Radiat Res, 1996 Jan, 145(1), 39 - 46 Induction and repair of chromosome aberrations in scid cells measured by premature chromosome condensation; Evans JW et al.; Severe combined immunodeficient (scid) murine cells, which are defective in both repair of DNA double-strand breaks and V(D)J recombination, are deficient in DNA-dependent protein kinase (DNA-PK), a protein which forms an activated complex with the DNA end-binding Ku proteins (p80 and p70) upon association with damaged DNA . Xrs 5 cells are deficient in the Ku p80 protein and also fail to form an active DNA-PK repair complex . Since both scid and xrs cells are defective in the same protein complex, we compared the kinetics of chromosome repair in scid cells to results published previously for xrs 5 cells . C.B-17 cells, scid cells and scid cells complemented with a single human chromosome 8 were irradiated with 6 Gy and allowed to repair from 0-24 h before fusion to HeLa cells for chromosome condensation . Breaks and dicentrics were visualized by fluorescence in situ hybridization . All cells had the same initial amount of chromosome damage, but scid cells had a slower rate of rejoining, more unrejoined breaks and more dicentrics than C.B-17 and scid cells with human chromosome 8 . The scid cells appear to respond differently than xrs 5 cells, despite both cells lacking an essential component of the same DNA repair complex. Mol Cell Biol, 1996 Jan, 16(1), 328 - 37 Three classes of mutations in the A subunit of the CCAAT-binding factor CBF delineate functional domains involved in the three-step assembly of the CBF-DNA complex; Sinha S et al.; The mammalian CCAAT-binding factor CBF (also called NF-Y or CP1) consists of three subunits, CBF-A, CBF-B, and CBF-C, all of which are required for DNA binding and present in the CBF-DNA complex . In this study we first established the stoichiometries of the CBF subunits, both in the CBF molecule and in the CBF-DNA complex, and showed that one molecule of each subunit is present in the complex . To begin to understand the interactions between the CBF subunits and DNA, we performed a mutational analysis of the CBF-A subunit . This analysis identified three classes of mutations in the segment of CBF-A that is conserved in Saccharomyces cerevisiae and mammals . Analysis of the first class of mutants revealed that a major part of the conserved segment was essential for interactions with CBF-C to form a heterodimeric CBF-A/CBF-C complex . The second class of mutants identified a segment of CBF-A that is necessary for interactions between the CBF-A/CBF-C heterodimer and CBF-B to form a CBF heterotrimer . The third class defined a domain of CBF-A involved in binding the CBF heterotrimer to DNA . The second and third classes of mutants acted as dominant negative mutants inhibiting the formation of a complex between the wild-type CBF subunits and DNA . The segment of CBF-A necessary for DNA binding showed sequence homology to a segment of CBF-C . Interestingly, these sequences in CBF-A and CBF-C were also homologous to the sequences in the histone-fold motifs of histones H2B and H2A, respectively, and to the archaebacterial histone-like protein HMf-2 . We discuss the functional domains of CBF-A and the properties of CBF in light of these sequence homologies and propose that an ancient histone-like motif in two CBF subunits controls the formation of a heterodimer between these subunits and the assembly of a sequence-specific DNA-protein complex. Mol Cell Biol, 1996 Jan, 16(1), 247 - 57 Regulation of the G-protein-coupled alpha-factor pheromone receptor by phosphorylation; Chen Q et al.; The alpha-factor pheromone receptor activates a G protein signaling cascade that stimulates MATa yeast cells to undergo conjugation . The cytoplasmic C terminus of the receptor is not necessary for G protein activation but instead acts as a regulatory domain that promotes adaptation to alpha-factor . The role of phosphorylation in regulating the alpha-factor receptor was examined by mutating potential phosphorylation sites . Mutation of the four most distal serine and threonine residues in the receptor C terminus to alanine caused increased sensitivity to alpha-factor and a delay in recovering from a pulse of alpha-factor . 32PO4 labeling experiments demonstrated that the alanine substitution mutations decreased the in vivo phosphorylation of the receptor . Phosphorylation apparently alters the regulation of G protein activation, since neither receptor number nor affinity for ligand was significantly altered by mutation of the distal phosphorylation sites . Furthermore, mutation of the distal phosphorylation sites in a receptor mutant that fails to undergo ligand-stimulated endocytosis caused increased sensitivity to alpha-factor, which suggests that regulation by phosphorylation can occur at the cell surface and is independent of endocytosis . Mutation of the distal serine and threonine residues of the receptor also caused a slight defect in alpha-factor-induced morphogenesis, but the defect was not as severe as the morphogenesis defect caused by truncation of the cytoplasmic C terminus of the receptor . These distal residues in the C terminus play a special role in receptor regulation, since mutation of the next five adjacent serine and threonine residues to alanine did not affect the sensitivity to alpha-factor . Altogether, these results indicate that phosphorylation plays an important role in regulating alpha-factor receptor function. Gene, 1995 Dec 29, 167(1-2), 337 - 8 Nucleotide sequence of the Aspergillus niger srpA gene; Thompson SA et al.; The Aspergillus niger (An) gene srpA, encoding a protein with homology to the signal recognition particle (SRP) 54-kDa protein from Saccharomyces cerevisiae (Sc), has been isolated and the nucleotide sequence determined . The putative An srpA gene is comprised of two exons of 78 and 1527 bp separated by a 49-bp intron, and encodes a protein of 534 amino acids that is 53% identical to Sc SRP54. Gene, 1995 Dec 29, 167(1-2), 303 - 6 Cloning and characterization of a member of the MST subfamily of Ste20-like kinases; Creasy CL et al.; We have identified a second human homology of the yeast Ste20 protein kinase family, which we designate MST2 . MST2 is most similar to the previously identified MST1 protein kinase (78% identity, 88% similarity) . Northern analysis indicates that MST2 mRNA is expressed at high levels in adult kidney, skeletal and placental tissues and at very low levels in adult heart, lung, liver and brain tissues . An in vitro kinase assay indicates that MST2 can phosphorylate an exogenous substrate, as well as itself, and phospho-amino-acid analysis indicates that it is a serine/threonine protein kinase . The identification of MST2 suggests that there may be subfamilies of Ste20-like protein kinases and that MST1 and MST2 may define one of these subfamilies. Gene, 1995 Dec 29, 167(1-2), 197 - 201 Isolation and characterization of a cDNA encoding Arabidopsis thaliana 3-hydroxy-3-methylglutaryl-coenzyme A synthase; Montamat F et al.; An 1.7-kb Arabidopsis thaliana (At) cDNA was isolated by complementation of a bap1 mutation affecting the transport of branched-chain amino acids (aa) in the yeast Saccharomyces cerevisiae . The determination of the nucleotide (nt) sequence revealed an open reading frame of 1383 nt which may encode a protein of 461 aa with a predicted molecular mass of 51,038 Da . The deduced aa sequence exhibited strong similarities with mammalian 3-hydroxy-3-methylglutaryl-coenzyme A synthase (HMGS) sequences . Although former biochemical studies have suggested that acetoacetyl-coenzyme A thiolase (AACT) and HMGS activities were carried by a single protein in plants, complementation studies and measurements of enzymatic activities clearly showed that the At HMGS is devoid of AACT activity. J Biol Chem, 1995 Dec 29, 270(52), 30914 - 8 Identification of Epstein-Barr virus nuclear antigen 1 protein domains that direct interactions at a distance between DNA-bound proteins; Laine A et al.; The EBNA1 protein of Epstein-Barr virus (EBV) binds to and activates DNA replication from the EBV latent origin of replication, oriP, via a direct interaction with the two noncontiguous subelements of oriP . The EBNA1 molecules bound to the oriP subelements interact efficiently with each other by a DNA looping mechanism . We have previously mapped a region of EBNA1 (termed the looping region) that is required to mediate the interaction of the EBNA1 molecules bound to the oriP subelements . We now demonstrate that two fragments of this region of EBNA1, which consist largely of an eight amino acid repeat, can mediate homotypic interactions when transferred to another DNA-binding protein . Protein interactions mediated by the EBNA1 looping region appear to be dependent on DNA binding since these interactions were detected between DNA-bound forms of the proteins only. FEBS Lett, 1995 Dec 27, 377(3), 505 - 11 Protein disulfide isomerase mutant lacking its isomerase activity accelerates protein folding in the cell; Hayano T et al.; We investigated the effect of protein disulfide isomerase (PDI) on in vivo protein folding of human lysozyme (h-LZM) in a specially constructed yeast coexpression system . Coexpression with PDI increased the amounts of intracellular h-LZM with the native conformation, leading to an increase in h-LZM secretion . The results indicated that PDI is a real catalyst of protein folding in the cell . The secretion of h-LZM increased even when both active sites of PDI were disrupted, suggesting that the effect of PDI resulted from a function other than the formation of disulfide bonds . This is the first finding that PDI without isomerase activity accelerates protein folding in vivo. Biochemistry, 1995 Dec 26, 34(51), 16543 - 51 Is the continuity of the domains required for the correct folding of a two-domain protein? Ritco-Vonsovici M, Minard P, Desmadril M, Yon JM. The role of domains in protein folding has been widely studied and discussed . Nevertheless, it is not clear whether the continuity of the domains in a protein is an essential requirement in determining the folding pathway . Previous studies have shown that the isolated structural domains of the two-domain monomeric enzyme, yeast phosphoglycerate kinase (yPGK), are able to fold independently into a quasinative structure, but they neither reassociate nor generate a functional enzyme {Minard, P., Hall, L., Betton, J . M., Missiakas, D., & Yon, J . M . (1989) Protein Eng . 3, 55-60; Fairbrother, W . J., Bowen, D., Hall, L., Williams, R . J . P . (1989) Eur . J . Biochem . 184, 617-625; Missiakas, D., Betton, J . M., Minard, P., & Yon, J . M . (1990) Biochemistry 29, 8683-8689} . In the present work, two circularly permuted variants of the yPGK gene were constructed . The natural adjacent chain termini were directly connected and the new extremities were created within the N-domain (at residues 71 and 72) or the C-domain (at residues 291 and 292), respectively . These two proteins were overexpressed and purified . They exhibit 14% and 23% of the wild-type enzyme activity, respectively . The two mutants fold in a compact conformation with slight changes in the secondary and tertiary structure probably related to the presence of a heterogeneous population of molecules . The unfolding studies reveal a large decrease in stability . From the present data it appears that, although the circular permutations induce some perturbations in the structure and stability of the protein, the continuity of the domains is not required for the protein to reach a native-like and functional structure. Science, 1995 Dec 22, 270(5244), 2008 - 11 Identification of a member of the MAPKKK family as a potential mediator of TGF-beta signal transduction; Yamaguchi K et al.; The mitogen-activated protein kinase (MAPK) pathway is a conserved eukaryotic signaling module that converts receptor signals into various outputs . MAPK is activated through phosphorylation by MAPK kinase (MAPKK), which is first activated by MAPKK kinase (MAPKKK) . A genetic selection based on a MAPK pathway in yeast was used to identify a mouse protein kinase (TAK1) distinct from other members of the MAPKKK family . TAK1 was shown to participate in regulation of transcription by transforming growth factor-beta (TGF-beta) . Furthermore, kinase activity of TAK1 was stimulated in response to TGF-beta and bone morphogenetic protein . These results suggest that TAK1 functions as a mediator in the signaling pathway of TGF-beta superfamily members. J Biol Chem, 1995 Dec 22, 270(51), 30567 - 70 Uncoupled packaging of targeting and cargo molecules during transport vesicle budding from the endoplasmic reticulum; Yeung T et al.; Formation of vesicular intermediates in protein transport between the endoplasmic reticulum and the Golgi apparatus involves a mechanism that sorts and packages two classes of molecules into transport vesicles: targeting molecules, which are required for targeting and consumption of vesicular intermediates, and cargo proteins . In order to examine the importance of cargo in this packaging reaction, we developed an in vitro assay that quantifies vesicle formation based on segregation of targeting molecules . Here we document that endoplasmic reticulum devoid of cargo proteins is competent in the formation and release of targeting molecule-containing vesicles in a fashion indistinguishable from its normal counterpart . This observation implies that packaging of cargo proteins may be uncoupled from the recruitment of targeting molecules during vesicle budding from the endoplasmic reticulum . Using the same assay, we demonstrate that the packaging of targeting molecules into vesicles is not dependent on the lumenal chaperone, BiP (Kar2p). J Biol Chem, 1995 Dec 22, 270(51), 30408 - 14 Identification of a family of closely related human ubiquitin conjugating enzymes; Jensen JP et al.; Two very closely related human E2 ubiquitin conjugating enzymes, UbfH5B and UbcH5C, have been identified . These enzymes are products of distinct genes and are 88-89% identical in amino acid sequence to the recently described human E2, UbcH5 (now designated UbcH5A), UbcH5A-C are homologous to a family of five ubiquitin conjugating enzymes from Arabidopsis thaliana, AtUBC8-12 . They are also closely related to Saccharomyces cerevisiae ScUBC4 and ScUBC5, which are involved in the stress response, and play a central role in the targeting of short-lived regulatory proteins for degradation . mRNAs encoding UbcH5A-C were co-expressed in all cell lines and tissues evaluated, with UbcH5C transcripts generally expressed at the highest levels . Analysis of Southern blots suggests that there are likely to be other related members of this family . Both UbcH5B and UbcH5C form thiol ester adducts with ubiquitin, and have activities similar to UbcH5A and AtUBC8 in the conjugation of ubiquitin to target proteins in the presence of the human ubiquitin protein ligase E6-AP . These results establish the existence of a highly conserved, and widely expressed, family of human ubiquitin conjugating enzymes. J Biol Chem, 1995 Dec 22, 270(51), 30377 - 83 Calf 5' to 3' exo/endonuclease must slide from a 5' end of the substrate to perform structure-specific cleavage; Murante RS et al.; Calf 5' to 3' exo/endonuclease, the counterpart of the human FEN-1 and yeast RTH-1 nucleases, performs structure-specific cleavage of both RNA and DNA and is implicated in Okazaki fragment processing and DNA repair . The substrate for endonuclease activity is a primer annealed to a template but with a 5' unannealed tail . The results presented here demonstrate that the nuclease must enter the 5' end of the unannealed tail and then slide to the region of hybridization where the cleavage occurs . The presence of bound protein or a primer at any point on the single-stranded tail prevents cleavage . However, biotinylation of a nucleotide at the 5' end or internal to the tail does not prevent cleavage . The sliding process is bidirectional . If the nuclease slides onto the tail, later binding of a primer to the tail traps the nuclease between the primer binding site and the cleavage site, preventing the nuclease from departing from the 5' end . A model for 5' entry, sliding, and cleavage is presented . The possible role of this unusual mechanism in Okazaki fragment processing, DNA repair, and protection of the replication fork from inappropriate endonucleolytic cleavage is presented. Proc Natl Acad Sci U S A, 1995 Dec 19, 92(26), 12490 - 4 UME6 is a central component of a developmental regulatory switch controlling meiosis-specific gene expression; Steber CM et al.; The UME6 gene of Saccharomyces cerevisiae was identified as a mitotic repressor of early meiosis-specific gene expression . It encodes a Zn2Cys6 DNA-binding protein which binds to URS1, a promoter element needed for both mitotic repression and meiotic induction of early meiotic genes . This paper demonstrates that a complete deletion of UME6 causes not only vegetative derepression of early meiotic genes during vegetative growth but also a significant reduction in induction of meiosis-specific genes, accompanied by a severe defect in meiotic progression . After initiating premeiotic DNA synthesis the vast majority of cells (approximately 85%) become arrested in prophase and fail to execute recombination; a minority of cells (approximately 15%) complete recombination and meiosis I, and half of these form asci . Quantitative analysis of the same early meiotic transcripts that are vegetatively derepressed in the ume6 mutant, SPO11, SPO13, IME2, and SPO1, indicates a low level of induction in meiosis above their vegetative derepressed levels . In addition, the expression of later meiotic transcripts, SPS2 and DIT1, is significantly delayed and reduced . The expression pattern of early meiotic genes in ume6-deleted cells is strikingly similar to that of early meiotic genes with promoter mutations in URS1 . These results support the view that UME6 and URS1 are part of a developmental switch that controls both vegetative repression and meiotic induction of meiosis-specific genes. Proc Natl Acad Sci U S A, 1995 Dec 19, 92(26), 12151 - 5 Phospholipase D signaling is essential for meiosis; Rose K et al.; Phospholipid metabolism plays an important role in cellular regulation by generating second messengers for signal transduction . Many stimuli activate a phospholipase D, which catalyzes the hydrolysis of phosphatidylcholine, producing phosphatidic acid and choline . Here we report that the yeast SP014 gene, which is essential for meiosis {Honigberg, S . M., Conicella, C . & Esposito, R . E . (1992) Genetics 130, 703-716}, encodes a phospholipase D . SP014 RNA and protein activity are induced during late meiotic prophase, and the enzyme has properties similar to mammalian phosphatidylinositol 4,5-bisphosphate-regulated phospholipase D . Characterization of an unusual allele of SP014 defines regions of the protein important for enzyme catalysis and regulation . These results implicate phospholipase D signaling in regulating cellular differentiation. Experientia, 1995 Dec 18, 51(12), 1110 - 5 Consensus phylogeny of Dictyostelium; Loomis WF et al.; The evolutionary relationship of Dictyostelium discoideum to the yeasts, fungi, plants, and animals is considered on the basis of physiological, morphological and molecular characteristics . Previous analyses of five proteins indicated that Dictyostelium diverged after the yeasts but before the metazoan radiation . However, analyses of the small ribosomal subunit RNA indicated divergence prior to the yeasts . We have extended the molecular phylogenetic analyses to six more proteins and find consistent evidence for a more recent common ancestor with metazoans than yeast . A consensus phylogeny generated from these new results by both distance matrix and parsimony analyses establishes Dictyostelum's place in evolution between the yeasts Saccharomyces cerevisiae and Schizzosaccharomyces pombe and the worm Caenorhabditis elegans. J Biol Chem, 1995 Dec 15, 270(50), 29983 - 90 Evidence for an anti-parallel orientation of the ligand-activated human androgen receptor dimer; Langley E et al.; Domain interactions of the human androgen receptor (AR) dimer were investigated using a protein-protein interaction assay in which the NH2- and carboxyl-terminal regions of human AR were fused to the Saccharomyces cerevisiae GAL4 DNA-binding domain and herpes simplex virus VP16 transactivation domain to produce chimeric proteins . Transcriptional activation of a GAL4 luciferase reporter vector up to 100-fold was greater than Fos/Jun leucine zipper binding, indicating stable AR interaction between AR NH2-terminal residues 1-503 and steroid-binding domain residues 624-919 that was specific for and dependent on androgen binding to the steroid-binding domain and was inhibited by anti-androgen binding . Deletion mutagenesis within the NH2-terminal region indicated transactivation domain residues 142-337 were not required for dimerization, whereas deletions near the NH2 terminus (delta 14-150) or NH2-terminal to the DNA-binding domain (delta 339-499) reduced or eliminated the AR interaction, respectively . An NH2-/NH2-terminal interaction was also observed, but no interaction was detected between ligand-free or bound steroid-binding domains . The results indicate that high affinity androgen binding promotes interactions between the NH2-terminal and steroid-binding domains of human AR, raising the possibility of an androgen-induced anti-parallel AR dimer. J Biol Chem, 1995 Dec 15, 270(50), 29848 - 53 The mitochondrial protein import machinery . Role of ATP in dissociation of the Hsp70.Mim44 complex; von Ahsen O et al.; Interaction of preproteins with the heat shock protein Hsp70 in the mitochondrial matrix is required for driving protein transport across the mitochondrial inner membrane . Binding of mt-Hsp70 to the protein Mim44 of the inner membrane import site seems to be an essential part of an ATP-dependent reaction cycle . However, the available results on the role played by ATP are controversial . Here we demonstrate that the mt-Hsp70.Mim44 complex contains ADP and that a nonhydrolyzable analog of ATP dissociates the mt-Hsp70.Mim44 complex in the presence of potassium ions . The previously reported requirement of ATP hydrolysis for complex dissociation was due to the use of a nonphysiological concentration of sodium ions . In the presence of potassium ions, mt-Hsp70 undergoes a conformational change that is not observed with a mutant Hsp70 defective in binding to Mim44 . The mutant Hsp70 is able to bind substrate proteins, differentiating binding to Mim44 from binding to substrate proteins . We conclude that binding of ATP, not hydrolysis, is required to dissociate the mt-Hsp70.Mim44 complex and that the reaction cycle includes an ATP-induced conformational change of mt-Hsp70. Cell, 1995 Dec 15, 83(6), 915 - 24 Coronin involved in phagocytosis: dynamics of particle-induced relocalization visualized by a green fluorescent protein Tag; Maniak M et al.; Coronin is a protein involved in cell locomotion and cytokinesis of Dictyostelium discoideum . Here we show that coronin is strongly enriched in phagocytic cups formed in response to particle attachment . A fusion of coronin with green fluorescent protein (GFP) accumulates in the cups within less than 1 min upon attachment of a particle and is gradually released from the phagosome within 1 min after engulfment is completed . Phagocytic cup formation competes with leading edge formation and can be interrupted at any stage . When the cup regresses, coronin dissociates from the site of accumulation . TRITC-labeled yeast cells have been used to assay phagocytosis quantitatively in wild-type and coronin-null cells . In the mutant, the rate of uptake is reduced to about one third, which shows that coronin contributes to the efficiency of phagocytosis to about the same extent as it improves the speed of cell locomotion. Biochim Biophys Acta, 1995 Dec 14, 1245(3), 317 - 24 Dielectric behavior of non-spherical cells in culture; Asami K et al.; In order to study dielectric behavior of non-spherical cells growing in suspension culture, a dielectric theory has been developed based on the shell-ellipsoid model that is a conducting ellipsoid covered with a thin insulating shell . The theory predicts three dielectric relaxations for a suspension of ellipsoidal cells with three different semiaxes . For prolate spheroidal cells with two different semiaxes that show two dielectric relaxations the effect of the axial ratio on the dielectric relaxations was examined in detail . The low-frequency relaxation attributed to the component along the major axis strongly depends on the axial ratio, while the high-frequency relaxation due to the component along the minor axis is rather insensitive to the axial ratio . The theory is also applicable to simulation of dielectric behavior of yeast cells in synchronized and asynchronized culture by assuming that budding yeast cells are prolate spheroids. Mol Gen Genet, 1995 Dec 10, 249(4), 447 - 55 Identification of a centromeric activity in the autonomously replicating TRA region allows improvement of the host-vector system for Candida maltosa; Ohkuma M et al.; A centromeric activity was identified in the previously isolated 3.8 kb DNA fragment that carries an autonomously replicating sequence (ARS) from the yeast Candida maltosa . Plasmids bearing duplicated copies of the centromeric DNA (dicentric plasmids) were physically unstable and structural rearrangements of the dicentric plasmids occurred frequently in the transformed cells . The centromeric DNA activity was dissociated from the ARS, which is 0.2 kb in size, and was delimited to a fragment at least 325 bp in length . The centromeric DNA region included the consensus sequences of CDEI (centromeric DNA element I) and an AT-rich CDEII-like region of Saccharomyces cerevisiae but had no homology to the functionally critical CDEIII consensus . A plasmid bearing the whole 3.8 kb fragment was present in 1-2 copies per cell and was maintained stably even under non-selective culture conditions, while a plasmid having only the 0.2 kb ARS was unstable and accumulated to high copy numbers . The high-copy-number plasmid allowed us to overexpress a gene to a high level, which had never been attained before, under the control of both constitutive and inducible promoters in C . maltosa. Science, 1995 Dec 8, 270(5242), 1671 - 4 Separation of origin recognition complex functions by cross-species complementation; Ehrenhofer-Murray AE et al.; Transcriptional silencing at the HMRa locus of Saccharomyces cerevisiae requires the function of the origin recognition complex (ORC), the replication initiator of yeast . Expression of a Drosophila melanogaster Orc2 complementary DNA in the yeast orc2-1 strain, which is defective for replication and silencing, complemented the silencing defect but not the replication defect; this result indicated that the replication and silencing functions of ORC were separable . The orc2-1 mutation mapped to the region of greatest homology between the Drosophila and yeast proteins . The silent state mediated by DmOrc2 was epigenetic; it was propagated during mitotic divisions in a relatively stable way, whereas the nonsilent state was metastable . In contrast, the silent state was erased during meiosis. J Mol Biol, 1995 Dec 8, 254(4), 657 - 67 Crystal structure of a bZIP/DNA complex at 2.2 A: determinants of DNA specific recognition; Keller W et al.; The X-ray structure of the GCN4-bZIP protein bound to DNA containing the ATF/CREB recognition sequence has been refined at 2.2 A . The water-mediated interactions between the basic domain and DNA are revealed, and combined with a more accurate description of the direct contacts, further clarify how binding specificity is achieved . Water molecules extend the interactions of both invariant basic domain residues, asparagine 235 and arginine 243, beyond their direct base contacts . The slight bending of the basic domain alpha-helix around the DNA facilitates the linking of arginine 241, 243 and 245 to main-chain carbonyl oxygen atoms via water molecules, apparently stabilizing interactions with the DNA. J Biol Chem, 1995 Dec 8, 270(49), 29433 - 8 Hsp90 mutants disrupt glucocorticoid receptor ligand binding and destabilize aporeceptor complexes; Bohen SP; In order to attain competence to respond to hormone, certain steroid hormone receptors must be assembled into hetero-oligomeric aporeceptor complexes, containing Hsp90 and other proteins . Members of the Hsp90 gene family are highly conserved, strongly expressed, and required for viability in eukaryotic organisms . To elucidate the role of Hsp90 in the activity of steroid hormone receptors in vivo, four Hsp90 mutatns, which cause defects in glucocorticoid receptor (GR) signaling, but support the viability of Saccharomyces cerevisiae, were previously isolated (Bohen, S . P., and Yamamoto, K . R . (1993) Proc . Natl . Acad . Sci . U.S.A . 90, 11424-11428) . In this study, I characterize the effects of the Hsp90 mutants on GR ligand response, ligand binding activity, and aporeceptor complex stability . The mutants fall into two classes . Three of the Hsp90 mutants cause defects in GR ligand binding in vivo and form aporeceptor complexes that are unstable in vitro, relative to those containing wild-type Hsp90 . The other mutant affects GR signaling, but aporeceptor complexes with this mutant are not defective for ligand binding or stability . These findings indicate that the binding of Hsp90 to GR in the aporeceptor complex is insufficient to induce a high ligand affinity conformation, rather the high ligand affinity to GR requires a specific interaction with Hsp90, which is altered by certain Hsp90 mutants. J Biol Chem, 1995 Dec 8, 270(49), 29151 - 61 Determining the requirements for cooperative DNA binding by Swi5p and Pho2p (Grf10p/Bas2p) at the HO promoter; Brazas RM et al.; SW15 encodes a zinc finger DNA binding protein required for the transcription of the Saccharomyces cerevisiae HO gene, and PHO2 encodes a homeodomain DNA binding protein . In vitro biochemical studies using purified Swi5p and Pho2p proteins have demonstrated that Swi5p and Pho2p bind cooperatively to the HO promoter . In this report we investigate the regions of the Swi5p and Pho2p proteins required for cooperative DNA binding . The analysis of each protein gives a similar result: the zinc finger or homeodomain DNA binding domains are each sufficient for in vitro DNA binding, but additional regions of each protein are required for cooperative DNA binding . In vitro and in vivo experiments were conducted with promoters with altered spacing between the Pho2p and Swi5p binding sites . Mutations that increased the distance between the two binding sites had minimal effects on either in vitro cooperative DNA binding or in vivo upstream activating sequence activity . These observations suggest that the interaction domains of Swi5p and Pho2p are flexible and can tolerate an increase in distance between the two binding sites . The mechanism of the cooperative DNA binding by Swi5p and Pho2p is discussed. J Biol Chem, 1995 Dec 8, 270(49), 29071 - 4 A conserved binding motif defines numerous candidate target proteins for both Cdc42 and Rac GTPases; Burbelo PD et al.; Rho, Rac, and Cdc42 are small GTPases that regulate the formation of a variety of actin structures and the assembly of associated integrin complexes, but little is known about the target proteins that mediate their effects . Here we have used a motif-based search method to identify putative effector proteins for Rac and Cdc42 . A search of the GenBankTM data base for similarity with the minimum Cdc42/Rac interactive binding (CRIB) region of a potential effector protein p65PAK has identified over 25 proteins containing a similar motif from a range of different species . These candidate Cdc42/Rac-binding proteins include family members of the mixed lineage kinases (MLK), a novel tyrosine kinase from Drosophila melanogaster (DPR2), a human protein MSE55, and several novel yeast and Caenorhabditis elegans proteins . Two murine p65PAK isoforms and a candidate protein from C . elegans, F09F7.5, interact strongly with the GTP form of both Cdc42 and Rac, but not Rho in a filter binding assay . Three additional candidate proteins, DPR2, MSE55, and MLK3 showed binding to the GTP form of Cdc42 and weaker binding with Rac, and again no interaction with Rho . These results indicate that proteins containing the CRIB motif bind to Cdc42 and/or Rac in a GTP-dependent manner, and they may, therefore, participate in downstream signaling. J Biol Chem, 1995 Dec 8, 270(49), 29055 - 8 Evidence that aspartic acid 301 is a critical substrate-contact residue in the active site of cytochrome P450 2D6; Ellis SW et al.; Model building studies have intimated a role for aspartic acid 301 in the substrate binding of cytochrome P450 2D6 (CYP2D6) . We have tested this hypothesis by generating a range of CYP2D6 mutants substituting a variety of amino acids at this site . The mutant proteins, which included substitution with a negatively charged glutamic acid residue or neutral asparagine, alanine, or glycine residues, were expressed in Saccharomyces cerevisiae . In addition, a mutant where aspartic acid 301 was deleted was also tested . All the mutants expressed approximately equivalent amounts of recombinant apoprotein and, apart from the alanine 301 and the aspartic acid 301 deletion mutants, gave carbon monoxide difference spectra of similar magnitude to the wild type . In the cases of the alanine and deletion mutants, the amount of holoprotein was significantly reduced or absent relative to the amount of apoprotein, indicating restricted heme incorporation . The glutamic acid mutant was shown to have similar catalytic properties to the wild type enzyme toward the substrates debrisoquine and metoprolol; however, some differences in regioselectivity and ligand binding were observed . The mutants containing neutral amino acids at position 301 exhibited marked reductions in catalytic activity . At low substrate concentrations little, if any, activity toward debrisoquine and metoprolol was measured . However, at a higher substrate concentration (2 mM) some activity was observed (about 10-20% of wild type levels) . Consistent with the above findings, the debrisoquine-induced spin changes in the mutant proteins were markedly reduced . These data collectively demonstrate that aspartic acid 301 plays an important role in determining the substrate specificity and activity of CYP2D6 and provide experimental evidence supporting the role of this amino acid in forming an electrostatic interaction between the basic nitrogen atom in CYP2D6 substrates and the carboxylate group of aspartic acid 301. Biochim Biophys Acta, 1995 Dec 7, 1259(3), 199 - 202 Cloning and expression of human cDNA encoding phosphatidylinositol transfer protein beta; Tanaka S et al.; cDNA encoding the beta isoform of human phosphatidylinositol transfer protein was cloned from a human brain cDNA library . The deduced sequence of the protein comprised 271 amino acids with a calculated molecular mass of 31,539 Da, and showed 98.1% identity to that of the beta isoform of rat phosphatidylinositol transfer protein . The cDNA hybridized to a 3.4-kb mRNA, which was widely expressed in various human tissues including brain. Biochem Biophys Res Commun, 1995 Dec 5, 217(1), 21 - 7 Molecular characterisation of recombinant green fluorescent protein by fluorescence correlation microscopy; Terry BR et al.; The cDNA for the green fluorescent protein (GFP) of Aequorea victoria has been expressed in transformed cells of Saccharomyces cerevisiae and the recombinant GFP isolated . Protonation and deprotonation of the cloned and purified GFP produced major effects on its spectral absorption characteristics with an increase in pH enhancing the fluorescence emission of the GFP more than twofold . Finally, molecular characterisation of GFP by fluorescence correlation microscopy in a minimal target volume of 1 fL yielded a translational diffusion coefficient (DT) of 8.7 x 10(-7) cm2.sec-1, equivalent to a Stokes radius of 2.82nm for a monodisperse globular protein of 27kDa. Proc Natl Acad Sci U S A, 1995 Dec 5, 92(25), 11916 - 20 GAIP, a protein that specifically interacts with the trimeric G protein G alpha i3, is a member of a protein family with a highly conserved core domain; De Vries L et al.; Using the yeast two-hybrid system we have identified a human protein, GAIP (G Alpha Interacting Protein), that specifically interacts with the heterotrimeric GTP-binding protein G alpha i3 . Interaction was verified by specific binding of in vitro-translated G alpha i3 with a GAIP-glutathione S-transferase fusion protein . GAIP is a small protein (217 amino acids, 24 kDa) that contains two potential phosphorylation sites for protein kinase C and seven for casein kinase 2 . GAIP shows high homology to two previously identified human proteins, GOS8 and 1R20, two Caenorhabditis elegans proteins, CO5B5.7 and C29H12.3, and the FLBA gene product in Aspergillus nidulans--all of unknown function . Significant homology was also found to the SST2 gene product in Saccharomyces cerevisiae that is known to interact with a yeast G alpha subunit (Gpa1) . A highly conserved core domain of 125 amino acids characterizes this family of proteins . Analysis of deletion mutants demonstrated that the core domain is the site of GAIP's interaction with G alpha i3 . GAIP is likely to be an early inducible phosphoprotein, as its cDNA contains the TTTTGT sequence characteristic of early response genes in its 3'-untranslated region . By Northern analysis GAIP's 1.6-kb mRNA is most abundant in lung, heart, placenta, and liver and is very low in brain, skeletal muscle, pancreas, and kidney . GAIP appears to interact exclusively with G alpha i3, as it did not interact with G alpha i2 and G alpha q . The fact that GAIP and Sst2 interact with G alpha subunits and share a common domain suggests that other members of the GAIP family also interact with G alpha subunits through the 125-amino-acid core domain. Biochemistry, 1995 Dec 5, 34(48), 15829 - 37 Binding of phenylarsenoxide to Arg-tRNA protein transferase is independent of vicinal thiols; Li J et al.; Reversible enzyme inhibition by phenylarsenoxides is generally taken to indicate the presence of functionally important vicinal thiol groups . Arginyl aminoacyl-tRNA transferase from eukaryotes is potently inhibited by phenylarsenoxides and possesses one or more essential sulfhydryl groups (Li, J., & Pickart C . M . (1995) Biochemistry 34, 139-147} . To map the putative Cys residues that mediate arsenoxide binding to the transferase from Saccharomyces cerevisiae, we systematically mutagenized the 15 Cys residues of the transferase, singly and in combination, to Ala (13 Cys) or Ser (2 Cys) . Six mutant enzymes, encompassing all 15 Cys residues of the transferase, were characterized in detail . The results revealed that Cys-20, Cys-23, and Cys-94 and/or Cys-95 were important for activity, since mutations at these positions reduced activity by 100-fold (Cys-94 and Cys-95 were mutated simultaneously) . Surprisingly, however, all of the mutant enzymes retained the ability to bind a radioiodinated phenylarsenoxide derivative, with undiminished stoichiometry and affinity . All of the mutant enzymes also remained susceptible to irreversible reaction with a bifunctional phenylarsenoxide bearing a paraalkyl halide substituent . Prior reaction of the enzyme with the bifunctional reagent blocked subsequent binding of the radiolabeled phenylarsenoxide, indicating that these two reagents bind at a single common site . These results indicate that high-affinity binding of trivalent arsenicals can occur by a thiol-independent mechanism. Cell Growth Differ, 1995 Dec, 6(12), 1541 - 7 Differential expression of gas and gadd genes at distinct growth arrest points during adipocyte development; Shugart EC et al.; The characterization of growth arrest-associated genes has revealed that cells actively suppress mitotic growth in response to extracellular signals . Mouse 3T3-L1 cells growth arrest at multiple distinct points during terminal differentiation to adipocytes . We examined the expression of growth arrest-specific (gas) and growth arrest- and DNA damage-inducible (gadd) genes as a function of 3T3-L1 growth arrest and adipocyte development . These growth arrest-associated genes are differentially expressed throughout adipocyte development . Some of the gas/gadd genes are preferentially expressed in a subset of growth arrest states . In contrast, gas1 and gas3 are expressed in serum-starved adipoblasts, contact-inhibited adipoblasts, and post-mitotic adipocytes . However, in post-mitotic adipocytes, gas1 and gas3 are induced in response to nutrient deprivation, not altered growth status . gas6 is an exception to the general concordance of mitotic growth arrest and gas/gadd expression in that gas6 is preferentially expressed during the clonal expansion of postconfluent adipoblasts . Combined, the expression patterns indicate that growth arrest-associated genes are regulated by numerous signal transduction pathways throughout a discrete developmental transition. EMBO J, 1995 Dec 1, 14(23), 6043 - 57 The sorting signal of cytochrome b2 promotes early divergence from the general mitochondrial import pathway and restricts the unfoldase activity of matrix Hsp70; Gartner F et al.; Cytochrome b2 is imported into mitochondria and sorted to the intermembrane space by a bipartite N-terminal presequence, which is a matrix targeting sequenced followed by an intermembrane space sorting signal . The N-terminus of the mature protein forms a folded heme binding domain that depends on the unfoldase function of matrix (mt) Hsp70 for import . We report that the distance between the presequence and the heme binding domain is critical for the ability of mt-Hsp70 to promote import of the domain . Hybrid proteins with 40 or more amino acids between the presequence and the heme binding domain are arrested in the import machinery . The translocation arrest can be overcome by unfolding of the preprotein or by inactivation of the intermembrane space sorting signal . Moreover, the sorting signal prevents backsliding of the precursor polypeptide in the import site in the initial import step, when the signal has not made contact with the matrix . The results indicate that the sorting signal interacts with component(s) of the inner membrane/intermembrane space during the initial import step and promotes an early divergence of b2 preproteins from the general matrix import pathway, precluding an unfolding role for mt-Hsp70 in the translocation of most of the mature portions of a preprotein . We propose a sorting model of cytochrome b2 which explains the apparently divergent previous results by a unifying hypothesis. EMBO J, 1995 Dec 1, 14(23), 5892 - 907 FKBP12-rapamycin target TOR2 is a vacuolar protein with an associated phosphatidylinositol-4 kinase activity; Cardenas ME et al.; In complex with the immunophilin FKBP12, the natural product rapamycin inhibits signal transduction events required for G1 to S phase cell cycle progression in yeast and mammalian cells . Genetic studies in yeast first implicated the TOR1 and TOR2 proteins as targets of the FKBP12-rapamycin complex . We report here that the TOR2 protein is membrane associated and localized to the surface of the yeast vacuole . Immunoprecipitated TOR2 protein contains readily detectable phosphatidylinositol-4 (PI-4) kinase activity attributable to either a TOR2 intrinsic activity or to a PI-4 kinase tightly associated with TOR2 . Importantly, we find that rapamycin stimulates FKBP12 binding to wild-type TOR2 but not to a rapamycin-resistant TOR2-1 mutant protein . Surprisingly, FKBP12-rapamycin binding does not markedly inhibit the PI kinase activity associated with TOR2, but does cause a delocalization of TOR2 from the vacuolar surface, which may deprive the TOR2-associated PI-4 kinase activity of its in vivo substrate . Several additional findings indicate that vacuolar localization is important for TOR2 function and, conversely, that TOR2 modulates vacuolar morphology and segregation . These studies demonstrate that TOR2 is an essential, highly conserved component of a signal transduction pathway regulating cell cycle progression conserved from yeast to man. EMBO J, 1995 Dec 1, 14(23), 5824 - 32 Rme1, a negative regulator of meiosis, is also a positive activator of G1 cyclin gene expression; Toone WM et al.; Control of G1 cyclin expression in Saccharomyces cerevisiae is mediated primarily by the transcription factor SBF (Swi4/Swi6) . In the absence of Swi4 and Swi6 cell viability is lost, but can be regained by ectopic expression of the G1 cyclin encoding genes, CLN1 or CLN2 . Here we demonstrate that the RME1 (regulator of meiosis) gene can also bypass the normally essential requirement for SBF . RME1 encodes a zinc finger protein which is able to repress transcription of IME1 (inducer of meiosis) and thereby inhibit cells from entering meiosis . We have found that expression of RME1 from a high copy number plasmid can specifically induce CLN2 expression . Deletion of RME1 alone shows no discernible effect on vegetative growth, however, deletion of RME1 in a swi6 delta swi4ts strain results in a lowering of the non-permissive temperature for viability . This suggests that Rme1 plays a significant but ancillary role in SBF in inducing CLN2 expression . We show that Rme1 interacts directly with the CLN2 promoter and have mapped the region of the CLN2 promoter required for Rme1-dependent activation . Consistent with Rme1 having a cell cycle role in G1, we have found that RME1 mRNA is synthesized periodically in the cell cycle, with maximum accumulation occurring at the M/G1 boundary . Thus Rme1 may act both to promote mitosis, by activating CLN2 expression, and prevent meiosis, by repressing IME1 expression. Naunyn Schmiedebergs Arch Pharmacol, 1995 Dec, 353(1), 116 - 21 Cytochromes of the P450 2C subfamily are the major enzymes involved in the O-demethylation of verapamil in humans; Busse D et al.; The calcium channel blocker verapamil {2,8-bis-(3,4-dimethoxyphenyl)-6-methyl-2-isopropyl-6-azaoctanitrile+ ++} undergoes extensive biotransformation in man . We have previously demonstrated cytochrome P450 (CYP) 3A4 and 1A2 to be the enzymes responsible for verapamil N-dealkylation (formation of D-617 {2-(3,4-dimethoxyphenyl)-5-methylamino-2-isopropylvaleronitrile}, and verapamil N-demethylation (formation of norverapamil {2,8-bis-(3,4-dimethoxyphenyl)-2-isopropyl-6-azaoctanitrile}), while there was no involvement of CYP3A4 and CYP1A2 in the third initial metabolic step of verapamil, which is verapamil O-demethylation . This pathway yields formation of D-703 {2-(4-hydroxy-3-methoxyphenyl)-8-(3,4-dimethoxyphenyl)-6-methyl-2-isopro pyl-6-azaoctanitrile} and D-702 {2-(3,4-dimethoxyphenyl)-8-(4-hydroxy-3-methoxyphenyl)-6-methyl-2-isopro pyl-6-azaoctanitrile} . The enzymes catalyzing verapamil O-demethylation have not been characterized so far . We have therefore identified and characterized the enzymes involved in verapamil O-demethylation in humans by using the following in vitro approaches: (I) characterization of O-demethylation kinetics in the presence of the microsomal fraction of human liver, (II) inhibition of verapamil O-demethylation by specific antibodies and selective inhibitors and (III) investigation of metabolite formation in microsomes obtained from yeast strain Saccharomyces cerevisiae W(R), that was genetically engineered for stable expression of human CYP2C8, 2C9 and 2C18 . In human liver microsomes (n=4), the intrinsic clearance (CLint), as derived from the ratio of Vmax/Km, was significantly higher for O-demethylation to D-703 compared to formation of D-702 following incubation with racemic verapamil (13.9 +/- 1.0 vs 2.4 +/- 0.6 ml*min-1*g-1, mean+/-SD; p<0.05), S-verapamil (16.8 +/- 3.3 vs 2.2 +/- 1.2 ml* min-1*g-1, p<0.05) and R-verapamil (12.1 +/- 2.9 vs 3.6 +/- 1.3 ml*min-1*g-1; p<0.05), thus indicating regioselectivity of verapamil O-demethylation process . The CLint of D-703 formation in human liver microsomes showed a modest but significant degree of stereoselectivity (p<0.05) with a S/R-ratio of 1.41 +/- 0.17 . Anti-LKM2 (anti-liver/kidney microsome) autoantibodies (which inhibit CYP2C9 and 2C19) and sulfaphenazole (a specific CYP2C9 inhibitor) reduced the maximum rate of formation of D-703 by 81.5 +/- 4.5% and 45%, that of D-702 by 52.7 +/- 7.5% and 72.5%, respectively . Both D-703 and D-702 were formed by stably expressed CYP2C9 and CYP2C18, whereas incubation with CYP2C8 selectively yielded D-703 . In conclusion, our results show that enzymes of the CYP2C subfamily are mainly involved in verapamil O-demethylation . Verapamil therefore has the potential to interact with other drugs which inhibit or induce these enzymes. Yeast, 1995 Dec, 11(15), 1519 - 23 The sequence of an 11.1 kb DNA fragment between ADH4 and ADE5 on the left arm of chromosome VII reveals the presence of eight open reading frames; Vandenbol M et al.; The complete sequence of a 11, 132 bp segment located on the left arm of chromosome VII of Saccharomyces cerevisiae has been determined and analysed . Eight open reading frames (ORFs) of at least 100 amino acids were identified . Five show similarities to known amino acid sequences . Another ORF encoding the chromosome segregation protein CSE1 is not entirely located in our sequenced fragment and is incomplete at its C-terminus . The two remaining ORFs do not display similarities to known sequences. Curr Opin Genet Dev, 1995 Dec, 5(6), 786 - 91 DNA excision repair and transcription: implications for genome evolution; Sullivan DT; The past two years have seen a substantial increase in knowledge regarding the enzymology of DNA excision repair . These data support a growing body of information which suggests that transcribed nucleotide sequences are preferentially subject to excision repair . It is possible that these mechanisms, or related ones, are relevant to the molecular evolution of sequences that appear not to evolve according to models which do not take into account regional sequence differences in the extent of DNA repair. Curr Opin Genet Dev, 1995 Dec, 5(6), 756 - 67 The elusive centromere: sequence divergence and functional conservation; Sunkel CE et al.; The centromere is an essential cis-acting structure present in the chromosomes of all eukaryotes, central to the mechanism that ensures proper segregation during meiosis and mitosis . Molecular characterization of centromeres in the budding and fission yeasts has advanced significantly over the last few years due to their relatively small size and the availability of functional assays . However, identification and characterization of centromeric sequences from multicellular organisms has proven to be slow and difficult in the absence of direct functional tests . Molecular data have recently become available on the centromere of Drosophila, making it possible to bridge a long-standing gap in our knowledge on the general structure of centromeres . An evaluation of the available data from yeast to man suggests that centromere sequence and centromere sequence organization have diverged significantly, even amongst different chromosomes of a single organism; however, overall centromere organization and kinetochore components might be significantly more conserved than thought previously. Curr Opin Genet Dev, 1995 Dec, 5(6), 746 - 55 Return of the H-word (heterochromatin); Lohe AR et al.; Recent advances in studies of yeast, Drosophila and humans have renewed interest in heterochromatin . These recent studies have demonstrated the interspersion and rapid spread of transposable elements into Drosophila heterochromatin; documented the requirement of heterochromatic genes for heterochromatin; identified heterochromatin-like regions in yeast chromosomes; confirmed an important role for satellite DNA in human centromere function; and suggested potential functions for heterochromatin-associated proteins. Mol Biochem Parasitol, 1995 Dec, 75(1), 87 - 97 Pyruvate dehydrogenase complex from the primitive insect trypanosomatid, Crithidia fasciculata: dihydrolipoyl dehydrogenase-binding protein has multiple lipoyl domains; Diaz F et al.; The pyruvate dehydrogenase complex (PDC) has been purified to apparent homogeneity from the insect trypanosomatid, Crithidia fasciculata, a member of the most primitive eukaryotic group to contain mitochondria . Separation of the purified PDC by SDS-PAGE yielded five bands of 70 (p70), 60 (p60), 55, 46 and 36.5 kDa, which appeared to correspond to dihydrolipoyl dehydrogenase binding protein (E3BP), dihydrolipoyl transacetylase (E2), E3, E1 alpha and E1 beta, respectively . The purified complex did not exhibit endogenous PDHa kinase activity . p70 was much less abundant than p60 . Polyclonal antisera raised against p70 did not cross-react with p60, and antisera raised against p60 did not cross-react with p70, suggesting that p60 did not arise from p70 by proteolysis . Both p70 and p60 contained similar amino terminal sequences . Both sequences contained the MPALSP motif similar to sequences present in both E3BP and E2 from other sources . Incubation of the purified PDC with {2-14C}pyruvate in the absence of CoA resulted in the acetylation of both p70 and p60, suggesting that both proteins contained lipoyl domains, but the specific incorporation of label into p70 was significantly greater than for p60 . Limited proteolysis of the acetylated complex with trypsin yielded two major fragments derived from p60 of 35 and 30 kDa, corresponding to E2L and E2I, and one major acetylated fragment of 58 kDa derived from p70 . Therefore, these results suggest that p70 is an E3BP and given its apparent M(r) and degree of acetylation, it contains multiple lipoyl domains. Eur J Cell Biol, 1995 Dec, 68(4), 377 - 86 Cytosolic and nuclear localization of protein phosphatase 2C beta 1 in COS and BHK cells; Wenk J et al.; We have recently elucidated the structure of an isoform of the Mg(2+)-dependent protein phosphatase 2C (PP2C beta 1) from rat liver (Wenk, J., H.-I . Trompeter, K.-G . Pettrich, P . T . W . Cohen, D . G . Campbell, G . Mieskes, FEBS Lett . 297, 135-138 (1992)) . In the present study the subcellular distribution of PP2C beta 1 was investigated in COS and BHK cells . We modified the PP2C beta 1 cDNA with a c-myc tag coding sequence at either its 3' or its 5' end end to distinguish the plasmid derived PP2C beta 1 from the endogenous phosphatase and to examine the influence of the modification on the enzymatic activity . Transient transfections of COS or BHK cells with pCMV2 derived vectors containing these constructs showed that the expressed hybrid protein with the lowest activity (N-terminal tagged < untagged < C-terminal tagged PP2C beta 1) was expressed to the highest level and vice versa . These experiments point to a possible toxic effect or a selection disadvantage after overexpression of PP2C beta 1 . In immunofluorescence studies with antibodies specific for the PP2C beta isoform, all overexpressed proteins showed the expected cytoplasmic as well as a significant nuclear localization . The nuclei remained stained even after selective perforation of the plasma membrane with digitonin and washing out the cytosolic PP2C beta 1 . We conclude that PP2C beta 1 has obligatory and important functions in metabolic as well as in nuclear processes. Mol Endocrinol, 1995 Dec, 9(12), 1645 - 54 Functional comparison of the Mus musculus molossinus and Mus musculus domesticus Sry genes; Dubin RA et al.; The Sry gene functions as a genetic switch initiating testicular development of the indifferent mammalian gonad . The Mus musculus molossinus Sry open reading frame (ORF) encodes a 395-amino acid transcription factor (mSry) that specifically binds and bends DNA through its N-terminal HMG domain and activates transcription through its long C-terminal (residues 144-366) glutamine/histidine-rich activation domain . The M . m . domesticus Sry ORF encodes a highly homologous, truncated protein (dSry) of approximately 230 amino acids, and the molecular basis for truncation is a point mutation that creates an amber stop codon within the activation domain . The mSry protein activates transcription of a Sry-responsive reporter gene in HeLa cells, but dSry does not . Gene swapping and in vitro DNA binding experiments revealed that lack of transcriptional activation by dSry was not the result of polymorphisms within the first 137 amino acids of the protein . Direct analysis of the C-terminal glutamine/histidine-rich domain revealed that dSry lacked a functional transcriptional activation domain . Fusion of the GAL4 DNA-binding domain to the C-terminal deletion mutants of the GAL4-mSry chimeric protein indicated that residues 263-345 of the glutamine/histidine-rich domain were necessary for high level transactivation . Furthermore, readthrough of the premature amber stop codon by transfer RNA suppression resulted in a strong GAL4-dSry transactivator . This demonstrated that the premature stop codon is the only polymorphism responsible for the inability of the dSry glutamine/histidine-rich region to transactivate. Mol Reprod Dev, 1995 Dec, 42(4), 477 - 85 Dynamics and organization of MAP kinase signal pathways; Errede B et al.; In the budding yeast, Saccharomyces cerevisiae, four separate but structurally related mitogen-activated protein kinase (MAPK) activation pathways are known . The best understood of these regulates mating . Pheromone binding to receptor informs cells of the proximity of a mating partner and induces differentiation to a mating competent state . The MAPK activation cascade mediating this signal is made up of Ste11 (a MEK kinase {MEKK}), Ste7 (a MAPK/ERK kinase {MEK}), and the redundant MAPK-related Fus3 and Kss1 enzymes . Another MAPK activation pathway is important for cell integrity and regulates cell wall construction . This cascade consists of Bck1 (a MEKK), the redundant Mkk1 and Mkk2 enzymes (MEKs), and Mpk1 (a MAPK) . We exploited these two pathways to learn about the coordination and signal transmission fidelity of MAPK activation cascades . Two lines of evidence suggest that the activities of the mating and cell integrity pathways are coordinated during mating differentiation . First, cells deficient in Mpk1 are susceptible to lysis when they make a mating projection in response to pheromone . Second, Mpk1 activation during pheromone induction coincides with projection formation . The mechanism underlying this coordination is still unknown to us . Our working model is that projection formation generates a mobile second messenger for activation of the cell integrity pathway . Analysis of a STE7 mutation gave us some unanticipated but important insights into parameters important for fidelity of signal transmission . The Ste7 variant has a serine to proline substitution at position 368 . Ste7-P368 has higher basal activity than the wild-type enzyme but still requires Ste11 for its function . Additionally, the proline substitution enables the variant to transmit the signal from mammalian Raf expressed in yeast . This novel activity suggests that Ste7-P368 is inherently more permissive than Ste7 in its interactions with MEKKs . Yet, Ste7-P368 cross function in the cell integrity pathway occurs only when it is highly overproduced or when Ste5 is missing . This behavior suggests that Ste5, which has been proposed to be a tether for the kinases in the mating pathway, contributes to Ste7 specificity and fidelity of signal transmission. Mol Reprod Dev, 1995 Dec, 42(4), 459 - 67 Transcriptional regulation by MAP kinases; Davis RJ; Tyrosine kinase growth factor receptors activate MAP kinase by a complex mechanism involving the SH2/3 protein Grb2, the exchange protein Sos, and Ras . The GTP-bound Ras protein binds to the Raf kinase and initiates a protein kinase cascade that leads to MAP kinase activation . Three MAP kinase kinase kinases have been described--c-Raf, c-Mos, and Mekk--that phosphorylate and activate Mek, the MAP kinase kinase . Activated Mek phosphorylates and activates MAP kinase . Subsequently, the activated MAP kinase translocates into the nucleus where many of the physiological targets of the MAP kinase signal transduction pathway are located . These substrates include transcription factors that are regulated by MAP kinase phosphorylation (e.g., Elk-1, c-Myc, c-Jun, c-Fos, and C/EBP beta) . Thus the MAP kinase pathway represents a significant mechanism of signal transduction by growth factor receptors from the cell surface to the nucleus that results in the regulation of gene expression . Three MAP kinase homologs have been identified in the rat: Erk1, Erk2, and Erk3 . Human MAP kinases that are similar to the rat Erk kinases have also been identified by molecular cloning . The human Erk1 protein kinase has been shown to be widely expressed as a 44-kDa protein in many tissues . The human Erk2 protein kinase is a 41-kDa protein that is expressed ubiquitously . In contrast, a human Erk3-related protein kinase has been found to be expressed at a high level only in heart muscle and brain . The loci of these MAP kinase genes are widely distributed within the human genome: erk2 at 22q11.2; erk1 at 16p11.2; and ek3-related at 18q12-21 . In the yeast Saccharomyces cerevisiae, five MAP kinase gene homologs have been described: smkl, mpk1, hog1, fus3, and kss1 . Together, these kinases are a more diverse group than the human erks that have been identified . Thus the erks are likely to represent only one subgroup of a larger human MAP kinase gene family . A candidate for this extended family of MAP kinases is the c-Jun NH2-terminal kinase (Jnk), which binds to and phosphorylates the transcription factor c-Jun at the activating sites Ser-63 and Ser-73 . Evidence is presented here to demonstrate that Jnk is a distant relative of the MAP kinase group that is activated by dual phosphorylation at Tyr and Thr. Genetika, 1995 Dec, 31(12), 1605 - 13 {Heterologous induction of the retrotransposon Ty1: reverse transcriptase plays a key role in initiating the retrotransposition cycle}; Reznik NL et al.; A new method was developed to study the mechanism of initiation of the retrotransposition cycle: retrotransposons of Drosophila melanogaster, gypsy, copia, and 17.6 were expressed in yeast under the control of potent yeast promoters . Expression of retrotransposons induced formation of viruslike particles (VLPs) associated with full-length Ty1 RNA and DNA sequences . This phenomenon was termed heterologous induction . When the gene for reverse transcriptase of human immunodeficiency virus (HIV) was expressed in yeast, the same results were obtained . These data allowed us to assume the excess of active reverse transcriptase to play the central role in induction of transposition . Possible mechanisms of induction of Ty1 transposition by homologous and heterologous elements are discussed. RNA, 1995 Dec, 1(10), 1009 - 17 Determination of nucleotide distances in RNA by means of copper phenanthroline-generated hydroxyl radical cleavage pattern; Hermann T et al.; In contrast to the commonly used Fe(II)-EDTA, bis(orthophenanthroline)-copper(I) (OP-Cu) first generates hydroxyl radicals after binding to RNA . Due to diffusion, the hydroxyl radicals can cleave neighboring nucleotides in a distance r of up to 1.5 nm to the OP-Cu binding site . Using the known structure of tRNAPhe as a reference, we show that the hydroxyl radical cleavage pattern generated by a specifically bound OP-Cu shows a 1/r dependence on the distance of the cleaved nucleotide to the OP-Cu binding site . We propose that OP-Cu is a suitable probe for obtaining data on the distances between nucleotides in RNA, which can be used in modeling the structure of the examined RNA . However, this information is restricted to about three to four bases surrounding an OP-Cu binding site. Antimicrob Agents Chemother, 1995 Dec, 39(12), 2708 - 17 Deletion of the Candida glabrata ERG3 and ERG11 genes: effect on cell viability, cell growth, sterol composition, and antifungal susceptibility; Geber A et al.; We have cloned and sequenced the structural genes encoding the delta 5,6 sterol desaturase (ERG3 gene) and the 14 alpha-methyl sterol demethylase (ERG11 gene) from Candida glabrata L5 (leu2) . Single and double mutants of these genes were created by gene deletion . The phenotypes of these mutants, including sterol profiles, aerobic viabilities, antifungal susceptibilities, and generation times, were studied . Strain L5D (erg3 delta::LEU2) accumulated mainly ergosta-7,22-dien-3 beta-ol, was aerobically viable, and remained susceptible to antifungal agents but had a slower generation time than its parent strain . L5LUD (LEU2 erg11 delta::URA3) strains required medium supplemented with ergosterol and an anaerobic environment for growth . A spontaneous aerobically viable mutant, L5LUD40R (LEU erg11 delta::URA3), obtained from L5LUD (LEU2 erg11 delta::URA3), was found to accumulate lanosterol and obtusifoliol, was resistant to azole antifungal agents, demonstrated some increase in resistance to amphotericin B, and exhibited a 1.86-fold increase in generation time in comparison with L5 (leu2) . The double-deletion mutant L5DUD61 (erg3 delta::LEU2 erg11 delta::URA3) was aerobically viable, produced mainly 14 alpha-methyl fecosterol, and had the same antifungal susceptibility pattern as L5LUD40R (LEU2 erg11 delta::URA3), and its generation time was threefold greater than that of L5 (leu2) . Northern (RNA) analysis revealed that the single-deletion mutants had a marked increase in message for the undeleted ERG3 and ERG11 genes . These results indicate that differences in antifungal susceptibilities and the restoration of aerobic viability exist between the C . glabrata ergosterol mutants created in this study and those sterol mutants with similar genetic lesions previously reported for Saccharomyces cerevisiae. Mol Biol Cell, 1995 Dec, 6(12), 1833 - 42 A role for Hsp90 in retinoid receptor signal transduction; Holley SJ et al.; The ubiquitous heat shock protein Hsp90 appears to participate directly in the function of a broad range of cellular signal transduction components, including steroid hormone receptors; however, an evolutionarily related subclass of intracellular receptors, exemplified by the retinoid receptors RAR and RXR, had been inferred from biochemical studies to function independently of Hsp90 . To examine this issue genetically, we measured mammalian and avian retinoid receptor activity in a Saccharomyces cerevisiae strain in which the expression of the yeast Hsp90 homologue could be conditionally repressed approximately 20-fold relative to wild type . We tested transcriptional activation by RAR or RXR-RAR, from two types of retinoic acid response elements, triggered by three different agonist ligands . In every condition, we found that activation was severely compromised under conditions of low Hsp90 expression . We showed that the defect was in signal transduction rather than transcription activation per se, and that high affinity hormone binding was abolished in extracts of cells producing low levels of Hsp90 . We suggest that Hsp90 may function in at least one step of signal transduction by all members of the intracellular receptor superfamily. Mol Biol Cell, 1995 Dec, 6(12), 1641 - 58 Cell cycle-regulated phosphorylation of Swi6 controls its nuclear localization; Sidorova JM et al.; The Swi6 transcription factor, required for G1/S-specific gene expression in Saccharomyces cerevisiae, is highly phosphorylated in vivo . Within the limits of resolution of the peptide analysis, the synchrony, and the time intervals tested, serine 160 appears to be the only site of phosphorylation in Swi6 that varies during the cell cycle . Serine 160 resides within a Cdc28 consensus phosphorylation site and its phosphorylation occurs at about the time of maximal transcription of Swi6- and Cdc28-dependent genes containing SCB or MCB elements . However, phosphorylation at this site is not Cdc28-dependent, nor does it control G1/S-specific transcription . The role of the cell cycle-regulated phosphorylation is to control the subcellular localization of Swi6 . Phosphorylation of serine 160 persists from late G1 until late M phase, and Swi6 is predominantly cytoplasmic during this time . Aspartate substitution for serine 160 inhibits nuclear localization throughout the cycle . Swi6 enters the nucleus late in M phase and throughout G1, when serine 160 is hypophosphorylated . Alanine substitution at position 160 allows nuclear entry of Swi6 throughout the cell cycle . GFP fusions with the N-terminal one-third of Swi6 display the same cell cycle-regulated localization as Swi6. Arch Microbiol, 1995 Dec, 164(6), 414 - 9 The alpha-D-mannan core of a complex cell-wall heteroglycan of Trichoderma reesei is responsible for beta-glucosidase activation; Rath J et al.; A heteroglycan responsible for the binding of the enzyme beta-1,4-D-glucosidase (EC 3.2.1.21) to fungal cell walls was isolated from cell walls of the filamentous fungus Trichoderma reesei . The heteroglycan, composed of mannose, galactose, glucose, and glucuronic acid, also activated beta-1,4-D-glucosidase, beta-1,4-D-xylosidase and N-acetyl-beta-1,4-D-glucosaminidase activity in vitro . The structural backbone of this heteroglycan was prepared by acid hydrolysis and gel filtration . The molecular structure of the core of the heteroglycan was determined by NMR studies as a linear alpha-1,6-D-mannan . The mannan core obtained by acid degradation stimulated the beta-glucosidase activity by 90% . Several glycosidases from Aspergillus niger were also activated by the T . reesei heteroglycan . The beta-glucosidase of Trichoderma was activated by mannan from Saccharomyces cerevisiae to a comparable extent. Plant J, 1995 Dec, 8(6), 949 - 57 Tobacco mitotic cyclins: cloning, characterization, gene expression and functional assay; Setiady YY et al.; Three cyclin cDNA clones (Ntcyc25, Ntcyc27, Ntcyc29) have been isolated from tobacco (Nicotiana tabacum) using the PCR cloning method . The encoded Ntcyc cyclins were highly homologous to mitotic cyclins . In synchronized tobacco suspension cultured cells, the mRNA levels of Ntcyc25 and Ntcyc27 were detectable through S, G2 and M phases, while the Ntcyc29 mRNA was detected from G2 to M phase . These expression patterns classified the Ntcyc25 and Ntcyc27 into A-type cyclin and Ntcyc29 into B-type cyclin . The three genes were expressed in growing tobacco cultured cells but ceased to be expressed when cells entered the stationary phase, indicating that the expression of these cyclin genes was well correlated with cell growth . The N-terminal truncated Ntcyc25 and Ntcyc29 cDNAs were able to rescue G1 cyclin mutant of Saccharomyces cerevisiae, while the full-length of Ntcyc27 protein could also partially perform G1 cyclin functions. Chemosphere, 1995 Dec, 31(11-12), 4515 - 29 Metabolism of the herbicide chlortoluron by human cytochrome P450 3A4; Mehmood Z et al.; Studies were carried out to investigate the metabolism of herbicide chlortoluron in the microsomal fractions and whole cells of Saccharomyces cerevisiae expressing human cytochrome P450 3A4 . Both whole cells and microsomal fractions of yeast expressing human cytochrome P450 3A4 exhibited a typical dithionite-reduced, CO-difference absorbance spectrum with maximum absorbance at 448 nm . Chlortoluron produced a type I binding spectrum with cytochrome P450 3A4 with a Ks value of 200 microM . Chlortoluron was metabolised into four metabolites; hydroxylated-N-monodemethylated, hydroxylated ring methylated, N-didemethylated and N-monodemethylated products . Chlortoluron metabolism was absolutely dependent on NADPH and no metabolism was observed in control transformants. Trends Biochem Sci, 1995 Dec, 20(12), 511 - 6 Finding prospective partners in the library: the two-hybrid system and phage display find a match; Allen JB et al.; The two-hybrid system uses the efficacy of yeast genetic assays to identify protein-protein interactions . It permits the rapid cloning of genes encoding products that interact with a given protein of interest . Also being developed are phage display methods that allow direct physical selection of binding proteins . These methods have significantly altered strategies for analysing signaling and regulatory pathways. J Cell Biol, 1995 Dec, 131(6 Pt 2), 1831 - 8 The tyrosine kinase substrate eps15 is constitutively associated with the plasma membrane adaptor AP-2; Benmerah A et al.; The ubiquitous eps15 protein was initially described as a substrate of the EGF receptor kinase . Its functions are not yet delineated and this work provides evidence for its possible role in endocytosis . A novel anti-eps15 antibody, 6G4, coimmunoprecipitated proteins of molecular mass 102 kD . In human cells, these proteins were identified as the alpha- and beta-adaptins of the AP-2 complex on the basis of their NH2-terminal sequence and their immunoreactivity with anti-alpha- and anti-beta-adaptin antibodies but not with anti-gamma-adaptin antibody . In addition, the anti-eps15 antibody coimmunoprecipitated metabolically labeled polypeptides with molecular mass of 50 and 17 kD, comparable to those of the two other components of the AP-2 complex, mu2 and sigma 2 . Constitutive association of eps15 with AP-2 was confirmed by two sets of experiments . First, eps15 was detected in immunoprecipitates of anti-alpha- and anti-beta-adaptin antibodies . Second, alpha- and beta- but not gamma-adaptins were precipitated by a glutathione-S-transferase eps15 fusion protein . The association of eps15 with AP-2 was ubiquitous and conserved between species, since it was observed in human lymphocytes and epithelial cells and in murine NIH3T3 fibroblasts . Our results are in keeping with a recent study showing homology between the NH2-terminal domains of eps15 and the product of the gene END3, involved in clathrin-mediated endocytosis of the pheromone alpha factor in Saccharomyces cerevisiae, and suggest a possible role for eps15 in clathrin-mediated endocytosis in mammals. Exp Parasitol, 1995 Dec, 81(4), 445 - 52 Onchocerca volvulus: identification and characterization of an immunogenic eggshell protein (Oveg1); Tume C et al.; A major antigen recognized by human sera in Onchocerca volvulus infections is a parasite eggshell protein . The cDNA clone for this antigen was isolated from a lambda gt11 O . volvulus cDNA library using antisera from patients with high microfilarial counts . Sequence analysis of the cDNA clone predicts a polyglutamine repeat near the 5' end of the cDNA, and a motif of four arginines near the 3' end, reminiscent of that found in many regulatory proteins . The cDNA was subcloned into a yeast expression vector and reagent quantities of recombinant antigen produced in Saccharomyces cerevisiae . Antisera produced to the recombinant purified protein localized the antigen to the eggshell of developing microfilariae within the adult female uterus . No other sites of Oveg1 expression were noted in adult worms, but labeling was seen in internal membrane structures of L3 larvae . Sera from infected chimps recognized Oveg1 only after infections became patent . Sera from infected humans showed reactivity to Oveg1 that varied from 39 to 95%, depending upon the geographic location. FASEB J, 1995 Dec, 9(15), 1518 - 26 Flavoprotein structure and mechanism . 8 . Structure-function relations for old yellow enzyme; Karplus PA et al.; The past 5 years have seen tremendous progress in our knowledge of old yellow enzyme (OYE) as a number of OYEs have been cloned and expressed, a high-resolution crystal structure has been determined for one of these, and new substrates have been found that can be turned over by the enzyme . Together these studies do not yet define the physiological role of OYE, but they lead to significant new insights into the enzymatic properties and structure-function relations of OYE. Virology, 1995 Dec 1, 214(1), 215 - 21 Interference with replication of two double-stranded RNA viruses by production of N-terminal fragments of capsid polypeptides; Yao W et al.; It is possible to interfere with the replication of a number of plant RNA viruses by systemic production of viral capsid polypeptides or RNA-dependent RNA polymerases, or by production of untranslatable portions of viral plus strands or minus strands . Interference can occur by a number of mechanisms . We have discovered that the Saccharomyces cerevisiae double-stranded RNA viruses ScVL1 and ScVLa, which exist as permanent persistent infections of their host cells, can be cured very efficiently by production of N-terminal fragments of their capsid polypeptides . These totiviruses produce only two polypeptides: a capsid polypeptide (Cap) and a Cap-Pol fusion polypeptide with RNA-dependent RNA polymerase activity . Three types of interference can be detected: interference due to overproduction of both Cap and Cap-Pol, interference due to overproduction of Cap (and consequent distortion of the Cap to Cap-Pol ratio), and interference due to negative complementation by N-terminal fragments of Cap . Some N-terminal fragments of Cap appear to be incorporated into viral particles, but only in the presence of a complete Cap protein . We postulate that incorporation of N-terminal fragments of Cap results in the formation of defective particles. Mol Cell Biol, 1995 Dec, 15(12), 7143 - 51 Plk is an M-phase-specific protein kinase and interacts with a kinesin-like protein, CHO1/MKLP-1; Lee KS et al.; PLK (STPK13) encodes a murine protein kinase closely related to those encoded by the Drosophila melanogaster polo gene and the Saccharomyces cerevisiae CDC5 gene, which are required for normal mitotic and meiotic divisions . Affinity-purified antibody generated against the C-terminal 13 amino acids of Plk specifically recognizes a single polypeptide of 66 kDa in MELC, NIH 3T3, and HeLa cellular extracts . The expression levels of both poly(A)+ PLK mRNA and its encoded protein are most abundant about 17 h after serum stimulation of NIH 3T3 cells . Plk protein begins to accumulate at the S/G2 boundary and reaches the maximum level at the G2/M boundary in continuously cycling cells . Concurrent with cyclin B-associated cdc2 kinase activity, Plk kinase activity sharply peaks at the onset of mitosis . Plk enzymatic activity gradually decreases as M phase proceeds but persists longer than cyclin B-associated cdc2 kinase activity . Plk is localized to the area surrounding the chromosomes in prometaphase, appears condensed as several discrete bands along the spindle axis at the interzone in anaphase, and finally concentrates at the midbody during telophase and cytokinesis . Plk and CHO1/mitotic kinesin-like protein 1 (MKLP-1), which induces microtubule bundling and antiparallel movement in vitro, are colocalized during late M phase . In addition, CHO1/MKLP-1 appears to interact with Plk in vivo and to be phosphorylated by Plk-associated kinase activity in vitro. Mol Cell Biol, 1995 Dec, 15(12), 7098 - 105 Mitochondrial GrpE modulates the function of matrix Hsp70 in translocation and maturation of preproteins; Laloraya S et al.; Mitochondrial GrpE (Mge1p) is a mitochondrial cochaperone essential for viability of the yeast Saccharomyces cerevisiae . To study the role of Mge1p in the biogenesis of mitochondrial proteins, we isolated a conditional mutant allele of MGE1 which conferred a temperature-sensitive growth phenotype and led to the accumulation of mitochondrial preproteins after shifting of the cells to the restrictive temperature . The mutant Mge1 protein was impaired in its interaction with the matrix heat shock protein mt-Hsp70 . The mutant mitochondria showed a delayed membrane translocation of preproteins, and the maturation of imported proteins was impaired, as evidenced by the retarded second proteolytic processing of a preprotein in the matrix . Moreover, the aggregation of imported proteins was decreased in the mutant mitochondria . The mutant Mge1p differentially modulated the interaction of mt-Hsp70 with preproteins compared with the wild type, resulting in decreased binding to preproteins in membrane transit and enhanced binding to fully imported proteins . We conclude that the interaction of Mge1p with mt-Hsp70 promotes the progress of the Hsp70 reaction cycle, which is essential for import and maturation of mitochondrial proteins. Mol Cell Biol, 1995 Dec, 15(12), 7059 - 66 Mechanism of differential utilization of the his3 TR and TC TATA elements; Iyer V et al.; The yeast his3 promoter region contains two TATA elements, TC and TR, that are differentially utilized in constitutive his3 transcription and Gcn4-activated his3 transcription . TR contains the canonical TATAAA sequence, whereas TC is an extended region that lacks a conventional TATA sequence and does not support transcription in vitro . Surprisingly, differential his3 TATA-element utilization does not depend on specific properties of activator proteins but, rather, is determined by the overall level of his3 transcription . At low levels of transcription, the upstream TC is preferentially utilized, even though it is inherently a much weaker TATA element than TR . The TATA elements are utilized equally at intermediate levels, whereas TR is strongly preferred at high levels of transcription . This characteristic behavior can be recreated by replacing TC with moderately functional derivatives of a conventional TATA element, suggesting that TC is a collection of weak TATA elements . Analysis of promoters containing two biochemically defined TATA elements indicates that differential utilization occurs when the upstream TATA element is weaker than the downstream element . In other situations, the upstream TATA element is preferentially utilized in a manner that is independent of the overall level of transcription . Thus, in promoters containing multiple TATA elements, relative utilization not only depends on the quality and arrangement of the TATA elements but can vary with the overall level of transcriptional stimulation . We suggest that differential TATA utilization results from the combination of an intrinsic preference for the upstream element and functional saturation of weak TATA elements at low levels of transcriptional stimulation. Mol Cell Biol, 1995 Dec, 15(12), 7032 - 42 Distinct roles for two purified factors in transcription of Xenopus mitochondrial DNA; Antoshechkin I et al.; Transcription of Xenopus laevis mitochondrial DNA (xl-mtDNA) by the mitochondrial RNA polymerase requires a dissociable factor . This factor was purified to near homogeneity and identified as a 40-kDa protein . A second protein implicated in the transcription of mtDNA, the Xenopus homolog of the HMG box protein mtTFA, was also purified to homogeneity and partially sequenced . The sequence of a cDNA clone encoding xl-mtTFA revealed a high degree of sequence similarity to human and Saccharomyces cerevisiae mtTFA . xl-mtTFA was not required for basal transcription from a minimal mtDNA promoter, and this HMG box factor could not substitute for the basal factor, which is therefore designated xl-mtTFB . An antibody directed against the N terminus of xl-mtTFA did not cross-react with xl-mtTFB . xl-mtTFA is an abundant protein that appears to have at least two functions in mitochondria . First, it plays a major role in packaging mtDNA within the organelle . Second, DNase I footprinting experiments identified preferred binding sites for xl-mtTFA within the control region of mtDNA next to major mitochondrial promoters . We show that binding of xl-mtTFA to a site separating the two clusters of bidirectional promoters selectively stimulates specific transcription in vitro by the basal transcription machinery, comprising mitochondrial RNA polymerase and xl-mtTFB. Mol Cell Biol, 1995 Dec, 15(12), 6971 - 8 Cotranscriptional splicing of a group I intron is facilitated by the Cbp2 protein; Lewin AS et al.; The nuclear CBP2 gene encodes a protein essential for the splicing of a mitochondrial group I intron in Saccharomyces cerevisiae . This intron (bI5) is spliced autocatalytically in the presence of high concentrations of magnesium and monovalent salt but requires the Cbp2 protein for splicing under physiological conditions . Addition of Cbp2 during RNA synthesis permitted cotranscriptional splicing . Splicing did not occur in the transcription buffer in the absence of synthesis . The Cbp2 protein appeared to modify the folding of the intron during RNA synthesis: pause sites for RNA polymerase were altered in the presence of the protein, and some mutant transcripts that did not splice after transcription did so during transcription in the presence of Cbp2 . Cotranscriptional splicing also reduced hydrolysis at the 3' splice junction . These results suggest that Cbp2 modulates the sequential folding of the ribozyme during its synthesis . In addition, splicing during transcription led to an increase in RNA synthesis with both T7 RNA polymerase and mitochondrial RNA polymerase, implying a functional coupling between transcription and splicing. Mol Cell Biol, 1995 Dec, 15(12), 6854 - 63 SOK2 may regulate cyclic AMP-dependent protein kinase-stimulated growth and pseudohyphal development by repressing transcription; Ward MP et al.; Yeast cyclic AMP (cAMP)-dependent protein kinase (PKA) activity is essential for growth and cell cycle progression . Dependence on PKA function can be partially relieved by overexpression of a gene, SOK2, whose product has significant homology with several fungal transcription factors (StuA from Aspergillus nidulans and Phd1 from Saccharomyces cerevisiae) that are associated with cellular differentiation and development . Deletion of SOK2 is not lethal but exacerbates the growth defect of strains compromised for PKA activity . Alterations in Sok2 protein production also affect the expression of genes involved in several other PKA-regulated processes, including glycogen accumulation (GAC1) and heat shock resistance (SSA3) . These results suggest SOK2 plays a general regulatory role in the PKA signal transduction pathway . Expression of the PKA catalytic subunit genes is unaltered by deletion or overexpression of SOK2 . Because homozygous sok2/sok2 diploid strains form pseudohyphae at an accelerated rate, the Sok2 protein may inhibit the switch from unicellular to filamentous growth, a process that is dependent on cAMP . Thus, the product of SOK2 may act downstream of PKA to regulate the expression of genes important in growth and development. Mol Cell Biol, 1995 Dec, 15(12), 6736 - 45 Seven-up inhibits ultraspiracle-based signaling pathways in vitro and in vivo; Zelhof AC et al.; Seven-up (Svp), the Drosophila homolog of the chicken ovalbumin upstream transcription factor (COUP-TF); Ultraspiracle (Usp), the Drosophila homolog of the retinoid X receptor; and the ecdysone receptor are all members of the nuclear/steroid receptor superfamily . COUP-TF negatively regulates hormonal signaling involving retinoid X receptor in tissue culture systems . Here we demonstrate that Svp, like COUP-TF, can modulate Ultraspiracle-based hormonal signaling both in vitro and in vivo . Transfection assays in CV-1 cells demonstrate that Seven-up can inhibit ecdysone-dependent transactivation by the ecdysone receptor complex, a heterodimeric complex of Usp and ecdysone receptor . This repression depends on the dose of Svp and occurs with two different Drosophila ecdysone response elements . Ectopic expression of Svp in vivo induces lethality during early metamorphosis, the time of maximal ecdysone responsiveness . Concomitant overexpression of Usp rescues the larvae from the lethal effects of Svp . DNA binding studies show that Svp can bind to various direct repeats of the sequence AGGTCA but cannot bind to one of the ecdysone response elements used in the transient transfection assays . Our results suggest that Svp-mediated repression can occur by both DNA binding competition and protein-protein interactions. Cancer Res, 1995 Dec 1, 55(23), 5540 - 4 The oncogene qin codes for a transcriptional repressor; Li J et al.; The retroviral oncogene qin codes for a protein that belongs to the winged helix family of transcriptional regulators . The Qin protein is localized in the nucleus and binds to the same DNA consensus sequence as rat brain factor 1 (BF-1) . Cellular Qin shows greater affinity to DNA than does viral Qin . Alone or fused to the DNA-binding domain of the yeast GAL4 protein, both Qin proteins act as transcriptional repressors . The major transcriptional repression domain maps to the region of amino acids 252-395 of viral Qin. J Biol Chem, 1995 Dec 1, 270(48), 28834 - 8 Inhibition of Ras/Raf interaction by anti-oncogenic mutants of neurofibromin, the neurofibromatosis type 1 (NF1) gene product, in cell-free systems; Mori S et al.; The neurofibromatosis type 1 (NF1) gene encodes a protein, neurofibromin, containing GTPase-activating protein-related domain (GRD) that stimulates intrinsic GTPase activity of Ras protein . By screening a randomly mutagenized NF1-GRD library in Saccharomyces cerevisiae, we isolated two NF1-GRD mutants (NF201 and NF204) with single amino acid substitutions, which suppress the heat shock-sensitive phenotype of the RAS2(G19V) mutant . The NF1-GRD mutants also suppress the oncogenic Ras-induced transformation of NIH 3T3 mouse fibroblasts (Nakafuku, M., Nagamine, M., Ohtoshi, A., Tanaka, K., Toh-e, A., and Kaziro, Y . (1993) Proc . Natl . Acad . Sci . U.S.A . 90, 6706-6710) . In this paper, we investigated the molecular mechanism of inhibition of the transforming Ras-specific function by the NF1-GRD mutants in mammalian cells . In human embryonic kidney (HEK) 293 cells, the mutant NF1-GRDs attenuated the stimulation of mitogen-activated protein kinase by Ras(G12V), but not by platelet-derived growth factor . In cell-free systems, purified recombinant NF1-GRD mutants showed an inhibitory effect on the association of Ras.guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) with Raf at several times lower concentrations than the wild type . Furthermore, it was revealed that the binding affinity of the mutant NF1-GRDs toward Ras.GTP gamma S is approximately 5-10 times higher than the wild type . These results suggest that the mutant NF1-GRDs tightly bind to an oncogenic Ras in its GTP-bound active conformation and block the interaction between Ras and its effector, Raf. J Virol, 1995 Dec, 69(12), 7857 - 67 Cellular factors required for papillomavirus DNA replication; Melendy T et al.; In vitro replication of papillomavirus DNA has been carried out with a combination of purified proteins and partially purified extracts made from human cells . DNA synthesis requires the viral E1 protein and the papillomavirus origin of replication . The E2 protein stimulates DNA synthesis in a binding site-independent manner . Papillomavirus DNA replication is also dependent on the cellular factors replication protein A, replication factor C, and proliferating-cell nuclear antigen as well as a phosphocellulose column fraction (IIA) . Fraction IIA contains DNA polymerase alpha-primase and DNA polymerase delta . Both of these polymerases are essential for papillomavirus DNA replication in vitro . However, unlike the case with T-antigen-dependent replication from the simian virus 40 origin, purified DNA polymerase alpha-primase and delta cannot efficiently replace fraction IIA in the replication reaction . Hence, additional cellular factors seem to be required for papillomavirus DNA replication . Interestingly, replication factor C and proliferating-cell nuclear antigen are more stringently required for DNA synthesis in the papillomavirus system than in the simian virus 40 in vitro system . These distinctions indicate that there must be mechanistic differences between the DNA replication systems of papillomavirus and simian virus 40. J Virol, 1995 Dec, 69(12), 7845 - 50 Plus-strand strong-stop DNA synthesis in retrotransposon Ty1; Lauermann V et al.; Reverse transcription in the yeast retrotransposon Ty1 follows the general "rules" of retroviral replication overall . However, some details of the retroviral and Ty1 reverse transcription processes are different . We have identified and determined the structure of plus-strand strong-stop DNA and examined the effect of polypurine tract deletion mutations on its synthesis . Furthermore, we have defined the stop signal for plus-strand strong-stop DNA synthesis as an unusual 2'-O-ribosylated nucleotide in the primer tRNA . Full-length plus-strand strong-stop DNA, following strand transfer, would have a terminal 2-base mismatch with minus-strand DNA . These findings indicate that the mechanism of plus-strand strong-stop DNA transfer in Ty1 differs from that of the retroviral transfer and suggest that full-length plus-strand strong-stop DNA is not a direct intermediate in Ty1 retrotransposition. Biochem Pharmacol, 1995 Nov 27, 50(11), 1775 - 82 Validation of the (omega-1)-hydroxylation of lauric acid as an in vitro substrate probe for human liver CYP2E1; Amet Y et al.; The (omega-1)-hydroxylation of lauric acid (11-OH-LA), a model substrate of fatty acids, was previously shown to be due to CYP2E1 in rat liver microsomes . The present study examined changes in hepatic CYP2E1 content and 11-OH-LA in a panel of 29 human liver microsomes . The 11-OH-LA activity was strongly correlated with the CYP2E1 content, quantitated by immunoblot (r = 0.75) and with four monooxygenase activities known to be mediated by CYP2E1: chlorzoxazone-6-hydroxylation (r = 0.73), 4-nitrophenol hydroxylation (r = 0.84), N-nitrosodimethylamine demethylation (r = 0.79) and n-butanol oxidation (r = 0.73) . The (omega-1)-hydroxylation of lauric acid was inhibited by ethanol (Ki = 3.5 mM), acetone (IC50 = 10 mM) dimethylsulfoxide, chlorzoxazone (competitive inhibitors of CYP2E1), diethyldithiocarbamate, and diallylsulfide (both selective mechanism-based inactivators of CYP2E1) . The weak value of ethanol Ki on the (omega-1)-hydroxylation of lauric acid suggested that low levels of alcohol could modify fatty acid metabolism in the liver . Furafylline and gestodene, suicide substrates of CYP1A and CYP3A4, respectively, did not modify the 11-hydroxylation of lauric acid . Polyclonal antibody directed against rat CYP2E1 inhibited the formation of 11-OH-LA without affecting 12-OH-LA activity . Taken together, these results suggest that CYP2E1 is involved in the (omega-1)-hydroxylation of lauric acid in human liver microsomes, and omega-hydroxylation is mediated by another enzyme . Finally, the use of yeasts and mammalian cells genetically engineered for expression of 9 human P450s demonstrated that CYP2E1 was the one enzyme involved in the (omega-1)-hydroxylation of lauric acid. J Biol Chem, 1995 Nov 24, 270(47), 28126 - 32 Prevention of in vitro protein thermal aggregation by the Sulfolobus solfataricus chaperonin . Evidence for nonequivalent binding surfaces on the chaperonin molecule; Guagliardi A et al.; We have studied the effects of the Sulfolobus solfataricus chaperonin on the aggregation and inactivation upon heating of four model enzymes: chicken egg white lysozyme (one 14.4-kDa chain), yeast alpha-glucosidase (one 68.5-kDa chain), chicken liver malic enzyme (four 65-kDa subunits), and yeast alcohol dehydrogenase (four 37.5-kDa subunits) . When the proteins were heated in the presence of an equimolar amount of chaperonin, 1) the aggregation was prevented in all solutions; 2) the inactivation profiles of the single-chain enzymes were comparable with those detected in the absence of the chaperonin, and enzyme activities were regained in the solutions heated in the presence of the chaperonin upon ATP hydrolysis (78 and 55% activity regains for lysozyme and alpha-glucosidase, respectively); 3) the inactivation of the tetrameric enzymes was completely prevented, whereas the activities decreased in the absence of the chaperonin . We demonstrate by gel filtration chromatography that the chaperonin interacted with the structures occurring during thermal denaturation of the model proteins and that the interaction with the single-chain proteins (but not that with the tetrameric proteins) was reversed upon ATP hydrolysis . The chaperonin had nonequivalent surfaces for the binding of the model proteins upon heating: the thermal denaturation intermediates of the single-chain proteins share Surfaces I, while the thermal denaturation intermediates of the tetrameric proteins share Surfaces II . ATP binding to the chaperonin induced a conformation that lacked Surfaces I and carried Surfaces II . These data support the concept that chaperonins protect native proteins against thermal aggregation by two mechanistically distinct strategies (an ATP-dependent strategy and an ATP-independent strategy), and provide the first evidence that a chaperonin molecule bears functionally specialized surfaces for the binding of the protein substrates. J Biol Chem, 1995 Nov 24, 270(47), 28010 - 3 Signal transduction by the formyl peptide receptor . Studies using chimeric receptors and site-directed mutagenesis define a novel domain for interaction with G-proteins; Amatruda TT 3rd et al.; The binding of small peptide ligands to high affinity chemoattractant receptors on the surface of neutrophils and monocytes leads to activation of heterotrimeric G-proteins, stimulation of phosphatidylinositol-phospholipase C (PI-PLC), and subsequently to the inflammatory response . It was recently shown (Amatruda, T . T., Gerard, N . P., Gerard, C., and Simon, M . I . (1993) J . Biol . Chem . 268, 10139-10144) that the receptor for the chemoattractant peptide C5a specifically interacts with G alpha 16, a G-protein alpha subunit of the Gq class, to trigger ligand-dependent stimulation of PI-PLC in transfected cells . In order to further characterize this chemoattractant peptide signal transduction pathway, we transfected cDNAs encoding the formylmethionylleucylphenylalanine receptor (fMLPR) into COS cells and measured the production of inositol phosphates . Ligand-dependent activation of PI-PLC was seen in COS cells transfected with the fMLPR and G alpha 16 and stimulated with fMLP but not in cells transfected with receptor alone or with receptor plus G alpha q . Chimeric receptors in which the N-terminal extracellular domain, the second intracellular domain, or the intracellular C-terminal tail of the fMLP receptor was replaced with C5a receptor domains (Perez, H . D., Holmes, R., Vilander, L . R., Adams, R . R., Manzana, W., Jolley, D., and Andrews, W . H . (1993) J . Biol . Chem . 268, 2292-2295) were capable of ligand-dependent activation of PI-PLC when co-transfected with G alpha 16 . A chimeric receptor exchanging the first intracellular domain of the fMLPR was constitutively activated, stimulating PI-PLC in the absence of ligand . Constitutive activation of PI-PLC, to a level 233% of that seen in cells transfected with wild-type fMLP receptors, was dependent on G alpha 16 . Site-directed mutagenesis of the first intracellular domain of the fMLPR (amino acids 54-62) reveals this to be a domain necessary for ligand-dependent activation of G alpha 16 . These results suggest that different receptors which mediate similar biochemical responses may utilize distinct mechanisms to activate G-proteins . Differences among the signaling pathways triggered by chemoattractant factor receptors suggest an opportunity for pharmacologic modifications of the inflammatory response. J Biol Chem, 1995 Nov 24, 270(47), 28003 - 5 Termination as a factor in "quality control" during ribosome biogenesis; Lee Y et al.; In eukaryotes, nascent rDNA and 5 S rRNA gene transcripts undergo 3'-end processing after termination . Mutations in which terminator sequences in these ribosomal RNA genes are deleted completely result in highly unstable transcripts, which are not properly processed and integrated into stable ribosome structure . Mutations that retard RNA processing by extending the 3' external transcribed spacer or by introducing additional secondary structure in the spacers have a similar effect on stable transcript integration . The results indicate that proper termination coupled with efficient rRNA processing acts as a "quality control" process, which helps to ensure that only normal rRNA precursors are effectively processed and assembled into active ribosomes. J Biol Chem, 1995 Nov 24, 270(47), 27995 - 8 Cdc42 and PAK-mediated signaling leads to Jun kinase and p38 mitogen-activated protein kinase activation; Bagrodia S et al.; The PAK family of protein kinases has been suggested as a potential target of the Cdc42 and Rac GTPases based on studies in vitro . We show that PAK-3 is activated by Cdc42 in vivo . Both, activated (GTPase-defective) Cdc42 and a constitutively active PAK-3 mutant stimulated the activity of Jun kinase 1 (JNK1) in transfected cells . Activated Cdc42 also stimulated the activity of the related p38 mitogen-activated protein kinase but was a less effective activator of ERK2 . The effect of Cdc42 on JNK activity was similar to that of the potent inflammatory cytokine interleukin-1 (IL-1) . The observation that a dominant-negative Cdc42 mutant inhibited IL-1 activation of JNK1 indicates a role for Cdc42 in IL-1 signaling . These results suggest that Cdc42 and PAK may mediate the effects of cytokines on transcriptional regulation. Genomics, 1995 Nov 20, 30(2), 320 - 8 A YAC contig encompassing the XRCC5 (Ku80) DNA repair gene and complementation of defective cells by YAC protoplast fusion; Blunt T et al.; The Chinese hamster ovary xrs mutants are sensitive to ionizing radiation, defective in DNA double-strand break rejoining, and unable to carry out V(D)J recombination effectively . Recently, the gene defective in these mutants, XRCC5, has been shown to encode Ku80, a component of the Ku protein and DNA-dependent protein kinase . We present here a YAC contig involving 25 YACs mapping to the region 2q33-q34, which encompasses the XRCC5 gene . Eight new markers for this region of chromosome 2 are identified . YACs encoding the Ku80 gene were transferred to xrs cells by protoplast fusion, and complementation of all the defective phenotypes has been obtained with two YACs . We discuss the advantages and disadvantages of this approach as a strategy for cloning human genes complementing defective rodent cell lines. Gene, 1995 Nov 20, 165(2), 333 - 4 Cloning, sequencing and expression of the gene encoding a major allotypic preprocarboxypeptidase A from bovine pancreas; Goo JH et al.; A cDNA encoding one of two major allotypic forms of bovine pancreatic preprocarboxypeptidase A (preproCPA) has been cloned, and its entire nucleotide (nt) sequence determined . The cloned cDNA contains a 26-nt 5'-noncoding region, a 1260-nt open reading frame and a 51-nt 3'-noncoding region . The amino acid (aa) sequence deduced from the gene sequence contains Ile179, Ala228 and Val305 at allotypic aa residues . The procarboxypeptidase A (proCPA) was produced in yeast and secreted into the medium . Upon treatment with trypsin, proCPA generated an active enzyme with the same size as the mature carboxypeptidase A (CPA), as analyzed by use of anti-CPA polyclonal antibody. Gene, 1995 Nov 20, 165(2), 273 - 7 Differential expression of CAP and CAP2 in adult rat tissues; Swiston J et al.; We previously reported the identification of the human CAP and CAP2 genes which encode proteins related to the yeast adenylyl cyclase (CYR)-associated CAP protein . The rat CAP homolog, MCH1, has also been previously cloned . We have cloned a cDNA encoding the rat homolog of CAP2 . Rat CAP/MCH1 and CAP2 are 63% identical to each other . Using the reverse transcription-polymerase chain reaction (RT-PCR) method, we have examined CAP/MCH1 and CAP2 mRNA levels in various adult rat tissues . Our results show a dramatic difference in the pattern of expression of these two genes . Consistent with previous reports, we detected CAP/MCH1 mRNA in all tissues examined; however, levels vary substantially between tissues . In particular, we found that CAP/MCH1 mRNA are present at relatively high levels in spleen, testes and lung, at moderate levels in brain, kidney, liver and small intestine, and at significantly lower levels in heart, skeletal muscle and skin . We have also investigated the levels of CAP/MCH1 in rat tissues by immunoblotting with a polyclonal antibody raised against a human CAP::GST fusion protein . In general, we find that the CAP/MCH1 mRNA levels reflect the amount of CAP/MCH1 found in different tissues . In contrast, CAP2 transcripts were present at relatively high levels in testes, at moderate levels in brain, heart and skeletal muscle, at lower levels in lung, skin, kidney and small intestine, and were undetectable in liver or spleen . The differences between the sequences and expression patterns of CAP/MCH1 and CAP2 are significant and suggest that these proteins have distinct functional roles. Gene, 1995 Nov 20, 165(2), 203 - 6 Cloning of the Candida glabrata TRP1 and HIS3 genes, and construction of their disruptant strains by sequential integrative transformation; Kitada K et al.; The Candida glabrata (Cg) TRP1 and HIS3 genes have been isolated by complementation of the Saccharomyces cerevisiae (Sc) trp1 and his3 mutants, respectively . Cg TRP1 encodes a polypeptide of 217 amino acids (aa), whose aa sequence is 58% identical to that of Sc TRP1 . Cg HIS3 encodes a polypeptide of 210 aa, whose aa sequence is 73% identical to that of the Sc HIS3 . Both Cg TRP1 and HIS3 were disrupted by sequential integrative transformation where the Sc URA3 was used as a selection marker for transformation . The resulting auxotrophic strain of his3- and trp1- was used to examine the ability of the Sc genes to complement the Cg mutations; Sc HIS3 and TRP1 complemented the Cg his3- and trp1- mutations, respectively. FEBS Lett, 1995 Nov 20, 375(3), 299 - 303 Up-regulation of protein serine/threonine phosphatase type 2C during 1 alpha,25-dihydroxyvitamin D3-induced monocytic differentiation of leukemic HL-60 cells; Nishikawa M et al.; Treatment with 20 nM 1 alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3) caused a progressive increase in the activity of Mg(2+)-dependent protein serine/threonine phosphatase type 2C (PP2C) in subcellular fractions of HL-60 cells, whereas PP2C activity was relatively constant throughout all-trans retinoic acid-induced (1 microM) granulocytic differentiation . The increase in PP2C activity appeared to parallel the 1,25(OH)2D3-induced phenotypic and functional changes in HL-60 cells . Immunoblot and Northern blot analysis indicated that the increase in PP2C activity corresponded to the increased expression of PP2C protein, which was preceded by an increase in the level of mRNA for PP2C beta . No mRNA for PP2C alpha was detected in resting or 1,25(OH)2D3-stimulated HL-60 cells . These results suggest that the increased expression of PP2C is related with the 1,25(OH)2D3-induced monocytic differentiation of HL-60 cells. FEBS Lett, 1995 Nov 20, 375(3), 220 - 2 The presence of a hydroxyl group at the C-1 atom of the transketolase substrate molecule is necessary for the enzyme to perform the transferase reaction; Meshalkina LE et al.; Transketolase catalyzes the transfer of an aldehyde residue from keto sugars to aldo sugars . The intermediate product is dihydroxyethylthiamine pyrophosphate (DHETPP) . In the absence of an acceptor substrate, the reaction is stopped at this stage and DHETPP does not undergo subsequent transformations . Pyruvate decarboxylase catalyses pyruvate decarboxylation to yield free aldehyde . The intermediate product is hydroxyethylthiamine pyrophosphate (HETPP) . It differs from DHETPP only in that it has no hydroxyl at the C-2 atom of the aldehyde residue . We have shown that transketolase can bind HETPP and split the aldehyde residue from it . This fact suggests that the path of the reaction is determined by the absence (in HETPP) or presence (in DHETPP) of a hydroxyl group . In the former case the reaction will yield free aldehyde, in the latter the aldehyde residue will be transferred onto an acceptor substrate. J Chromatogr A, 1995 Nov 17, 716(1-2), 215 - 20 Analysis by capillary electrophoresis-laser-induced fluorescence detection of oligosaccharides produced from enzyme reactions; Le X et al.; Six structurally similar, fluorescently labeled oligosaccharides were baseline resolved by capillary electrophoresis (CE); laser induced fluorescence (LIF) detection gave detection limits of 50 molecules for the oligosaccharides . A simple design of the LIF detector that incorporates the advantages of high sensitivity, stability and ease of operation is described . The system was used to monitor enzyme products formed during the incubation of yeast cells with alpha-D-Glc(1-->2)alpha-D-Glc(1-->3)alpha-D-glc-O(CH2)8CONHCH2CH2NHCO - tetramethylrhodamine . This fluorescent trisaccharide is enzymatically hydrolyzed to fluorescent disaccharide, monosaccharide and the free linker arm that is used to conjugate the saccharides with the fluorophore tetramethylrhodamine. Cell, 1995 Nov 17, 83(4), 529 - 38 A group II intron RNA is a catalytic component of a DNA endonuclease involved in intron mobility; Zimmerly S et al.; The mobility (homing) of the yeast mitochondrial DNA group II intron al2 occurs via target DNA-primed reverse transcription at a double-strand break in the recipient DNA . Here, we show that the site-specific DNA endonuclease that makes the double-strand break is a ribonucleoprotein complex containing the al2-encoded reverse transcriptase protein and excised al2 RNA . Remarkably, the al2 RNA catalyzes cleavage of the sense strand of the recipient DNA, while the al2 protein appears to cleave the antisense strand . The RNA-catalyzed sense strand cleavage occurs via a partial reverse splicing reaction in which the protein component stabilizes the active intron structure and appears to confer preference for DNA substrates . Our results demonstrate a biologically relevant ribozyme reaction with a substrate other than RNA. J Biol Chem, 1995 Nov 17, 270(46), 27812 - 6 Two residues that may ligate Ca2+ in transmembrane domain six of the plasma membrane Ca(2+)-ATPase; Adebayo AO et al.; In order to identify Ca2+ ligands in the putative transmembrane domain 6 of the plasma membrane Ca2+ pump, amino acids Asn879, Met882, Asp883, and Ser887 were singly altered . Asn879, Met882, and Asp883 were chosen because the corresponding amino acids have been proposed as Ca2+ ligands in the sarcoplasmic reticulum Ca2+ pump (Clarke, D . M., Loo, T . W., and MacLennan, D . H . (1990) J . Biol . Chem . 265, 6262-6267) . For the alterations, a fully active truncated version of the pump was used, because the interaction of Ca2+ with the pump could be studied without interference from calmodulin binding . The mutants at Asn and Asp did not carry out ATP-supported Ca2+ uptake and formed no acylphosphate from {gamma-32P}ATP, suggesting that, like the corresponding amino acids in the sarcoplasmic reticulum Ca2+ pump, these two are Ca2+ ligands . However, all the mutants at the position of Met882 showed some activity . Indeed, the Met882--> Ile mutant was fully active at a saturating Ca2+ concentration and only the K1/2 for Ca2+ activation was shifted slightly upward . Converting the Met to Thr (which is the corresponding residue in the sarcoplasmic reticulum Ca2+ pump) reduced the activity to 20% of the wild type, further emphasizing the differences between the two Ca2+ pumps . The mutant Ser887--> Ala was expressed in greater amounts than, and had a specific activity about 50% higher than, the wild type, indicating that this serine also could not be a Ca2+ ligand and could not replace the missing Thr at position Met882. J Biol Chem, 1995 Nov 17, 270(46), 27531 - 7 TOR mutations confer rapamycin resistance by preventing interaction with FKBP12-rapamycin; Lorenz MC et al.; The antifungal, immunosuppressive compound rapamycin arrests the cell cycle in G1 in both yeast cells and T-lymphocytes . Previous genetic studies in yeast identified mutations in three genes, FPR1 (FKBP12), TOR1, and TOR2, which confer rapamycin resistance, and genetic findings implicated the TOR proteins as direct targets of FKBP12-rapamycin . Consistent with this model, we find that modulating TOR1 and TOR2 expression alters rapamycin sensitivity . We describe several TOR2 mutations that confer rapamycin resistance . These mutations prevent FKBP12-rapamycin binding to TOR2, as assayed with the two-hybrid system . We find that TOR1 and the mammalian TOR homologue (mTOR) also bind FKBP12-rapamycin, and mutations corresponding to those in TOR2 similarly block FKBP12-rapamycin binding . We demonstrate that FKBP12 prolyl isomerase activity is not required for FKBP12-rapamycin binding to TOR and that a composite protein-drug surface contacts the TOR proteins . These studies confirm that the TOR proteins are direct targets of FKBP12-rapamycin, reveal that drug-resistant mutations prevent this association, and define structural features of these complexes. J Biol Chem, 1995 Nov 17, 270(46), 27475 - 80 Role of acidic residues in the interaction of NADPH-cytochrome P450 oxidoreductase with cytochrome P450 and cytochrome c; Shen AL et al.; Site-directed mutagenesis of the acidic clusters 207Asp-Asp-Asp209 and 213Glu-Glu-Asp215 of NADPH-cytochrome P450 oxidoreductase demonstrates that both cytochrome c and cytochrome P450 interact with this region; however, the sites and mechanisms of interaction of the two substrates are clearly distinct . Substitutions in the first acidic cluster did not affect cytochrome c or ferricyanide reductase activity, but substitution of asparagine for aspartate at position 208 reduced cytochrome P450-dependent benzphetamine N-demethylase activity by 63% with no effect on KP450m or KNADPHm . Substitutions in the second acidic cluster affected cytochrome c reduction but not benzphe