domain . The KAP-1-RBCC mediates a highly specific, direct interaction with the KRAB domain, and it appears to function as an integrated, possibly cooperative structural unit wherein each sub-domain contributes to oligomerization and/or ligand recognition . These observations provide the first principles for RBCC domain-mediated protein-protein interaction and have implications for identifying new ligands for RBCC domain proteins .

Pflugers Arch, 2000, 439(3 Suppl), R94 - 6
Interaction trap experiment with CDC6; Zavec PB et al.; CDC6 is an essential gene of yeast Saccharomyces cerevisiae . Although DNA sequence of the gene is available for a long time, biochemical function of Cdc6 protein in the cell cycle remains unclear . Using the interaction trap experiment we were looking for proteins interacting specifically with Cdc6 . Four gene products interacting with Cdc6 were detected . By sequence analysis we found that ECM11 codes for the protein involved in the cell wall synthesis, YNL201 codes for the protein of unknown function, probably involved in the carbon metabolism, YOR279 codes for protein of completely unknown function with no significant similarity with any known protein, and the interaction with Ty1 retrotransposition element was also found . The strongest interaction with Cdc6 bait measured as beta-galactosidase activity was observed with ECM11 and YNL201; YOR279 interacts slightly weaker . The weakest beta-galactosidase activity was obtained by Ty1A element . The strongest suppression of cdc6-1 mutation was observed by Ty1A element, the slight one with ECM11 and YNL201 but no suppression of thermosensitive mutation was detected for YOR279.

Trends Cell Biol, 2000 Feb, 10(2), 60 - 7
Turning on ARF: the Sec7 family of guanine-nucleotide-exchange factors; Jackson CL et al.; ARF proteins are important regulators of membrane dynamics and protein transport within the eukaryotic cell . The Sec7 domain is approximately 200 amino acids in size and stimulates guanine-nucleotide exchange on members of the ARF class of small GTPases . The members of one subclass of Sec7-domain proteins are direct targets of the secretion-inhibiting drug brefeldin A, which blocks the exchange reaction by trapping a reaction intermediate in an inactive, abortive complex . A separate subclass of Sec7-domain proteins is involved in signal transduction and possess a domain that mediates membrane binding in response to extracellular signals.

J Biol Chem, 2000 Feb 4, 275(5), 3377 - 81
The transcriptional co-activator ADA5 is required for HAC1 mRNA processing in vivo; Welihinda AA et al.; Accumulation of unfolded proteins in the endoplasmic reticulum (ER) activates signaling pathways to induce transcription of a number of genes encoding ER protein chaperones and-folding catalysts . In Saccharomyces cerevisiae this transcriptional induction is mediated by an increase in the synthesis of the transcription factor Hac1p . The transmembrane receptor Ire1p/Ern1p containing a Ser/Thr protein kinase and endoribonuclease activity transmits the unfolded protein response (UPR) from the ER to the nucleus . Activation of Ire1p kinase induces its endoribonuclease activity to cleave unspliced HAC1 mRNA and generate exon fragments that are subsequently ligated by tRNA ligase (RLG1) . Whereas unspliced HAC1 mRNA is poorly translated, spliced HAC1 mRNA is efficiently translated . Subunits of the yeast transcriptional co-activator complex SAGA also play a role in the UPR . Deletion of GCN5, ADA2, or ADA3 reduces, and deletion of ADA5 completely abolishes, the UPR . Although HAC1 mRNA requires only Ire1p and Rlg1p in vitro, we demonstrate that ADA5 is required for the IRE1/RLG1-dependent splicing reaction of HAC1 mRNA in vivo . In addition, Ada5p interacts with Ire1p . These results suggest that subcomponents of transcriptional co-activator complexes may be involved in RNA processing events.

J Biol Chem, 2000 Feb 4, 275(5), 3051 - 6
Molecular cloning and analysis of strictosidine beta-D-glucosidase, an enzyme in terpenoid indole alkaloid biosynthesis in Catharanthus roseus; Geerlings A et al.; Strictosidine beta-D-glucosidase (SGD) is an enzyme involved in the biosynthesis of terpenoid indole alkaloids (TIAs) by converting strictosidine to cathenamine . The biosynthetic pathway toward strictosidine is thought to be similar in all TIA-producing plants . Somewhere downstream of strictosidine formation, however, the biosynthesis diverges to give rise to the different TIAs found . SGD may play a role in creating this biosynthetic diversity . We have studied SGD at both the molecular and enzymatic levels . Based on the homology between different plant beta-glucosidases, degenerate polymerase chain reaction primers were designed and used to isolate a cDNA clone from a Catharanthus roseus cDNA library . A full-length clone gave rise to SGD activity when expressed in Saccharomyces cerevisiae . SGD shows approximately 60% homology at the amino acid level to other beta-glucosidases from plants and is encoded by a single-copy gene . Sgd expression is induced by methyl jasmonate with kinetics similar to those of two other genes acting prior to Sgd in TIA biosynthesis . These results show that coordinate induction of the biosynthetic genes forms at least part of the mechanism for the methyl jasmonate-induced increase in TIA production . Using a novel in vivo staining method, subcellular localization studies of SGD were performed . This showed that SGD is most likely associated with the endoplasmic reticulum, which is in accordance with the presence of a putative signal sequence, but in contrast to previous localization studies . This new insight in SGD localization has significant implications for our understanding of the complex intracellular trafficking of metabolic intermediates during TIA biosynthesis.

J Biol Chem, 2000 Feb 4, 275(5), 3037 - 41
Enkephalins are transported by a novel eukaryotic peptide uptake system; Hauser M et al.; We have identified an oligopeptide transporter in the yeast Saccharomyces cerevisiae which mediates the uptake of tetra- and pentapeptides, including the endogenous opioids leucine enkephalin (Tyr-Gly-Gly-Phe-Leu) and methionine enkephalin (Tyr-Gly-Gly-Phe-Met) . The transporter is encoded by the gene OPT1 . Yeast expressing OPT1 can utilize enkephalins to satisfy amino acid auxotrophic requirements for growth . The transport of radiolabeled leucine enkephalin exhibits saturable kinetics, with a K(m) of 310 microM . Transport activity is optimum at acidic pH and sensitive to reagents which uncouple oxidative phosphorylation, suggesting an energy dependence on the proton gradient . Growth, transport, and chromatographic data indicate that leucine enkephalin is not hydrolyzed in the extracellular medium and as such is translocated intact across the cell membrane . The system is specific for tetra- and pentapeptides and can be inhibited by the opioid receptor antagonists naloxone and naltrexone . To date, this is the first example of a eukaryotic transport system which can use enkephalins as a substrate, opening the possibility that a homologue exists in higher eukaryotes.

J Cell Sci, 2000 Feb, 113 ( Pt 4), 663 - 72
Expression and nuclear localization of BLM, a chromosome stability protein mutated in Bloom's syndrome, suggest a role in recombination during meiotic prophase; Moens PB et al.; Bloom's syndrome (BS) is a recessive human genetic disorder characterized by short stature, immunodeficiency and elevated risk of malignancy . BS cells have genomic instability and an increased frequency of sister chromatid exchange . The gene mutated in BS, BLM, encodes a 3'-5' helicase (BLM) with homology to bacterial recombination factor, RecQ . Human males homozygous for BLM mutations are infertile and heterozygous individuals display increased frequencies of structural chromosome abnormalities in their spermatozoa . Also, mutations in the Saccharomyces cerevisiae homolog of BLM, Sgs1, cause a delay in meiotic nuclear division and a reduction in spore viability . These observations suggest that BLM may play a role during meiosis . Our antibodies raised against the C terminus of the human protein specifically recognize both mouse and human BLM in western blots of cell lines and in successive developmental stages of spermatocytes, but fail to detect BLM protein in a cell line with a C-terminally truncated protein . BLM protein expression and location are detected by immunofluorescence and immunoelectron microscopy as discrete foci that are sparsely present on early meiotic prophase chromosome cores, later found abundantly on synapsed cores, frequently in combination with the recombinases RAD51 and DMC1, and eventually as pure BLM foci . The colocalization of RAD51/DMC1 with BLM and the statistically significant excess of BLM signals in the synapsed pseudoautosomal region of the X-Y chromosomes, which is a recombinational hot spot, provide indications that BLM protein may function in the meiotic recombination process.

Plant J, 1999 Dec, 20(6), 707 - 11
The different pH optima and substrate specificities of extracellular and vacuolar invertases from plants are determined by a single amino-acid substitution; Goetz M et al.; Different plant invertase isoenzymes are characterized by a single amino-acid difference in a conserved sequence, the WEC-P/V-D box . A proline residue is present in this sequence motif of extracellular invertase sequences, whereas a valine is found at the same position of vacuolar invertase sequences . The role of this distinct difference was studied by substituting the proline residue of extracellular invertase CIN1 from Chenopodium rubrum with a valine residue, by site-directed mutagenesis . The mutated gene was heterologously expressed in an invertase-deficient Saccharomyces cerevisiae strain . The single amino-acid difference was shown to be the molecular basis for two enzymatic properties of invertase isoenzymes, for both the pH optimum and the substrate specificity . A proline in the WEC-P/V-D box determines the more acidic pH optimum and the higher cleavage rate of raffinose of extracellular invertases, compared to vacuolar invertases that have a valine residue at this position.

Plant J, 1999 Dec, 20(6), 641 - 52
The tomato I-box binding factor LeMYBI is a member of a novel class of myb-like proteins; Rose A et al.; The RBCS3A gene of tomato belongs to a small gene family consisting of five members . Although the RBCS1, RBCS2 and RBCS3A promoters contain closely related cis regulatory sequences, the expression patterns of the genes are different . Whereas the RBCS1 and RBCS2 genes are expressed in both leaves and young fruit, the RBCS3A promoter is highly active in leaves, but not in young fruit . This lack of transcription could be due to a mutation in the RBCS3A promoter creating the so-called F-box, a protein binding site located between the activating cis elements, the I-box and G-box . In order to identify proteins that bind to the RBCS3A I-box/F-box region, the yeast one-hybrid system was used . One clone, LeMYBI was isolated which contains strong similarity to plant myb transcription factors . The encoded LeMYBI protein is at least 188 amino acids in length and contains two myb-like domains located at the amino terminus and close to the carboxy terminus, separated by a negatively charged domain . The protein contains a SHAQKYF amino acid signature motif in the second myb-like repeat, which is highly conserved in a number of recently identified plant myb-related genes, thus defining a new class of plant DNA-binding proteins . LeMYBI binds specifically to the I-box sequence of the RBCS1, RBCS2 and RBCS3A promoters, therefore representing the first cloned I-box binding factor . LeMYBI acts as a transcriptional activator in yeast and plants, and binds to the I-box with a DNA-binding domain located in the carboxyterminal domain.

Plant J, 1999 Dec, 20(5), 553 - 61
Plant interstitial telomere motifs participate in the control of gene expression in root meristems; Tremousaygue D et al.; The promoters of Arabidopsis eEF1A genes contain a telomere motif, the telo-box, associated with an activating sequence, the tef-box . Database searches indicated the presence of telo-boxes in the 5' region of numerous genes encoding components of the translational apparatus . By using several promoter constructs we demonstrate that the telo-box is required for the expression of a beta-glucoronidase gene in root primordia of transgenic Arabidopsis . This effect was observed when a telo-box was inserted upstream or downstream from the transcription initiation site, and occurred in synergy with the tef-box . These results clearly indicate that interstitial telomere motifs in plants are involved in control of gene expression . South-western screening of a lambdaZAP library with a double-stranded Arabidopsis telomere motif resulted in characterization of a protein related to the conserved animal protein Puralpha . The possibility of a regulation process similar to that achieved by the Rap1p in Saccharomyces cerevisiae is discussed.

Mol Microbiol, 2000 Jan, 35(2), 415 - 27
Fus3 controls Ty1 transpositional dormancy through the invasive growth MAPK pathway; Conte D Jr et al.; Fus3, the mitogen-activated protein kinase (MAPK) of the mating pheromone response pathway, inhibits a post-translational step of Ty1 retrotransposition . Fus3 also inhibits haploid invasive growth by blocking cross-activation of invasive growth gene expression by the pheromone response signal cascade . Here, we show that Fus3 kinase activity and dosage co-ordinately regulate Ty1 transposition and invasive growth . A chromosomal copy of the kinase-defective fus3-K42R allele fails to inhibit either Ty1 transposition or invasive growth . When overexpressed, kinase-defective Fus3 weakly inhibits both Ty1 transposition and invasive growth, but is much less inhibitory than wild-type Fus3 expressed at the same level . Moreover, increasing the dosage of wild-type Fus3 intensifies the inhibition of both Ty1 transposition and invasive growth . To demonstrate that Fus3 regulates Ty1 transposition via its negative regulation of the invasive growth pathway, we show by epistatic analysis that the invasive growth pathway transcription factors Ste12 and Tec1 are both required for Fus3-mediated inhibition of Ty1 transposition . When haploid invasive growth is stimulated by high-copy expression of TEC1, by expression of the dominant hypermorphic allele STE11-4 or by deletion of HOG1, Ty1 transposition is concomitantly activated . In summary, these results demonstrate that the haploid invasive growth pathway activates Ty1 transposition at both transcriptional and post-transcriptional levels and that Fus3 inhibits Ty1 transposition by inhibiting the invasive growth pathway.

Mol Microbiol, 2000 Jan, 35(2), 397 - 406
Induction of neutral trehalase Nth1 by heat and osmotic stress is controlled by STRE elements and Msn2/Msn4 transcription factors: variations of PKA effect during stress and growth; Zahringer H et al.; Saccharomyces cerevisiae neutral trehalase, encoded by NTH1, controls trehalose hydrolysis in response to multiple stress conditions, including nutrient limitation . The presence of three stress responsive elements (STREs, CCCCT) in the NTH1 promoter suggested that the transcriptional activator proteins Msn2 and Msn4, as well as the cAMP-dependent protein kinase (PKA), control the stress-induced expression of Nth1 . Here, we give direct evidence that Msn2/Msn4 and the STREs control the heat-, osmotic stress- and diauxic shift-dependent induction of Nth1 . Disruption of MSN2 and MSN4 abolishes or significantly reduces the heat- and NaCl-induced increases in Nth1 activity and transcription . Stress-induced increases in activity of a lacZ reporter gene put under control of the NTH1 promoter is nearly absent in the double mutant . In all instances, basal expression is also reduced by about 50% . The trehalose concentration in the msn2 msn4 double mutant increases less during heat stress and drops more slowly during recovery than in wild-type cells . This shows that Msn2/Msn4-controlled expression of enzymes of trehalose synthesis and hydrolysis help to maintain trehalose concentration during stress . However, the Msn2/Msn4-independent mechanism exists for heat control of trehalose metabolism . Site-directed mutagenesis of the three STREs (CCCCT changed to CATCT) in NTH1 promoter fused to a reporter gene indicates that the relative proximity of STREs to each other is important for the function of NTH1 . Elimination of the three STREs abolishes the stress-induced responses and reduces basal expression by 30% . Contrary to most STRE-regulated genes, the PKA effect on the induction of NTH1 by heat and sodium chloride is variable . During diauxic growth, NTH1 promoter-controlled reporter activity strongly increases, as opposed to the previously observed decrease in Nth1 activity, suggesting a tight but opposite control of the enzyme at the transcriptional and post-translational levels . Apparently, inactive trehalase is accumulated concomitant with the accumulation of trehalose . These results might help to elucidate the general connection between control by STREs, Msn2/Msn4 and PKA and, in particular, how these components play a role in control of trehalose metabolism.

Genes Cells, 2000 Jan, 5(1), 29 - 41
Regulation of interaction of the acetyltransferase region of p300 and the DNA-binding domain of Sp1 on and through DNA binding; Suzuki T et al.; BACKGROUND: The coactivator p300 acts as a transcriptional adaptor for many DNA-binding activators . The finding that p300 possesses intrinsic acetyltransferase activity which, by chemically modifying histone tails affects the nucleosomal environment and transcription, has greatly advanced our understanding of its function . Subsequent recent studies have shown that non-histone proteins are also acetylated . However, one central question which has remained unanswered is how the coactivator/acetyltransferase interacts with DNA-binding activators to modulate their actions . RESULTS: Here we have demonstrated physical and functional interaction between the acetyltransferase region of p300 and the DNA-binding domain (DBD) of the transcription factor Sp1 . This interaction stimulates DNA binding by the DBD of Sp1, which is mediated primarily by physical interaction rather than acetylation, despite acetylation of the DBD of Sp1 by the acetyltransferase region of p300 . Furthermore, DNA binding by the DBD of Sp1 inhibits both its association and acetylation by the acetyltransferase region of p300 . CONCLUSIONS: These findings suggest a new role for p300 in regulating promoter access by DNA-binding activators through multiple regulatory interactions.

Genes Cells, 2000 Jan, 5(1), 1 - 8
Cell cycle mechanisms of sister chromatid separation; roles of Cut1/separin and Cut2/securin; Yanagida M; The correct transmission of chromosomes from mother to daughter cells is fundamental for genetic inheritance . Separation and segregation of sister chromatids in growing cells occurs in the cell cycle stage called 'anaphase' . The basic process of sister chromatid separation is similar in all eukaryotes: many gene products required are conserved . In this review, the roles of two proteins essential for the onset of anaphase in fission yeast, Cut2/securin and Cut1/separin, are discussed with regard to cell cycle regulation, and compared with the postulated roles of homologous proteins in other organisms . Securin, like mitotic cyclins, is the target of the anaphase promoting complex (APC)/cyclosome and is polyubiquitinated before destruction in a manner dependent upon the destruction sequence . The anaphase never occurs properly in the absence of securin destruction . In human cells, securin is an oncogene . Separin is a large protein (MW approximately 180 kDa), the C-terminus of which is conserved, and is thought to be inhibited by association with securin at the nonconserved N-terminus . In the budding yeast, Esp1/separin is thought to be a component of proteolysis against Scc1, an essential subunit of cohesin which is thought to link duplicated sister chromatids up to the anaphase . Whether fission yeast Cut1/separin is also implicated in proteolysis of cohesin is discussed.

Biochemistry, 2000 Feb 1, 39(4), 712 - 7
The first 28 N-terminal amino acid residues of human heart muscle carnitine palmitoyltransferase I are essential for malonyl CoA sensitivity and high-affinity binding; Shi J et al.; Heart/skeletal muscle carnitine palmitoyltransferase I (M-CPTI) is 30-100-fold more sensitive to malonyl CoA inhibition than the liver isoform (L-CPTI) . To determine the role of the N-terminal region of human heart M-CPTI on malonyl CoA sensitivity and binding, a series of deletion mutations were constructed ranging in size from 18 to 83 N-terminal residues . All of the deletions except Delta83 were active . Mitochondria from the yeast strains expressing Delta28 and Delta39 exhibited a 2.5-fold higher activity compared to the wild type, but were insensitive to malonyl CoA inhibition and had complete loss of high-affinity malonyl CoA binding . The high-affinity site (K(D1), B(max1)) for binding of malonyl CoA to M-CPTI was completely abolished in the Delta28, Delta39, Delta51, and Delta72 mutants, suggesting that the decrease in malonyl CoA sensitivity observed in these mutants was due to the loss of the high-affinity binding entity of the enzyme . Delta18 showed only a 4-fold loss in malonyl CoA sensitivity but had activity and high-affinity malonyl CoA binding similar to the wild type . Replacement of the N-terminal domain of L-CPTI with the N-terminal domain of M-CPTI does not change the malonyl CoA sensitivity of the chimeric L-CPTI, suggesting that the amino acid residues responsible for the differing sensitivity to malonyl CoA are not located in this N-terminal region . These results demonstrate that the N-terminal residues critical for activity and malonyl CoA sensitivity in M-CPTI are different from those of L-CPTI.

Biochemistry, 2000 Feb 1, 39(4), 701 - 11
Role of highly conserved residues in the reaction catalyzed by recombinant Delta7-sterol-C5(6)-desaturase studied by site-directed mutagenesis; Taton M et al.; The role of 15 residues in the reaction catalyzed by Arabidopsis thaliana Delta7-sterol-C5(6)-desaturase (5-DES) was investigated using site-directed mutagenesis and expression of the mutated enzymes in an erg3 yeast strain defective in 5-DES . The mutated desaturases were assayed in vivo by sterol analysis and quantification of Delta5,7-sterols . In addition, the activities of the recombinant 5-DESs were examined directly in vitro in the corresponding yeast microsomal preparations . One group of mutants was affected in the eight evolutionarily conserved histidine residues from three histidine-rich motifs . Replacement of these residues by leucine or glutamic acid completely eliminated the desaturase activity both in vivo and in vitro, in contrast to mutations at seven other conserved residues . Thus, mutants H203L, H222L, H222E, P201A, G234A, and G234D had a 5-DES activity reduced to 2-20% of the wild-type enzyme, while mutants K115L, P175V, and P175A had a 5-DES activity and catalytical efficiency (V/K) that was similar to that of the wild-type . Therefore, these residues are not essential for the catalysis but contribute to the activity through conformational or other effects . One possible function for the histidine-rich motifs would be to provide the ligands for a presumed catalytic Fe center, as previously proposed for a number of integral membrane enzymes catalyzing desaturations and hydroxylations {Shanklin et al . (1994) Biochemistry 33, 12787-12794} . Another group of mutants was affected in residue 114 based on previous in vivo observations in A . thaliana indicating that mutant T114I was deficient in 5-DES activity . We show that the enzyme T114I has an 8-fold higher Km and 10-fold reduced catalytic efficiency . Conversely, the functionally conservative substituted mutant enzyme T114S displays a 28-fold higher Vmax value and an 8-fold higher Km value than the wild-type enzyme . Consequently, V/K for T114S was 38-fold higher than that for T114I . The data suggest that Thr 114 is involved in stabilization of the enzyme-substrate complex with a marked discrimination between the ground-state and the transition state of a rate-controlling step in the catalysis by the 5-DES.

Crit Rev Eukaryot Gene Expr, 1999, 9(3-4), 231 - 43
Histone acetyltransferase complexes and their link to transcription; Howe L et al.; Early studies revealing the relationship between the state of histone acetylation and gene transcription were largely indirect . Increasing information regarding the enzymes that catalyze transcription linked acetylation is beginning to clarify this issue . This review attempts to relate previous data regarding the distribution of histone acetylation within different chromatin regions with recent data regarding the substrate specificity, subunit composition, and recruitment of the known histone acetyltransferase complexes.

Science, 2000 Jan 28, 287(5453), 661 - 4
Creating a protein-based element of inheritance; Li L et al.; Proteins capable of self-perpetuating changes in conformation and function (known as prions) can serve as genetic elements . To test whether novel prions could be created by recombinant methods, a yeast prion determinant was fused to the rat glucocorticoid receptor . The fusion protein existed in different heritable functional states, switched between states at a low spontaneous rate, and could be induced to switch by experimental manipulations . The complete change in phenotype achieved by transferring a prion determinant from one protein to another confirms the protein-only nature of prion inheritance and establishes a mechanism for engineering heritable changes in phenotype that should be broadly applicable.

Biotechniques, 2000 Jan, 28(1), 102 - 7
Fifty-one kilobase HSV-1 plasmid vector can be packaged using a helper virus-free system and supports expression in the rat brain; Wang X et al.; Herpes simplex virus type 1 (HSV-1) plasmid vectors have a number of attractive features for gene transfer into neurons . In particular, the large size of the HSV-1 genome suggests that HSV-1 vectors might be designed to accommodate large inserts . We now report the construction and characterization of a 51 kb HSV-1 plasmid vector . This vector was efficiently packaged into HSV-1 particles using a helper virus-free packaging system . The structure of the packaged vector DNA was verified by both Southern blot and PCR analyses . A vector stock was microinjected into the rat striatum, the rats were sacrificed at 4 days after gene transfer, and numerous X-gal positive striatal cells were observed . This 51 kb vector was constructed using general principles that may support the routine construction of large vectors . Potential applications of such HSV-1 vectors include characterizing large promoter fragments or genomic clones and co-expressing multiple genes.

Bioessays, 2000 Jan, 22(1), 48 - 56
clk-1, mitochondria, and physiological rates; Branicky R et al.; Mutations in the C . elegans maternal-effect gene clk-1 are highly pleiotropic, affecting the duration of diverse developmental and behavioral processes . They result in an average slowing of embryonic and post-embryonic development, adult rhythmic behaviors, reproduction, and aging.(1) CLK-1 is a highly conserved mitochondrial protein,(2,3) but even severe clk-1 mutations affect mitochondrial respiration only slightly.(3) Here, we review the evidence supporting the regulatory role of clk-1 in physiological timing . We also discuss possible models for the action of CLK-1, in particular, one proposing that CLK-1 is involved in the coordination of mitochondrial and nuclear function . BioEssays 22:48-56, 2000 .

Biotechnol Bioeng, 2000 Mar 5, 67(5), 616 - 22
Alginate hydrogel microspheres and microcapsules prepared by spinning disk atomization; Senuma Y et al.; Our spinning disk atomization (SDA) can, relative to other existing techniques, produce micron-sized particles with very narrow size distribution . The aim of this work is to present this technology for the production of alginate microspheres and microcapsules . We atomized and gelled aqueous alginate solutions into very narrowly dispersed microspheres with sizes ranging from 300 to 600 microm . Here, the interest is to produce, at high rate, particles of a given size with a narrow size distribution and also to show a new method of encapsulation using SDA . The viscosity and flow rate contributions in the drop formation is qualitatively analyzed to show how they affect droplet size . In addition, a technique for high degree of encapsulation is presented in which yeast is used as a model system . The production of yeast-loaded microspheres by SDA shows the potential of the technique for biotechnology applications .

Biochemistry (Mosc), 1999 Dec, 64(12), 1382 - 90
Prions: infectious proteins with genetic properties; Ter-Avanesyan MD et al.; The data on prions--proteinaceous infectious agents--are briefly summarized . Prions cause several incurable neurodegenerative diseases in mammals, while in lower eukaryotes the prion properties of proteins may be responsible for the inheritance of some phenotypic traits . The novel experimental models for finding and studying proteins with prion properties based on the yeast Saccharomyces cerevisiae and the fungus Podospora anserina are described . The significance of the prion phenomenon for biology and medicine is discussed.

Biochemistry (Mosc), 1999 Dec, 64(12), 1367 - 72
Novel function of the eukaryotic polypeptide-chain releasing factor 3 (eRF3/GSPT) in the mRNA degradation pathway; Hoshino S et al.; The mammalian GTP-binding protein GSPT, whose carboxy-terminal sequence is homologous to the eukaryotic elongation factor EF1alpha, binds to the polypeptide chain releasing factor eRF1 to function as eRF3 in translation termination . However, the amino-terminal domain of GSPT, which contains a prion-like sequence, is not required for the binding . Instead, the amino-terminal domain is capable of binding to the carboxy-terminal domain of polyadenylate-binding protein (PABP), whose amino terminus is associating with the poly(A) tail of mRNAs, presumably for their stabilization . Interestingly, multimerization of PABP with poly(A), which is ascribed to the action of its carboxy-terminal domain, was completely inhibited by the interaction with the amino-terminal domain of GSPT . This may facilitate shortening of the poly(A) tail of mRNAs by an RNase . Thus, GSPT/eRF3 appears to function not only as a stimulator of eRF1 in the translation termination but also as an initiator of the mRNA degradation machinery . Further physiological and cell biological approaches will be necessary to show whether our current in vitro findings on GSPT/eRF3 indeed reflect its bifunctional properties in living cells.

FEBS Lett, 2000 Jan 21, 466(1), 183 - 6
Identification of the two histidine residues responsible for the inhibition by malonyl-CoA in peroxisomal carnitine octanoyltransferase from rat liver; Morillas M et al.; Carnitine octanoyltransferase (COT), an enzyme that facilitates the transport of medium chain fatty acids through peroxisomal membranes, is inhibited by malonyl-CoA . cDNAs encoding full-length wild-type COT and one double mutant variant from rat peroxisomal COT were expressed in Saccharomyces cerevisiae . Both expressed forms were expressed similarly in quantitative terms and exhibited full enzyme activity . The wild-type-expressed COT was inhibited by malonyl-CoA like the liver enzyme . The activity of the enzyme encoded by the double mutant H131A/H340A was completely insensitive to malonyl-CoA in the range assayed (2-200 microM) . These results indicate that the two histidine residues, H131 and H340, are the sites responsible for inhibition by malonyl-CoA . Another mutant variant, H327A, abolishes the enzyme activity, from which it is concluded that it plays an important role in catalysis.

Mol Cell Biol, 2000 Feb, 20(4), 1321 - 8
Regulatory interactions between the Reg1-Glc7 protein phosphatase and the Snf1 protein kinase; Sanz P et al.; Protein phosphatase 1, comprising the regulatory subunit Reg1 and the catalytic subunit Glc7, has a role in glucose repression in Saccharomyces cerevisiae . Previous studies showed that Reg1 regulates the Snf1 protein kinase in response to glucose . Here, we explore the functional relationships between Reg1, Glc7, and Snf1 . We show that different sequences of Reg1 interact with Glc7 and Snf1 . We use a mutant Reg1 altered in the Glc7-binding motif to demonstrate that Reg1 facilitates the return of the activated Snf1 kinase complex to the autoinhibited state by targeting Glc7 to the complex . Genetic evidence indicated that the catalytic activity of Snf1 negatively regulates its interaction with Reg1 . We show that Reg1 is phosphorylated in response to glucose limitation and that this phosphorylation requires Snf1; moreover, Reg1 is dephosphorylated by Glc7 when glucose is added . Finally, we show that hexokinase PII (Hxk2) has a role in regulating the phosphorylation state of Reg1, which may account for the effect of Hxk2 on Snf1 function . These findings suggest that the phosphorylation of Reg1 by Snf1 is required for the release of Reg1-Glc7 from the kinase complex and also stimulates the activity of Glc7 in promoting closure of the complex.

Mol Cell Biol, 2000 Feb, 20(4), 1194 - 205
RAD51 is required for the repair of plasmid double-stranded DNA gaps from either plasmid or chromosomal templates; Bartsch S et al.; DNA double-strand breaks may be induced by endonucleases, ionizing radiation, chemical agents, and mechanical forces or by replication of single-stranded nicked chromosomes . Repair of double-strand breaks can occur by homologous recombination or by nonhomologous end joining . A system was developed to measure the efficiency of plasmid gap repair by homologous recombination using either chromosomal or plasmid templates . Gap repair was biased toward gene conversion events unassociated with crossing over using either donor sequence . The dependence of recombinational gap repair on genes belonging to the RAD52 epistasis group was tested in this system . RAD51, RAD52, RAD57, and RAD59 were required for efficient gap repair using either chromosomal or plasmid donors . No homologous recombination products were recovered from rad52 mutants, whereas a low level of repair occurred in the absence of RAD51, RAD57, or RAD59 . These results suggest a minor pathway of strand invasion that is dependent on RAD52 but not on RAD51 . The residual repair events in rad51 mutants were more frequently associated with crossing over than was observed in the wild-type strain, suggesting that the mechanisms for RAD51-dependent and RAD51-independent events are different . Plasmid gap repair was reduced synergistically in rad51 rad59 double mutants, indicating an important role for RAD59 in RAD51-independent repair.

Mol Cell Biol, 2000 Feb, 20(4), 1095 - 103
Kinetics of ribosomal pausing during programmed -1 translational frameshifting; Lopinski JD et al.; In the Saccharomyces cerevisiae double-stranded RNA virus, programmed -1 ribosomal frameshifting is responsible for translation of the second open reading frame of the essential viral RNA . A typical slippery site and downstream pseudoknot are necessary for this frameshifting event, and previous work has demonstrated that ribosomes pause over the slippery site . The translational intermediate associated with a ribosome paused at this position is detected, and, using in vitro translation and quantitative heelprinting, the rates of synthesis, the ribosomal pause time, the proportion of ribosomes paused at the slippery site, and the fraction of paused ribosomes that frameshift are estimated . About 10% of ribosomes pause at the slippery site in vitro, and some 60% of these continue in the -1 frame . Ribosomes that continue in the -1 frame pause about 10 times longer than it takes to complete a peptide bond in vitro . Altering the rate of translational initiation alters the rate of frameshifting in vivo . Our in vitro and in vivo experiments can best be interpreted to mean that there are three methods by which ribosomes pass the frameshift site, only one of which results in frameshifting.

J Cell Biol, 2000 Jan 24, 148(2), 353 - 62
A role for myosin-I in actin assembly through interactions with Vrp1p, Bee1p, and the Arp2/3 complex; Evangelista M et al.; Type I myosins are highly conserved actin-based molecular motors that localize to the actin-rich cortex and participate in motility functions such as endocytosis, polarized morphogenesis, and cell migration . The COOH-terminal tail of yeast myosin-I proteins, Myo3p and Myo5p, contains an Src homology domain 3 (SH3) followed by an acidic domain . The myosin-I SH3 domain interacted with both Bee1p and Vrp1p, yeast homologues of human WASP and WIP, adapter proteins that link actin assembly and signaling molecules . The myosin-I acidic domain interacted with Arp2/3 complex subunits, Arc40p and Arc19p, and showed both sequence similarity and genetic redundancy with the COOH-terminal acidic domain of Bee1p (Las17p), which controls Arp2/3-mediated actin nucleation . These findings suggest that myosin-I proteins may participate in a diverse set of motility functions through a role in actin assembly.

Development, 2000 Feb, 127(4), 881 - 92
OVO transcription factors function antagonistically in the Drosophila female germline; Andrews J et al.; OVO controls germline and epidermis differentiation in flies and mice . In the Drosophila germline, alternative OVO-B and OVO-A isoforms have a common DNA-binding domain, but different N-termini . We show that these isoforms are transcription factors with opposite regulatory activities . Using yeast one-hybrid assays, we identified a strong activation domain within a common region and a counteracting repression domain within the OVO-A-specific region . In flies, OVO-B positively regulated the ovarian tumor promoter, while OVO-A was a negative regulator of the ovarian tumor and ovo promoters . OVO-B isoforms supplied ovo(+) function in the female germline and epidermis, while OVO-A isoforms had dominant-negative activity in both tissues . Moreover, elevated expression of OVO-A resulted in maternal-effect lethality while the absence of OVO-A resulted in maternal-effect sterility . Our data indicate that tight regulation of antagonistic OVO-B and OVO-A isoforms is critical for germline formation and differentiation.

Endocrinology, 2000 Feb, 141(2), 621 - 8
Potential involvement of FRS2 in insulin signaling; Delahaye L et al.; Shp-2 is implicated in several tyrosine kinase receptor signaling pathways . This phosphotyrosine phosphatase is composed of a catalytic domain in its C-terminus and two SH2 domains in its N-terminus . Shp-2 becomes activated upon binding through one or both SH2 domains to tyrosine phosphorylated molecules such as Shc or insulin receptor substrates . We were interested in finding a new molecule(s), tyrosine phosphorylated by the insulin receptor (IR), that could interact with Shp-2 . To do so, we screened a human placenta complementary DNA (cDNA) library with the SH2 domain-containing part of Shp-2 using a modified yeast two-hybrid system . In this system we induce or repress the expression of a constitutive active IR beta-subunit . When expressed, IR phosphorylates proteins produced from the library that can then associate with Shp-2 . Using this approach, we isolated FRS2 as a potential target for tyrosine phosphorylation by the IR . After cloning the entire cDNA, we found that 1) in the yeast two-hybrid system, FRS2 interacts with Shp-2 in a fashion dependent on the presence of the IR; and 2) in the PC12/IR cell-line, insulin leads to an increase in FRS2 association with the phosphatase . We next wanted to determine whether FRS2 could be a direct substrate for IR . In an in vitro kinase assay we found that wheat-germ agglutinin-purified IR phosphorylates glutathione-S-transferase-FRS2 fusion protein . Finally, in intact cells we show that insulin stimulates tyrosine phosphorylation of endogenous FRS2 . In summary, by screening a two-hybrid cDNA library, we have isolated FRS2 as a possible substrate for IR . We found that IR can directly phosphorylate FRS2 . Moreover, in intact cells insulin stimulates tyrosine phosphorylation of FRS2 and its subsequent association with Shp-2 . Taken together these results suggest that FRS2 could participate in insulin signaling by recruiting Shp-2 and, hence, could function as a docking molecule similar to insulin receptor substrate proteins.

Endocrinology, 2000 Feb, 141(2), 571 - 80
Two estrogen receptor (ER) isoforms with different estrogen dependencies are generated from the trout ER gene; Pakdel F et al.; A characteristic of all estrogen receptors (ER) cloned from fish to date is the lack of the first 37-42 N-terminal amino acids specific to the A domain . Here we report the isolation and characterization from trout ovary of a full-length complementary DNA (cDNA) clone encoding an N-terminal variant form of the rainbow trout ER (rtER) . Sequence analysis of open reading frame of this cDNA predicts a 622-amino acid protein . The C-terminal region of this protein, from amino acid position 45 to the end, was very similar to the previously reported rtER (referred to as the short form, or rtER(S)) . In contrast, this novel rtER cDNA (referred to as the long form, or rtER(L)) contains an additional in-frame ATG initiator codon that adds 45 residues to the N-terminal region of the protein . This new N-terminal region may represent the A domain of ER found in tetrapod species . The first 227 bp of this new cDNA were similar to the 3'-end intronic sequence of the rtER gene intron 1 . These data together with S1 nuclease, primer extension, and RT-PCR experiments demonstrate that the rtER(L) represents a second isoform of rtER that arises from an alternative promoter within the first intron of the gene . Transcripts encoding both rtER forms were expressed in the liver . In vitro translation of the rtER(L) cDNA produced 2 proteins with molecular masses of 71 and 65 kDa, whereas rtER(S) cDNA produced 1 65-kDa protein . Interestingly, Western blot analysis with a specific antibody against the C-terminal region of rtER revealed 2 receptor forms of 65 and 71 kDa in trout liver nuclear extracts, in agreement with the presence of the 2 distinct classes of rtER messenger RNA in this tissue . Functional analysis of both rtER isoforms revealed that although rtER(S) consistently exhibited a basal (estrogen-independent) trans-activation activity that could be further increased in the presence of estrogens, the novel isoform rtER(L) is characterized by a strict estrogen-dependent transcriptional activity . These data suggest that the additional 45 residues at the N-terminal region of rtER(L) clearly modify the hormone-independent trans-activation function of the receptor.

Yeast, 2000 Feb, 16(3), 267 - 76
Disruption and functional analysis of six ORFs on chromosome IV: YDR013w, YDR014w, YDR015c, YDR018c, YDR020c, YDR021w (FAL1); Dardalhon M et al.; The disruption of six novel yeast genes has been realized in two genetic backgrounds . Six open reading frames (ORFs) from chromosome IV, YDR013w, YDR014w, YDR015c, YDR018c, YDR020c and YDR021w, were disrupted using the KanMX4 marker and PCR-targeting with long flanking regions homologous (LFH) to the target locus . The deletants were verified at the molecular level, using PCR and Southern analysis . Sporulation and tetrad analysis revealed that ORFs YDR013w and YDR021w (also known as FAL1) are essential genes . Microscopical observations showed that ydr013wDelta haploid cells were blocked after one or two cell cycles and presented heterogeneous bud sizes . The ydr021wDelta haploid cells gave rise to microcolonies of about 20 cells . The other four ORFs are non-essential . Basic phenotypic analysis of the non-lethal deletant strains did not reveal any significant differences in cell morphology, growth on different media and temperatures, sporulation and mating efficiency between parental and mutant strains in the FY1679 background .

Nucleic Acids Res, 2000 Feb 15, 28(4), 917 - 24
Characterization of the enzymatic activity of hChlR1, a novel human DNA helicase; Hirota Y et al.; Recently, we cloned two highly related human genes, hChlR1 ( DDX11 ) and hChlR2 ( DDX12 ), which appear to be homologs of the Saccharomyces cerevisiae CHL1 gene . Nucleotide sequence analysis suggests that these genes encode new members of the DEAH family of DNA helicases . While the enzymatic activity of CHL1 has not been characterized, the protein is required for the maintenance of high fidelity chromosome segregation in yeast . Here we report that the hChlR1 protein is a novel human DNA helicase . We have expressed and purified hChlR1 using a baculovirus system and analyzed its enzymatic activity . The recombinant hChlR1 protein possesses both ATPase and DNA helicase activities that are strictly dependent on DNA, divalent cations and ATP . These activities are abolished by a single amino acid substitution in the ATP-binding domain . The hChlR1 protein can unwind both DNA/DNA and RNA/DNA substrates . It has a preference for movement in the 5'-->3' direction on short single-stranded DNA templates . However, unlike other DNA helicases, the hChlR1 DNA helicase can translocate along single-stranded DNA in both directions when substrates have a very long single-stranded DNA region . The enzymatic activities of hChlR1 suggest that DNA helicases are required for maintaining the fidelity of chromosome segregation.

Biophys Chem, 2000 Jan 24, 83(3), 223 - 37
The main role of the sequence-dependent DNA elasticity in determining the free energy of nucleosome formation on telomeric DNAs; Filesi I et al.; Using a competitive reconstitution assay, we measured the free energy spent in nucleosome formation of eight telomeric DNAs, differing in sequence and/or in length . The obtained values are in satisfactorily good agreement with those derived from a theoretical model that allows the calculation of the free energy of nucleosome formation on the basis of sequence-dependent DNA elasticity, using a statistical thermodynamic approach . Both theoretical and experimental evaluations show that telomeres are characterized by the highest free energies of nucleosome formation among all the DNA sequences so far studied . The free energy of nucleosome formation varies according to the different telomeric sequences and the length of the fragments . Theoretical analysis and experimental mapping by lambda exonuclease show that telomeric nucleosomes occupy multiple positions spaced every telomeric repeat . Sequence-dependent DNA elasticity appears as the main determinant of the stability of telomeric nucleosomes and their multiple translational positioning.

Structure Fold Des, 1999 Dec 15, 7(12), 1557 - 66
The three-dimensional structure of the HRDC domain and implications for the Werner and Bloom syndrome proteins; Liu Z et al.; BACKGROUND: The HRDC (helicase and RNaseD C-terminal) domain is found at the C terminus of many RecQ helicases, including the human Werner and Bloom syndrome proteins . RecQ helicases have been shown to unwind DNA in an ATP-dependent manner . However, the specific functional roles of these proteins in DNA recombination and replication are not known . An HRDC domain exists in both of the human RecQ homologues that are implicated in human disease and may have an important role in their function . RESULTS: We have determined the three-dimensional structure of the HRDC domain in the Saccharomyces cerevisiae RecQ helicase Sgs1p by nuclear magnetic resonance (NMR) spectroscopy . The structure resembles auxiliary domains in bacterial DNA helicases and other proteins that interact with nucleic acids . We show that a positively charged region on the surface of the Sgs1p HRDC domain can interact with DNA . Structural similarities to bacterial DNA helicases suggest that the HRDC domain functions as an auxiliary domain in RecQ helicases . Homology models of the Werner and Bloom HRDC domains show different surface properties when compared with Sgs1p . CONCLUSIONS: The HRDC domain represents a structural scaffold that resembles auxiliary domains in proteins that are involved in nucleic acid metabolism . In Sgs1p, the HRDC domain could modulate the helicase function via auxiliary contacts to DNA . However, in the Werner and Bloom syndrome helicases the HRDC domain may have a role in their functional differences by mediating diverse molecular interactions.

Nature, 1999 Nov 11, 402(6758), 203 - 7
APC(Cdc20) promotes exit from mitosis by destroying the anaphase inhibitor Pds1 and cyclin Clb5; Shirayama M et al.; Ubiquitin-mediated proteolysis due to the anaphase-promoting complex/cyclosome (APC/C) is essential for separation of sister chromatids, requiring degradation of the anaphase inhibitor Pds1, and for exit from mitosis, requiring inactivation of cyclin B Cdk1 kinases . Exit from mitosis in yeast involves accumulation of the cyclin kinase inhibitor Sic1 as well as cyclin proteolysis mediated by APC/C bound by the activating subunit Cdh1/Hct1 (APC(Cdh1)) . Both processes require the Cdc14 phosphatase, whose release from the nucleolus during anaphase causes dephosphorylation and thereby activation of Cdh1 and accumulation of another protein, Sic1 (refs 4-7) . We do not know what determines the release of Cdc14 and enables it to promote Cdk1 inactivation, but it is known to be dependent on APC/C bound by Cdc20 (APC(Cdc20)) (ref . 4) . Here we show that APC(Cdc20) allows activation of Cdc14 and promotes exit from mitosis by mediating proteolysis of Pds1 and the S phase cyclin Clb5 in the yeast Saccharomyces cerevisiae . Degradation of Pds1 is necessary for release of Cdc14 from the nucleolus, whereas degradation of Clb5 is crucial if Cdc14 is to overwhelm Cdk1 and activate its foes (Cdh1 and Sic1) . Remarkably, cells lacking both Pds1 and Clb5 can proliferate in the complete absence of Cdc20.

Cancer Res, 2000 Jan 1, 60(1), 28 - 34
Tumor progression is accompanied by significant changes in the levels of expression of polyamine metabolism regulatory genes and clusterin (sulfated glycoprotein 2) in human prostate cancer specimens; Bettuzzi S et al.; Using Northern blotting, the expression levels of the genes for polyamine metabolism regulatory proteins and clusterin have been measured in a series of 23 human prostate cancers (CaPs) dissected from radical prostatectomy specimens . Patient matched, nontumor tissue was dissected from benign areas of the gland . The results indicate that transcripts encoding ornithine decarboxylase (ODC), ODC antizyme, adenosylmethionine decarboxylase, and spermidine/spermine N1-acetyltransferase (SSAT) were significantly higher, whereas clusterin (sulfated glycoprotein 2) mRNA was significantly lower in tumors compared with the benign tissue . All mRNA levels were compared with those of histone H3 and growth arrest-specific gene 1, markers of cell proliferation and cell quiescence, respectively, and glyceraldehyde 3-phosphate dehydrogenase, a housekeeping gene . In poorly differentiated and locally invasive CaPs and in tumors with unfavorable prognosis or total prostate-specific antigen (PSA) levels > 10.0 ng/ml at diagnosis, an overall increase in the levels of H3 mRNA and a decrease in growth arrest-specific gene 1 mRNA was detected, indicative of higher proliferation activity, whereas the differences in expression levels for the polyamine metabolism and clusterin genes were higher . ODC and SSAT changes were positively correlated in normal tissue but not in high-grade cancer, whereas ODC antizyme and SSAT changes were positively correlated in more malignant CaPs but not in normal tissue . Tumor classification based on the changes in expression levels of all of the genes studied could be correlated to differentiation grade and local invasiveness classification systems in 72.2 and 83.3% of the cases, respectively . In a 1-year follow-up period, three patients whose CaPs ranked as less aggressive according to clinical staging, but classified as advanced cancers with the proposed molecular classification, showed increases in total PSA levels, indicative of tumor relapse . Thus, molecular classification, based on gene expression, may enhance the available prognostic tools for prostate tumors.

J Biol Chem, 2000 Jan 28, 275(4), 2795 - 803
Correlation between in vitro stability and in vivo performance of anti-GCN4 intrabodies as cytoplasmic inhibitors; Worn A et al.; A cellular assay system for measuring the activity of cytoplasmically expressed anti-GCN4 scFv fragments directed against the Gcn4p dimerization domain was established in the budding yeast Saccharomyces cerevisiae . The inhibitory potential of different constitutively expressed anti-GCN4 scFv intrabodies was monitored by measuring the activity of beta-galactosidase expressed from a GCN4-dependent reporter gene . The in vivo performance of these scFv intrabodies in specifically decreasing reporter gene activity was related to their in vitro stability, measured by denaturant-induced equilibrium unfolding . A framework-engineered stabilized version showed significantly improved activity, while a destabilized point mutant of the anti-GCN4 wild-type showed decreased effects in vivo . These results indicate that stability engineering can result in improved performance of scFv fragments as intrabodies . Increasing the thermodynamic stability appears to be an essential factor for improving the yield of functional scFv in the reducing environment of the cytoplasm, where the conserved intradomain disulfides of antibody fragments cannot form.

J Biol Chem, 2000 Jan 28, 275(4), 2705 - 12
Probing the structure of the PI-SceI-DNA complex by affinity cleavage and affinity photocross-linking; Hu D et al.; The PI-SceI protein is an intein-encoded homing endonuclease that initiates the mobility of its gene by making a double strand break at a single site in the yeast genome . The PI-SceI protein splicing and endonucleolytic active sites are separately located in each of two domains in the PI-SceI structure . To determine the spatial relationship between bases in the PI-SceI recognition sequence and selected PI-SceI amino acids, the PI-SceI-DNA complex was probed by photocross-linking and affinity cleavage methods . Unique solvent-accessible cysteine residues were introduced into the two PI-SceI domains at positions 91, 97, 170, 230, 376, and 378, and the mutant proteins were modified with either 4-azidophenacyl bromide or iron (S)-1-(p-bromoacetamidobenzyl)-ethylenediaminetetraacetate (FeBABE) . The phenyl azide-coupled proteins cross-linked to the PI-SceI target sequence, and the FeBABE-modified proteins cleaved the DNA proximal to the derivatized amino acid . The results suggest that an extended beta-hairpin loop in the endonuclease domain that contains residues 376 and 378 contacts the major groove near the PI-SceI cleavage site . Conversely, residues 91, 97, and 170 in the protein splicing domain are in close proximity to a distant region of the substrate . To interpret our results, we used a new PI-SceI structure that is ordered in regions of the protein that bind DNA . The data strongly support a model of the PI-SceI-DNA complex derived from this structure.

J Biol Chem, 2000 Jan 28, 275(4), 2627 - 35
PAR1 thrombin receptor-G protein interactions . Separation of binding and coupling determinants in the galpha subunit; Swift S et al.; Signal transfer between the protease-activated PAR1 thrombin receptor and membrane-associated heterotrimeric G proteins is mediated by protein-protein interactions . We constructed a yeast signaling system that resolves domain-specific functions of binding from coupling in the Galpha subunit . The endogenous yeast Galpha subunit, Gpa1, does not bind to PAR1 and served as a null structural template . N- and C-terminal portions of mammalian G(i2) and G(16) were substituted back into the Gpa1 template and gain-of-function assessed . The C-terminal third of G(16), but not of G(i2), provides sufficient interactions for coupling to occur with PAR1 . The N-terminal two-thirds of G(i2) also contains sufficient determinants to bind and couple to PAR1 and overcome the otherwise negative or missing interactions supplied by the C-terminal third of Gpa1 . Replacement of the N-terminal alpha-helix of G(i2), residues 1-34, with those of Gpa1 abolishes coupling but not binding to PAR1 or to betagamma subunits . These data support a model that the N-terminal alphaN helix of the Galpha subunit is physically interposed between PAR1 and the Gbeta subunit and directly assists in transferring the signal between agonist-activated receptor and G protein.

J Biol Chem, 2000 Jan 28, 275(4), 2613 - 8
Steady-state and rapid kinetic analysis of topoisomerase II trapped as the closed-clamp intermediate by ICRF-193; Morris SK et al.; DNA topoisomerase II uses a complex, sequential mechanism of ATP hydrolysis to catalyze the transport of one DNA duplex through a transient break in another . ICRF-193 is a catalytic inhibitor of topoisomerase II that is known to trap a closed-clamp intermediate form of the enzyme . Using steady-state and rapid kinetic ATPase and DNA transport assays, we have analyzed how trapping this intermediate by the drug perturbs the topoisomerase II mechanism . The drug has no effect on the rate of the first turnover of decatenation but potently inhibits subsequent turnovers with an IC(50) of 6.5 +/- 1 microM for the Saccharomyces cerevisiae enzyme . This drug inhibits the ATPase activity of topoisomerase II by an unusual, mixed-type mechanism; the drug is not a competitive inhibitor of ATP, and even at saturating concentrations of drug, the enzyme continues to hydrolyze ATP, albeit at a reduced rate . Topoisomerase II that was specifically isolated in the drug-bound, closed-clamp form continues to hydrolyze ATP, indicating that the enzyme clamp does not need to re-open to bind and hydrolyze ATP . When rapid-quench ATPase assays were initiated by the addition of ATP, the drug had no effect on the sequential hydrolysis of either the first or second ATP . By contrast, when the drug was prebound, the enzyme hydrolyzed one labeled ATP at the uninhibited rate but did not hydrolyze a second ATP . These results are interpreted in terms of the catalytic mechanism for topoisomerase II and suggest that ICRF-193 interacts with the enzyme bound to one ADP.

J Biol Chem, 2000 Jan 28, 275(4), 2513 - 9
Identification of a human brain-specific isoform of mammalian STE20-like kinase 3 that is regulated by cAMP-dependent protein kinase; Zhou TH et al.; A novel isoform of mammalian STE20-like kinase 3 (MST3) with a different 5' coding region from MST3, termed MST3b, was identified by searching through expressed sequence tag data base and obtained by rapid amplification of cDNA 5'-ends . MST3b was assigned to the long arm of human chromosome 13, D13S159-D13S280, by use of the National Center for Biotechnology Information sequence-tagged sites data base . Reverse transcription-polymerase chain reaction and Northern blot analysis with a probe derived from 5' distinct sequence of MST3b revealed that the expression of MST3b mRNA is restricted to the brain, in contrast to ubiquitous distribution of MST3 transcript . Western analysis confirmed the brain-specific expression of MST3b protein . In situ hybridization of rat brain sections with a MST3b-specific probe indicated that MST3b is widely expressed in different brain regions, with especially high expression in hippocampus and cerebral cortex . When expressed in human embryonic kidney 293 (HEK293) cells, MST3b effectively phosphorylated myelin basic protein, as well as undergoing autophosphorylation . Interestingly, expression of MST3, but not MST3b, in HEK293 cells was able to activate the endogenous p42/44 mitogen-activated protein kinase (MAPK) up to 4-fold, whereas neither isoform activated p38 MAPK under the same conditions . Further experiments demonstrated that MST3b, but not MST3, was effectively phosphorylated by activation of cyclic AMP-dependent protein kinase (PKA) in both in vivo and in vitro assays . The mutation of Thr-18 into Ala in MST3b (T18A), a putative PKA phosphorylation site that is absent in MST3, abolished its phosphorylation by PKA . Consequently, expression of the T18A mutant in HEK293 cells led to partial activation of p42/44 MAPK, indicating that MST3b is under the regulation of PKA . Taken together, our data provide evidence that the two isoforms of STE20-like kinase 3 are differentially distributed and regulated.

J Biol Chem, 2000 Jan 28, 275(4), 2410 - 4
The human Cdc14 phosphatases interact with and dephosphorylate the tumor suppressor protein p53; Li L et al.; The yeast Cdc14 phosphatase has been shown to play an important role in cell cycle regulation by dephosphorylating proteins phosphorylated by the cyclin-dependent kinase Cdc28/clb . We recently cloned two human orthologs of the yeast CDC14, termed hCDC14A and -B, the gene products of which share approximately 80% amino acid sequence identity within their N termini and phosphatase domains . Here we report that the hCdc14A and hCdc14B proteins interact with the tumor suppressor protein p53 both in vitro and in vivo . This interaction is dependent on the N termini of the hCdc14 proteins and the C terminus of p53 . Furthermore, the hCdc14 phosphatases were found to dephosphorylate p53 specifically at the p34(Cdc2)/clb phosphorylation site (p53-phosphor-Ser(315)) . Our findings that hCdc14 is a cyclin-dependent kinase substrate phosphatase suggest that it may play a role in cell cycle control in human cells . Furthermore, the identification of p53 as a substrate for hCdc14 indicates that hCdc14 may regulate the function of p53.

J Biol Chem, 2000 Jan 28, 275(4), 2399 - 403
Protein phosphatase 2Calpha dephosphorylates axin and activates LEF-1-dependent transcription; Strovel ET et al.; The Dishevelled (Dvl) gene family encodes cytoplasmic proteins that are necessary for Wnt signal transduction . Utilizing the yeast two-hybrid system, we identified protein phosphatase 2Calpha (PP2C) as a Dvl-PDZ domain-interacting protein . PP2C exists in a complex with Dvl, beta-catenin, and Axin, a negative regulator of Wnt signaling . In a Wnt-responsive LEF-1 reporter gene assay, expression of PP2C activates transcription and also elicits a synergistic response with beta-catenin and Wnt-1 . In addition, PP2C expression relieves Axin-mediated repression of LEF-1-dependent transcription . PP2C utilizes Axin as a substrate both in vitro and in vivo and decreases its half-life . These results indicate that PP2C is a positive regulator of Wnt signal transduction and mediates its effects through the dephosphorylation of Axin.

Ann Ist Super Sanita, 1999, 35(2), 185 - 92
{Adrenoleukodystrophy: genetics, phenotypes, pathogenesis, and treatment}; Di Biase A et al.; Adrenoleukodystrophy is a genetically determined disorder inherited as an X-linked recessive trait due to the defective peroxisomal oxydation of very long chain fatty acids (VLCFA) . It is hallmarked by demyelination of the central nervous system and adrenal insufficiency . Even though the studies concerning the molecular basis of the disease are in progress, the role of VLCFA in the demyelination is still unclear . In this paper we report the most recent knowledges about genetics, pathogenesis and treatment of this disorder . 117 cases have been recognized in Italy in the period 1985-1997, but many cases could be missing due to the heterogeneus clinical manifestations that vary from mild to very severe forms . To control the devastating course of this disease two therapeutic approaches are under evaluation: bone marrow transplantation and dietary treatment based on a mixture of glyceroyl trioleate and glyceroyl trierucate . Nevertheless this dietary treatment provides normalization of plasma VLCFA, no significant modification of the natural course of the disease has been demonstrated . For what concerns bone marrow transplantation, in recent years a more accurate selection of patients and donors has been giving favourable results, but some strict criteria should be respected.

J Virol, 2000 Feb, 74(4), 1892 - 9
Characterization of the interaction between the interferon-induced protein P56 and the Int6 protein encoded by a locus of insertion of the mouse mammary tumor virus; Guo J et al.; For determining cellular functions of the interferon-inducible human cytoplasmic protein P56, we undertook a Saccharomyces cerevisiae two-hybrid screen that identified Int6 as a P56-interacting protein . That the interaction also occurs in human cells was confirmed by coimmunoprecipitation and the observed cytoplasmic displacement of nuclear Int6 upon coexpression of P56 . Because Int6 has been claimed to be both a cytoplasmic and a nuclear protein, we investigated the structural basis of this discrepancy . By mutational analyses, we showed that the Int6 protein contains a bipartite nuclear localization signal and a nuclear export signal at the far end of the amino terminus . The 20 amino-terminal residues of Int6, when they were attached to a different nuclear protein, were sufficient to translocate that protein to the cytoplasm . Within this region, replacement of any of the three leucine residues with alanine destroyed the function of the export signal . The specific domain of P56 that is required for its interaction with Int6 was mapped using the yeast two-hybrid assay and a mammalian coimmunoprecipitation assay . Both assays demonstrated that the C-terminal region of P56 containing three specific tetratricopeptide motifs is required for this interaction . In contrast, removal of an internal domain of P56 enhanced the interaction, as quantified by the two-hybrid assay.

J Virol, 2000 Feb, 74(4), 1736 - 41
Hepatitis C virus core protein interacts with 14-3-3 protein and activates the kinase Raf-1; Aoki H et al.; Persistent hepatitis C virus (HCV) infection is a major cause of chronic liver dysfunction in humans and is epidemiologically closely associated with the development of human hepatocellular carcinoma . Among HCV components, core protein has been reported to be implicated in cell growth regulation both in vitro and in vivo, although mechanisms explaining those effects are still unclear . In the present study, we identified that members of the 14-3-3 protein family associate with HCV core protein . 14-3-3 protein bound to HCV core protein in a phosphoserine-dependent manner . Introduction of HCV core protein caused a substantial increase in Raf-1 kinase activity in HepG2 cells and in a yeast genetic assay . Furthermore, the HCV core-14-3-3 interaction was essential for Raf-1 kinase activation by HCV core protein . These results suggest that HCV core protein may represent a novel type of Raf-1 kinase-activating protein through its interaction with 14-3-3 protein and may contribute to hepatocyte growth regulation.

Ross Fiziol Zh Im I M Sechenova, 1999 Jul, 85(7), 984 - 96
{Prion phenomenon in medicine and biology}; Ter-Avanesian MD et al.; The data obtained suggest that the fatal changes in brain tissue associated with the prion diseases, are initiated by a conformational rearrangement of constitutively expressed cellular protein PrP . Possible mechanisms of such a conversion of this protein are discussed . Existence of the proteins with the prion properties in low eukaryotes may determine the unusual mechanisms of the "protein" inheritance . A new experimental model for studying the proteins with the prion properties in the yeast Saccharomyces cerevisiae, is described.

J Clin Invest, 2000 Jan, 105(2), 233 - 9
Deficiency of dolichol-phosphate-mannose synthase-1 causes congenital disorder of glycosylation type Ie; Imbach T et al.; Congenital disorders of glycosylation (CDG), formerly known as carbohydrate-deficient glycoprotein syndromes, lead to diseases with variable clinical pictures . We report the delineation of a novel type of CDG identified in 2 children presenting with severe developmental delay, seizures, and dysmorphic features . We detected hypoglycosylation on serum transferrin and cerebrospinal fluid beta-trace protein . Lipid-linked oligosaccharides in the endoplasmic reticulum of patient fibroblasts showed an accumulation of the dolichyl pyrophosphate Man(5)GlcNAc(2) structure, compatible with the reduced dolichol-phosphate-mannose synthase (DolP-Man synthase) activity detected in these patients . Accordingly, 2 mutant alleles of the DolP-Man synthase DPM1 gene, 1 with a 274C>G transversion, the other with a 628delC deletion, were detected in both siblings . Complementation analysis using DPM1-null murine Thy1-deficient cells confirmed the detrimental effect of both mutations on the enzymatic activity . Furthermore, mannose supplementation failed to improve the glycosylation status of DPM1-deficient fibroblast cells, thus precluding a possible therapeutic application of mannose in the patients . Because DPM1 deficiency, like other subtypes of CDG-I, impairs the assembly of N-glycans, this novel glycosylation defect was named CDG-Ie.

Biochemistry, 2000 Jan 25, 39(3), 567 - 74
An accessory DNA binding motif in the zinc finger protein Adr1 assists stable binding to DNA and can be replaced by a third finger; Young ET et al.; The DNA binding domain of Adr1, the protein derived from alcohol dehydrogenase regulatory gene 1, is unusual for zinc finger proteins in that it consists of two classical Cys2His2 zinc fingers and an amino-terminal proximal accessory region termed PAR . PAR is unstructured in the free protein and becomes structured in the DNA-bound form . We investigated the role of PAR in DNA binding using molecular and biochemical approaches, and its importance for activation in vivo, using Adr1-dependent reporter genes . PAR was unimportant for DNA binding when a third finger was added to Adr1, and its importance was diminished but not eliminated by mutations in finger two that increased DNA binding affinity . The kinetic rate constants for three Adr1 proteins containing or lacking PAR were determined by surface plasmon resonance . PAR increased the on rate and decreased the off rate for specific DNA sites for Adr1 containing wild-type fingers one and two . Surprisingly, PAR had no significant effect on the kinetic rate constants when a third finger was present, or when single-stranded DNA was used as the substrate for DNA binding . A mutant form of Adr1-F1F2 in which finger 2 makes three base-specific contacts with DNA had a higher affinity for DNA than Adr1 containing three fingers, yet the mutant protein still depended on PAR for optimal binding affinity . The ability to activate transcription in vivo was correlated with a low dissociation rate, suggesting that stabilizing an activator at the promoter might be rate-limiting for transcription in vivo . PAR may have evolved to lend additional stability to DNA-Adr1 complexes encompassing short binding sites . In addition, PAR may have a role in transcription at a step after DNA binding since deletion of PAR from Adr1 with three fingers decreased activation in vivo but had no effect on DNA binding kinetics.

Biochemistry, 2000 Jan 25, 39(3), 548 - 56
Protein-directed DNA structure II . Raman spectroscopy of a leucine zipper bZIP complex; Benevides JM et al.; Mechanisms of transcription may involve protein-directed changes in DNA structure and DNA-directed changes in protein structure . We have employed Raman spectroscopy to characterize vibrational signatures associated with such induced molecular fitting for two classes of transcription factors-the basic leucine-zipper (bZIP) motif and the high-mobility-group (HMG) box-each with a DNA target site . Results for bZIP are described here; findings for the HMG-box are reported in the preceding paper in this issue {Benevides, J . M., Chan, G., Lu, X.-J., Olson, W . K., Weiss, M . A., and Thomas, G . J., Jr . (2000) Biochemistry 39, 537-547} . The yeast activator GCN4 provides a well-studied example of bZIP recognition, wherein B-DNA serves essentially as a template for protein folding . Analysis of Raman spectra of the 57-residue GCN4 bZIP domain, its AP-1 binding site, and their specific complex confirms a DNA-induced increase in alpha-helicity, attributable to folding of GCN4 basic arms with virtually no change in B-DNA structure, consistent with previous X-ray and NMR structure determinations . The absence of DNA perturbations in the bZIP model contrasts sharply with the HMG box, where DNA structure perturbations predominate . The bZIP and HMG-box models represent two opposing extremes in a range of induced fits identifiable by Raman spectroscopy . Previously characterized lambda repressor/operator complexes {Benevides, J . M., Weiss, M . A., and Thomas, G . J . (1994) J . Biol . Chem . 269, 10869-10878} occupy an intermediate position within this range . A comprehensive tabulation of Raman markers proposed as diagnostic of different protein/DNA recognition motifs is presented . The results are analyzed in terms of available DNA crystal structures (Nucleic Acid Database) to identify details of DNA conformation that correlate with specific Raman recognition markers.

J Gen Virol, 2000 Jan, 81(Pt 1), 209 - 18
Interactions in vivo between the proteins of infectious bursal disease virus: capsid protein VP3 interacts with the RNA-dependent RNA polymerase, VP1; Tacken MG et al.; Little is known about the intermolecular interactions between the viral proteins of infectious bursal disease virus (IBDV) . By using the yeast two-hybrid system, which allows the detection of protein-protein interactions in vivo, all possible interactions were tested by fusing the viral proteins to the LexA DNA-binding domain and the B42 transactivation domain . A heterologous interaction between VP1 and VP3, and homologous interactions of pVP2, VP3, VP5 and possibly VP1, were found by co-expression of the fusion proteins in Saccharomyces cerevisiae . The presence of the VP1-VP3 complex in IBDV-infected cells was confirmed by co-immunoprecipitation studies . Kinetic analyses showed that the complex of VP1 and VP3 is formed in the cytoplasm and eventually is released into the cell-culture medium, indicating that VP1-VP3 complexes are present in mature virions . In IBDV-infected cells, VP1 was present in two forms of 90 and 95 kDa . Whereas VP3 initially interacted with both the 90 and 95 kDa proteins, later it interacted exclusively with the 95 kDa protein both in infected cells and in the culture supernatant . These results suggest that the VP1-VP3 complex is involved in replication and packaging of the IBDV genome.

Exp Cell Res, 2000 Feb 1, 254(2), 309 - 20
Intraperoxisomal localization of very-long-chain fatty acyl-CoA synthetase: implication in X-adrenoleukodystrophy; Smith BT et al.; X-adrenoleukodystrophy (X-ALD) is a demyelinating disorder characterized by the accumulation of saturated very-long-chain (VLC) fatty acids (>C(22:0)) due to the impaired activity of VLC acyl-CoA synthetase (VLCAS) . The gene responsible for X-ALD was found to code for a peroxisomal integral membrane protein (ALDP) that belongs to the ATP binding cassette superfamily of transporters . To understand the function of ALDP and how ALDP and VLCAS interrelate in the peroxisomal beta-oxidation of VLC fatty acids we investigated the peroxisomal topology of VLCAS protein . Antibodies raised against a peptide toward the C-terminus of VLCAS as well as against the N-terminus were used to define the intraperoxisomal localization and orientation of VLCAS in peroxisomes . Indirect immunofluorescent and electron microscopic studies show that peroxisomal VLCAS is localized on the matrix side . This finding was supported by protease protection assays and Western blot analysis of isolated peroxisomes . To further address the membrane topology of VLCAS, Western blot analysis of total membranes or integral membranes prepared from microsomes and peroxisomes indicates that VLCAS is a peripheral membrane-associated protein in peroxisomes, but an integral membrane in microsomes . Moreover, peroxisomes isolated from cultured skin fibroblasts from X-ALD patients with a mutation as well as a deletion in ALDP showed a normal amount of VLCAS . The consequence of VLCAS being localized to the luminal side of peroxisomes suggests that ALDP may be involved in stabilizing VLCAS activity, possibly through protein-protein interactions, and that loss or alterations in these interactions may account for the observed loss of peroxisomal VLCAS activity in X-ALD .

Genes Dev, 2000 Jan 1, 14(1), 55 - 66
Isolation of a novel histone deacetylase reveals that class I and class II deacetylases promote SMRT-mediated repression; Kao HY et al.; The transcriptional corepressor SMRT functions by mediating the repressive effect of transcription factors involved in diverse signaling pathways . The mechanism by which SMRT represses basal transcription has been proposed to involve the indirect recruitment of histone deacetylase HDAC1 via the adaptor mSin3A . In contrast to this model, a two-hybrid screen on SMRT-interacting proteins resulted in the isolation of the recently described HDAC5 and a new family member termed HDAC7 . Molecular and biochemical results indicate that this interaction is direct and in vivo evidence colocalizes SMRT, mHDAC5, and mHDAC7 to a distinct nuclear compartment . Surprisingly, HDAC7 can interact with mSin3A in yeast and in mammalian cells, suggesting association of multiple repression complexes . Taken together, our results provide the first evidence that SMRT-mediated repression is promoted by class I and class II histone deacetylases and that SMRT can recruit class II histone deacetylases in a mSin3A-independent fashion.

Genes Dev, 2000 Jan 1, 14(1), 45 - 54
Nuclear receptor corepressors partner with class II histone deacetylases in a Sin3-independent repression pathway; Huang EY et al.; Transcriptional repression mediated by corepressors N-CoR and SMRT is a critical function of nuclear hormone receptors, and is dysregulated in human myeloid leukemias . At the present time, these corepressors are thought to act exclusively through an mSin3/HDAC1 complex . Surprisingly, however, numerous biochemical studies have not detected N-CoR or SMRT in mSin3- and HDAC1-containing complexes . Each corepressor contains multiple repression domains (RDs), the significance of which is unknown . Here we show that these RDs are nonredundant, and that one RD, which is conserved in N-CoR and SMRT, represses transcription by interacting directly with class II HDAC4 and HDAC5 . Endogenous N-CoR and SMRT each associate with HDAC4 in a complex that does not contain mSin3A or HDAC1 . This is the first example of a single corepressor utilizing distinct domains to engage multiple HDAC complexes . The alternative HDAC complexes may mediate specific repression pathways in normal as well as leukemic cells.

Antimicrob Agents Chemother, 2000 Feb, 44(2), 368 - 77
Discovery of novel antifungal (1,3)-beta-D-glucan synthase inhibitors; Onishi J et al.; The increasing incidence of life-threatening fungal infections has driven the search for new, broad-spectrum fungicidal agents that can be used for treatment and prophylaxis in immunocompromised patients . Natural-product inhibitors of cell wall (1,3)-beta-D-glucan synthase such as lipopeptide pneumocandins and echinocandins as well as the glycolipid papulacandins have been evaluated as potential therapeutics for the last two decades . As a result, MK-0991 (caspofungin acetate; Cancidas), a semisynthetic analogue of pneumocandin B(o), is being developed as a broad-spectrum parenteral agent for the treatment of aspergillosis and candidiasis . This and other lipopeptide antifungal agents have limited oral bioavailability . Thus, we have sought new chemical structures with the mode of action of lipopeptide antifungal agents but with the potential for oral absorption . Results of natural-product screening by a series of newly developed methods has led to the identification of four acidic terpenoid (1,3)-beta-D-glucan synthase inhibitors . Of the four compounds, the in vitro antifungal activity of one, enfumafungin, is comparable to that of L-733560, a close analogue of MK-0991 . Like the lipopeptides, enfumafungin specifically inhibits glucan synthesis in whole cells and in (1,3)-beta-D-glucan synthase assays, alters the morphologies of yeasts and molds, and produces a unique response in Saccharomyces cerevisiae strains with point mutations in FKS1, the gene which encodes the large subunit of glucan synthase.

J Cell Sci, 2000 Feb, 113 ( Pt 3), 451 - 9
Developmentally regulated activity of CRM1/XPO1 during early Xenopus embryogenesis; Callanan M et al.; In this work, we have investigated the role of CRM1/XPO1, a protein involved in specific export of proteins and RNA from the nucleus, in early Xenopus embryogenesis . The cloning of the Xenopus laevis CRM1, XCRM1, revealed remarkable conservation of the protein during evolution (96.7% amino acid identity between Xenopus and human) . The protein and mRNA are maternally expressed and are present during early embryogenesis . However, our data show that the activity of the protein is developmentally regulated . Embryonic development is insensitive to leptomycin B, a specific inhibitor of CRM1, until the neurula stage . Moreover, the nuclear localization of CRM1 changes concomitantly with the appearance of the leptomycin B sensitivity . These data suggest that CRM1, present initially in an inactive form, becomes functional before the initiation of the neurula stage during gastrula-neurula transition, a period known to correspond to a critical transition in the pattern of gene expression . Finally, we confirmed the gastrula-neurula transition-dependent activation of CRM1 by pull-down experiments as well as by the study of the intracellular localization of a green fluorescent protein tagged with a nuclear export signal motif during early development . This work showed that the regulated activity of CRM1 controls specific transitions during normal development and thus might be a key regulator of early embryogenesis.

Proc Natl Acad Sci U S A, 2000 Jan 18, 97(2), 716 - 21
Heterochromatic deposition of centromeric histone H3-like proteins; Henikoff S et al.; Centromeres of most organisms are embedded within constitutive heterochromatin, the condensed regions of chromosomes that account for a large fraction of complex genomes . The functional significance of this centromere-heterochromatin relationship, if any, is unknown . One possibility is that heterochromatin provides a suitable environment for assembly of centromere components, such as special centromeric nucleosomes that contain distinctive histone H3-like proteins . We describe a Drosophila H3-like protein, Cid (for centromere identifier) that localizes exclusively to fly centromeres . When the cid upstream region drives expression of H3 and H2B histone-green fluorescent protein fusion genes in Drosophila cells, euchromatin-specific deposition results . Remarkably, when the cid upstream region drives expression of yeast, worm, and human centromeric histone-green fluorescent protein fusion proteins, localization is preferentially within Drosophila pericentric heterochromatin . Heterochromatin-specific localization also was seen for yeast and worm centromeric proteins constitutively expressed in human cells . Preferential localization to heterochromatin in heterologous systems is unexpected if centromere-specific or site-specific factors determine H3-like protein localization to centromeres . Rather, the heterochromatic state itself may help localize centromeric components.

Nature, 2000 Jan 6, 403(6765), 108 - 12
Nuclear pore complexes in the organization of silent telomeric chromatin; Galy V et al.; The functional regulation of chromatin is closely related to its spatial organization within the nucleus . In yeast, perinuclear chromatin domains constitute areas of transcriptional repression . These 'silent' domains are defined by the presence of perinuclear telomere clusters . The only protein found to be involved in the peripheral localization of telomeres is Yku70/Yku80 . This conserved heterodimer can bind telomeres and functions in both repair of DNA double-strand breaks and telomere maintenance . These findings, however, do not address the underlying structural basis of perinuclear silent domains . Here we show that nuclear-pore-complex extensions formed by the conserved TPR homologues Mlp1 and Mlp2 are responsible for the structural and functional organization of perinuclear chromatin . Loss of MLP2 results in a severe deficiency in the repair of double-strand breaks . Furthermore, double deletion of MLP1 and MLP2 disrupts the clustering of perinuclear telomeres and releases telomeric gene repression . These effects are probably mediated through the interaction with Yku70 . Mlp2 physically tethers Yku70 to the nuclear periphery, thus forming a link between chromatin and the nuclear envelope . We show, moreover, that this structural link is docked to nuclear-pore complexes through a cleavable nucleoporin, Nup145 . We propose that, through these interactions, nuclear-pore complexes organize a nuclear subdomain that is intimately involved in the regulation of chromatin metabolism.

Trends Biochem Sci, 2000 Jan, 25(1), 15 - 9
The many HATs of transcription coactivators; Brown CE et al.; Histone acetylation is closely linked to gene transcription . The identification of histone acetyltransferases (HATs) and the large multiprotein complexes in which they reside has yielded important insights into how these enzymes regulate transcription . The demonstration that HAT complexes interact with sequence-specific activator proteins illustrates how these complexes target specific genes . In addition to histones, some HATs can acetylate non-histone proteins suggesting multiple roles for these enzymes.

EMBO J, 2000 Jan 17, 19(2), 187 - 98
Monoubiquitin carries a novel internalization signal that is appended to activated receptors; Shih SC et al.; Ubiquitin modification of signal transducing receptors at the plasma membrane is necessary for rapid receptor internalization and downregulation . We have investigated whether ubiquitylation alters a receptor cytoplasmic tail to reveal a previously masked internalization signal, or whether ubiquitin itself carries an internalization signal . Using an alpha-factor receptor-ubiquitin chimeric protein, we demonstrate that monoubiquitin can mediate internalization of an activated receptor that lacks all cytoplasmic tail sequences . Furthermore, fusion of ubiquitin in-frame to the stable plasma membrane protein Pma1p stimulates endocytosis of this protein . Ubiquitin does not carry a functional tyrosine- or di-leucine-based internalization signal . Instead, the three-dimensional structure of the folded ubiquitin polypeptide carries an internalization signal that consists of two surface patches surrounding the critical residues Phe4 and Ile44 . We conclude that ubiquitin functions as a novel regulated internalization signal that can be appended to a plasma membrane protein to trigger downregulation.

J Exp Biol, 2000 Feb, 203(Pt 3), 447 - 57
Identification of a ubiquitous family of membrane proteins and their expression in mouse brain; Grossman TR et al.; A family of genes encoding membrane proteins with a unique structure has been identified in DNA and cDNA clones of various eukaryotes ranging from yeast to human . The nucleotide sequences of three novel cDNAs from Drosophila melanogaster and mouse were determined . The amino acid sequences of the two mouse proteins have human homologs . The gene (TMS1) encoding the yeast member of this family was disrupted, and the resulting mutant showed no significant phenotype under several stress conditions . The expression of the mouse genes TMS-1 and TMS-2 was examined by in situ hybridization of sections from brain, liver, kidney, heart and testis of an adult mouse as well as in a 1-day-old whole mouse . While the expression of TMS-2 was found to be restricted to the central nervous system, TMS-1 was also expressed in kidney and testis . The expression of TMS-1 and TMS-2 in the brain overlapped and was localized to areas associated with glutamatergic excitatory neurons, such as the hippocampus and cerebral cortex . High-magnification analysis indicated that both mRNAs are expressed in neurons . Semiquantitative analysis of mRNA expression was performed in various parts of the brain . The conservation, unique structure and localization in the mammalian brain of this novel protein family suggest an important biological role.

Mol Biol Cell, 2000 Jan, 11(1), 339 - 54
Functions and functional domains of the GTPase Cdc42p; Kozminski KG et al.; Cdc42p, a Rho family GTPase of the Ras superfamily, is a key regulator of cell polarity and morphogenesis in eukaryotes . Using 37 site-directed cdc42 mutants, we explored the functions and interactions of Cdc42p in the budding yeast Saccharomyces cerevisiae . Cytological and genetic analyses of these cdc42 mutants revealed novel and diverse phenotypes, showing that Cdc42p possesses at least two distinct essential functions and acts as a nodal point of cell polarity regulation in vivo . In addition, mapping the functional data for each cdc42 mutation onto a structural model of the protein revealed as functionally important a surface of Cdc42p that is distinct from the canonical protein-interacting domains (switch I, switch II, and the C terminus) identified previously in members of the Ras superfamily . This region overlaps with a region (alpha5-helix) recently predicted by structural models to be a specificity determinant for Cdc42p-protein interactions.

Mol Biol Cell, 2000 Jan, 11(1), 277 - 86
The N terminus of the transmembrane protein BP180 interacts with the N-terminal domain of BP230, thereby mediating keratin cytoskeleton anchorage to the cell surface at the site of the hemidesmosome; Hopkinson SB et al.; In epidermal cells, the keratin cytoskeleton interacts with the elements in the basement membrane via a multimolecular junction called the hemidesmosome . A major component of the hemidesmosome plaque is the 230-kDa bullous pemphigoid autoantigen (BP230/BPAG1), which connects directly to the keratin-containing intermediate filaments of the cytoskeleton via its C terminus . A second bullous pemphigoid antigen of 180 kDa (BP180/BPAG2) is a type II transmembrane component of the hemidesmosome . Using yeast two-hybrid technology and recombinant proteins, we show that an N-terminal fragment of BP230 can bind directly to an N-terminal fragment of BP180 . We have also explored the consequences of expression of the BP230 N terminus in 804G cells that assemble hemidesmosomes in vitro . Unexpectedly, this fragment disrupts the distribution of BP180 in transfected cells but has no apparent impact on the organization of endogenous BP230 and alpha6beta4 integrin . We propose that the BP230 N terminus competes with endogenous BP230 protein for BP180 binding and inhibits incorporation of BP180 into the cell surface at the site of the hemidesmosome . These data provide new insight into those interactions of the molecules of the hemidesmosome that are necessary for its function in integrating epithelial and connective tissue types.

Mol Biol Cell, 2000 Jan, 11(1), 227 - 39
ATPase-defective mammalian VPS4 localizes to aberrant endosomes and impairs cholesterol trafficking; Bishop N et al.; The yeast vacuolar sorting protein Vps4p is an ATPase required for endosomal trafficking that couples membrane association to its ATPase cycle . To investigate the function of mammalian VPS4 in endosomal trafficking, we have transiently expressed wild-type or ATPase-defective human VPS4 (hVPS4) in cultured cells . Wild-type hVPS4 was cytosolic, whereas a substantial fraction of hVPS4 that was unable to either bind or hydrolyze ATP was localized to membranes, including those of specifically induced vacuoles . Vacuoles were exclusively endocytic in origin, and subsets of enlarged vacuoles stained with markers for each stage of the endocytic pathway . Sorting of receptors from the early endosome to the recycling compartment or to the trans-Golgi network was not significantly affected, and no mutant hVPS4 associated with these compartments . However, many hVPS4-induced vacuoles were substantially enriched in cholesterol relative to the endosomal compartments of untransfected cells, indicating that expression of mutant hVPS4 gives rise to a kinetic block in postendosomal cholesterol sorting . The phenotype described here is largely consistent with the defects in vacuolar sorting associated with class E vps mutants in yeast, and a role for mammalian VPS4 is discussed in this context.

Mol Biol Cell, 2000 Jan, 11(1), 141 - 52
Mutants of the Yarrowia lipolytica PEX23 gene encoding an integral peroxisomal membrane peroxin mislocalize matrix proteins and accumulate vesicles containing peroxisomal matrix and membrane proteins; Brown TW et al.; pex mutants are defective in peroxisome assembly . The mutant strain pex23-1 of the yeast Yarrowia lipolytica lacks morphologically recognizable peroxisomes and mislocalizes all peroxisomal matrix proteins investigated preferentially to the cytosol . pex23 strains accumulate vesicular structures containing both peroxisomal matrix and membrane proteins . The PEX23 gene was isolated by functional complementation of the pex23-1 strain and encodes a protein, Pex23p, of 418 amino acids (47,588 Da) . Pex23p exhibits high sequence similarity to two hypothetical proteins of the yeast Saccharomyces cerevisiae . Pex23p is an integral membrane protein of peroxisomes that is completely, or nearly completely, sequestered from the cytosol . Pex23p is detected at low levels in cells grown in medium containing glucose, and its levels are significantly increased by growth in medium containing oleic acid, the metabolism of which requires intact peroxisomes.

Mol Biol Cell, 2000 Jan, 11(1), 103 - 16
Tim18p is a new component of the Tim54p-Tim22p translocon in the mitochondrial inner membrane; Kerscher O et al.; The mitochondrial inner membrane contains two separate translocons: one required for the translocation of matrix-targeted proteins (the Tim23p-Tim17p complex) and one for the insertion of polytopic proteins into the mitochondrial inner membrane (the Tim54p-Tim22p complex) . To identify new members of the Tim54p-Tim22p complex, we screened for high-copy suppressors of the temperature-sensitive tim54-1 mutant . We identified a new gene, TIM18, that encodes an integral protein of the inner membrane . The following genetic and biochemical observations suggest that the Tim18 protein is part of the Tim54p-Tim22p complex in the inner membrane: multiple copies of TIM18 suppress the tim54-1 growth defect; the tim18::HIS3 disruption is synthetically lethal with tim54-1; Tim54p and Tim22p can be coimmune precipitated with the Tim18 protein; and Tim18p, along with Tim54p and Tim22p, is detected in an approximately 300-kDa complex after blue native electrophoresis . We propose that Tim18p is a new component of the Tim54p-Tim22p machinery that facilitates insertion of polytopic proteins into the mitochondrial inner membrane.

J Biol Chem, 2000 Jan 21, 275(3), 2137 - 46
A novel mechanism of cell killing by anti-topoisomerase II bisdioxopiperazines; Jensen LH et al.; Bisdioxopiperazines are a unique class of topoisomerase II inhibitors that lock topoisomerase II at a point in the enzyme reaction cycle where the enzyme forms a closed clamp around DNA . We examined cell killing by ICRF-187 and ICRF-193 in yeast cells expressing human topoisomerase II alpha (htop-IIalpha) . Expression of htop-IIalpha in yeast cells sensitizes them to both ICRF-187 and ICRF-193, compared with cells expressing yeast topoisomerase II . ICRF-193 is still able to exert growth inhibition in the presence of genes encoding both ICRF-193-resistant and ICRF-193-sensitive htop-IIalpha enzymes, indicating that sensitivity to bisdioxopiperazines is dominant . Killing by ICRF-193 occurs more rapidly, than the killing in yeast cells due to a temperature-sensitive yeast topoisomerase II incubated at the non-permissive temperature . These results are reminiscent of a top-II poison such as etoposide . However, the killing caused by ICRF-193 and ICRF-187 is not enhanced by mutations in the RAD52 pathway . The levels of drug-induced DNA cleavage observed with htop-IIalpha in vitro is insufficient to explain the sensitivity induced by this enzyme in yeast cells . Finally, arrest of cells in G(1) does not protect cells from ICRF-193 lethality, a result inconsistent with killing mechanisms due to catalytic inhibition of top-II or stabilization of a cleavable complex . We suggest that the observed pattern of cell killing is most consistent with a poisoning of htop-II by ICRF-193 by a novel mechanism . The accumulation of closed clamp conformations of htop-II induced by ICRF-193 that are trapped on DNA might interfere with transcription, or other DNA metabolic processes, resulting in cell death.

J Biol Chem, 2000 Jan 21, 275(3), 1541 - 50
Activation of DNA-dependent protein kinase by single-stranded DNA ends; Hammarsten O et al.; DNA-dependent protein kinase (DNA-PK) is involved in joining DNA double-strand breaks induced by ionizing radiation or V(D)J recombination . The kinase is activated by DNA ends and composed of a DNA binding subunit, Ku, and a catalytic subunit, DNA-PK(CS) . To define the DNA structure required for kinase activation, we synthesized a series of DNA molecules and tested their interactions with purified DNA-PK(CS) . The addition of unpaired single strands to blunt DNA ends increased binding and activation of the kinase . When single-stranded loops were added to the DNA ends, binding was preserved, but kinase activation was severely reduced . Obstruction of DNA ends by streptavidin reduced both binding and activation of the kinase . Significantly, short single-stranded oligonucleotides of 3-10 bases were capable of activating DNA-PK(CS) . Taken together, these data indicate that kinase activation involves a specific interaction with free single-stranded DNA ends . The structure of DNA-PK(CS) contains an open channel large enough for double-stranded DNA and an adjacent enclosed cavity with the dimensions of single-stranded DNA . The data presented here support a model in which duplex DNA binds to the open channel, and a single-stranded DNA end is inserted into the enclosed cavity to activate the kinase.

Mol Biol Rep, 1999 Dec, 26(4), 261 - 7
Efficiency of expression of transfected genes depends on the cell cycle; Marenzi S et al.; Lipofection, a lipid-mediated DNA transfection procedure, was used to transfect synchronized L929 mouse fibroblast cells with a reporter plasmid containing the bacterial chloramphenicol acetyltransferase gene . The efficiency of gene expression was investigated on transfection of cells at different stages of the cell cycle . Our data show that expression of the reporter gene was minimal when transfection was performed in G0-phase and parallel experimental data disproved the possibility that the reduced expression observed was due to differential uptake at different times in the cell cycle . Investigation into the condensation state of the plasmid has shown that the low chloramphenicol acetyltransferase gene expression could be a direct consequence of the packaging of the plasmid into condensed chromatin when transfection occurs in G0-phase . The inactivation of the reporter gene is not reversed by growth of the cells in high serum or by treatment with Trichostatin A, a specific inhibitor of histone deacetylase, suggesting that the inactive chromatin formed in G0-phase cells lacks associated histone acetylase activity . In contrast, the high activity seen when cells in S-phase are transfected is enhanced even further by treatment with Trichostatin A.

Nat Struct Biol, 2000 Jan, 7(1), 62 - 71
D/H amide kinetic isotope effects reveal when hydrogen bonds form during protein folding; Krantz BA et al.; We have exploited a procedure to identify when hydrogen bonds (H-bonds) form under two-state folding conditions using equilibrium and kinetic deuterium/hydrogen amide isotope effects . Deuteration decreases the stability of equine cytochrome c and the dimeric and crosslinked versions of the GCN4-p1 coiled coil by approximately 0 . 5 kcal mol-1 . For all three systems, the decrease in equilibrium stability is reflected by a decrease in refolding rates and a near equivalent increase in unfolding rates . This apportionment indicates that approximately 50% of the native H-bonds are formed in the transition state of these helical proteins . In contrast, an alpha/beta protein, mammalian ubiquitin, exhibits a small isotope effect only on unfolding rates, suggesting its folding pathway may be different . These four proteins recapitulate the general trend that approximately 50% of the surface buried in the native state is buried in the transition state, leading to the hypothesis that H-bond formation in the transition state is cooperative, with alpha-helical proteins forming a number of H-bonds proportional to the amount of surface buried in the transition state.

Nat Struct Biol, 2000 Jan, 7(1), 11 - 3
May the driving force be with you--whatever it is; Cavanagh J et al.; Changes in the atomic coordinate fluctuations contribute to the entropy of biomolecular processes such as complex formation . Characterizations of such changes in proteins reveal that the response may be dramatically different between the backbone and the side chains, and further resolve enthalpy-entropy compensation at the molecular level.

Eur J Biochem, 2000 Jan, 267(2), 434 - 49
Cdc20 protein contains a destruction-box but, unlike Clb2, its proteolysisis not acutely dependent on the activity of anaphase-promoting complex; Goh PY et al.; Both chromosome segregation and the final exit from mitosis require a ubiquitin-protein ligase called anaphase-promoting complex (APC) or cyclosome . This multiprotein complex ubiquitinates various substrates, such as the anaphase inhibitor Pds1 and mitotic cyclins, and thus targets them for proteolysis by the 26S proteasome . The ubiquitination by APC is dependent on the presence of a destruction-box sequence in the N-terminus of target proteins . Recent reports have strongly suggested that Cdc20, a WD40 repeat-containing protein required for nuclear division in the budding yeast Saccharomyces cerevisiae, is essential for the APC-mediated proteolysis . To understand the function of CDC20, we have studied its regulation in some detail . The expression of the CDC20 gene is cell-cycle regulated such that it is transcribed only during late S phase and mitosis . Although the protein is unstable to some extent through out the cell cycle, its degradation is particularly enhanced in G1 . Cdc20 contains a destruction box sequence which, when mutated or deleted, stabilizes it considerably in G1 . Surprisingly, we find that while the inactivation of APC subunits Cdc16, Cdc23 or Cdc27 results in stabilization of the mitotic cyclin Clb2 in G1, the proteolytic destruction of Cdc20 remains largely unaffected . This suggests the existence of proteolytic mechanisms in G1 that can degrade destruction-box containing proteins, such as Cdc20, in an APC-independent manner.

Protein Sci, 1999 Dec, 8(12), 2645 - 54
Thermal stability of hydrophobic heme pocket variants of oxidized cytochrome c; Liggins JR et al.; Microcalorimetry has been used to measure the stabilities of mutational variants of yeast iso-1 cytochrome c in which F82 and L85 have been replaced by other hydrophobic amino acids . Specifically, F82 has been replaced by Y and L85 by A . The double mutant F82Y,L85A iso-1 has also been studied, and the mutational perturbations are compared to those for the two single mutants, F82Y iso-1 and L85A iso-1 . Results are interpreted in terms of known crystallographic structures . The data show that (1) the destabilization of the mutant proteins is similar in magnitude to that which is theoretically predicted by the more obvious mutation-induced structural effects; (2) the free energy of destabilization of the double mutant, F82Y,L85A iso-1, is less than the sum of those of the two single mutants, almost certainly because, in the double mutant, the -OH group of Y82 is able to protrude into the cavity formed by the L85A substitution . The more favorable structural accommodation of the new -OH group in the double mutant leads to additional stability through (1) further decreases in the volumes of internal cavities and (2) formation of an extra protein-protein hydrogen bond.

Plant Physiol, 2000 Jan, 122(1), 157 - 68
Calmodulin activation of an endoplasmic reticulum-located calcium pump involves an interaction with the N-terminal autoinhibitory domain; Hwang I et al.; To investigate how calmodulin regulates a unique subfamily of Ca(2+) pumps found in plants, we examined the kinetic properties of isoform ACA2 identified in Arabidopsis . A recombinant ACA2 was expressed in a yeast K616 mutant deficient in two endogenous Ca(2+) pumps . Orthovanadate-sensitive (45)Ca(2+) transport into vesicles isolated from transformants demonstrated that ACA2 is a Ca(2+) pump . Ca(2+) pumping by the full-length protein (ACA2-1) was 4- to 10-fold lower than that of the N-terminal truncated ACA2-2 (Delta2-80), indicating that the N-terminal domain normally acts to inhibit the pump . An inhibitory sequence (IC(50) = 4 microM) was localized to a region within valine-20 to leucine-44, because a peptide corresponding to this sequence lowered the V(max) and increased the K(m) for Ca(2+) of the constitutively active ACA2-2 to values comparable to the full-length pump . The peptide also blocked the activity (IC(50) = 7 microM) of a Ca(2+) pump (AtECA1) belonging to a second family of Ca(2+) pumps . This inhibitory sequence appears to overlap with a calmodulin-binding site in ACA2, previously mapped between aspartate-19 and arginine-36 (J.F . Harper, B . Hong, I . Hwang, H.Q . Guo, R . Stoddard, J.F . Huang, M.G . Palmgren, H . Sze inverted question mark1998 J Biol Chem 273: 1099-1106) . These results support a model in which the pump is kept "unactivated" by an intramolecular interaction between an autoinhibitory sequence located between residues 20 and 44 and a site in the Ca(2+) pump core that is highly conserved between different Ca(2+) pump families . Results further support a model in which activation occurs as a result of Ca(2+)-induced binding of calmodulin to a site overlapping or immediately adjacent to the autoinhibitory sequence.

Development, 2000 Feb, 127(3), 573 - 83
Investigating the function of follicular subpopulations during Drosophila oogenesis through hormone-dependent enhancer-targeted cell ablation; Han DD et al.; Although it is known that the establishment of polarity during Drosophila oogenesis is initiated by signalling from the oocyte to the overlying follicle cells, much less is understood about the role of specific follicular subpopulations . One powerful approach for addressing this question, toxigenic cell ablation of specific subpopulations, has not previously been applicable to studying follicular subpopulations because many of the genes and Gal4 enhancer trap insertions that are expressed in the ovary are also expressed at earlier times in development . To overcome this problem, we have utilized a fusion protein between Gal4 and the human estrogen receptor to achieve hormone-dependent, tissue-specific gene expression of UAS-linked transgenes in flies . We used this system to study the role of the polar subpopulations of follicle cells during oogenesis by expressing within them a modified form of diphtheria toxin that causes cell death . Our results confirmed previous functions ascribed to these cells, and also demonstrated a previously undescribed role for the border cells in facilitating the migration of the anterior Fasciclin III-expressing polar pair cells to the edge of the oocyte.

Toxicol Appl Pharmacol, 2000 Jan 1, 162(1), 22 - 33
Issues arising when interpreting results from an in vitro assay for estrogenic activity; Beresford N et al.; Concern about possible adverse effects caused by the inadvertent exposure of humans and wildlife to endocrine-active chemicals, has led some countries to develop an in vivo-in vitro screening program for endocrine effects . In this paper, a previously described estrogen-inducible recombinant yeast strain (Saccharomyces cerevisiae) is used to investigate a number of issues that could potentially lead to the mislabeling of chemicals as endocrine disruptors . The chemicals studied were: 17beta-estradiol, dihydrotestosterone, testosterone, estradiol-3-sulfate, 4-nonylphenol, 4-tert-octylphenol, 4-tert-butylphenol, bisphenol-A, methoxychlor, 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane, butyl benzyl phthalate, 4-hydroxytamoxifen, and ICI 182,780 . Alterations in assay methodology (for example, incubation time, initial yeast cell number, and the use of different solvents) did not affect the potency of bisphenol-A and 4-nonylphenol relative to 17beta-estradiol, but did alter the apparent potency of butyl benzyl phthalate . Other issues (including the metabolic activation of methoxychlor, the chemical purity of a steroid metabolite and unusual chemical artifacts observed with alkylphenolic chemicals) which affect data interpretation are described . Many of the issues raised will also affect other in vitro assays for endocrine activity, and some will be relevant to the interpretation of data from in vivo assays . These examples illustrate that considerable care and thought must be applied when interpreting results derived from any single assay . Only by using a suite of assays will we minimize the chances of wrongly labeling chemicals as endocrine disruptors .

Mutat Res, 1999 Dec 6, 430(2), 299 - 305
Space radiation effects and microgravity; Kiefer J et al.; Humans in space are exposed both to space radiation and microgravity . The question whether radiation effects are modified by microgravity is an important aspect in risk estimation . No interaction is expected at the molecular level since the influence of gravity is much smaller than that of thermal motion . Influences might be expected, however, at the cellular and organ level . For example, changes in immune competence could modify the development of radiogenic cancers . There are no data so far in this area . The problem of whether intracellular repair of radiation-induced DNA lesions is changed under microgravity conditions was recently addressed in a number of space experiments . The results are reviewed; they show that repair processes are not modified by microgravity.

J Cell Biol, 2000 Jan 10, 148(1), 115 - 26
Filamin 2 (FLN2): A muscle-specific sarcoglycan interacting protein; Thompson TG et al.; Mutations in genes encoding for the sarcoglycans, a subset of proteins within the dystrophin-glycoprotein complex, produce a limb-girdle muscular dystrophy phenotype; however, the precise role of this group of proteins in the skeletal muscle is not known . To understand the role of the sarcoglycan complex, we looked for sarcoglycan interacting proteins with the hope of finding novel members of the dystrophin-glycoprotein complex . Using the yeast two-hybrid method, we have identified a skeletal muscle-specific form of filamin, which we term filamin 2 (FLN2), as a gamma- and delta-sarcoglycan interacting protein . In addition, we demonstrate that FLN2 protein localization in limb-girdle muscular dystrophy and Duchenne muscular dystrophy patients and mice is altered when compared with unaffected individuals . Previous studies of filamin family members have determined that these proteins are involved in actin reorganization and signal transduction cascades associated with cell migration, adhesion, differentiation, force transduction, and survival . Specifically, filamin proteins have been found essential in maintaining membrane integrity during force application . The finding that FLN2 interacts with the sarcoglycans introduces new implications for the pathogenesis of muscular dystrophy.

Oncogene, 1999 Dec 20, 18(55), 7883 - 99
DNA damage-induced cell cycle checkpoints and DNA strand break repair in development and tumorigenesis; Dasika GK et al.; Several newly identified tumor suppressor genes including ATM, NBS1, BRCA1 and BRCA2 are involved in DNA double-strand break repair (DSBR) and DNA damage-induced checkpoint activation . Many of the gene products involved in checkpoint control and DSBR have been studied in great detail in yeast . In addition to evolutionarily conserved proteins such as Chk1 and Chk2, studies in mammalian cells have identified novel proteins such as p53 in executing checkpoint control . DSBR proteins including Mre11, Rad50, Rad51, Rad54, and Ku are present in yeast and in mammals . Many of the tumor suppressor gene products interact with these repair proteins as well as checkpoint regulators, thus providing a biochemical explanation for the pleiotropic phenotypes of mutant cells . This review focuses on the proteins mediating G1/S, S, and G2/M checkpoint control in mammalian cells . In addition, mammalian DSBR proteins and their activities are discussed . An intricate network among DNA damage signal transducers, cell cycle regulators and the DSBR pathways is illustrated . Mouse knockout models for genes involved in these processes have provided valuable insights into their function, establishing genomic instability as a major contributing factor in tumorigenesis.

Biol Bull, 1999 Dec, 197(3), 341 - 7
Microinjection of an antibody to the Ku protein arrests development in sea urchin embryos; Kanungo J et al.; Ku is the regulatory subunit of the DNA-dependent protein kinase (DNA-PK) . This enzyme plays a role in DNA repair, recombination, and transcription . It is composed of a large catalytic subunit (p460), and a regulatory heterodimer, the Ku protein, which consists of 86-kDa and 70-kDa subunits . These various components of the enzyme have been found in both eggs and embryos of the sea urchin . When variable amounts of a specific monoclonal antibody to the Ku protein (Ku 162) were injected into one cell of a 2-cell embryo of Lytechinus pictus, they caused a dose-dependent developmental arrest of the injected cell . The non-injected cell continued to develop normally . In contrast, injection of an antibody (N3H10) raised against the 70-kDa subunit of the Ku protein had no effect on development when injected into 2-cell-stage embryos . Co-injection of purified DNA-PK with the antibody reversed the antibody-mediated inhibition of development . In the fertilized egg and during the early stages of development, the DNA-PK was localized largely in the cytoplasm, but in later developmental stages, it assumed a nuclear location . On the basis of these results, we postulate that the injection of the Ku antibody either prevents the translocation of the DNA-PK into the nucleus or interferes with its enzymatic activity either in the nucleus or in the cytoplasm . In either case, the results suggest that DNA-PK plays an important role in regulating the early stages of embryogenesis in this primitive organism.

Nucleic Acids Res, 2000 Dec 15, 28(24), 4846 - 55
Characterization of the B lymphocyte-induced maturation protein-1 (Blimp-1) gene, mRNA isoforms and basal promoter; Tunyaplin C et al.; Blimp-1 is a transcriptional repressor that is both required and sufficient to trigger terminal differentiation of B lymphocytes and monocyte/macrophages . Here we report the organization of the mouse Blimp-1 gene, an analysis of Blimp-1 homologs in different species, the characterization of Blimp-1 mRNA isoforms and initial studies on the transcription of Blimp-1 . The murine Blimp-1 gene covers approximately 23 kb and contains eight exons . There are Blimp-1 homologs in species evolutionarily distant from mouse (Caenorhabditis elegans and Drosophila melanogaster) but no homolog was found in the unicellular yeast Saccharomyces cerevisiae . The three major Blimp-1 mRNA isoforms result from the use of different polyadenylation sites and do not encode different proteins . Run-on transcription analyses were used to show that the developmentally regulated expression of Blimp-1 mRNA in B cells is determined by transcription initiation . Multiple Blimp-1 transcription initiates sites were mapped near an initiator element and a region conferring basal promoter activity has been identified.

Mol Cell Biol, 2000 Feb, 20(3), 1055 - 62
Peri-implantation lethality in mice lacking the Sm motif-containing protein Lsm4; Hirsch E et al.; Small nuclear ribonucleoproteins (snRNPs) are particles present only in eukaryotic cells . They are involved in a large variety of RNA maturation processes, most notably in pre-mRNA splicing . Several of the proteins typically found in snRNPs contain a sequence signature, the Sm domain, conserved from yeast to mammals . By using a promoter trap strategy to target actively transcribed loci in murine embryonic stem cells, a new murine gene encoding an Sm motif-containing protein was identified . Database searches revealed that it is the mouse orthologue of Lsm4p, a protein found in yeast and human cells and putatively associated with U6 snRNA . Introduction of the geo reporter gene cassette under the control of the murine Lsm4 (mLsm4) endogenous promoter showed that the gene was ubiquitously transcribed in embryonic and adult tissues . The insertion of the geo cassette disrupted the mLsm4 allele, and homozygosity for the mutation led to a recessive embryonic lethal phenotype . mLsm4-null zygotes survived to the blastocyst stages, implanted into the uterus, but died shortly thereafter . The early death of mLsm4p-null mice suggests that the role of mLsm4p in splicing is essential and cannot be compensated by other Lsm proteins.

Mol Cell Biol, 2000 Feb, 20(3), 1021 - 9
PR48, a novel regulatory subunit of protein phosphatase 2A, interacts with Cdc6 and modulates DNA replication in human cells; Yan Z et al.; Initiation of DNA replication in eukaryotes is dependent on the activity of protein phosphatase 2A (PP2A), but specific phosphoprotein substrates pertinent to this requirement have not been identified . A novel regulatory subunit of PP2A, termed PR48, was identified by a yeast two-hybrid screen of a human placental cDNA library, using human Cdc6, an essential component of prereplicative complexes, as bait . PR48 binds specifically to an amino-terminal segment of Cdc6 and forms functional holoenzyme complexes with A and C subunits of PP2A . PR48 localizes to the nucleus of mammalian cells, and its forced overexpression perturbs cell cycle progression, causing a G(1) arrest . These results suggest that dephosphorylation of Cdc6 by PP2A, mediated by a specific interaction with PR48, is a regulatory event controlling initiation of DNA replication in mammalian cells.

Mol Cell Biol, 2000 Feb, 20(3), 786 - 96
The function of DNA polymerase alpha at telomeric G tails is important for telomere homeostasis; Adams Martin A et al.; Telomere length control is influenced by several factors, including telomerase, the components of telomeric chromatin structure, and the conventional replication machinery . Although known components of the replication machinery can influence telomere length equilibrium, little is known about why mutations in certain replication proteins cause dramatic telomere lengthening . To investigate the cause of telomere elongation in cdc17/pol1 (DNA polymerase alpha) mutants, we examined telomeric chromatin, as measured by its ability to repress transcription on telomere-proximal genes, and telomeric DNA end structures in pol1-17 mutants . pol1-17 mutants with elongated telomeres show a dramatic loss of the repression of telomere-proximal genes, or telomeric silencing . In addition, cdc17/pol1 mutants grown under telomere-elongating conditions exhibit significant increases in single-stranded character in telomeric DNA but not at internal sequences . The single strandedness is manifested as a terminal extension of the G-rich strand (G tails) that can occur independently of telomerase, suggesting that cdc17/pol1 mutants exhibit defects in telomeric lagging-strand synthesis . Interestingly, the loss of telomeric silencing and the increase in the sizes of the G tails at the telomeres temporally coincide and occur before any detectable telomere lengthening is observed . Moreover, the G tails observed in cdc17/pol1 mutants incubated at the semipermissive temperature appear only when the cells pass through S phase and are processed by the time cells reach G(1) . These results suggest that lagging-strand synthesis is coordinated with telomerase-mediated telomere maintenance to ensure proper telomere length control.

Mol Cell Biol, 2000 Feb, 20(3), 749 - 54
Cdc37 promotes the stability of protein kinases Cdc28 and Cak1; Farrell A et al.; In the budding yeast Saccharomyces cerevisiae, Cdc37 is required for the productive formation of Cdc28-cyclin complexes . The cdc37-1 mutant arrests at Start with low levels of Cdc28 protein, which is predominantly unphosphorylated at Thr169, fails to bind cyclin, and has little protein kinase activity . We show here that Cdc28 and not cyclin is specifically defective in the cdc37-1 mutant and that Cdc37 likely does not act as an assembly factor for Cdc28-cyclin complex formation . We have also found that the levels and activity of the protein kinase Cak1 are significantly reduced in the cdc37-1 mutant . Pulse-chase analysis indicates that Cdc28 and Cak1 proteins are both destabilized when Cdc37 function is absent during but not after translation . In addition, Cdc37 promotes the production of Cak1, but not that of Cdc28, when coexpressed in insect cells . We conclude that budding yeast Cdc37, like its higher eukaryotic homologs, promotes the physical integrity of multiple protein kinases, perhaps by virtue of a cotranslational role in protein folding.

Genetics, 2000 Jan, 154(1), 273 - 84
Quantitative analysis of gene function in the Drosophila embryo; Tracey WD Jr et al.; The specific functions of gene products frequently depend on the developmental context in which they are expressed . Thus, studies on gene function will benefit from systems that allow for manipulation of gene expression within model systems where the developmental context is well defined . Here we describe a system that allows for genetically controlled overexpression of any gene of interest under normal physiological conditions in the early Drosophila embryo . This regulated expression is achieved through the use of Drosophila lines that express a maternal mRNA for the yeast transcription factor GAL4 . Embryos derived from females that express GAL4 maternally activate GAL4-dependent UAS transgenes at uniform levels throughout the embryo during the blastoderm stage of embryogenesis . The expression levels can be quantitatively manipulated through the use of lines that have different levels of maternal GAL4 activity . Specific phenotypes are produced by expression of a number of different developmental regulators with this system, including genes that normally do not function during Drosophila embryogenesis . Analysis of the response to overexpression of runt provides evidence that this pair-rule segmentation gene has a direct role in repressing transcription of the segment-polarity gene engrailed . The maternal GAL4 system will have applications both for the measurement of gene activity in reverse genetic experiments as well as for the identification of genetic factors that have quantitative effects on gene function in vivo.

J Cell Biochem, 1999, Suppl 32-33, 141 - 8
Control of histone modifications; Davie JR et al.; A role for histone modifications in transcription processes and the remodeling of chromatin structure has been established . This review highlights the recent advances made in studies on histone acetyltransferases, histone deacetylases, histone kinases, and protein phosphatases, as well as their roles in transcriptional activation and repression . Coactivators with histone acetyltransferase activity stimulate transcription, whereas corepressors with histone deacetylase activity repress transcription . Families of histone acetyltransferases and deacetylases have been identified . We have learned that their substrates are not limited to histones but also include transcription factors and architectural proteins . Studies on the composition of multiprotein complexes with histone acetyltransferase or histone deacetylase have revealed mechanisms by which these complexes are recruited to specific genomic sites that are transcriptionally active, silenced, or being repaired . A new and exciting development, presented in this review, is the role of signal transduction pathways in the phosphorylation of histone H3 and the expression of immediate-early genes . J . Cell . Biochem . Suppls . 32/33:141-148, 1999 .

J Cell Biochem, 1999, Suppl 32-33, 32 - 40
Dual functions for transcriptional regulators: myth or reality?
St-Arnaud R.
Several transcriptional regulatory molecules have been described that appear to possess dual function in separate cellular compartments . It remains unclear whether the proteins really exert dual functions, or which of the transcriptional regulatory role or the cytoplasm-associated function is the physiologically relevant action of the protein . This review will briefly describe the cases at hand and attempt to sort the true bifunctional proteins from the aritfactual trespassers . J . Cell . Biochem . Suppls . 32/33:32-40, 1999 .

Mol Gen Genet, 1999 Dec, 262(4-5), 668 - 76
A new transcriptional activator for amylase genes in Aspergillus; Petersen KL et al.; We have cloned a regulatory gene for amylase synthesis in Aspergillus oryzae . This gene, amyR, encodes a 604-amino acid transcriptional activator with a Cys6 zinc cluster, that shows extensive homology to the DNA binding domain of GAL4 from Saccharomyces cerevisiae . The DNA binding domain of amyR binds to two types of sequences found in a number of promoters from Aspergillus genes coding for starch-degrading enzymes . One type of binding site is characterized by two CGG triplets separated by eight nucleotides . The other type has only one CGG triplet, which is followed by the sequence AAATTTAA.

J Neurosci, 2000 Jan 1, 20(1), 283 - 93
The bHLH gene hes1 as a repressor of the neuronal commitment of CNS stem cells; Nakamura Y et al.; Hes1 is one of the basic helix-loop-helix transcription factors that regulate mammalian CNS development, and its loss- and gain-of-function phenotypes indicate that it negatively regulates neuronal differentiation . Here we report that Hes1(-/-) mice expressed both early (TuJ1 and Hu) and late (MAP2 and Neurofilament) neuronal markers prematurely, and that there were approximately twice the normal number of neurons in the Hes1(-/-) brain during early neural development . However, immunochemical analyses of sections and dissociated cells using neural progenitor markers, including nestin, failed to detect any changes in Hes1(-/-) progenitor population . Therefore, further characterization of neural progenitor cells that discriminated between multipotent and monopotent cells was performed using two culture methods, low-density culture, and a neurosphere assay . We demonstrate that the self-renewal activity of multipotent progenitor cells was reduced in the Hes1(-/-) brain, and that their subsequent commitment to the neuronal lineage was accelerated . The Hes1(-/-) neuronal progenitor cells were functionally abnormal, in that they divided, on average, only once, and then generated two neurons, (instead of one progenitor cell and one neuron), whereas wild-type progenitor cells divided more . In addition, some Hes1(-/-) progenitors followed an apoptotic fate . The overproduction of neurons in the early Hes1(-/-) brains may reflect this premature and immediate generation of neurons as well as a net increase in the number of neuronal progenitor cells . Taken together, we conclude that Hes1 is important for maintaining the self-renewing ability of progenitors and for repressing the commitment of multipotent progenitor cells to a neuronal fate, which is critical for the correct number of neurons to be produced and for the establishment of normal neuronal function.

J Virol, 2000 Feb, 74(3), 1275 - 85
An African swine fever virus ERV1-ALR homologue, 9GL, affects virion maturation and viral growth in macrophages and viral virulence in swine; Lewis T et al.; The African swine fever virus (ASFV) genome contains a gene, 9GL, with similarity to yeast ERV1 and ALR genes . ERV1 has been shown to function in oxidative phosphorylation and in cell growth, while ALR has hepatotrophic activity . 9GL encodes a protein of 119 amino acids and was highly conserved at both nucleotide and amino acid levels among all ASFV field isolates examined . Monospecific rabbit polyclonal antibody produced to a glutathione S-transferase-9GL fusion protein specifically immunoprecipitated a 14-kDa protein from macrophage cell cultures infected with the ASFV isolate Malawi Lil-20/1 (MAL) . Time course analysis and viral DNA synthesis inhibitor experiments indicated that p14 was a late viral protein . A 9GL gene deletion mutant of MAL (Delta9GL), exhibited a growth defect in macrophages of approximately 2 log(10) units and had a small-plaque phenotype compared to either a revertant (9GL-R) or the parental virus . 9GL affected normal virion maturation; virions containing acentric nucleoid structures comprised 90 to 99% of all virions observed in Delta9GL-infected macrophages . The Delta9GL virus was markedly attenuated in swine . In contrast to 9GL-R infection, where mortality was 100%, all Delta9GL-infected animals survived infection . With the exception of a transient fever response in some animals, Delta9GL-infected animals remained clinically normal and exhibited significant 100- to 10,000-fold reductions in viremia titers . All pigs previously infected with Delta9GL survived infection when subsequently challenged with a lethal dose of virulent parental MAL . Thus, ASFV 9GL gene deletion mutants may prove useful as live-attenuated ASF vaccines.

Blood, 2000 Jan 15, 95(2), 633 - 8
Transforming activity of receptor tyrosine kinase tyro3 is mediated, at least in part, by the PI3 kinase-signaling pathway; Lan Z et al.; Protein tyrosine phosphorylation is an integral part of cytokine-induced proliferation and differentiation of hematopoietic cells . The authors previously reported cloning and characterization of the receptor tyrosine kinase Tif, also termed Tyro3 . Using the yeast 2-hybrid technology, they recently identified that the p85 subunit of phosphatidylinositol 3-kinase (PI3 kinase) interacted with the cytoplasmic domain of Tyro3 . On treatment with epidermal growth factor (EGF), NIH3T3 cells expressed EGFR/Tyro3 (a fusion receptor with the extracellular domain from epidermal growth factor receptor and the transmembrane and cytoplasmic domains from Tyro3), and EGFR/Tyro3 was rapidly phosphorylated on tyrosine residues . The interaction between Tyro3 and p85 was also confirmed by glutathione S-transferase (GST) pull-down experiments . Co-immunoprecipitation followed by Western blot analysis revealed that PI3 kinase was associated with and phosphorylated by the activated Tyro3 . Tyro3-associated PI3 kinase exhibited an enhanced kinase activity . In addition, EGF treatment of EGFR/Tyro3-expressing cells led to enhanced phosphorylation of Akt, a downstream component of PI3 kinase . Treatment of NIH3T3 cells expressing a full length of rat Tyro-3, but not NIH3T3 cells, with protein S also resulted in phosphorylation of Akt . Soft agar colony assays showed that the addition of EGF to EGFR/Tyro3-transfected cells, but not to the parental NIH3T3 cells, resulted in a concentration-dependent increase in the formation of anchorage-independent colonies . Tyro3-mediated transformation of NIH3T3 cells was significantly blocked by wortmannin, a PI3 kinase-specific inhibitor . Results of these combined studies strongly suggested that the oncogenic transforming ability of Tyro3 was mediated at least in part by the PI3 kinase pathway . (Blood . 2000;95:633-638)

J Biol Chem, 2000 Jan 14, 275(2), 1405 - 13
The noncatalytic domain of protein-tyrosine phosphatase-PEST targets paxillin for dephosphorylation in vivo; Shen Y et al.; The noncatalytic domain of protein-tyrosine phosphatase (PTP)-PEST contains a binding site for the focal adhesion-associated protein paxillin . This binding site has been narrowed to a 52-residue sequence that is composed of two nonoverlapping, weak paxillin binding sites . The PTP-PEST binding site on paxillin has been mapped to the two carboxyl-terminal LIM (lin11, isl-1, and mec-3) domains . Transient expression of PTP-PEST reduced tyrosine phosphorylation of p130(cas), as anticipated . A PTP-PEST mutant defective for binding p130(cas) does not cause a reduction in its tyrosine phosphorylation in vivo . Expression of PTP-PEST also caused a reduction of phosphotyrosine on paxillin . Expression of mutants of PTP-PEST with deletions in the paxillin-binding site did not associate with paxillin in vivo and failed to cause a reduction in the phosphotyrosine content of paxillin . These results demonstrate that paxillin can serve as a PTP-PEST substrate in vivo and support the model that a noncatalytic domain interaction recruits paxillin to PTP-PEST to facilitate its dephosphorylation.

J Biol Chem, 2000 Jan 14, 275(2), 1112 - 8
Targeting and subcellular localization of Toxoplasma gondii catalase . Identification of peroxisomes in an apicomplexan parasite; Kaasch AJ et al.; We sought to identify and characterize peroxisomes in the apicomplexan parasite Toxoplasma gondii . To initiate this process, we first cloned and sequenced the gene for T . gondii catalase (EC 1 . 11.1.6), a marker enzyme for peroxisomes in eukaryotic cells . The gene predicts a protein of 57.2 kDa and 502 amino acids and has a strong homology to other eukaryotic catalases . A polyclonal antiserum raised against a glutathione S-transferase fusion protein recognized a single band with a molecular mass of 63 kDa by immunoblot . By immunofluorescence T . gondii catalase is present primarily in a punctate staining pattern anterior to the parasite nucleus . This compartment is distinguishable from other parasite organelles, namely micronemes, rhoptries, dense granules, and the apicoplast . Cytochemical visualization of catalase using diaminobenzidine precipitation gives a vesicular staining pattern anterior to the nucleus at the light level and round, vesicular structures with an estimated diameter of 100-300 nm by electron microscopy . T . gondii catalase has a putative C-terminal peroxisomal targeting signal in the last 3 amino acids (-AKM) . Expression of T . gondii catalase in mammalian cells results in peroxisomal localization, whereas a construct lacking the targeting signal remains in the cytosol . Furthermore, addition of -AKM to the C terminus of chloramphenicol acetyltransferase is sufficient to target this protein to peroxisomes . These results provide the first evidence for peroxisomes in Apicomplexan parasites.

J Biol Chem, 2000 Jan 14, 275(2), 1007 - 14
Loss of in vitro metal ion binding specificity in mutant copper-zinc superoxide dismutases associated with familial amyotrophic lateral sclerosis; Goto JJ et al.; The presence of the copper ion at the active site of human wild type copper-zinc superoxide dismutase (CuZnSOD) is essential to its ability to catalyze the disproportionation of superoxide into dioxygen and hydrogen peroxide . Wild type CuZnSOD and several of the mutants associated with familial amyotrophic lateral sclerosis (FALS) (Ala(4) --> Val, Gly(93) --> Ala, and Leu(38) --> Val) were expressed in Saccharomyces cerevisiae . Purified metal-free (apoproteins) and various remetallated derivatives were analyzed by metal titrations monitored by UV-visible spectroscopy, histidine modification studies using diethylpyrocarbonate, and enzymatic activity measurements using pulse radiolysis . From these studies it was concluded that the FALS mutant CuZnSOD apoproteins, in direct contrast to the human wild type apoprotein, have lost their ability to partition and bind copper and zinc ions in their proper locations in vitro . Similar studies of the wild type and FALS mutant CuZnSOD holoenzymes in the "as isolated" metallation state showed abnormally low copper-to-zinc ratios, although all of the copper acquired was located at the native copper binding sites . Thus, the copper ions are properly directed to their native binding sites in vivo, presumably as a result of the action of the yeast copper chaperone Lys7p (yeast CCS) . The loss of metal ion binding specificity of FALS mutant CuZnSODs in vitro may be related to their role in ALS.

J Biol Chem, 2000 Jan 14, 275(2), 840 - 6
GABA receptor rho1 subunit interacts with a novel splice variant of the glycine transporter, GLYT-1; Hanley JG et al.; Ionotropic gamma-aminobutyric acid (GABA(A) and GABA(C)) receptors mediate fast synaptic inhibition in the central nervous system . GABA(C) receptors are expressed predominantly in the retina on bipolar cell axon terminals, and are thought to mediate feedback inhibition from GABAergic amacrine cells . Utilizing the yeast two-hybrid system, we previously identified MAP1B as a binding partner of the GABA(C) receptor rho1 subunit . Here we describe the isolation of an additional rho1 interacting protein: a novel C-terminal variant of the glycine transporter GLYT-1 . We show that GLYT-1 exists as four alternatively spliced mRNAs which encode proteins expressing one of two possible intracellullar N- and C-terminal domains . Variants containing the novel C terminus efficiently transport glycine when expressed in COS cells, but with unusual kinetics . We have confirmed the interaction between the novel C terminus and rho1 subunit and demonstrated binding in heterologous cells . This interaction may be crucial for the integration of GABAergic and glycinergic neurotransmission in the retina.

J Biol Chem, 2000 Jan 14, 275(2), 801 - 8
SAC1 encodes a regulated lipid phosphoinositide phosphatase, defects in which can be suppressed by the homologous Inp52p and Inp53p phosphatases; Hughes WE et al.; The yeast protein Sac1p is involved in a range of cellular functions, including inositol metabolism, actin cytoskeletal organization, endoplasmic reticulum ATP transport, phosphatidylinositol-phosphatidylcholine transfer protein function, and multiple-drug sensitivity . The activity of Sac1p and its relationship to these phenotypes are unresolved . We show here that the regulation of lipid phosphoinositides in sac1 mutants is defective, resulting in altered levels of all lipid phos- phoinositides, particularly phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate . We have identified two proteins with homology to Sac1p that can suppress drug sensitivity and also restore the levels of the phosphoinositides in sac1 mutants . Overexpression of truncated forms of these suppressor genes confirmed that suppression was due to phosphoinositide phosphatase activity within these proteins . We have now demonstrated this activity for Sac1p and have characterized its specificity . The in vitro phosphatase activity and specificity of Sac1p were not altered by some mutations . Indeed, in vivo mutant Sac1p phosphatase activity also appeared unchanged under conditions in which cells were drug-resistant . However, under different growth conditions, both drug sensitivity and the phosphatase defect were manifest . It is concluded that SAC1 encodes a novel lipid phosphoinositide phosphatase in which specific mutations can cause the sac1 phenotypes by altering the in vivo regulation of the protein rather than by destroying phosphatase activity.

Anal Biochem, 2000 Jan 15, 277(2), 214 - 20
Studies on the fluorescence reaction between nucleic acid and the complex of cobalt(II) with 5-(3-fluoro-4-chlorophenylazo)-8-sulfonamidoquinoline and its applications; Cao QE et al.; Based on the enhancement of the fluorescence intensity of the complex of 5-(3-fluoro-4-chlorophenylazo)-8-sulfonamidoquinoline (FCPBSQ) with Co(2+) by nucleic acid in the presence of Tween 80 and in the weakly basic medium, a fluorescence method for the determination of nucleic acid was proposed . The calibration graphs for the determination of denatured calf thymus DNA (ct DNA), fish sperm DNA (fs DNA), and yeast RNA (yt RNA) were obtained in concentration ranges 0.050-4.0, 0.10-3.0, and 0.050-3.0 microg/ml with limits of detection of 0.010, 0.030, and 0.020 microg/ml, respectively . The method has been satisfactorily used for the determination of DNA in wheat cell extraction and ct DNA, fs DNA, and yt RNA in synthetic samples . Investigations on the binding mode by the Scatchard plots method suggested that both intercalation and electrostatic binding modes existed in this system .

J Inorg Biochem, 1999 Oct, 77(1-2), 71 - 81
Specificity of platinum-DNA adduct repair; Chaney SG et al.; Cell lines with resistance to cisplatin and carboplatin often retain sensitivity to platinum complexes with different carrier ligands (e.g., oxaliplatin and JM216) . HeLa cell extracts were shown to excise cisplatin, oxaliplatin, and JM216 adducts with equal efficiency, suggesting that nucleotide excision repair does not contribute to the carrier-ligand specificity of platinum resistance . We have shown previously that the extent of replicative bypass in vivo is influenced by the carrier ligand of the platinum adducts . The specificity of replicative bypass may be determined by the DNA polymerase complexes that catalyze translesion synthesis past Pt-DNA adducts, by the mismatch-repair system that removes newly synthesized DNA opposite Pt-DNA adducts, and/or by DNA damage-recognition proteins that bind to the Pt-DNA adducts and block translesion synthesis . Primer extension on DNA templates containing site-specifically placed cisplatin, oxaliplatin, or JM216 Pt-GG adducts revealed that the eukaryotic DNA polymerases beta, zeta, gamma and HIV-1 RT had a similar specificity for translesion synthesis past Pt-DNA adducts (oxaliplatin > or = cisplatin > JM216) . In addition, defects in the mismatch-repair proteins hMSH6 and hMLH1 led to increased replicative bypass of cisplatin adducts, but not of oxaliplatin adducts . Finally, primer extension assays performed in the presence of HMG1, which is known to recognize cisplatin-damaged DNA, revealed that inhibition of translesion synthesis by HMG1 also depended on the carrier ligand of the Pt-DNA adduct (cisplatin > oxaliplatin = JM216) . These studies show that DNA polymerases, the mismatch-repair system and damage-recognition proteins can all impart specificity to replicative bypass of Pt-DNA adducts . Replicative bypass, in turn, may influence the carrier-ligand specificity of resistance.

Ter Arkh, 1999, 71(11), 41 - 5
{Association of TAP1/TAP2 gene polymorphism with inflammatory urogenital diseases combined with joint diseases}; Sartakova ML et al.; AIM: To study association of TAP1/TAP2 gene polymorphism with urogenital infections combined with joint lesions . MATERIALS AND METHODS: Of 139 patients examined 45 ones had inflammation caused by Chlamydia trachomatis (17 had joint disease), 42 had Mycoplasma hominis infection (joint lesions in 17 cases) . Method of amplification was used on the basis of specific primers (ARMS) . RESULTS: Patients infected with C . trachomatis significantly more frequently had allele TAP1-02011 and TAP2-0201 (RR = 18.5, p < 0.01 and RR = 4.61, p < 0.05, respectively) . Joint lesion in patients with chlamydial infection was associated with allele TAP1-02011 (RR = 11.92, p < 0.05) . In mycoplasmosis association with joint lesions there is a significant link of joint syndrome with heterogeneous combination threonine/alanine in gene TAP2 position 565 (RR = 4.22, p < 0.05) . CONCLUSION: The findings can be used for predicting the joint syndrome development in patients with urogenital infection.

Rapid Commun Mass Spectrom, 2000, 14(1), 12 - 7
Analysis of non-covalent protein complexes up to 290 kDa using electrospray ionization and ion trap mass spectrometry; Wang Y et al.; Non-covalently-bound subunit complexes of proteins have been measured by an ion trap mass spectrometer equipped with an orthogonal electrospray ionization source . For the analysis of the generated molecular ions with high mass/charge ratios, the mass/charge range of the ion trap was extended by increasing its radio frequency (rf) voltage to 15 kV (V(0-p)) and by resonant ion ejection . Ions of the non-covalent dimer of bovine serum albumin (BSA), as well as of subunit complexes of alcohol dehydrogenase (ADH) from bakers' yeast and from horse liver, have been detected at mass/charge values between 3000-9000 Th . The maximum observed molecular weight was that of a non-covalently-bound subunit-octamer of bakers' yeast ADH (two non-covalently-bound subunit-tetramers) at ca . 290 kDa .

Biochem Biophys Res Commun, 2000 Jan 7, 267(1), 418 - 22
NXP-1, a human protein related to Rad21/Scc1/Mcd1, is a component of the nuclear matrix; Sadano H et al.; Nuclear matrix is a complex intranuclear network supposed to be involved in the various nuclear functions . In order to identify the nuclear matrix proteins, we isolated a cDNA clone from a human placenta cDNA library . This clone was partially represented a known cDNA clone HA1237 . HA1237 encoded a 631-amino-acid peptide, which we designated NXP-1 . NXP-1 was related to yeast Rad21/Scc1/Mcd1, Xenopus XRAD21, and mouse PW29, and identical with HR21spA isolated from a human testis cDNA library . We developed a polyclonal antibody to the purified NXP-1 bacterially expressed as a fusion protein with GST . Western blot analysis with anti-NXP-1 polyclonal antibody showed nuclear matrix localization of NXP-1 in HeLa cells . Indirect immunofluorescence staining also showed nuclear and nuclear matrix localization of the NXP-1 . Results of in vitro binding assays employing nuclear matrix preparations indicated that the N-terminal region (16-128 amino acid) of NXP-1 has an important role in nuclear matrix distribution .

Mol Phylogenet Evol, 1999 Dec, 13(3), 483 - 92
Use of atp6 in fungal phylogenetics: an example from the boletales; Kretzer AM et al.; Complete nucleotide sequences have been determined for atp6 from Suillus luteus and cox3 from Suillus sinuspaulianus (Boletales, Hymenomycetes, Basidiomycota), which code for ATPase subunit 6 and cytochrome oxidase subunit 3, respectively . These sequences were used to design PCR primers for the amplification of partial atp6 and cox3 sequences from other members of the Boletales and outgroup taxa . In atp6 and cox3 from Russula rosacea, one of the outgroup taxa, we observed a number of in-frame TGA(trp) codons, which imply a Neurospora crassa-type mitochondrial code in R . rosacea and possibly in basidiomycetes in general . Interestingly, however, most basidiomycetes other than R . rosacea appear to strongly prefer the TGG(trp) codon, which is unusual, given the strong A + T bias in fungal mitochondrial genomes . Pairwise comparisons were performed between atp6 sequences from increasingly divergent fungal lineages, and results show that all three codon positions become saturated in substitutions after an estimated divergence time of approx 300 Ma . This means that atp6 is likely to provide phylogenetic resolution within fungal classes but not at higher taxonomic levels . Also, because of the strong A + T bias in fungal mitochondrial genomes, A/T transversions were found to be more common than any other type of substitution, resulting in transversions being about two to three times more common in most pairwise sequence comparisons . Finally, atp6 sequences were used to infer phylogenetic relationships between 27 taxa from the Boletales and 4 outgroup taxa . Analyses were performed (i) on nucleotide sequence data using parsimony (successive approximation) as well as maximum likelihood methods and (ii) on deduced amino acid sequences using distance methods based on empirical substitution probabilities . Results from the various analyses are largely concordant with each other as well as with prior analyses of partial mitochondrial large-subunit rDNA (mtLSU rDNA) . Analysis of the combined atp6 and mtLSU rDNA sequences results in increased bootstrap support for several key branches . Relationships that have been resolved for the first time in the current analysis are discussed .

FEBS Lett, 1999 Dec 3, 462(3), 329 - 34
Differential gene expression of mammalian SPO11/TOP6A homologs during meiosis; Shannon M et al.; As the initiator of DNA double-strand breaks during meiosis in Saccharomyces cerevisiae, the SPO11 protein is essential for recombination . Similarity between SPO11 and archaebacterial TOP6A proteins points to evolutionary specialization of a DNA cleavage function for meiotic recombination . To determine whether this extends to mammals, we isolated and characterized mouse and human SPO11 cDNAs . Mammalian SPO11 genes were found to be expressed at high levels only in testis, wherein mouse Spo11 transcript is restricted primarily to meiotic germ cells and is maximally expressed at midpachynema . Mouse Spo11 is located near the distal end of chromosome 2, while human SPO11 is found in the homologous position of chromosome 20q13.2-13.3, a region that is amplified in some breast cancers . Sequence homology and differential expression together support a highly conserved role for SPO11 in the enzymatic cleavage of DNA that accompanies meiotic recombination.

FEBS Lett, 1999 Dec 3, 462(3), 249 - 53
Isolation and characterization of a calendic acid producing (8,11)-linoleoyl desaturase; Fritsche K et al.; For the biosynthesis of calendic acid a (8,11)-linoleoyl desaturase activity has been proposed . To isolate this desaturase, PCR-based cloning was used . The open reading frame of the isolated full-length cDNA is a 1131 bp sequence encoding a protein of 377 amino acids . For functional identification the cDNA was expressed in Saccharomyces cerevisiae, and formation of calendic acid was analyzed by RP-HPLC . The expression of the heterologous enzyme resulted in a significant amount of calendic acid presumably esterified within phospholipids . The results presented here identify a gene encoding a new type of (1,4)-acyl lipid desaturase.

FEBS Lett, 1999 Dec 3, 462(3), 246 - 8
Inhibition of transketolase by p-hydroxyphenylpyruvate; Solovjeva ON et al.; The effect of p-hydroxyphenylpyruvate, a natural analogue of transketolase substrate, on the catalytic activity of the enzyme was investigated . p-Hydroxyphenylpyruvate proved to be a reversible and competitive inhibitor of transketolase with respect to substrate; it was also able to displace thiamine diphosphate from holotransketolase . The data suggest that p-hydroxyphenylpyruvate participates in the regulation of tyrosine biosynthesis by influencing the catalytic activity of transketolase.

Life Sci, 1999, 65(26), 2829 - 35
Somatostatin analogs stimulate DNA-dependent protein kinase activity in human gastric tumoral cell-line HGT1; Sadji Z et al.; DNA-dependent protein kinase catalytic subunit (DNA-PKcs) phosphorylates, in the presence of double-stranded DNA, several transcription-, replication- and repair -factors . Its interaction with the DNA-binding regulatory component Ku (p86-/p70-Ku) is required for stabilization and activity . We have previously shown that p86-Ku behaves as a specific receptor for the growth inhibitory tetradecapeptide, somatostatin . In this work, we investigate a possible regulation by somatostatin analogs, of DNA-PK activity in the human gastric tumoral HGT1/clone6 cell-line . We demonstrate that a 48 h-preincubation of cells with octreotide or RC-160, stimulates DNA-PK activity by 8 and 10 fold with ED50s of 1 and 0.1 nM, respectively . These stimulations appearing only after 3 h were inhibited by cycloheximide . They were not observed in a cell clone which was transfected by a cDNA encoding p86-Ku antisense . This study demonstrates the existence of a new somatostatin signaling pathway involving the stimulation of DNA-PK activity.

Yeast, 2000 Jan 15, 16(1), 57 - 64
Evolutionary complementation for polymerase II CTD function; Stiller JW et al.; The C-terminal domain (CTD) of the largest subunit (RPB1) of eukaryotic RNA polymerase II is essential for pol II function and has been shown to play a number of important roles in the mRNA transcription cycle . The CTD is composed of a tandemly repeated heptapeptide that is conserved in yeast, animals, plants and several protistan organisms . Some eukaryotes, however, have what appear to be degenerate or deviant CTD regions, and others have no CTD at all . The functional and evolutionary implications of this variation among RPB1 C-termini is largely unexplored . We have transformed yeast cells with a construct consisting of the yeast RPB1 gene with 25 heptads from the primitive protist Mastigamoeba invertens in place of the wild-type CTD . The Mastigamoeba heptads differ from the canonical CTD by the invariable presence of alanines in place of threonines at position 4, and in place of serines at position 7 of each heptad . Despite this double substitution, mutants are viable even under conditions of temperature and nutrient stress . These results provide new insights into the relative functional importance of several of the conserved CTD residues, and indicate that in vivo expression of evolutionary variants in yeast can provide important clues for understanding the origin, evolution and function of the pol II CTD .

Biotechnol Bioeng, 2000 Feb 5, 67(3), 300 - 11
Performance modeling and simulation of biochemical process sequences with interacting unit operations; Groep ME et al.; Many biochemical processes consist of a sequence of operations for which optimal operating conditions (setpoints) have to be determined . If such optimization is performed for each operation separately with respect to objectives defined for each operation individually, overall process performance is likely to be suboptimal . Interactions between unit operations have to be considered, and a unique objective has to be defined for the whole process . This paper shows how a suitable optimization problem can be formulated and solved to obtain the best overall set of operating conditions for a process . A typical enzyme production process has been chosen as an example . In order to arrive at a demonstrative model for the entire sequence of unit operations, it is shown how interaction effects may be accommodated in the models . Optimal operating conditions are then determined subject to a global process objective and are shown to be different from those resulting from optimization of each separate operation . As this strategy may result in an economic benefit, it merits further research into interaction modeling and performance optimization .

Glycoconj J, 1999 Jul, 16(7), 327 - 36
UDP-N-Acetyl-alpha-D-glucosamine as acceptor substrate of beta-1,4-galactosyltransferase . Enzymatic synthesis of UDP-N-acetyllactosamine; Elling L et al.; The capacity of UDP-N-acetyl-alpha-D-glucosamine (UDP-GlcNAc) as an in vitro acceptor substrate for beta-1,4-galactosyltransferase (beta4GalT1, EC 2.4.1.38) from human and bovine milk and for recombinant human beta4GalT1, expressed in Saccharomyces cerevisiae, was evaluated . It turned out that each of the enzymes is capable to transfer Gal from UDP-alpha-D-galactose (UDP-Gal) to UDP-GlcNAc, affording Gal(beta1-4)GlcNAc(alpha1-UDP (UDP-LacNAc) . Using beta4GalT1 from human milk, a preparative enzymatic synthesis of UDP-LacNAc was carried out, and the product was characterized by fast-atom bombardment mass spectrometry and 1H and 13C NMR spectroscopy . Studies with all three beta4GalTs in the presence of alpha-lactalbumin showed that the UDP-LacNAc synthesis is inhibited and that UDP-alpha-D-glucose is not an acceptor substrate . This is the first reported synthesis of a nucleotide-activated disaccharide, employing a Leloir glycosyltransferase with a nucleotide-activated monosaccharide as acceptor substrate . Interestingly, in these studies beta4GalT1 accepts an alpha-glycosidated GlcNAc derivative . The results imply that beta4GalT1 may be responsible for the biosynthesis of UDP-LacNAc, previously isolated from human milk.

Proc Natl Acad Sci U S A, 2000 Jan 4, 97(1), 262 - 7
Knowledge-based analysis of microarray gene expression data by using support vector machines; Brown MP et al.; We introduce a method of functionally classifying genes by using gene expression data from DNA microarray hybridization experiments . The method is based on the theory of support vector machines (SVMs) . SVMs are considered a supervised computer learning method because they exploit prior knowledge of gene function to identify unknown genes of similar function from expression data . SVMs avoid several problems associated with unsupervised clustering methods, such as hierarchical clustering and self-organizing maps . SVMs have many mathematical features that make them attractive for gene expression analysis, including their flexibility in choosing a similarity function, sparseness of solution when dealing with large data sets, the ability to handle large feature spaces, and the ability to identify outliers . We test several SVMs that use different similarity metrics, as well as some other supervised learning methods, and find that the SVMs best identify sets of genes with a common function using expression data . Finally, we use SVMs to predict functional roles for uncharacterized yeast ORFs based on their expression data.

J Clin Microbiol, 2000 Jan, 38(1), 227 - 35
Typing of Candida glabrata in clinical isolates by comparative sequence analysis of the cytochrome c oxidase subunit 2 gene distinguishes two clusters of strains associated with geographical sequence polymorphisms; Sanson GF et al.; We tested whether comparative sequence analysis of the mitochondrion-encoded cytochrome c oxidase subunit 2 gene (COX2) could be used to distinguish intraspecific variants of Candida glabrata . Mitochondrial genes are suitable for investigation of close phylogenetic relationships because they evolve much faster than nuclear genes, which in general exhibit very limited intraspecific variation . For this survey we used 11 clinical isolates of C . glabrata from three different geographical locations in Brazil, 10 isolates from one location in the United States, 1 American Type Culture Collection strain as an internal control, and the published sequence of strain CBS 138 . The complete coding region of COX2 was amplified from total cellular DNA, and both strands were sequenced twice for each strain . These sequences were aligned with published sequences from other fungi, and the numbers of substitutions and phylogenetic relationships were determined . Typing of these strains was done by using 17 substitutions, with 8 being nonsynonymous and 9 being synonymous . Also, cDNAs made from purified mitochondrial polyadenylated RNA were sequenced to confirm that our sequences correspond to the expressed copies and not nuclear pseudogenes and that a frameshift mutation exists in the 3' end of the coding region (position 673) relative to the Saccharomyces cerevisiae sequence and the previously published C . glabrata sequence . We estimated the average evolutionary rate of COX2 to be 11.4% sequence divergence/10(8) years and that phylogenetic relationships of yeasts based on these sequences are consistent with rRNA sequence data . Our analysis of COX2 sequences enables typing of C . glabrata strains based on 13 haplotypes and suggests that positions 51 and 519 indicate a geographical polymorphism that discriminates strains isolated in the United States and strains isolated in Brazil . This provides for the first time a means of typing of Candida strains that cause infections by use of direct sequence comparisons and the associated divergence estimates.

Cell, 1999 Dec 23, 99(7), 691 - 702
A role for presenilin-1 in nuclear accumulation of Ire1 fragments and induction of the mammalian unfolded protein response; Niwa M et al.; The unfolded protein response (UPR) mediates signaling from the endoplasmic reticulum to the nucleus . In yeast, a key regulatory step in the UPR is the spliceosome-independent splicing of HAC1 mRNA encoding a UPR-specific transcription factor, which is initiated by the transmembrane kinase/endoribonuclease Ire1 . We show that yeast HAC1 mRNA is correctly spliced in mammalian cells upon UPR induction and that mammalian Ire1 can precisely cleave both splice junctions . Surprisingly, UPR induction leads to proteolytic cleavage of Ire1, releasing fragments containing the kinase and nuclease domains that accumulate in the nucleus . Nuclear localization and UPR induction are reduced in presenilin-1 knockout cells . These results suggest that the salient features of the UPR are conserved among eukaryotic cells and that presenilin-1 controls Ire1 proteolysis in mammalian cells.

Mol Cell, 1999 Nov, 4(5), 735 - 43
A C/EBP beta isoform recruits the SWI/SNF complex to activate myeloid genes; Kowenz-Leutz E et al.; The activation of many genes requires the concerted effort of two or more transcription factors . Although C/EBP beta is known to cooperate with Myb to induce transcription of the granulocyte-specific mim-1 gene, the molecular mechanism of this cooperativity is undefined . We show that the N terminus of the full-length C/EBP beta isoform, which is essential for induction of the mim-1 gene in chromatin, interacts specifically with the SWI/SNF complex . Grafting this domain onto Myb generates a chimeric activator that recruits SWI/SNF and induces mim-1 transcription in the absence of C/EBP beta . Interaction between C/EBP beta and SWI/SNF is essential for activating a subgroup of resident target genes in chromatin and may represent a major determinant of combinatorial gene regulation in eukaryotes.

Mol Cell, 1999 Nov, 4(5), 725 - 34
Acetylation of MyoD directed by PCAF is necessary for the execution of the muscle program; Sartorelli V et al.; p300/CBP and PCAF coactivators have acetyltransferase activities and regulate transcription, cell cycle progression, and differentiation . They are both required for MyoD activity and muscle differentiation . Nevertheless, their roles must be different since the acetyltransferase activity of PCAF but not of p300 is involved in controlling myogenic transcription and differentiation . Here, we provide a molecular explanation of this phenomenon and report that MyoD is directly acetylated by PCAF at evolutionarily conserved lysines . Acetylated MyoD displays an increased affinity for its DNA target . Importantly, conservative substitutions of acetylated lysines with nonacetylatable arginines impair the ability of MyoD to stimulate transcription and to induce muscle conversion indicating that acetylation of MyoD is functionally critical.

FEBS Lett, 1999 Dec 31, 464(3), 189 - 93
Endogenous mutations in human uncoupling protein 3 alter its functional properties; Brown AM et al.; Human uncoupling protein (UCP3) is a mitochondrial transmembrane carrier that uncouples oxidative phosphorylation and is a candidate gene for obesity . Expression of native human UCP3 mutations in yeast showed complete loss (R70W), significant reduction (R143X), or no effect (V102I and IVS6+1G > A) on the uncoupling activity of UCP3 . It is concluded that certain mutations in UCP3 alter its functional impact on membrane potential (deltaphi), possibly conferring susceptibility to develop metabolic diseases.

Proc Natl Acad Sci U S A, 2000 Jan 4, 97(1), 430 - 5
Epstein-Barr virus nuclear protein 2 interacts with p300, CBP, and PCAF histone acetyltransferases in activation of the LMP1 promoter; Wang L et al.; The Epstein-Barr virus (EBV) nuclear protein 2 (EBNA2) and herpes simplex virion protein 16 (VP16) acidic domains that mediate transcriptional activation now are found to have affinity for p300, CBP, and PCAF histone acetyltransferases (HATs) . Transcriptionally inactive point mutations in these domains lack affinity for p300, CBP, or PCAF . P300 and CBP copurify with the principal HAT activities that bind to EBNA2 or VP16 acidic domains through velocity sedimentation and anion-exchange chromatography . EBNA2 binds to both the N- and C-terminal domains of p300 and coimmune-precipitates from transfected 293T cells with p300 . In EBV-infected Akata Burkitt's tumor cells that do not express the EBV encoded oncoproteins EBNA2 or LMP1, p300 expression enhances the ability of EBNA2 to up-regulate LMP1 expression . Through its intrinsic HAT activity, PCAF can further potentiate the p300 effect . In 293 T cells, P300 and CBP (but not PCAF) can also coactivate transcription mediated by the EBNA2 or VP16 acidic domains and HAT-negative mutants of p300 have partial activity . Thus, the EBNA2 and VP16 acidic domains can utilize the intrinsic HAT or scaffolding properties of p300 to activate transcription.

Proc Natl Acad Sci U S A, 2000 Jan 4, 97(1), 61 - 6
Competition between a sterol biosynthetic enzyme and tRNA modification in addition to changes in the protein synthesis machinery causes altered nonsense suppression; Benko AL et al.; The Saccharomyces cerevisiae Mod5 protein catalyzes isopentenylation of A to i(6)A on tRNAs in the nucleus, cytosol, and mitochondria . The substrate for Mod5p, dimethylallyl pyrophosphate, is also a substrate for Erg20p that catalyzes an essential step in sterol biosynthesis . Changing the distribution of Mod5p so that less Mod5p is present in the cytosol decreases i(6)A on cytosolic tRNAs and alters tRNA-mediated nonsense suppression . We devised a colony color/growth assay to assess tRNA-mediated nonsense suppression and used it to search for genes, which, when overexpressed, affect nonsense suppression . We identified SAL6, TEF4, and YDL219w, all of which likely affect nonsense suppression via alteration of the protein synthesis machinery . We also identified ARC1, whose product interacts with aminoacyl synthetases . Interestingly, we identified ERG20 . Midwestern analysis showed that yeast cells overproducing Erg20p have reduced levels of i(6)A on tRNAs . Thus, Erg20p appears to affect nonsense suppression by competing with Mod5p for substrate . Identification of ERG20 reveals that yeast have a limited pool of dimethylallyl pyrophosphate . It also demonstrates that disrupting the balance between enzymes that use dimethylallyl pyrophosphate as substrate affects translation.

Proc Natl Acad Sci U S A, 2000 Jan 4, 97(1), 55 - 60
Signal recognition particle components in the nucleolus; Politz JC et al.; The signal recognition particle (SRP) is a ribonucleoprotein composed of an Alu domain and an S domain . The S domain contains unique sequence SRP RNA and four SRP proteins: SRP19, SRP54, SRP68, and SRP72 . SRP interacts with ribosomes to bring translating membrane and secreted proteins to the endoplasmic reticulum (ER) for proper processing . Additionally, SRP RNA is a member of a family of small nonribosomal RNAs found recently in the nucleolus, suggesting that the nucleolus is more plurifunctional than previously realized . It was therefore of interest to determine whether other SRP components localize to this intranuclear site . In transfected rat fibroblasts, green fluorescent protein fusions of SRP19, SRP68, and SRP72 localized to the nucleolus, as well as to the cytoplasm, as expected . SRP68 also accumulated in the ER, consistent with its affinity for the ER-bound SRP receptor . SRP54 was detected in the cytoplasm as a green fluorescent protein fusion and in immunofluorescence studies, but was not detected in the nucleolus . In situ hybridization experiments also revealed endogenous SRP RNA in the nucleolus . These results demonstrate that SRP RNA and three SRP proteins visit the nucleolus, suggesting that partial SRP assembly, or another unidentified activity of the SRP components, occurs at the nucleolus . SRP54 apparently interacts with nascent SRP beyond the nucleolus, consistent with in vitro reconstitution experiments showing that SRP19 must bind to SRP RNA before SRP54 binds . Our findings support the notion that the nucleolus is the site of assembly and/or interaction between the family of ribonucleoproteins involved in protein synthesis, in addition to ribosomes themselves.

Appl Environ Microbiol, 2000 Jan, 66(1), 363 - 8
Characterization of the kexin-like maturase of Aspergillus niger; Jalving R et al.; Secreted yields of foreign proteins may be enhanced in filamentous fungi through the use of translational fusions in which the target protein is fused to an endogenous secreted carrier protein . The fused proteins are usually separated in vivo by cleavage of an engineered Kex2 endoprotease recognition site at the fusion junction . We have cloned the kexin-encoding gene of Aspergillus niger (kexB) . We constructed strains that either overexpressed KexB or lacked a functional kexB gene . Kexin-specific activity doubled in membrane-protein fractions of the strain overexpressing KexB . In contrast, no kexin-specific activity was detected in the similar protein fractions of the kexB disruptant . Expression in this loss-of-function strain of a glucoamylase human interleukin-6 fusion protein with an engineered Kex2 dibasic cleavage site at the fusion junction resulted in secretion of unprocessed fusion protein . The results show that KexB is the endoproteolytic proprotein processing enzyme responsible for the processing of (engineered) dibasic cleavage sites in target proteins that are transported through the secretion pathway of A . niger.

J Biol Chem, 2000 Jan 7, 275(1), 651 - 6
A mechanism of repression by acute myeloid leukemia-1, the target of multiple chromosomal translocations in acute leukemia; Lutterbach B et al.; AML1 is one of the most frequently translocated genes in human leukemia . Here we demonstrate that acute myeloid leukemia-1 (AML-1) (Runx-1) represses transcription from a native promoter, p21(Waf1/Cip1) . Unexpectedly, this repression did not require interactions with the Groucho co-repressor . To define the mechanism of repression, we asked whether other co-repressors could interact with AML-1 . We demonstrate that AML-1 interacts with the mSin3 co-repressors . Moreover, endogenous AML-1 associated with endogenous mSin3A in mammalian cells . A deletion mutant of AML-1 that did not interact with mSin3A failed to repress transcription . The AML-1/mSin3 association suggests a mechanism of repression for the chromosomal translocation fusion proteins that disrupt AML-1.

J Biol Chem, 2000 Jan 7, 275(1), 386 - 9
Regulation and reversibility of vacuolar H(+)-ATPase; Hirata T et al.; Arabidopsis thaliana vacuolar H(+)-translocating pyrophosphatase (V-PPase) was expressed functionally in yeast vacuoles with endogenous vacuolar H(+)-ATPase (V-ATPase), and the regulation and reversibility of V-ATPase were studied using these vacuoles . Analysis of electrochemical proton gradient (DeltamuH) formation with ATP and pyrophosphate indicated that the proton transport by V-ATPase or V-PPase is not regulated strictly by the proton chemical gradient (DeltapH) . On the other hand, vacuolar membranes may have a regulatory mechanism for maintaining a constant membrane potential (DeltaPsi) . Chimeric vacuolar membranes showed ATP synthesis coupled with DeltamuH established by V-PPase . The ATP synthesis was sensitive to bafilomycin A(1) and exhibited two apparent K(m) values for ADP . These results indicate that V-ATPase is a reversible enzyme . The ATP synthesis was not observed in the presence of nigericin, which dissipates DeltapH but not DeltaPsi, suggesting that DeltapH is essential for ATP synthesis.

J Biol Chem, 2000 Jan 7, 275(1), 343 - 50
Mlp2p, a component of nuclear pore attached intranuclear filaments, associates with nic96p; Kosova B et al.; A fraction of the yeast nucleoporin Nic96p is localized at the terminal ring of the nuclear basket . When Nic96p was affinity purified from glutaraldehyde-treated spheroplasts, it was found to be associated with Mlp2p . Mlp2p, together with Mlp1p, are the yeast Tpr homologues, which form the nuclear pore-attached intranuclear filaments (Strambio-de-Castillia, C., Blobel, G., and Rout, M . P . (1999) J . Cell Biol . 144, 839-855) . Double disruption mutants of MLP1 and MLP2 are viable and apparently not impaired in nucleocytoplasmic transport . However, overproduction of MLP1 causes nuclear accumulation of poly(A)(+) RNA in a chromatin-free area of the nucleus.

Genes Dev, 1999 Dec 15, 13(24), 3271 - 9
The TOR signaling cascade regulates gene expression in response to nutrients; Cardenas ME et al.; Rapamycin inhibits the TOR kinases, which regulate cell proliferation and mRNA translation and are conserved from yeast to man . The TOR kinases also regulate responses to nutrients, including sporulation, autophagy, mating, and ribosome biogenesis . We have analyzed gene expression in yeast cells exposed to rapamycin using arrays representing the whole yeast genome . TOR inhibition by rapamycin induces expression of nitrogen source utilization genes controlled by the Ure2 repressor and the transcriptional regulator Gln3, and globally represses ribosomal protein expression . gln3 mutations were found to confer rapamycin resistance, whereas ure2 mutations confer rapamycin hypersensitivity, even in cells expressing dominant rapamycin-resistant TOR mutants . We find that Ure2 is a phosphoprotein in vivo that is rapidly dephosphorylated in response to rapamycin or nitrogen limitation . In summary, our results reveal that the TOR cascade plays a prominent role in regulating transcription in response to nutrients in addition to its known roles in regulating translation, ribosome biogenesis, and amino acid permease stability.

Genes Dev, 1999 Dec 15, 13(24), 3209 - 16
Mechanism of corepressor binding and release from nuclear hormone receptors; Nagy L et al.; The association of transcription corepressors SMRT and N-CoR with retinoid and thyroid receptors results in suppression of basal transcriptional activity . A key event in nuclear receptor signaling is the hormone-dependent release of corepressor and the recruitment of coactivator . Biochemical and structural studies have identified a universal motif in coactivator proteins that mediates association with receptor LBDs . We report here the identity of complementary acting signature motifs in SMRT and N-CoR that are sufficient for receptor binding and ligand-induced release . Interestingly, the motif contains a hydrophobic core (PhixxPhiPhi) similar to that found in NR coactivators . Surprisingly, mutations in the amino acids that directly participate in coactivator binding disrupt the corepressor association . These results indicate a direct mechanistic link between activation and repression via competition for a common or at least partially overlapping binding site.

Nat Genet, 2000 Jan, 24(1), 5 - 6
Grass-roots genomics; Johnston M et al.; In these heady days of genomic enterprise, the wet-bench geneticist may wonder what the future holds in store for those who explore biology in the laboratory . We argue that the goal of genomics is to serve geneticists.

Carbohydr Res, 1999 Oct 15, 321(3-4), 143 - 56
Synthesis and glycosidase inhibitory activity of 5-thioglucopyranosylamines . Molecular modeling of complexes with glucoamylase; Randell KD et al.; The synthesis of a series of 5-thio-D-glucopyranosylarylamines by reaction of 5-thio-D-glucopyranose pentaacetate with the corresponding arylamine and mercuric chloride catalyst is reported . The products were obtained as anomeric mixtures of the tetraacetates which can be separated and crystallized . The tetraacetates were deprotected to give alpha/beta mixtures of the parent compounds which were evaluated as inhibitors of the hydrolysis of maltose by glucoamylase G2 (GA) . A transferred NOE NMR experiment with an alpha/beta mixture of 7 in the presence of GA showed that only the alpha isomer is bound by the enzyme . The Ki values, calculated on the basis of specific binding of the alpha isomers, are 0.47 mM for p-methoxy-N-phenyl-5-thio-D-glucopyranosylamine (7), 0.78 mM for N-phenyl-5-thio-D-glucopyranosylamine (8), 0.27 mM for p-nitro-N-phenyl-5-thio-D-glucopyranosylamine (9) and 0.87 mM for p-trifluoromethyl-N-phenyl-5-thio-D-glucopyranosylamine (10), and the K(m) values for the substrates maltose and p-nitrophenyl alpha-D-glucopyranoside are 1.2 and 3.7 mM, respectively . Methyl 4-amino-4-deoxy-4-N-(5'-thio-alpha-D-glucopyranosyl)-alpha-D-glucopyrano side (11) is a competitive inhibitor of GA wild-type (Ki 4 microM) and the active site mutant Trp120-->Phe GA (Ki 0.12 mM) . Compounds 7, 8, and 11 are also competitive inhibitors of alpha-glucosidase from brewer's yeast, with Ki values of 1.05 mM, > 10 mM, and 0.5 mM, respectively . Molecular modeling of the inhibitors in the catalytic site of GA was used to probe the ligand-enzyme complementary interactions and to offer insight into the differences in inhibitory potencies of the ligands.

J Cell Biol, 1999 Dec 27, 147(7), 1503 - 18
An actin-binding protein of the Sla2/Huntingtin interacting protein 1 family is a novel component of clathrin-coated pits and vesicles; Engqvist-Goldstein AE et al.; The actin cytoskeleton has been implicated in endocytosis, yet few molecules that link these systems have been identified . Here, we have cloned and characterized mHip1R, a protein that is closely related to huntingtin interacting protein 1 (Hip1) . These two proteins are mammalian homologues of Sla2p, an actin binding protein important for actin organization and endocytosis in yeast . Sequence alignments and secondary structure predictions verified that mHip1R belongs to the Sla2 protein family . Thus, mHip1R contains an NH(2)-terminal domain homologous to that implicated in Sla2p's endocytic function, three predicted coiled-coils, a leucine zipper, and a talin-like actin-binding domain at the COOH terminus . The talin-like domain of mHip1R binds to F-actin in vitro and colocalizes with F-actin in vivo, indicating that this activity has been conserved from yeast to mammals . mHip1R shows a punctate immunolocalization and is enriched at the cell cortex and in the perinuclear region . We concluded that the cortical localization represents endocytic compartments, because mHip1R colocalizes with clathrin, AP-2, and endocytosed transferrin, and because mHip1R fractionates biochemically with clathrin-coated vesicles . Time-lapse video microscopy of mHip1R-green fluorescence protein (GFP) revealed a blinking behavior similar to that reported for GFP-clathrin, and an actin-dependent inward movement of punctate structures from the cell periphery . These data show that mHip1R is a component of clathrin-coated pits and vesicles and suggest that it might link the endocytic machinery to the actin cytoskeleton.

J Cell Biol, 1999 Dec 27, 147(7), 1493 - 502
Adenine nucleotide translocase-1, a component of the permeability transition pore, can dominantly induce apoptosis; Bauer MK et al.; Here, we describe the isolation of adenine nucleotide translocase-1 (ANT-1) in a screen for dominant, apoptosis-inducing genes . ANT-1 is a component of the mitochondrial permeability transition complex, a protein aggregate connecting the inner with the outer mitochondrial membrane that has recently been implicated in apoptosis . ANT-1 expression led to all features of apoptosis, such as phenotypic alterations, collapse of the mitochondrial membrane potential, cytochrome c release, caspase activation, and DNA degradation . Both point mutations that impair ANT-1 in its known activity to transport ADP and ATP as well as the NH(2)-terminal half of the protein could still induce apoptosis . Interestingly, ANT-2, a highly homologous protein could not lead to cell death, demonstrating the specificity of the signal for apoptosis induction . In contrast to Bax, a proapoptotic Bcl-2 gene, ANT-1 was unable to elicit a form of cell death in yeast . This and the observed repression of apoptosis by the ANT-1-interacting protein cyclophilin D suggest that the suicidal effect of ANT-1 is mediated by specific protein-protein interactions within the permeability transition pore.

J Cell Biol, 1999 Dec 27, 147(7), 1443 - 56
Export of a cysteine-free misfolded secretory protein from the endoplasmic reticulum for degradation requires interaction with protein disulfide isomerase; Gillece P et al.; Protein disulfide isomerase (PDI) interacts with secretory proteins, irrespective of their thiol content, late during translocation into the ER; thus, PDI may be part of the quality control machinery in the ER . We used yeast pdi1 mutants with deletions in the putative peptide binding region of the molecule to investigate its role in the recognition of misfolded secretory proteins in the ER and their export to the cytosol for degradation . Our pdi1 deletion mutants are deficient in the export of a misfolded cysteine-free secretory protein across the ER membrane to the cytosol for degradation, but ER-to-Golgi complex transport of properly folded secretory proteins is only marginally affected . We demonstrate by chemical cross-linking that PDI specifically interacts with the misfolded secretory protein and that mutant forms of PDI have a lower affinity for this protein . In the ER of the pdi1 mutants, a higher proportion of the misfolded secretory protein remains associated with BiP, and in export-deficient sec61 mutants, the misfolded secretory protein remain bounds to PDI . We conclude that the chaperone PDI is part of the quality control machinery in the ER that recognizes terminally misfolded secretory proteins and targets them to the export channel in the ER membrane.

J Cell Biol, 1999 Dec 27, 147(7), 1371 - 8
Mitotic regulators govern progress through steps in the centrosome duplication cycle; Vidwans SJ et al.; Centrosome duplication is marked by discrete changes in centriole structure that occur in lockstep with cell cycle transitions . We show that mitotic regulators govern steps in centriole replication in Drosophila embryos . Cdc25(string), the expression of which initiates mitosis, is required for completion of daughter centriole assembly . Cdc20(fizzy), which is required for the metaphase-anaphase transition, is required for timely disengagement of mother and daughter centrioles . Stabilization of mitotic cyclins, which prevents exit from mitosis, blocks assembly of new daughter centrioles . Common regulation of the nuclear and centrosome cycles by mitotic regulators may ensure precise duplication of the centrosome.

Genes Chromosomes Cancer, 2000 Feb, 27(2), 124 - 9
Prevalence of germline truncating mutations in ATM in women with a second breast cancer after radiation therapy for a contralateral tumor; Shafman TD et al.; Patients treated with conservative surgery and radiation therapy for early-stage breast cancer develop a contralateral breast cancer at a rate of approximately 0.75% per year . Ataxia-telangiectasia (AT) is an autosomal recessive disease that is characterized by increased sensitivity to ionizing radiation (IR) and cancer susceptibility . Epidemiologic studies have suggested that AT carriers, who comprise 1% of the population, may be at an increased risk for developing breast cancer, particularly after exposure to IR . To test this hypothesis, we analyzed blood samples from 57 patients who developed a contralateral breast cancer at least 6 months after completion of radiation therapy for an initial breast tumor . A cDNA-based truncation assay in yeast was used to test for heterozygous mutations in the ATM gene, which is responsible for AT . No mutations were detected . Our findings fail to support the hypothesis that AT carriers account for a significant fraction of breast cancer cases arising in women after exposure to radiation . Genes Chromosomes Cancer 27:124-129, 2000 .

Mol Cell Endocrinol, 1999 Oct 25, 156(1-2), 159 - 68
Estrogen receptor variants ERdelta5 and ERdelta7 down-regulate wild-type estrogen receptor activity; Wang H et al.; The estrogen receptor (ER) plays a key role in mediating the effect of estrogens . It is the primary target for endocrine therapy for many diseases, including breast cancer . The ER contains six domains that are associated with distinct functions; the presence of all six domains is required for ligand-dependent receptor activity . ER variants, reported in breast tumors and other neoplasms, usually lack one or more domains or a part of a domain . Such deletions can have dramatic effects on ER activity, cellular response to hormone, and response to hormonal therapy . We used simple and rapid yeast systems to understand more clearly how ER variants alter the response of wild-type ER (wtER) to estrogen and antiestrogens . We co-expressed ER variant, ERdelta5 or ERdelta7, with wtER in yeast containing an ERE-LacZ reporter . We found that ERdelta5 and ERdelta7 decreased the response of wtER to 1 nM 17beta-estradiol by 41-43 and 24-34%, respectively . Alone, ERdelta5 displayed weak hormone-independent transcriptional activity that was not affected by tamoxifen or ICI 182,780 . ERdelta7, in contrast, showed no constitutive activity and no response to ligands . To further understand whether ERdelta5 and ERdelta7 affect wtER activity by forming a variant:wtER heterodimer, we used the yeast two-hybrid system . The protein-protein interaction results showed that ERdelta5 and ERdelta7 could form neither homodimers with themselves nor heterodimers with wtER . This finding suggests that the influence of ERdelta5 and ERdelta7 on wtER is not mediated by suppressing wtER through heterodimerization.

Cell, 1999 Dec 10, 99(6), 589 - 601
Golgi membranes are absorbed into and reemerge from the ER during mitosis; Zaal KJ et al.; Quantitative imaging and photobleaching were used to measure ER/Golgi recycling of GFP-tagged Golgi proteins in interphase cells and to monitor the dissolution and reformation of the Golgi during mitosis . In interphase, recycling occurred every 1.5 hr, and blocking ER egress trapped cycling Golgi enzymes in the ER with loss of Golgi structure . In mitosis, when ER export stops, Golgi proteins redistributed into the ER as shown by quantitative imaging in vivo and immuno-EM . Comparison of the mobilities of Golgi proteins and lipids ruled out the persistence of a separate mitotic Golgi vesicle population and supported the idea that all Golgi components are absorbed into the ER . Moreover, reassembly of the Golgi complex after mitosis failed to occur when ER export was blocked . These results demonstrate that in mitosis the Golgi disperses and reforms through the intermediary of the ER, exploiting constitutive recycling pathways . They thus define a novel paradigm for Golgi genesis and inheritance.

Annu Rev Cell Dev Biol, 1999, 15, 661 - 703
{PSI+}: an epigenetic modulator of translation termination efficiency; Serio TR et al.; The {PSI+} factor of the yeast Saccharomyces cerevisiae is an epigenetic regulator of translation termination . More than three decades ago, genetic analysis of the transmission of {PSI+} revealed a complex and often contradictory series of observations . However, many of these discrepancies may now be reconciled by a revolutionary hypothesis: protein conformation-based inheritance (the prion hypothesis) . This model predicts that a single protein can stably exist in at least two distinct physical states, each associated with a different phenotype . Propagation of one of these traits is achieved by a self-perpetuating change in the protein from one form to the other . Mounting genetic and biochemical evidence suggests that the determinant of {PSI+} is the nuclear encoded Sup35p, a component of the translation termination complex . Here we review the series of experiments supporting the yeast prion hypothesis and provide another look at the 30 years of work preceding this theory in light of our current state of knowledge.

Annu Rev Cell Dev Biol, 1999, 15, 63 - 80
Cooperation between microtubule- and actin-based motor proteins; Brown SS; Organelle transport has been proposed to proceed in two steps: long-range transport along microtubules and local delivery via actin filaments . This model is supported by recent studies of pigment transport in several cell types and transport in neurons, and in several cases, class V myosin has been implicated as the actin-based motor . Mutations in mice (dilute) and yeast (myo2) have also implicated this class of myosin in organelle transport, and genetic interactions in yeast have indicated that a kinesin-related protein (Smy1p) plays a supporting role . This link between members of two different motor superfamilies has now taken a surprising turn: There is evidence for a physical interaction between class V myosins and kinesin or Smy1p in both mice and yeast.

Proc Natl Acad Sci U S A, 1999 Dec 21, 96(26), 15044 - 9
Varying the number of telomere-bound proteins does not alter telomere length in tel1Delta cells; Ray A et al.; Yeast telomere DNA consists of a continuous, approximately 330-bp tract of the heterogeneous repeat TG(1-3) with irregularly spaced, high affinity sites for the protein Rap1p . Yeast monitor, or count, the number of telomeric Rap1p C termini in a negative feedback mechanism to modulate the length of the terminal TG(1-3) repeats, and synthetic telomeres that tether Rap1p molecules adjacent to the TG(1-3) tract cause wild-type cells to maintain a shorter TG(1-3) tract . To identify trans-acting proteins required to count Rap1p molecules, these same synthetic telomeres were placed in two short telomere mutants: yku70Delta (which lack the yeast Ku70 protein) and tel1Delta (which lack the yeast ortholog of ATM) . Although both mutants maintain telomeres with approximately 100 bp of TG(1-3), only yku70Delta cells maintained shorter TG(1-3) repeats in response to internal Rap1p molecules . This distinct response to internal Rap1p molecules was not caused by a variation in Rap1p site density in the TG(1-3) repeats as sequencing of tel1Delta and yku70Delta telomeres showed that both strains have only five to six Rap1p sites per 100-bp telomere . In addition, the tel1Delta short telomere phenotype was epistatic to the unregulated telomere length caused by deletion of the Rap1p C-terminal domain . Thus, the length of the TG(1-3) repeats in tel1Delta cells was independent of the number of the Rap1p C termini at the telomere . These data indicate that tel1Delta cells use an alternative mechanism to regulate telomere length that is distinct from monitoring the number of telomere binding proteins.

Proc Natl Acad Sci U S A, 1999 Dec 21, 96(26), 14990 - 5
Caenorhabditis elegans mediator complexes are required for developmental-specific transcriptional activation; Kwon JY et al.; Mediator proteins are required for transcriptional regulation of most genes in yeast . Mammalian Mediator homologs also function as transcriptional coactivators in vitro; however, their physiological role in gene-specific transcription is not yet known . To determine the role of Mediator proteins in the development of complex organisms, we purified putative Mediator complexes from Caenorhabditis elegans and analyzed their phenotypes in vivo . C . elegans Mediator homologs were assembled into two multiprotein complexes . RNA interference assays showed that the CeMed6, CeMed7, and CeMed10/CeNut2 gene products are required for the expression of developmentally regulated genes, but are dispensable for expression of the ubiquitously expressed genes tested in this study . Therefore, the gene-specific function of Mediator as an integrator of transcriptional regulatory signals is evolutionarily conserved and is essential for C . elegans development.

Proc Natl Acad Sci U S A, 1999 Dec 21, 96(26), 14967 - 72
Methylation of histone H3 at lysine 4 is highly conserved and correlates with transcriptionally active nuclei in Tetrahymena; Strahl BD et al.; Studies into posttranslational modifications of histones, notably acetylation, have yielded important insights into the dynamic nature of chromatin structure and its fundamental role in gene expression . The roles of other covalent histone modifications remain poorly understood . To gain further insight into histone methylation, we investigated its occurrence and pattern of site utilization in Tetrahymena, yeast, and human HeLa cells . In Tetrahymena, transcriptionally active macronuclei, but not transcriptionally inert micronuclei, contain a robust histone methyltransferase activity that is highly selective for H3 . Microsequence analyses of H3 from Tetrahymena, yeast, and HeLa cells indicate that lysine 4 is a highly conserved site of methylation, which to date, is the major site detected in Tetrahymena and yeast . These data document a nonrandom pattern of H3 methylation that does not overlap with known acetylation sites in this histone . In as much as H3 methylation at lysine 4 appears to be specific to macronuclei in Tetrahymena, we suggest that this modification pattern plays a facilitatory role in the transcription process in a manner that remains to be determined . Consistent with this possibility, H3 methylation in yeast occurs preferentially in a subpopulation of H3 that is preferentially acetylated.

Proc Natl Acad Sci U S A, 1999 Dec 21, 96(26), 14961 - 6
Transforming growth factor beta targeted inactivation of cyclin E:cyclin-dependent kinase 2 (Cdk2) complexes by inhibition of Cdk2 activating kinase activity; Nagahara H et al.; Transforming growth factor beta (TGF-beta)-mediated G(1) arrest previously has been shown to specifically target inactivation of cyclin D:cyclin-dependent kinase (Cdk) 4/6 complexes . We report here that TGF-beta-treated human HepG2 hepatocellular carcinoma cells arrest in G(1), but retain continued cyclin D:Cdk4/6 activity and active, hypophosphorylated retinoblastoma tumor suppressor protein . Consistent with this observation, TGF-beta-treated cells failed to induce p15(INK4b), down-regulate CDC25A, or increase levels of p21(CIP1), p27(KIP1), and p57(KIP2) . However, TGF-beta treatment resulted in the specific inactivation of cyclin E:Cdk2 complexes caused by absence of the activating Thr(160) phosphorylation on Cdk2 . Whole-cell lysates from TGF-beta-treated cells showed inhibition of Cdk2 Thr(160) Cdk activating kinase (CAK) activity; however, cyclin H:Cdk7 activity, a previously assumed mammalian CAK, was not altered . Saccharomyces cerevisiae contains a genetically and biochemically proven CAK gene, CAK1, that encodes a monomeric 44-kDa Cak1p protein unrelated to Cdk7 . Anti-Cak1p antibodies cross-reacted with a 45-kDa human protein with CAK activity that was specifically down-regulated in response to TGF-beta treatment . Taken together, these observations demonstrate that TGF-beta signaling mediates a G(1) arrest in HepG2 cells by targeting Cdk2 CAK and suggests the presence of at least two mammalian CAKs: one specific for Cdk2 and one for Cdk4/6.

Proc Natl Acad Sci U S A, 1999 Dec 21, 96(26), 14899 - 904
DNA double-strand break repair proteins are required to cap the ends of mammalian chromosomes; Bailey SM et al.; Recent findings intriguingly place DNA double-strand break repair proteins at chromosome ends in yeast, where they help maintain normal telomere length and structure . In the present study, an essential telomere function, the ability to cap and thereby protect chromosomes from end-to-end fusions, was assessed in repair-deficient mouse cell lines . By using fluorescence in situ hybridization with a probe to telomeric DNA, spontaneously occurring chromosome aberrations were examined for telomere signal at the points of fusion, a clear indication of impaired end-capping . Telomeric fusions were not observed in any of the repair-proficient controls and occurred only rarely in a p53 null mutant . In striking contrast, chromosomal end fusions that retained telomeric sequence were observed in nontransformed DNA-PK(cs)-deficient cells, where they were a major source of chromosomal instability . Metacentric chromosomes created by telomeric fusion became even more abundant in these cells after spontaneous immortalization . Restoration of repair proficiency through transfection with a functional cDNA copy of the human DNA-PK(cs) gene reduced the number of fusions compared with a negative transfection control . Virally transformed cells derived from Ku70 and Ku80 knockout mice also displayed end-to-end fusions . These studies demonstrate that DNA double-strand break repair genes play a dual role in maintaining chromosomal stability in mammalian cells, the known role in repairing incidental DNA damage, as well as a new protective role in telomeric end-capping.

Proc Natl Acad Sci U S A, 1999 Dec 21, 96(26), 14866 - 70
Rapamycin-modulated transcription defines the subset of nutrient-sensitive signaling pathways directly controlled by the Tor proteins; Hardwick JS et al.; The immunosuppressant rapamycin inhibits Tor1p and Tor2p (target of rapamycin proteins), ultimately resulting in cellular responses characteristic of nutrient deprivation through a mechanism involving translational arrest . We measured the immediate transcriptional response of yeast grown in rich media and treated with rapamycin to investigate the direct effects of Tor proteins on nutrient-sensitive signaling pathways . The results suggest that Tor proteins directly modulate the glucose activation and nitrogen discrimination pathways and the pathways that respond to the diauxic shift (including glycolysis and the citric acid cycle) . Tor proteins do not directly modulate the general amino acid control, nitrogen starvation, or sporulation (in diploid cells) pathways . Poor nitrogen quality activates the nitrogen discrimination pathway, which is controlled by the complex of the transcriptional repressor Ure2p and activator Gln3p . Inhibiting Tor proteins with rapamycin increases the electrophoretic mobility of Ure2p . The work presented here illustrates the coordinated use of genome-based and biochemical approaches to delineate a cellular pathway modulated by the protein target of a small molecule.

Proc Natl Acad Sci U S A, 1999 Dec 21, 96(26), 14759 - 64
Crystal structure of the Sec18p N-terminal domain; Babor SM et al.; Yeast Sec18p and its mammalian orthologue N-ethylmaleimide-sensitive fusion protein (NSF) are hexameric ATPases with a central role in vesicle trafficking . Aided by soluble adapter factors (SNAPs), Sec18p/NSF induces ATP-dependent disassembly of a complex of integral membrane proteins from the vesicle and target membranes (SNAP receptors) . During the ATP hydrolysis cycle, the Sec18p/NSF homohexamer undergoes a large-scale conformational change involving repositioning of the most N terminal of the three domains of each protomer, a domain that is required for SNAP-mediated interaction with SNAP receptors . Whether an internal conformational change in the N-terminal domains accompanies their reorientation with respect to the rest of the hexamer remains to be addressed . We have determined the structure of the N-terminal domain from Sec18p by x-ray crystallography . The Sec18p N-terminal domain consists of two beta-sheet-rich subdomains connected by a short linker . A conserved basic cleft opposite the linker may constitute a SNAP-binding site . Despite structural variability in the linker region and in an adjacent loop, all three independent molecules in the crystal asymmetric unit have the identical subdomain interface, supporting the notion that this interface is a preferred packing arrangement . However, the linker flexibility allows for the possibility that other subdomain orientations may be sampled.

Mol Cell Biol, 2000 Jan, 20(2), 634 - 47
Inhibition of TATA-binding protein function by SAGA subunits Spt3 and Spt8 at Gcn4-activated promoters; Belotserkovskaya R et al.; SAGA is a 1.8-MDa yeast protein complex that is composed of several distinct classes of transcription-related factors, including the adaptor/acetyltransferase Gcn5, Spt proteins, and a subset of TBP-associated factors . Our results indicate that mutations that completely disrupt SAGA (deletions of SPT7 or SPT20) strongly reduce transcriptional activation at the HIS3 and TRP3 genes and that Gcn5 is required for normal HIS3 transcriptional start site selection . Surprisingly, mutations in Spt proteins involved in the SAGA-TBP interaction (Spt3 and Spt8) cause derepression of HIS3 and TRP3 transcription in the uninduced state . Consistent with this finding, wild-type SAGA inhibits TBP binding to the HIS3 promoter in vitro, while SAGA lacking Spt3 or Spt8 is not inhibitory . We detected two distinct forms of SAGA in cell extracts and, strikingly, one lacks Spt8 . Conditions that induce HIS3 and TRP3 transcription result in an altered balance between these complexes strongly in favor of the form without Spt8 . These results suggest that the composition of SAGA may be dynamic in vivo and may be regulated through dissociable inhibitory subunits.

Mol Cell Biol, 2000 Jan, 20(2), 604 - 16
A nuclear 3'-5' exonuclease involved in mRNA degradation interacts with Poly(A) polymerase and the hnRNA protein Npl3p; Burkard KT et al.; Inactivation of poly(A) polymerase (encoded by PAP1) in Saccharomyces cerevisiae cells carrying the temperature-sensitive, lethal pap1-1 mutation results in reduced levels of poly(A)(+) mRNAs . Genetic selection for suppressors of pap1-1 yielded two recessive, cold-sensitive alleles of the gene RRP6 . These suppressors, rrp6-1 and rrp6-2, as well as a deletion of RRP6, allow growth of pap1-1 strains at high temperature and partially restore the levels of poly(A)(+) mRNA in a manner distinct from the cytoplasmic mRNA turnover pathway and without slowing a rate-limiting step in mRNA decay . Subcellular localization of an Rrp6p-green fluorescent protein fusion shows that the enzyme residues in the nucleus . Phylogenetic analysis and the nature of the rrp6-1 mutation suggest the existence of a highly conserved 3'-5' exonuclease core domain within Rrp6p . As predicted, recombinant Rrp6p catalyzes the hydrolysis of a synthetic radiolabeled RNA in a manner consistent with a 3'-5' exonucleolytic mechanism . Genetic and biochemical experiments indicate that Rrp6p interacts with poly(A) polymerase and with Npl3p, a poly(A)(+) mRNA binding protein implicated in pre-mRNA processing and mRNA nuclear export . These findings suggest that Rrp6p may interact with the mRNA polyadenylation system and thereby play a role in a nuclear pathway for the degradation of aberrantly processed precursor mRNAs.

Mol Cell Biol, 2000 Jan, 20(2), 556 - 62
The essential cofactor TRRAP recruits the histone acetyltransferase hGCN5 to c-Myc; McMahon SB et al.; The c-Myc protein functions as a transcription factor to facilitate oncogenic transformation; however, the biochemical and genetic pathways leading to transformation remain undefined . We demonstrate here that the recently described c-Myc cofactor TRRAP recruits histone acetylase activity, which is catalyzed by the human GCN5 protein . Since c-Myc function is inhibited by recruitment of histone deacetylase activity through Mad family proteins, these opposing biochemical activities are likely to be responsible for the antagonistic biological effects of c-Myc and Mad on target genes and ultimately on cellular transformation.

EMBO J, 1999 Oct 15, 18(20), 5761 - 77
The RNA export factor Gle1p is located on the cytoplasmic fibrils of the NPC and physically interacts with the FG-nucleoporin Rip1p, the DEAD-box protein Rat8p/Dbp5p and a new protein Ymr 255p; Strahm Y et al.; Gle1p is an essential, nuclear pore complex (NPC)-associated RNA export factor . In a screen for high copy suppressors of a GLE1 mutant strain, we identified the FG-nucleoporin Rip1p and the DEAD-box protein Rat8p/Dbp5p, both of which have roles in RNA export; we also found Ymr255p/Gfd1p, a novel inessential protein . All three high copy suppressors interact with the C-terminal domain of Gle1p; immunoelectron microscopy localizations indicate that Gle1p, Rip1p and Rat8p/Dbp5p are present on the NPC cytoplasmic fibrils; Rip1p was also found within the nucleoplasm and on the nuclear baskets . In vivo localizations support the hypothesis that Rip1p contributes to the association of Gle1p with the pore and that Gle1p, in turn, provides a binding site for Rat8p/Dbp5p at the NPC . These data are consistent with the view that Gle1p, Rip1p, Rat8p/Dbp5p and Ymr255p/Gfd1p associate on the cytoplasmic side of the NPC to act in a terminal step of RNA export . We also describe a human functional homologue of Rip1p, called hCG1, which rescues Rip1p function in yeast, consistent with the evolutionary conservation of this NPC-associated protein.

Curr Biol, 1999 Dec 16-30, 9(24), 1501 - 4
The nonhomologous DNA end joining pathway is important for chromosome stability in primary fibroblasts; Karanjawala ZE et al.; There are two types of chromosome instability, structural and numerical, and these are important in cancer . Many structural abnormalities are likely to involve double-strand DNA (dsDNA) breaks . Nonhomologous DNA end joining (NHEJ) and homologous recombination are the major pathways for repairing dsDNA breaks . NHEJ is the primary pathway for repairing dsDNA breaks throughout the G0, G1 and early S phases of the cell cycle {1} . Ku86 and DNA ligase IV are two major proteins in the NHEJ pathway . We examined primary dermal fibroblasts from mice (wild type, Ku86(+/-), Ku86(-/-), and DNA ligase IV(+/-)) for chromosome breaks . Fibroblasts from Ku86(+/-) or DNA ligase IV(+/-) mice have elevated frequencies of chromosome breaks compared with those from wild-type mice . Fibroblasts from Ku86(-/-) mice have even higher levels of chromosome breaks . Primary pre-B cells from the same animals did not show significant accumulation of chromosome breaks . Rather the pre-B cells showed increased cell death . These studies demonstrate that chromosome breaks arise frequently and that NHEJ is required to repair this constant spontaneous damage.

J Biol Chem, 1999 Dec 31, 274(53), 37750 - 4
Signal peptides having standard and nonstandard cleavage sites can be processed by Imp1p of the mitochondrial inner membrane protease; Chen X et al.; We have performed a site-directed mutagenesis study showing that residues comprising the type I signal peptidase signature in the two catalytic subunits of the yeast inner membrane protease, Imp1p and Imp2p, are functionally important, consistent with the idea that these subunits contain a serine/lysine catalytic dyad . Previous studies have shown that Imp1p cleaves signal peptides having asparagine at the -1 position, which deviates from the typical signal peptide possessing a small uncharged amino acid at this position . To determine whether asparagine is responsible for the nonoverlapping substrate specificities exhibited by the inner membrane protease subunits, we have substituted asparagine with 19 amino acids in the Imp1p substrate i-cytochrome (cyt) b(2) . The resulting signal peptides containing alanine, serine, cysteine, leucine, and methionine can be cleaved efficiently by Imp1p . The remaining mutant signal peptides are cleaved inefficiently or not at all . Surprisingly, none of the amino acid changes results in the recognition of i-cyt b(2) by Imp2p, whose natural substrate, i-cyt c(1), has alanine at the -1 position . The data demonstrate that (i) although the -1 residue is important in substrates recognized by Imp1p, signal peptides having standard and nonstandard cleavage sites can be processed by Imp1p, and (ii) a -1 asparagine does not govern the substrate specificity of the inner membrane protease subunits.

J Biol Chem, 1999 Dec 31, 274(53), 37575 - 82
Post-translational regulation of Adr1 activity is mediated by its DNA binding domain; Sloan JS et al.; ADR1 encodes a transcriptional activator that regulates genes involved in carbon source utilization in Saccharomyces cerevisiae . ADR1 is itself repressed by glucose, but the significance of this repression for regulating target genes is not known . To test if the reduction in Adr1 levels contributes to glucose repression of ADH2 expression, we generated yeast strains in which the level of Adr1 produced during growth in glucose-containing medium is similar to that present in wild-type cells grown in the absence of glucose . In these Adr1-overproducing strains, ADH2 expression remained tightly repressed, and UAS1, the element in the ADH2 promoter that binds Adr1, was sufficient to maintain glucose repression . Post-translational modification of Adr1 activity is implicated in repression, since ADH2 derepression occurred in the absence of de novo protein synthesis . The N-terminal 172 amino acids of Adr1, containing the DNA binding and nuclear localization domains, fused to the Herpesvirus VP16-encoded transcription activation domain, conferred regulated expression at UAS1 . Nuclear localization of an Adr1-GFP fusion protein was not glucose-regulated, suggesting that the DNA binding domain of Adr1 is sufficient to confer regulated expression on target genes . A Gal4-Adr1 fusion protein was unable to confer glucose repression at GAL4-dependent promoters, suggesting that regulation mediated by ADR1 is specific to UAS1.

Plant Mol Biol, 1999 Nov, 41(4), 563 - 73
His-Asp phosphotransfer possibly involved in the nitrogen signal transduction mediated by cytokinin in maize: molecular cloning of cDNAs for two-component regulatory factors and demonstration of phosphotransfer activity in vitro; Sakakibara H et al.; Implication of His-to-Asp and/or Asp-to-His (His-Asp) phosphorelay has been recently reported in signal transduction pathways initiated by ethylene and cytokinin . These signaling systems are generally composed of sensor His-protein kinases, His-containing phosphotransfer (HPt) domains, and response regulator domains . In this study, we isolated maize cDNAs, designated as ZmRR2 and ZmHP2, which encode a response regulator domain and HPt domain, respectively, and we identified their His-to-Asp phosphotransfer activity in vitro . The putative translated product of ZmRR2 was highly similar to that of ZmRR1 (78% identity), a maize response regulator homologue . The putative translated product of ZmHP2 showed similarity to that of HPt domains from Arabidopsis thaliana (AHP1-AHP3: 44 to 47% identity) and Saccharomyces cerevisiae (Ypdlp: 24% identity) . In vitro experiments demonstrated that the putative signaling factors can transfer the phosphoryl group from His-80 of ZmHP2 to Asp-90 of ZmRRs . Treating detached leaves with t-zeatin or supplying inorganic nitrogen to the whole plant induced the accumulation of ZmRR1 and ZmRR2 transcripts . On the other hand, the steady-state transcript level of ZmHP2 was not affected by cytokinin or inorganic nitrogen sources . These results indicate that His-Asp phosphotransfer may be involved in the transduction of nitrogen signals mediated by cytokinin, and that multiple response regulators participate in the signaling pathways.

Curr Opin Genet Dev, 1999 Dec, 9(6), 688 - 94
Origin of multicellular eukaryotes - insights from proteome comparisons; Aravind L et al.; The complete genomes of the yeast Saccharomyces cerevisiae and the nematode worm Caenorhabditis elegans have recently become available allowing the comparison of the complete protein sets of a unicellular and multicellular eukaryote for the first time . These comparisons reveal some striking trends in terms of expansions or extensive shuffling of specific domains that are involved in regulatory functions and signaling . Similar comparisons with the available sequence data from the plant Arabidopsis thaliana produce consistent results . These observations have provided useful insights regarding the origin of multicellular organisms.

Nature, 1999 Dec 9, 402(6762), 689 - 92
The TOR signalling pathway controls nuclear localization of nutrient-regulated transcription factors; Beck T et al.; The rapamycin-sensitive TOR signalling pathway in Saccharomyces cerevisiae activates a cell-growth program in response to nutrients such as nitrogen and carbon . The TOR1 and TOR2 kinases (TOR) control cytoplasmic protein synthesis and degradation through the conserved TAP42 protein . Upon phosphorylation by TOR, TAP42 binds and possibly inhibits type 2A and type-2A-related phosphatases; however, the mechanism by which TOR controls nuclear events such as global repression of starvation-specific transcription is unknown . Here we show that TOR prevents transcription of genes expressed upon nitrogen limitation by promoting the association of the GATA transcription factor GLN3 with the cytoplasmic protein URE2 . The binding of GLN3 to URE2 requires TOR-dependent phosphorylation of GLN3 . Phosphorylation and cytoplasmic retention of GLN3 are also dependent on the TOR effector TAP42, and are antagonized by the type-2A-related phosphatase SIT4 . TOR inhibits expression of carbon-source-regulated genes by stimulating the binding of the transcriptional activators MSN2 and MSN4 to the cytoplasmic 14-3-3 protein BMH2 . Thus, the TOR signalling pathway broadly controls nutrient metabolism by sequestering several transcription factors in the cytoplasm.

Nature, 1999 Dec 9, 402(6762), 672 - 6
Induction of autophagy and inhibition of tumorigenesis by beclin 1; Liang XH et al.; The process of autophagy, or bulk degradation of cellular proteins through an autophagosomic-lysosomal pathway, is important in normal growth control and may be defective in tumour cells . However, little is known about the genetic mediators of autophagy in mammalian cells or their role in tumour development . The mammalian gene encoding Beclin 1, a novel Bcl-2-interacting, coiled-coil protein, has structural similarity to the yeast autophagy gene, apg6/vps30, and is mono-allelically deleted in 40-75% of sporadic human breast cancers and ovarian cancers . Here we show, using gene-transfer techniques, that beclin 1 promotes autophagy in autophagy-defective yeast with a targeted disruption of agp6/vps30, and in human MCF7 breast carcinoma cells . The autophagy-promoting activity of beclin 1 in MCF7 cells is associated with inhibition of MCF7 cellular proliferation, in vitro clonigenicity and tumorigenesis in nude mice . Furthermore, endogenous Beclin 1 protein expression is frequently low in human breast epithelial carcinoma cell lines and tissue, but is expressed ubiquitously at high levels in normal breast epithelia . Thus, beclin 1 is a mammalian autophagy gene that can inhibit tumorigenesis and is expressed at decreased levels in human breast carcinoma . These findings suggest that decreased expression of autophagy proteins may contribute to the development or progression of breast and other human malignancies.

Development, 2000 Jan, 127(2), 237 - 44
Glia maintain follower neuron survival during Drosophila CNS development; Booth GE et al.; While survival of CNS neurons appears to depend on multiple neuronal and non-neuronal factors, it remains largely unknown how neuronal survival is controlled during development . Here we show that glia regulate neuronal survival during formation of the Drosophila embryonic CNS . When glial function is impaired either by mutation of the glial cells missing gene, which transforms glia toward a neuronal fate, or by targeted genetic glial ablation, neuronal death is induced non-autonomously . Pioneer neurons, which establish the first longitudinal axon fascicles, are insensitive to glial depletion whereas the later extending follower neurons die . This differential requirement of neurons for glia is instructive in patterning and links control of cell number with axon guidance during CNS development.

Anal Biochem, 1999 Dec 15, 276(2), 242 - 50
The separation and direct detection of ceramides and sphingoid bases by normal-phase high-performance liquid chromatography and evaporative light-scattering detection; McNabb TJ et al.; Sphingolipids are an important class of lipids due to their role as biologically active molecules and as intracellular second messengers . Sphingolipid metabolites are involved in a wide variety of important biological processes including signal transduction and growth regulation . Simple, quantitative analytical methods are needed to assay these complex lipids, in order to study their biological functions . The current methods used to quantify ceramides and long-chain sphingoid bases are primarily based on derivatization with uv or fluorescent tags and with radioactive-based enzymatic assays . A method was developed to separate ceramides and sphingoid bases by normal-phase high-performance liquid chromatography and detect them directly with evaporative light-scattering detection . Ceramides and the sphingoid bases phytosphingosine, dihydrosphingosine, sphingosine, and sphingosine 1-phosphate were resolved with a rapid and quantitative assay in the nanomole range . Yeast extracts grown to various time points were assayed for ceramide and sphingoid bases using a simple, isocratic HPLC system . Both ceramide and phytosphingosine, the primary sphingoid base present in yeast cell extracts, were detected in yeast cell extracts . Phytosphingosine was resolved as a sharp peak with the addition of triethylamine and formic acid modifiers to a chloroform/ethanol mobile phase . This method demonstrates the first direct assay of both ceramides and sphingoid bases .

Curr Opin Cell Biol, 1999 Dec, 11(6), 717 - 25
Cytokinesis: an emerging unified theory for eukaryotes?
Hales KG, Bi E, Wu JQ, Adam JC, Yu IC, Pringle JR.
In animal and fungal cells, cytokinesis involves an actomyosin ring that forms and contracts at the division plane . Important new details have emerged concerning the composition, assembly, and dynamics of these contractile rings . In addition, recent advances suggest that targeted membrane addition is a central feature of cytokinesis in animal cells - as it is in fungi and plants - and the coordination of actomyosin ring function with targeted exocytosis at the cleavage plane is being explored . Important new information has also emerged about the spatial and temporal regulation of cytokinesis, especially in relation to the function of the spindle midzone in animal cells and the control of cytokinesis by GTPase systems.

Curr Opin Cell Biol, 1999 Dec, 11(6), 708 - 16
Progression into and out of mitosis; Zachariae W; Progression through mitosis is controlled by cyclin-dependent kinases, which drive cells into metaphase, and by the anaphase-promoting complex/cyclosome, a ubiquitin ligase that triggers sister chromatid separation and exit from mitosis . Recent work has shown how the mutual regulation between cyclin-dependent kinases and the anaphase-promoting complex/cyclosome ensures that cell-cycle events occur in the right order . The analysis of complexes required for sister chromatid cohesion and chromosome condensation has revealed how cyclin-dependent kinases and the anaphase-promoting complex/cyclosome control the behaviour of chromosomes.

Mutat Res, 1999 Dec 7, 435(3), 225 - 32
Senescent human fibroblasts have elevated Ku86 proteolytic cleavage activity; Jeng YW et al.; A proteolytic activity capable of cleaving the Ku86 subunit of Ku protein to two polypeptides, with molecular masses of 69 and 17 kDa in vitro, is present in a human diploid fibroblast (HDF) cell line . The activity is elevated in late-passaged and senescent cells, and the cleaved 69-kDa product seems able to form complex with Ku70 to bind DNA ends . However, further studies indicate that cleavage of Ku86 could be inhibited by including leupeptin in the extraction buffer, and no 69 kDa variant was evident in the cell . In fact, the levels of Ku86, Ku70 and DNA-end binding activity of Ku remained unchanged during replicative senescence . Thus, this study reveals an intriguing protease in HDFs, and also indicates that inconsistent results of Ku86 expression will be obtained if the protease activity is not completely inhibited.

FEBS Lett, 1999 Dec 17, 463(3), 245 - 9
An in vivo assay for the identification of target proteases which cleave membrane-associated substrates; Steiner H et al.; Proteases not only play a fundamental role in numerous physiological processes, but are also involved in several human diseases including Alzheimer's disease (AD) . A key protease implicated in AD is the so far unidentified gamma-secretase, which cleaves the membrane-bound beta-amyloid precursor protein (betaAPP) at the C-terminus of its amyloid domain within the membrane to release the neurotoxic amyloid beta-peptide . In order to allow the isolation of proteases, which specifically cleave membrane-bound substrates within or in the vicinity of a transmembrane domain, we developed a reporter gene assay in Saccharomyces cerevisiae . This assay may allow the identification of genes encoding target proteases that specifically cleave membrane bound substrates by transforming expression libraries.

Nucleic Acids Res . 2000 Jan 15;28(2):e6.
A method for preparing genomic DNA that restrains branch migration of Holliday junctions; Allers T et al.; The Holliday junction is a central intermediate in genetic recombination . This four-stranded DNA structure is capable of spontaneous branch migration, and is lost during standard DNA extraction protocols . In order to isolate and characterize recombination intermediates that contain Holliday junctions, we have developed a rapid protocol that restrains branch migration of four-way DNA junctions . The cationic detergent hex-adecyltrimethylammonium bromide is used to lyse cells and precipitate DNA . Manipulations are performed in the presence of the cations hexamine cobalt(III) or magnesium, which stabilize Holliday junctions in a stacked-X configuration that branch migrates very slowly . This protocol was evaluated using a sensitive assay for spontaneous branch migration, and was shown to preserve both artificial Holliday junctions and meiotic recombination intermediates containing four-way junctions.

Nucleic Acids Res, 2000 Jan 15, 28(2), 620 - 5
The N-terminal region of DNA polymerase delta catalytic subunit is necessary for holoenzyme function; Schumacher SB et al.; Genetic and biochemical studies have shown that DNA polymerase delta (Poldelta) is the major replicative Pol in the eukaryotic cell . Its functional form is the holoenzyme composed of Poldelta, proliferating cell nuclear antigen (PCNA) and replication factor C (RF-C) . In this paper, we describe an N-terminal truncated form of DNA polymerase delta (DeltaN Poldelta) from calf thymus . The DeltaN Poldelta was stimulated as the full-length Poldelta by PCNA in a RF-C-independent Poldelta assay . However, when tested for holoenzyme function in a RF-C-dependent Poldelta assay in the presence of RF-C, ATP and replication protein A (RP-A), the DeltaN Poldelta behaved differently . First, the DeltaN Poldelta lacked holoenzyme functions to a great extent . Second, product size analysis and kinetic experiments showed that the holoenzyme containing DeltaN Poldelta was much less efficient and synthesized DNA at a much slower rate than the holoenzyme containing full-length Poldelta . The present study provides the first evidence that the N-terminal part of the large subunit of Poldelta is involved in holo-enzyme function.

Nucleic Acids Res, 2000 Jan 15, 28(2), 534 - 43
Oligonucleotide-conjugated beads for transdominant genetic experiments; Feldhaus MJ et al.; Transdominant genetics using expression libraries can identify proteins and peptides that affect cell division . In conjunction with these libraries, oligo-nucleotide-conjugated beads and flow cytometry were used to test a strategy that potentially expands the range of such genetic studies . The experimental approach involved creation of tagged expression libraries, introduction of these libraries into cells, growth of the cultured cells for several generations and recovery on oligonucleotide-conjugated beads of sequences that encode growth-modulatory proteins or peptides . Experiments in Saccharomyces cerevisiae demonstrating the feasibility of the strategy are presented.

Biochemistry, 1999 Dec 21, 38(51), 16876 - 81
Equilibrium thermodynamics of a physiologically-relevant heme-protein complex; Wang X et al.; We used isothermal titration calorimetry to study the equilibrium thermodynamics for formation of the physiologically-relevant redox protein complex between yeast ferricytochrome c and yeast ferricytochrome c peroxidase . A 1:1 binding stoichiometry was observed, and the binding free energies agree with results from other techniques . The binding is either enthalpy- or entropy-driven depending on the conditions, and the heat capacity change upon binding is negative . Increasing the ionic strength destabilizes the complex, and both the binding enthalpy and entropy increase . Increasing the temperature stabilizes the complex, indicating a positive van't Hoff binding enthalpy, yet the calorimetric binding enthalpy is negative (-1.4 to -6.2 kcal mol(-)(1)) . We suggest that this discrepancy is caused by solvent reorganization in an intermediate state . The measured enthalpy and heat capacity changes are in reasonable agreement with the values estimated from the surface area change upon complex formation . These results are compared to those for formation of the horse ferricytochrome c/yeast ferricytochrome c peroxidase complex . The results suggest that the crystal and solution structures for the yeast complex are the same, while the crystal and solution structures for horse cytochrome c/yeast cytochrome c peroxidase are different.

Biochemistry, 1999 Dec 21, 38(51), 16831 - 9
Applicability of urea in the thermodynamic analysis of secondary and tertiary RNA folding; Shelton VM et al.; The equilibrium folding of a series of self-complementary RNA duplexes and the unmodified yeast tRNA(Phe) is studied as a function of urea and Mg(2+) concentration with optical spectroscopies and chemical modification under isothermal conditions . Via application of standard methodologies from protein folding, the folding free energy and its dependence on urea concentration, the m value, are determined . The free energies of the RNA duplexes obtained from the urea titrations are in good agreement with those calculated from thermal melting studies {Freier, S . I., et al . (1986) Proc . Natl . Acad . Sci . U.S.A . 83, 9373} . The m value correlates with the length of the RNA duplex and is not sensitive to ionic conditions and temperature . The folding of the unmodified yeast tRNA(Phe) can be described by two Mg(2+)-dependent transitions, the second of which corresponds to the formation of the native tertiary structure as confirmed by hydroxyl radical protection and partial nuclease digestion . Both transitions are sensitive to urea and have m values of 0.94 and 1.70 kcal mol(-)(1) M(-)(1), respectively . Although the precise chemical basis of urea denaturation of RNA is uncertain, the m values for the duplexes and tRNA(Phe) are proportional to the amount of the surface area buried in the folding transition . This proportionality, 0.099 cal mol(-)(1) M(-)(1) A(-)(2), is very similar to that observed for proteins, 0.11 cal mol(-)(1) M(-)(1) A(-)(2) {Myers, J., Pace, N., and Scholtz, M . (1995) Protein Sci . 4, 2138} . These results indicate that urea titration can be used to measure both the free energy and the magnitude of an RNA folding transition.

RNA, 1999 Dec, 5(12), 1615 - 31
Splicing factor SF1 from Drosophila and Caenorhabditis: presence of an N-terminal RS domain and requirement for viability; Mazroui R et al.; Splicing factor SF1 contributes to the recognition of the 3' splice site by interacting with U2AF65 and binding to the intron branch site during the formation of the early splicing complex E . These interactions and the essential functional domains of SF1 are highly conserved in Saccharomyces cerevisiae . We have isolated cDNAs encoding SF1 from Drosophila (Dm) and Caenorhabditis (Ce) . The encoded proteins share the U2AF65 interaction domain, a hnRNP K homology domain, and one or two zinc knuckles required for RNA binding as well as Pro-rich C-terminal sequences with their yeast and mammalian counterparts . In contrast to SF1 in other species, DmSF1 and CeSF1 are characterized by an N-terminal region enriched in Ser, Arg, Lys, and Asp residues with homology to the RS domains of several splicing proteins . These domains mediate protein-protein or protein-RNA interactions, suggesting an additional role for DmSF1 and CeSF1 in pre-mRNA splicing . Human (Hs), fly, and worm SF1 interact equally well with HsU2AF65 or the Drosophila homolog DmU2AF50 . Moreover, DmSF1 lacking its N terminus is functional in prespliceosome formation in a HeLa splicing system, emphasizing the conserved nature of interactions at an early step in spliceosome assembly . The Ce-SF1 gene is located in a polycistronic transcription unit downstream of the genes encoding U2AF35 (uaf-2) and a cyclophilin (cyp-13), implying the coordinate transcriptional regulation of these genes . Injection of double-stranded RNA into C . elegans results in embryonic lethality; thus, the SF1 gene is essential not only in yeast but also in at least one metazoan.

RNA, 1999 Dec, 5(12), 1597 - 604
Human Nop5/Nop58 is a component common to the box C/D small nucleolar ribonucleoproteins; Lyman SK et al.; We have identified an apparent human homolog of the yeast Nop5/Nop58 protein . hNop5/Nop58 codes for a protein of predicted molecular weight 59.6 kDa and is 46.8% identical to Saccharomyces cerevisiae Nop5/Nop58 . Immunofluorescent staining with antibodies against hNop5/Nop58 indicate that it is localized primarily to the nucleolus, and coimmunoprecipitation from nuclear extracts demonstrates that hNop5/Nop58 interacts with the box C/D family of snoRNAs . Thus, hNop5/Nop58 is a common component of the box C/D snoRNPs, and joins fibrillarin as the second such component identified and characterized in metazoans.

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