Nucleic Acids Res, 1995 Aug 25, 23(16), 3161 - 7 Cleavage of tRNA with imidazole and spermine imidazole constructs: a new approach for probing RNA structure; Vlassov VV et al.; Hydrolysis of RNA in imidazole buffer and by spermine-imidazole conjugates has been investigated . The RNA models were yeast tRNA(Asp) and a transcript derived from the 3'-terminal sequence of tobacco mosaic virus RNA representing a minihelix capable of being enzymatically aminoacylated with histidine . Imidazole buffer and spermine-imidazole conjugates in the presence of free imidazole cleave phosphodiester bonds in the folded RNAs in a specific fashion . Imidazole buffer induces cleavages preferentially in single-stranded regions because nucleotides in these regions have more conformational freedom and can assume more easily the geometry needed for formation of the hydrolysis intermediate state . Spermine-imidazole constructs supplemented with free imidazole cleave tRNA(Asp) within single-stranded regions after pyrimidine residues with a marked preference for pyrimidine-A sequences . Hydrolysis patterns suggest a cleavage mechanism involving an attack by the imidazole residue of the electrostatically bound spermine-imidazole and by free imidazole at the most accessible single-stranded regions of the RNA . Cleavages in a viral RNA fragment recapitulating a tRNA-like domain were found in agreement with the model of this molecule that accounts for its functional properties, thus illustrating the potential of the imidazole-derived reagents as structural probes for solution mapping of RNAs . The cleavage reactions are simple to perform, provide information reflecting the state of the ribose-phosphate backbone of RNA and can be used for mapping single- and double-stranded regions in RNAs. Cell, 1995 Aug 25, 82(4), 545 - 54 Group II intron mobility occurs by target DNA-primed reverse transcription; Zimmerly S et al.; Mobile group II introns encode reverse transcriptases and insert site specifically into intronless alleles (homing) . Here, in vitro experiments show that homing of the yeast mtDNA group II intron aI2 occurs by reverse transcription at a double-strand break in the recipient DNA . A site-specific endonuclease cleaves the antisense strand of recipient DNA at position +10 of exon 3 and the sense strand at the intron insertion site . Reverse transcription of aI2-containing pre-mRNA is primed by the antisense strand cleaved in exon 3 and results in cotransfer of the intron and flanking exon sequences . Remarkably, the DNA endonuclease that initiates homing requires both the aI2 reverse transcriptase protein and aI2 RNA . Parallels in their reverse transcription mechanisms raise the possibility that mobile group II introns were ancestors of nuclear non-long terminal repeat retrotransposons and telomerases. J Mol Biol, 1995 Aug 25, 251(4), 493 - 506 The role of DNA bending in Flp-mediated site-specific recombination; Luetke KH et al.; The Flp recombinase of the 2 micron plasmid of Saccharomyces cerevisiae binds to a recognition target site, induces DNA bending and catalyses DNA cleavage and strand exchange to bring about recombination . The minimal Flp recognition target site contains two Flp binding sequences flanking an 8 bp core region; binding of Flp results in the formation of two Flp:DNA complexes (complexes I and II) . Binding of a Flp monomer to a single symmetry element generates a DNA bend of about 60 degrees (a type I bend), whereas binding of two Flp monomers to the FRT site generates a DNA bend of > 144 degrees (a type II bend) . We have used circular permutation analysis to locate the centre of the type I and type II DNA bends induced by Flp, and the Flp peptides P27 (27 kDa; amino acid residues 124 to 346) and P32 (32 kDa; amino acid residues 124 to 423) . The location of the centre of the type I bend depends upon whether the substrate contains one or two Flp binding elements . When the substrate contains one symmetry element, the centre of the type I bend induced by Flp is located at the core-distal end of the b element . However, it is located at the core-proximal end of the b element when the substrate contains two Flp-binding elements . The P27 and P32 peptides, which lack the NH2-terminal 13 kDa region of Flp, do not show this behaviour . We deduce that the 13 kDa region of Flp is critical for the positioning of the type I bend centre on a minimal Flp recognition site . We propose a model in which a single molecule of Flp interacts with two symmetry elements to account for these results . The centre of the type II bend induced by Flp is in the middle of the core region . We used ligation-defective Flp proteins to determine the location of the type II bend centres in complexes where either the top or bottom strand was cleaved . The bend centres of such complexes depend upon which strand is cleaved . We propose a model which associates the position of Flp-induced type II bends with a defined order of strand exchanges in the recombination reaction. J Biol Chem, 1995 Aug 25, 270(34), 19786 - 90 Interaction of the ribosomal protein, L5, with protein phosphatase type 1; Hirano K et al.; The two-hybrid system was used to screen for binding proteins of type 1 protein phosphatase . Two plasmids were constructed, one containing the cDNA of the delta isoform of the type 1 catalytic subunit and the other containing a chicken gizzard cDNA library . Yeast (Y190) were transformed with the plasmids and screened for interacting species . 35 positive clones were categorized into 19 gene groups . Most of these were not identified . One clone, however, contained a sequence identical to the C-terminal portion of the chicken ribosomal protein L5 and corresponded to nucleotide residues 606-975 . L5 was isolated from rat liver ribosomes as the L5.5 S RNA complex . This activated phosphatase activity of a myosin-bound phosphatase and the isolated type 1 catalytic subunit using phosphorylated myosin light chains and phosphorylase alpha as substrates . In addition, it was found that phosphatase sedimented with ribosomal subunits containing L5 but did not sediment with those deficient in L5 . These data indicate that L5 binds to the catalytic subunit of the type 1 protein phosphatase and may act as a target molecule for phosphatase in ribosomal function or other cell mechanisms. Biochem Biophys Res Commun, 1995 Aug 24, 213(3), 803 - 14 The possible involvement of DNA-dependent protein kinase in the phosphorylation of P1 protein in cell-free DNA replication of Xenopus eggs; Someya A et al.; We prepared two types of antibodies: one directed against an oligopeptide corresponding to the C-terminal portion of P1 protein and the other against an oligopeptide corresponding to the C-terminal portion of the 80-kDa subunit of the Ku antigen (p80 Ku) essential for DNA-dependent protein kinase (DNA-PK) activity . Immunoprecipitation and immunoblot of sperm nuclei preincubated in Xenopus egg extracts by anti-P1 antibody showed that Xenopus P1 protein is a phosphoprotein with two phosphorylated forms: a hyperphosphorylated form extractable with Triton X-100 and a hypophosphorylated form resistant to Triton X-100 . The immunodepletion of extracts with anti-p80 Ku IgG-bound beads caused the hyperphosphorylated form to disappear but hardly affected the hypophosphorylated form of P1 protein . DNA replication was stimulated by immunodepletion of the extract with anti-p80 Ku IgG-bound beads . These findings suggest that DNA-PK down-regulates DNA replication through inhibition of hyperphosphorylation of P1 protein during S phase in this cell-free system. Nature, 1995 Aug 24, 376(6542), 705 - 9 Reconstitution of the initial steps of mitochondrial protein import; Hachiya N et al.; We have reconstituted the initial steps of mitochondrial protein import with a purified precursor protein, a purified, ATP-dependent, cytosolic chaperone selective for mitochondrial precursors (mitochondrial import stimulating factor; MSF), and either intact mitochondria or intact or solubilized mitochondrial outer membranes . We show that the precursor-MSF complex first binds to the Mas37p/Mas70p subunits of the mitochondrial import receptor . After ATP-dependent release of MSF, the precursor is transferred from Mas37p/Mas70p to the Mas20p/Mas22p subunits of the receptor, and finally delivered to the import channel in the outer membrane . Import in the absence of the MSF bypasses Mas37p/Mas70p . The ATP-mediated transfer of a precursor from MSF to specific subunits of the import receptor is similar to the GTP-mediated transfer of precursors from the signal recognition particle to its receptor on the endoplasmic reticulum. Nature, 1995 Aug 24, 376(6542), 690 - 5 A new family of outwardly rectifying potassium channel proteins with two pore domains in tandem; Ketchum KA et al.; Potassium channels catalyse the permeation of K+ ions across cellular membranes and are identified by a common structural motif, a highly conserved signature sequence of eight amino acids in the P domain of each channel's pore-forming alpha-subunit . Here we describe a novel K+ channel (TOK1) from Saccharomyces cerevisiae that contains two P domains within one continuous polypeptide . Xenopus laevis oocytes expressing the channel exhibit a unique, outwardly rectifying, K(+)-selective current . The channel is permeable to outward flow of ions at membrane potentials above the K+ equilibrium potential; its conduction-voltage relationship is thus sensitive to extracellular K+ ion concentration . In excised membrane patches, external divalent cations block the channel in a voltage-dependent manner, and their removal in this configuration allows inward channel current . These attributes are similar to those described for inwardly rectifying K+ channels, but in the opposite direction, a previously unrecognized channel behaviour . Our results identify a new class of K+ channel which is distinctive in both its primary structure and functional properties . Structural homologues of the channel are present in the genome of Caenorhabditis elegans. Biochemistry, 1995 Aug 22, 34(33), 10365 - 75 The substrate binding site of human liver cytochrome P450 2C9: an approach using designed tienilic acid derivatives and molecular modeling; Mancy A et al.; Biochemical experiments, using the well-defined human liver CYP2C9 expressed in yeast, and molecular modeling techniques were used to derive a predictive model for substrates of CYP2C9 . The ability of 10 2-aroylthiophenes related to tienilic acid to act as substrates for CYP2C9 was studied . Four of them were original compounds that were synthesized and completely characterized by several spectroscopic techniques . In these 10 compounds various chemical functions, such as ester, amide, alcohol, phenol, ether or tetrazole functions, replaced the OCH2COOH function of tienilic acid . Among them, only the derivatives containing an acidic function (carboxylic acids, phenol, and tetrazole whose pKaS are 4.8, 6.3, and 3.8, respectively) underwent a 5-hydroxylation of their thiophene ring like tienilic acid . Despite their close structural analogy with tienilic acid, all of the other compounds not only did not undergo any 5-hydroxylation of their thiophene ring but also failed to act as inhibitors of CYP2C9 . These results strongly suggested that the presence, at pH 7.4, of a negative charge on the substrate is a very important feature in its recognition by CYP2C9 . In fact, the four new substrates of CYP2C9 described in this study, a carboxylic acid, phenol, and tetrazole derivative, each of which is related to tienilic acid, and the antiinflammatory drug, suprofen (with Km between 12 and 130 microM and kcat between 0.2 and 1.3 min-1), as well as almost all CYP2C9 substrates reported in the literature, exhibit a pKa below 7 (except phenytoin whose pKa is 8.1) . They mainly exist as anions at physiological pH . By using molecular modeling techniques, 12 CYP2C9 substrates were superimposed with respect to their hydroxylation site and fitted onto templates, which were rigid molecules such as (S)-warfarin and phenytoin . It was thus possible to arrange them in order that all their anionic sites were at a distance around 4 A from a common point (a putative cationic site of the protein) in space . These results provide a model of the substrate binding site of CYP2C9, in which substrates interact through their anionic site A- with a cationic residue of the CYP2C9 protein C+ . In that model, the distance between the hydroxylation site (Hy) and the anionic site (A-) is 7.8 +/- 1.6 A, and the <HyA-C+ angle is 82 +/- 15 degrees. FEBS Lett, 1995 Aug 21, 370(3), 264 - 8 NTR1 encodes a high affinity oligopeptide transporter in Arabidopsis; Rentsch D et al.; Hterologous complementation of yeast mutants has enabled the isolation of genes encoding several families of amino acid transporters . Among them, NTR1 codes for a membrane protein with weak histidine transport activity . However, at the sequence level, NTR1 is related to rather non-specific oligopeptide transporters from a variety of species including Arabidopsis and to the Arabidopsis nitrate transporter CHL1 . A yeast mutant deficient in oligopeptide transport was constructed allowing to show that NTR1 functions as a high affinity, low specificity peptide transporter . In siliques NTR1-expression is restricted to the embryo, implicating a role in the nourishment of the developing seed. Mol Gen Genet, 1995 Aug 21, 248(3), 260 - 9 Regulation of cell wall beta-glucan assembly: PTC1 negatively affects PBS2 action in a pathway that includes modulation of EXG1 transcription; Jiang B et al.; Analysis of genes involved in yeast cell wall beta-glucan assembly has led to the isolation of EXG1, PBS2 and PTC1 . EXG1 and PBS2 were isolated as genes that, when expressed from multicopy plasmids, led to a dominant killer toxin-resistant phenotype . The PTC1 gene was cloned by functional complementation of the calcofluor white-hypersensitive mutant cwh47-1 . PTC1/CWH47 is the structural gene for a type 2C serine/threonine phosphatase, EXG1 codes for an exo-beta-glucanase, and PBS2 encodes a MAP kinase kinase in the Pbs2p-Hog1p signal transduction pathway . Overexpression of EXG1 on a 2 mu plasmid led to reduction in a cell wall beta 1,6-glucan and caused killer resistance in wild type cells; while the exg1 delta mutant displayed modest increases in killer sensitivity and beta 1,6-glucan levels . Disruption of PTC1/CWH47 and overexpression of PBS2 gave rise to similar beta-glucan related phenotypes, with higher levels of EXG1 transcription, increased exo-beta-glucanase activity, reduced beta 1,6-glucan levels, and resistance to killer toxin . Genetic analysis revealed that loss of function of the PBS2 gene was epistatic to PTC1/CWH47 disruption, indicating a functional role for the Ptc1p/Cwh47p phosphatase in the Pbs2p-Hog1p signal transduction pathway . These results suggest that Ptc1p/Cwh47p and Pbs2p play opposing regulatory roles in cell wall glucan assembly, and that this is effected in part by modulating Exg1p activity. Arch Biochem Biophys, 1995 Aug 20, 321(2), 493 - 500 Farnesyl pyrophosphate synthase from white lupin: molecular cloning, expression, and purification of the expressed protein; Attucci S et al.; Plants produce a variety of sesquiterpenoid compounds with diverse biological functions, whose synthesis is initiated by farnesyl pyrophosphate synthase {EC 2.5.1.1, EC 2.5.1.10} . The lack of availability of molecular tools to analyze this enzyme has, thus far, prevented the study of its expression and regulation in plants . A DNA fragment corresponding to a portion of the farnesyl pyrophosphate synthase gene was amplified by the polymerase chain reaction using was amplified by the polymerase chain reaction using degenerate primers designed from two highly conserved domains (FLV(A/L)DD(I/M)MD and FQIQDDYLD) found in eukaryotic farnesyl pyrophosphate synthase sequences . A clone, pS19, of a 438-bp PCR fragment was used to screen a white lupin root cDNA library . Two full-length cDNA clones (pFPS1 and pFPS2) were isolated and sequenced, and one of them (pFPS2) was expressed in a bacterial system and the enzyme protein encoded by the clone was purified . The 1345-bp insert of pFPS2 contains a 1026-bp open reading frame, encoding a 342-amino-acid peptide with a calculated molecular mass of 39,310 Da . The deduced amino acid sequence of lupin farnesyl pyrophosphate synthase pFPS2 shares 90 and 79% identity with those from Lupinus albus (pFPS1) and Arabidopsis thaliana, respectively, 51% with the yeast enzyme, and 44% identity with those from rat and human . The overexpressed protein, which was purified to near homogeneity, displayed both dimethylallyl transferase and geranyl transferase activities . Polyclonal antibodies raised against the purified protein immunorecognized a ca 39-kDa protein in lupin root extracts. J Mol Biol, 1995 Aug 18, 251(3), 448 - 67 Prediction of quaternary structure of coiled coils . Application to mutants of the GCN4 leucine zipper; Vieth M et al.; Using a simplified protein model, the equilibrium between different oligomeric species of the wild-type GCN4 leucine zipper and seven of its mutants have been predicted . Over the entire experimental concentration range, agreement with experiment is found in five cases, while in two cases agreement is found over a portion of the concentration range . These studies demonstrate a methodology for predicting coiled coil quaternary structure and allow for the dissection of the interactions responsible for the global fold . In agreement with the conclusion of Harbury et al., the results of the simulations indicate that the pattern of hydrophobic and hydrophilic residues alone is insufficient to define a protein's three-dimensional structure . In addition, these simulations indicate that the degree of chain association is determined by the balance between specific side-chain packing preferences and the entropy reduction associated with side-chain burial in higher-order multimers. J Biol Chem, 1995 Aug 18, 270(33), 19337 - 44 Characterization of physical interactions of the putative transcriptional adaptor, ADA2, with acidic activation domains and TATA-binding protein; Barlev NA et al.; RNA polymerase II transcription requires functional interactions between activator proteins bound to upstream DNA sites and general factors bound to the core promoter . Accessory transcription factors, such as adaptors and coactivators, have important, but still unclear, roles in the activation process . We tested physical interactions of the putative adaptor ADA2 with activation domains derived from acidic activator proteins and with certain general transcription factors . ADA2 associated with the herpesvirus VP16 and yeast GCN4 activation domains but not with the activation domain of yeast HAP4, which previously was shown to be independent of ADA2 function in vivo and in vitro . Furthermore, the amino terminus of ADA2 directly interacted with the VP16 activation domain, suggesting that ADA2 provides determinants for interaction between activation domains and the adaptor complex . Both TATA-binding protein (TBP) and TFIIB have previously been shown to interact directly with the VP16 activation domain in vitro (Stringer, K . F., Ingles, C . J., and Greenblatt, J . (1990) Nature 345, 783-786; Lin, Y . S., Ha, I., Maldonado, E., Reinberg, D., and Green, M . R . (1991) Nature 353, 569-571) . Interestingly, when binding was tested between VP16 and these general factors in yeast nuclear extracts, both factors interacted with VP16, but only the TBP/VP16 association was dependent on ADA2 . In addition, ADA2 physically associated with TBP, but not with TFIIB . These results suggest that the role of ADA2 in transcriptional activation is to promote physical interaction between activation domains and TBP. J Biol Chem, 1995 Aug 18, 270(33), 19269 - 76 Induction of the acyl-coenzyme A synthetase gene by fibrates and fatty acids is mediated by a peroxisome proliferator response element in the C promoter; Schoonjans K et al.; The long-chain acyl-coenzyme A synthetase (ACS) gene gives rise to three transcripts containing different first exons preceded by specific regulatory regions A, B, and C . Exon-specific oligonucleotide hybridization indicated that only A-ACS mRNA is expressed in rat liver . Fibrate administration induced liver C-ACS strongly and A-ACS mRNA to a lesser extent . B-ACS mRNA remained undetectable . In primary rat hepatocytes and Fa-32 hepatoma cells C-ACS mRNA increased after treatment with fenofibric acid, alpha-bromopalmitate, tetradecylthioacetic acid, or alpha-linolenic acid . Nuclear run-on experiments indicated that fenofibric acid and alpha-bromopalmitate act at the transcriptional level . Transient transfections showed a 3.4-, 2.3-, and 2.2-fold induction of C-ACS promoter activity after fenofibric acid, alpha-bromopalmitate, and tetradecylthioacetic acid, respectively . Unilateral deletion and site-directed mutagenesis identified a peroxisome proliferator activator receptor (PPAR)-responsive element (PPRE) mediating the responsiveness to fibrates and fatty acids . This ACS PPRE contains three imperfect half sites spaced by 1 and 3 oligonucleotides and binds PPAR.retinoid X receptor heterodimers in gel retardation assays . In conclusion, the regulation of C-ACS mRNA expression by fibrates and fatty acids is mediated by PPAR.retinoid X receptor heterodimers interacting through a PPRE in the C-ACS promoters . PPAR therefore occupies a key position in the transcriptional control of a pivotal enzyme controlling the channeling of fatty acids into various metabolic pathways. Nature, 1995 Aug 17, 376(6541), 606 - 8 Mechanism of DNA-binding enhancement by the human T-cell leukaemia virus transactivator Tax; Baranger AM et al.; Tax protein activates transcription of the human T-cell leukaemia virus type I (HTLV-I) genome through three imperfect cyclic AMP-responsive element (CRE) target sites located within the viral promoter . Previous work has shown that Tax interacts with the bZIP element of proteins that bind the CRE target site to promote peptide dimerization, suggesting an association between Tax and bZIP coiled coil . Here we show that the site of interaction with Tax is not the coiled coil, but the basic segment . This interaction increases the stability of the GCN4 bZIP dimer by 1.7 kcal mol-1 and the DNA affinity of the dimer by 1.9 kcal mol-1 . The differential effect of Tax on several bZip-DNA complexes that differ in peptide sequence or DNA conformation suggests a model for Tax action based on stabilization of a distinct DNA-bound protein structure . This model may explain how Tax interacts with transcription factors of considerable sequence diversity to alter patterns of gene expression. Nature, 1995 Aug 17, 376(6541), 602 - 5 Recognition of bZIP proteins by the human T-cell leukaemia virus transactivator Tax; Perini G et al.; Human T-cell leukaemia virus type I (HTLV-I) Tax protein increases the DNA binding of many cellular transcription factors that contain a basic region-leucine zipper (bZIP) DNA-binding domain . bZIP domains comprise a leucine-rich dimerization motif and a basic region that mediates DNA contact . How Tax recognizes diverse bZIPs is not understood . Here we show that no specific sequence of the leucine zipper is required for a Tax response . In contrast, the basic region is essential for the Tax-mediated DNA-binding increase, which can be eliminated by single substitutions of several conserved amino acids . Surprisingly, Tax alters the relative affinity of a bZIP for different DNA binding sites . Thus, through recognition of the conserved basic region . Tax increases DNA binding and modifies DNA site selection . Tax provides a model for how a single auxiliary factor can regulate multiple sequence-specific DNA-binding proteins. Genes Dev, 1995 Aug 15, 9(16), 2053 - 64 Asymmetry in active complexes of FLP recombinase; Qian XH et al.; The FLP recombinase promotes a site-specific recombination reaction in the 2mu plasmid of yeast . The protein-DNA complex that carries out the reaction is asymmetric . Three FLP monomers bound to specific FLP-recognition sequences are required to efficiently carry out one set of reciprocal DNA cleavage and strand exchange events on a Holliday junction substrate . If a fourth monomer plays an auxiliary role in the reaction, it is bound without sequence specificity . The data suggest a modified model for cleavage of DNA in trans by the FLP recombinase that might help reconcile some seemingly conflicting resulted obtained with integrase class recombinases. Genes Dev, 1995 Aug 15, 9(16), 1978 - 91 HMG17 is a chromatin-specific transcriptional coactivator that increases the efficiency of transcription initiation; Paranjape SM et al.; We have examined the effect of HMG17 on transcription by RNA polymerase II by the assembly and analysis of HMG17-containing chromatin templates consisting of regularly spaced nucleosomal arrays . Structural analysis of the chromatin indicated that HMG17 is incorporated into chromatin in a physiological manner with the full complement of core histones . The transcriptional studies revealed that HMG17 stimulates transcription in conjunction with the sequence-specific activator GAL4-VP16 . This effect was observed with chromatin, but not with non-nucleosomal templates, and required the presence of HMG17 during chromatin assembly . The incorporation of HMG17 into chromatin resulted in a 7- to 40-fold stimulation of GAL4-VP16-activated transcription to levels that were comparable to those observed with histone-free DNA templates . In contrast, transcription from HMG17-containing chromatin was not detectable in the absence of GAL4-VP16 or with a GAL4 derivative {GAL4(1-147)} lacking the VP16 activation domain . Finally, the incorporation of HMG17 into chromatin was found to increase the efficiency of transcription initiation, but not the extent of transcriptional elongation . Thus, HMG17 is a chromatin-specific transcriptional coactivator that increases the efficiency of initiation of transcription by RNA polymerase II. Proc Natl Acad Sci U S A, 1995 Aug 15, 92(17), 8011 - 5 An integral membrane component of coatomer-coated transport vesicles defines a family of proteins involved in budding; Stamnes MA et al.; We have isolated a major integral membrane protein from Golgi-derived coatomer-coated vesicles . This 24-kDa protein, p24, defines a family of integral membrane proteins with homologs present in yeast and humans . In addition to sequence similarity, all p24 family members contain a motif with the characteristic heptad repeats found in coiled coils . When the yeast p24 isoform, yp24A, is knocked out in a strain defective for vesicle fusion, a dramatic reduction in the accumulation of transport vesicles is observed . Together, these results indicate a role for this protein family in the budding of coatamer-coated and other species of coated vesicles. Proc Natl Acad Sci U S A, 1995 Aug 15, 92(17), 7892 - 6 14-3-3 proteins associate with cdc25 phosphatases; Conklin DS et al.; The cdc25 phosphatases play key roles in cell cycle progression by activating cyclin-dependent kinases . Two members of the 14-3-3 protein family have been isolated in a yeast two-hybrid screen designed to identify proteins that interact with the human cdc25A and cdc25B phosphatases . Genes encoding the human homolog of the 14-3-3 epsilon protein and the previously described 14-3-3 beta protein have been isolated in this screening . 14-3-3 proteins constitute a family of well-conserved eukaryotic proteins that were originally isolated in mammalian brain preparations and that possess diverse biochemical activities related to signal transduction . We present evidence that indicates that cdc25 and 14-3-3 proteins physically interact both in vitro and in vivo . 14-3-3 protein does not, however, affect the phosphatase activity of cdc25A . Raf-1, which is known to bind 14-3-3 proteins, has recently been shown to associate with cdc25A and to stimulate its phosphatase activity . 14-3-3 protein, however, has no effect on the cdc25A-kinase activity of Raf-1 . Instead, 14-3-3 may facilitate the association of cdc25 with Raf-1 in vivo, participating in the linkage between mitogenic signaling and the cell cycle machinery. Proc Natl Acad Sci U S A, 1995 Aug 15, 92(17), 7677 - 80 A single GAL4 dimer can maximally activate transcription under physiological conditions; Xu HE et al.; Most eukaryotic promoters contain multiple binding sites for one or more transcriptional activators that interact in a synergistic manner . A common view is that synergism is a manifestation of the need for many contacts between activators and the general transcription machinery that a single activator presumably cannot fulfill . In this model, various combinations of protein-protein interactions control the level of gene expression . However, we show here that under physiological conditions, a single binding site and presumably GAL4 can activate transcription to the maximum possible level in vivo . Synergistic effects in this natural system are shown to be consistent with cooperative DNA binding . These results point to DNA occupancy as the major element in fine tuning gene expression in the galactose regulon. Structure, 1995 Aug 15, 3(8), 823 - 33 The solution structure and backbone dynamics of the fibronectin type I and epidermal growth factor-like pair of modules of tissue-type plasminogen activator; Smith BO et al.; BACKGROUND: The thrombolytic serine protease tissue-type plasminogen activator (t-PA) is a classical modular protein consisting of three types of domain in addition to the serine protease domain: F1 (homologous to fibronectin type I); G (epidermal growth factor-like) and kringle . Biochemical data suggest that the F1 and G modules play a major role in the binding of t-PA to fibrin and to receptors on hepatocytes . RESULTS: We have derived the solution structure of the F1 and G pair of modules from t-PA by two- and three-dimensional NMR techniques, in combination with dynamical simulated annealing calculations . We have also obtained information about the molecule's backbone dynamics through measurement of amide 15N relaxation parameters . CONCLUSIONS: Although the F1 and G modules each adopt their expected tertiary structure, the modules interact intimately to bury a hydrophobic core, and the inter-module linker makes up the third strand of the G module's major beta-sheet . The new structural results allow the interpretation of earlier mutational data relevant to fibrin-binding and hepatocyte-receptor binding. Cell, 1995 Aug 11, 82(3), 453 - 61 DNA strand exchange mediated by a RAD51-ssDNA nucleoprotein filament with polarity opposite to that of RecA; Sung P et al.; Yeast RAD51 gene functions in genetic recombination and DNA double-strand break repair . In vitro, in the presence of ATP and replication protein A, RAD51 protein pairs single-stranded DNA (ssDNA) with homologous double-stranded DNA (dsDNA) and catalyzes strand exchange between the synapsed DNA partners . Electron microscopic analyses show that RAD51 forms helical filaments on both ssDNA and dsDNA, in which the DNA is highly extended . However, results presented here indicate that only the RAD51-ssDNA nucleoprotein filament is functionally relevant . Strand exchange is arrested when heterology is encountered in the duplex partner, and analysis of the configuration of the terminal joint thus formed reveals that pairing and strand exchange initiate at the 5' end of the complementary strand in the linear duplex, a reaction polarity opposite to that of the bacterial prototype RecA. Nature, 1995 Aug 10, 376(6540), 530 - 3 A human nucleoporin-like protein that specifically interacts with HIV Rev; Fritz CC et al.; The Rev protein of human immunodeficiency virus type 1 (HIV-1) facilitates the nuclear export of unspliced and partly spliced viral RNAs . Rev contains an RNA binding domain, required for interaction with HIV-1 RNA, and an effector domain, required for RNA-bound Rev to function . The Rev effector domain is believed to interact with a cellular cofactor required for the Rev response and thus HIV-1 replication . Here we report the use of a yeast two-hybrid screen to clone human Rev interacting protein (hRIP), which specifically interacts with the Rev effector domain . This hRIP protein has homology with nucleoporins, a class of proteins that mediate nucleocytoplasmic transport . These and other properties of hRIP are those expected of a Rev cellular cofactor. Genomics, 1995 Aug 10, 28(3), 470 - 6 Cloning of the cDNA for the human ATP synthase OSCP subunit (ATP5O) by exon trapping and mapping to chromosome 21q22.1-q22.2; Chen H et al.; Exon trapping was used to clone portions of potential genes from human chromosome 21 . One trapped sequence showed striking homology with the bovine and rat ATP synthase OSCP (oligomycin sensitivity conferring protein) subunit . We subsequently cloned the full-length human ATP synthase OSCP cDNA (GDB/HGMW approved name ATP50) from infant brain and muscle libraries and determined its nucleotide and deduced amino acid sequence (EMBL/GenBank Accession No . X83218) . The encoded polypeptide contains 213 amino acids, with more than 80% identity to bovine and murine ATPase OSCP subunits and over 35% identity to Saccharomyces cerevisiae and sweet potato sequences . The human ATP5O gene is located at 21q22.1-q22.2, just proximal to D21S17, in YACs 860G11 and 838C7 of the Chumakov et al . (Nature 359:380, 1992) YAC contig . The gene is expressed in all human tissues examined, most strongly in muscle and heart . This ATP5O subunit is a key structural component of the stalk of the mitochondrial respiratory chain F1F0-ATP synthase and as such may contribute in a gene dosage-dependent manner to the phenotype of Down syndrome (trisomy 21). Gene, 1995 Aug 8, 161(1), 119 - 23 Cloning and analysis of cDNA encoding an elongation factor 1 alpha from the dimorphic fungus Histoplasma capsulatum; Shearer G Jr; The cDNA encoding translation elongation factor 1 alpha (EF-1 alpha) was isolated from the dimorphic fungus, Histoplasma capsulatum (Hc), an important pathogen of man . A cDNA library was probed with the tef1 gene from the fungus Mucor racemosus . Ten independent clones were isolated, all with similar restriction patterns . The longest clone (1.96 kb) was sequenced . Southern blot analysis revealed that the Hc tef1 gene was present as a single copy . A single transcript of approx . 2300 nucleotides was found in total RNA from both the yeast and mold forms of the organism . Comparison of the deduced 460-amino-acid Hc EF-1 alpha protein to EF-1 alpha proteins from other species of fungi revealed the greatest degree of similarity to proteins from the filamentous ascomycetes Podospora anserina and Trichoderma reesei . Phylogenetic tree analysis of fungal tef genes indicated that Hc is most closely related to filamentous ascomycetes and most distantly related to the budding yeast Saccharomyces cerevisiae. Biochemistry, 1995 Aug 8, 34(31), 9977 - 84 Thermodynamic and kinetic characterization of the binding of the TATA binding protein to the adenovirus E4 promoter; Petri V et al.; A thermodynamic analysis of the binding of the TATA binding protein (TBP) from Saccharomyces cerevisiae to the adenovirus E4 promoter was conducted using quantitative DNase I "footprint" titration techniques . These studies were conducted to provide a foundation for studies of TBP structure-function relations and its assembly into transcription preinitiation complexes . The binding of TBP to the E4 promoter is well described by the Langmuir binding polynomial, suggesting that no linked equilibria contribute to the binding reaction under the conditions examined . Van't Hoff analysis yielded a nonlinear dependence on temperature with the TBP-E4 promoter interaction displaying maximal affinity at 30 degrees C . An unusually negative value of the apparent standard heat capacity change, delta Cp degrees = -3.5 +/- 0.5 kcal/mol.K, was determined from these data . The dependence of the TBP-E4 promoter interaction on {KCl} indicates that 3.6 +/- 0.3 K+ ions are displaced upon complex formation . Within experimental error, no linkage of proton binding with the TBP-E4 promoter interaction is detectable between pH 5.9 and 8.7 . Rates of association of TBP for the E4 promoter were obtained using a novel implementation of a quench-flow device and DNase I "footprinting" techniques . The value determined for the second-order rate constant at pH 7.4, 100 mM KCl, 5 mM MgCl2, 1 mM CaCl2, 30 degrees C (ka = 5.2 +/- 0.5) x 10(5) M-1 s-1) confirms the results obtained by Hawley and co-workers {Hoopes, B.C., LeBlanc, J.F., & Hawley, D.K . (1992) J . Biol . Chem . 267, 11539-11547} and extends them through TBP concentrations of 636 nM.(ABSTRACT TRUNCATED AT 250 WORDS) FEBS Lett, 1995 Aug 7, 369(2-3), 153 - 7 Repression of transcriptional activity by heterologous KRAB domains present in zinc finger proteins; Vissing H et al.; We report the characterization of three novel members of the KRAB-domain containing C2-H2 zinc finger family (ZNF133, 136 and 140) . KRAB (Kruppel-associated box) is an evolutionarily conserved protein domain found N-terminally with respect to the zinc finger repeats that encodes the DNA binding domain . ZNF133 and ZNF140 have both the KRAB A- and KRAB B-boxes present at their N-terminus, whereas ZNF136 contains only the KRAB A-box . We have previously demonstrated that the KRAB domains derived from ZNF133 and ZNF140 are potent transcriptional repression domains {Margolin et al . (1994) Proc . Natl . Acad . Sci . USA 91, 4509-4513} . The KRAB domain from ZNF136, containing only subdomain A, is a considerable weaker suppression domain; however, when fused to the heterologous KRAB B subdomain of ZNF10 (KOX1) the two subdomains from a KRAB domain which induces repression as potently as previously reported KRAB domains. Biochem Biophys Res Commun, 1995 Aug 4, 213(1), 175 - 80 Kinetic studies on cyclophellitol analogues--mechanism-based inactivators; Tai VW et al.; The (1R,6S)- and (1R,2S,6S)-diastereoisomers of cyclophellitol were found to be effective irreversible inactivators of alpha-D-glucosidase and alpha-D-mannosidase, respectively . The (1R,6S)-diastereoisomer inactivates brewers yeast alpha-D-glucosidase according to pseudo-first order kinetics with inactivation constants of Ki = 26.9 microM, ki = 0.401 min-1 while the (1R,2S,6S)-diastereoisomer inactivates jack beans alpha-D-mannosidase in a similar manner with Ki = 120 microM, ki = 2.85 min-1 . The irreversibility of these compounds was evidenced by the lack of reactivation upon dialysis of the inactivated enzyme. Biochem Biophys Res Commun, 1995 Aug 4, 213(1), 147 - 53 Loop-size spacings between CGCG clusters in long segments of human DNA; Avril N et al.; The CGCG tetranucleotides are clustered inside the CpG islands in the genomes of vertebrates . In order to study the distribution of the islands in the human chromosome we have mapped the loci sensitive to the CGCG specific restriction nuclease, in a 1.5 Mb long DNA segment cloned as Yeast Artificial Chromosome (YAC) . The sites most sensitive to Bsh 1236 I nuclease show chromosomal loop-size spacing . This result, as well as the result of nucleotide sequence analysis of long genomic segments, suggests that the CGCG are organised in clusters (not always undermethylated) which are coincident with GC peaks on the sine wave-like curve representing DNA composition along the mammalian chromosome. J Biol Chem, 1995 Aug 4, 270(31), 18388 - 95 Expression cloning of lfc, a novel oncogene with structural similarities to guanine nucleotide exchange factors and to the regulatory region of protein kinase C; Whitehead I et al.; In order to identify cDNAs that can induce oncogenic transformation, a retroviral vector was used to transfer a library of cDNAs from the murine 32D hemopoietic cell line into NIH 3T3 fibroblasts . We have identified and recovered a provirus containing a 1.8-kilobase pair cDNA whose expression causes morphological transformation in NIH 3T3 cells . The transforming cDNA contains a complete open reading frame that encodes a protein (designated Lfc) with a region of sequence similarity to the product of the lbc oncogene . This region includes a domain that is characteristic of the CDC24 family of guanine nucleotide exchange factors in tandem with a pleckstrin homology (PH) domain . The Lfc protein is distinguished from Lbc by a 150-amino acid NH2-terminal extension that contains a cysteine- and histidine-rich domain similar to the diacylglycerol-binding site (zinc butterfly) found in protein kinase C . NH2- and COOH-terminal deletion analysis revealed that both the PH and putative guanine nucleotide exchange factor domains are required, but the zinc butterfly is dispensable, for transformation . Although the removal of the PH domain of the Lfc protein completely eliminated its ability to transform NIH 3T3 cells, replacement of this domain with an isoprenylation site restored all of its transforming activity . This suggests that a PH domain-dependent recruitment of the Lfc protein to the cellular membrane is a necessary step for cellular transformation . The lfc gene is expressed in a broad range of tissues as well as in a variety of hemopoietic and non-hemopoietic cell lines . Lfc appears to be a new member of a growing family of proteins that are likely to act as activators of Ras-like proteins in a developmental or cell-lineage specific manner. J Biol Chem, 1995 Aug 4, 270(31), 18198 - 200 Effect of cellular location on the function of ferrochelatase; Prasad AR et al.; Ferrochelatase, the terminal enzyme of the heme biosynthetic pathway, is a nuclear encoded protein that is synthesized in the cytoplasm in a precursor form and then is translocated to the matrix side of the inner mitochondrial membrane . Since the product of the enzymatic reaction, protoheme IX, is utilized almost exclusively in the cytoplasmic compartment or on the cytoplasmic side of the inner mitochondrial membrane, it was of interest to determine if the intracellular location of ferrochelatase-deficient strain of the yeast Saccharomyces cerevisiae vectors that coded for full-length ferrochelatase and a truncated form of the enzyme that lacked the mitochondrial targeting sequence were expressed . Both of these transformed cells produce approximately equal total amounts of ferrochelatase, as determined by enzyme assays and Western blot analysis, but only with the full-length construct was ferrochelatase properly localized . In cells containing the truncated construct, ferrochelatase activity was found in all membrane fractions but was not located on the matrix side of the inner mitochondrial membrane . Cells containing either construct produced heme, although the amount of heme synthesized by cells with the truncated construct was significantly less . Interestingly in cells with improperly localized ferrochelatase the amount of b-type cytochrome decreased by 80% as opposed to c- and a-type cytochromes where the decreases were only 60 and 40%, respectively. Mol Cell Biochem, 1995 Aug-Sep, 149-150, 203 - 12 Towards the molecular basis for the regulation of mitochondrial dehydrogenases by calcium ions; Nichols BJ et al.; In mammalian cells, increases in calcium concentration cause increases in oxidative phosphorylation . This effect is mediated by the activation of four mitochondrial dehydrogenases by calcium ions; FAD-glycerol 3-phosphate dehydrogenase, pyruvate dehydrogenase, NAD-isocitrate dehydrogenase and oxoglutarate dehydrogenase . FAD-glycerol 3-phosphate dehydrogenase, being located on the outer surface of the inner mitochondrial membrane, is exposed to fluctuations in cytoplasmic calcium concentration . The other three enzymes are located within the mitochondrial matrix . While the kinetic properties of all of these enzymes are well characterised, the molecular basis for their regulation by calcium is not . This review uses information derived from calcium binding studies, analysis of conserved calcium binding motifs and comparison of amino acid sequences from calcium sensitive and non-sensitive enzymes to discuss how the recent cloning of several subunits from the four dehydrogenases enhances our understanding of the ways in which these enzymes bind calcium . FAD-glycerol 3-phosphate dehydrogenase binds calcium ions through a domain which is part of the polypeptide chain of the enzyme . In contrast, it is possible that the calcium sensitivity of the other three dehydrogenases may involve separate calcium binding subunits. Curr Genet, 1995 Aug, 28(3), 274 - 9 RAD58 (XRS4)--a new gene in the RAD52 epistasis group; Chepurnaya OV et al.; The RAD58 (XRS4) gene of Saccharomyces cerevisiae has been previously identified as a DNA repair gene . In this communication, we show that RAD58 also encodes an essential meiotic function . The spore inviability of rad58 strains is not rescued by a spo13 mutation . The rad50 mutation suppresses spore inviability of a spo13 rad58 strain suggesting that RAD58 acts after RAD50 in meiotic recombination . The rad58-4 mutation does not prevent mitotic recombination events . Haploid rad58 cells fail to carry out G2-repair of gamma-induced lesions, whereas rad58/rad58 diploids are able to perform some diploid-specific repair of these lesions. Mol Biotechnol, 1995 Aug, 4(1), 25 - 43 Preparation, manipulation, and pulse strategy for one-dimensional pulsed-field gel electrophoresis (ODPFGE); Noolandi J et al.; The underlying principles for zero-integrated-field electrophoresis (ZIFE) pulses and more general forward-biased pulse schemes are reviewed for one-dimensional pulsed-field gel electrophoresis (ODPFGE) separations of large DNA molecules . Detailed descriptions of materials, preparation protocols, hardware requirements, and procedures are given . A variety of gel pictures for known yeast DNA markers are shown. Am J Physiol, 1995 Aug, 269(2 Pt 2), F180 - 9 Subcellular distribution and membrane association of Rho-related small GTP-binding proteins in kidney cortex; Boivin D et al.; We have examined the subcellular distribution of Rho-related small GTP-binding proteins in the kidney . RhoA, CDC42, and Rac1 small GTP-binding proteins were found to be expressed at high levels in rat outer kidney cortex . Western blot analysis showed that these proteins were predominantly associated with brush-border and basolateral plasma membranes, with the exception of Rac1 which was localized predominantly in the mitochondria . RhoA and CDC42 were also found in the cytosol, and a small fraction was associated with cytoskeletal elements . A GDP-dissociation inhibitor specific for the Rho family (RhoGDI) was also identified and found to be located exclusively in the cytosol . Upon fractionation of kidney cytosol with anion-exchange chromatography, RhoA and CDC42 proteins eluted in two major well-resolved peaks that coeluted with the RhoGDI protein, suggesting that they form heterodimers . Association of RhoA and CDC42 with RhoGDI was further suggested by coelution of these proteins with RhoGDI at an estimated size of approximately 45 kDa after gel-filtration chromatography . However, a second peak of RhoA eluted as a 20-kDa protein, indicating that not all RhoA is complexed to RhoGDI . Addition of RhoA- and CDC42-enriched fractions to purified membranes from kidney cortex resulted in their translocation to the membranes and their carboxyl methylation . Both processes were stimulated by guanosine 5'-O-(3-thiotriphosphate) . Methylation inhibitors had no effect on the translocation of RhoA to membranes, suggesting that this covalent modification is not required for association to the membrane.(ABSTRACT TRUNCATED AT 250 WORDS) FASEB J, 1995 Aug, 9(11), 1043 - 50 Mechanisms of DNA damage recognition in mammalian nucleotide excision repair; Naegeli H; The ability of nucleotide excision repair (NER) to process multiple forms of DNA damage is highly dependent on the precision by which DNA modifications are located in the genome . Studies of mammalian NER have shown that this system eliminates a wide range of chemically and structurally distinct DNA lesions whereby some types of damage are repaired at higher rates than others . Although the biochemical basis for this broad but heterogeneous response to DNA damage is poorly understood, recent discoveries in closely related areas of DNA metabolism indicate that selectivity for specific sites is achieved through the assembly of nucleoprotein complexes, in which DNA is frequently bent and unwound . In many cases, selectivity may be further enhanced by the action of specialized DNA helicases . These principles in protein-DNA recognition suggest a hypothetical mechanism of damage recognition that accounts for the wide substrate range of mammalian NER and also accommodates its preference for specific DNA lesions. J Cell Biol, 1995 Aug, 130(4), 797 - 805 Wortmannin causes mistargeting of procathepsin D . evidence for the involvement of a phosphatidylinositol 3-kinase in vesicular transport to lysosomes; Davidson HW; At present little is known of the biochemical machinery controlling transport of newly synthesized lysosomal hydrolases from the trans-Golgi network (TGN) to endosomes . The demonstration that Vps34p (a protein required for targeting soluble hydrolases to the vacuole in Saccharomyces cerevisiae) is a phosphatidylinositol 3-kinase (PI3-K) suggested the possibility that a homologous enzyme might be involved in the equivalent step in mammalian cells . Using the PI3-K inhibitors wortmannin and LY294002, I provide evidence to support this hypothesis . Treatment of K-562 cells with wortmannin induced secretion of procathepsin D, with half-maximal inhibition of accurate targeting to lysosomes at 10-20 nM . Kinetic analysis indicated that a late Golgi (TGN) step was affected, and that other constitutive vesicular transport events were not . The M6P recognition signal was still generated in the presence of wortmannin suggesting that the drug was directly inhibiting export of the receptor-ligand complex from the TGN, while removal of the drug led to a rapid restoration of accurate sorting . At the concentrations used, wortmannin and LY294002 are presently accepted to be specific inhibitors of PI3-K . I conclude that these data implicate such an enzyme in the trafficking of M6P-receptor-ligand complexes from the TGN towards lysosomes. J Cell Biol, 1995 Aug, 130(4), 781 - 96 Role for phosphatidylinositol 3-kinase in the sorting and transport of newly synthesized lysosomal enzymes in mammalian cells; Brown WJ et al.; Previous work with the yeast Saccharomyces cerevisiae has demonstrated a role for a phosphatidylinositol-specific PI 3-kinase, the product of the VPS34 gene, in the targeting of newly synthesized proteins to the vacuole, an organelle functionally equivalent to mammalian lysosomes (Schu, P . V., K . Takegawa, M . J . Fry, J . H . Stack, M . D . Waterfield, and S . D . Emr . 1993 . Science {Wash . DC} . 260:88-91) . The activity of Vps34p kinase is significantly reduced by the PI 3-kinase inhibitors wortmannin, a fungal metabolite, and LY294002, a quercetin analog (Stack, J . H., and S . D . Emr . 1994 . J . Biol . Chem . 269:31552-31562) . We show here that at concentrations which inhibit VPS34-encoded PI 3-kinase activity, wortmannin also inhibits the processing and delivery of newly synthesized cathepsin D to lysosomes in mammalian cells with half-maximal inhibition of delivery occurring at 100 nM wortmannin . As a result of wortmannin action, newly synthesized, unprocessed cathepsin D is secreted into the media . Moreover, after accumulation in the trans-Golgi network (TGN) at 20 degrees C, cathepsin D was rapidly missorted to the secretory pathway after addition of wortmannin and shifting to 37 degrees C . At concentrations that inhibited lysosomal enzyme delivery, both wortmannin and LY294002 caused a highly specific dilation of mannose 6-phosphate receptor (M6PR)-enriched vesicles of the prelysosome compartment (PLC), which swelled to approximately 1 micron within 15 min after treatment . With increasing time, the inhibitors caused a significant yet reversible change in M6PR distribution . By 3 h of treatment, the swollen PLC vacuoles were essentially depleted of receptors and, in addition, there was a fourfold loss of receptors from the cell surface . However, M6PRs were still abundant in the TGN . These results are most consistent with the interpretation that PI 3-kinase regulates the trafficking of lysosomal enzymes by interfering with a M6PR-dependent sorting event in the TGN . Moreover, they provide evidence that trafficking of soluble hydrolases to mammalian lysosomes and yeast vacuoles rely on similar regulatory mechanisms. FEBS Lett, 1995 Aug 1, 369(1), 93 - 6 COPII: a membrane coat that forms endoplasmic reticulum-derived vesicles; Barlowe C; Vesicle budding from the endoplasmic reticulum (ER) has been reconstituted with washed membranes and three soluble proteins: Sec13 complex, Sec23 complex and the small GTPase Sar1p . The proteins that drive this cell-free vesicle budding reaction form an approximately 10 nm thick electron dense coat on ER-derived vesicles . Although the overall mechanism of membrane budding driven by various cytoplasmic coats appears similar, the constituents of this new membrane coat are molecularly distinct from the non-clathrin coat (COP) involved in intra-Golgi transport and the clathrin-containing coats . The new vesicle coat has been termed COPII. EMBO J, 1995 Aug 1, 14(15), 3766 - 76 A universally conserved region of the largest subunit participates in the active site of RNA polymerase III; Dieci G et al.; The largest subunits of the three eukaryotic nuclear RNA polymerase present extensive sequence homology with the beta' subunit of the bacterial enzymes over five major co-linear regions . Region d is the most highly conserved and contains a motif, (Y/F)NADFDGD(E/Q)M(N/A), which is invariant in all multimeric RNA polymerases . An extensive mutagenesis of that region in yeast RNA polymerase III led to a vast majority (16/22) of lethal single-site substitutions . A few conditional mutations were also obtained . One of them, rpc160-112, corresponds to a double substitution (T506I, N509Y) and has a slow growth phenotype at 25 degrees C . RNA polymerase III from the mutant rpc160-112 was severely impaired in its ability to transcribe a tRNA gene in vitro . The transcription defect did not originate from a deficiency in transcription complex formation and RNA chain initiation, but was mainly due to a reduced elongation rate . Under conditions of substrate limitation, the mutant enzyme showed increased pausing at the intrinsic pause sites of the SUP4 tRNA gene and an increased rate of slippage of nascent RNA, as compared with the wild-type enzyme . The enzyme defect was also detectable with poly{d(A-T)} as template, in the presence of saturating DNA, ATP and UTP concentrations . The mutant enzyme behavior is best explained by a distortion of the active site near the growing point of the RNA product. EMBO J, 1995 Aug 1, 14(15), 3645 - 53 Functionality and specific membrane localization of transport GTPases carrying C-terminal membrane anchors of synaptobrevin-like proteins; Ossig R et al.; Ras-related guanine nucleotide-binding proteins of the Ypt/Rab family fulfill a pivotal role in vesicular protein transport both in yeast and in mammalian cells . Proper functioning of these proteins involves their cycling between a GTP- and a GDP-bound state as well as their reversible association with specific membranes . Here we show that the yeast Ypt1 and Sec4 proteins, essential components of the vesicular transport machinery, allow unimpaired vesicular transport when permanently fixed to membranes by membrane-spanning domains replacing their two C-terminal cysteine residues . Membrane detachment of the GTPases therefore is not obligatory for transport vesicle docking to or fusion with an acceptor membrane . It was also found that the membrane anchors derived from different synaptobrevin-related proteins have targeting information and direct the chimeric GTPases to different cellular compartments, presumably from the endoplasmic reticulum via the secretory pathway. Biochem J, 1995 Aug 1, 309 ( Pt 3), 1009 - 14 Cloning and expression of cDNAs for the beta subunit of eukaryotic initiation factor-2B, the guanine nucleotide exchange factor for eukaryotic initiation factor-2; Craddock BL et al.; A key control point in the initiation of protein synthesis in mammalian cells is the recycling of eukaryotic initiation factor (eIF)-2 by the guanine nucleotide exchange factor eIF-2B . In mammalian cells, eIF-2B is a complex of five different subunits termed epsilon, delta, gamma, beta and alpha . To clone cDNAs for the beta subunit of rabbit eIF-2B, amino acid sequence data was first obtained and used to design redundant oligonucleotide primers for use in PCR . PCR products were used to screen a rabbit liver cDNA library in lambda gt11 to obtain full-length cDNAs for eIF-2B beta . The cDNAs were sequenced completely on both strands and revealed an open reading frame encoding a predicted 351-amino acid polypeptide of 39.0 kDa . The molecular mass and pI (5.99) of the predicted protein agree well with the properties of eIF-2B beta purified from rabbit reticulocytes . In vitro transcription/-translation of the cDNAs gave rise to a product that migrated at a position indistinguishable from that of this subunit of the purified protein . The amino acid sequence shows a high degree of similarity to that of GCD7, a Saccharomyces cerevisiae protein thought to be equivalent to mammalian eIF-2B beta . Northern-blot analysis revealed a single major mRNA species for eIF-2B beta in each of the four rabbit tissues tested. J Clin Invest, 1995 Aug, 96(2), 1131 - 6 Mitochondrial respiration scavenges extramitochondrial superoxide anion via a nonenzymatic mechanism; Guidot DM et al.; We determined that mitochondrial respiration reduced cytosolic oxidant stress in vivo and scavenged extramitochondrial superoxide anion (O2-.) in vitro . First, Saccharomyces cerevisiae deficient in both the cytosolic antioxidant cupro-zinc superoxide dismutase (Cu,Zn-SOD) and electron transport (Rho0 state) grew poorly (P < 0.05) in 21% O2 compared with parent yeast and yeast deficient only in electron transport or Cu,Zn-SOD, whereas anaerobic growth was the same (P > 0.05) in all yeast . Second, isolated yeast and mammalian mitochondria scavenged extramitochondrial O2- . generated by xanthine/xanthine oxidase . Yeast mitochondria scavenged 42% more (P < 0.05) extramitochondrial O2- . during pyruvate/malate-induced respiration than in the resting state . Addition of either antimycin (respiratory chain inhibitor) or FCCP (respiratory chain uncoupler) prevented increased O2- . scavenging . Mitochondria isolated from yeast deficient in the mitochondrial manganous superoxide dismutase (Mn-SOD) increased (P < 0.05) O2- . scavenging 56% during respiration . This apparent SOD activity, expressed in units of SOD activity per milligram of mitochondrial protein, was the same (9 +/- 0.6 vs . 10 +/- 1.0; P = 0.43) as the O2- . scavenging of mitochondria with Mn-SOD, suggesting that respiration-dependent mitochondrial O2- . scavenging was nonenzymatic . Finally, isolated rat liver and lung mitochondria also increased (P < 0.05) O2- . scavenging during respiration . We speculate that respiring mitochondria, via the protonmotive pump, present a polarized, proton-rich surface that enhances nonenzymatic dismutation of extramitochondrial O2- . and that this is a previously unrecognized function of mitochondrial respiration with potential physiological ramifications. Mol Cell Biol, 1995 Aug, 15(8), 4497 - 506 The histidyl-tRNA synthetase-related sequence in the eIF-2 alpha protein kinase GCN2 interacts with tRNA and is required for activation in response to starvation for different amino acids; Wek SA et al.; Protein kinase GCN2 is a multidomain protein that contains a region homologous to histidyl-tRNA synthetases juxtaposed to the kinase catalytic moiety . Previous studies have shown that in response to histidine starvation, GCN2 phosphorylates eukaryotic initiation factor 2 (eIF-2), to induce the translational expression of GCN4, a transcriptional activator of genes subject to the general amino acid control . It was proposed that the synthetase-related sequences of GCN2 stimulate the activity of the kinase by interacting directly with uncharged tRNA that accumulates during amino acid limitation . In addition to histidine starvation, expression of GCN4 is also regulated by a number of other amino acid limitations . Questions that we posed in this report are whether uncharged tRNA is the most direct regulator of GCN2 and whether the function of this kinase is required to recognize each of the different amino acid starvation signals . We show that GCN2 phosphorylation of eIF-2, and the resulting general amino acid control pathway, is stimulated in response to starvation for each of several different amino acids, in addition to histidine limitation . Cells containing a defective aminoacyl-tRNA synthetase also stimulated GCN2 phosphorylation of eIF-2 in the absence of amino acid starvation, indicating that uncharged tRNA levels are the most direct regulator of GCN2 kinase . Using a Northwestern blot (RNA binding) assay, we show that uncharged tRNA can bind to the synthetase-related domain of GCN2 . Mutations in the motif 2 sequence conserved among class II synthetases, including histidyl-tRNA synthetases, impair the ability of this synthetase-related domain to bind tRNA and abolish GCN2 phosphorylation of eIF-2 required to stimulate the general amino acid control response . These in vivo and in vitro experiments indicate that synthetase-related sequences regulate GCN2 kinase function by monitoring the levels of multiple uncharged tRNAs that accumulate during amino acid limitations. Mol Cell Biol, 1995 Aug, 15(8), 4479 - 88 Studies of point mutants define three essential paired nucleotides in the domain 5 substructure of a group II intron; Boulanger SC et al.; Domain 5 (D5) is a highly conserved, largely helical substructure of group II introns that is essential for self-splicing . Only three of the 14 base pairs present in most D5 structures (A2.U33, G3.U32, and C4.G31) are nearly invariant . We have studied effects of point mutations of those six nucleotides on self-splicing and in vivo splicing of aI5 gamma, an intron of the COXI gene of Saccharomyces cerevisiae mitochondria . Though none of the point mutations blocked self-splicing under one commonly used in vitro reaction condition, the most debilitating mutations were at G3 and G4 . Following mitochondrial Biolistic transformation, it was found that mutations at A2, G3, and C4 blocked respiratory growth and splicing while mutations at the other sites had little effect on either phenotype . Intra-D5 second-site suppressors showed that pairing between nucleotides at positions 2 and 33 and 4 and 31 is especially important for D5 function . At the G3.U32 wobble pair, the mutant A.U pair blocks splicing, but a revertant of that mutant that can form an A+.C base pair regains some splicing . A dominant nuclear suppressor restores some splicing to the G3A mutant but not the G3U mutant, suggesting that a purine is required at position 3 . These findings are discussed in terms of the hypothesis of Madhani and Guthrie (H . D . Madhani and C . Guthrie, Cell 71:803-817, 1992) that helix 1 formed between yeast U2 and U6 small nuclear RNAs may be the spliceosomal cognate of D5. Mol Cell Biol, 1995 Aug, 15(8), 4309 - 18 The carboxyl-terminal transactivation domain of heat shock factor 1 is negatively regulated and stress responsive; Shi Y et al.; We have characterized a stress-responsive transcriptional activation domain of mouse heat shock factor 1 (HSF1) by using chimeric GAL4-HSF1 fusion proteins . Fusion of the GAL4 DNA-binding domain to residues 124 to 503 of HSF1 results in a chimeric factor that binds DNA yet lacks any transcriptional activity . Transactivation is acquired upon exposure to heat shock or by deletion of a negative regulatory domain including part of the DNA-binding-domain-proximal leucine zippers . Analysis of a collection of GAL4-HSF1 deletion mutants revealed the minimal region for the constitutive transcriptional activator to map within the extreme carboxyl-terminal 108 amino acids, corresponding to a region rich in acidic and hydrophobic residues . Loss of residues 395 to 425 or 451 to 503, which are located at either end of this activation domain, severely diminished activity, indicating that the entire domain is required for transactivation . The minimal activation domain of HSF1 also confers enhanced transcriptional response to heat shock or cadmium treatment . These results demonstrate that the transcriptional activation domain of HSF1 is negatively regulated and that the signal for stress induction is mediated by interactions between the amino-terminal negative regulator and the carboxyl-terminal transcriptional activation domain. Mol Cell Biol, 1995 Aug, 15(8), 4291 - 302 Mutations in RAD27 define a potential link between G1 cyclins and DNA replication; Vallen EA et al.; The yeast Saccharomyces cerevisiae has three G1 cyclin (CLN) genes with overlapping functions . To analyze the functions of the various CLN genes, we examined mutations that result in lethality in conjunction with loss of cln1 and cln2 . We have isolated alleles of RAD27/ERC11/YKL510, the yeast homolog of the gene encoding flap endonuclease 1, FEN-1.cln1 cln2 rad27/erc11 cells arrest in S phase; this cell cycle arrest is suppressed by the expression of CLN1 or CLN2 but not by that of CLN3 or the hyperactive CLN3-2 . rad27/erc11 mutants are also defective in DNA damage repair, as determined by their increased sensitivity to a DNA-damaging agent, increased mitotic recombination rates, and increased spontaneous mutation rates . Unlike the block in cell cycle progression, these phenotypes are not suppressed by CLN1 or CLN2 . CLN1 and CLN2 may activate an RAD27/ERC11-independent pathway specific for DNA synthesis that CLN3 is incapable of activating . Alternatively, CLN1 and CLN2 may be capable of overriding a checkpoint response which otherwise causes cln1 cln2 rad27/erc11 cells to arrest . These results imply that CLN1 and CLN2 have a role in the regulation of DNA replication . Consistent with this, GAL-CLN1 expression in checkpoint-deficient, mec1-1 mutant cells results in both cell death and increased chromosome loss among survivors, suggesting that CLN1 overexpression either activates defective DNA replication or leads to insensitivity to DNA damage. J Virol, 1995 Aug, 69(8), 4683 - 92 Substrate specificity of Ty1 integrase; Moore SP et al.; Integration of the Saccharomyces cerevisiae retrotransposon Ty1 requires the element-encoded integrase (IN) protein, which is a component of cytoplasmic virus-like particles (VLPs) . Using purified recombinant Ty1 IN and an oligonucleotide integration assay based on Ty1 long terminal repeat sequences, we have compared IN activity on substrates having either wild-type or altered donor ends . IN showed a marked preference for blunt-end substrates terminating in an A:T pair over substrates ending in a G:C pair or a 3' dideoxyadenosine . VLP activity on representative substrates also showed preference for donor strands which have an adenosine terminus . Staggered-end substrates showed little activity when nucleotides were removed from the end of the wild-type donor strand, but removal of one nucleotide from the complementary strand did not significantly diminish activity . Removal of additional nucleotides from the complementary strand reduced activity to minimal detection levels . These results suggest that the sequence specificity of Ty1 IN is not stringent in vitro . The absence of Ty1 IN-mediated 3' dinucleotide cleavage, a characteristic of retroviral integrases, was demonstrated by using selected substrates . In addition to the forward reaction, both recombinant IN and VLP-associated IN carry out the reverse disintegration reaction with long terminal repeat-based dumbbell substrates . Disintegration activity exhibits sequence preferences similar to those observed for the forward reaction. J Nat Prod, 1995 Aug, 58(8), 1174 - 84 Mechanism-based antitumor screening of Caribbean marine organisms: isolation and structure determination of novel diterpenoids from the gorgonian Eunicea tourneforti; Govindan M et al.; As part of a collaborative research effort between the University of the Virgin Islands and Virginia Polytechnic Institute and State University, we carried out the extraction and bioassay of 87 marine organisms in a mechanism-based assay involving genetically altered yeast strains . Of these, nineteen showed differential activity between the mutant and wild-type yeast strains indicating the presence of potential DNA interacting agents . We now report the isolation and characterization of five new diterpenoids, 2-6, together with the previously known diterpenoid 1, from the bioactive extracts of the gorgonian Eunicea tourneforti forma atra . The structures of the isolated compounds were determined by employing a variety of one- and two-dimensional nmr methods. J Lipid Res, 1995 Aug, 36(8), 1664 - 75 Developmental regulation of the catalytic subunit of the apolipoprotein B mRNA editing enzyme (APOBEC-1) in human small intestine; Giannoni F et al.; Apolipoprotein (apo) B mRNA editing is a site-specific cytidine deamination reaction responsible for the production of apoB-48 in mammalian small intestine . This process is mediated by an enzyme complex that includes the catalytic subunit, APOBEC-1 . In the present study, it is shown that the developmental regulation of apoB mRNA editing in fetal human small intestine is closely mirrored by accumulation of APOBEC-1 mRNA . Similar results were obtained using Caco-2 cells, the data further suggesting that culture of these cells under conditions previously shown to promote differentiation produce an earlier and more marked induction of APOBEC-1 mRNA abundance . Complementary analysis of APOBEC-1 protein accumulation using immunocytochemical localization reveals its appearance to be temporally coordinated with the accumulation of APOBEC-1 mRNA and its distribution to be confined to villus-associated enterocytes . Previous studies demonstrated a close temporal association between the development of triglyceride synthesis and apoB mRNA editing in the rat liver and small intestine . Analysis of fatty acid CoA ligase, monoacylglycerol acyltransferase, and diacylglycerol acyltransferase activity in preparations of human liver and small intestine demonstrates activity of all three enzymes in the late first and early second trimester, suggesting that certain aspects of complex lipid biosynthesis in the human fetal small intestine and liver are regulated developmentally . The cues that modulate the post-transcriptional regulation of fetal human small intestinal apoB gene expression may thus include both temporal programming and events related to the emergence of lipid transport capability. Curr Biol, 1995 Aug 1, 5(8), 859 - 61 Transcriptional regulation . Flipping the Myc switch; Bernards R; When certain cells differentiate, Myc in Myc-Max heterodimers is replaced by Mad or Mxi, generating heterodimers that suppress transcription by interacting with the repressor Sin3. Curr Biol, 1995 Aug 1, 5(8), 854 - 8 Proteolysis . The proteasome: a protein-degrading organelle? Rubin DM, Finley D. The crystal structure of the proteasome suggests that degradation of ubiquitin-protein conjugates is achieved by unfolding the protein substrate and translocating it through a channel into a peptidase-containing chamber. Curr Biol, 1995 Aug 1, 5(8), 822 - 5 Aging . Silence is golden; Shore D; A pioneering genetic analysis of aging in yeast has revealed that a protein complex known to play an essential role in transcriptional silencing at mating-type loci and telomeres also controls aging and stress resistance. Hum Mol Genet, 1995 Aug, 4(8), 1411 - 9 Friedreich's ataxia: a defect in signal transduction? Carvajal JJ, Pook MA, Doudney K, Hillermann R, Wilkes D, al-Mahdawi S, Williamson R, Chamberlain S. We have previously assigned the mutation causing Friedreich's ataxia (FRDA) to 9q13 by genetic linkage and fluorescent in situ hybridization analysis, and identified recombination events which position the gene centromeric to D9S5 . We report here the extension of a yeast artificial chromosome contig to span the 860 kb interval immediately proximal to this marker, which includes the D9S886 and D9S887/888 loci reported to flank the FRDA locus, and the construction of a high resolution cosmid contig initiated from the D9S888 locus . Exon trapping and cDNA library screening strategies have resulted in the isolation of a candidate gene which traverses the centromeric boundary of the FRDA critical region . The gene spans a genomic interval greater than 220 kb with at least two of the coding exons located proximal to the D9S887/888 loci . Expression is complex, with multiple transcripts detected in a variety of tissues and evidence of alternative splicing and developmental control . The predicted amino acid sequence for the 2.7 kb transcript reported here shows a marked homology to the deduced amino acid sequence of the Saccharomyces cerevisiae MSS4 protein, proposed to function within the phosphoinositide cycle, suggesting a potential role for the human homologue in signal transduction . Whilst no evidence for mutation has been detected in this transcript, the sequence represents only one of the shorter alternatively spliced species identified by Northern analysis and direct sequencing . This gene remains a strong candidate for FRDA. Mol Biol Cell, 1995 Aug, 6(8), 1049 - 59 Domains required for CENP-C assembly at the kinetochore; Lanini L et al.; Chromosomes segregate at mitosis along microtubules attached to the kinetochore, an organelle that assembles at the centromere . Despite major advances in defining molecular components of the yeast segregation apparatus, including discrete centromere sequences and proteins of the kinetochore, relatively little is known of corresponding elements in more complex eukaryotes . We show here that human CENP-C, a human autoantigen previously localized to the kinetochore, assembles at centromeres of divergent species, and that the specificity of this targeting is maintained by an inherent destruction mechanism that prevents the accumulation of CENP-C and toxicity of mistargeted CENP-C . The N-terminus of CENP-C is not only required for CENP-C destruction but renders unstable proteins that otherwise possess long half-lives . The conserved targeting of CENP-C is underscored by the discovery of significant homology between regions of CENP-C and Mif2, a protein of Saccharomyces cerevisiae required for the correct segregation of chromosomes . Mutations in the Mif2 homology domain of CENP-C impair the ability of CENP-C to assemble at the kinetochore . Together, these data indicate that essential elements of the chromosome segregation apparatus are conserved in eukaryotes. Curr Opin Biotechnol, 1995 Aug, 6(4), 419 - 24 Interfacial metal-binding site design; Matthews DJ; In recent years, much attention has focused on the characterization of metal-binding sites in natural metalloproteins and the design of novel metal-binding motifs . As a result, it is now possible to harness the high specificity and potency of metal-ion binding to modulate intermolecular interactions . Some encouraging results have been obtained using designed metal-binding sites in such diverse applications as the stabilization of artificial peptide assembly, regulation of membrane channels, control of enzyme activity and enhancement of hormone-receptor interactions. Curr Opin Biotechnol, 1995 Aug, 6(4), 411 - 8 Depletion and replacement of protein metal ligands; Barrick D; Recently, site-directed mutagenesis has been applied to protein-derived metal ligands in a way that permits the replacement in trans of protein ligands . The chemical diversity of ligands available using this method far exceeds that attainable using standard mutagenesis . Non-conservative ligand replacement can yield novel metalloproteins with altered ligand-binding, enzymatic activities, and spectroscopic properties . Conservative ligand substitution, or 'ligand detachment', allows the structural and functional effects of the covalent linkage between the ligand and the protein to be evaluated; this linkage is often proposed to play a critical role in modulating the structure and reactivity of the metal center . Furthermore, this method can be exploited to study the details of molecular recognition at the structural, thermodynamic, and dynamic levels. Biosci Biotechnol Biochem, 1995 Aug, 59(8), 1596 - 7 Primary structure of an allergenic peptide occurring in the chymotryptic hydrolysate of gluten; Watanabe M et al.; An allergenic peptide was isolated from the chymotryptic hydrolysate of gluten by gel filtration and HPLC . The primary structure of the peptide was determined as Ser-Gln-Gln-Gln-Gln- Pro-Pro-Phe-Ser-Gln-Gln-Gln-Pro-Pro-Phe-Ser-Gln-Gln-Gln-Gln-Pro-Pro-Phe- Ser- Gln-Gln-Gln-Gln-Pro-Pro-Phe-Ser-Gln-Gln-Gln-Pro-Pro-Phe . The amino acid sequence similarity shows that the peptide originated from a low-molecular-weight glutenin chain. Biosci Biotechnol Biochem, 1995 Aug, 59(8), 1444 - 9 Cloning and nucleotide sequence of the calmodulin-encoding gene (cmdA) from Aspergillus oryzae; Yasui K et al.; A cDNA and genomic gene encoding calmodulin were isolated from Aspergillus oryzae using a part of the calmodulin gene from A . nidulans as a hybridization probe . The gene was in a 3.4-kb SphI fragment and Southern-blot analysis of genomic DNA suggested the existence of a single copy of the calmodulin gene in A . oryzae . The nucleotide sequence analysis showed that the gene consists of five introns and six exons . Although the nucleotide sequence homology with that of A . nidulans was not so high (68%), the deduced amino acid sequence was 100% and 84% identical with calmodulin of A . nidulans and chicken, respectively . The cDNA encoding A . oryzae calmodulin was expressed under the control of the GAL1 promoter in the calmodulin null mutant (cmd1) of yeast, Saccharomyces cerevisiae, and could function as a calmodulin gene. Mol Cell Biol, 1995 Aug, 15(8), 4466 - 78 Stereochemical selectivity of group II intron splicing, reverse splicing, and hydrolysis reactions; Podar M et al.; We have previously shown, using phosphorothioate substitutions at splice site, that both transesterification steps of group II intron self-splicing proceed, by stereochemical inversion, with an Sp but not an Rp phosphorothioate . Under alternative reaction conditions or with various intron fragments, group II introns can splice following hydrolysis at the 5' splice site and can also hydrolyze the bond between spliced exons (the spliced-exon reopening reaction) . In this study, we have determined the stereochemical specificities of all of the major model hydrolytic reactions carried out by the aI5 gamma intron from Saccharomyces cerevisiae mitochondria . For all substrates containing exon 1 and most of the intron, the stereospecificity of hydrolysis is the same as for the step 1 transesterification reaction . In contrast, the spliced-exon reopening reaction proceeds with an Rp but not an Sp phosphorothioate at the scissile bond, as does true reverse splicing . Thus, by stereochemistry, this reaction appears to be related to the reverse of step 2 of self-splicing . Finally, a substrate RNA that contains the first exon and nine nucleotides of the intron, when reacted with the intron ribozyme, releases the first exon regardless of the configuration of the phosphorothioate at the 5' splice site, suggesting that this substrate can be cleaved by either the step 1 or the step 2 reaction site . Our findings clarify the relationships of these model reactions to the transesterification reactions of the intact self-splicing system and permit new studies to be interpreted more rigorously. Curr Opin Cell Biol, 1995 Aug, 7(4), 536 - 43 The role of coat proteins in the biosynthesis of secretory proteins; Salama NR et al.; The biosynthesis of secretory proteins requires vesicle-mediated transport between the organelles of the secretory pathway . Biochemical and genetic analysis of the secretory pathway has identified two non-clathrin coats--COPI and COPII--that drive the formation of vesicles that mediate transport between the endoplasmic reticulum and the Golgi apparatus, and through the compartments of the Golgi . Recently, a molecular description of the subunits of these coats and the development of biochemical reagents to study their function has yielded new information on how these proteins share the task of organizing vesicle traffic early in the secretory pathway. RNA, 1995 Aug, 1(6), 610 - 23 Purification and characterization of the Upf1 protein: a factor involved in translation and mRNA degradation; Czaplinski K et al.; mRNA degradation is an important control point in the regulation of gene expression and has been shown to be linked to the process of translation . One clear example of this linkage is the observation that nonsense mutations in a gene can accelerate the decay of the corresponding mRNA . In the yeast Saccharomyces cerevisiae, the product of the UPF1 gene, harboring zinc finger, NTP hydrolysis, and helicase motifs, was shown to be a trans-acting factor in this decay pathway . A UPF1 gene disruption results in stabilization of nonsense-containing mRNAs and leads to a nonsense suppression phenotype . As a first step toward understanding the molecular and biochemical mechanism of nonsense-mediated mRNA decay, we have purified Upf1p from a yeast extract and characterized its nucleic acid-dependent NTPase activity, helicase activity, and nucleic acid binding properties . The results presented in this paper demonstrate that Upf1p contains both RNA- and DNA-dependent ATPase activities and RNA and DNA helicase activities . In the absence of ATP, Upf1p binds to single-stranded RNA or DNA, whereas hydrolysis of ATP facilitates its release from single-stranded nucleic acid . Based on these results, the role of Upf1p's biochemical activities in mRNA decay and translation are discussed. RNA, 1995 Aug, 1(6), 584 - 97 Prp16p, Slu7p, and Prp8p interact with the 3' splice site in two distinct stages during the second catalytic step of pre-mRNA splicing; Umen JG et al.; For the second catalytic step of pre-mRNA splicing to occur, a 3' splice site must be selected and juxtaposed with the 5' exon . Four proteins, Prp16p, Slu7p, Prp17p, Prp18p, and an integral spliceosomal protein, Prp8p, are known to be required for the second catalytic step . prp8-101, an allele of PRP8 defective in 3' splice site recognition, exhibits specific genetic interactions with mutant alleles of the other second step splicing factors . The prp8-101 mutation also results in decreased crosslinking of Prp8p to the 3' splice site . To determine the role of the step-two-specific proteins in 3' splice site recognition and in binding of Prp8p to the 3' splice site, we performed crosslinking studies in mutant and immunodepleted extracts . Our results suggest an ordered pathway in which, after the first catalytic step, Prp16p crosslinks strongly to the 3' splice site and Prp8p and Slu7p crosslink weakly . ATP hydrolysis by Prp16p affects a conformational change that reduces the crosslinking of Prp16p with the 3' splice site and allows stronger crosslinking of Prp8p and Slu7p . Thus, the 3' splice site appears to be recognized in two stages during the second step of splicing . Strong 3' splice site crosslinking of Prp8p and Slu7p also requires the functions of Prp17p and Prp18p . Therefore, Prp8p and Slu7p interact with the 3' splice site at the latest stage of splicing prior to the second catalytic step that can currently be defined, and may be at the active site. Appl Environ Microbiol, 1995 Aug, 61(8), 2885 - 90 Functional analysis of the threonine- and serine-rich Gp-I domain of glucoamylase I from Aspergillus awamori var . kawachi; Semimaru T et al.; Glucoamylase I (GAI) from Aspergillus awamori var . kawachi hydrolyzes raw starch efficiently and is composed of three functional domains: the amino-terminal catalytic GAI' domain (A-1 to V-469), the threonine- and serine-rich O-glycosylated Gp-I domain (A-470 to V-514), and the carboxy-terminal raw starch-binding Cp domain (A-515 to R-615) . In order to investigate the role of the Gp-I domain, an additional repeat of Gp-I and internal deletions of the entire Gp-I sequence or parts of the Gp-I sequence were introduced within Gp-I . All mutant genes as well as the wild-type gene were inserted into a yeast-secretion vector, YEUp3H alpha, and expressed in Saccharomyces cerevisiae . Wild-type GAI expressed in yeast cells (GAY), GAGpI, having an extra Gp-I, and GA delta 470-493, lacking the A-470-to-T-493 sequences of Gp-I, were successfully secreted into the culture medium . On the other hand, GA delta 470-507, lacking A-470 to S-507, and GA delta GpI, lacking the entire Gp-I (A-470-to-V-514) sequence, failed to be secreted and remained in the yeast cells . The carbohydrate content of GAGpI was 1.2 times higher than that of GAY and 2.4 times higher than that of the original GAI . The raw starch digestibility of GAGpI was almost the same as that of GAY but was 1.5 times faster than that of GAI.(ABSTRACT TRUNCATED AT 250 WORDS) Proteins, 1995 Aug, 22(4), 392 - 403 Effects of pH and high ionic strength on the adsorption and activity of native and mutated cellobiohydrolase I from Trichoderma reesei; Reinikainen T et al.; Cellobiohydrolase I (CBHI) is the major cellulase of Trichoderma reesei . The enzyme contains a discrete cellulose-binding domain (CBD), which increases its binding and activity on crystalline cellulose . We studied cellulase-cellulose interactions using site-directed mutagenesis on the basis of the three-dimensional structure of the CBD of CBHI . Three mutant proteins which have earlier been produced in Saccharomyces cerevisiae were expressed in the native host organism . The data presented here support the hypothesis that a conserved tyrosine (Y492) located on the flat and more hydrophilic surface of the CBD is essential for the functionality . The data also suggest that the more hydrophobic surface is not directly involved in the CBD function . The pH dependence of the adsorption revealed that electrostatic repulsion between the bound proteins may also control the adsorption . The binding of CBHI to cellulose was significantly affected by high ionic strength suggesting that the interaction with cellulose includes a hydrophobic effect . High ionic strength increased the activity of the isolated core and of mutant proteins on crystalline cellulose, indicating that once productively bound, the enzymes are capable of solubilizing cellulose even with a mutagenized or with no CBD. Lipids, 1995 Aug, 30(8), 763 - 70 Inhibition of neutral cholesteryl ester hydrolase by the glycolytic enzyme enolase . Is this a secondary function of enolase? Shand JH, West DW. There is an accumulation of the glycolytic enzyme enolase and of cholesteryl esters in macrophages that have been converted into "foam" cells . In this study, we questioned whether enolase could be involved in this accumulation of cholesteryl esters by inhibiting the activity of neutral cholesteryl ester hydrolases . Enolase from both yeast and rabbit muscle were incubated with three different cholesteryl ester hydrolases and were shown to inhibit the hydrolysis of cholesteryl esters . Inhibition was dependent on the concentration of enolase and appeared to occur through binding of the enolase to the cholesteryl ester . Nevertheless, the yeast and rabbit muscle enolases differed in their efficiency of inhibition and in their mechanism of action . Purification of commercial enolase preparations by gel-filtration yielded single proteins with the same inhibitory activities as the originals, indicating that the inhibition was not due to the presence of an impurity . Partially purified alpha alpha- and gamma gamma-isoforms of the enzyme from rat brain also appear to have inhibitory effects on cholesteryl ester hydrolysis . Negative control of the hydrolytic phase of the cholesterol/cholesteryl ester cycle may be a secondary function of enolases which correlates with the accumulation of cholesteryl esters in a number of neuro-degenerative and demyelinating diseases. J Biotechnol, 1995 Jul 31, 41(2-3), 121 - 9 Efficient low redundancy large-scale DNA sequencing at EMBL; Voss H et al.; An efficient low redundancy DNA sequencing strategy should allow high accuracy determination of the consensus sequence on both strands of a DNA fragment from a minimal number of sequencing reactions with minimal overlap . At EMBL we developed a directed strategy for cosmid-scale sequencing based on primer walking, whereas most other sequencing projects of this scale rely on the random 'shotgun' strategy . In our strategy, highly accurate raw data are obtained from automated double-stranded Sanger dideoxy sequencing with inexpensive walking primers (8 to 10 $ per primer), T7 DNA polymerase and internal labelling by fluorescein-15- dATP on A.L.F . DNA sequencers (Pharmacia Biotech) . The use of 60-cm long glass plates enables reading length of up to 1000 bases . Comparing various random and directed sequencing strategies in the course of the European Community yeast genome sequencing project on cosmids from chromosomes IX, XI and XV, primer walking was found to be the strategy resulting in the lowest possible redundancy of 2.6 to 2.8 . Future development of the sequencing strategy is based on the new EMBL 2-dye sequencing device for simultaneous sequencing on both strands, and implementation of an initial limited random sequencing phase to reduce the number of walking primers required by a factor of 3, while still maintaining a low redundancy of approx . 3. Cell, 1995 Jul 28, 82(2), 221 - 30 Protein facilitation of group I intron splicing by assembly of the catalytic core and the 5' splice site domain; Weeks KM et al.; The yeast mitochondrial group I intron b15 undergoes self-splicing at high Mg2+ concentrations, but requires the splicing factor CBP2 for reaction under physiological conditions . Chemical accessibility and UV cross-linking experiments now reveal that self-processing is slow because functional elements are not properly positioned in an active tertiary structure . Folding energy provided by CBP2 drives assembly of two RNA domains that comprise the catalytic core and meditates association of an approximately 100 nt 5' domain that contains the 5' splice site . Thus, the protein assembles RNA secondary structure elements into a specific three-dimensional array while the RNA provides the catalytic center . The division of labor between RNA and protein illustrated by this simple system reveals principles applicable to complex ribonucleoprotein assemblies such as the spliceosome and ribosome. Biochem Biophys Res Commun, 1995 Jul 26, 212(3), 1098 - 106 The possible involvement of replication-related proteins with a DEAD-box-like motif in cell-free DNA replication of Xenopus eggs; Someya A et al.; Two types of antibodies were prepared: one directed against an oligopeptide specific to P1 protein, a mammalian homologue of yeast MCM3, and the other against an oligopeptide with a DEAD box motif, which is a highly conserved sequence in the P1 protein family . Immunoprecipitation of the eluate from anti-P1 family IgG-bound beads, which had been incubated in Xenopus egg extracts, with anti-P1 IgG-bound beads revealed that three proteins were coprecipitated . Two proteins remained in the supernatant after the immunoprecipitation of the eluate from anti-P1 family IgG-bound beads with anti-P1 IgG-bound beads . The immunodepleted extracts with anti-P1 family IgG-bound beads showed much lower DNA replication activity than did mock-treated extracts . Recovery of replication was achieved by supplementing the depleted extracts with both the eluate from anti-P1 IgG-bound beads and the supernatant obtained after the immunoprecipitation of the eluate with anti-P1 IgG-bound beads but not by supplementing the extracts with only the proteins eluted from anti-P1 IgG-bound beads . These findings suggest that some proteins containing a DEAD-box-like motif as well as mammalian homologues of yeast MCM2, MCM3 and CDC46 play an important role in cell-free DNA replication of Xenopus eggs. Nucleic Acids Res, 1995 Jul 25, 23(14), 2629 - 35 Cloning of cDNAs encoding the 160 kDa subunit of the bovine cleavage and polyadenylation specificity factor; Jenny A et al.; 3'-processing of mRNA precursors depends on several protein factors . One of them, cleavage and polyadenylation specificity factor (CPSF) is required for the cleavage of the mRNA precursor or as well as for the tail elongation reaction . We have obtained complementary DNA encoding the 160 kDa subunit, which had previously been shown to interact with the AAUAAA polyadenylation signal . The cDNAs code for an open reading frame of 1444 amino acids . The translated protein has a calculated molecular weight of 161 kDa and a predicted pl of 6.2 . Polyclonal antibodies raised against a bacterially expressed fragment of the cDNA recognise 160 kDa subunit of purified calf thymus CPSF . The sequence contains a possible nuclear localisation signal but none of the known RNA binding motifs . It does, however, show sequence similarities to a UV-damaged DNA binding protein (UVdDb). Biochim Biophys Acta, 1995 Jul 25, 1263(1), 39 - 44 Mutational analysis of human U6 RNA: stabilizing the intramolecular helix blocks the spliceosomal assembly pathway; Wolff T et al.; U6 RNA undergoes several conformational transitions during the spliceosome cycle: after the interaction with U4, the singular form of U6 is converted into the U4-U6 base-paired form, and within the spliceosome, the U4-U6 duplex isomerizes into the active U6-U2 conformation . The secondary structure of the singular form contains an extended 3' stem-loop, the upper part of which (intramolecular helix) most likely reforms in the spliceosome . We have previously shown in the mammalian splicing complementation system that the loop and the three adjacent, highly conserved base pairs of the intramolecular helix function during both the U4-U6 interaction and the first step of splicing . Here we demonstrate that the balanced stability of the lower, less conserved part of the 3' stem-loop is also critical for U4-U6 interaction; however, no specific splicing function could be detected in this region . The analysis of the heterologous interaction between mammalian U4 snRNP and yeast U6 RNA derivatives suggests that there are--in addition to the 3' loop and the stability of the intramolecular helix--specific sequence determinants in the 3' terminal domain of U6 that are important for efficient U4/U6 snRNP assembly. J Biol Chem, 1995 Jul 21, 270(29), 17442 - 56 A proteolytic pathway that recognizes ubiquitin as a degradation signal; Johnson ES et al.; Previous work has shown that a fusion protein bearing a "nonremovable" N-terminal ubiquitin (Ub) moiety is short-lived in vivo, the fusion's Ub functioning as a degradation signal . The proteolytic system involved, termed the UFD pathway (Ub fusion degradation), was dissected in the yeast Saccharomyces cerevisiae by analyzing mutations that perturb the pathway . Two of the five genes thus identified, UFD1 and UFD5, function at post-ubiquitination steps in the UFD pathway . UFD3 plays a role in controlling the concentration of Ub in a cell: ufd3 mutants have greatly reduced levels of free Ub, and the degradation of Ub fusions in these mutants can be restored by overexpressing Ub . UFD2 and UFD4 appear to influence the formation and topology of a multi-Ub chain linked to the fusion's Ub moiety . UFD1, UFD2, and UFD4 encode previously undescribed proteins of 40, 110, and 170 kDa, respectively . The sequence of the last approximately 280 residues of Ufd4p is similar to that of E6AP, a human protein that binds to both the E6 protein of oncogenic papilloma viruses and the tumor suppressor protein p53, whose Ub-dependent degradation involves E6AP . UFD5 is identical to the previously identified SON1, isolated as an extragenic suppressor of sec63 alleles that impair the transport of proteins into the nucleus . UFD5 is essential for activity of both the UFD and N-end rule pathways (the latter system degrades proteins that bear certain N-terminal residues) . We also show that a Lys --> Arg conversion at either position 29 or position 48 in the fusion's Ub moiety greatly reduces ubiquitination and degradation of Ub fusions to beta-galactosidase . By contrast, the ubiquitination and degradation of Ub fusions to dihydrofolate reductase are inhibited by the UbR29 but not by the UbR48 moiety . ufd4 mutants are unable to ubiquitinate the fusion's Ub moiety at Lys29, whereas ufd2 mutants are impaired in the ubiquitination at Lys48 . These and related findings suggest that Ub-Ub isopeptide bonds in substrate-linked multi-Ub chains involve not only the previously identified Lys48 but also Lys29 of Ub, and that structurally different multi-Ub chains have distinct functions in Ub-dependent protein degradation. J Biol Chem, 1995 Jul 21, 270(29), 17317 - 20 A transient GCN4 mRNA destabilization follows GCN4 translational derepression; Kyrpides N et al.; Studies based on experimental strategies that utilized either inhibitors or structural alterations point to the existence of an inverse relationship between translation and stability of a given mRNA . In this study we have investigated the potential link between translation and stability of the yeast GCN4 mRNA whose translational rates change with respect to amino acid availability . We observed that under conditions favoring its translation, the steady state levels of the GCN4 mRNA were decreased, but this was not due to a measurable alternation in its decay rate . We have demonstrated that an extensive destabilization of this message is intimately coupled with its increased access to heavy polysomes, which occurs transiently in the process of translational derepression . This transient change in the stability is what readjusts the steady state levels of the GCN4 mRNA . This study demonstrates in vivo the existence of a mechanism of mRNA degradation that is coupled with the process of translation. Genomics, 1995 Jul 20, 28(2), 286 - 90 Physical and genetic mapping of the CMT4A locus and exclusion of PMP-2 as the defect in CMT4A; Othmane KB et al.; We have previously localized one form of the autosomal recessive Charcot-Marie-Tooth disease type 4 (CMT4A) to a 5-cM region of chromosome 8q13-q21 . We now report the formation of a 7-Mb YAC contig spanning the region . This contig was used to map nine additional microsatellites and six STSs to this region, and subsequent haplotype analysis has narrowed the CMT4A flanking interval to less than 1 cM . In addition, using SSCP and our physical map, we have demonstrated that the myelin protein PMP-2, mapped by FISH to this region, is not the defect in CMT4A. Mol Cell Biochem, 1995 Jul 19, 148(2), 191 - 8 Partial purification and characterization of a soluble protein phosphatase from Leishmania donovani promastigotes; Nandi S et al.; A soluble protein phosphatase from the promastigote form of the parasitic protozoan Leishmania donovani was partially purified using Sephadex G-100, DEAE-cellulose and again Sephadex G-100 columns . The partially purified enzyme showed a native molecular weight of about 42,000 in both Sephadex G-100 and sucrose density gradient centrifugation . The sedimentation constant, stokes radius and frictional ratio were found to be 3.43S, 2.8 nm and 1.20 respectively . The enzyme preferentially utilized phosphohistone as the best exogenous substrate . Mg2+ ions were essential for enzyme activity; among other metal ions Mn2+ can replace Mg2+ to a certain extent whereas Ca2+, Co2+ and Zn2+ could not substitute for Mg2+ . The pH optimum of the enzyme was 6.5-7.5 and the temperature optimum 37 degrees C . The apparent Km for phosphohistone was 7.14 microM . ATP, ADP, inorganic phosphate and pyrophosphate had inhibitory effect on the enzyme activity whereas no inhibition was observed with sodium tartrate and okadaic acid . These results suggest that L . donovani promastigotes possess a protein phosphatase which has similar characteristics with the mammalian protein phosphatase 2C. Proc Natl Acad Sci U S A, 1995 Jul 18, 92(15), 7036 - 40 Green fluorescent protein as a vital marker and reporter of gene expression in Drosophila; Yeh E et al.; We have used the green fluorescent protein (GFP) from the jellyfish Aequorea victoria as a vital marker/reporter in Drosophila melanogaster . Transgenic flies were generated in which GFP was expressed under the transcriptional control of the yeast upstream activating sequence that is recognized by GAL4 . These flies were crossed to several GAL4 enhancer trap lines, and expression of GFP was monitored in a variety of tissues during development using confocal microscopy . Here, we show that GFP could be detected in freshly dissected ovaries, imaginal discs, and the larval nervous system without prior fixation or the addition of substrates or antibodies . We also show that expression of GFP could be monitored in intact living embryos and larvae and in cultured egg chambers, allowing us to visualize dynamic changes in gene expression during real time. EMBO J, 1995 Jul 17, 14(14), 3520 - 7 The coactivator p15 (PC4) initiates transcriptional activation during TFIIA-TFIID-promoter complex formation; Kaiser K et al.; We have analyzed the mechanisms underlying stimulation of transcription by the activator GAL4-AH and the recombinant coactivator p15 (PC4) . We show that p15 binds to both double-stranded and single-stranded DNA . Analyses of deletion mutants correlates binding to double-stranded DNA with the ability to mediate activator-dependent transcription . Consistent with this finding, phosphorylation of p15 by casein kinase II inhibits binding to double-stranded DNA and the activity of p15 . The functional characterization suggests interactions of p15 with both DNA and components of the TFIID complex . GAL4-AH functions in concert with p15 during formation of TFIIA-TFIID-promoter (DA) complexes, as concluded from order-of-addition experiments . At limiting TFIID concentrations, the number of DA complexes is enhanced . The activator also stimulates transcription moderately after DA complex formation, independently of the concentrations of general transcription factors. EMBO J, 1995 Jul 17, 14(14), 3434 - 44 Hsp78, a Clp homologue within mitochondria, can substitute for chaperone functions of mt-hsp70; Schmitt M et al.; Hsp78 is a Clp homologue within mitochondria of Saccharomyces cerevisiae . Deletion of HSP78 does not cause any detectable changes in wild type cells, but results in a petite phenotype in the ssc1-3 mutant strain carrying a temperature-sensitive allele of mt-hsp70 . When overexpressed in the ssc1-3 mutant strain, hsp78 suppresses the defect in mitochondrial protein import under permissive conditions in vitro and interacts directly with newly imported polypeptide chains . As a molecular chaperone, hsp78 prevents the aggregation of misfolded proteins in the matrix of mitochondria under conditions of impaired mt-hsp70 function . However, unlike misfolded proteins associated with mt-hsp70, hsp78-bound polypeptides are not efficiently degraded by the ATP-dependent PIM1 protease . Thus, hsp78 can partially substitute for mt-hsp70 functions in the assembly of mitochondria and may be part of a salvage pathway if mt-hsp70 is limiting. Eur J Biochem, 1995 Jul 15, 231(2), 405 - 13 Stability, activity and structure of adenylate kinase mutants; Spuergin P et al.; Sequence/structure relationships have been explored by site-directed mutagenesis using a structurally known adenylate kinase . In particular the effects of helix capping and nonpolar core expansion on thermodynamic stability have been analyzed . Six point mutations were produced and characterized by SDS/PAGE, native PAGE, isoelectric focussing, electrophoretic titration, enzyme kinetics, and X-ray structure analysis . Heat-denaturation experiments yielded melting temperatures Tm and melting enthalpy changes delta Hm . The heat capacity change delta Cp of the wild-type enzyme was determined by guanidine hydrochloride denaturation in conjunction with Tm and delta Hm . Using the wild-type delta Cp value, Gibbs free energy changes delta G at room temperature were calculated for all mutants . Four mutants were designed for helix capping stabilization, but only one of them showed such an effect . Because of electrostatic interference with the induced-fit motion, one mutant decreased the catalytic activity strongly . Two mutants expanded nonpolar cores causing destabilization . The mutant with the lower stability could be crystallized and subjected to an X-ray analysis at 223-pm resolution which showed the structural changes . The enzyme was stabilized by adding a -Pro-His-His tail to the C-terminal alpha-helix for nickel-chelate chromatography . This addition constitutes a helix cap . Taken together, the results demonstrate that stabilization by helix capping is difficult to achieve because the small positive effect is drowned by adverse mutational disruption . Further addition of atoms to nonpolar cores destabilized the protein, although the involved geometry changes were very small, demonstrating the importance of efficient packing. Eur J Biochem, 1995 Jul 15, 231(2), 370 - 80 The basic subdomain of the c-Jun oncoprotein . A joint CD, Fourier-transform infrared and NMR study; Krebs D et al.; The structural properties of the basic subdomain of the basic zipper (bZIP) protein c-Jun were examined by joint means of 1H-NMR, CD and Fourier-transform infrared (FTIR) spectroscopies . The basic subdomain (residues 252-281 in c-Jun) is responsible for sequence-specific recognition of DNA . A modified basic subdomain bSD (residues 1-35) and its N-terminal part and C-terminal part fragments (NP, residues 1-19; and, CP, residues 16-35) were prepared by solid-phase synthesis and purified by HPLC . In aqueous solution, in the absence of DNA, bSD behaved mostly as an unstructured peptide characterized by only 5% alpha helix . However, upon mixing bSD and a specific DNA fragment, i.e . a CRE(cAMP-responsive element)-containing hexadecanucleotide, the alpha helix was stabilized to an extent of 20% at 20 degrees C or 35% at 2 degrees C . At the same time, no significant change could be detected in the DNA spectra . Addition of trifluoroethanol to an aqueous bSD sample resulted in an increase of the alpha-helix content so that about 60% of alpha helix was found at a ratio of 75% trifluoroethanol (20 degrees C) . These effects were reflected in both CD and FTIR measurements . Changes shown by the CD spectra during the process suggested a mechanism dominated by a two-state helix/unordered transition . NMR data, namely alpha H chemical shifts, NOE cross-peaks and NH temperature coefficients provided indications for extended or nascent helix structures within four short stretches dispersed along the sequence for c-Jun bSD, contrasting with the unique and continuous stretch reported for Gcn4 (yeast general control protein 4) bSD in aqueous solution . Trifluoroethanol stabilized the alpha-helix structure mainly at these four sites . The malleability of the basic subdomain of c-Jun was emphasized in relation to its ability to fit the DNA helix in adopting an alpha-helix structure . The complex formation apparently requires substantial conformational change from the peptide and only little from the oligonucleotide. Genes Dev, 1995 Jul 15, 9(14), 1716 - 27 Histone H4 and the maintenance of genome integrity; Megee PC et al.; The normal progression of Saccharomyces cerevisiae through nuclear division requires the function of the amino-terminal domain of histone H4 . Mutations that delete the domain, or alter 4 conserved lysine residues within the domain, cause a marked delay during the G2+M phases of the cell cycle . Site-directed mutagenesis of single and multiple lysine residues failed to map this phenotype to any particular site; the defect was only observed when all four lysines were mutated . Starting with a quadruple lysine-to-glutamine substitution allele, the insertion of a tripeptide containing a single extra lysine residue suppressed the G2+M cell cycle defect . Thus, the amino-terminal domain of histone H4 has novel genetic functions that depend on the presence of lysine per se, and not a specific primary peptide sequence . To determine the nature of this function, we examined H4 mutants that were also defective for G2/M checkpoint pathways . Disruption of the mitotic spindle checkpoint pathway had no effect on the phenotype of the histone amino-terminal domain mutant . However, disruption of RAD9, which is part of the pathway that monitors DNA integrity, caused precocious progression of the H4 mutant through nuclear division and increased cell death . These results indicate that the lysine-dependent function of histone H4 is required for the maintenance of genome integrity, and that DNA damage resulting from the loss of this function activates the RAD9-dependent G2/M checkpoint pathway. Genes Dev, 1995 Jul 15, 9(14), 1709 - 15 Cell proliferation and DNA replication defects in a Drosophila MCM2 mutant; Treisman JE et al.; The yeast MCM2, MCM3, and MCM5/CDC46 genes are required for DNA replication and have been proposed to act as factors that license the DNA for one and only one round of replication per cell cycle . We have identified a Drosophila gene, DmMCM2, that is highly homologous to MCM2 . A P-element insertion into this gene, which prevents its transcription, inhibits proliferation of cells in the imaginal discs and central nervous system (CNS) and causes an apparent prolongation of S phase in the embryonic and larval CNS . DmMCM2 is expressed in the embryo in a pattern corresponding to that of S-phase cells . These results suggest that DmMCM2 plays a role in the regulation of DNA replication analogous to that of its yeast counterpart. Genes Dev, 1995 Jul 15, 9(14), 1781 - 96 GCD10, a translational repressor of GCN4, is the RNA-binding subunit of eukaryotic translation initiation factor-3; Garcia-Barrio MT et al.; GCN4 mRNA is translated by a reinitiation mechanism involving four short upstream open reading frames (uORFs) in its leader sequence . Decreasing the activity of eukaryotic initiation factor-2 (eIF-2) by phosphorylation inhibits general translation in yeast but stimulates GCN4 expression by allowing ribosomes to scan past the uORFs and reinitiate at GCN4 instead . GCD10 was first identified genetically as a translational repressor of GCN4 . We show here that GCD10 is an essential protein of 54.6 kD that is required in vivo for the initiation of total protein synthesis . GCD10 binds RNA in vitro and we present strong biochemical evidence that it is identical to the RNA-binding subunit of yeast initiation factor-3 (eIF-3) . eIF-3 is a multisubunit complex that stimulates translation initiation in vitro at several different steps . We suggest that gcd10 mutations decrease the ability of eIF-3 to stimulate binding of eIF-2/GTP/Met-tRNA(iMet) ternary complexes to small ribosomal subunits in vivo . This would explain why mutations in eIF-3 mimic eIF-2 alpha phosphorylation in allowing ribosomes to bypass the uORFs and reinitiate at GCN4 . Our results indicate that GCN4 expression provides a sensitive in vivo assay for the function of eIF-3 in initiation complex formation. J Mol Biol, 1995 Jul 14, 250(3), 315 - 26 Repression of vertebrate RNA polymerase III transcription by DNA binding proteins located upstream from the transcription start site; McBryant SJ et al.; Derivatives of yeast tRNA and Xenopus tRNA and 5 S RNA genes have been constructed in which natural 5' flanking sequences have been replaced by the binding sites for either the yeast transcription activator protein GCN4 or the three amino-terminal zinc fingers of the Xenopus factor TFIIA (zf1-3) . The binding sites for these proteins have been placed at various distances upstream from the start site for transcription initiation in the parent genes . Each of these plasmid DNAs is actively transcribed in both an unfractionated transcription extract prepared from unfertilized Xenopus eggs and in a reconstituted Xenopus transcription system . Binding of the test proteins to plasmid DNAs harboring the cognate binding sites severely represses transcription when these binding sites are located less than approximately 40 base-pairs upstream from the transcription start site . The DNA-binding proteins are without effect on the transcription of plasmids lacking binding sites or when the binding sites are located further upstream . Assembly of DNA templates into a complete transcription complex prior to addition of the DNA-binding proteins prevents repression . Proteins present in a fraction containing TFIIIB are necessary for this reversal of repression . These data suggest that vertebrate TFIIIB binds upstream from class III genes and this binding can be prevented by occlusion of the TFIIIB binding site by the test proteins GCN4 and zf1-3. Cell, 1995 Jul 14, 82(1), 121 - 30 TOR kinase domains are required for two distinct functions, only one of which is inhibited by rapamycin; Zheng XF et al.; The rapamycin-sensitive signaling pathway is required to transduce specific mitogenic signals to the cell cycle machinery responsible for G1 progression . Genetic studies in yeast identified two related genes on this pathway, TOR1 and TOR2, thought to encode novel phosphatidylinositol kinases . We now show that an intact kinase domain is required for the G1 cell cycle functions of both proteins, for the ability of a mutation in a neighboring FKBP12-rapamycin-binding domain of the TOR1 protein to inhibit the growth of yeast cells when overexpressed, and for the essential function of the TOR2 protein . The G1 function of both TOR proteins is sensitive to rapamycin, but the essential function of TOR2 is not . Thus, FKBP12-rapamycin does not appear t