S. cerevisiae

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 to inhibit the kinase activity of TOR proteins in a general way; instead, it may interfere selectively with TOR protein binding to or phosphorylation of G1 effectors.

Biochemistry, 1995 Jul 11, 34(27), 8869 - 75
Mechanism of inhibition of proliferating cell nuclear antigen-dependent DNA synthesis by the cyclin-dependent kinase inhibitor p21; Podust VN et al.; It is known that the direct binding of the cyclin-dependent kinase (Cdk) inhibitor p21, also called Cdk-interacting protein 1 (p21), to proliferating cell nuclear antigen (PCNA) results in the inhibition of PCNA-dependent DNA synthesis . We provide evidence that p21 first inhibits the replication factor C-catalyzed loading of PCNA onto DNA and second prevents the binding of DNA polymerase delta core to the PCNA clamp assembled on DNA . The second effect contributes most to the inhibition of pol delta holoenzyme activity . p21 primarily inhibited the DNA synthesis resulting from multiple reassembly of DNA polymerase delta holoenzyme . On the other hand, an ability of the PCNA clamp to translocate along double-stranded DNA was not affected by p21 . These data were confirmed with a mutant of p21 that is unable to bind PCNA and therefore neither inhibited clamp assembly nor prevented the loading of DNA polymerase delta core onto DNA . Our data suggest that p21 does not discriminate in vitro "repair" and "replication" DNA synthesis based on template length but does act preferentially on polymerization which encounters obstacles to progress.

J Mol Biol, 1995 Jul 7, 250(2), 169 - 80
Evidence for functional interaction between the HIV-1 Tat transactivator and the TATA box binding protein in vivo; Veschambre P et al.; Tat strongly activates transcription of the HIV-1 provirus by stimulating both initiation and elongation . This transactivator binds to the TAR RNA element, but can also associate with cellular transcription factors, interacting with upstream promoter sequences . To achieve a better understanding of the role of Tat in the assembly of the transcriptional initiation complex in the living cell, we have examined how the activity of this protein is modified when the general transcription factor involved in the first step of this process, TBP, is overexpressed . The activity of Tat, either wild-type or fused to the DNA binding domain of GAL4 (GBTat), was tested using reporter constructs containing GAL4 binding sites upstream of a minimal promoter corresponding to the HIV-1 TATA box, with or without the TAR element . We found that overexpression of TBP led to a dramatic increase in the activity of the GBTat protein . In order to activate GBTat, TBP must be able to interact with the TATA box . Analysis of several Tat mutants indicated that both the cysteine-rich and the core domains of this transactivator are necessary and sufficient to activate transcription when TBP is overexpressed . In vitro experiments showed that Tat binds specifically to TBP . There was a correlation between the ability of different Tat mutants to bind TBP and their capacity to activate transcription in vivo . With the natural HIV-1 promoter, overexpression of TBP first stimulated and then suppressed the Tat-induced activity . This inhibition was abrogated by an increase in the intracellular levels of Tat . These experimental data indicate that Tat stimulates initiation of transcription by interacting with TBP in vivo.

J Biol Chem, 1995 Jul 7, 270(27), 16315 - 20
Substrate specificity and expression profile of amino acid transporters (AAPs) in Arabidopsis; Fischer WN et al.; Three amino acid transporter genes (AAP3-5) were isolated from Arabidopsis by complementation of a yeast mutant defective in histidine uptake . Transport is driven against a concentration gradient and sensitive to protonophores . Analysis of the substrate specificity demonstrates that the carriers have a broad substrate specificity covering the major transport forms of reduced nitrogen, i.e . glutamine and glutamate . The transporters have similar affinities for glutamate, glutamine, and alanine but differ with respect to valine, phenylalanine, histidine, arginine, and lysine . AAP3 and AAP5 efficiently transport arginine and lysine and are involved in basic amino acid transport . The predicted polypeptides of 53 kDa are highly hydrophobic with 12 putative membrane-spanning regions and show significant homologies to Arabidopsis amino acid transporters AAP1 and AAP2 . Each of the genes has a different organ-specific expression in the plant . AAP3 is exclusively expressed in roots and AAP4 mainly in source leaves, stems, and flowers, whereas AAP5 is found in all tissues . The specific distribution in the plant and the different substrate specificities of AAP transporters may indicate that tissues differ both qualitatively and quantitatively regarding import or export of amino acids.

J Biol Chem, 1995 Jul 7, 270(27), 15954 - 7
Functional roles for the pleckstrin and Dbl homology regions in the Ras exchange factor Son-of-sevenless; McCollam L et al.; Activation of p21ras by receptor tyrosine kinases is thought to result from recruitment of guanine nucleotide exchange factors such as Son-of-sevenless (Sos) to plasma membrane receptor substrates via adaptor proteins such as Grb2 . This hypothesis was tested in the present studies by evaluating the ability of truncation and deletion mutants of Drosophila (d)Sos to enhance {32P}GTP loading of p21ras when expressed in 32P-labeled COS or 293 cells . The dSos catalytic domain (residues 758-1125), expressed without the dSos NH2-terminal (residues 1-757) or adaptor-binding COOH-terminal (residues 1126-1596) regions, exhibits intrinsic exchange activity as evidenced by its rescue of mutant Saccharomyces cerevisiae deficient in endogenous GTP/GDP exchange activity . Here we show that this dSos catalytic domain fails to affect GTP p21ras levels when expressed in cultured mammalian cells unless the NH2-terminal domain is also present . Surprisingly, the COOH-terminal, adaptor binding domain of dSos was not sufficient to confer p21ras exchange activity to the Sos catalytic domain in these cells in the absence of the NH2-terminal domain . This function of promoting catalytic domain activity could be localized by mutational analysis to the pleckstrin and Dbl homology sequences located just NH2-terminal to the catalytic domain . The results demonstrate a functional role for these pleckstrin and Dbl domains within the dSos protein, and suggest the presence of unidentified cellular elements that interact with these domains and participate in the regulation of p21ras.

J Biol Chem, 1995 Jul 7, 270(27), 15950 - 3
Targeting peptides transiently block a mitochondrial channel; Lohret TA et al.; The effects of synthetic targeting peptides on the activity of the multiple conductance channel (MCC) of mouse and yeast mitochondria were investigated using patch-clamp techniques . Amino-terminal targeting peptides of two inner membrane proteins reversibly decreased the open probability and mean open time of MCC . One of these targeting peptides had no effect on two other voltage-dependent mitochondrial channels . Furthermore, the effects induced by the two targeting peptides on MCC were not elicited by two peptides of an outer membrane protein . The specific interactions of targeting peptides with MCC suggest that this channel may be involved in protein import across the inner mitochondrial membrane.

Science, 1995 Jul 7, 269(5220), 75 - 8
The TBP-TFIIA interaction in the response to acidic activators in vivo; Stargell LA et al.; A yeast TBP mutant (N2-1) is described here that is defective specifically in responding to acidic activators in vivo . N2-1 does not support activation by Gal4, Ace1, and Gcn4, but appears unaffected for constitutive transcription, repression by the Cyc8-Tup1 and Not complexes, and transcription by polymerase I (Pol) and Pol III . In vitro, N2-1 fails to interact with TFIIA, but it associates normally with a TATA element, an acidic activation domain, and TFIIB . Fusion of the small subunit of TFIIA to N2-1 restores activation function in vivo . Thus, an efficient interaction between TBP and TFIIA is required for transcriptional activation in vivo.

Oncogene, 1995 Jul 6, 11(1), 119 - 30
Wild type PAX3 protein and the PAX3-FKHR fusion protein of alveolar rhabdomyosarcoma contain potent, structurally distinct transcriptional activation domains; Bennicelli JL et al.; Alveolar rhabdomyosarcoma (ARMS) is characterized cytogenetically by a t(2;13)(q35;q14) chromosomal translocation involving two transcription factor genes: PAX3 and FKHR . ARMS cells express a PAX3-FKHR fusion protein containing the complete N-terminal, DNA-binding domain of PAX3 and the C-terminus of FKHR . Recently we demonstrated that PAX3-FKHR is a more potent transcriptional activator than PAX3 despite impaired binding to canonical PAX3 binding sites . Therefore, we propose that the gene fusion results in switching of PAX3 and FKHR transactivation domains with distinct structure, potency or function . To compare the PAX3 and putative PAX3-FKHR transactivation domains, we fused C-terminal test fragments to the heterologous GAL4 DNA-binding domain and tested activation of a reporter gene co-transfected into four cell types . GAL4-PAX3 and GAL4-PAX3-FKHR were found to be potent activators exhibiting different concentration-dependent transactivation profiles and distinct structural motifs . Deletion mapping demonstrated essential acidic and/or serine/threonine-rich domains in the extreme 3' ends of their respective coding regions and positive modifying elements in adjacent 5' sequences . These data demonstrate that PAX3 and PAX3-FKHR contain structurally distinct transcriptional activation domains and suggest that a consequent difference in function is important for oncogenesis.

Biochim Biophys Acta, 1995 Jul 6, 1237(1), 95 - 8
Isolation of the vma-4 gene encoding the 26 kDa subunit of the Neurospora crassa vacuolar ATPase; Bowman EJ et al.; We have isolated the vma-4 gene, which encodes a 25,746 Dalton subunit of the vacuolar ATPase, from Neurospora crassa . The gene contains two introns and was mapped to the left arm of linkage group I . Comparison of the predicted amino acid sequence with homologous proteins from Saccharomyces cerevisiae, Manduca sexta, and Bos taurus showed only 25% sequence identity . However, computer-assisted predictions of secondary structures gave similar results for all four proteins . Analysis of the sequence and the available biochemical data indicated that the vma-4 gene product may play the same structural role in the vacuolar ATPase as does the gamma-subunit in F-type ATPases.

Gene, 1995 Jul 4, 159(2), 283 - 4
Identification of a new member of the human eIF-5A gene family; Koettnitz K et al.; Using an oligodeoxynucleotide generated by rapid PCR amplification of 5'-cDNA ends (5'-RACE) as a detection probe, we have isolated a new genomic clone encoding the human eukaryotic initiation factor 5A (eIF-5A) . Sequence analysis revealed that the eIF-5A coding region is identical to the corresponding cDNA but interrupted by three introns . In a plasmid shuffle experiment we show functional replacement of the essential homologous gene in Saccharomyces cerevisiae by this human eIF-5A.

EMBO J, 1995 Jul 3, 14(13), 3236 - 46
Interactions between the terminal bases of mammalian introns are retained in inosine-containing pre-mRNAs; Deirdre A et al.; Nuclear pre-mRNA splicing has a fundamentally similar two-step mechanism to that employed by group II self-splicing introns . It is believed that nuclear pre-mRNA splicing involves a network of RNA-RNA interactions which form the catalytic core of the active spliceosome . We show here a non-Watson-Crick interaction between the first and last guanosine residues of a mammalian intron . As in Saccharomyces cerevisiae, substitution of the conserved guanosines at the 5' and 3' splice sites by A and C respectively, specifically suppresses step 2 splicing defects resulting from the individual mutations . No other combination of terminal nucleotides was able to restore splicing . We additionally provide independent evidence for an indirect interaction between other nucleotides of the consensus splice sites during step 2 of splicing . Substitution of the nucleotide in the +3 position of the 5' splice site affects competition between closely spaced AG dinucleotides at the 3' splice site, although the interaction is not via direct differential base pairing . Finally, we show that complete substitution of guanosine residues by inosine in a pre-mRNA has only a modest effect upon step 2 of splicing, although earlier spliceosome assembly steps are impaired . Predictions can thus be made about the precise configuration of the non-Watson-Crick interaction between the terminal residues.

EMBO J, 1995 Jul 3, 14(13), 3184 - 99
GCN20, a novel ATP binding cassette protein, and GCN1 reside in a complex that mediates activation of the eIF-2 alpha kinase GCN2 in amino acid-starved cells; Vazquez de Aldana CR et al.; GCN2 is a protein kinase that phosphorylates the alpha-subunit of translation initiation factor 2 (eIF-2) and thereby stimulates translation of GCN4 mRNA in amino acid-starved cells . We isolated a null mutation in a previously unidentified gene, GCN20, that suppresses the growth-inhibitory effect of eIF-2 alpha hyperphosphorylation catalyzed by mutationally activated forms of GCN2 . The deletion of GCN20 in otherwise wild-type strains impairs derepression of GCN4 translation and reduces the level of eIF-2 alpha phosphorylation in vivo, showing that GCN20 is a positive effector of GCN2 kinase function . In accordance with this conclusion, GCN20 was co-immunoprecipitated from cell extracts with GCN1, another factor required to activate GCN2, and the two proteins interacted in the yeast two-hybrid system . We conclude that GCN1 and GCN20 are components of a protein complex that couples the kinase activity of GCN2 to the availability of amino acids . GCN20 is a member of the ATP binding cassette (ABC) family of proteins and is closely related to ABC proteins identified in Caenorhabditis elegans, rice and humans, suggesting that the function of GCN20 may be conserved among diverse eukaryotic organisms.

Biochim Biophys Acta, 1995 Jul 3, 1250(1), 1 - 8
Radiation-induced inactivation of flavocytochrome b2 in dilute aqueous solution; Bhattacharya D et al.; Effect of gamma radiation on flavocytochrome b2 in dilute aqueous solution was studied . A study of the effect of the radiolytically produced inorganic free-radical anions such as I2.-, Br2.- and (SCN)2.- on the enzyme activity indicates the involvement of cysteine and tyrosine residues in the catalytic activity of flavocytochrome b2 . The changes in kinetic parameters, i.e., Michaelis-Menten constant Km and maximal velocity Vmax, due to irradiation under different conditions suggest that radiation induced enzyme inactivation is the result of destruction of active-site residues as well as modification of the substrate binding site . Fluorescence studies of unirradiated and irradiated enzyme reveal that FMN (flavin mononucleotide) is inaccessible to water radicals.

Proc Natl Acad Sci U S A, 1995 Jul 3, 92(14), 6587 - 91
A genetic screen identifies cellular factors involved in retroviral -1 frameshifting; Lee SI et al.; To identify cellular factors that function in -1 ribosomal frameshifting, we have developed assays in the yeast Saccharomyces cerevisiae to screen for host mutants in which frameshifting is specifically affected . Expression vectors have been constructed in which the mouse mammary tumor virus gag-pro frameshift region is placed upstream of the lacZ gene or the CUP1 gene so that the reporters are in the -1 frame relative to the initiation codon . These vectors have been used to demonstrate that -1 frameshifting is recapitulated in yeast in response to retroviral mRNA signals . Using these reporters, we have isolated spontaneous host mutants in two complementation groups, ifs1 and ifs2, in which frameshifting is increased 2-fold . These mutants are also hypersensitive to antibiotics that target the 40S ribosomal subunit . We have cloned the IFS1 gene and shown that it encodes a previously undescribed protein of 1091 aa with clusters of acidic residues in the carboxyl-terminal region . Haploid cells lacking 82% of the IFS1 open reading frame are viable and phenotypically identical to ifs1-1 mutants . This approach could help identify potential targets for antiretroviral agents.

Glycobiology, 1995 Jul, 5(5), 463 - 72
Molecular cloning of eukaryotic glycoprotein and glycolipid glycosyltransferases: a survey; Field MC et al.; The rapidity with which molecular sequence data are gathered continues to grow . The result is that, for many workers, it is increasingly difficult to keep abreast of the current state of play of molecular cloning, even for those genes that encode proteins of special interest . The clear success of the various worldwide genome projects has made this even more apparent, and by the end of 1996 the complete determination of the nucleotide sequences of the genomes of two eukaryotes, Saccharomyces cerevisiae and Caenorhabditis elegans, will have either been completed or will be nearing completion . This article is an attempt to provide, in an easily accessible format, a compilation of genes and cDNAs that have been sequenced and deposited in GenBank that encode transferase enzymes involved in eukaryotic glycoprotein or glycolipid biosynthesis . The full sequence information can be easily retrieved from a databank, e.g . GenBank, using the relevant accession number(s).

J Biochem (Tokyo), 1995 Jul, 118(1), 13 - 7
Thermosensitivity of green fluorescent protein fluorescence utilized to reveal novel nuclear-like compartments in a mutant nucleoporin NSP1; Lim CR et al.; Tagging proteins with the green fluorescent protein (GFP) from Aequorea victoria is a good means of analyzing protein localization in living cells . Nevertheless, GFP and a chimeric protein, GFP-nucleoplasmin, expressed in Saccharomyces cerevisiae were less fluorescent at high culture temperatures . Proteins synthesized at a low temperature retained their fluorescence despite a shift to a higher temperature . Hence, when a temperature-sensitive nsp1 mutant expressing GFP-nucleoplasmin was cultured at 23 degrees C and then shifted to 35 degrees C, we were able to exclusively monitor the localization of the protein synthesized prior to the temperature shift . This protein accumulated in novel nuclear-like compartments devoid of DNA.

Biochem Mol Biol Int, 1995 Jul, 36(4), 817 - 26
Purification and characterization of hepatic oligosaccharyltransferase; Kumar V et al.; Oligosaccharyltransferase transfers a preformed oligosaccharide from a dolichol carrier molecule to specific asparaginyl residues of proteins synthesized in the endoplasmic reticulum . We have isolated a protein complex with this activity from chicken liver microsomes with 850 fold purification . The purification procedure involved removal of peripheral and lumenal proteins, solubilization of the membranes by non-ionic detergent and glycerol gradient centrifugation . The complex was purified further by ion-exchange and gel filtration chromatography . SDS-PAGE analysis of the final preparation revealed 3 major protein bands, two bands with an approximate molecular weight of 65-kDa and one band of approximately 50-kDa . Endoglycosidase H digestion of the purified subunits indicated the presence of carbohydrate on the 65-I subunit . No carbohydrate was detected in the 65-II subunit or the 50-kDa subunit . Amino acid sequence analysis of the intact protein subunits and internal peptides generated by cynogen bromide digestion, identified the 65-kDa subunits as ribophorin I and II . The 50-kDa subunit has 25% homology with a yeast membrane protein (Wbplp) which is essential for oligosaccharyltransferase activity in Saccharomyces cerevisiae.

Mol Cell Biol, 1995 Jul, 15(7), 3917 - 25
Mutational analysis of Hsp90 function: interactions with a steroid receptor and a protein kinase; Nathan DF et al.; Hsp90 is a protein chaperone whose functions are focused on a specific set of target proteins . The nature of Hsp90's interactions with these proteins is poorly understood . To provide tools for examining these interactions, we have isolated eight broadly distributed temperature-sensitive (ts) point mutations in the Hsp90 gene (HSP82) of Saccharomyces cerevisiae . The mutants fall into two distinct classes . One has a classic ts phenotype, with nearly wild-type activity at 25 degrees C and a precipitous loss of function at 34 degrees C . The remaining seven mutants, in contrast, cause a general reduction in Hsp90 function and are ts because they do not provide the high level of function required for growth at high temperatures . The effects of these mutants on two target proteins, a transcription factor (glucocorticoid receptor) and a tyrosine kinase (pp60v-src), provided several insights on Hsp90 function . First, Hsp90 is not only required to help the glucocorticoid receptor achieve a hormone-activable state, it is continuously required to maintain that state . Second, Hsp90's function in the maturation of pp60v-src involves separable roles in protein accumulation and kinase activation . Thus, Hsp90 is an integral component of both the steroid receptor and kinase signaling pathways . Finally, all eight point mutants affect the activation of both the glucocorticoid receptor and pp60v-src, indicating that Hsp90 promotes the activity of these very different target proteins through common mechanisms.

Mol Cell Biol, 1995 Jul, 15(7), 3479 - 86
Human type II receptor for bone morphogenic proteins (BMPs): extension of the two-kinase receptor model to the BMPs; Liu F et al.; Bone morphogenic proteins (BMPs) are universal regulators of animal development . We report the identification and cloning of the BMP type II receptor (BMPR-II), a missing component of this receptor system in vertebrates . BMPR-II is a transmembrane serine/threonine kinase that binds BMP-2 and BMP-7 in association with multiple type I receptors, including BMPR-IA/Brk1, BMPR-IB, and ActR-I, which is also an activin type I receptor . Cloning of BMPR-II resulted from a strong interaction of its cytoplasmic domain with diverse transforming growth factor beta family type I receptor cytoplasmic domains in a yeast two-hybrid system . In mammalian cells, however, the interaction of BMPR-II is restricted to BMP type I receptors and is ligand dependent . BMPR-II binds BMP-2 and -7 on its own, but binding is enhanced by coexpression of type I BMP receptors . BMP-2 and BMP-7 can induce a transcriptional response when added to cells coexpressing ActR-I and BMPR-II but not to cells expressing either receptor alone . The kinase activity of both receptors is essential for signaling . Thus, despite their ability to bind to type I and II receptors receptors separately, BMPs appear to require the cooperation of these two receptors for optimal binding and for signal transduction . The combinatorial nature of these receptors and their capacity to crosstalk with the activin receptor system may underlie the multifunctional nature of their ligands.

J Cell Biol, 1995 Jul, 130(1), 29 - 39
Functional characterization of the 180-kD ribosome receptor in vivo; Wanker EE et al.; A cDNA encoding the 180-kD canine ribosome receptor (RRp) was cloned and sequenced . The deduced primary structure indicates three distinct domains: an NH2-terminal stretch of 28 uncharged amino acids representing the membrane anchor, a basic region (pI = 10.74) comprising the remainder of the NH2-terminal half and an acidic COOH-terminal half (pI = 4.99) . The most striking feature of the amino acid sequence is a 10-amino acid consensus motif, NQGKKAEGAP, repeated 54 times in tandem without interruption in the NH2-terminal positively charged region . We postulate that this repeated sequence represents a ribosome binding domain which mediates the interaction between the ribosome and the ER membrane . To substantiate this hypothesis, recombinant full-length ribosome receptor and two truncated versions of this protein, one lacking the potential ribosome binding domain, and one lacking the COOH terminus, were expressed in Saccharomyces cerevisiae . Morphological and biochemical analyses showed all proteins were targeted to, and oriented correctly in the ER membrane . In vitro ribosome binding assays demonstrated that yeast microsomes containing the full-length canine receptor or one lacking the COOH-terminal domain were able to bind two to four times as many human ribosomes as control membranes lacking a recombinant protein or microsomes containing a receptor lacking the NH2-terminal basic domain . Electron micrographs of these cells revealed that the expression of all receptor constructs led to a proliferation of perinuclear ER membranes known as "karmellae." Strikingly, in those strains which expressed cDNAs encoding a receptor containing the putative ribosome binding domain, the induced ER membranes (examined in situ) were richly studded with ribosomes . In contrast, karmellae resulting from the expression of receptor cDNA lacking the putative ribosome binding domain were uniformly smooth and free of ribosomes . Cell fractionation and biochemical analyses corroborated the morphological characterization . Taken together these data provide further evidence that RRp functions as a ribosome receptor in vitro, provide new evidence indicating its functionality in vivo, and in both cases indicate that the NH2-terminal basic domain is essential for ribosome binding.

J Virol, 1995 Jul, 69(7), 4037 - 44
Transactivation activity of Meq, a Marek's disease herpesvirus bZIP protein persistently expressed in latently infected transformed T cells; Qian Z et al.; Marek's disease virus (MDV) is an avian herpesvirus that induces a variety of diseases, including T-cell lymphomas, in chickens . In latently infected, transformed lymphoid cells, very few viral transcripts or proteins are detected . We previously described a gene, meq (MDV EcoQ), which is persistently expressed in MDV-transformed tumor samples and cell lines . meq codes for a 339-amino-acid protein with a basic-leucine zipper domain near its N terminus and a proline-rich domain near its C terminus . The basic-leucine zipper domain shows homology with Jun/Fos family proteins, whereas the proline-rich domain resembles that of the WT-1 tumor suppressor protein . These structural features raise the possibility that Meq functions as a transcription factor in regulating viral latency or oncogenesis . In this report, we show that the proline-rich domain is a potent transcription activator when fused to the yeast (Saccharomyces cerevisiae) Gal4(1-147) DNA-binding domain . The transactivation activity maps to the C-terminal 130 amino acids, with the last 33 amino acids essential . In the absence of these 33 amino acids, a two-and-one-half proline-rich repeat structure was found to exhibit repression activity . We further show that Meq is able to dimerize not only with itself but also with c-Jun . Meq/c-Jun heterodimers bind to an AP1-like sequence in the meq promoter region with an affinity much greater than that of Meq/Meq or c-Jun/c-Jun homodimers . Cotransfection chloramphenicol acetyltransferase assays suggest that the Meq/c-Jun heterodimers can up-regulate Meq expression in both chicken embryo fibroblasts and F9 cells . Our data provide the first biochemical evidence that Meq is a transcriptional factor and identify c-Jun as one of Meq's interacting partners.

Genetics, 1995 Jul, 140(3), 965 - 72
DNA synthesis errors associated with double-strand-break repair; Strathern JN et al.; Repair of a site-specific double-strand DNA break (DSB) resulted in increased reversion frequency for a nearby allele . Site-specific DSBs were introduced into the genome of Saccharomyces cerevisiae by the endonuclease encoded by the HO gene . Expression of the HO gene from a galactose-inducible promoter allowed efficient DNA cleavage at a single site in large populations of cells . To determine whether the DNA synthesis associated with repair of DSBs has a higher error rate than that associated with genome duplication, HO-induced DSBs were generated 0.3 kb from revertible alleles of trp1 . The reversion rate of the trp1 alleles was approximately 100-fold higher among cells that had experienced an HO cut than among uninduced cells . The reverted allele was found predominantly on the chromosome that experienced the DNA cleavage.

Protein Sci, 1995 Jul, 4(7), 1272 - 8
Mutational analysis of phenylalanine beta 85 in the valine beta 6 acceptor pocket during hemoglobin S polymerization; Adachi K et al.; Hemoglobin (Hb) S containing Leu, Ala, Thr, or Trp substitutions at beta 85 were made and expressed in yeast in an effort to evaluate the role of Phe-beta 85 in the acceptor pocket during polymerization of deoxy Hb S . The four Hb S variants have the same electrophoretic mobility as Hb S, and these beta 85 substitutions do not significantly affect heme-globin interactions and tetramer helix content . Hb S containing Trp-beta 85 had decreased oxygen affinity, whereas those with Leu-, Ala-, and Thr-beta 85 had increased oxygen affinity . All four supersaturated beta 85 variants polymerized with a delay time as does deoxy Hb S . This is in contrast to deoxy Hb S containing Phe-beta 88, Ala-beta 88, Glu-beta 88, or Glu-beta 85, which polymerized with no clear delay time (Adachi K, Konitzer P, Paulraj CG, Surrey S, 1994, J Biol Chem 269:17477-17480; Adachi K, Reddy LR, Surrey S, 1994, J Biol Chem 269:31563-31566) . Leu substitution at beta 85 accelerated deoxy Hb S polymerization, whereas Ala, Thr, or Trp substitution inhibited polymerization . The length of the delay time and total polymer formed for these beta 85 Hb S variants depended on hemoglobin concentration in the same fashion as for deoxy Hb S: the higher the concentration, the shorter the delay time and the more polymer formed . Critical concentrations required for polymerization of deoxy Hb SF veta 85L, Hb SF beta 85A, Hb SF beta 85T, and Hb SF beta 85W are 0.65-, 2.2-, 2.5- and 3-fold higher, respectively, than Hb S.(ABSTRACT TRUNCATED AT 250 WORDS)

Biosci Biotechnol Biochem, 1995 Jul, 59(7), 1221 - 8
Cloning, sequencing, and heterologous expression of a gene coding for Arthromyces ramosus peroxidase; Sawai-Hatanaka H et al.; To understand the relationship between the structure and functions of the peroxidase of Arthromyces ramosus, a novel taxon of hyphomycete, and the evolutionary relationship of the A.ramosus peroxidase (ARP) with the other peroxidases, we isolated complementary and genomic DNA clones encoding ARP and characterized them . The sequence analyses of the ARP and cDNA coding for ARP showed that a mature ARP consists of 344 amino acids with a N-terminal pyroglutamic acid preceded by a signal peptide of 20 amino acid residues . The amino acid sequence of ARP was 99% identical to that of the peroxidase of Coprinus cinereus, a basidiomycete, and also had very high similarities (41-43% identity) to those of basidiomycetous lignin peroxidases, although we could find no lignin peroxidase activities for ARP when assayed with lignin model compounds . We could identified His184 and His56 as proximal and distal ligands to heme, respectively, and Arg52 as an essential Arg . Comparison of the sequences of complementary and genomic DNAs found that protein-encoding DNA is interrupted by 14 intervening sequences . The ARP cDNA was expressed in the yeast Saccharomyces cerevisiae under the promoter of the glyceraldehyde 3-phosphate dehydrogenase gene, yielding 0.02 units/ml of a secreted active peroxidase.

Bioessays, 1995 Jul, 17(7), 609 - 20
In vivo biochemistry: physical monitoring of recombination induced by site-specific endonucleases; Haber JE; The recombinational repair of chromosomal double-strand breaks (DSBs) is of critical importance to all organisms, who devote considerable genetic resources to ensuring such repair is accomplished . In Saccharomyces cerevisiae, DSB-mediated recombination can be initiated synchronously by the conditional expression of two site-specific endonucleases, HO or I-Scel . DNA undergoing recombination can then be extracted at intervals and analyzed . Recombination initiated by meiotic-specific DSBs can be followed in a similar fashion . This type of 'in vivo biochemistry' has been used to describe several discrete steps in two different homologous recombination pathways: gene conversion and single-strand annealing . The roles of specific proteins during recombination can be established by examining DNA in strains deleted for the corresponding gene . These same approaches are now becoming available for the study of recombination in both higher plants and animals . Physical monitoring can also be used to analyze nonhomologous recombination events, whose mechanisms appear to be conserved from yeast to mammals.

Biochem J, 1995 Jul 1, 309 ( Pt 1), 167 - 73
Variable effects of maturity-onset-diabetes-of-youth (MODY)-associated glucokinase mutations on substrate interactions and stability of the enzyme; Liang Y et al.; Mutations in the human glucokinase (GK) gene are thought to cause maturity-onset diabetes of youth (MODY) by leading to the production of enzymes with reduced catalytic activities and increased glucose Km values . However, in some cases the diabetic phenotype is more severe than might be predicted from these apparent kinetic effects alone . To determine whether these mutations might also effect other characteristics of the enzyme, nine MODY-associated mutants were expressed as fusion proteins with Schistosoma japonicum glutathione S-transferase (GST) and compared with three wild-type human GK isoforms that were also expressed in the same manner . Three GST-GK isoforms (liver 1, liver 2 and islet) were kinetically indistinguishable from each other and from purified rat liver GK . Noteworthy is a glucose-induced fit effect for the interaction of trinitrophenyl (TNP)-ATP with GST-GK, whereby glucose significantly increased the affinity of TNP-ATP binding to GST-GK without changing the stoichiometry of binding . The nine MODY-associated mutations studied either showed diminished catalytic activity, substrate affinities, allosteric regulation, or stability of the fusion enzyme . We conclude that: (1) Gly261 and Lys414 are important for ATP binding; (2) Val203 may be essential for a glucose-induced fit effect; and (3) the stability of fusion protein may be significantly reduced when Glu300 is replaced by Lys . These results suggest that, in addition to effects on the Km and Vmax . of GK, a decrease in the ATP-binding affinity or stability of the mutated enzyme may also contribute to a reduction of GK activity in individuals with GK-MODY . In the B-cell this would have the effect of blunting glucose-stimulated insulin release, thereby contributing to the diabetic phenotype.

Int J Hyperthermia, 1995 Jul-Aug, 11(4), 459 - 88
Heat shock proteins, thermotolerance, and their relevance to clinical hyperthermia; Li GC et al.; Mammalian cells, when exposed to a non-lethal heat shock, have the ability to acquire a transient resistance to subsequent exposures at elevated temperatures, a phenomenon termed thermotolerance . The mechanism(s) for the development of thermotolerance is not well understood, but earlier experimental evidence suggests that protein synthesis may play a role in its manifestation . On the molecular level, heat shock activates a specific set of genes, so-called heat shock genes, and results in the preferential synthesis of heat shock proteins . The heat shock response, specifically the regulation, expression and functions of heat shock proteins, has been extensively studied in the past decades and has attracted the attention of a wide spectrum of investigators ranging from molecular and cell biologists to radiation and hyperthermia oncologists . There is much data supporting the hypothesis that heat shock proteins play important roles in modulating cellular responses to heat shock, and are involved in the development of thermotolerance . This review summarizes some current knowledge on thermotolerance and the functions of heat shock proteins, especially hsp70 . The relationship between thermotolerance development and hsp70 synthesis in tumours and in normal tissues is examined . The possibility of using hsp70 as an indicator for thermotolerance is discussed.

Genomics, 1995 Jul 1, 28(1), 32 - 8
cDNA, gene structure, and chromosomal localization of human GAR1 (CNCG3L), a homolog of the third subunit of bovine photoreceptor cGMP-gated channel; Ardell MD et al.; A unique glutamic acid-rich protein was previously identified in bovine rod photoreceptors (Sugimoto et al., 1991, Proc . Natl . Acad . Sci . USA 88: 3116-3119) and later suggested to be a third subunit (gamma) of the rod cGMP-gated cation channel (Chen et al., 1994, Proc . Natl . Acad . Sci . USA 91: 11757-11761) . Here, we report on the characterization of the GAR1 gene encoding a human homolog of bovine gamma . Sequence analysis of cDNA clones encoding human gamma revealed an open reading frame predicting a protein of 299 amino acids (approximately 32 kDa), half the size of the bovine gamma subunit . Comparison of the N-terminal half of bovine gamma with the predicted human gamma sequence revealed 90% identity within the first 31 amino acids, and only 60% homology was found throughout the remainder of the protein sequence . As in bovine gamma, the predicted isoelectric point of the human protein is very acidic despite the absence of the bovine C-terminal glutamic acid-rich domain . The integrity of the cDNA sequence was confirmed by analysis of several overlapping genomic clones that span the GAR1 gene . The protein coding region of the gene consists of 12 exons spanning approximately 11 kb with exon sequence identical to that of the cDNA clones . PCR of somatic cell hybrid DNA with primer pairs that amplify a portion of the GAR1 gene (locus designation CNCG3L) demonstrate localization to chromosome 16 . The location of the gene was further delimited by fluorescence in situ hybridization placing the gene at 16q13.(ABSTRACT TRUNCATED AT 250 WORDS)

Curr Biol, 1995 Jul 1, 5(7), 775 - 83
Phosphatidylinositol transfer protein dictates the rate of inositol trisphosphate production by promoting the synthesis of PIP2; Cunningham E et al.; BACKGROUND: Phosphatidylinositol transfer protein (PI-TP), which has the ability to transfer phosphatidylinositol (PI) from one membrane compartment to another, is required in the inositol lipid signalling pathway through phospholipase C-beta (PLC-beta) that is regulated by GTP-binding protein(s) in response to extracellular signals . Here, we test the hypothesis that the principal role of PI-TP is to couple sites of lipid hydrolysis to sites of synthesis, and so to replenish depleted substrate for PLC-beta . RESULTS: We have designed an experimental protocol that takes advantage of the different rates of release of endogenous PI-TP and PLC-beta from HL60 cells permeabilized with streptolysin O . We have examined the kinetics of stimulated inositol lipid hydrolysis in cells depleted of PI-TP, but not of endogenous PLC-beta, in the presence and absence of exogenous PI-TP . Linear time-courses were observed in the absence of any added protein, and the rate was accelerated by PI-TP using either guanosine 5'{gamma-thio}-triphosphate (GTP gamma S) or the receptor-directed agonist fMetLeuPhe as activators . In addition, depletion from the cells of both PI-TP and PLC-beta isoforms by extended permeabilization (40 minutes) allowed us to control the levels of PLC-beta present in the cells . Once again, PI-TP increased the rates of reactions . To identify whether the role of PI-TP was to make available the substrate phosphatidylinositol bisphosphate (PIP2) for the PLC, we examined the synthesis of PIP2 in cells depleted of PI-TP . We found that PI-TP was essential for the synthesis of PIP2 . CONCLUSIONS: The predicted function of PI-TP in inositol lipid signalling is the provision of substrate for PLC-beta from intracellular sites where PI is synthesized . We propose that PI-TP is in fact a co-factor in inositol lipid signalling and acts by interacting with the inositol lipid kinases . We hypothesize that the preferred substrate for PLC-beta is not the lipid that is resident in the membrane but that provided through PI-TP.

Lipids, 1995 Jul, 30(7), 633 - 40
Kinetic analysis of cardiolipin synthase: a membrane enzyme with two glycerophospholipid substrates; Schlame M et al.; Mitochondrial cardiolipin synthase catalyzes the transfer of a phosphatidyl moiety from phosphatidyl-CMP (PtdCMP) to phosphatidylglycerol (PtdGro) in the presence of specific divalent cations . The synthase was solubilized from Saccharomyces cerevisiae mitochondria and purified about 300-fold . The partially enzyme was part of a medium-size, mixed micelle which had to bind to a foreign substrate/detergent micelle before catalysis could occur . The kinetics of cardiolipin synthase were studied by changing the molar fraction of substrate in the micelles . The enzyme obeyed Michaelis-Menten kinetics in relation to PtdCMP with a Km of 0.03 mol% . PtdGro caused sigmoidal kinetics with a low apparent affinity . It is speculated that it was involved in docking the enzyme to the substrate/detergent micelle . Cardiolipin synthase did not catalyze isotope exchange between {14C}CMP and PtdCMP, virtually excluding a ping-pong catalytic mechanism . Mg2+ stimulated the activity by increasing the turnover number rather than the substrate affinity, a mechanism which was also found for the Co(2+)-activation of rat liver cardiolipin synthase . It is concluded that a direct association of the metal ion and the enzyme forms the active cardiolipin synthase which has a very high affinity for PtdCMP and a lower affinity for PtdGro.

Z Naturforsch {C}, 1995 Jul-Aug, 50(7-8), 535 - 42
Membrane association of the Rieske iron-sulfur protein; Szczepaniak A et al.; The mode of membrane attachment of the Rieske iron-sulfur proteins from cytochrome b6f complex of pea thylakoids and from cytochrome bc1 complex of yeast mitochondria has been studied using biochemical approaches . The relative sensitivity of the Rieske protein to trypsin in the thylakoid membrane shows that all trypsin sites of the Rieske protein are on the lumen side of the thylakoid membrane . In contrast to cytochrome f the chloroplast Rieske protein was extracted from thylakoids using chaotropic agents (NaSCN, urea), an alkaline pH and relatively low concentrations of Trinon X-100 . The cytochrome bc1 complex Rieske protein from mitochondrial membranes of yeast was also released by NaSCN and alkaline treatment . The results presented here led us to the conclusion that the mitochondrial and chloroplast Rieske proteins are extrinsic and that their association with the rest of the complex involves hydrophobic interactions.

Yeast, 1995 Jul, 11(9), 865 - 71
Sequence and functional analysis of a 7.2 kb DNA fragment containing four open reading frames located between RPB5 and CDC28 on the right arm of chromosome II; Rose M et al.; In a coordinated approach, several laboratories sequenced Saccharomyces cerevisiae chromosome II during the European BRIDGE project . Here we report on the sequence and functional analysis of a 7217 bp fragment located on the right arm of chromosome II between RPB5 and CDC28 . The fragment contains four open reading frames probably encoding proteins of 79.2 kDa (corresponding gene YBR156c), 12.1 kDa (YBR157c), 62.7 kDa (YBR158w) and 38.7 kDa (YBR159w) . All four open reading frames encode new proteins, as concluded from data base searches . The respective genes were destroyed by gene replacement in one allele of diploid cells . After sporulation and tetrad analysis, the resulting mutant haploid strains were investigated . No phenotype with respect to spore germination, viability, carbohydrate utilization, and growth was found for YBR157c, encoding the smallest open reading frame investigated . Gene replacement within the YBR156c gene encoding a highly basic and possibly nuclear located protein was lethal . Ybr158 revealed similarities to the Grrl (Cat80) protein with respect to the leucine-rich region . Cells harboring a mutation in the YBR158w gene showed strongly reduced growth as compared to the wild-type cells . The protein predicted from YBR159w shared 33% identical amino acid residues with the human estradiol 17-beta-hydroxysterol dehydrogenase 3 . Haploid ybr159c mutants were only able to grow at reduced temperatures, but even under these conditions the mutants grew slower than wild-type strains.

J Biol Chem, 1995 Jun 30, 270(26), 15832 - 7
The tumor suppressor maspin does not undergo the stressed to relaxed transition or inhibit trypsin-like serine proteases . Evidence that maspin is not a protease inhibitory serpin; Pemberton PA et al.; The role of tumor suppressor proteins in the development of malignancy has made the understanding of their molecular mechanisms of action of great importance . Maspin is a tumor suppressor produced by a number of cell types of epithelial origin . Exogenous recombinant maspin has been shown to block the growth, motility, and invasiveness of breast tumor cell lines in vitro and in vivo . Although belonging to the the serine proteinase inhibitor (serpin) superfamily of proteins, the molecular mechanism of maspin is currently unknown . Here we show that the reactive site loop of maspin exists in an exposed conformation that does not require activation by cofactors . The reactive site loop of maspin, however, does not act as an inhibitor of proteinases such as chymotrypsin, elastase, plasmin, thrombin, and trypsin but rather as a substrate . Maspin is also unable to inhibit tissue and urokinase type plasminogen activators . Stability studies show that maspin cannot undergo the stressed-relaxed transition typical of proteinase-inhibitory serpins, and the protein is capable of spontaneous polymerization induced by changes in pH . It is likely, therefore, that maspin is structurally more closely related to ovalbumin and angiotensinogen, and its tumor suppressor activity is independent of a latent or intrinsic trypsin-like serine proteinase-inhibitory activity.

J Biol Chem, 1995 Jun 30, 270(26), 15739 - 46
Expression, domain structure, and enzymatic properties of an active recombinant human DNA topoisomerase II beta; Austin CA et al.; Human cells express two genetically distinct isoforms of DNA topoisomerase II, alpha and beta, which catalyze ATP-dependent DNA strand passage and are an important antitumor drug target . Here we report for the first time the successful overexpression of human topoisomerase II beta in yeast by cloning a topoisomerase II beta cDNA in a yeast shuttle vector under the control of a galactose-inducible promoter . Recombinant human topoisomerase II beta (residues 46-1621 fused to the first 5 residues of yeast topoisomerase II) was purified to homogeneity, yielding an enzymatically active polypeptide in sufficient quantity to allow analysis of its domain structure and comparison with that of recombinant human topoisomerase II alpha . Partial digestion of beta with either trypsin or protease SV8 generated fragments of approximately 130, 90, 62, and 45-50 kDa, arising from cleavage at three limited and discrete regions of the protein (A, B, and C) indicating the presence of at least four structural domains . Recombinant human topoisomerase II alpha and beta induced DNA breakage which was promoted by a variety of agents . Isoform differences in drug-induced DNA breakage were observed . These studies of human topoisomerase II beta in concert with alpha should aid the determination of their individual roles in cancer chemotherapy and should facilitate the design, targeting, and testing of cytotoxic antitumor agents.

Yeast, 1995 Jun 30, 11(8), 775 - 81
Analysis of a 42.5 kb DNA sequence of chromosome X reveals three tRNA genes and 14 new open reading frames including a gene most probably belonging to the family of ubiquitin-protein ligases; Huang ME et al.; We have sequenced a 42,500 bp stretch located on chromosome X of Saccharomyces cerevisiae between the genes MET3 and CDC8 . This stretch contains 24 open reading frames (ORFs) of at least 100 amino acids . Ten of these correspond to previously published sequences, whereas of the 14 remaining ORFs, only one, GTD892, has significant similarity to proteins from yeast or other organisms . It may belong to the family of ubiquitin-protein ligases and be involved in the ubiquitin-dependent proteolytic pathway . In addition, three tRNA genes were recognized, two of which had not been hitherto localized.

Cell, 1995 Jun 30, 81(7), 1137 - 46
The small GTP-binding proteins Rac1 and Cdc42 regulate the activity of the JNK/SAPK signaling pathway; Coso OA et al.; c-Jun amino-terminal kinases (JNKs) and mitogen-activated protein kinases (MAPKs) are closely related; however, they are independently regulated by a variety of environmental stimuli . Although molecules linking growth factor receptors to MAPKs have been recently identified, little is known about pathways controlling JNK activation . Here, we show that in COS-7 cells, activated Ras effectively stimulates MAPK but poorly induces JNK activity . In contrast, mutationally activated Rac1 and Cdc42 GTPases potently activate JNK without affecting MAPK, and oncogenic guanine nucleotide exchange factors for these Rho-like proteins selectively stimulate JNK activity . Furthermore, expression of inhibitory molecules for Rho-related GTPases and dominant negative mutants of Rac1 and Cdc42 block JNK activation by oncogenic exchange factors or after induction by inflammatory cytokines and growth factors . Taken together, these findings strongly support a critical role for Rac1 and Cdc42 in controlling the JNK signaling pathway.

Nature, 1995 Jun 29, 375(6534), 806 - 9
A SNARE-like protein required for traffic through the Golgi complex; Banfield DK et al.; The secretory pathway of eukaryotic cells comprises several distinct membrane-bound compartments which are interconnected by transport vesicles that pinch off from one membrane and fuse with the next . Targeting of these vesicles is mediated in part by interactions between integral membrane proteins on the vesicles and target organelles (soluble NSF attachment protein receptors (SNAREs)), termed v-SNAREs and t-SNAREs, respectively . SNAREs required for endoplasmic reticulum (ER)-Golgi transport and for fusion of vesicles with the plasma membrane are already known . Here we identify two yeast membrane proteins that show genetic interactions with Sed5p, which is the t-SNARE for ER-Golgi traffic . One of these membrane proteins, Sft1p, is structurally similar to the known v-SNAREs and is required for transport from an early to a later Golgi compartment . Our results indicate that a single t-SNARE can control more than one transport step, and provide the first candidate for a SNARE involved in intra-Golgi traffic.

Mol Gen Genet, 1995 Jun 25, 247(6), 661 - 9
RanBP1, a Ras-like nuclear G protein binding to Ran/TC4, inhibits RCC1 via Ran/TC4; Hayashi N et al.; A human protein that is 92% identical and 97% homologous at the amino acid level to RanBP1 from mouse was identified by the two-hybrid method, using two types of target cDNAs fused to sequences encoding the GAL4 DNA-binding domain . The target cDNAs encoded the human Ran/TC4 and human RCC1 proteins, respectively . An in vitro binding experiment showed that RanBP1 binds to RCC1 with the aid of Ran . Partially purified, GST-fused RanBP1 inhibited RCC1-stimulated guanine nucleotide release from Ran in vitro . Consistent with this in vitro finding, overproduction of human RanBP1 was detrimental to growth of tsBN2, a temperature-sensitive BHK21 hamster cell line defective in the RCC1 gene, and inhibited the growth of the Saccharomyces cerevisiae rcc1 mutants prp20, mtr1 and srm1 . The specific effect of RanBP1 on rcc1- cells was confirmed by the finding that overproduction of RanBP1 induces significant levels of expression of a FUS1-lacZ gene and an increase in mating efficiencies in a ste3, pheromone receptor-deficient yeast mutant . This phenotype is similar to the srm1, a mutant isolated as a suppressor that restores mating to receptorless mutants . These findings indicate that RanBP1 negatively regulates RCC1.

Nucleic Acids Res, 1995 Jun 25, 23(12), 2277 - 86
Functional domains of the heavy metal-responsive transcription regulator MTF-1; Radtke F et al.; Metallothioneins (MTs) constitute a class of low molecular weight, cysteine-rich, metal binding proteins which are regulated at the level of gene transcription in response to heavy metals and other adverse treatments . We have previously cloned a zinc finger factor (MTF-1) that binds specifically to heavy metal-responsive DNA sequence elements in metallothionein promoters and shown that this factor is essential for basal and heavy metal-induced transcription . Here we report that the C-terminal part of MTF-1 downstream of the DNA binding zinc fingers harbours three different transactivation domains, namely an acidic domain, a proline-rich domain and a domain rich in serine and threonine . When fused to the heterologous DNA binding domain of the yeast factor GAL4 these activation domains function constitutively, i.e . transcription of a GAL4-driven reporter gene is not induced by heavy metals . In search of the region(s) responsible for metal induction, external and internal deletion mutations of mouse and human MTF-1 and chimeric variants thereof were tested with a reporter gene driven by a metal-responsive promoter . The N-terminal part of MTF-1 containing the zinc fingers, which are dependent on zinc for efficient DNA binding, can indeed confer a limited (3- to 4-fold) zinc-responsive transcription when fused to the heterologous activation domain of the viral VP16 protein . Another region containing the acidic and proline-rich activation domains also contributes to metal inducibility, but only in the context of intact MTF-1 . This indicates that the activity of MTF-1 results from a complex interplay of different functional domains.

J Biol Chem, 1995 Jun 23, 270(25), 15277 - 84
A constitutive heat shock element-binding factor is immunologically identical to the Ku autoantigen; Kim D et al.; Analysis of the heat shock element (HSE)-binding proteins in extracts of rodent cells, during heat shock and their post-heat shock recovery, indicates that the regulation of heat shock response involves a constitutive HSE-binding factor (CHBF), in addition to the heat-inducible heat shock factor HSF1 . We purified the CHBF to apparent homogeneity from HeLa cells using column chromatographic techniques including an HSE oligonucleotide affinity column . The purified CHBF consists of two polypeptides with apparent molecular masses of 70 and 86 kDa . Immunoblot and gel mobility shift analysis verify that CHBF is identical or closely related to the Ku autoantigen . The DNA binding characteristics of CHBF to double-stranded or single-stranded DNA are similar to that of Ku autoantigen . In gel mobility shift analysis using purified CHBF and recombinant human HSF1, CHBF competes with HSF1 for the binding of DNA sequences containing HSEs in vitro . Furthermore, when Rat-1 cells were co-transfected with human Ku expression vectors and the hsp70-promoter-driven luciferase reporter gene, thermal induction of luciferase is significantly suppressed relative to cells transfected with only the hsp70-luciferase construct . These data suggest a role of CHBF (or Ku protein) in the regulation of heat response in vivo.

J Biol Chem, 1995 Jun 23, 270(25), 15130 - 6
Transactivation of LAP/NF-IL6 is mediated by an acidic domain in the N-terminal part of the protein; Trautwein C et al.; LAP/NF-IL6 is a member of the C/EBP family of transcriptional activators and has been shown to be involved in the regulation of the acute-phase response . We have previously shown that phosphorylation of the liver-enriched transcriptional activator protein (LAP) Ser-105 enhances the activation of LAP-dependent genes . To identify the region which is important for gene activation, a series of LAP mutants were constructed, and domain swapping experiments with the DNA-binding domain of GAL4 were performed . These experiments point to an acidic region located between amino acids 21 and 105 of LAP/NF-IL6 which activates genes independent of the DNA-binding domain and the leucine zipper of LAP/NF-IL6 . Computer-assisted predictions reveal two regions, a helical and a hydrophobic region in the transactivation domain, which could be important in mediating the direct interaction with the basal machinery . Site-directed mutagenesis of acidic residues in both regions demonstrates that the hydrophobic region located between amino acids 85 and 95 is the likely motif for the interaction with the basal machinery . Our results demonstrate that a hydrophobic region in the acidic transactivation domain of LAP/NF-IL6 seems to be relevant in mediating gene activation of LAP-dependent genes.

J Biol Chem, 1995 Jun 23, 270(25), 14998 - 5004
Topoisomerase II binds to ellipticine in the absence or presence of DNA . Characterization of enzyme-drug interactions by fluorescence spectroscopy; Froelich-Ammon SJ et al.; Although a number of drugs currently in use for the treatment of human cancers act by stimulating topoisomerase II-mediated DNA breakage, little is known regarding interactions between these agents and the enzyme . To further define the mechanism of drug action, interactions between ellipticine (an intercalative drug with clinical relevance) and yeast topoisomerase II were characterized . By utilizing a yeast genetic system, topoisomerase II was identified as the primary cellular target of the drug . Furthermore, ellipticine did not inhibit enzyme-mediated DNA religation, suggesting that it stimulates DNA breakage by enhancing the forward rate of cleavage . Finally, ellipticine binding to DNA, topoisomerase II, and the enzyme-DNA complex was assessed by steady-state and frequency domain fluorescence spectroscopy . As determined by changes in fluorescence intensity and emission maximum wavelength, and by lifetime analysis, only the protonated species of ellipticine bound to a double-stranded 40-mer oligonucleotide containing a topoisomerase II cleavage site (KD approximately 65 nM) . In contrast, predominantly deprotonated ellipticine bound to the enzyme.DNA complex (KD approximately 1.5 microM) or to the enzyme in the absence of nucleic acids (KD approximately 160 nM) . These findings suggest that ellipticine interacts directly with topoisomerase II and that the enzyme dictates the ionic state of the drug in the ternary complex . A model is presented in which the topoisomerase II.ellipticine.DNA complex is formed via initial drug binding to either the enzyme or DNA.

J Biol Chem, 1995 Jun 23, 270(25), 14875 - 83
Isolation and characterization of human casein kinase I epsilon (CKI), a novel member of the CKI gene family; Fish KJ et al.; The casein kinase I (CKI) gene family is a rapidly enlarging group whose members have been implicated in the control of cytoplasmic and nuclear processes, including DNA replication and repair . We report here the cloning and characterization of a novel isoform of CKI from a human placental cDNA library . The cDNA for this isoform, hCKI epsilon, predicts a basic polypeptide of 416 amino acids and a molecular mass of 47.3 kDa . It encodes a core kinase domain of 285 amino acids and a carboxyl-terminal tail of 123 amino acids . The kinase domain is 53-98% identical to the kinase domains of other CKI family members and is most closely related to the delta isoform . Localization of the hCKI epsilon gene to chromosome 22q12-13 and the hCKI delta gene to chromosome 17q25 confirms that these are distinct genes in the CKI family . Northern blot analysis shows that hCKI epsilon is expressed in multiple human cell lines . Recombinant hCKI epsilon is an active enzyme that phosphorylates known CKI substrates including a CKI-specific peptide substrate and is inhibited by CKI-7, a CKI-specific inhibitor . A budding yeast isoform of CKI, HRR25, has been implicated in DNA repair responses . Expression of hCKI epsilon but not hCKI alpha rescued the slow-growth phenotype of a Saccharomyces cerevisiae strain with a deletion of HRR25 . Human CKI epsilon is a novel CKI isoform with properties that overlap those of previously described CKI isoforms.

J Biotechnol, 1995 Jun 21, 40(3), 155 - 62
Construction and heterologous expression of a synthetic copy of the cutinase cDNA from Fusarium solani pisi; van Gemeren IA et al.; A copy of the cutinase cDNA from Fusarium solani pisi was constructed starting from synthetic oligonucleotides . For this construction three separate cassettes were made, which were subsequently assembled to form the cutinase gene . Heterologous expression of the synthetic cutinase gene and the subsequent secretion of the recombinant enzyme was achieved in Saccharomyces cerevisiae and Aspergillus awamori.

Biochemistry, 1995 Jun 20, 34(24), 7941 - 8
Eukaryotic acidic phosphoproteins interact with the ribosome through their amino-terminal domain; Jose MP et al.; Variable-size fragments of the four yeast acidic ribosomal protein genes rpYP1 alpha, rpYP1 beta, rpYP2 alpha and rpYP2 beta were fused to the LacZ gene in the vector series YEp356-358 . The constructs were used to transform wild-type Saccharomyces cerevisiae and several gene-disrupted strains lacking different acidic ribosomal protein genes . The distribution of the chimeric proteins between the cytoplasm and the ribosomes, tested as beta-galactosidase activity, was estimated . Hybrid proteins containing around a minimum of 65-75 amino acids from their amino-terminal domain are able to bind to the ribosomes in the presence of the complete native proteins . Hybrid proteins containing no more than 36 amino terminal amino acids bind to the ribosomes in the absence of a competing native protein . The fused YP1-beta-galactosidase proteins are also able to form a complex with the native YP2 type proteins, promoting their binding to the ribosome . The stability of the hybrid polypeptides seems to be inversely proportional to the size of their P protein fragment . These results indicate that only the amino-terminal domain of the eukaryotic P proteins is needed for the P1-P2 complex formation required for interaction with the ribosome . The highly conserved P protein carboxyl end is not implicated in the binding to the particles and is exposed to the medium.

Proc Natl Acad Sci U S A, 1995 Jun 20, 92(13), 6157 - 60
Activation of mammalian retinoid X receptors by the insect growth regulator methoprene; Harmon MA et al.; We report that methoprene and its derivatives can stimulate gene transcription in vertebrates by acting through the retinoic acid-responsive transcription factors, the retinoid X receptors (RXRs) . Methoprene is an insect growth regulator in domestic and agricultural use as a pesticide . At least one metabolite of methoprene, methoprene acid, directly binds to RXR and is a transcriptional activator in both insect and mammalian cells . Unlike the endogenous RXR ligand, 9-cis-retinoic acid, this activity is RXR-specific; the methoprene derivatives do not activate the retinoic acid receptor pathway . Methoprene is a juvenile hormone analog that acts to retain juvenile characteristics during insect growth, preventing metamorphosis into an adult, and it has been shown to have ovicidal properties in some insects . Thus, a pesticide that mimics the action of juvenile hormone in insects can also activate a mammalian retinoid-responsive pathway . This finding provides a basis through which the potential bioactivity of substances exposed to the environment may be reexamined and points the way for discovery of new receptor ligands in both insects and vertebrates.

Proc Natl Acad Sci U S A, 1995 Jun 20, 92(13), 6047 - 51
Protease footprinting reveals a surface on transcription factor TFIIB that serves as an interface for activators and coactivators; Hori R et al.; Transcriptional stimulation by the model activator GAL4-VP16 (a chimeric protein consisting of the DNA-binding domain of the yeast activator GAL4 and the acidic activation domain of the herpes simplex virus protein VP16) involves a series of poorly understood protein-protein interactions between the VP16 activation domain and components of the RNA polymerase II general transcription machinery . One of these interactions is the VP16-mediated binding and recruitment of transcription factor TFIIB . However, TATA box-binding protein (TBP)-associated factors (TAFs), or coactivators, are required for this interaction to culminate in productive transcription complex assembly, and one such TAF, Drosophila TAF40, reportedly forms a ternary complex with VP16 and TFIIB . Due to TFIIB's central role in gene activation, we sought to directly visualize the surfaces of this protein that mediate formation of the ternary complex . We developed an approach called protease footprinting in which the broad-specificity proteases chymotrypsin and alkaline protease were used to probe binding of 32P-end-labeled TFIIB to GAL4-VP16 or TAF40 . Analysis of the cleavage products revealed two regions of TFIIB protected by VP16 from protease attack, one of which overlapped with a region protected by TAF40 . The close proximity of the VP16 and TAF40 binding sites on the surface of TFIIB suggests that this region could act as a regulatory interface mediating the effects of activators and coactivators on transcription complex assembly.

J Mol Biol, 1995 Jun 16, 249(4), 693 - 9
A single amino acid change in a pathway-specific transcription factor results in differing degrees of constitutivity, hyperinducibility and derepression of several structural genes; Oestreicher N et al.; unYc462 is a gain-of-function mutation in the purine catabolism positive regulatory gene of Aspergillus nidulans . This allele leads to a constitutive, hyperinducible and derepressed expression of a least three genes controlled by uaY, and this occurs at different levels depending on the target gene . The uaYc462 allele was mapped physically in relation to known loss-of-function alleles and sequenced . uaYc462 is a one-base change in codon 222, resulting in a serine to leucine change . We propose that this mutation maps in a functional domain involved, directly or indirectly, in the interaction of UaY with other components of the transcriptional apparatus . A sequence similar to the motif surrounding serine 222 may play similar roles in the PPR1 and ADR1 proteins of Saccharomyces cerevisiae.

Eur J Biochem, 1995 Jun 15, 230(3), 926 - 933
Detection and partial purification of a cruciform-resolving activity (X-solvase) from nuclear extracts of mouse B-cells; Solaro P et al.; We have identified a cruciform-resolving enzyme (X-solvase) in nuclear extracts from mouse B-cells, called EMX1, by using an exonuclease-resistant cruciform DNA as a substrate . The cruciform was a 104-nt oligonucleotide that spontaneously adopted a branched conformation with four arms, each arm protected by a terminal loop of five T residues . A ligatable nick was left in one arm . After ligation, the covalently closed substrate was used to follow an 1800-fold purification of the mouse X-solvase (EMX1) from crude nuclear extracts by chromatography on DEAE-cellulose, MonoQ and heparin-Sepharose . The purest fractions containing EMX1 show high specificity for cruciform DNA . The cleavage pattern is indistinguishable from that found in the same substrates after treatment with endonuclease VII from phage T4 or endonuclease X3 from the yeast Saccharomyces cerevisiae . EMX1 and yeast endonuclease X3 were also found to be sensitive to anti-(endonuclease VII) antibodies which inhibited their reactions with cruciform DNAs in vitro.

EMBO J, 1995 Jun 15, 14(12), 2772 - 83
Targets of immunophilin-immunosuppressant complexes are distinct highly conserved regions of calcineurin A; Cardenas ME et al.; The immunosuppressive complexes cyclophilin A-cyclosporin A (CsA) and FKBP12-FK506 inhibit calcineurin, a heterodimeric Ca(2+)-calmodulin-dependent protein phosphatase that regulates signal transduction . We have characterized CsA- or FK506-resistant mutants isolated from a CsA-FK506-sensitive Saccharomyces cerevisiae strain . Three mutations that confer dominant CsA resistance are single amino acid substitutions (T350K, T350R, Y377F) in the calcineurin A catalytic subunit CMP1 . One mutation that confers dominant FK506 resistance alters a single residue (W430C) in the calcineurin A catalytic subunit CMP2 . In vitro and in vivo, the CsA-resistant calcineurin mutants bind FKBP12-FK506 but have reduced affinity for cyclophilin A-CsA . When introduced into the CMP1 subunit, the FK506 resistance mutation (W388C) blocks binding by FKBP12-FK506, but not by cyclophilin A-CsA . Co-expression of CsA-resistant and FK506-resistant calcineurin A subunits confers resistance to CsA and to FK506 but not to CsA plus FK506 . Double mutant calcineurin A subunits (Y377F, W388C CMP1 and Y419F, W430C CMP2) confer resistance to CsA, to FK506 and to CsA plus FK506 . These studies identify cyclophilin A-CsA and FKBP12-FK506 binding targets as distinct, highly conserved regions of calcineurin A that overlap the binding domain for the calcineurin B regulatory subunit.

Yeast, 1995 Jun 15, 11(7), 691 - 6
Localization of the FAR3 gene: genetic mapping and molecular cloning using a chromosome walk-'n'-roll strategy; Horecka J; FAR3 is a newly-discovered yeast gene required specifically for pheromone-mediated cell cycle arrest . I have used strains harboring the far3-1 mutation to map the gene to the right arm of chromosome XIII, establishing the gene order CEN13-LYS7-MCM1-FAR3 . I cloned the FAR3 gene based on its genetic map position using a strategy that combined chromosome walking and a related technique termed 'chromosome rolling' . In addition to the genetic and physical localization of FAR3, I present data that suggest corrections to the tentative map positions of VAN1 and ARG80.

Experientia, 1995 Jun 14, 51(6), 569 - 71
Covalent immobilization as a stimulus of cell wall composition changes; Jirku V; Covalent immobilization of yeast cells by an activated diamine spacer is accompanied by increased levels of cell wall proteins, lipids, amino sugars, amino acids and acid phosphatase leakage, and by altered composition of mannoproteins . The observed changes in cell wall composition are attributed to the effect of cell-solid surface contact.

Mol Gen Genet, 1995 Jun 10, 247(5), 571 - 8
Transcriptional interference caused by GCN4 overexpression reveals multiple interactions mediating transcriptional activation; Tavernarakis N et al.; Overproduction of Gcn4p in yeast cells resulted in the inhibition of transcription from promoters controlled by the GAL4 or dA:dT elements . We have demonstrated that this effect is mediated through the activation domain of Gcn4p and that the function of the transcriptional activator at the affected promoter is impaired . The inhibitory effect of Gcn4p and that the function of the transcriptional activator at the affected promoter is impaired . The inhibitory effect of Gcn4p on these promoters persisted in yeast strains disrupted for the ADA2 and/or GCN5 genes, whose products are required for only part of the transcriptional activation capacity of Gcn4p and other activators, but was alleviated by overexpression of gamma TFIIB . These results support the hypothesis that general transcription factors become unavailable at certain promoters when an activator is overexpressed and strongly imply the existence of an Ada2p/Gcn5p-independent pathway of communication between acidic activators and the basic transcription machinery . In a genetic screen, we have isolated a mutation which neutralises the squelching effects of Gcn4p . This AFR1-1 (activation function reduced) mutation is dominant, it affects the transcriptional activation properties of a number of activators and results in lethality when combined with a gcn5 disruption . Our results suggest that the AFR1 gene product is involved in the mediation of transcriptional activation.

J Mol Biol, 1995 Jun 9, 249(3), 535 - 44
The KIN28 gene is required both for RNA polymerase II mediated transcription and phosphorylation of the Rpb1p CTD; Valay JG et al.; Kin28p, associated with cyclin Ccl1p, is a putative cyclin-dependent kinase (CDK) of the p34cdc2 family in Saccharomyces cerevisiae . Search for mutations co-lethal (syn mutations) with a kin28 thermosensitive mutation (kin28-ts3) has uncovered genetic interactions between gene KIN28 and genes RAD3, SIN4, STI1 and CDC37 . The genetic interaction between KIN28 and the CDC37 cell division cycle gene suggests that a connection exists between the activity of CDK-Kin28p and cell-cycle progression . Both RAD3 and SIN4 gene products are implicated in the RNA polymerase II transcription process . Here we show that RNA polymerase II transcription is drastically reduced in a kin28-ts mutant, at restrictive temperature . This impairment correlates with a markedly decreased phosphorylation of the C-terminal domain (CTD) of the largest subunit of RNA polymerase II (Rpb1p) . Thus, the Kin28 gene product is required in vivo for RNA polymerase II phosphorylation and transcriptional activity as recently suggested by experiments using an in vitro reconstituted system.

Biochem Biophys Res Commun, 1995 Jun 6, 211(1), 92 - 9
Involvement of the peptide sensitive channel in the translocation of basic peptides into mitochondria; Juin P et al.; The Peptide Sensitive Channel (PSC), a cationic channel of the mitochondrial outer membrane, is blocked by several highly basic peptides . Among these peptides, the most active are pCOX IV (1-12)Y, a mitochondrial addressing peptide and dynorphin B (1-13), a peptide unrelated to mitochondrial physiology . The voltage-dependent characteristics of the block duration of the PSC induced by these peptides and the fact that these peptides are imported into mitochondria in an in vitro assay suggest the involvement of the PSC in peptide translocation into mitochondria . We have analyzed the interaction of Mast Cell Degranulating peptide (MCD), a disulfide rich basic peptide, with yeast and mammalian mitochondria . Electrophysiological experiments with native and reduced forms of this peptide (nMCD and rMCD) showed an interaction of both forms with the yeast PSC . On the other hand, only rMCD blocked the electrical activity of the bovine adrenal cortex PSC . Similarly, although both forms inhibited the import of dynorphin B (1-13) into yeast mitochondria, only rMCD inhibited this import in bovine mitochondria . The correlation between electrophysiological and biochemical data strongly suggest that dynorphin B is translocated across the outer membrane at the level of the PSC.

Am J Med Genet, 1995 Jun 5, 57(2), 304 - 6
Erythrocyte membrane reacylation in juvenile neuronal ceroid-lipofuscinosis: measurement of membrane-bound carnitine palmitoyl transferase, acyl-CoA synthetase, and lysophospholipid: acyl-CoA acyltransferase activities; Bennett MJ et al.; In order to study the biochemical mechanisms responsible for the membrane fatty acid deficiency in juvenile neuronal ceroid-lipofuscinosis, we have analyzed the reacylation pathway in isolated erythrocyte membranes in 5 patients . We studied membrane carnitine palmitoyl transferase, and developed a combined assay to study acyl-CoA synthetase and lysophospholipid acyl-CoA acyltransferase activities . There were no significant differences between control and patient membranes, suggesting that abnormalities in these 3 putative candidate enzymes are not responsible for the disease.

J Biol Chem, 1995 Jun 2, 270(22), 13017 - 21
Interactions between the subunits of casein kinase II; Gietz RD et al.; Casein kinase II (CKII) is a protein serine/threonine kinase known to control the activity of a variety of regulatory nuclear proteins . This enzyme has a tetrameric structure composed of two catalytic (alpha and/or alpha ') subunits and two beta subunits . We have examined the subunit composition of tetrameric complexes of purified bovine CKII by immunoprecipitation using alpha, alpha ', or beta subunit-specific antibodies . These experiments indicate that the enzyme can exist as homotetramers (i.e., alpha 2 beta 2 or alpha 2' beta 2) as well as heterotetramers (i.e . alpha alpha ' beta 2) . To further examine subunit interactions between the alpha, alpha ', or beta subunits of CKII, we have utilized the yeast two-hybrid system (Fields, S . and Song, O . (1989) Nature 340: 245-246) . For these studies, each subunit of human CKII was expressed in yeast as a fusion with the DNA binding domain or with the transcriptional activation domain of the yeast GAL4 transcriptional activator . These studies demonstrate that the alpha or alpha ' subunits of CKII can interact with the beta subunits of CKII, but not with other alpha or alpha ' subunits . By comparison, the beta subunits of CKII can interact with alpha, alpha ', or beta subunits . These results indicate that the CKII holoenzyme forms because of the ability of beta subunits to dimerize, bringing two heterodimers (alpha beta or alpha ' beta) into a tetrameric complex.

Science, 1995 Jun 2, 268(5215), 1362 - 5
Role of the protein chaperone YDJ1 in establishing Hsp90-mediated signal transduction pathways; Kimura Y et al.; The substrate-specific protein chaperone Hsp90 (heat shock protein 90) from Saccharomyces cerevisiae functions in diverse signal transduction pathways . A mutation in YDJ1, a member of the DnaJ chaperone family, was recovered in a synthetic-lethal screen with Hsp90 mutants . In an otherwise wild-type background, the ydj1 mutation exerted strong and specific effects on three Hsp90 substrates, derepressing two (the estrogen and glucocorticoid receptors) and reducing the function of the third (the tyrosine kinase p60v-src) . Analysis of one of these substrates, the glucocorticoid receptor, indicated that Ydj1 exerts its effects through physical interaction with Hsp90 substrates.

J Bioenerg Biomembr, 1995 Jun, 27(3), 311 - 30
Experimental and theoretical analysis of the interaction between cytochrome c and cytochrome b5; Mauk AG et al.; Experimental and theoretical investigation of the interaction of cytochrome c and cytochrome b5 performed over nearly twenty years has produced considerable insight into the manner in which these proteins recognize and bind to each other . The results of these studies and the experimental and theoretical strategies that have been developed to achieve these results have significant implications for understanding the behavior of similar complexes formed by more complex and less-well characterized electron transfer proteins . The current review provides a comprehensive summary and critical evaluation of the literature on which the current status of our understanding of the interaction of cytochrome c and cytochrome b5 is based . The general issues related to the study of electron transfer complexes of this type are discussed and some new directions for future investigation of such systems are considered.

Curr Genet, 1995 Jun, 28(1), 60 - 6
A point mutation in the 5'-untranslated leader that affects translational activation of the mitochondrial COX3 mRNA; Costanzo MC et al.; The 613-base 5'-untranslated leader (5'-UTL) of the Saccharomyces cerevisiae mitochondrial COX3 mRNA contains the target of an mRNA-specific translational activator complex composed of at least three nuclearly encoded proteins . We have genetically mapped a collection of cox3 point mutations, using a set of defined COX3 deletions, and found one to be located in the region coding the 5'-UTL . The strain carrying this allele was specifically defective in translation of the COX3 mRNA . Nucleotide-sequence analysis showed that the allele was in fact a double mutation comprised of a single-base insertion in the 5'-UTL (T inserted between bases -428 and -427 with respect to the start of translation) and a G to A substitution at +3 that changed the ATG initiation codon to ATA . Both mutations were required to block translation completely . The effects of the ATG to ATA mutation alone (cox3-1) had previously been analyzed in this laboratory: it reduces, but does not eliminate, translation, causing a slow respiratory growth phenotype . The T insertion in the 5'-UTL had no detectable respiratory growth phenotype as a single mutation . However, the 5'-UTL insertion mutation enhanced the respiratory defective phenotype of missense mutations in pet54, one of the COX3-specific translational-activator genes . This phenotypic enhancement suggests that the -400 region of the 5'-UTL, where the mutation is located, is important for Pet54p-COX3 mRNA interaction.

Curr Genet, 1995 Jun, 28(1), 19 - 25
The evolutionary relationships between homologs of ribosomal YL8 protein and YL8-like proteins; Mizuta K et al.; We previously reported the sequence of YL8A, one of the two genes encoding yeast ribosomal protein YL8 . With the aim of conducting an evolutionary study we have cloned and sequenced a second gene, YL8B . The disruption of both genes is lethal . Unlike other duplicated ribosomal protein genes, each open reading frame is interrupted by two introns containing long conserved sequences . A comparison of nucleotide and amino-acid sequences reveals that the duplication of the YL8 gene must have occurred very recently . Alignment and phylogenetic analysis of the amino-acid sequences of YL8-related proteins from various species show the existence not only of YL8 ribosomal proteins but also of a family of YL8-like proteins . These are present in at least three species of yeast and seem to be functionally distinct from ribosomal proteins.

Protein Eng, 1995 Jun, 8(6), 575 - 82
Identification and elimination by site-directed mutagenesis of thermolabile aspartyl bonds in Aspergillus awamori glucoamylase; Chen HM et al.; Both native Aspergillus niger glucoamylase and wild-type Aspergillus awamori glucoamylase expressed in Saccharomyces cerevisiae, which have identical primary structures, undergo hydrolysis at aspartyl bonds at low pH values and elevated temperatures . In native A.niger enzyme the Asp126-Gly127 bond was preferentially cleaved at pH 3.5, while at pH 4.5 cleavage of the Asp257-Pro258 and Asp293-Gly294 bonds was dominant . In wild-type A.awamori glucoamylase, cleavage of the latter was dominant at both pH 3.5 and 4.5 . Site-directed mutations Asp126-->Glu and Gly127-->Ala in wild-type enzyme decreased specific activities by approximately 60 and 30%, respectively, and increased irreversible thermoinactivation rates 3- to 4-fold at pH 4.5 . Replacement of Asp257 with Glu and Asp293 with Glu or Gln decreased specific activities by approximately 20%, but greatly reduced cleavage of the Asp257-Pro258 and Asp293-Gly294 bonds . The Asp257-->Glu mutant was produced very slowly and was more thermostable than wild-type glucoamylase at pH 4.5 up to 70 degrees C . Replacement of Asp293 with either Glu or Gln significantly raised protein production and slightly increased thermostability at pH 3.5 and 4.5, but not at pH 5.5.

Appl Environ Microbiol, 1995 Jun, 61(6), 2341 - 5
A serine (threonine) protein kinase confers fungicide resistance in the phytopathogenic fungus Ustilago maydis; Orth AB et al.; A mutant of Ustilago maydis (VR43) with single-gene resistance to the dicarboximide fungicide vinclozolin was previously isolated and characterized . A genomic library was constructed, and an 8.7-kb resistance-conferring fragment was isolated by sib selection . Sequencing this fragment, we identified an 1,218-bp open reading frame, which, if disrupted by deletion, no longer confers resistance . Analyses of the data in GenBank demonstrated a high degree of homology between the product of the 1,218-bp open reading frame, referred to as the adr-1 gene, and Ser (Thr) protein kinases.

J Cell Biol, 1995 Jun, 129(6), 1473 - 89
The overgrown hematopoietic organs-31 tumor suppressor gene of Drosophila encodes an Importin-like protein accumulating in the nucleus at the onset of mitosis; Torok I et al.; The tumor suppressor gene overgrown hematopoietic organs-31 (oho31) of Drosophila encodes a protein with extensive homology to the Importin protein of Xenopus (50% identity), the related yeast SRP1 protein, and the mammalian hSRP1 and RCH1 proteins . A strong reduction in the expression of oho31 by a P element inserted in the 5' untranslated region of the oho31 transcript or a complete inactivation of oho31 by imprecise P element excision leads to malignant development of the hematopoietic organs and the genital disc, as shown by their growth autonomy in transplantation assays . We have cloned the oho31 gene of Drosophila melanogaster and determined its nucleotide sequence . The gene encodes a phosphoprotein of 522 amino acids made of three domains: a central hydrophobic domain of eight repeats of 42-44 amino acids each, displaying similarity to the arm motif found in junctional and nucleopore complex proteins, and flanked by two hydrophilic NH2- and COOH-terminal domains . Immunostaining revealed that the OHO31 protein is supplied maternally and rapidly degraded during the first 13 nuclear divisions . Thereafter, the OHO31 protein is predominantly expressed, albeit at reduced levels, in proliferating tissues . During the interphase of early embryonic cell cycles, the OHO31 protein is present in the cytoplasm and massively accumulates in the nucleus at the onset of mitosis in late interphase and prophase . The nuclear import of OHO31 is, however, less pronounced during later developmental stages . These results suggest that, similar to Importin, OHO31 may act as a cytosolic factor in nuclear transport . Moreover, the cell cycle-dependent accumulation of OHO31 in the nucleus indicates that this protein may be required for critical nuclear reactions occurring at the onset of mitosis.

J Cell Biol, 1995 Jun, 129(6), 1433 - 45
BM28, a human member of the MCM2-3-5 family, is displaced from chromatin during DNA replication; Todorov IT et al.; We have recently cloned and characterized a human member (BM28) of the MCM2-3-5 family of putative relication factors (Todorov, I.T., R . Pepperkok, R.N . Philipova, S . Kearsey, W . Ansorge, and D . Werner . 1994 . J . Cell Sci . 107:253-265) . While this protein is located in the nucleus throughout interphase, we report here a dramatic alteration in its nuclear binding during the cell cycle . BM28 is retained in the nucleus after Triton X-100 extraction in G1 and early S phase cells, but is progressively lost as S phase proceeds, and little BM28 is retained in detergent-extracted G2 nuclei . BM28 that is resistant to extraction in G1 nuclei is removed by DNase I digestion, suggesting that the protein is chromatin associated . In addition, we present evidence for variations in the electrophoretic mobility of BM28 that may reflect posttranslational modifications of BM28 during the cell cycle . During mitosis, BM28 is present as a fast-migrating form, but on entry into G1, the protein is converted into a slow-migrating form . With the onset of S phase, the slow-migrating form is progressively converted into the fast form . BM28 is phosphorylated at all stages of the cell cycle, but during interphase the fast form is hyperphosphorylated compared with the slow form . These apparent changes in modification may reflect or effect changes in the nuclear binding of BM28 . The behavior of BM28 is not dissimilar to related proteins in Saccharomyces cerevisiae, such as Mcm2p, which are excluded from the nucleus after DNA replication . We speculate that BM28 may be involved in the control that limits eukaryotic DNA replication to one round per cell cycle.

EMBO J, 1995 Jun 1, 14(11), 2602 - 12
Extensive interactions of PRP8 protein with the 5' and 3' splice sites during splicing suggest a role in stabilization of exon alignment by U5 snRNA; Teigelkamp S et al.; Precursor RNAs containing 4-thiouridine at specific sites were used with UV-crosslinking to map the binding sites of the yeast protein splicing factor PRP8 . PRP8 protein interacts with a region of at least eight exon nucleotides at the 5' splice site and a minimum of 13 exon nucleotides and part of the polypyrimidine tract in the 3' splice site region . Crosslinking of PRP8 to mutant and duplicated 3' splice sites indicated that the interaction is not sequence specific, nor does it depend on the splice site being functional . Binding of PRP8 to the 5' exon was established before step 1 and to the 3' splice site region after step 1 of splicing . These interactions place PRP8 close to the proposed catalytic core of the spliceosome during both transesterification reactions . To date, this represents the most extensive mapping of the binding site(s) of a splicing factor on the substrate RNA . We propose that the large binding sites of PRP8 stabilize the intrinsically weaker interactions of U5 snRNA with both exons at the splice sites for exon alignment by the U5 snRNP.

EMBO J, 1995 Jun 1, 14(11), 2570 - 9
Poly(dA:dT), a ubiquitous promoter element that stimulates transcription via its intrinsic DNA structure; Iyer V et al.; Many yeast promoters contain homopolymeric dA:dT sequences that affect nucleosome formation in vitro and are required for wild-type levels of transcription in vivo . Here, we show that poly(dA:dT) is a novel promoter element whose function depends on its intrinsic structure, not its interaction with sequence-specific, DNA-binding proteins . First, poly(dA:dT) stimulates Gcn4-activated transcription in a manner that is length dependent and inversely related to intracellular Gcn4 levels . Second, Datin, the only known poly(dA:dT)-binding protein, behaves as a repressor through poly(dA:dT) sequences . Third, poly(dG:dC), a structurally dissimilar homopolymer that also affects nucleosomes, has transcriptional properties virtually identical to those of poly(dA:dT) . Three probes of chromatin structure including HinfI endonuclease cleavage in vivo indicate that poly(dA:dT) increases accessibility of the Gcn4 binding site and adjacent sequences in physiological chromatin . These observations suggest that, by virtue of its intrinsic structure, poly(dA:dT) locally affects nucleosomes and increases the accessibility of transcription factors bound to nearby sequences.

Biochem J, 1995 Jun 1, 308 ( Pt 2), 405 - 9
Cloning and expression of the carboxypeptidase gene from Aspergillus saitoi and determination of the catalytic residues by site-directed mutagenesis; Chiba Y et al.; Carboxypeptidase from Aspergillus saitoi removes acidic, neutral and basic amino acids as well as proline from the C-terminal position at pH 2-5 . cpdS, a cDNA encoding A . saitoi carboxypeptidase, was cloned and expressed . Analysis of the 1816-nucleotide sequence revealed a single open reading frame coding for 523 amino acids . When A . saitoi carboxypeptidase cDNA was expressed in yeast cells, carboxypeptidase activity was detected in the cell extract and was immunostained with a 72 kDa protein with polyclonal anti-(A . saitoi carboxypeptidase) serum . The recombinant enzyme treated with glycopeptidase F migrated with an apparent molecular mass of 60 kDa on SDS/PAGE, which was the same as that of the de-N-glycosylated carboxypeptidase from A . saitoi . Site-directed mutagenesis of the cpdS indicated that Ser-153, Asp-357 and His-436 residues were essential for the enzymic catalysis . It can be concluded that A . saitoi carboxypeptidase has a catalytic triad comprising Asp-His-Ser and is a member of serine carboxypeptidase family (EC 3.4.16.1).

Nature, 1995 Jun 1, 375(6530), 421 - 4
MCM3 complex required for cell cycle regulation of DNA replication in vertebrate cells; Madine MA et al.; An intact nuclear membrane restricts DNA replication to only one round in each cell cycle, apparently by excluding an essential replication-licensing factor throughout interphase . A family of related yeast replication proteins, MCM2, 3 and 5 (also called, after cell-division cycle, CDC46), resemble licensing factor, entering the nucleus only during mitosis . We have cloned a Xenopus homologue of MCM3 (XMCM3) and raised antibodies against expressed protein . Immunodepletion of Xenopus egg extracts removes a complex of MCM2, 3 and 5 homologues and inhibits replication of Xenopus sperm nuclei or permeable G2 HeLa nuclei . However, G1 HeLa nuclei still replicate efficiently . Mock-depleted extracts replicate all three templates . XMCM3 accumulates in nuclei before replication but anti-XMCM3 staining decreases during replication . These results can explain why replicated nuclei are unable to reinitiate replication in a single cell cycle.

Mol Cell Biol, 1995 Jun, 15(6), 3442 - 9
Synergistic activation of ADH2 expression is sensitive to upstream activation sequence 2 (UAS2) orientation, copy number and UAS1-UAS2 helical phasing; Donoviel MS et al.; The alcohol dehydrogenase 2 (ADH2) gene of Saccharomyces cerevisiae is under stringent glucose repression . Two cis-acting upstream activation sequences (UAS) that function synergistically in the derepression of ADH2 gene expression have been identified . UAS1 is the binding site for the transcriptional regulator Adr1p . UAS2 has been shown to be important for ADH2 expression and confers glucose-regulated, ADR1-independent activity to a heterologous reporter gene . An analysis of point mutations within UAS2, in the context of the entire ADH2 upstream regulatory region, showed that the specific sequence of UAS2 is important for efficient derepression of ADH2, as would be expected if UAS2 were the binding site for a transcriptional regulatory protein . In the context of the ADH2 upstream regulatory region, including UAS1, working in concert with the ADH2 basal promoter elements, UAS2-dependent gene activation was dependent on orientation, copy number, and helix phase . Multimerization of UAS2, or its presence in reversed orientation, resulted in a decrease in ADH2 expression . In contrast, UAS2-dependent expression of a reporter gene containing the ADH2 basal promoter and coding sequence was enhanced by multimerization of UAS2 and was independent of UAS2 orientation . The reduced expression caused by multimerization of UAS2 in the native promoter was observed only in the presence of ADR1 . Inhibition of UAS2-dependent gene expression by Adr1p was also observed with a UAS2-dependent ADH2 reporter gene . This inhibition increased with ADR1 copy number and required the DNA-binding activity of Adr1p . Specific but low-affinity binding of Adr1p to UAS2 in vitro was demonstrated, suggesting that the inhibition of UAS2-dependent gene expression observed in vivo could be a direct effect due to Adr1p binding to UAS2.

Mol Cell Biol, 1995 Jun, 15(6), 3129 - 37
Cell cycle-regulated transcription of the CLB2 gene is dependent on Mcm1 and a ternary complex factor; Maher M et al.; Clb2 is the major B-type mitotic cyclin required for entry into mitosis in the budding yeast Saccharomyces cerevisiae . We showed that accumulation of CLB2 transcripts in G2 cells is controlled at the transcriptional level and identified a 55-bp upstream activating sequence (UAS) containing an Mcm1 binding site as being necessary and sufficient for cell cycle regulation . Sequences within the cell cycle-regulated UAS were shown to bind Mcm1 in vitro, and mutation which abolished Mcm1-dependent DNA binding activity eliminated cell cycle-regulated transcription in vivo . A second protein with no autonomous DNA binding activity was also recruited into Mcm1-UAS complexes, generating a ternary complex . A point mutation in the CLB2 UAS which blocked ternary complex formation, but still allowed Mcm1 to bind, resulted in loss of cell cycle regulation in vivo, suggesting that the ternary complex factor is also important in control of CLB2 transcription . We discuss the possibility that the CLB2 gene is coregulated with other genes known to be regulated with the same periodicity and suggest that Mcm1 and the ternary complex factor may coordinately regulate several other G2-regulated transcripts.

Mol Cell Biol, 1995 Jun, 15(6), 3119 - 28
Rpa4, a homolog of the 34-kilodalton subunit of the replication protein A complex; Keshav KF et al.; Replication protein A (RPA) is a complex of three polypeptides of 70, 34, and 13 kDa isolated from diverse eukaryotes . The complex is a single-stranded DNA-binding protein essential for simian virus 40-based DNA replication in vitro and for viability in the yeast Saccharomyces cerevisiae . We have identified a new 30-kDa human protein which interacts with the 70- and 13-kDa subunits of RPA, with a yeast two-hybrid/interaction trap method . This protein, Rpa4, has 47% identity with Rpa2, the 34-kDa subunit of RPA . Rpa4 associates with the 70- and 13-kDa subunits to form a trimeric complex capable of binding to single-stranded DNA . Rpa4 is preferentially expressed in placental and colon mucosa tissues . In the placenta, Rpa4 is more abundant than the 70-kDa Rpa1 subunit and is not associated with either Rpa1 or with any other single-stranded DNA-binding protein . In proliferating cells in culture, Rpa4 is considerably less abundant than Rpa1 and Rpa2 . Northern (RNA) blot analysis suggest that there are alternatively processed forms of the RPA4 mRNA, and Southern blot analysis indicates that beside RPA4 there may be other members of the RPA2 gene family.

Mol Cell Biol, 1995 Jun, 15(6), 3082 - 9
E2F-5, a new E2F family member that interacts with p130 in vivo; Hijmans EM et al.; E2F DNA binding sites are found in a number of genes whose expression is tightly regulated during the cell cycle . The activity of E2F transcription factors is regulated by association with specific repressor molecules that can bind and inhibit the E2F transactivation domain . For E2F-1, E2F-2, and E2F-3, the repressor is the product of the retinoblastoma gene, pRb . E2f-4 interacts with pRb-related p107 and not with pRb itself . Recently, a cDNA encoding a third member of the retinoblastoma gene family, p130, was isolated . p130 also interacts with E2F DNA binding activity, primarily in the G0 phase of the cell cycle . We report here the cloning of a fifth member of the E2F gene family . The human E2F-5 cDNA encodes a 346-amino-acid protein with a predicted molecular mass of 38 kDa . E2F-5 is more closely related to E2F-4 (78% similarity) than to E2F-1 (57% similarity) . E2F-5 resembles the other E2Fs in that it binds to a consensus E2F site in a cooperative fashion with DP-1 . By using a specific E2F-5 antiserum, we found that under physiological conditions, E2F-5 interacts preferentially with p130.

Mol Cell Biol, 1995 Jun, 15(6), 3072 - 81
Mammalian DNA polymerase auxiliary proteins: analysis of replication factor C-catalyzed proliferating cell nuclear antigen loading onto circular double-stranded DNA; Podust LM et al.; To understand the mechanism of action of the two eukaryotic replication auxiliary proteins proliferating cell nuclear antigen (PCNA) and replication factor C (RF-C), we constructed a plasmid for producing PCNA which could be 32P labelled in vitro . This allowed us to analyze the assembly of the auxiliary proteins directly on DNA and to examine this process in the absence of DNA synthesis . By using closed circular double-stranded DNA or gapped circular DNA for protein-DNA complex formation, the following results were obtained, (i) RF-C can load PCNA in an ATP-dependent manner directly on double-stranded DNA, and no 3'-OH ends are required for this reaction; (ii) the RF-C-PCNA complex assembled on closed circular DNA differs from those assembled on gapped or nicked circular DNA; (iii) the stable RF-C-PCNA complex can be assembled on circular but not on linear DNA; and (iv) only gapped DNA can partially retain the assembled RF-C-PCNA complex upon the linearization of the template . We propose that RF-C first binds unspecifically to double-stranded DNA in the presence of ATP and then loads PCNA onto DNA to yield a protein complex able to track along DNA . The RF-C-PCNA complex could slide along the template until it encounters a 3'-OH primer-template junction, where it is likely transformed into a competent clamp . The latter complex, finally, might still be able to slide along double-stranded DNA.

J Exp Med, 1995 Jun 1, 181(6), 2049 - 58
Absence of autoantigen Ku in mature human neutrophils and human promyelocytic leukemia line (HL-60) cells and lymphocytes undergoing apoptosis; Ajmani AK et al.; The Ku autoantigen is a heterodimer of 70- and 80-kD proteins recognized by autoantibodies from patients with systemic lupus erythematosus and related diseases that is the DNA-binding component of a DNA-dependent protein kinase . The catalytic activity of DNA-dependent protein kinase is carried by a 350-kD subunit (p350) . In light of the recently described role of Ku in repairing double-strand DNA breaks, we investigated the regulation of Ku and p350 levels in neutrophils, a terminally differentiated cell type destined to undergo apoptosis . Since the appearance of double-strand DNA breaks is characteristic of apoptosis, we were interested in the possibility that Ku might oppose programmed cell death . Analysis of peripheral blood cells by flow cytometry using anti-Ku and anti-p350 monoclonal antibodies revealed that neutrophils were unstained, whereas resting (G0) lymphocytes were positive . The absence of Ku in mature neutrophils was confirmed by Western blotting and enzyme-linked immunosorbent assay for Ku antigen . In contrast, the human promyelocytic leukemia line, HL-60, which undergoes differentiation toward neutrophils after dimethylsulfoxide treatment, was positive for Ku and p350 . In view of the short lifespan of neutrophils and the prolonged half-life of Ku and p350 (> 5 d), these data suggested that Ku was actively degraded during myeloid differentiation . Analysis of HL-60 cells by flow cytometry revealed that Ku staining was bimodal . Cells in G1/G0, S, or G2/M were all stained positively, whereas cells with a subdiploid DNA content characteristic of apoptosis were Ku negative . Similar results were obtained with phytohemagglutin-stimulated human lymphocytes . These data suggest that the Ku antigen is actively degraded in both myeloid cells destined to undergo apoptosis and apoptotic lymphocytes, raising the possibility that degradation of Ku may help to prevent the inappropriate repair of fragmented nuclear DNA during apoptosis.

Nat Struct Biol, 1995 Jun, 2(6), 450 - 7
Deconstruction of GCN4/GCRE into a monomeric peptide-DNA complex; Stanojevic D et al.; Here we describe a system that enables short peptides to bind DNA sequence-specifically . Linking the peptide covalently to DNA through a disulphide bond eliminates the unfavourable energetic cost of diffusion and thus potentiates the peptide-DNA interaction . By this approach we have deconstructed the GCN4/DNA complex into its elemental DNA recognition units . We find that the GCN4 basic region contacts the two half-sites with very different affinities and propose that this thermodynamic asymmetry plays a role in differential regulation of gene expression . Specific binding of the peptide to DNA stabilizes the disulphide bond toward reduction suggesting a novel approach to the discovery of new DNA-binding specificities.

Curr Opin Cell Biol, 1995 Jun, 7(3), 414 - 20
Eukaryotic DNA replication; Wang TA et al.; Recent discoveries suggest that the initiation of eukaryotic DNA replication involves at least two steps--one occurring near the completion of mitosis and the other at the onset of S phase--that bring about the ordered assembly of initiator proteins at the origin . The identification and characterization of components involved in promoting or antagonizing each of these steps has provided a preliminary understanding of how replication initiation is regulated so precisely during the cell cycle.

Curr Opin Cell Biol, 1995 Jun, 7(3), 371 - 5
Interplay between chromatin structure and transcription; Kornberg RD et al.; Research on the interplay between chromatin and transcription has progressed along three lines during the past year . Evidence has been reported for disruption of nucleosomes by transcriptional regulatory proteins in cell-free systems; displacement of the histone octamer during transcription has been conclusively demonstrated; and insights into transcriptional repression by heterochromatin have been gained from studies of silent mating loci and telomeres in yeast.

Curr Opin Cell Biol, 1995 Jun, 7(3), 344 - 51
Comparing transcriptional initiation by RNA polymerases I and III; Geiduschek EP et al.; We comment on the current understanding of transcriptional initiation by RNA polymerases I and III, and look for common modes of operation of these enzymes, emphasizing selected recent developments . These include definitive experiments on the constitution of the human RNA polymerase I transcription factor SL1/TIF-IB, the development of a genetic system for analyzing the function of RNA polymerase I in yeast, the elucidation of the structure of the human snRNA gene transcription factor SNAPc, and initial stages of mapping the protein-protein interactions involved in the assembly of transcriptional initiation complexes.

Curr Opin Cell Biol, 1995 Jun, 7(3), 329 - 36
The SMC family: from chromosome condensation to dosage compensation; Hirano T et al.; Recent genetic analyses in yeasts and biochemical studies in vertebrate cells have led to the discovery of a family of putative ATPases that play a fundamental role in chromosome condensation and segregation in mitosis . One of the members was also found to be involved in dosage compensation in Caenorhabditis elegans, providing a new link between global regulation of gene expression and chromosome structure . This unique family of proteins may control higher-order chromosome dynamics by regulated self-assembly or mechanochemical activity.

Curr Opin Cell Biol, 1995 Jun, 7(3), 319 - 24
The nucleolus: an organelle formed by the act of building a ribosome; Melese T et al.; Recent evidence corroborates the idea that the structure of the nucleolus need not be strictly maintained for proper function, suggesting that the organelle is composed of supramolecular assemblies formed during rRNA synthesis . More controversial is whether the nucleolus exists in the absence of rRNA synthesis and whether it interacts with the nuclear scaffold . The simultaneous and highly integrative nature of building a ribosome is reflected in the numerous observations showing that proteins involved in all aspects of ribosomal biogenesis affect pre-rRNA processing . The identification of several new nucleolar proteins without an obvious role in pre-rRNA metabolism may provide the field with long sought after assembly factors that might be key players in eukaryotic ribosome biogenesis.

J Ind Microbiol, 1995 Jun, 14(6), 523 - 30
Relationships among the genera Ashbya, Eremothecium, Holleya and Nematospora determined from rDNA sequence divergence; Kurtzman CP; Species of the genera Ashbya, Eremothecium, Holleya, and Nematospora were compared from extent of divergence in a 580-nucleotide region near the 5' end of the large subunit (26S) ribosomal DNA gene . The four genera are closely related and comprise a subclade of the hemiascomycetes . Because the taxa show little divergence, it is proposed that all be placed in the genus Eremothecium . The family Eremotheciaceae, fam . nov., is proposed.

J Ind Microbiol, 1995 Jun, 14(6), 456 - 60
The phylogenetic relationships of Eeniella nana Smith, Batenburg-van der Vegte et Scheffers based on the partial sequences of 18S and 26S ribosomal RNAs (Candidaceae); Yamada Y et al.; Two strains of Eeniella nana were examined for their partial base sequences of 18S and 26S rRNAs . In the partial base sequences of 18S rRNA (positions 1451 through 1618, 168 bases) the strains of E . nana have five, five, four and eleven base differences with those of Dekkera bruxellensis (type species), D . anomala (and Brettanomyces anomalus), D . naardenensis and D . custersiana, respectively . In the 26S rRNA partial base sequencings (positions 1611 through 1835, 225 bases and positions 493 through 622, 130 bases) the base differences were 46, 43, 34 and 40 and the percent similarities were 53-54, 51-54, 56-57 and 51-53, respectively . The sequence data obtained are discussed phylogenetically and taxonomically, especially on retention of the generic name Eeniella.

Hum Mol Genet, 1995 Jun, 4(6), 1007 - 14
YAC cloning Mus musculus telomeric DNA: physical, genetic, in situ and STS markers for the distal telomere of chromosome 10; Kipling D et al.; Three Mus musculus DBA/2 YAC libraries were constructed using a half-YAC telomere cloning vector . This functional complementation approach yields libraries which include terminal restriction fragments of the mouse genome . Screening all three libraries led to the isolation of 32 independent clones which carry linear YACs containing the mouse terminal repeat sequence, (TTAGGG)n . These YACs provide a resource to isolate regions of the mouse genome close to chromosome termini and excluded from existing conventional YAC libraries . To demonstrate their utility, a hybridization probe was isolated from Mtel-1, the first (TTAGGG)n-containing YAC isolated . This probe detects a approximately 70 kb Kpnl fragment in the mouse genome which is sensitive to pretreatment with BAL31 exonuclease . A PCR-based genetic marker generated from the sequence of this probe maps 4.4 cM from the most distal anchor locus on chromosome 10 in the EUCIB interspecific backcross . STS primers for this locus, D10Hgu1, were used to isolate YAC 110F4 from a commercially available mouse YAC library . Fluorescence in situ hybridization demonstrates that YAC 110F4 hybridizes to the distal telomere of chromosome 10 . Clones in this collection of telomere YACs therefore partially overlap clones in conventional YAC libraries, and thus the previously unavailable terminal regions of the mouse genome can now be linked with the developing mouse STS YAC contig . Genetic markers such as D10Hgu1 allow the ends of the mouse genetic map to be defined, thus closing the map.

Plant Cell, 1995 Jun, 7(6), 759 - 71
cycMs3, a novel B-type alfalfa cyclin gene, is induced in the G0-to-G1 transition of the cell cycle; Meskiene I et al.; Cyclins are key regulators of the cell cycle in all eukaryotes . We have previously isolated two B-type cyclin genes, cycMs1 and cycMs2, from alfalfa that are primarily expressed during the G2-to-M phase transition and are most likely mitotic cyclin genes . Here, we report the isolation of a novel alfalfa cyclin gene, termed cycMs3 (for cyclin Medicago sativa), by selecting for mating type alpha-pheromone-induced cell cycle arrest suppression in yeast . The central region of the predicted amino acid sequence of the cycMs3 gene is most similar to the cyclin box of yeast B-type and mammalian A- and B-type cyclins . In situ hybridization showed that cycMs3 mRNA can be detected only in proliferating cells and not in differentiated alfalfa cells . When differentiated G0-arrested cells were induced to reenter the cell cycle in the G1 phase and resume cell division by treatment with plant hormones, cycMs3 transcript levels increased long before the onset of DNA synthesis . In contrast, histone H3-1 mRNA and cycMs2 transcripts were not observed before DNA replication and mitosis, respectively . In addition, cycMs3 mRNA was found in all stages of the cell cycle in synchronously dividing cells, whereas the cycMs2 and histone H3-1 genes showed a G2-to-M phase- or S phase-specific transcription pattern, respectively . These data suggest that the role of cyclin CycMs3 differs from that of CycMs1 and CycMs2 . We propose that CycMs3 helps control reentry of quiescent G0-arrested cells into the G1 phase of the cell cycle.

Plant Cell, 1995 Jun, 7(6), 677 - 88
Processing and secretion of a virally encoded antifungal toxin in transgenic tobacco plants: evidence for a Kex2p pathway in plants; Kinal H et al.; Ustilago maydis is a fungal pathogen of maize . Some strains of U . maydis encode secreted polypeptide toxins capable of killing other susceptible strains of U . maydis . We show here that one of these toxins, the KP6 killer toxin, is synthesized by transgenic tobacco plants containing the viral toxin cDNA under the control of a cauliflower mosaic virus promoter . The two components of the KP6 toxin, designated alpha and beta, with activity and specificity identical to those found in toxin secreted by U . maydis cells, were isolated from the intercellular fluid of the transgenic tobacco plants . The beta polypeptide from tobacco was identical in size and N-terminal sequence to the U . maydis KP6 beta polypeptide . Processing of the KP6 preprotoxin in U . maydis requires a subtilisin-like processing protease, Kex2p, which is present in both animal and fungal cells and is required for processing of (among other things) small secreted polypeptide hormones and secreted toxins . Our findings present evidence for Kex2p-like processing activity in plants . The systemic production of this viral killer toxin in crop plants may provide a new method of engineering biological control of fungal pathogens in crop plants.

Trends Genet, 1995 Jun, 11(6), 235 - 41
Genetic approaches to nuclear pore structure and function; Doye V et al.; The major features of nucleocytoplasmic transport through nuclear pore complexes are conserved from yeast to humans . This transport machinery, which includes the 125 MDa nuclear pore complex structure, has been molecularly dissected in the yeast Saccharomyces cerevisiae by genetic approaches . Here, we summarize the genetic analyses used to elucidate structure-function relationships within this large supramolecular assembly.

Free Radic Res, 1995 Jun, 22(6), 519 - 31
Purification, N-terminal amino acid sequence and partial characterization of a Cu,Zn superoxide dismutase from the pathogenic fungus Aspergillus fumigatus; Holdom MD et al.; A superoxide dismutase (SOD) has been purified to homogeneity from the fungal pathogen Aspergillus fumigatus using a combination of cell homogenization, isoelectric focusing and gel filtration FPLC . The N-terminal amino acid sequence of the purified enzyme demonstrated substantial homology to known Cu,Zn superoxide dismutases for a range of organisms, including Neurospora crassa and Saccharomyces cerevisiae . The enzyme subunit has a pI of 5.9, a relative molecular mass of 19 kDa and a spectral absorbance maximum of 550nm . The non reduced enzyme has a relative molecular mass of 95 kDa . The enzyme remained active after prolonged incubation at 70 degrees C and was pH insensitive in the range 7-11 . Potassium cyanide and diethyldithiocarbamate, known Cu,Zn SOD inhibitors, caused inhibition of the purified enzyme at working concentrations of 0.25 mM, whilst sodium azide and o-phenanthroline demonstrated inhibition at higher concentrations (10-30 mM) . SOD activity was also detectable in culture filtrate of A . fumigatus . This enzyme may have a potential role as a virulence factor in the avoidance of neutrophil and phagocyte oxidative burst killing mechanisms.

Trends Biochem Sci, 1995 Jun, 20(6), 231 - 5
On the origin of a silencer; Dillin A et al.; Although there are several compelling pieces of evidence suggesting that transcription can promote DNA replication, there are few studies that indicate a role for DNA replication in controlling transcription . Here, we discuss the role of the HMR-E silencer, an origin of replication that plays a crucial role in the regulation of expression of mating-type genes in the yeast Saccharomyces cerevisiae.

Exp Mycol, 1995 Jun, 19(2), 160 - 2
Molecular cloning and sequence of the cytoplasmic ribosomal protein S15a gene from Agaricus bisporus; Schaap PJ et al.; We have isolated an Agaricus bisporus cDNA which encodes an open reading frame of 130 amino acids . A comparison with the Genbank database shows that the deduced amino acid sequence of this open reading frame is highly homologous to the small subunit ribosomal proteins S15a of Brassica napus and Drosophila melanogaster and to the small subunit ribosomal proteins S24 of Strongylocentrotus purpuratus and Saccharomyces cerevisiae.

FASEB J, 1995 Jun, 9(9), 726 - 35
The MAPK signaling cascade; Seger R et al.; The transmission of extracellular signals into their intracellular targets is mediated by a network of interacting proteins that regulate a large number of cellular processes . Cumulative efforts from many laboratories over the past decade have allowed the elucidation of one such signaling mechanism, which involves activations of several membranal signaling molecules followed by a sequential stimulation of several cytoplasmic protein kinases collectively known as mitogen-activated protein kinase (MAPK) signaling cascade . Up to six tiers in this cascade contribute to the amplification and specificity of the transmitted signals that eventually activate several regulatory molecules in the cytoplasm and in the nucleus to initiate cellular processes such as proliferation, differentiation, and development . Moreover, because many oncogenes have been shown to encode proteins that transmit mitogenic signals upstream of this cascade, the MAPK pathway provides a simple unifying explanation for the mechanism of action of most, if not all, nonnuclear oncogenes . The pattern of MAPK cascade is not restricted to growth factor signaling and it is now known that signaling pathways initiated by phorbol esters, ionophors, heat shock, and ligands for seven transmembrane receptors use distinct MAPK cascades with little or no cross-reactivity between them . In this review we emphasize primarily the first MAPK cascade to be discovered that uses the MEK and ERK isoforms and describe their involvement in different cellular processes.

Bioorg Med Chem, 1995 Jun, 3(6), 777 - 84
Sequence-specific DNA binding by covalently constrained peptide dimers of the basic leucine zipper protein GCN4; Okagami M et al.; DNA binding of covalently bonded peptide dimers was studied by using enantiomeric and C2-symmetric templates as a dimerization module . Amino acid sequence of the peptide is derived from that of DNA contact region of the basic leucine zipper protein GCN4 . These peptide dimers were designed to possess different constraints with respect to the orientation of two peptides . The basic region peptides were covalently linked to the enantiomeric template at the C-terminal ends . Two peptides are arranged either in a right-handed or left-handed geometry depending on the chirality of the template . The GCN4 basic region dimers with both right-handed and left-handed geometries show equal affinity to the native GCN4 binding DNA sequences, 5'-ATGACTCAT-3' and 5'-ATGACGTCAT-3', as revealed by the gel mobility shift assay . Specific recognition of the palindromic DNA sequence by the peptide dimers was confirmed by the DNase I footprinting . Circular dichroism spectroscopic study indicates that the basic region peptides bound the target DNA sequence in a helical conformation . The degree to which a chiral constraint effects may depend on the geometry of two DNA binding domains in the parent protein-DNA complex and on a position to apply the chiral constraint.

Thyroid, 1995 Jun, 5(3), 207 - 11
Screening for PIT1 abnormality by PCR direct sequencing method; Irie Y et al.; PIT1 abnormality is defined as a genetic abnormality in the PIT1 gene, which encodes a pituitary specific transcription factor Pit-1/GHF-1.PIT1 abnormality has been reported in several patients displaying either complete or incomplete deficiency of thyrotropin (TSH), growth hormone (GH), and prolactin (PRL) in either familial or sporadic cases . To see if there are abnormalities in the PIT1 gene in patients with incomplete TSH, GH, and PRL deficiency, we utilized a PCR direct sequencing method to determine the Pit-1/GHF-1 coding sequence . A total of 15 patients, 1 patient from a family with TSH and GH deficiency, 3 patients with TSH, GH, and PRL deficiency, and 11 patients treated with both human GH (hGH) and thyroid hormone were studied . In one patient of combined pituitary hormone deficiency, the Arg-271-Trp mutation was detected . Since both of the parents did not harbor this mutation, it is a de novo germ line mutation . No mutation was detected in the other patients, showing that PIT1 abnormality is not a frequent cause of GH deficiency.

Mol Biol Cell, 1995 Jun, 6(6), 741 - 56
The origin recognition complex in silencing, cell cycle progression, and DNA replication; Loo S et al.; This report describes the isolation of ORC5, the gene encoding the fifth largest subunit of the origin recognition complex, and the properties of mutants with a defective allele of ORC5 . The orc5-1 mutation caused temperature-sensitive growth and, at the restrictive temperature, caused cell cycle arrest . At the permissive temperature, the orc5-1 mutation caused an elevated plasmid loss rate that could be suppressed by additional tandem origins of DNA replication . The sequence of ORC5 revealed a potential ATP binding site, making Orc5p a candidate for a subunit that mediates the ATP-dependent binding of ORC to origins . Genetic interactions among orc2-1 and orc5-1 and other cell cycle genes provided further evidence for a role for the origin recognition complex (ORC) in DNA replication . The silencing defect caused by orc5-1 strengthened previous connections between ORC and silencing, and combined with the phenotypes caused by orc2 mutations, suggested that the complex itself functions in both processes.

Bioessays, 1995 Jun, 17(6), 471 - 80
Cyclin-dependent protein kinases: key regulators of the eukaryotic cell cycle; Nigg EA; Passage through the cell cycle requires the successive activation of different cyclin-dependent protein kinases (CDKs) . These enzymes are controlled by transient associations with cyclin regulatory subunits, binding of inhibitory polypeptides and reversible phosphorylation reactions . To promote progression towards DNA replication, CDK/cyclin complexes phosphorylate proteins required for the activation of genes involved in DNA synthesis, as well as components of the DNA replication machinery . Subsequently, a different set of CDK/cyclin complexes triggers the phosphorylation of numerous proteins to promote the profound structural reorganizations that accompany the entry of cells into mitosis . At present, much research is focused on elucidating the links between CDK/cyclin complexes and signal transduction pathways controlling cell growth, differentiation and death . In future, a better understanding of the cell cycle machinery and its deregulation during oncogenesis may provide novel opportunities for the diagnostic and therapeutic management of cancer and other proliferation-related diseases.

Brain Behav Immun, 1995 Jun, 9(2), 129 - 48
Pyrogens specifically disrupt the acquisition of a task involving cognitive processing in the rat; Aubert A et al.; In addition to changes in body temperature and other metabolic and physiological responses corresponding to immune activation, pyrogens can induce profound behavioral changes referred to collectively as sickness behavior . One feature of sickness behavior, sometimes reported in clinical settings, but rarely exposed to experimental analysis, is depressed cognitive functioning . The present series of five experiments sought to demonstrate the existence of specific cognitive deficits in rats, independently of any confounding performance effects of pyrogen injections . The behavioral task used, called autoshaping, consisted of presenting hungry Wistar rats with a stimulus (introduction of a retractable lever) that predicted food delivery . Control rats quickly learned to press the lever, although this response does not influence the probability of food delivery . When pyrogens (250 micrograms/kg lipopolysaccharide, 4 micrograms/rat interleukin-1 beta, or 300 mg/rat yeast) were injected to rats during acquisition of this task, they severely disrupted acquisition while the pyrogen was active . The same treatments were, however, without effect on performance when injected later, when performance had stabilized . It is argued that these results demonstrate specific, performance-independent effects of pyrogens on the cognitive processes needed for the acquisition of this task . The results are discussed in terms of the relationship between these effects and the cytokines induced in the brain by pyrogens, and in terms of the exact nature of the cognitive process likely to be affected.

Am J Pathol, 1995 Jun, 146(6), 1302 - 8
Proliferation-associated nuclear antigen Ki-S1 is identical with topoisomerase II alpha . Delineation of a carboxy-terminal epitope with peptide antibodies; Boege F et al.; Proliferation-linked expression of the nuclear Ki-S1 antigen is a significant prognostic indicator in mammary carcinomas . Here, we show staining of a protein of 170 kd by Ki-S1 antibody in immunoblots of Saccharomyces cerevisiae expressing human topoisomerase II alpha but not in the parental strain . In HL-60 cells containing both isoforms of human topoisomerase II, Ki-S1 antibody binds selectively to the 170-kd isoenzyme in a similar fashion as peptide-antibodies directed against amino acid residues 1 to 15 or 1512 to 1530 of human topoisomerase II alpha . Conversely, antibodies directed against carboxyl-terminal sequences of human topoisomerase II beta selectively stain a 180-kd protein . The immunoreactive pattern of V8 endoproteinase restriction digests of human topoisomerase II alpha was identical for Ki-S1-antibody and peptide-antibodies directed against residues 1512 to 1530 but different for peptide-antibodies directed against residues 1 to 15 . The Rf values of the smallest fragment commonly recognized by Ki-S1 antibody and the carboxy terminus-specific peptide-antibody place the Ki-S1 epitope within the last 495 carboxyl-terminal amino acid residues of topoisomerase II alpha.

RNA, 1995 Jun, 1(4), 425 - 36
RNA structural patterns and splicing: molecular basis for an RNA-based enhancer; Libri D et al.; Efficient splicing of the 325-nt yeast (Saccharomyces cerevisiae) rp51b intron requires the presence of two short interacting sequences located 200 nt apart . We used the powerful technique of randomization-selection to probe the overall structure of the intron and to investigate its role in pre-mRNA splicing . We identified a number of alternative RNA-RNA interactions in the intron that promote efficient splicing, and we showed that similar base pairings can also improve splicing efficiency in artificially designed introns . Only a very limited amount of structural information is necessary to create or maintain such a mechanism . Our results suggest that the base pairing contributes transiently to the spliceosome assembly process, most likely by complementing interactions between splicing factors . We propose that splicing enhancement by structure represents a general mechanism operating in large yeast introns that evolutionarily preceded the protein-based splicing enhancers of higher eukaryotes.

RNA, 1995 Jun, 1(4), 391 - 406
Branch-point attack in group II introns is a highly reversible transesterification, providing a potential proofreading mechanism for 5'-splice site selection; Chin K et al.; By examining the first step of group II intron splicing in the absence of the second step, we have found that there is an interplay of three distinct reactions at the 5'-splice site: branching, reverse branching, and hydrolytic cleavage . This approach has yielded the first kinetic parameters describing eukaryotic branching and establishes that group II intron catalysis can proceed on a rapid timescale . The efficient reversibility of the first step is due to increased conformational organization in the branched intermediate and it has several important mechanistic implications . Reversibility in the first step requires that the second step of splicing serve as a kinetic trap, thus driving splicing to completion and coordinating the first and second step of splicing . Facile reverse branching also provides the intron with a proofreading mechanism to control the fidelity of 5'-splice site selection and it provides a kinetic basis for the apparent mobility of group II introns.

RNA, 1995 Jun, 1(4), 375 - 90
The final stages of spliceosome maturation require Spp2p that can interact with the DEAH box protein Prp2p and promote step 1 of splicing; Roy J et al.; Pre-mRNA processing occurs by assembly of splicing factors on the substrate to form the spliceosome followed by two consecutive RNA cleavage-ligation reactions . The Prp2 protein hydrolyzes ATP and is required for the first reaction (Yean SL, Lin RJ, 1991, Mol Cell Biol 11:5571-5577; Kim SH, Smith J, Claude A, Lin RJ, 1992, EMBO J 11:2319-2326) . The Saccharomyces cerevisiae SPP2 gene was previously identified as a high-copy suppressor of temperature-sensitive prp2 mutants (Last RL, Maddock JR, Woolford JL Jr, 1987, Genetics 117:619-631) . We have characterized the function of Spp2p in vivo and in vitro . Spp2p is an essential protein required for the first RNA cleavage reaction in vivo . Depletion of Spp2p from yeast cells results in accumulation of unspliced pre-mRNAs . A temperature-sensitive spp2-1 mutant accumulates pre-mRNAs in vivo and is unable to undergo the first splicing reaction in vitro . However, spliceosomal complexes are assembled in extracts prepared from the mutant . We show that Spp2p function is required after spliceosome assembly but prior to the first reaction . Spp2p associates with the spliceosome before the first RNA cleavage reaction and is likely to be released from the spliceosome following ATP hydrolysis by Prp2p . The Prp2 and Spp2 proteins are capable of physically interacting with each other . These results suggest that Spp2p interacts with Prp2p in the spliceosome prior to the first cleavage-ligation reaction . Spp2p is the first protein that has been found to interact with a DEAD/H box splicing factor.

J Mol Biol, 1995 May 26, 249(1), 11 - 28
Addition of a 29 residue carboxyl-terminal tail converts a simple HMG box-containing protein into a transcriptional activator; Dairaghi DJ et al.; Human mitochondrial transcription factor A (h-mtTFA) is essential for initiation of transcription from the two promoters located in the displacement-loop region of human mitochondrial DNA . This 25 kDa protein contains two tandem, HMG box DNA-binding domains separated by a 27 amino acid residue linker region and followed by a 25 residue carboxyl-terminal tail; both the linker and tail are rich in basic amino acid residues . Mutational analysis of h-mtTFA revealed that the tail region is important for specific DNA recognition and essential for transcriptional activation . The critical role of the human tail in transcription was confirmed by constructing chimeric proteins that exchanged similar regions between h-mtTFA and its Saccharomyces cerevisiae homolog, sc-mtTFA . Wild-type sc-mtTFA is unable to activate transcription from the human mitochondrial light-strand promoter (LSP) . Addition of the human tail region to sc-mtTFA conferred LSP-specific promoter activation . In all of the different h-mtTFA mutations tested, transcriptional activation was correlated with specific DNA-binding activity, suggesting that these two functions may be inseparable, a situation entirely consistent with previous mutational analyses of human mitochondrial promoters.

Science, 1995 May 26, 268(5214), 1188 - 90
Requirement for phosphatidylinositol transfer protein in epidermal growth factor signaling; Kauffmann-Zeh A et al.; Stimulation of phosphatidylinositol-4,5-bisphosphate (PIP2) hydrolysis is a widespread mechanism for receptor-mediated signaling in eukaryotes . Cytosolic phosphatidylinositol transfer protein (PITP) is necessary for guanosine triphosphate (GTP)-dependent hydrolysis of PIP2 by phospholipase C-beta (PLC-beta), but the role of PITP is unclear . Stimulation of phospholipase C-gamma (PLC-gamma) in A431 human epidermoid carcinoma cells treated with epidermal growth factor (EGF) required PITP . Stimulation of PI-4 kinase in cells treated with EGF also required PITP . Coprecipitation studies revealed an EGF-dependent association of PITP with the EGF receptor, with PI-4 kinase, and with PLC-gamma.

J Biol Chem, 1995 May 26, 270(21), 12697 - 703
Transcriptional activation by the mouse Ah receptor . Interplay between multiple stimulatory and inhibitory functions; Ma Q et al.; The aromatic hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that mediates cellular responses to the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) . We cloned AhR cDNA from C57BL/6 mouse liver and verified by transfection that it encodes a functional protein . Analyses of deletion mutants indicate that the carboxyl half of AhR contains several types of transactivation domain, which function independently of domains that mediate TCDD recognition, DNA binding, and heterodimerization with the Ah receptor nuclear translocator (Arnt) protein . The transactivation domains function independently of each other, display different levels of activity, and act synergistically when linked . In addition, AhR contains an 82-amino acid domain that inhibits transactivation . The inhibitory domain displays specificity, in that it blocks the transactivating functions of AhR and Arnt, but not that of the herpes simplex protein VP16 . The inhibitory activity depends upon the cell type in which AhR is expressed, implying that a cell-specific protein mediates the effect.

J Biol Chem, 1995 May 26, 270(21), 12485 - 90
Interaction between the nucleocapsid protein and the phosphoprotein of human parainfluenza virus 3 . Mapping of the interacting domains using a two-hybrid system; Zhao H et al.; A two-hybrid system was used to study interaction in vivo between the nucleocapsid protein (NP) and the phosphoprotein (P) of human parainfluenza virus type 3 (HPIV-3) . Two plasmids, one containing the amino terminus of P fused to the DNA-binding domain of the yeast transactivator, GAL4, and the other containing the amino terminus of NP fused to the herpesvirus transactivator, VP16, were transfected in COS-1 cells along with a chloramphenicol acetyltransferase (CAT) reporter plasmid containing GAL4 DNA-binding sites . A specific and high-affinity interaction between NP and P was observed as measured by the activation of the CAT gene . Mapping of the domains in P (603 amino acids) involved in the association with NP revealed that NH2-terminal 40 and COOH-terminal 20 amino acids are important for such association . Interestingly, a stretch of NH2-terminal amino acids as short as 63-403 interacted with NP more than the wild type, reaching greater than 2.5-fold as measured by the CAT assay . These results suggest that a domain is present in P that negatively regulates its interaction with NP . Deletion of NH2-terminal 40 and COOH-terminal 160 amino acids of NP reduced the CAT activity by more than 95% . These results underscore the important differences between negative strand RNA viruses with respect to interactions between these two viral proteins involved in gene expression.

Mol Cell Biochem, 1995 May 24, 146(2), 121 - 6
Characterization of the HeLa cell single-stranded DNA-dependent ATPase/DNA helicase II; Vishwanatha JK et al.; A single-stranded DNA-dependent ATPase activity, consisting of two subunits of 83 kDa (p90) and 68 kDa (p70), was previously purified from HeLa cells (Vishwanatha, J.K . and Baril, E.F . (1990) Biochem 29, 8753-8759) . Homology of the two subunits of single-stranded DNA-dependent ATPase with the human Ku protein (Cao et al . (1994) Biochem 33, 8548-8557) and identity of the Ku protein as the human DNA helicase II (Tuteja et al . (1994) EMBO J . 13, 4991-5001) have been reported recently . Using antisera raised against the subunits of the HDH II, we confirm that the Hela single-stranded DNA-dependent ATPase is the HDH II . Similar to the activity reported for Ku protein, ssDNA-dependent ATPase binds to double-stranded DNA and the DNA-protein complex detected by gel mobility shift assay consists of both the ATPase subunits . The p90 subunit is predominantly nuclear and is easily dissociated from chromatin . The p70 is distributed in cytosol and nucleus, and a fraction of the nuclear p70 protein is found to be associated with the nuclear matrix . Both the p90 and p70 subunits of the ATPase are present in G1 and S phase of the cell cycle and are rapidly degraded in the G2/M phase of the cell cycle.

Proc Natl Acad Sci U S A, 1995 May 23, 92(11), 5067 - 71
Genetic and biochemical dissection of protein linkages in the cadherin-catenin complex; Jou TS et al.; The cadherin-catenin complex is important for mediating homotypic, calcium-dependent cell-cell interactions in diverse tissue types . Although proteins of this complex have been identified, little is known about their interactions . Using a genetic assay in yeast and an in vitro protein-binding assay, we demonstrate that beta-catenin is the linker protein between E-cadherin and alpha-catenin and that E-cadherin does not bind directly to alpha-catenin . We show that a 25-amino acid sequence in the cytoplasmic domain of E-cadherin and the amino-terminal domain of alpha-catenin are independent binding sites for beta-catenin . In addition to beta-catenin and plakoglobin, another member of the armadillo family, p120 binds to E-cadherin . However, unlike beta-catenin, p120 does not bind alpha-catenin in vitro, although a complex of p120 and endogenous alpha-catenin could be immunoprecipitated from cell extracts . In vitro protein-binding assays using recombinant E-cadherin cytoplasmic domain and alpha-catenin revealed two catenin pools in cell lysates: an approximately 1000- to approximately 2000-kDa complex bound to E-cadherin and an approximately 220-kDa pool that did not contain E-cadherin . Only beta-catenin in the approximately 220-kDa pool bound exogenous E-cadherin . Delineation of these molecular linkages and the demonstration of separate pools of catenins in different cell lines provide a foundation for examining regulatory mechanisms involved in the assembly and function of the cadherin-catenin complex.

Proc Natl Acad Sci U S A, 1995 May 23, 92(11), 5027 - 31
Regulation of the polarization of T cells toward antigen-presenting cells by Ras-related GTPase CDC42; Stowers L et al.; The mechanisms by which cells rapidly polarize in the direction of external signals are not understood . Helper T cells, when contacted by an antigen-presenting cell, polarize their cytoskeletons toward the antigen-presenting cell within minutes . Here we show that, in T cells, the mammalian Ras-related GTPase CDC42 (the homologue of yeast CDC42, a protein involved in budding polarity) can regulate the polarization of both actin and microtubules toward antigen-presenting cells but is not involved in other T-cell signaling processes such as those which culminate in interleukin 2 production . Although T-cell polarization appears dispensable for signaling leading to interleukin 2 production, polarization may direct lymphokine secretion towards the correct antigen-presenting cell in a crowded cellular environment . Inhibitor experiments suggest that phosphatidylinositol 3-kinase is required for cytoskeletal polarization but that calcineurin activity, known to be important for other aspects of signaling, is not . Apparent conservation of CDC42 function between yeast and T cells suggests that this GTPase is a general regulator of cytoskeletal polarity in many cell types.

Biochemistry, 1995 May 23, 34(20), 6573 - 80
Site-specific cross-linking as a method for studying intramolecular electron transfer; Pappa HS et al.; Site-directed mutagenesis has been used to introduce cysteine residues into yeast cytochrome c peroxidase and yeast cytochrome c for the purpose of forming site-specific cross-linked intermolecular complexes . This enables the formation of well-defined homogeneous covalently linked complexes for the purpose of relating structure to intramolecular electron transfer . Two complexes have been prepared and analyzed . Complex I has an engineered cysteine at position 290 near the C-terminus of the peroxidase linked to the naturally occurring Cys102 near the C-terminus of yeast cytochrome c . This complex exhibits undetectable rates of intramolecular electron transfer . Complex II has Cys290 of the peroxidase linked to the engineered Cys73 of cyt c . This complex was designed to mimic the crystal structure of the peroxidase-cytochrome c noncovalent complex {Pelletier & Kraut (1992) Science 258, 1748-1755} . Stopped-flow studies show that complex II carries out intramolecular electron transfer from ferrocytochrome c to peroxidase compound I at a rate of approximately 500-800 s-1 . This indicates that the binding orientation observed in the crystal structure is competent in rapid intramolecular electron transfer.

Proc Natl Acad Sci U S A, 1995 May 23, 92(11), 4947 - 51
Identification of an 11-kDa FKBP12-rapamycin-binding domain within the 289-kDa FKBP12-rapamycin-associated protein and characterization of a critical serine residue; Chen J et al.; Complexed with its intracellular receptor, FKBP12, the natural product rapamycin inhibits G1 progression of the cell cycle in a variety of mammalian cell lines and in the yeast Saccharomyces cerevisae . Previously, a mammalian protein that directly associates with FKBP12-rapamycin has been identified and its encoding gene has been cloned from both human (designated FRAP) {Brown, E.J., Albers, M.W., Shin, T.B., Ichikawa, K., Keith, C.T., Lane, W.S . & Schreiber, S.L . (1994) Nature (London) 369, 756-758} and rat (designated RAFT) {Sabatini, D.M., Erdjument-Bromage, H., Lui, M., Tempst, P . & Snyder, S.H . (1994) Cell 78, 35-43} . The full-length FRAP is a 289-kDa protein containing a putative phosphatidylinositol kinase domain . Using an in vitro transcription/translation assay method coupled with proteolysis studies, we have identified an 11-kDa FKBP12-rapamycin-binding domain within FRAP . This minimal binding domain lies N-terminal to the kinase domain and spans residues 2025-2114 . In addition, we have carried out mutagenesis studies to investigate the role of Ser2035, a potential phosphorylation site for protein kinase C within this domain . We now show that the FRAP Ser2035-->Ala mutant displays similar binding affinity when compared with the wild-type protein, whereas all other mutations at this site, including mimics of phosphoserine, abolish binding, presumably due to either unfavorable steric interactions or induced conformational changes.

Mol Gen Genet, 1995 May 20, 247(4), 471 - 81
Isolation and characterization of extragenic mutations affecting the expression of the uroporphyrinogen decarboxylase gene (HEM12) in Sacharomyces cerevisiae; Zoladek T et al.; Uroporphyrinogen decarboxylase (Uro-d; EC 4.1.1.37), the fifth enzyme in the heme biosynthetic pathway, which catalyzes the sequential decarboxylation of uroporphyrinogen to coproporphyrinogen, is encoded by the HEM12 gene in Saccharomyces cerevisiae . The HEM12 gene is transcribed into a major short mRNA and a minor longer one, approximately 1.35 and 1.55 kb, respectively, in size, and that differ in the 5' untranslated region . "Uroporphyric" mutants, which have no mutations in the HEM12 gene but accumulate uroporphyrinogen, a phenotype characteristic of partial Uro-d deficiency, were investigated . Genetic analysis showed that the mutant phenotype depends on the combined action of two unlinked mutations, udt1 and either ipa1, ipa2, or ipa3 . ipa1 is tightly linked to HEM12 . The mutation udt1 apparently acts specifically on the HEM12 gene, and causes a six to tenfold decrease in the levels of the short HEM12 mRNA, in the beta-galactosidase activity of a HEM12-lacZ fusion, in immunodetectable protein and enzyme activity . But heme synthesis is normal and porphyrin accumulation was modest . The mutations ipa1, ipa2, and ipa3 had no phenotype on their own, but they caused an increase in porphyrin accumulation in a udt1 background . This multiplicity of genetic factors leading to uroporphyric yeast cells closely resembles the situation in human porphyria cutanea tarda.

J Biol Chem, 1995 May 19, 270(20), 12235 - 41
Phosphorylation-dependent interaction of the cytoplasmic domains of the type I and type II transforming growth factor-beta receptors; Chen RH et al.; Transforming growth factor-beta (TGF-beta) transduces signals through its type I and type II receptors . Both receptor types have previously been shown to interact in a heteromeric complex in the presence of TGF-beta . We have now characterized these interactions between both receptor types using a combination of yeast two-hybrid interaction assays and coimmunoprecipitation analyses . Our results indicate a direct association between the cytoplasmic domains of the two receptor types . Mutation analysis of these cytoplasmic domains reveals that this direct interaction requires kinase activity and, thus, depends on phosphorylation, probably via a transphosphorylation mechanism . Furthermore, the two receptor types already have an inherent affinity for each other in the absence of TGF-beta, and the heteromeric complex can be detected in coimmunoprecipitations under these conditions . Taken together, our results reveal a novel mechanism of receptor complex formation, whereby two different cytoplasmic domains directly associate with each other . This interaction may play a major role in activation of serine/threonine kinase receptors.

J Biol Chem, 1995 May 19, 270(20), 12152 - 61
Three different rearrangements in a single intron truncate sterol regulatory element binding protein-2 and produce sterol-resistant phenotype in three cell lines . Role of introns in protein evolution; Yang J et al.; The cholesterol analogue 25-hydroxycholesterol kills animal cells by blocking the proteolytic activation of two sterol-regulated transcription factors designated sterol regulatory element binding protein-1 and -2 (SREBP-1 and SREBP-2) . These proteins, each approximately 1150 amino acids in length, are embedded in the membranes of the nucleus and endoplasmic reticulum by virtue of hydrophobic COOH-terminal segments . In cholesterol-depleted cells the proteins are cleaved to release soluble NH2-terminal fragments of approximately 480 amino acids that enter the nucleus and activate genes encoding the low density lipoprotein receptor and enzymes of cholesterol synthesis . 25-Hydroxycholesterol blocks this cleavage, and cells die of cholesterol deprivation . We previously described a mutant 25-hydroxycholesterol-resistant hamster cell line (SRD-1 cells) in which the SREBP-2 gene had undergone a recombination between the intron following codon 460 and an intron in an unrelated gene . The SREBP-2 sequence terminated at residue 460, eliminating the membrane attachment domain and producing a constitutively active factor that no longer required proteolysis and thus was not inhibited by 25-hydroxycholesterol . Here, we report that two additional sterol-resistant cell lines (SRD-2 and SRD-3) have also undergone genomic rearrangements in the intron following codon 460 of the SREBP-2 gene . Although the molecular rearrangements differ in the three mutant lines, each leads to the production of a constitutively active transcription factor whose SREBP-2 sequence terminates at residue 460 . These findings provide a dramatic illustration of the advantage that introns provide in allowing proteins to gain new functions in response to new environmental challenges.

J Biol Chem, 1995 May 19, 270(20), 11962 - 9
Structure-function analysis of Bcl-2 protein . Identification of conserved domains important for homodimerization with Bcl-2 and heterodimerization with Bax; Hanada M et al.; The Bcl-2 protein is a suppressor of programmed cell death that homodimerizes with itself and forms heterodimers with a homologous protein Bax, a promoter of cell death . Expression of Bax in Saccharomyces cerevisiae as a membrane-bound fusion protein results in a lethal phenotype that is suppressible by co-expression of Bcl-2 . Functional analysis of deletion mutants of human Bcl-2 in yeast demonstrated the presence of at least three conserved domains that are required to suppress Bax-mediated cytotoxicity, termed domains A (amino acids 11-33), B (amino acids 138-154), and C (amino acids 188-196) . In vitro binding experiments using GST-Bcl-2 fusion proteins demonstrated that Bcl-2(delta B) and Bcl-2(delta C) deletion mutants had a markedly impaired ability to heterodimerize with Bax but retained the ability to homodimerize with wild-type Bcl-2 . In contrast, Bcl-2(delta A) and an NH2-terminal deletion mutant Bcl-2(delta 1-82) retained Bax binding activity in vitro but failed to suppress Bax-mediated cytotoxicity in yeast . Sequences downstream of domain C in the region 197-218 also were shown to be required for Bax-binding in vitro and anti-death function in yeast . Analysis of Bcl-2/Bcl-2 homodimerization using both in vitro binding assays as well as a yeast two-hybrid method provided evidence in support of a head-to-tail model for Bcl-2/Bcl-2 homodimerization and revealed that sequences within the NH2-terminal A domain interact with a structure that requires the presence of both the carboxyl B and C domains in combination . In addition to further delineating structural features within Bcl-2 that are required for homo-dimerization, the findings reported here support the hypothesis that Bcl-2 promotes cell survival by binding directly to Bax but suggest that ability to bind Bax can be insufficient for anti-cell death function.

Cell, 1995 May 19, 81(4), 505 - 12
FADD, a novel death domain-containing protein, interacts with the death domain of Fas and initiates apoptosis; Chinnaiyan AM et al.; Using the cytoplasmic domain of Fas in the yeast two-hybrid system, we have identified a novel interacting protein, FADD, which binds Fas and Fas-FD5, a mutant of Fas possessing enhanced killing activity, but not the functionally inactive mutants Fas-LPR and Fas-FD8 . FADD contains a death domain homologous to the death domains of Fas and TNFR-1 . A point mutation in FADD, analogous to the lpr mutation of Fas, abolishes its ability to bind Fas, suggesting a death domain to death domain interaction . Overexpression of FADD in MCF7 and BJAB cells induces apoptosis, which, like Fas-induced apoptosis, is blocked by CrmA, a specific inhibitor of the interleukin-1 beta-converting enzyme . These findings suggest that FADD may play an important role in the proximal signal transduction of Fas.

J Biol Chem, 1995 May 12, 270(19), 11646 - 53
Homology requirements for ligation and strand exchange by the FLP recombinase; Zhu XD et al.; The FLP recombinase of the 2-microns plasmid of Saccharomyces cerevisiae belongs to the integrase family whose members form a covalent bond between a conserved tyrosine of the recombinase and the 3'-phosphoryl group at the site of cleavage . Ligation takes place when the 5'-OH generated during the cleavage step attacks the phosphotyrosine bond and reforms a phosphodiester bond . When the incoming 5'-OH is from the partner duplex, strand exchange occurs . The FLP recognition target (FRT) contains two inverted 13-base pair (bp) FLP binding sequences that surround an 8-bp core region . It has been shown that heterology in the core regions of the recombinase FLP recognition target sites can dramatically impair recombination . Therefore, it was of interest to study the homology requirements of the core sequence for FLP-mediated ligation . Using nicked duplex substrates containing mismatches in the core sequence, we have demonstrated that the FLP ligation reaction can tolerate mismatches at all positions in the 8-bp core except the position immediately adjacent to the cleavage site . Using half-FRT substrates that contain a single-stranded core sequence, we showed that 4 base pairs adjacent to the cleavage site in the core are required for FLP to execute ligation with a single-stranded oligonucleotide . FLP is also able to ligate the protruding single strand on a half-FRT site to the opposite strand to form a hairpin . We have studied the effect of the base composition of the protruding 8-nucleotide single strand upon the efficiency of hairpin ligation . These studies revealed the importance of intrastrand complementarity in the formation of hairpin by FLP . Hence we conclude that the homology in the position adjacent to the cleavage site is most important, and the degree of the homology required is dependent on the nature of the ligation assay.

Biochem Pharmacol, 1995 May 11, 49(9), 1335 - 9
Kinetic characteristics of rat liver peroxisomal nafenopin-CoA ligase; Roberts BJ et al.; In this study we have demonstrated that rat hepatic peroxisomes catalyse the formation of nafenopin-CoA . The process is mediated by apparent high affinity (Km 6.7 microM), low capacity (Vmax 0.31 nmol/mg/min) and low affinity, high capacity isoforms . Palmitic acid (Ki 1.1 microM), R(-) ibuprofen (Ki 7.9 microM), ciprofibrate (Ki 60.2 microM) and clofibric acid (Ki 86.8 microM) competitively inhibited nafenopin-CoA formation catalysed by the apparent high affinity isoform . An antibody raised against the microsomal palmitoyl-CoA ligase inhibited the equivalent peroxisomal enzyme significantly (P < 0.001) but did not inhibit peroxisomal nafenopin-CoA ligase activity . These data suggest that nafenopin-CoA formation is catalysed by a peroxisomal CoA ligase which differs from the peroxisomal long chain fatty acid-CoA ligase in relation to its xenobiotic/antibody inhibitor profile and kinetic characteristics.

Arch Biochem Biophys, 1995 May 10, 319(1), 267 - 73
Isolation and characterization of a new cDNA clone belonging to the cytochrome P450 2C gene subfamily in hamsters; Sakuma T et al.; Cytochrome P450s are heme-containing proteins which evolved from an ancestral gene to form a large superfamily of enzymes . We previously isolated three distinct CYP2C cDNAs, namely CYP2C25, 2C26, and 2C27, from liver cDNA libraries of male and female Syrian hamsters . In the present study, we isolated another cDNA clone, assigned as CYP2C28, from the same male cDNA library . This cDNA consisted of 1556 nucleotides and encoded a polypeptide of 490 amino acids . CYP2C28 showed relatively low identities to other hamster CYP2C forms (71.4-72.6% in amino acid sequences) and showed the highest similarity to rat CYP2C24 (88.7%), while the other hamster CYP2C forms showed high similarities among themselves (> 90.2%) . CYP2C28 protein expressed in yeast catalyzed the N-demethylation of aminopyrine and benzphetamine, but did not catalyze the hydroxylation of tolbutamide, testosterone, and progesterone . Northern blot analysis demonstrated that CYP2C28 mRNA was expressed in hamster livers 1 week after birth, when the other hamster CYP2C forms were not detectable . The level of CYP2C28 mRNA increased to 3 weeks and then decreased with time in males . The level in females was lower than that in males . In adult hamsters, CYP2C28 was induced almost threefold by administration of phenobarbital but not affected by 3-methycholanthrene . On the other hand, administration of pregnenolone 16 alpha-carbonitrile induced CYP2C28 in females but not in males . These results indicate that CYP2C28 is a unique form in the hamster CYP2C subfamily.

Virology, 1995 May 10, 209(1), 19 - 28
Transcriptional activation by DNA-binding derivatives of HSV-1 VP16 that lack the carboxyl-terminal acidic activation domain; Popova B et al.; The herpes simplex virus transactivator VP16 directs the assembly of a multicomponent protein-DNA complex with cellular components Oct-1 and VCAF-1, contributing a potent carboxyl-terminal acidic activation domain that is essential for activation of gene expression in mammalian cells . We show here that VP16, devoid of this acidic activation domain, functions as a strong transcriptional activator in the yeast Saccharomyces cerevisiae when appended onto a heterologous GAL4 DNA binding domain, as determined by measuring activation of a resident GAL1:lacZ reporter gene . Deletion analysis indicated that sequences contained within the amino-terminal 369 amino acids of VP16 were necessary for transactivation by truncated VP16 . Activation by truncated VP16 in yeast was comparable to that observed with a hybrid protein consisting of the GAL4 DNA binding domain linked to the VP16 acidic activation domain . Similar GAL4-VP16 hybrid proteins were only marginally active in mammalian cells . Sequence requirements for transactivation by truncated VP16 can be demarcated from domains of VP16 that are required for interaction with VCAF-1 and for protein-DNA complex formation with Oct-1 . Our findings indicate that VP16 contains additional sequences upstream of the acidic activation domain that may play a direct role in transactivation.

Proc Natl Acad Sci U S A, 1995 May 9, 92(10), 4701 - 5
LEM1, an ATP-binding-cassette transporter, selectively modulates the biological potency of steroid hormones; Kralli A et al.; The rat glucocorticoid receptor confers hormone-dependent transcriptional enhancement when expressed in yeast, thereby enabling the genetic identification of nonreceptor proteins that function in the hormone signal-transduction pathway . We isolated a yeast mutant, lem1, with increased sensitivity to dexamethasone and triamcinolone acetonide; responsiveness to a third agonist, deoxycorticosterone, is unaffected . Cloning of wild-type LEM1 revealed a putative transport protein of the ATP-binding cassette family . Dexamethasone accumulation is increased in lem1 cells, suggesting that wild-type LEM1 decreases dexamethasone potency by exporting this ligand . LEM1 appears to affect certain steroids and not others . We propose that transporters like LEM1 can selectively modulate the intracellular levels of steroid hormones . Differential activities of such transporters in mammalian cells might regulate hormone availability and thereby hormone signaling in a cell-type specific manner.

Proc Natl Acad Sci U S A, 1995 May 9, 92(10), 4651 - 5
Cdc37 is required for association of the protein kinase Cdc28 with G1 and mitotic cyclins; Gerber MR et al.; Studies of the temperature-sensitive cdc37-1 mutant of Saccharomyces cerevisiae suggest that Cdc37 is required for passage through the G1 phase of the cell cycle, but its precise function is not known . We have investigated the role of Cdc37 in the regulation of the cyclin-dependent protein kinase Cdc28 . We find that G1 arrest in the cdc37-1 mutant is accompanied by a decrease in the Cdc28 activity associated with the G1 cyclin Cln2 . This defect appears to be caused by a decrease in the binding of Cdc28 and Cln2 . cdc37-1 mutants also exhibit a defect in the binding and activation of Cdc28 by the mitotic cyclin Clb2 . Thus Cdc37 may be a regulator that is required for the association of Cdc28 with multiple cyclins.

Proc Natl Acad Sci U S A, 1995 May 9, 92(10), 4587 - 90
General requirement for RNA polymerase II holoenzymes in vivo; Thompson CM et al.; Yeast RNA polymerase II holoenzymes have been described that consist of RNA polymerase II, a subset of general transcription factors, and nine SRB regulatory proteins . The feature that distinguishes the RNA polymerase II holoenzymes from other forms of RNA polymerase II in the cell is their tight association with SRB proteins . We investigated the fraction of genes that require SRB proteins in vivo by examining the effect of temperature-sensitive mutations in SRB genes on transcription by RNA polymerase II . Upon transfer to the restrictive temperature, there is a rapid and general shutdown of mRNA synthesis in srb mutant cells . These data, combined with the observation that essentially all of the SRB protein in cells is tightly associated with RNA polymerase II molecules, argue that SRB-containing holoenzymes are the form of RNA polymerase II recruited to most promoters in the cell.

Proc Natl Acad Sci U S A, 1995 May 9, 92(10), 4512 - 6
Suppression of heat-induced hsp70 expression by the 70-kDa subunit of the human Ku autoantigen; Li GC et al.; Expression of the 70-kDa polypeptide of human Ku autoantigen in rat cells is shown to suppress specifically the induction of hsp70 upon heat shock . Thermal induction of other heat shock proteins is not significantly affected, nor is the state of phosphorylation or the DNA-binding ability of the heat shock transcription factor HSF1 . These findings support a model in which hsp70 gene expression is controlled by a second regulatory factor in addition to the positive activator HSF1 . The Ku autoantigen, or a protein closely related to it, is likely to be involved in the regulation of hsp70 expression.

Proc Natl Acad Sci U S A, 1995 May 9, 92(10), 4437 - 41
Heat shock protein hsp90 regulates dioxin receptor function in vivo; Whitelaw ML et al.; The dioxin (aryl hydrocarbon) receptor is a ligand-dependent basic helix-loop-helix (bHLH) factor that binds to xenobiotic response elements of target promoters upon heterodimerization with the bHLH partner factor Arnt . Here we have replaced the bHLH motif of the dioxin receptor with a heterologous DNA-binding domain to create fusion proteins that mediate ligand-dependent transcriptional enhancement in yeast (Saccharomyces cerevisiae) . Previously, our experiments indicated that the ligand-free dioxin receptor is stably associated with the 90-kDa heat shock protein, hsp90 . To investigate the role of hsp90 in dioxin signaling we have studied receptor function in a yeast strain where hsp90 expression can be down-regulated to about 5% relative to wild-type levels . At low levels of hsp90, ligand-dependent activation of the chimeric dioxin receptor construct was almost completely inhibited, whereas the activity of a similar chimeric construct containing the structurally related Arnt factor was not affected . Moreover, a chimeric dioxin receptor construct lacking the central ligand- and hsp90-binding region of the receptor showed constitutive transcriptional activity in yeast that was not impaired upon down-regulation of hsp90 expression levels . Thus, these data suggest that hsp90 is a critical determinant of conditional regulation of dioxin receptor function in vivo via the ligand-binding domain.

Proc Natl Acad Sci U S A, 1995 May 9, 92(10), 4274 - 8
Molecular cloning and characterization of cDNA encoding the alpha subunit of the rat protein synthesis initiation factor eIF-2B; Flowers KM et al.; Eukaryotic initiation factor 2B (eIF-2B) is an essential component of the pathway of peptide-chain initiation in mammalian cells, yet little is known about its molecular structure and regulation . To investigate the structure, regulation, and interactions of the individual subunits of eIF-2B, we have begun to clone, characterize, and express the corresponding cDNAs . We report here the cloning and characterization of a 1510-bp cDNA encoding the alpha subunit of eIF-2B from a rat brain cDNA library . The cDNA contains an open reading frame of 918 bp encoding a polypeptide of 305 aa with a predicted molecular mass of 33.7 kDa . This cDNA recognizes a single RNA species approximately 1.6 kb in length on Northern blots of RNA from rat liver . The predicted amino acid sequence contains regions identical to the sequences of peptides derived from bovine liver eIF-2B alpha subunit . Expression of this cDNA in vitro yields a peptide which comigrates with natural eIF-2B alpha in SDS/polyacrylamide gels . The predicted amino acid sequence exhibits 42% identity to that deduced for the Saccharomyces cerevisiae GCN3 protein, the smallest subunit of yeast eIF-2B . In addition, expression of the rat cDNA in yeast functionally complements a gcn3 deletion for the inability to induce histidine biosynthetic genes under the control of GCN4 . These results strongly support the hypothesis that mammalian eIF-2 alpha and GCN3 are homologues . Southern blots indicate that the eIF-2B alpha cDNA also recognizes genomic DNA fragments from several other species, suggesting significant homology between the rat eIF-2B alpha gene and that from other species.

Cancer Lett, 1995 May 8, 91(2), 169 - 75
The inhibition of protein prenyltransferases by oxygenated metabolites of limonene and perillyl alcohol; Gelb MH et al.; The monoterpenes limonene and perillyl alcohol are effective therapeutic agents against advanced rat mammary cancer . Limonene is currently undergoing clinical testing in cancer patients . These monoterpenes and their oxygenated metabolites have been previously shown to inhibit protein prenylation in cultured cells . Since farnesylation of ras protein is critical for its ability to cause oncogenic transformation, inhibition of protein prenylation may be the basis of the anti-tumor effects of limonene and perillyl alcohol . In this study we test the ability of limonene and its oxygenated analogs to inhibit protein prenylation enzymes in vitro . Limonene and perillyl alcohol and their major in vivo metabolite, perillic acid, are weak inhibitors of both mammalian and yeast protein farnesyl transferase (PFT) and protein geranylgeranyl transferase (PGGT) . In contrast, a minor metabolite of both limonene and perillyl alcohol, perillic acid methyl ester, is a potent inhibitor of both enzymes . Perillic acid methyl ester is a competitive inhibitor of yeast PFT with respect to farnesyl pyrophosphate . These studies suggest that if the inhibition of protein prenylation is a mechanism for limonene's and perillyl alcohol's anti-cancer activities, these monoterpenes may be prodrugs that are converted into pharmacologically-active substances by metabolic modification.

FEBS Lett, 1995 May 8, 364(2), 207 - 10
Purification and characterization of human deoxyhypusine synthase from HeLa cells; Klier H et al.; Post-translational modification of a specific lysine residue in eukaryotic initiation factor 5A is essential for cell viability . The amino acid hypusine, which is the product of this modification, is derived in two subsequent enzyme-catalyzed reactions . We have purified and characterized the enzyme responsible for the first step in hypusine modification, deoxyhypusine synthase, from HeLa cells . The human enzyme is multimeric with a native apparent molecular weight of 150,000 consisting of subunits of 41,000 . The amino acid sequences of its peptide fragments share high sequence identity with a hypothetical protein (YHRO68w) on chromosome VIII of Saccharomyces cerevisiae . This protein appears to be the deoxyhypusine synthase of yeast.

J Biol Chem, 1995 May 5, 270(18), 10976 - 81
TATA-binding protein residues implicated in a functional interplay between negative cofactor NC2 (Dr1) and general factors TFIIA and TFIIB; Kim TK et al.; The TATA-binding protein (TBP) plays a key role in transcription initiation . Several negative cofactors (NC1, NC2, and Dr1) are known to interact with TBP in a manner that prevents productive interactions of transcription factors TFIIA and TFIIB with promoter-bound TBP . To gain insights into the regulatory interplay on the surface of TBP, we have employed mutant forms of TBP to identify amino acid residues important for interactions with the negative regulatory cofactor NC2 and the general factor TFIIB . The results show the involvement of distinct domains of TBP in these interactions . Residues (Lys-133, Lys-145, and Lys-151) in the basic repeat region are important for interactions with NC2, as well as with TFIIA (Buratowski, S., and Zhou, H . (1992) Science 255, 1130-1132; Lee, D . K., DeJong, J., Hashimoto, S., Horikoshi, M., and Roeder, R . G . (1992) Mol . Cell . Biol . 12, 5189-5196), whereas a residue (Leu-189) in the second stirrup-like loop spanning S2' and S3' is required for interaction with TFIIB . In addition, we demonstrate that NC2 is identical to the previously cloned negative cofactor Dr1 . The implications of these results for TBP structure and function are discussed.

J Biol Chem, 1995 May 5, 270(18), 10514 - 24
A Ca(2+)-binding chimera of human lysozyme and bovine alpha-lactalbumin that can form a molten globule; Pardon E et al.; In contrast to lysozymes, which undergo two-state thermal denaturation, the Ca(2+)-free form of the homologous alpha-lactalbumins forms an intermediate "molten globule" state . To understand this difference, we have produced a chimera of human lysozyme and bovine alpha-lactalbumin . In the synthetic gene of the former the sequence coding for amino acid residues 76-102 was replaced by that for bovine alpha-lactalbumin 72-97, which represents the Ca(2+)-binding loop and the central helix C . The chimeric protein, LYLA1, expressed in Saccharomyces cerevisiae was homogeneous on electrophoresis and mass spectrometry . Its Ca2+ binding constant was 2.50 (+/- 0.04) x 10(8) M-1, and its muramidase activity 10% of that of human lysozyme . One-dimensional NMR spectroscopy indicated the presence of a compact, well structured protein . From two-dimensional NMR spectra, main chain resonances for 118 of a total of 129 residues could be readily assigned . Nuclear Overhauser effect analysis and hydrogen-deuterium exchange measurements indicated the presence and persistence of all expected secondary structure elements . Thermal denaturation, measured by circular dichroism, showed a single transition temperature for the Ca2+ form at 90 degrees C, whereas unfolding of the apo form occurred at 73 degrees C in the near-UV and 81 degrees C in the far-UV range . These observations illustrate that by transplanting the central part of bovine alpha-lactalbumin, we have introduced into human lysozyme two important properties of alpha-lactalbumins, i.e . stabilization through Ca2+ binding and molten globule behavior.

Cell, 1995 May 5, 81(3), 359 - 68
Contact with a component of the polymerase II holoenzyme suffices for gene activation; Barberis A et al.; In yeast strains bearing the point mutation called GAL11P (for potentiator), certain GAL4 derivatives lacking any classical activating region work as strong activators . The P mutation confers upon GAL11, a component of the RNA polymerase II holoenzyme, the ability to interact with a portion of the dimerization region of GAL4 . The region of GAL11 affected by the P mutation is evidently functionally inert in ordinary cells, suggesting that this mutation is of no functional significance beyond creating an artificial target for the GAL4 dimerization fragment . From these observations and further analyses of GAL11, we propose that a single activator-holoenzyme contact can trigger gene activation simply by recruiting the latter to DNA.

Plant Physiol, 1995 May, 108(1), 285 - 94
Induction of apoplastic invertase of Chenopodium rubrum by D-glucose and a glucose analog and tissue-specific expression suggest a role in sink-source regulation; Roitsch T et al.; Photoautotrophic suspension-culture cells of Chenopodium rubrum that were shifted to mixotrophic growth by adding glucose were used as model system to investigate the influence of the source-sink transition in higher plants on the expression and enzyme activities of intracellular and extracellular invertases . The complete cDNA coding for an extracellular invertase was cloned and sequenced from C . rubrum, and its identity has been proven by heterologous expression in Saccharomyces cerevisiae . The higher activity of extracellular invertase after preincubation in the presence of glucose was paralleled by an increased expression of the corresponding gene . The induction by glucose could be mimicked by the nonmetabolizable glucose analog 6-deoxyglucose . Both enzyme activity and mRNA level of extracellular invertase showed a sink-tissue-specific distribution in plants . The activity of neutral and acidic intracellular invertases were not affected by preincubation of autotrophic tissue cultures with sugars, nor did they show a tissue-specific distribution in plants . The data suggest that apoplastic invertase not only has an important function in phloem unloading and carbohydrate partitioning between source and sink tissues but may also have a role in establishing metabolic sinks.

J Mol Evol, 1995 May, 40(5), 499 - 508
The class II aminoacyl-tRNA synthetases and their active site: evolutionary conservation of an ATP binding site; Eriani G et al.; Previous sequence analyses have suggested the existence of two distinct classes of aminoacyl-tRNA synthetase . The partition was established on the basis of exclusive sets of sequence motifs (Eriani et al . {1990} Nature 347:203-306) . X-ray studies have now well defined the structural basis of the two classes: the class I enzymes share with dehydrogenases and kinases the classic nucleotide binding fold called the Rossmann fold, whereas the class II enzymes possess a different fold, not found elsewhere, built around a six-stranded antiparallel beta-sheet . The two classes of synthetases catalyze the same global reaction that is the attachment of an amino acid to the tRNA, but differ as to where on the terminal adenosine of the tRNA the amino acid is placed: class I enzymes act on the 2' hydroxyl whereas the class II enzymes prefer the 3' hydroxyl group . The three-dimensional structure of aspartyl-tRNA synthetase from yeast, a typical class II enzyme, is described here, in relation to its function . The crucial role of the sequence motifs in substrate binding and enzyme structure is high-lighted . Overall these results underline the existence of an intimate evolutionary link between the aminoacyl-tRNA synthetases, despite their actual structural diversity.

Eur J Biochem, 1995 May 1, 229(3), 669 - 74
Photoaffinity labeling of protoporphyrinogen oxidase, the molecular target of diphenylether-type herbicides; Camadro JM et al.; Diphenylether-type herbicides are extremely potent inhibitors of protoporphyrinogen oxidase, a membrane-bound enzyme involved in the heme and chlorophyll biosynthesis pathways . Tritiated acifluorfen and a diazoketone derivative of tritiated acifluorfen were specifically bound to a single class of high-affinity binding sites on yeast mitochondrial membranes with apparent dissociation constants of 7 nM and 12.5 nM, respectively . The maximum density of specific binding sites, determined by Scatchard analysis, was 3 pmol.mg-1 protein . Protoporphyrinogen oxidase specific activity was estimated to be 2500 nmol protoporphyrinogen oxidized h-1.mol-1 enzyme . The diazoketone derivative of tritiated acifluorfen was used to specifically photolabel yeast protoporphyrinogen oxidase . The specifically labeled polypeptide in wild-type mitochondrial membranes had an apparent molecular mass of 55 kDa, identical to the molecular mass of the purified enzyme . This photolabeled polypeptide was not detected in a protoporphyrinogen-oxidase-deficient yeast strain, but the membranes contained an equivalent amount of inactive immunoreactive protoporphyrinogen oxidase protein.

J Bacteriol, 1995 May, 177(10), 2887 - 91
Translation and M1 double-stranded RNA propagation: MAK18 = RPL41B and cycloheximide curing; Carroll K et al.; MAK18 is one of nearly 30 chromosomal genes of Saccharomyces cerevisiae necessary for propagation of the killer toxin-encoding M1 double-stranded RNA satellite of the L-A double-stranded RNA virus . We have cloned and sequenced MAK18 and find that it is identical to RPL41B, one of the two genes encoding large ribosomal subunit protein L41 . The mak18-1 mutant is deficient in 60S subunits, which we suggest results in a preferential decrease in translation of viral poly(A)-deficient mRNA . We have reexamined the curing of M1 by low concentrations of cycloheximide (G . R . Fink and C . A . Styles, Proc . Natl . Acad . Sci . USA 69:2846-2849, 1972), which is known to act on ribosomal large subunit protein L29 . We find that when M1 is supported by L-A proteins made from the poly(A)+ mRNA of a cDNA clone of L-A, cycloheximide does not decrease the M1 copy number, consistent with our hypothesis.

EMBO J, 1995 May 1, 14(9), 2089 - 98
Sm and Sm-like proteins belong to a large family: identification of proteins of the U6 as well as the U1, U2, U4 and U5 snRNPs; Seraphin B; Several small nuclear RNAs (snRNAs), including the spliceosomal U1, U2, U4 and U5 snRNAs, are associated with Sm proteins . These eight small proteins form a heteromeric complex that binds to snRNAs and plays a major role in small nuclear ribonucleoprotein (snRNP) biogenesis and transport . These proteins are also a major target for autoantibodies in the human disease systemic lupus erythematosus . By sequence comparison I have shown that all the known Sm proteins share a common structural motif which might explain their immunological cross-reactivity . Database searches using this motif uncovered a large number of Sm-like proteins from plants, animals and fungi . These proteins have been grouped in at least 13 different subfamilies . Genes encoding divergent yeast members were cloned and used to produce tagged fusion proteins . Some of these proteins are canonical Sm proteins as they associate with the yeast U1, U2, U4/U6 and U5 snRNAs . Surprisingly, one Sm-like protein was found to be a component of the U6 snRNP . These findings have implications for the structure of the Sm protein complex, spliceosomal snRNP evolution, snRNA transport and modification as well as the involvement of Sm proteins in systemic lupus erythematosus.

EMBO J, 1995 May 1, 14(9), 2066 - 75
Identification and characterization of Uss1p (Sdb23p): a novel U6 snRNA-associated protein with significant similarity to core proteins of small nuclear ribonucleoproteins; Cooper M et al.; The SDB23 gene of Saccharomyces cerevisiae was isolated in a search for high copy-number suppressors of mutations in a cell cycle gene, DBF2, SDB23 encodes a 21,276 Da protein with significant sequence similarity to characterized mammalian snRNP core proteins . Examination of multiple sequence alignments of snRNP core proteins with Sdb23p indicates that all of these proteins share a number of highly conserved residues, and identifies a novel motif for snRNP core proteins . Sdb23p is essential for cell viability and is required for nuclear pre-mRNA splicing both in vivo and in vitro . Extracts prepared from Sdb23p-depleted cells are unable to support splicing and have vastly reduced levels of U6 snRNA . The stability of U1, U2, U4 and U5 spliceosomal snRNAs is not affected by the loss of Sdb23p . Antibodies raised against Sdb23p strongly coimmunoprecipitate free U6 snRNA and U4/U6 base-paired snRNAs . These results establish that SDB23 encodes a novel U6 snRNA-associated protein that is essential for the stability of U6 snRNA . We therefore propose the more logical name USS1 (U-Six SnRNP) for this gene.

EMBO J, 1995 May 1, 14(9), 2020 - 33
The N-terminal part of TIF1, a putative mediator of the ligand-dependent activation function (AF-2) of nuclear receptors, is fused to B-raf in the oncogenic protein T18; Le Douarin B et al.; Nuclear receptors (NRs) bound to response elements mediate the effects of cognate ligands on gene expression . Their ligand-dependent activation function, AF-2, presumably acts on the basal transcription machinery through intermediary proteins/mediators . We have isolated a mouse nuclear protein, TIF1, which enhances RXR and RAR AF-2 in yeast and interacts in a ligand-dependent manner with several NRs in yeast and mammalian cells, as well as in vitro . Remarkably, these interactions require the amino acids constituting the AF-2 activating domain conserved in all active NRs . Moreover, the oestrogen receptor (ER) AF-2 antagonist hydroxytamoxifen cannot promote ER-TIF1 interaction . We propose that TIF1, which contains several conserved domains found in transcriptional regulatory proteins, is a mediator of ligand-dependent AF-2 . Interestingly, the TIF1 N-terminal moiety is fused to B-raf in the mouse oncoprotein T18.

EMBO J, 1995 May 1, 14(9), 1970 - 8
A novel serine kinase activated by rac1/CDC42Hs-dependent autophosphorylation is related to PAK65 and STE20; Martin GA et al.; We identified three proteins in neutrophil cytosol of molecular size 65, 62 and 68 kDa which interact in a GTP-dependent manner with rac1 and CDC42Hs, but not with rho . Purification of p65 and subsequent peptide sequencing revealed identity to rat brain PAK65 and to yeast STE20 kinase domains . Based on these sequences we screened a human placenta library and cloned the full-length cDNA . The complete amino acid sequence of the human cDNA shares approximately identity with rat brain PAK65; within the kinase domain the human protein shares > 95% and approximately 63% identity with rat PAK65 and yeast STE20 respectively . The new human (h)PAK65 mRNA is ubiquitously expressed and hPAK65 protein is distinct from either human or rat brain PAK65 . Recombinant hPAK65 exhibits identical specificity to the endogenous p65; both can bind rac1 and CDC42Hs in a GTP-dependent manner . The GTP-bound forms of rac1 and CDC42Hs induce autophosphorylation of hPAK65 on serine residues only . hPAK65 activated by either rac1 or CDC42Hs is phosphorylated on the same sites . Induction of hPAK65 autophosphorylation by rac1 or CDC42Hs stimulates hPAK65 kinase activity towards myelin basic protein and once hPAK65 is activated, rac1 or CDC42Hs are no longer required to keep it active . The affinities of rac/CDC42Hs for the non-phosphorylated and phosphorylated hPAK65 were similar . hPAK65 had only a marginal effect on the intrinsic GTPase activity of CDC42Hs, but significantly affected the binding and GAP activity of p190 . These data are consistent with a model in which hPAK65 functions as an effector molecule for rac1 and CDC42Hs.

Mol Cell Biol, 1995 May, 15(5), 2839 - 48
GAL4 interacts with TATA-binding protein and coactivators; Melcher K et al.; A major goal in understanding eukaryotic gene regulation is to identify the target(s) of transcriptional activators . Efforts to date have pointed to various candidates . Here we show that a 34-amino-acid peptide from the carboxy terminus of GAL4 is a strong activation domain (AD) and retains at least four proteins from a crude extract: the negative regulator GAL80, the TATA-binding protein (TBP), and the putative coactivators SUG1 and ADA2 . TFIIB was not retained . Concentrating on TBP, we demonstrate in in vitro binding assays that its interaction with the AD is specific, direct, and salt stable up to at least 1.6 M NaCl . The effects of mutations in the GAL4 AD on transcriptional activation in vivo correlate with their affinities to TBP . A point mutation (L114K) in yeast TBP, which has been shown to compromise the mutant protein in both binding to the VP16 AD domain and activated transcription in vitro, reduces the affinity to the GAL4 AD to the same degree as to the VP16 AD . This suggests that these two prototypic activators make similar contacts with TBP.

Mol Cell Biol, 1995 May, 15(5), 2612 - 24
Human cyclin E, a nuclear protein essential for the G1-to-S phase transition; Ohtsubo M et al.; Cyclin E was first identified by screening human cDNA libraries for genes that would complement G1 cyclin mutations in Saccharomyces cerevisiae and has subsequently been found to have specific biochemical and physiological properties that are consistent with it performing a G1 function in mammalian cells . Most significantly, the cyclin E-Cdk2 complex is maximally active at the G1/S transition, and overexpression of cyclin E decreases the time it takes the cell to complete G1 and enter S phase . We have now found that mammalian cells express two forms of cyclin E protein which differ from each other by the presence or absence of a 15-amino-acid amino-terminal domain . These proteins are encoded by alternatively spliced mRNAs and are localized to the nucleus during late G1 and early S phase . Fibroblasts engineered to constitutively overexpress either form of cyclin E showed elevated cyclin E-dependent kinase activity and a shortened G1 phase of the cell cycle . The overexpressed cyclin E protein was detected in the nucleus during all cell cycle phases, including G0 . Although the cyclin E protein could be overexpressed in quiescent cells, the cyclin E-Cdk2 complex was inactive . It was not activated until 6 to 8 h after readdition of serum, 4 h earlier than the endogenous cyclin E-Cdk2 . This premature activation of cyclin E-Cdk2 was consistent with the extent of G1 shortening caused by cyclin E overexpression . Microinjection of affinity-purified anti-cyclin E antibodies during G1 inhibited entry into S phase, whereas microinjection performed near the G1/S transition was ineffective . These results demonstrate that cyclin E is necessary for entry into S phase . Moreover, we found that cyclin E, in contrast to cyclin D1, was required for the G1/S transition even in cells lacking retinoblastoma protein function . Therefore, cyclins E and D1 control two different transitions within the human cell cycle.

Appl Environ Microbiol, 1995 May, 61(5), 1923 - 30
Tri6 encodes an unusual zinc finger protein involved in regulation of trichothecene biosynthesis in Fusarium sporotrichioides; Proctor RH et al.; In Fusarium sporotrichioides, several genes required for biosynthesis of the trichothecene mycotoxin T-2 toxin are closely linked . Further characterization of this gene cluster has revealed a gene, Tri6, that specifies a 217-amino-acid protein with regions similar to Cys2His2 zinc finger proteins . Temporal expression of Tri6 is similar to that of trichothecene biosynthetic pathway genes . Analysis of Tri6 transcripts indicated that transcription is initiated in two regions and that within each region there may be at least four initiation sites . Disruption of Tri6 resulted in a mutant that did not produce trichothecenes but that did accumulate low levels of the trichothecene precursor trichodiene . The Tri6 mutant was unable to convert six trichothecene biosynthetic intermediates to T-2 toxin, and transcription of two biosynthetic genes, Tri4 and Tri5, was greatly reduced in the mutant relative to the wild type . In addition, the product of Tri6 functioned as a transcriptional activator in Saccharomyces cerevisiae when fused to the DNA binding region of GAL4 . These results indicate that Tri6 encodes a protein involved in the transcriptional regulation of trichothecene biosynthetic genes in F . sporotrichioides.

J Clin Microbiol, 1995 May, 33(5), 1104 - 7
Quality control guidelines for National Committee for Clinical Laboratory Standards recommended broth macrodilution testing of amphotericin B, fluconazole, and flucytosine; Pfaller MA et al.; Amphotericin B, fluconazole, and flucytosine (5FC) were tested in a multilaboratory study to establish quality control (QC) guidelines for yeast antifungal susceptibility testing . Ten candidate QC strains were tested in accordance with National Committee for Clinical Laboratory Standards M27-P guidelines against the three antifungal agents in each of six laboratories . Each laboratory was assigned a unique lot of RPMI 1640 broth medium as well as a lot of RPMI 1640 common to all of the laboratories . The candidate QC strains were tested 20 times each against the three antifungal agents in both unique and common lots of RPMI 1640 . A minimum of 220 MICs per drug per organism were generated during the study . Overall, 95% of the MICs of amphotericin B, fluconazole, and 5FC fell within the desired 3 log2-dilution range (mode +/- 1 log2 dilution) . Excellent performance with all three drugs was observed for Candida parapsilosis ATCC 22019 and C . krusei ATCC 6258 . With these strains, on-scale 3 log2-dilution ranges encompassing 96 to 99% of the MICs of all three drugs were established . These two strains are recommended for QC testing of amphotericin B, fluconazole, and 5FC . Reference ranges were also established for an additional four strains for use in method development and for training . Four strains failed to perform adequately for recommendation as either QC or reference strains.

Biophys J, 1995 May, 68(5), 1678 - 80
A method for anchoring round shaped cells for atomic force microscope imaging; Kasas S et al.; More and more researchers are interested in imaging living (Henderson, 1994) or fixed cells in their natural environment using the atomic force microscope (AFM) . However, the AFM tip interacts strongly with the sample, and its z range freedom is limited to a few micrometers . This means that the cells to be imaged have to be strongly attached to the substrate, and imaging is restricted to cells having a flattened shape . Here we propose a simple and inexpensive solution to overcome these limitations . The method we propose is trapping living round shaped cells in a Millipore filter with a pore size comparable to the dimensions of the cell . The highest part of some of the blocked cells protrude through the holes of the filter and can this way be easily observed using the AFM without detachment.

Int J Pept Protein Res, 1995 May, 45(5), 418 - 29
Studies on conformational consequences of i to i + 3 side-chain cyclization in model cyclic tetrapeptides; Rao MH et al.; In an effort to explore the effect of ring size on the biologically active conformation of cyclic analogs of the mating pheromone alpha-factor (WHWLQLKPGQPMY) from Saccharomyces cerevisiae, eight cyclic tetrapeptides corresponding to the KPGQ portion of alpha-factor were synthesized . These N-alpha-acetyl/carboxyl amide terminal cyclic tetrapeptides were prepared on a 4-methylbenzhydrylamine resin using orthogonal Boc, Fmoc, OFm and OtBut protecting groups and HOBt-DIPC accelerated active esters or urethane-protected N-carboxyanhydrides . On-resin cyclization of the side-chain amino and carboxyl groups of the first and fourth residues, respectively, was performed with the BOP reagent to generate lactams containing 14-18 atoms . HF cleavage resulted in two products, the desired cyclic tetrapeptide and a major side product . All peptides were purified to near homogeneity (> 99%) by using reversed-phase HPLC and were characterized by FBMS and 1H NMR . Certain constrained cyclic tetrapeptides appear to be a mixture of isomers at room temperature as evidenced by HPLC and NMR . The major side product has been identified as a cyclo dimer, obtained as a consequence of interchain cyclization on the resin . CD analysis in several solvents gives evidence that some of the cyclic tetrapeptides exist in beta-turn conformations.

Electrophoresis, 1995 May, 16(5), 876 - 8
Detection of nonfunctional overexpression gene products using two-dimensional gel electrophoresis with a narrowed pH range; Wohl T et al.; A commonly used technique in the analysis of yeast protein function is the overexpression of cloned genes . In the case that overexpression does not lead to an altered phenotype a stable synthesis of the protein has to be demonstrated . Here an example is shown where overexpression of the yeast HYP2 genes, coding for a hypusine-containing protein, was alternatively analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) alone or by two-dimensional (2-D) gel electrophoresis, using the narrowed pH range of 4.5-5.4 . The results of the SDS-PAGE suggested a stable overproduction of HYP gene product, whereas 2-D analysis revealed an accumulation of nonfunctional protein isoforms.

Electrophoresis, 1995 May, 16(5), 739 - 41
The use of genomic DNA probes for in-gel hybridization; Wohl T et al.; Hybridization within agarose gels using oligonucleotide probes has been described in several publications; genomic DNA probes, however, have been used rarely and only with limited success . Here we present a simple and convenient procedure for in-gel hybridization using radiolabeled genomic DNA fragments . The protocol was improved by the use of formamide in the hybridization as well as in the washing step . This method was compared with the conventional Southern blotting technique and was shown to produce good results in restriction pattern analysis, as well as in chromosomal localization with the help of pulsed field gel electrophoresis.

Curr Biol, 1995 May 1, 5(5), 496 - 9
Repair and recombination . How to make ends meet; Roth DB et al.; The repair of double-stranded breaks in DNA and the recombination of antibody gene V(D)J segments share a common pathway involving the Ku protein, which binds DNA ends, and its associated protein kinase.

Curr Biol, 1995 May 1, 5(5), 455 - 8
Molecular chaperones . Resurrection or destruction?
Horwich AL.
Recent studies implicate Hsp104/Clp family chaperones in both protein disaggregation and protein degradation . How do these homologous ring-shaped complexes function in such different ways?

Xenobiotica, 1995 May, 25(5), 469 - 76
Differential induction of rat hepatic microsomal and peroxisomal long-chain and nafenopin-CoA ligases by clofibric acid and di-(2-ethylhexyl)phthalate; Roberts BJ et al.; 1 . Activity of rat hepatic microsomal and peroxisomal long-chain (palmitoyl) and nafenopin-CoA ligases were studied following administration of either clofibric acid, di-(2-ethylhexyl)phthalate (DEHP) or phenobarbitone . 2 . Clofibric acid significantly induced the peroxisomal palmitoyl and nafenopin-CoA ligases, whilst no induction of the equivalent enzymes was observed in the microsomal fraction . 3 . DEHP induced only palmitoyl-CoA formation in peroxisomes, whilst all enzymes were refractory to phenobarbitone treatment . 4 . The enzyme-specific patterns of inductions both intra- and inter-organelle suggest that the palmitoyl and nafenopin-CoA ligases are under different regulatory control . 5 . Modulation of both the rate and extent of nafenopin- and palmitoyl-CoA formation was both agent and organelle specific . 6 . This study highlights the difficulty in delineating the individual roles of both fatty acyl-CoAs and xenobiotic-CoAs in peroxisome proliferation.

J Cell Sci, 1995 May, 108 ( Pt 5), 1817 - 29
Identification of three distinct peroxisomal protein import defects in patients with peroxisome biogenesis disorders; Slawecki ML et al.; Zellweger syndrome, neonatal adrenoleukodystrophy, infantile Refsum's disease, and classical rhizomelic chondrodysplasia punctata are lethal genetic disorders caused by defects in peroxisome biogenesis . We report here a characterization of the peroxisomal matrix protein import capabilities of fibroblasts from 62 of these peroxisome biogenesis disorder patients representing all ten known complementation groups . Using an immunofluorescence microscopy assay, we identified three distinct peroxisomal protein import defects among these patients . Type-1 cells have a specific inability to import proteins containing the PTS1 peroxisomal targeting signal, type-2 cells have a specific defect in import of proteins containing the PTS2 signal, and type-3 cells exhibit a loss of, or reduction in, the import of both PTS1 and PTS2 proteins . Considering that the common cellular phenotype of Zellweger syndrome, neonatal adrenoleukodystrophy and infantile Refsum's disease has been proposed to be a complete defect in peroxisomal matrix protein import, the observation that 85% (40/47) of the type-3 cell lines imported a low but detectable amount of both PTS1 and PTS2 proteins was surprising . Furthermore, different cell lines with the type-3 defect exhibited a broad spectrum of different phenotypes; some showed a complete absence of matrix protein import while others contained 50-100 matrix protein-containing peroxisomes per cell . We also noted certain relationships between the import phenotypes and clinical diagnoses: both type-1 cell lines were from neonatal adrenoleukodystrophy patients, all 13 type-2 cell lines were from classical rhizomelic chondrodysplasia punctata patients, and the type-3 import defect was found in the vast majority of Zellweger syndrome (22/22), neonatal adrenoleukodytrophy (17/19), and infantile Refsum's disease (7/7) patients . Our finding that all type-1 cell lines were from the second complementation group (CG2), all 13 type-2 cell lines were from CG11, and that cells from the eight remaining complementation groups only exhibit the type-3 defect indicates that mutations in particular genes give rise to the different types of peroxisomal protein import defects . This hypothesis is further supported by correlations between certain complementation groups and particular type-3 subphenotypes: all patient cell lines belonging to CG3 and CG10 showed a complete absence of peroxisomal matrix protein import while those from CG6, CG7, and CG8 imported some peroxisomal matrix proteins . However, the fact that cell lines from within particular complementation groups (CG1, CG4) could have different matrix protein import characteristics suggests that allelic heterogeneity also plays an important role in generating different import phenotypes in certain patients.(ABSTRACT TRUNCATED AT 400 WORDS)

Mutat Res, 1995 May, 328(2), 119 - 26
Altered metaphase chromosome structure in xrs-5 cells is not related to its radiation sensitivity or defective DNA break rejoining; Schwartz JL et al.; The chinese hamster ovary (CHO) cell line xrs-5 is a radiation-sensitive derivative of CHO-K1 cells . The xrs-5 cells have a defect in DNA double-strand break rejoining and show alterations in chromosome structure and nuclear morphology . The relationship between radiation sensitivity and metaphase chromosome morphology was examined in 12 'revertant' xrs-5 clones isolated following treatment with 5-azacytidine . nine of the clones were radioresistant while the other three retained xrs-5-like radiation sensitivity . Chromosome morphology reverted to CHO-K1-like characteristics in three of the radioresistant clones and one of the radiosensitive clones suggesting that the over-condensed metaphase chromosome morphology of xrs-5 cells does not underlie its radiation sensitivity . Radiation sensitivity did correlate with DNA double-strand break rejoining ability . The radioresistant clones showing the over-condensed xrs-5-like chromosome morphology were also slightly more sensitive to the topoisomerase II inhibitor etoposide (VP-16) than CHO-K1, suggesting that the over-condensed morphology might be due to alterations in the phosphorylation of chromatin proteins.

Mol Cell Biol, 1995 May, 15(5), 2500 - 8
Phosphotyrosine-dependent interaction of SHC and insulin receptor substrate 1 with the NPEY motif of the insulin receptor via a novel non-SH2 domain; Gustafson TA et al.; The SHC proteins have been implicated in insulin receptor (IR) signaling . In this study, we used the sensitive two-hybrid assay of protein-protein interaction to demonstrate that SHC interacts directly with the IR . The interaction is mediated by SHC amino acids 1 to 238 and is therefore independent of the Src homology 2 domain . The interaction is dependent upon IR autophosphorylation, since the interaction is eliminated by mutation of the IR ATP-binding site . In addition, mutational analysis of the Asn-Pro-Glu-Tyr (NPEY) motif within the juxtamembrane domain of the IR showed the importance of the Asn, Pro, and Tyr residues to both SHC and IR substrate 1 (IRS-1) binding . We conclude that SHC interacts directly with the IR and that phosphorylation of Tyr-960 within the IR juxtamembrane domain is necessary for efficient interaction . This interaction is highly reminiscent of that of IRS-1 with the IR, and we show that the SHC IR-binding domain can substitute for that of IRS-1 in yeast and COS cells . We identify a homologous region within the IR-binding domains of SHC and IRS-1, which we term the SAIN (SHC and IRS-1 NPXY-binding) domain, which may explain the basis of these interactions . The SAIN domain appears to represent a novel motif which is able to interact with autophosphorylated receptors such as the IR.

Stem Cells, 1995 May, 13 Suppl 1, 117 - 28
Cell cycle checkpoints and repair of ionizing radiation damage; Liu VF et al.; Following exposure to ionizing radiation (IR), normal cells activate a delay in any phase of the cell cycle in conjunction with DNA repair mechanisms . Cell cycle delay or arrest is a programmed response that is mutable by a variety of genetic changes . DNA repair mechanisms that are responsible for the repair of otherwise lethal IR-induced double-strand breaks (DSBs) operate in a parallel pathway . The formulation of this pathway has recently been investigated, and new information regarding several mutant cell lines that are unable to execute IR-induced DSB repair are summarized . The scid mutation and defects in Ku proteins have been characterized . Molecular readouts of the properties of IR repair have been identified, including the hyperphosphorylation of the 34 kDa subunit of replication protein A . In addition, we have identified features of the G1/S IR-induced checkpoint that can be influenced by p53 status, genetic background or the levels of cell cycle proteins . A further understanding of the players in these pathways is expected to lead to the identification of molecular markers for ionizing radiation damage . Examination of the changes in these proteins may be valuable in a clinical setting for documenting radiation exposure.

Zh Evol Biokhim Fiziol, 1995 May-Jun, 31(3), 245 - 57
{The search for homologous sequences in the primary structure of dolichol-coupled enzymes}; Shpakov AO; Primary structures of several dolichol-coupled enzymes, mammal and yeast N-acetylglucosaminylphosphote transferases (GPT), yeast mannosyl transferase and dolicholphosphomannosyl transferase were compared . The enzymes presented take part in the synthesis of oligosaccharide precursor used for N-glycosylation of proteins in endoplasmic reticulum of eukaryotic cells . For GPT family equalization of amino acid sequences (AAS) was conducted and the profile of invariability of the primary structure was calculated . At the comparison of AAS in the enzymes outlined with the ones in carbohydrate-binding proteins homologous segments were identified and their possible role was discussed.

Yeast, 1995 Apr 30, 11(5), 467 - 73
Cloning and sequencing of the LEU2 homologue gene of Schwanniomyces occidentalis; Iserentant D et al.; A gene that complements the leu2 mutation of Saccharomyces cerevisiae has been cloned from Schwanniomyces occidentalis . The gene codes for a protein of 379 amino acids . As expected for a Schwanniomyces gene, it has a high AT content, which is also reflected in the codon usage . The sequence homology with other known leu2 complementing genes is low.

J Biol Chem, 1995 Apr 28, 270(17), 10171 - 8
Distinct regions of Cu(I).ACE1 contact two spatially resolved DNA major groove sites; Dobi A et al.; The interaction between the Cu(I).ACE1 (CuACE1) transcription factor and its DNA binding site in the yeast metallothionein gene was studied by systematically altering the DNA sequence through base substitution, modification, and deletions as well as by altering the protein structure through chemical modification . We show here that CuACE1 is comprised of two distinct domains that contact DNA through minor groove interactions located between two major groove interaction sites . The minor groove interactions are shown to be critical for formation of a stable CuACE1.DNA complex . The NH2-terminal segment of ACE1 is shown to contact the 5'-most distal major groove site.

Nature, 1995 Apr 27, 374(6525), 822 - 3
Stimulation of RNA polymerase II transcription initiation by recruitment of TBP in vivo; Klages N et al.; Eukaryotic transcriptional activators may stimulate RNA polymerase II activity by promoting assembly of preinitiation complexes on promoters through their interactions with one or more components of the basal machinery . On the basis of its central role in initiating transcription-complex formation upon binding to the TATA box, the general transcription factor TFIID, which includes the TATA-binding protein (TBP) and several TBP-associated factors, has been implicated as a target for activators . Consistent with this idea, an increasing number of activators have been reported to bind directly to TBP . To assess the functional importance of these in vitro interactions for transcriptional regulation in vivo, we made use of a novel strategy in yeast to show that a physical interaction with TBP is sufficient for a sequence-specific DNA-binding protein to increase initiation of transcription by RNA polymerase II . These results imply that binding of TFIID to promoter elements is a limiting step in transcription complex assembly in vivo.

Biochem Biophys Res Commun, 1995 Apr 26, 209(3), 823 - 31
The association and involvement of some members of the P1 protein family in a cell-free DNA replication of Xenopus eggs; Someya A et al.; Two types of antibodies were prepared one directed against an oligopeptide specific to P1Cdc46, a mammalian homologue of yeast CDC46, and the other against an oligopeptide highly conserved in the P1 protein family . Immunoprecipitation with anti-P1Cdc46 antibody revealed that some members of the P1 protein family were coprecipitated with P1Cdc46 in the soluble fraction of Xenopus S phase extracts . Immunoblot analysis showed that all of the coprecipitated proteins reacted with the antibody against an oligopeptide, designated as a DEAD box motif, a highly conserved sequence in the P1 protein family . The immunodepleted extracts with anti-P1Cdc46 antibody-bound beads showed much lower activity of DNA replication than the mock-treated extracts . Recovery of replication was achieved by supplementing depleted extracts with the proteins eluted from anti-P1Cdc46 antibody-bound beads . These findings suggest that the proteins contained in the P1 protein family were associated in the extracts and that the multiprotein complex of the family plays an essential role in a cell-free DNA replication of Xenopus eggs.

Proc Natl Acad Sci U S A, 1995 Apr 25, 92(9), 4011 - 5
Human acetyl-CoA carboxylase: characterization, molecular cloning, and evidence for two isoforms; Abu-Elheiga L et al.; We have cloned and sequenced the cDNA coding for human HepG2 acetyl-CoA carboxylase (ACC; EC 6.4.1.2) . The sequence has an open reading frame of 7038 bp that encode 2346 amino acids (M(r), 264,737) . The C-terminal 2.6-kb sequence is very different from that recently reported for human ACC (Ha, J., Daniel, S., Kong, I.-S., Park, C.-K., Tae, H.-J . & Kim, K.-H . {1994} Eur . J . Biochem . 219, 297-306) . Northern blot analysis revealed that the ACC mRNA is approximately 10 kb in size and that its level varies among the tissues tested . Evidence is presented to show that the human ACC gene is 200-480 kbp in size and maps to chromosome 17q12 . We also provide evidence for the presence of another ACC-like gene with similarly sized mRNA but tissue-specific expression different from that of the ACC gene reported herein . That this second ACC-like gene encodes the 280-kDa carboxylase is not ruled out.

Proc Natl Acad Sci U S A, 1995 Apr 25, 92(9), 3963 - 7
A simple p53 functional assay for screening cell lines, blood, and tumors; Flaman JM et al.; Mutations in the p53 gene are implicated in the pathogenesis of half of all human tumors . We have developed a simple functional assay for p53 mutation in which human p53 expressed in Saccharomyces cerevisiae activates transcription of the ADE2 gene . Consequently, yeast colonies containing wild-type p53 are white and colonies containing mutant p53 are red . Since this assay tests the critical biological function of p53, it can distinguish inactivating mutations from functionally silent mutations . By combining this approach with gap repair techniques in which unpurified p53 reverse transcription-PCR products are cloned by homologous recombination in vivo it is possible to screen large numbers of samples and multiple clones per sample for biologically important mutations . This means that mutations can be detected in tumor specimens contaminated with large amounts of normal tissue . In addition, the assay detects temperature-sensitive mutants, which give pink colonies . We show here that this form of p53 functional assay can be used rapidly to detect germline mutations in blood samples, somatic mutations in tumors, and mutations in cell lines.

Proc Natl Acad Sci U S A, 1995 Apr 25, 92(9), 3884 - 7
Repressible cation-phosphate symporters in Neurospora crassa; Versaw WK et al.; The filamentous fungus Neurospora crassa possesses two nonhomologous high-affinity phosphate permeases, PHO-4 and PHO-5 . We have isolated separate null mutants of these permeases, allowing us to study the remaining active transporter in vivo in terms of phosphate uptake and sensitivity to inhibitors . The specificity for the cotransported cation differs for PHO-4 and PHO-5, suggesting that these permeases employ different mechanisms for phosphate translocation . Phosphate uptake by PHO-4 is stimulated 85-fold by the addition of Na+, which supports the idea that PHO-4 is a Na(+)-phosphate symporter . PHO-5 is unaffected by Na+ concentration but is much more sensitive to elevated pH than is PHO-4 . Presumably, PHO-5 is a H(+)-phosphate symporter . Na(+)-coupled symport is usually associated with animal cells . The finding of such a system in a filamentous fungus is in harmony with the idea that the fungal and animal kingdoms are more closely related to each other than either is to the plant kingdom.

J Biol Chem, 1995 Apr 21, 270(16), 9676 - 82
Multiple promoters in rat acyl-CoA synthetase gene mediate differential expression of multiple transcripts with 5'-end heterogeneity; Suzuki H et al.; Nucleotide sequence analysis of six independently isolated cDNAs for rat acyl-CoA synthetase (ACS) revealed three forms of ACS mRNA, designated form-A, -B, and -C mRNAs, which differ in their 5'-untranslated regions . Form-A mRNA was preferentially detected in normal and peroxisome-induced livers, whereas form-B mRNA was found in peroxisome-induced livers but not in normal livers and hearts, and form-C mRNA was preferentially found in normal hearts and peroxisome-induced livers . Analysis of two overlapping genomic clones for the rat ACS gene revealed that the three 5'-untranslated regions of the mRNAs are individually encoded by three different exons located within a 20-kilobase genomic fragment . The transcription start sites of the three forms of ACS mRNA were determined and nucleotide sequences of 5'-upstream regions of the three 5'-end exons were determined . The 5'-upstream regions were fused to the chloramphenicol acetyltransferase gene and transcription units of the three forms of ACS mRNAs were determined . These data indicate that the three forms of ACS mRNA with 5'-end heterogeneity are generated by alternative transcription from three promoters in the rat ACS gene.

Science, 1995 Apr 21, 268(5209), 436 - 9
Measurement of interhelical electrostatic interactions in the GCN4 leucine zipper; Lumb KJ et al.; The dimerization specificity of the bZIP transcription factors resides in the leucine zipper region . It is commonly assumed that electrostatic interactions between oppositely charged amino acid residues on different helices of the leucine zipper contribute favorably to dimerization specificity . Crystal structures of the GCN4 leucine zipper contain interhelical salt bridges between Glu20 and Lys15' and between Glu22 and Lys27' . 13C-nuclear magnetic resonance measurements of the glutamic acid pKa values at physiological ionic strength indicate that the salt bridge involving Glu22 does not contribute to stability and that the salt bridge involving Glu20 is unfavorable, relative to the corresponding situation with a neutral (protonated) Glu residue . Moreover, the substitution of Glu20 by glutamine is stabilizing . Thus, salt bridges will not necessarily contribute favorably to bZIP dimerization specificity and may indeed be unfavorable, relative to alternative neutral-charge interactions.

Virology, 1995 Apr 20, 208(2), 467 - 77
Weak transcriptional activation is sufficient for transformation by v-Myb; Engelke U et al.; The v-myb oncogene causes monoblastic leukemia in chickens and transforms avian myelomonocytic cells in vitro, v-Myb is a short-lived nuclear protein which binds to DNA in a sequence-specific manner and can activate gene expression in transient DNA transfections . Analysis of a series of v-Myb mutants has shown that the ability to activate transcription appears to be required for leukemic transformation . We have systematically investigated transcriptional activation by v-Myb and have made several new observations: (i) v-Myb is a very weak activator when compared to GAL4; (ii) very weak transcriptional activation by v-Myb is sufficient for transformation, whereas very strong transcriptional activation by a v-Myb-VP16 fusion protein is not; and (iii) v-Myb can activate transcription by two genetically distinct mechanisms, only one of which requires the presence of Myb-binding sites.

Arch Biochem Biophys, 1995 Apr 20, 318(2), 251 - 63
Interaction of Cu,Zn superoxide dismutase with hydrogen sulfide; Searcy DG et al.; Addition of HS- enhanced the O(2-)-scavenging activity of bovine erythrocyte Cu,Zn superoxide dismutase (EC 1.15.1.1) by about twofold . The positive effect was measured using a diverse selection of SOD activity assays, and cannot be an artifact restricted to any single technique . Km values for HS- varied in different assay techniques, but we estimate Km approximately 80 microM HS- . In contrast to HS-, other small molecules tested with SOD either had little effect or were inhibitory . Consumption of HS- and O2- occurred in nearly 1:1 mole ratio . The products were H2O2 and sulfane sulfur, such as either elemental sulfur or polysulfide . Binding of HS- to the enzyme was rapid, with k > 10(7) M-1 s-1 . The resulting complex exhibited a Cu-to-S charge-transfer absorbance band at 345 nm and an altered Cu(II) EPR spectrum . Taken together, these observations suggest that HS- binds at the catalytic Cu center of SOD and can be a genuine substrate of the enzyme.

Biochemistry, 1995 Apr 18, 34(15), 5054 - 9
Altering the regiospecificity of androstenedione hydroxylase activity in P450s 2a-4/5 by a mutation of the residue at position 481; Iwasaki M et al.; Mouse P450 2a-5 (coumarin 7-hydroxylase) acquires androstenedione (AD) hydroxylase activity by substituting Phe at position 209 with Asn . However, this mutant P450 2a-5 (F209N) and the corresponding mutant P450 2a-4 (L209N) exhibit different regiospecificites of androstenedione (AD) hydroxylase activity . While the former mutant catalyzes both AD 15 alpha- and 7 alpha-hydroxylase activities at similar rates, the latter mutant maintains the original high specificity of AD 15 alpha-hydroxylase activity . The AD hydroxylase activities in chimeric enzymes of the mutants L209N and F209N show that the regiospecificites are determined by the carboxy-terminal halves of the P450 molecules . Mutations at each of the four different residues within the carboxy-terminal halves indicate that the differences in regiospecificity are determined by the Val/Ala mutation at position 481 . As the size of the hydrophobic amino acid at position 481 becomes larger (Ala < Val < Ile), the regiospecificities toward the C15 position of the AD molecule are dramatically increased . The regiospecificity is also increased by placing positively-charged Arg at position 481, although the remaining 15 alpha-hydroxylase activity in this mutant is considerably lower than the other P450s . The results indicate that the size of the residue at position 481 is a key factor in regulating the regiospecificity of AD hydroxylase activity in the P450s . Modeling AD in the substrate-heme pocket of bacterial P450 101A provided further support that residue 481 may reside near the steroid molecule so as to possibly affect the AD hydroxylase activity.

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