Curr Opin Cell Biol, 1997 Aug, 9(4), 496 - 504 The diversity of Rab proteins in vesicle transport; Novick P et al.; Rab proteins have been primarily implicated in vesicle docking as regulators of SNARE pairing . Recent findings, however, indicate that their function in vesicle trafficking can go beyond this role, and a number of proteins, unrelated to each other, have been identified as putative Rab effectors . Although the GTPase switch of Rab proteins is highly conserved, functional mechanisms may be highly diversified among members of the Rab family. Curr Opin Cell Biol, 1997 Aug, 9(4), 488 - 95 Linking cargo to vesicle formation: receptor tail interactions with coat proteins; Kirchhausen T et al.; How soluble cargo molecules concentrate into budding vesicles is the subject of intensive current research . Clathrin-based vesiculation from the plasma membrane and the trans-Golgi network constitutes the best described system that supports this sorting process . Soluble ligands bind to specific transmembrane receptors which have been shown to interact directly with clathrin adaptor complexes, components of clathrin coats . At the same time, these clathrin adaptors facilitate clathrin coat assembly and probably regulate the recruitment of the rest of the coat components . Recent studies have looked at both the interaction of receptor tails with adaptors and the assembly of the clathrin coat . Progress has also been made in elucidating how soluble cargo molecules may be concentrated for exit from the endoplasmic reticulum. Curr Opin Cell Biol, 1997 Aug, 9(4), 484 - 7 Coatomer (COPI)-coated vesicles: role in intracellular transport and protein sorting; Cosson P et al.; Coatomer-coated vesicles have been proposed to play a role in many distinct steps of intracellular transport . Coatomer potentially plays a role in forward transport from the endoplasmic reticulum to the Golgi apparatus and through the Golgi apparatus . It may also function in retrograde transport and in the endocytic pathway . There are limitations to the various approaches used to study the role of coatomer, and looking at these helps us to better define the questions that remain to be answered. Curr Opin Cell Biol, 1997 Aug, 9(4), 477 - 83 COPII and secretory cargo capture into transport vesicles; Kuehn MJ et al.; Yeast cylosolic coat proteins (COPII) direct the formation of vesicles from the endoplasmic reticulum . The vesicles selectively capture both cargo molecules and the secretory machinery that is necessary for the fusion of the vesicle with the recipient compartment, the Golgi apparatus . Recent efforts have aimed to understand how proteins are selected for inclusion into these vesicles . A variety of cargo adaptors may concentrate and sort secretory and membrane proteins by direct or indirect interaction with a subset of coat protein subunits. Trends Genet, 1997 Aug, 13(8), 308 - 13 An uncertain silence; Sherman JM et al.; Transcriptional silencing is the most well-studied epigenetic phenomenon in yeast . In Saccharomyces cerevisiae, silencing has recently been found at previously unidentified loci . In addition to the silent mating-type loci and telomeres, genes within the ribosomal DNA and, perhaps, at undefined aging loci are silenced . Efficiency of silencing at different loci varies and is affected by competition between the loci and by the involvement of different factors in distinct protein complexes . The recent discovery of conserved gene families encoding proteins related to modulators of acetylation and deacetylation suggests mechanisms for differential regulation of silencing at known loci and the existence of additional, as yet undiscovered, silenced loci. Curr Biol, 1997 Aug 1, 7(8), 607 - 10 The prohibitin family of mitochondrial proteins regulate replicative lifespan; Coates PJ et al.; Cellular senescence is determined by multiple factors, including the genetic regulation of metabolism and responses to endogenous and exogenous stresses {1-4} . Recent studies implicate a limited number of gene products in elongating lifespan in yeast and Caenorhabditis elegans {2-4}; these include the C, elegans gene cik-1, a central regulator of metabolism {5}, and yeast RAS2, which controls the response to ultraviolet irradiation and other stresses {3} . Another gene postulated to effect senescence is PHB1, the yeast homologue of prohibitin {3}, a rodent gene initially identified as a potential regulator of growth arrest and tumour suppressor {6-8} . Highly conserved prohibitin homologues have been identified in mammals {9}, Drosophila {10}, C . elegans {9}, plants {11} and yeast . A second mammalian gene, encoding BAP37, a protein with sequence similarity to prohibitin, is thought to be involved in lymphocyte function {9} . Here, we show that the nuclear-encoded mammalian prohibitin and BAP37 proteins are present in mitochondria, are co-expressed, and interact physically with each other . Deletion of the Saccharomyces cerevisiae homologues, PHB1 and PHB2, results in a decreased replicative lifespan and a defect in mitochondrial membrane potential . Our observations highlight the relationship between the metabolic efficiency of cells and the ageing process, and provide evidence for its evolutionary conservation. Curr Biol, 1997 Aug 1, 7(8), R492 - 5 DNA repair: RAD alert; Ivanov EL et al.; Mammalian homologues of two important yeast genes involved in DNA double-strand break repair and recombination, RAD51 and RAD54, have been isolated . Knock-out mutations of the genes in mice reveal both reassuring similarities to, and surprising differences from, the analogous mutant phenotypes in yeast. Nat Struct Biol, 1997 Aug, 4(8), 626 - 8 Protein structural classes in five complete genomes; Frishman D et al.; The predicted distribution of globular proteins over folding types in five complete genomes differs from the tendencies observed in known protein structures . The ratio between the number of predicted membrane and globular proteins is conserved. Biophys J, 1997 Aug, 73(2), 1031 - 41 Thermal unfolding in a GCN4-like leucine zipper: 13C alpha NMR chemical shifts and local unfolding curves; Holtzer ME et al.; 13C alpha chemical shifts and site-specific unfolding curves are reported for 12 sites on a 33-residue, GCN4-like leucine zipper peptide (GCN4-lzK), ranging over most of the chain and sampling most heptad positions . Data were derived from NMR spectra of nine synthetic, isosequential peptides bearing 99% 13C alpha at sites selected to avoid spectral overlap in each peptide . At each site, separate resonances appear for unfolded and folded forms, and most sites show resonances for two folded forms near room temperature . The observed chemical shifts suggest that 1) urea-unfolded GCN4-lzK chains are randomly coiled; 2) thermally unfolded chains include significant transient structure, except at the ends; 3) the coiled-coli structure in the folded chains is atypical near the C-terminus; 4) only those interior sites surrounded by canonical interchain salt bridges fail to show two folded forms . Local unfolding curves, obtained from integrated resonance intensities, show that 1) sites differ in structure content and in melting temperature, so the equilibrium population must comprise more than two molecular conformations; 2) there is significant end-fraying, even at the lowest temperatures, but thermal unfolding is not a progressive unwinding from the ends; 3) residues 9-16 are in the lowest melting region; 4) heptad position does not dictate stability; 5) significant unfolding occurs below room temperature, so the shallow, linear decline in backbone CD seen there has conformational significance . It seems that only a relatively complex array of conformational states could underlie these findings. J Mol Biol, 1997 Aug 1, 270(5), 627 - 39 Molecular cloning and expression of the Caenorhabditis elegans klp-3, an ortholog of C terminus motor kinesins Kar3 and ncd; Khan ML et al.; Common to all eukaryotes, kinesins are cytoskeletal motor proteins that mediate intracellular transport on microtubule tracks, using ATP hydrolysis . A Caenorhabditis elegans cDNA clone corresponding to the klp-3 gene, encoding a novel kinesin, was isolated, and mapped on LGII . Northern blot analysis using the klp-3 cDNA probe reveals a 1.9 kb mRNA that is transcribed at a low level during development . Temporal and spatial expression of the klp-3::lacZ fusion gene is limited to the marginal cells in the pharynx, and a group of muscle cells in the posterior gut region . The nucleotide sequence of klp-3 has been deduced from the cDNA and nematode genome sequencing consortium data . Conceptual translation of the klp-3 gene reveals a kinesin-like protein with its conserved motor domain containing the ATP binding and microtubule binding sites located in the C terminus . KLP-3 shares extensive homology with the yeast Kar3 and Drosophila ncd kinesins, which have previously been shown to mediate chromosomal movement and segregation during meiosis and mitosis . Overexpression of the klp-3 gene partially rescues the lethal phenotype of the maternal lethal him-14 ts(it44) mutants at non-permissive temperatures, and reduces the incidence of males caused by non-disjunction of the X-chromosome . Similarly, expression of a klp-3 antisense RNA, under the control of a heat shock promoter, causes embryonic arrest, dead eggs and polyploid cells in transgenic lines, suggesting a critical role for the klp-3 function in chromosome segregation . Further analysis of the klp-3 gene in C . elegans may elucidate diverse functions of the C terminus mitotic motor proteins during development. J Biol Chem, 1997 Aug 1, 272(31), 19383 - 92 Evidence that intramolecular interactions are involved in masking the activation domain of transcriptional activator Leu3p; Wang D et al.; The Leu3 protein of Saccharomyces cerevisiae regulates the expression of genes involved in branched chain amino acid biosynthesis and in ammonia assimilation . It is modulated by alpha-isopropylmalate, an intermediate in leucine biosynthesis . In the presence of alpha-isopropylmalate, Leu3p is a transcriptional activator . In the absence of the signal molecule, the activation domain is masked, and Leu3p acts as a repressor . The recent discovery that Leu3p retains its regulatory properties when expressed in mammalian cells (Guo, H., and Kohlhaw, G . B . (1996) FEBS Lett . 390, 191-195) suggests that masking and unmasking of the activation domain occur without the participation of auxiliary proteins . Here we present experimental support for this notion and address the mechanism of masking . We show that modulation of Leu3p is exceedingly sensitive to mutations in the activation domain . An activation domain double mutant (D872N/D874N; designated Leu3-dd) was constructed that has the characteristics of a permanently masked activator . Using separately expressed segments containing either the DNA binding domain-middle region or the activation domain of wild type Leu3p (or Leu3-dd) in a modified yeast two-hybrid system, we provide direct evidence for alpha-isopropylmalate-dependent interaction between these segments . Finally, we use the phenotype of Leu3-dd-containing cells (slow growth in the absence of added leucine) to select for suppressor mutations that map to the middle region of Leu3-dd . The properties of nine such suppressors further support the idea that masking is an intramolecular process and suggest a means for mapping the surface involved in masking. Mol Cell Biol, 1997 Aug, 17(8), 4870 - 6 Identification of AUF1 (heterogeneous nuclear ribonucleoprotein D) as a component of the alpha-globin mRNA stability complex; Kiledjian M et al.; mRNA turnover is an important regulatory component of gene expression and is significantly influenced by ribonucleoprotein (RNP) complexes which form on the mRNA . Studies of human alpha-globin mRNA stability have identified a specific RNP complex (alpha-complex) which forms on the 3' untranslated region (3'UTR) of the mRNA and appears to regulate the erythrocyte-specific accumulation of alpha-globin mRNA . One of the protein activities in this multiprotein complex is a poly(C)-binding activity which consists of two proteins, alphaCP1 and alphaCP2 . Neither of these proteins, individually or as a pair, can bind the alpha-globin 3'UTR unless they are complexed with the remaining non-poly(C) binding proteins of the alpha-complex . With the yeast two-hybrid screen, a second alpha-complex protein was identified . This protein is a member of the previously identified A+U-rich (ARE) binding/degradation factor (AUF1) family of proteins, which are also known as the heterogeneous nuclear RNP (hnRNP) D proteins . We refer to these proteins as AUF1/hnRNP-D . Thus, a protein implicated in ARE-mediated mRNA decay is also an integral component of the mRNA stabilizing alpha-complex . The interaction of AUF1/hnRNP-D is more efficient with alphaCP1 relative to alphaCP2 both in vitro and in vivo, suggesting that the alpha-complex might be dynamic rather than a fixed complex . AUF1/hnRNP-D could, therefore, be a general mRNA turnover factor involved in both stabilization and decay of mRNA. Mol Cell Biol, 1997 Aug, 17(8), 4842 - 51 Molecular analysis of the SNF2/SWI2 protein family member MOT1, an ATP-driven enzyme that dissociates TATA-binding protein from DNA; Auble DT et al.; MOT1 is an essential Saccharomyces cerevisiae protein and a member of the SNF2/SWI2 family of ATPases . MOT1 functions by removing TATA-binding protein (TBP) from DNA, and as a consequence, MOT1 can regulate transcription both in vitro and in vivo . Here we describe the in vivo and in vitro activities of MOT1 deletion and substitution mutants . The results indicate that MOT1 is targeted to TBP both in vitro and in vivo via amino acids in its nonconserved N terminus . The conserved C-terminal ATPase of MOT1 appears to contribute to TBP-DNA complex recognition in the absence of ATP, but it appears to function primarily during the actual ATP-dependent dissociation reaction . Chimeric proteins in which homologous portions of SNF2/SWI2 have been substituted for the MOT1 ATPase can bind to TBP-DNA complexes but fail to dissociate these complexes in the presence of ATP, suggesting that the specificity of action of MOT1 is also conferred by the C-terminal ATPase . ATPase assays demonstrate that the MOT1 ATPase is activated by TBP . Thus, MOT1 undergoes at least two conformational changes: (i) an allosteric effect of TBP that mediates the activation of the MOT1 ATPase and (ii) an ATP-driven "power stroke" that causes TBP-DNA complex dissociation . These results provide a general framework for understanding how members of the SNF2/SWI2 protein family use ATP to modulate protein-DNA interactions to regulate many diverse processes in cells. Mol Cell Biol, 1997 Aug, 17(8), 4820 - 9 Two evolutionarily conserved repression domains in the Drosophila Kruppel protein differ in activator specificity; Hanna-Rose W et al.; To identify biologically functional regions in the product of the Drosophila melanogaster gene Kruppel, we cloned the Kruppel homolog from Drosophila virilis . Both the previously identified amino (N)-terminal repression region and the DNA-binding region of the D . virilis Kruppel protein are greater than 96% identical to those of the D . melanogaster Kruppel protein, demonstrating a selective pressure to maintain the integrity of each region during 60 million to 80 million years of evolution . An additional region in the carboxyl (C) terminus of Kruppel that was most highly conserved was examined further . A 42-amino-acid stretch within the conserved C-terminal region also encoded a transferable repression domain . The short, C-terminal repression region is a composite of three subregions of distinct amino acid composition, each containing a high proportion of either basic, proline, or acidic residues . Mutagenesis experiments demonstrated, unexpectedly, that the acidic residues contribute to repression function . Both the N-terminal and C-terminal repression regions were tested for the ability to affect transcription mediated by a variety of activator proteins . The N-terminal repression region was able to inhibit transcription in the presence of multiple activators . However, the C-terminal repression region inhibited transcription by only a subset of the activator proteins . The different activator specificities of the two regions suggest that they repress transcription by different mechanisms and may play distinct biological roles during Drosophila development. Mol Cell Biol, 1997 Aug, 17(8), 4687 - 95 Mutations in the conserved C-terminal sequence in thyroid hormone receptor dissociate hormone-dependent activation from interference with AP-1 activity; Saatcioglu F et al.; A short C-terminal sequence that is deleted in the v-ErbA oncoprotein and conserved in members of the nuclear receptor superfamily is required for normal biological function of its normal cellular counterpart, the thyroid hormone receptor alpha (T3R alpha) . We carried out an extensive mutational analysis of this region based on the crystal structure of the hormone-bound ligand binding domain of T3R alpha . Mutagenesis of Leu398 or Glu401, which are surface exposed according to the crystal structure, completely blocks or significantly impairs T3-dependent transcriptional activation but does not affect or only partially diminishes interference with AP-1 activity . These are the first mutations that clearly dissociate these activities for T3R alpha . Substitution of Leu400, which is also surface exposed, does not affect interference with AP-1 activity and only partially diminishes T3-dependent transactivation . None of the mutations affect ligand-independent transactivation, consistent with previous findings that this activity is mediated by the N-terminal domain of T3R alpha . The loss of ligand-dependent transactivation for some mutants can largely be reversed in the presence of GRIP1, which acts as a strong ligand-dependent coactivator for wild-type T3R alpha . There is excellent correlation between T3-dependent in vitro association of GRIP1 with T3R alpha mutants and their ability to support T3-dependent transcriptional activation . Therefore, GRIP1, previously found to interact with the glucocorticoid, estrogen, and androgen receptors, may also have a role in T3R alpha-mediated ligand-dependent transcriptional activation . When fused to a heterologous DNA binding domain, that of the yeast transactivator GAL4, the conserved C terminus of T3R alpha functions as a strong ligand-independent activator in both mammalian and yeast cells . However, point mutations within this region have drastically different effects on these activities compared to their effect on the full-length T3R alpha . We conclude that the C-terminal conserved region contains a recognition surface for GRIP1 or a similar coactivator that facilitates its interaction with the basal transcriptional apparatus . While important for ligand-dependent transactivation, this interaction surface is not directly involved in transrepression of AP-1 activity. Mol Cell Biol, 1997 Aug, 17(8), 4644 - 53 Probing the structure and function of the estrogen receptor ligand binding domain by analysis of mutants with altered transactivation characteristics; Eng FC et al.; We have developed a genetic screen for the yeast Saccharomyces cerevisiae to isolate estrogen receptor (ER) mutants with altered transactivation characteristics . Use of a "reverse" ER, in which the mutagenized ligand binding domain was placed at the N terminus of the receptor, eliminated the isolation of truncated constitutively active mutants . A library was screened with a low-affinity estrogen, 2-methoxyestrone (2ME), at concentrations 50-fold lower than those required for activation of the unmutagenized ER . Several mutants displaying enhanced sensitivity to 2ME were isolated . We further characterized a mutant carrying the substitution L536P, which was located immediately N terminal to the AF-2-activating domain of the receptor . Amino acid 536 corresponds to a ligand contact residue in retinoic acid receptor gamma, suggesting that key contact points are conserved among receptors . Introduction of L536P into the original ER cDNA isolate HE0, which contains the substitution G400V, rendered the receptor more sensitive to a variety of agonists . When introduced into the wild-type ER HEG0, L536P also rendered the receptor more sensitive to agonists, and, in addition, induced high levels of constitutive activity that could be inhibited by antiestrogens . Estrogens containing a keto substitution in the steroid D ring, but not those containing a hydroxyl group, were full agonists of L536P-HEG0 . Limited proteolytic analysis suggested that the L536P substitution, which is located immediately N terminal to the AF-2 domain, induces a conformational change in the ER that partially mimics binding by hormone . Both HEG0 and L536P-HEG0 formed complexes with hsp90 in vitro, indicating a lack of correlation between interaction with hsp90 in vitro and hormonal regulation of ER transactivation in vivo . This supports the idea that a factor(s) acting downstream of hsp90 is important for controlling activity of the hormone-free receptor. Mol Cell Biol, 1997 Aug, 17(8), 4474 - 89 Evidence that GCN1 and GCN20, translational regulators of GCN4, function on elongating ribosomes in activation of eIF2alpha kinase GCN2; Marton MJ et al.; In the yeast Saccharomyces cerevisiae, phosphorylation of translation initiation factor eIF2 by protein kinase GCN2 leads to increased translation of the transcriptional activator GCN4 in amino acid-starved cells . The GCN1 and GCN20 proteins are components of a protein complex required for the stimulation of GCN2 kinase activity under starvation conditions . GCN20 is a member of the ATP-binding cassette (ABC) family, most of the members of which function as membrane-bound transporters, raising the possibility that the GCN1/GCN20 complex regulates GCN2 indirectly as an amino acid transporter . At odds with this idea, indirect immunofluorescence revealed cytoplasmic localization of GCN1 and no obvious association with plasma or vacuolar membranes . In addition, a fraction of GCN1 and GCN20 cosedimented with polysomes and 80S ribosomes, and the ribosome association of GCN20 was largely dependent on GCN1 . The C-terminal 84% of GCN20 containing the ABCs was found to be dispensable for complex formation with GCN1 and for the stimulation of GCN2 kinase function . Because ABCs provide the energy-coupling mechanism for ABC transporters, these results also contradict the idea that GCN20 regulates GCN2 as an amino acid transporter . The N-terminal 15 to 25% of GCN20, which is critically required for its regulatory function, was found to interact with an internal segment of GCN1 similar in sequence to translation elongation factor 3 (EF3) . Based on these findings, we propose that GCN1 performs an EF3-related function in facilitating the activation of GCN2 by uncharged tRNA on translating ribosomes . The physical interaction between GCN20 and the EF3-like domain in GCN1 could allow for modulation of GCN1 activity, and the ABC domains in GCN20 may be involved in this regulatory function . A human homolog of GCN1 has been identified, and the portion of this protein most highly conserved with yeast GCN1 has sequence similarity to EF3 . Thus, similar mechanisms for the detection of uncharged tRNA on translating ribosomes may operate in yeast and human cells. Mol Cell Biol, 1997 Aug, 17(8), 4330 - 7 Cooperative binding interactions required for function of the Ty1 sterile responsive element; Baur M et al.; The Ste12p transcription factor controls the expression of Ty1 transposable element insertion mutations and genes whose products are required for mating in Saccharomyces cerevisiae . The binding site for Ste12p is a consensus DNA sequence known as a pheromone response element (PRE) . Upstream activating sequences (UASs) derived from known Ste12p-dependent genes have previously been characterized to require either multiple PREs or a single PRE coupled to a binding site for a second protein . The Ste12p-dependent UAS from Ty1, called a sterile response element (SRE), is of the second type and is comprised of a PRE and an adjacent TEA (TEF-1, Tec1, and AbaA motif) DNA consensus sequence (TCS) . In this report, we show by UV cross-linking analysis that two proteins, Ste12p and a protein with an apparent size of 72 kDa, directly contact the Ty1 SRE . Other experiments show that Tec1p is required for formation of the Ty1 SRE protein-DNA complex and is physically present in the complex . These results establish a direct role for Tec1p in the Ty1 SRE and yet another set of combinatorial interactions that achieve a qualitatively distinct mode of transcriptional regulation with Ste12p. Nucleic Acids Res, 1997 Aug 1, 25(15), 3042 - 50 NMR analysis of CYP1(HAP1) DNA binding domain-CYC1 upstream activation sequence interactions: recognition of a CGG trinucleotide and of an additional thymine 5 bp downstream by the zinc cluster and the N-terminal extremity of the protein; Vuidepot AL et al.; The DNA binding domain of the yeast transcriptional activator CYP1(HAP1) contains a zinc-cluster structure . The structures of the DNA binding domain-DNA complexes of two other zinc-cluster proteins (GAL4 and PPR1) have been studied by X-ray crystallography . Their binding domains present, besides the zinc cluster, a short linker peptide and a dimerization element . They recognize, as homodimers, two rotationally symmetric CGG trinucleotides, the linker peptide and the dimerization element playing a crucial role in binding specificity . Surprisingly, CYP1 recognizes degenerate forms of a direct repeat, CGGnnnTAnCGGnnnTA, and the role of its linker is under discussion . To better understand the binding specificity of CYP1, we have studied, by NMR, the interaction between the CYP1(55-126) peptide and two DNA fragments derived from the CYC1 upstream activation sequence 1B . Our data indicate that CYP1(55-126) interacts with a CGG and with a thymine 5 bp downstream . The CGG trinucleotide is recognized by the zinc cluster in the major groove, as for GAL4 and PPR1, and the thymine is bound in the minor groove by the N-terminal region, which possesses a basic stretch of arginyl and lysyl residues . This suggests that the CYP1(55-126) N-terminal region could play a role in the affinity and/or specificity of the interaction with its DNA targets, in contrast to GAL4 and PPR1. Nucleic Acids Res, 1997 Aug 1, 25(15), 2985 - 91 Conditional gene targeted deletion by Cre recombinase demonstrates the requirement for the double-strand break repair Mre11 protein in murine embryonic stem cells; Xiao Y et al.; Repair of DNA damage resulting in double-strand breaks (DSBs) is controlled by gene products executing homologous recombination or end-joining pathways . The MRE11 gene has previously been implicated in DSB repair in the yeast Saccharomyces cerevisiae . Here we have developed a methodology to study the roles of the murine Mre11 homolog in pluripotent embryonic stem cells . Using a gene targeting approach, a triple LoxP site cassette was inserted into a region of MRE11 genomic DNA flanking conserved phosphodiesterase motifs . The addition of Cre recombinase activity promotes deletions of three types that can be scored . We find that deletion at phosphodiesterase motif III encoded in the N-terminus of Mre11 is acheived in the presence of a wild-type MRE11 allele . However, when the wild-type MRE11 allele is inactivated by gene targeted insertion of a neo marker, only Cre recombination events that allow expression of wild-type Mre11 protein are observed . Therefore, Mre11 is required for normal cell proliferation . This methodology introduces a means to study important regions of essential genes in cell culture models. Nucleic Acids Res, 1997 Aug 1, 25(15), 2967 - 72 The role of a basic amino acid cluster in target site selection and non-specific binding of bZIP peptides to DNA; Metallo SJ et al.; The ability of a transcription factor to locate and bind its cognate DNA site in the presence of closely related sites and a vast array of non-specific DNA is crucial for cell survival . The CREB/ATF family of transcription factors is an important group of basic region leucine zipper (bZIP) proteins that display high affinity for the CRE site and low affinity for the closely related AP-1 site . Members of the CREB/ATF family share in common a cluster of basic amino acids at the N-terminus of their bZIP element . This basic cluster is necessary and sufficient to cause the CRE site to bend upon binding of a CREB/ATF protein . The possibility that DNA bending and CRE/AP-1 specificity were linked in CREB/ATF proteins was investigated using chimeric peptides derived from human CRE-BP1 (a member of the CREB/ATF family) and yeast GCN4, which lacks both a basic cluster and CRE/AP-1 specificity . Gain of function and loss of function experiments demonstrated that the basic cluster was not responsible for the CRE/AP-1 specificity displayed by all characterized CREB/ATF proteins . The basic cluster was, however, responsible for inducing very high affinity for non- specific DNA . It was further shown that basic cluster-containing peptides bind non-specific DNA in a random coil conformation . We postulate that the high non- specific DNA affinities of basic cluster-containing peptides result from cooperative electrostatic interactions with the phosphate backbone that do not require peptide organization. J Virol, 1997 Aug, 71(8), 5942 - 51 Two classes of human papillomavirus type 16 E1 mutants suggest pleiotropic conformational constraints affecting E1 multimerization, E2 interaction, and interaction with cellular proteins; Yasugi T et al.; Random mutagenesis of human papillomavirus type 16 (HPV16) E1 was used to generate E1 missense mutants defective for interaction with either hUBC9 or 16E1-BP, two cDNAs encoding proteins that have been identified by their ability to interact with HPV16 E1 in two-hybrid assays . hUBC9, the human counterpart of Saccharomyces cerevisiae UBC9, is a ubiquitin-conjugating enzyme known to be involved in cell cycle progression . 16E1-BP encodes a protein of no known function but does contain an ATPase signature motif . Eight hUBC9 or 16E1-BP interaction-defective HPV16 E1 missense mutants were identified and characterized for origin-dependent transient DNA replication, ATPase activity, and various protein-protein interaction phenotypes . Six of these mutant E1 proteins were significantly impaired for replication . Among these, two classes of replication-defective HPV16 E1 missense mutants were observed . One class, represented by the S330R replication-defective mutant (containing an S-to-R change at position 330), remained competent for all protein-protein interactions tested, with the exception of hUBC9 association . Furthermore, this mutant, unlike the other replication-defective HPV16 E1 missense mutants, had a strong dominant negative replication phenotype in transient-replication assays . The other class, represented by five of the missense mutants, was defective for multiple protein-protein interactions, usually including, but not limited to, the interaction defect for which each mutant was originally selected . In many cases, a single missense mutation in one region of HPV16 E1 had pleiotropic effects, even upon activities thought to be associated with other domains of HPV16 E1 . This suggests that E1 proteins are not modular but may instead be composed of multiple structurally and/or functionally interdependent domains. Gene, 1997 Jul 31, 194(2), 163 - 7 The sequence of palF, an environmental pH response gene in Aspergillus nidulans; Maccheroni W Jr et al.; To molecularly characterize the influence of external pH in the secretion of enzymes by filamentous fungi, we have cloned and sequenced the palF gene of Aspergillus nidulans (An) . An An wild-type (wt) chromosome VII-specific cosmid library was used to transform a palF15 mutant strain . Selection for complementation was done on medium containing beta-glycerolphosphate as the sole Pi source . Two cosmids were identified (W2G08 and W4G12) and further subcloning of one cosmid (W2G08) defined a 5-kb PstI genomic fragment, which fully complements the palF15 mutation . An internal fragment from the genomic clone recognized a single message of approx . 3.5 kb on Northern blot . cDNA clones were obtained from a lambda gt10 cDNA library and sequenced, showing a nucleotide (nt) sequence of 3311 base pairs (bp), with a 828 bp long 5'-untranslated region (UTR) . The major open reading frame (ORF) identified in the sequence codes for a putative 775 amino acid (aa) protein, which shares some similarity with two putative ORF products of Saccharomyces cerevisiae (Sc). Biochem Biophys Res Commun, 1997 Jul 30, 236(3), 559 - 64 Molecular cloning and characterization of mammalian homologues of the Drosophila retinal degeneration B gene; Aikawa Y et al.; Null mutations in the retinal degeneration B gene (rdgB) in flies result in an activity-dependent retinal degeneration . Here we report the isolation of the mouse and human homologues of rdgB gene that are strongly expressed in brain and moderately expressed in other tissues . The deduced amino acid sequences encoding a 1244 a.a protein bear a 96% similarity between mouse and human and resemble the Drosophila rdgB, particularly in the phosphatidylinositol transfer domain at the N-terminus and in six putative transmembrane domains at the C-terminus . Immunoblots with antiserum raised against a bacterially expressed fragment of the mouse rdgB showed the band with a molecular weight of about 170 kDa . Interestingly, a burst of mouse rdgB expression occurs on 17th day of gestation, suggesting a crucial role of the gene product in brain development at this particular stage . A gene, mpt-1, encoding for mouse rdgB was mapped to the proximal end of chromosome 19, which is the same location as Mvb-1, a gene locus encoding the modifier of mouse vibrator mutation (mv). J Cell Biol, 1997 Jul 28, 138(2), 255 - 70 Plasma membrane translocation of fluorescent-labeled phosphatidylethanolamine is controlled by transcription regulators, PDR1 and PDR3; Kean LS et al.; The transcription regulators, PDR1 and PDR3, have been shown to activate the transcription of numerous genes involved in a wide range of functions, including resistance to physical and chemical stress, membrane transport, and organelle function in Saccharomyces cerevisiae . We report here that PDR1 and PDR3 also regulate the transcription of one or more undetermined genes that translocate endogenous and fluorescent-labeled (M-C6-NBD-PE) phosphatidylethanolamine across the plasma membrane . A combination of fluorescence microscopy, fluorometry, and quantitative analysis demonstrated that M-C6-NBD-PE can be translocated both inward and outward across the plasma membrane of yeast cells . Mutants, defective in the accumulation of M-C6-NBD-PE, were isolated by selectively photokilling normal cells that accumulated the fluorescent phospholipid . This led to the isolation of numerous trafficking in phosphatidylethanolamine (tpe) mutants that were defective in intracellular accumulation of M-C6-NBD-PE . Complementation cloning and linkage analysis led to the identification of the dominant mutation TPE1-1 as a new allele of PDR1 and the semidominant mutation tpe2-1 as a new allele of PDR3 . The amount of endogenous phosphatidylethanolamine exposed to the outer leaflet of the plasma membrane was measured by covalent labeling with the impermeant amino reagent, trinitrobenzenesulfonic acid . The amount of outer leaflet phosphatidylethanolamine in both mutant strains increased four- to fivefold relative to the parent Tpe+ strain, indicating that the net inward flux of endogenous phosphatidylethanolamine as well as M-C6-NBD-PE was decreased . Targeted deletions of PDR1 in the new allele, PDR1-11, and PDR3 in the new allele, pdr3-11, resulted in normal M-C6-NBD-PE accumulation, confirming that PDR1-11 and pdr3-11 were gain-of-function mutations in PDR1 and PDR3, respectively . Both mutant alleles resulted in resistance to the drugs cycloheximide, oligomycin, and 4-nitroquinoline N-oxide (4-NQO) . However, a previously identified drug-resistant allele, pdr3-2, accumulated normal amounts of M-C6-NBD-PE, indicating allele specificity for the loss of M-C6-NBD-PE accumulation . These data demonstrated that PDR1 and PDR3 regulate the net rate of M-C6-NBD-PE translocation (flip-flop) and the steady-state distribution of endogenous phosphatidylethanolamine across the plasma membrane. J Biol Chem, 1997 Jul 25, 272(30), 18974 - 81 A complex consisting of human replication factor C p40, p37, and p36 subunits is a DNA-dependent ATPase and an intermediate in the assembly of the holoenzyme; Cai J et al.; Human replication factor C (hRFC) is a multi-subunit protein complex capable of supporting proliferating cell nuclear antigen (PCNA)-dependent DNA synthesis by DNA polymerases delta and epsilon . The hRFC complex consists of five different subunits with apparent molecular masses of 140, 40, 38, 37, and 36 kDa . We have previously reported the expression of a three-subunit core complex, consisting of the p40, p37, and p36 subunits following coupled in vitro transcription-translation of the cDNAs encoding these proteins (Uhlmann, F., Cai, J., Flores-Rozas, H., Dean, F . B., Finkelstein, J . , O'Donnell, M., and Hurwitz, J . (1996) Proc . Natl . Acad . Sci . U . S . A . 93, 6521-6526) . Here we describe the isolation of a stable complex composed of the p40, p37, and p36 subunits of hRFC from baculovirus-infected insect cells . The purified p40.p37.p36 complex, like the five-subunit RFC, contained DNA-dependent ATPase activity that was stimulated by PCNA, preferentially bound to primed DNA templates, interacted with PCNA, and was capable of unloading PCNA from singly-nicked circular DNA . In contrast to the five-subunit RFC, the three-subunit core complex did not load PCNA onto DNA . The p40 . p37.p36 complex inhibited the elongation of primed DNA templates catalyzed by the DNA polymerase delta holoenzyme . Incubation of the p40.p37.p36 complex with the hRFC p140 and p38 subunits formed the five-subunit hRFC complex that supported PCNA-dependent DNA synthesis by DNA polymerase delta. J Biol Chem, 1997 Jul 25, 272(30), 18725 - 31 Mitochondrial protein import . Tom40 plays a major role in targeting and translocation of preproteins by forming a specific binding site for the presequence; Rapaport D et al.; During preprotein transport across the mitochondrial outer membrane, the N-terminal presequence initially binds to a surface-exposed site, termed cis site, of the protein translocation complex of this membrane (the TOM complex) . The presequence then moves into the translocation pore and becomes exposed at the intermembrane space side . Membrane passage is driven by specific interaction of the presequence with the trans site . We have used chemical cross-linking to identify components in the vicinity of the translocating presequence . Preproteins bound to the surface-exposed cis site can be cross-linked via their N-terminal presequence to Tom20 and Tom22, demonstrating their direct association with this part of the preprotein . In addition, the presequence establishes an early contact to Tom40, a membrane-embedded protein of the TOM complex . Upon further entry of the preprotein into the translocation pore, the presequence loses its contact with Tom20/Tom22, but remains in firm association with Tom40 . Our study suggests that Tom40 plays an important function in guiding the presequence of a preprotein across the mitochondrial outer membrane . We propose that Tom40 forms a major part of the trans presequence binding site. Proc Natl Acad Sci U S A, 1997 Jul 22, 94(15), 8076 - 81 Ku70-deficient embryonic stem cells have increased ionizing radiosensitivity, defective DNA end-binding activity, and inability to support V(D)J recombination; Gu Y et al.; V(D)J recombination requires both lymphoid-specific and generally expressed enzymatic activities . All three known generally expressed activities involved in V(D)J recombination are also involved in DNA double-strand break repair (DSBR) . Two of these are components of the DNA-dependent protein kinase (DNA-PK) and include Ku80 and DNA-PK catalytic subunit (DNA-PKcs); the third, XRCC4, is a protein of unknown function . The Ku70 protein is an additional component of DNA-PK; Ku70 forms a heterodimer with Ku80 to generate the DNA end-binding component of the enzyme . To test putative functions for Ku70, we have used gene-targeted mutation to generate a murine embryonic stem cell line which lacks Ku70 expression . We find that the Ku70(-/-) cells produce no detectable Ku70 and very little Ku80, suggesting a direct interrelationship between their levels . Correspondingly, these cells lack the nonspecific DNA end-binding activity associated with Ku . Significantly, the Ku70(-/-) embryonic stem cells have markedly increased sensitivity to gamma-irradiation relative to Ku70(+/-) or wild-type embryonic stem cells . Furthermore, the Ku70(-/-) cells lack the ability to effectively rejoin signal and coding ends liberated in transiently introduced V(D)J recombination substrates by enforced RAG-1 and RAG-2 expression . We conclude that the Ku70 gene product is involved in DSBR and V(D)J recombination and confirm that the Ku70 gene can be classified as a member of the x-ray cross-complementation group 6 (XRCC6) . Potential differences between the Ku70(-/-) and Ku80(-/-) V(D)J recombination defects are discussed. Proc Natl Acad Sci U S A, 1997 Jul 22, 94(15), 8036 - 41 Synergistic and promoter-selective activation of transcription by recruitment of transcription factors TFIID and TFIIB; Gonzalez-Couto E et al.; Eukaryotic transcriptional activators may function by stimulating formation of RNA polymerase II preinitiation complexes at the core promoter of genes . In this case, their mode of action will intrinsically depend on how these complexes assemble on promoters in living cells, an issue that remains largely unexplored . Here we show that in yeast the basal transcription machinery is brought to the promoter in the form of at least two subcomplexes, TFIID and a complex comprising TFIIB and other essential components . Individual recruitment of either complex by artificial contact with a transcriptionally inactive, sequence-specific DNA-binding protein suffices to trigger transcriptional activation from a wild-type core promoter bearing the appropriate binding site . In contrast, activation from a promoter containing a weakened TATA element is only observed upon recruitment of TFIID . Tethering TFIIB on that promoter remains without effect, but the simultaneous recruitment of both components leads to strong synergistic activation . These findings suggest a simple mechanism whereby two activators that contact distinct subcomplexes of the basal machinery may stimulate transcription synergistically and differentially depending on the nature of the promoter. FEBS Lett, 1997 Jul 21, 412(1), 233 - 5 Sterol 22-desaturase, cytochrome P45061, possesses activity in xenobiotic metabolism; Kelly SL et al.; CYP61 was revealed in the sequencing of the yeast genome on chromosome XIII and was the last member of the CYP superfamily in yeast to be discovered . We show here that besides the housekeeping role in 22-desaturation during ergosterol biosynthesis the enzyme is also that responsible for benzo(a)pyrene metabolism/promutagen activation by yeast in genotoxicity assays . This enzyme may represent an ancestral activity for the superfamily which allowed xenobiotic metabolism for the first time. Biochim Biophys Acta, 1997 Jul 18, 1340(2), 187 - 204 Probing Flp: a new approach to analyze the structure of a DNA recognizing protein by combining the genetic algorithm, mutagenesis and non-canonical DNA target sites; Saxena P et al.; A topological and functional overview of a DNA recognition protein with unknown structure can be achieved by combining three different, but complementary approaches: modeling by the genetic algorithm, functional analysis of mutated variants, and testing the target DNA using non-canonical oligonucleotides . As an example we choose the Flp protein, a site-specific recombinase from Saccharomyces cerevisiae . We derive the topological outline including the DNA binding cleft, examine DNA binding regions by deletional and mutational analysis, and analyze the DNA binding site using 7-deazaadenine, 7-deazaguanine, inosine and 4-O-methylthymine as probes . The combined data offer a comprehensive sketch of a plausible protein architecture for Flp . The structure is detailed enough to verify the prediction accuracy for different peptide regions from pre-existing data and by new experimental design. Biochem Biophys Res Commun, 1997 Jul 18, 236(2), 248 - 52 Frequent somatic mutations of hMSH3 with reference to microsatellite instability in hereditary nonpolyposis colorectal cancers; Akiyama Y et al.; hMSH3 is one of the human DNA mismatch repair genes but has not yet been reported to be associated with hereditary nonpolyposis colorectal cancer . Recently, somatic mutation at a polyadenine tract, i.e., (A)8, in hMSH3 was reported in cancers with microsatellite instability (MI) . To clarify the tumorigenetic role of hMSH3, we screened for somatic mutations at the hMSH3 (A)8 repeat in 29 tumors from 23 hereditary nonpolyposis colorectal cancer patients . One or two A deletions in the (A)8 repeat were found in 11 (57.9%) of the 19 MI-positive tumors but not in 10 MI-negative ones, indicating secondary mutations after germline mutations of other mismatch repair genes . Moreover, the MI frequency of three or more nucleotide repeats was higher in hMSH3 (A)8-mutated tumor cells than in nonmutated ones (p<0.05) . These data suggest that a mutation of a mismatch repair gene enhances the frequency of another mismatch repair gene mutation, such as of hMSH3, resulting in severe MI. J Biol Chem, 1997 Jul 18, 272(29), 18467 - 72 Analysis of the functional domain of the rat liver mitochondrial import receptor Tom20; Iwahashi J et al.; Tom20 is an outer mitochondrial membrane protein and functions as a component of the import receptor complex for the cytoplasmically synthesized mitochondrial precursor proteins . It consists of the N-terminal membrane-anchor segment, the tetratricopeptide repeat (TPR) motif, a charged amino acids-rich linker segment between the membrane anchor and the TPR motif, and the C-terminal acidic amino acid cluster . To assess the functional significance of these segments in mammalian Tom20, we cloned rat Tom20 and expressed mutant rat Tom20 proteins in Deltatom20 yeast cells and examined their ability to complement the defects of respiration-driven growth and mitochondrial protein import . Tom20N69, a mutant consisting of the membrane anchor and the linker segments, was targeted to mitochondria and complemented the growth and import defects as efficiently as wild-type Tom20, whereas a mutant lacking the linker segment did not . In vitro protein import into mitochondria isolated from the complemented yeast cells revealed that the precursor targeted to yeast Tom70 was efficiently imported into the mitochondria via rat Tom20N69 . Thus the linker segment is essential for the function of rat Tom20, whereas the TPR motif and the C-terminal acidic amino acids are not. J Biol Chem, 1997 Jul 18, 272(29), 18341 - 9 A cryptic DNA binding domain at the COOH terminus of TFIIIB70 affects formation, stability, and function of preinitiation complexes; Huet J et al.; TFIIIC-dependent assembly of yeast TFIIIB on class III genes unmasks a high avidity of TFIIIB for DNA . TFIIIB contains TATA-binding protein (TBP), TFIIIB90/B", and TFIIIB70/Brf1, which is homologous to TFIIB . Using limited proteolysis, we have found that the COOH terminus of TFIIIB70 (residues 510-596) forms a protease-resistant domain that binds DNA tightly as seen by Southwestern, DNase I footprinting, and gel shift assays . Consistent with a role for this DNA binding activity, preinitiation complexes were formed less efficiently with truncated TFIIIB70 lacking the COOH-terminal domain and displayed an increased sensitivity to heparin . B' (TFIIIB70 + TBP).TFIIIC.DNA complexes were also particularly unstable . In addition, TFIIIB.TFIIIC.DNA complexes containing truncated TFIIIB70 were impaired in promoting transcription initiation. EMBO J, 1997 Jul 16, 16(14), 4433 - 40 Trans mRNA splicing in trypanosomes: cloning and analysis of a PRP8-homologous gene from Trypanosoma brucei provides evidence for a U5-analogous RNP; Lucke S et al.; In trypanosomes all mRNAs are generated through trans mRNA splicing, requiring the functions of the small nuclear RNAs U2, U4 and U6 . In the absence of conventional cis mRNA splicing, the structure and function of a U5-analogous snRNP in trypanosomes has remained an open question . In cis splicing, a U5 snRNP-specific protein component called PRP8 in yeast and p220 in man is a highly conserved, essential splicing factor involved in splice-site recognition and selection . We have cloned and sequenced a genomic region from Trypanosoma brucei, that contains a PRP8/p220-homologous gene (p277) coding for a 277 kDa protein . Using an antibody against a C-terminal region of the trypanosomal p277 protein, a small RNA of approximately 65 nucleotides could be specifically co-immunoprecipitated that appears to be identical with a U5 RNA (SLA2 RNA) recently identified by Dungan et al . (1996) . Based on sedimentation, immunoprecipitation and Western blot analyses we conclude that this RNA is part of a stable ribonucleoprotein (RNP) complex and associated not only with the p277 protein, but also with the common proteins present in the other trans-spliceosomal snRNPs . Together these results demonstrate that a U5-analogous RNP exists in trypanosomes and suggest that basic functions of the U5 snRNP are conserved between cis and trans splicing. Eur J Biochem, 1997 Jul 15, 247(2), 605 - 13 Disruption of three acid proteases in Aspergillus niger--effects on protease spectrum, intracellular proteolysis, and degradation of target proteins; van den Hombergh JP et al.; Three acid protease genes encoding two extracellular proteases (PEPA and PEPB) and one intracellular protease (PEPE) were disrupted in Aspergillus niger . Northern-blot analysis showed the absence of wild-type protease mRNAs in the disruptants while western-blot analysis proved the absence of the encoded proteases . Characterization of the residual proteolytic spectra in the disruptants indicated that the extracellular protease activity was reduced to 16% and 94% for the delta pepA and the delta pepB disruptants, repectively . In the delta pepE disruptant, the total intracellular proteolytic activity was reduced to 32% . Apart from the reduced intracellular pepstatin-inhibitable aspartyl protease activity, serine protease and serine carboxypeptidase activities were also significantly reduced in the delta pepE strain . This may indicate the presence of a cascade activation mechanism for several vacuolar proteases, triggered by the PEPE protein, similar to the situation in Saccharomyces cerevisiae . Disruption of a single protease gene had no effects on the transcription of other non-disrupted protease genes in A . niger . In supernatants of the disruptants, reduced degradation of a proteolytically very susceptible tester protein (PELB) was observed . By recombination, we also constructed delta pepA delta pepB, delta pepB delta pepE and delta pepA delta pepE double disruptants as well as a delta pepA delta pepB delta pepE triple disruptant, lacking all three acid protease activities . The in vitro residual PELB activity was the highest in the triple disruptant and the delta pepA delta pepB recombinant. Eur J Biochem, 1997 Jul 15, 247(2), 461 - 9 The role of the cap structure in RNA processing and nuclear export; Lewis JD et al.; The cap structure that is characteristic of all polymerase-II-transcribed RNAs has been shown to play an important role in many aspects of RNA metabolism including RNA processing, RNA nuclear transport, and translation initiation . The effects of the cap structure on these different processes is mediated by proteins that recognise and bind to it, and are therefore generically called cap-binding proteins . For example, the cap-binding protein eIF4E, in a complex with other proteins, mediates the effect of the cap on the initiation of translation . EIF-4E is predominantly localised in the cytoplasm . In the last five years, it has been demonstrated that a second cap-binding protein complex, which is mainly localised in the nucleus, mediates the stimulatory effects of the cap in nuclear processes such as pre-mRNA splicing, RNA 3'-end formation, and RNA nuclear export . The purpose of this review is to summarise our current knowledge on the role of the cap structure and of the cap-binding protein complex in nuclear RNA metabolism and present evidence that at least some processes may be coupled in vivo. Genes Dev, 1997 Jul 15, 11(14), 1801 - 11 The UNC-14 protein required for axonal elongation and guidance in Caenorhabditis elegans interacts with the serine/threonine kinase UNC-51; Ogura K et al.; Certain unc mutants in the nematode Caenorhabditis elegans, such as unc-14 and unc-51, show abnormal axonal elongation and axonal structures . We cloned the unc-51 gene previously and predicted that it encodes a novel serine/threonine protein kinase . In this study, we precisely localized the activity to rescue an unc-14 mutation . Also, we identified four cDNA clones encoded by the unc-14 rescuing region, in screens for proteins that bind to UNC-51 using a yeast two-hybrid system . A mutation site in the cDNA was identified for each of the six unc-14 mutants, establishing that the unc-14 gene was cloned . The unc-14 gene encodes a novel protein of 665 amino acids, and is coexpressed with the unc-51 gene in the cell bodies and axons of almost all neurons including DD/VD and hermaphrodite-specific neurons . Another clone recovered in the two-hybrid screen encodes a carboxy-terminal region of UNC-51 . Analysis using the yeast two-hybrid system suggested that a central region of UNC-14 bound to a carboxy-terminal region of UNC-51, and that the UNC-51 carboxy-terminal region oligomerized . In in vitro binding studies using recombinant fusion proteins, UNC-14 interacted with UNC-51 directly . We propose that UNC-51 protein kinase acts as an oligomer, and that UNC-14 is a regulator of UNC-51, in axonal elongation and guidance. Genes Dev, 1997 Jul 15, 11(14), 1786 - 800 Tam1, a telomere-associated meiotic protein, functions in chromosome synapsis and crossover interference; Chua PR et al.; The TAM1 gene of Saccharomyces cerevisiae is expressed specifically during meiosis and encodes a protein that localizes to the ends of meiotic chromosomes . In a tam1 null mutant, there is an increase in the frequency of chromosomes that fail to recombine and an associated increase in homolog nondisjunction at meiosis I . The tam1 mutant also displays an increased frequency of precocious separation of sister chromatids and a reduced efficiency of distributive disjunction . The defect in distributive disjunction may be attributable to overloading of the distributive system by the increased number of nonrecombinant chromosomes . Recombination is not impaired in the tam1 mutant, but crossover interference is reduced substantially . In addition, chromosome synapsis is delayed in tam1 strains . The combination of a defect in synapsis and a reduction in interference is consistent with previous studies suggesting a role for the synaptonemal complex in regulating crossover distribution . tam1 is the only known yeast mutant in which the control of crossover distribution is impaired, but the frequency of crossing over is unaffected . We discuss here possibilities for how a telomere-associated protein might function in chromosome synapsis and crossover interference. Cancer Res, 1997 Jul 15, 57(14), 2961 - 5 CaSm: an Sm-like protein that contributes to the transformed state in cancer cells; Schweinfest CW et al.; A novel gene encoding a protein containing Sm motif-like domains was found to have elevated expression in pancreatic cancer and in several cancer-derived cell lines . CaSm (for Cancer-associated Sm-like) mRNA is up-regulated in 87.5% (seven of eight) of pancreatic tumor/normal pairs . Similarly, cell lines from cancers originating in liver, ovary, lung, and kidney show increased CaSm expression compared to their normal tissue cognates . CaSm encodes a 133-amino acid open reading frame that contains the two Sm motifs found in the common snRNP proteins, with the greatest homology to the Sm G protein (60% similarity) . Two hypothetical proteins from Caenorhabditis elegans and Saccharomyces cerevisiae share even greater similarity (72.8 and 67.7%, respectively), suggesting a broad family of proteins containing Sm motifs . Antisense CaSm RNA is able to alter the transformed phenotype of pancreatic cancer cells by reducing their ability to form large colonies in soft agar when compared to untransfected cells . Therefore, CaSm expression appears to be necessary for maintenance of the transformed state. Cancer Res, 1997 Jul 15, 57(14), 2847 - 50 Heat inactivation of Ku autoantigen: possible role in hyperthermic radiosensitization; Burgman P et al.; Heat shock prior, during, or immediately after ionizing radiation synergistically increases cell killing, a phenomenon termed hyperthermic radiosensitization . Recently, we have shown a constitutive DNA-binding factor in rodent cells that is inactivated by heat shock to be identical to Ku autoantigen . Ku, consisting of an Mr 70,000 (Ku70) and an Mr 86,000 (Ku80) subunit, is a heterodimeric nuclear protein and is the DNA-binding regulatory component of the mammalian DNA-dependent protein kinase DNA-PK . Recent genetic and biochemical studies indicate the involvement of Ku and DNA-PK in DNA double-strand break repair and V(D)J recombination . On the basis of these findings, we propose that heat-induced loss of the DNA-binding activity of Ku may lead to hyperthermic radiosensitization . To test this hypothesis, we examined and compared the DNA-binding activity of Ku, the DNA-PK kinase activity, and hyperthermic radiosensitization in rodent cells immediately after heat shock and during post-heat shock recovery at 37 degrees C . Our results show that the heat-induced loss of Ku-DNA binding activity correlates well with an increased radiosensitivity of the heat-shocked cells, and furthermore, the loss of synergistic interaction between heat and radiation parallels the recovery of the DNA-binding activity of Ku . On the other hand, the heat-induced decrease of DNA-PK activity did not correlate with hyperthermic radiosensitization . Our data, for the first time, provide evidence for a role of Ku protein in modulating the cellular response to combined treatments of heat shock and ionizing radiation. Biochem J, 1997 Jul 15, 325 ( Pt 2), 299 - 301 Co-operation of phosphatidylinositol transfer protein with phosphoinositide 3-kinase gamma in the formylmethionyl-leucylphenylalanine-dependent production of phosphatidylinositol 3,4,5-trisphosphate in human neutrophils; Kular G et al.; Phosphoinositide 3-kinase (PI3K) and its product phosphatidylinositol 3,4,5-trisphosphate (PIP3) play an essential role in the regulation of neutrophil functions by the chemoattractant formylmethionyl-leucylphenylalanine (FMLP) . Here we show that permeabilization of human neutrophils leads to loss of cytosolic components, including PI3Kgamma, and causes the loss of FMLP-dependent production of PIP3 . FMLP-sensitive synthesis of PIP3 could be restored by reconstitution of permeabilized neutrophils with recombinant PI3Kgamma . Admixture of recombinant phosphatidylinositol transfer protein (PITP) to the reconstitution cocktail produced a further increase of PIP3 synthesis, whereas pertussis toxin suppressed the FMLP-dependent production of PIP3 . We conclude that FMLP-sensitive PIP3 formation in human neutrophils involves the FMLP receptor, heterotrimeric G-proteins of the Gi type, PI3Kgamma and PITP. J Cell Biol, 1997 Jul 14, 138(1), 119 - 30 Control of mitotic events by Nap1 and the Gin4 kinase; Altman R et al.; Little is known about the pathways used by cyclins and cyclin-dependent kinases to induce the events of the cell cycle . In budding yeast, a protein called Nap1 binds to the mitotic cyclin Clb2, and Nap1 is required for the ability of Clb2 to induce specific mitotic events, but the role played by Nap1 is unclear . We have used genetic and biochemical approaches to identify additional proteins that function with Nap1 in the control of mitotic events . These approaches have both identified a protein kinase called Gin4 that is required for the ability of Clb2 and Nap1 to promote the switch from polar to isotropic bud growth that normally occurs during mitosis . Gin4 is also required for the ability of Clb2 and Nap1 to promote normal progression through mitosis . The Gin4 protein becomes phosphorylated as cells enter mitosis, resulting in the activation of Gin4 kinase activity, and the phosphorylation of Gin4 is dependent upon Nap1 and Clb2 in vivo . Affinity chromatography experiments demonstrate that Gin4 binds tightly to Nap1, indicating that the functions of these two proteins are closely tied within the cell . These results demonstrate that the activation of Gin4 is under the control of Clb2 and Nap1, and they provide an important step towards elucidating the molecular pathways that link cyclin-dependent kinases to the events they control. J Cell Biol, 1997 Jul 14, 138(1), 65 - 80 A novel class of RanGTP binding proteins; Gorlich D et al.; The importin-alpha/beta complex and the GTPase Ran mediate nuclear import of proteins with a classical nuclear localization signal . Although Ran has been implicated also in a variety of other processes, such as cell cycle progression, a direct function of Ran has so far only been demonstrated for importin-mediated nuclear import . We have now identified an entire class of approximately 20 potential Ran targets that share a sequence motif related to the Ran-binding site of importin-beta . We have confirmed specific RanGTP binding for some of them, namely for two novel factors, RanBP7 and RanBP8, for CAS, Pse1p, and Msn5p, and for the cell cycle regulator Cse1p from Saccharomyces cerevisiae . We have studied RanBP7 in more detail . Similar to importin-beta, it prevents the activation of Ran's GTPase by RanGAP1 and inhibits nucleotide exchange on RanGTP . RanBP7 binds directly to nuclear pore complexes where it competes for binding sites with importin-beta, transportin, and apparently also with the mediators of mRNA and U snRNA export . Furthermore, we provide evidence for a Ran-dependent transport cycle of RanBP7 and demonstrate that RanBP7 can cross the nuclear envelope rapidly and in both directions . On the basis of these results, we propose that RanBP7 might represent a nuclear transport factor that carries an as yet unknown cargo, which could apply as well for this entire class of related RanGTP-binding proteins. J Cell Biol, 1997 Jul 14, 138(1), 37 - 44 Aminopeptidase I is targeted to the vacuole by a nonclassical vesicular mechanism; Scott SV et al.; The yeast vacuolar protein aminopeptidase I (API) is synthesized as a cytosolic precursor that is transported to the vacuole by a nonclassical targeting mechanism . Recent genetic studies indicate that the biosynthetic pathway that transports API uses many of the same molecular components as the degradative autophagy pathway . This overlap coupled with both in vitro and in vivo analysis of API import suggested that, like autophagy, API transport is vesicular . Subcellular fractionation experiments demonstrate that API precursor (prAPI) initially enters a nonvacuolar cytosolic compartment . In addition, subvacuolar vesicles containing prAPI were purified from a mutant strain defective in breakdown of autophagosomes, further indicating that prAPI enters the vacuole inside a vesicle . The purified subvacuolar vesicles do not appear to contain vacuolar marker proteins . Immunogold EM confirms that prAPI is localized in cytosolic and in subvacuolar vesicles in a mutant strain defective in autophagic body degradation . These data suggest that cytosolic vesicles containing prAPI fuse with the vacuole to release a membrane-bounded intermediate compartment that is subsequently broken down, allowing API maturation. Cell, 1997 Jul 11, 90(1), 109 - 19 FOG, a multitype zinc finger protein, acts as a cofactor for transcription factor GATA-1 in erythroid and megakaryocytic differentiation; Tsang AP et al.; The hematopoietic transcription factor GATA-1 is essential for development of the erythroid and megakaryocytic lineages . Using the conserved zinc finger DNA-binding domain of GATA-1 in the yeast two-hybrid system, we have identified a novel, multitype zinc finger protein, Friend of GATA-1 (FOG), which binds GATA-1 but not a functionally inactive mutant lacking the amino (N) finger . FOG is coexpressed with GATA-1 during embryonic development and in erythroid and megakaryocytic cells . Furthermore, FOG and GATA-1 synergistically activate transcription from a hematopoietic-specific regulatory region and cooperate during both erythroid and megakaryocytic cell differentiation . These findings indicate that FOG acts as a cofactor for GATA-1 and provide a paradigm for the regulation of cell type-specific gene expression by GATA transcription factors. Cell, 1997 Jul 11, 90(1), 87 - 96 Holliday junctions accumulate in replication mutants via a RecA homolog-independent mechanism; Zou H et al.; The Holliday junction recombination intermediate, an X-shaped DNA molecule (xDNA), was analyzed at rDNA in mitotically growing yeast . In wild-type cells, xDNA is only detected at S phase, suggesting that recombination is stimulated to repair replication-related lesions . A search for mutations that increase the level of xDNA uncovered a gene encoding a subunit of DNA polymerase alpha . Systematic examination of replication mutants revealed that defects in polymerase alpha and delta but not the epsilon complex stimulate the level of xDNA . These xDNAs are Holliday junctions and not replication intermediates . The level of Holliday junctions is greatly reduced in rad52 mutants, but surprisingly, not in mutants defective in the three known mitotically expressed yeast RecA homologs. Cell, 1997 Jul 11, 90(1), 65 - 75 Identification and structural characterization of the ATP/ADP-binding site in the Hsp90 molecular chaperone; Prodromou C et al.; Hsp90 molecular chaperones in eukaryotic cells play essential roles in the folding and activation of a range of client proteins involved in cell cycle regulation, steroid hormone responsiveness, and signal transduction . The biochemical mechanism of Hsp90 is poorly understood, and the involvement of ATP in particular is controversial . Crystal structures of complexes between the N-terminal domain of the yeast Hsp90 chaperone and ADP/ATP unambiguously identify a specific adenine nucleotide binding site homologous to the ATP-binding site of DNA gyrase B . This site is the same as that identified for the antitumor agent geldanamycin, suggesting that geldanamycin acts by blocking the binding of nucleotides to Hsp90 and not the binding of incompletely folded client polypeptides as previously suggested . These results finally resolve the question of the direct involvement of ATP in Hsp90 function. J Biol Chem, 1997 Jul 11, 272(28), 17776 - 83 Ykt6p, a prenylated SNARE essential for endoplasmic reticulum-Golgi transport; McNew JA et al.; Vesicular transport between secretory compartments requires specific recognition molecules called SNAREs . Here we report the identification of three putative SNAREs, p14 (Sft1p), p28 (Gos1p), and a detailed characterization of p26 (Ykt6p) . All three were originally isolated as interacting partners of the cis Golgi target membrane-associated SNARE Sed5p, when Sec18p (yeast NSF) was inactivated . YKT6 is an essential gene that codes for a novel vesicle-associated SNARE functioning at the endoplasmic reticulum-Golgi transport step in the yeast secretory pathway . Depletion of Ykt6p results in the accumulation of the p1 precursor (endoplasmic reticulum form) of the vacuolar enzyme carboxypeptidase Y and morphological abnormalities consistent with a defect in secretion . Membrane localization of Ykt6p is essential for protein function and is normally mediated by isoprenylation . However, replacement of the isoprenylation motif with a bona fide transmembrane anchor results in a functional protein confirming that membrane localization, but not isoprenylation per se, is required for function . Ykt6p and its homologues are highly conserved from yeast to human as demonstrated by the functional complementation of the loss of Ykt6p by its human counterpart . This is the first example of a human SNARE protein functionally replacing a yeast SNARE . This observation implies that the specific details of the vesicle targeting code, like the genetic code, are conserved in evolution. J Biol Chem, 1997 Jul 11, 272(28), 17410 - 5 N-terminal hydrophobic sorting signals of preproteins confer mitochondrial hsp70 independence for import into mitochondria; Gruhler A et al.; The requirement of mitochondrial hsp70 (mt-hsp70) for the import of a series of preproteins containing hydrophobic sorting signals into isolated yeast mitochondria was investigated . Here we demonstrate that the presence of such a sorting signal in proximity to the N-terminal matrix-targeting sequence of a preprotein can secure a translocating polypeptide chain in the import channel in a manner that does not require mt-hsp70 activity . Trapping the translocating chain in this fashion leads to efficient processing by the mitochondrial processing peptidase and to complete translocation across the outer mitochondrial membrane into the intermembrane space . These mt-hsp70-independent effects appear to be exerted at the level of the inner membrane through an interaction of the hydrophobic core of the sorting signal with component(s) of the translocase of the inner membrane . Hydrophobic sorting signals of inner membrane proteins inserted into the membrane from the matrix, as well as those of intermembrane space proteins, are capable of causing this mt-hsp70-independent stabilization, demonstrating that this phenomenon is not unique to those preproteins normally sorted to the intermembrane space. J Biol Chem, 1997 Jul 11, 272(28), 17258 - 62 The cytoplasmic loop between putative transmembrane segments 6 and 7 in sarcoplasmic reticulum Ca2+-ATPase binds Ca2+ and is functionally important; Falson P et al.; Limited proteolysis by proteinase K of rabbit SERCA1 Ca2+-ATPase generates a number of fragments which have been identified recently . Here, we have focused on two proteolytic C-terminal fragments, p20C and p19C, starting at Gly-808 and Asp-818, respectively . The longer peptide p20C binds Ca2+, as deduced from changes in migration rate by SDS-polyacrylamide gel electrophoresis performed in the presence of Ca2+ as well as from labeling with 45Ca2+ in overlay experiments . In contrast, the shorter peptide p19C, a proteolysis fragment identical to p20C but for 10 amino acids missing at the N-terminal side, did not bind Ca2+ when submitted to the same experiments . Two cluster mutants of Ca2+-ATPase, D813A/D818A and D813A/D815A/D818A, expressed in the yeast Saccharomyces cerevisiae, were found to have a very low Ca2+-ATPase activity . Region 808-818 is thus essential for both Ca2+ binding and enzyme activity, in agreement with similar results recently reported for the homologous gastric H+, K+-ATPase (Swarts, H . G . P., Klaassen, C . H . W., de Boer, M., Fransen, J . A . M . , and De Pont, J . J . H . H . M . (1996) J . Biol . Chem . 271, 29764-29772) . However, the accessibility of proteinase K to the peptidyl link between Leu-807 and Gly-808 clearly shows that the transmembrane segment M6 ends before region 808-818 . It is remarkable that critical residues for enzyme activity are located in a cytoplasmic loop starting at Gly-808. Science, 1997 Jul 11, 277(5323), 232 - 5 Murine model of Niemann-Pick C disease: mutation in a cholesterol homeostasis gene; Loftus SK et al.; An integrated human-mouse positional candidate approach was used to identify the gene responsible for the phenotypes observed in a mouse model of Niemann-Pick type C (NP-C) disease . The predicted murine NPC1 protein has sequence homology to the putative transmembrane domains of the Hedgehog signaling molecule Patched, to the cholesterol-sensing regions of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and SREBP cleavage-activating protein (SCAP), and to the NPC1 orthologs identified in human, the nematode Caenorhabditis elegans, and the yeast Saccharomyces cerevisiae . The mouse model may provide an important resource for studying the role of NPC1 in cholesterol homeostasis and neurodegeneration and for assessing the efficacy of new drugs for NP-C disease. Exp Cell Res, 1997 Jul 10, 234(1), 66 - 77 Characterization of a mutant profilin with reduced actin-binding capacity: effects in vitro and in vivo; Hajkova L et al.; We are investigating structure-function relationships in profilin and actin by site-specific mutagenesis using a yeast, Saccharomyces cerevisiae, expression system to produce wild-type and mutant proteins . This paper shows that deleting proline 96 and threonine 97, which are located close to the major actin binding site on profilin, did not significantly alter the interaction between profilin and phosphatidylinositol 4,5-bisphosphate, nor did it affect the profilin:poly(L-proline) interaction . The mutant protein, however, had a lower capacity to bind to actin in vitro than wild-type profilin, though it showed a slightly increased profilin-enhanced nucleotide exchange on the actin . When microinjected into Swiss 3T3 mouse fibroblasts or porcine aortic endothelial cells, the mutant profilin did not change the organization of the microfilament system like the wild-type profilin did . This provides further evidence that profilin controls microfilament organization in the cell by interacting directly with actin. Nature, 1997 Jul 10, 388(6638), 195 - 200 Tom5 functionally links mitochondrial preprotein receptors to the general import pore; Dietmeier K et al.; Most mitochondrial proteins are synthesized as preproteins on cytosolic polysomes and are subsequently imported into the organelle . The mitochondrial outer membrane contains a multisubunit preprotein translocase (Tom) which has receptors on the cytosolic side and a general import pore (GIP) in the membrane . Tom20-Tom22 and Tom70-Tom37 function as import receptors with a preference for preproteins that have amino-terminal presequences or internal targeting information, respectively . Tom40 is an essential constituent of the GIP, whereas Tom6 and Tom7 modulate the assembly and dissociation of the Tom machinery . Here we report the identification of Tom5, a small subunit that has a crucial role importing preproteins destined for all four mitochondrial subcompartments . Tom5 has a single membrane anchor and a cytosolic segment with a negative net charge, and accepts preproteins from the receptors and mediates their insertion into the GIP . We conclude that Tom5 represents a functional link between surface receptors and GIP, and is part of an 'acid chain' that guides the stepwise transport of positively charged mitochondrial targeting sequences. Biochem Biophys Res Commun, 1997 Jul 9, 236(1), 75 - 8 A novel fungal gene encoding chitin synthase with a myosin motor-like domain; Fujiwara M et al.; A csmA gene that encodes chitin synthase with a myosin motor-like domain was isolated from the filamentous fungus Aspergillus nidulans . Initially, we obtained the csmA as a homolog of the Aspergillus fumigatus chsE-partial fragment . A large open reading frame encoding a polypeptide of 1,852 a.a . was identified by determining the cDNA sequences . The chitin synthase conserved region was situated at the C-terminus and classified into class V as reported previously . On the other hand, the N-terminal region showed significant similarity to myosin motors and could not be classified into any types of myosins identified so far . Thus, it is suggested that this is the first report of unconventional myosin fused to a metabolic enzyme . The finding of this new type of chitin synthase gene suggests that localization of chitin synthesis may be guided by association with cytoskeletal structures. Proc Natl Acad Sci U S A, 1997 Jul 8, 94(14), 7308 - 13 The cluA- mutant of Dictyostelium identifies a novel class of proteins required for dispersion of mitochondria; Zhu Q et al.; The cluA gene of Dictyostelium discoideum encodes a novel 150-kDa protein . Disruption of cluA results in clustering of mitochondria near the cell center . This is a striking difference from normal cells, whose mitochondria are dispersed uniformly throughout the cytoplasm . The mutant cell populations also exhibit an increased frequency of multinucleated cells, suggesting an impairment in cytokinesis . Both phenotypes are reversed by transformation of cluA- cells with a plasmid carrying a constitutively expressed cluA gene . The predicted sequence of the cluA gene product is homologous to sequences encoded by open reading frames in the genomes of Saccharomyces cerevisiae and Caenorhabditis elegans, but not to any known protein . The only exception is a short region with some homology to the 42-residue imperfect repeats present in the kinesin light chain, which probably function in protein-protein interaction . These studies identify a new class of proteins that appear to be required for the proper distribution of mitochondria. Proc Natl Acad Sci U S A, 1997 Jul 8, 94(14), 7251 - 6 Multiprotein bridging factor 1 (MBF1) is an evolutionarily conserved transcriptional coactivator that connects a regulatory factor and TATA element-binding protein; Takemaru K et al.; Multiprotein bridging factor 1 (MBF1) is a transcriptional cofactor that bridges between the TATA box-binding protein (TBP) and the Drosophila melanogaster nuclear hormone receptor FTZ-F1 or its silkworm counterpart BmFTZ-F1 . A cDNA clone encoding MBF1 was isolated from the silkworm Bombyx mori whose sequence predicts a basic protein consisting of 146 amino acids . Bacterially expressed recombinant MBF1 is functional in interactions with TBP and a positive cofactor MBF2 . The recombinant MBF1 also makes a direct contact with FTZ-F1 through the C-terminal region of the FTZ-F1 DNA-binding domain and stimulates the FTZ-F1 binding to its recognition site . The central region of MBF1 (residues 35-113) is essential for the binding of FTZ-F1, MBF2, and TBP . When the recombinant MBF1 was added to a HeLa cell nuclear extract in the presence of MBF2 and FTZ622 bearing the FTZ-F1 DNA-binding domain, it supported selective transcriptional activation of the fushi tarazu gene as natural MBF1 did . Mutations disrupting the binding of FTZ622 to DNA or MBF1, or a MBF2 mutation disrupting the binding to MBF1, all abolished the selective activation of transcription . These results suggest that tethering of the positive cofactor MBF2 to a FTZ-F1-binding site through FTZ-F1 and MBF1 is essential for the binding site-dependent activation of transcription . A homology search in the databases revealed that the deduced amino acid sequence of MBF1 is conserved across species from yeast to human. Proc Natl Acad Sci U S A, 1997 Jul 8, 94(14), 7166 - 9 Interaction of human apurinic endonuclease and DNA polymerase beta in the base excision repair pathway; Bennett RA et al.; Mutagenic abasic (AP) sites are generated directly by DNA-damaging agents or by DNA glycosylases acting in base excision repair . AP sites are corrected via incision by AP endonucleases, removal of deoxyribose 5-phosphate, repair synthesis, and ligation . Mammalian DNA polymerase beta (Polbeta) carries out most base excision repair synthesis and also can excise deoxyribose 5-phosphate after AP endonuclease incision . Yeast two-hybrid analysis now indicates protein-protein contact between Polbeta and human AP endonuclease (Ape protein) . In vitro, binding of Ape protein to uncleaved AP sites loads Polbeta into a ternary complex with Ape and the AP-DNA . After incision by Ape, only Polbeta exhibits stable DNA binding . Kinetic experiments indicated that Ape accelerates the excision of 5'-terminal deoxyribose 5-phosphate by Polbeta . Thus, the two central players of the base excision repair pathway are coordinated in sequential reactions. Biochemistry, 1997 Jul 8, 36(27), 8253 - 60 Mutagenesis of the uncoupling protein of brown adipose tissue . Neutralization Of E190 largely abolishes pH control of nucleotide binding; Echtay KS et al.; For expression in Saccharomyces cerevisiae the cDNA of the uncoupling protein (UCP) of brown adipose tissue from hamster has been isolated and used to transform yeast cells . Optimized expression conditions yielded 2% of mitochondrial protein as UCP . UCP was isolated, avoiding copurification of ADP/ATP carrier and porin . Intrahelical E190, previously suggested to be the pH sensor for nucleotide binding, was neutralized to glutamine by mutagenesis . In binding titrations with {14C}guanosine 5'-triphosphate (GTP) and with fluorescent dansyl-GTP, near equal binding capacity for GTP was measured in wild-type (wt) and E190Q . The KD for GTP binding to UCP from yeast has the same strong pH dependence as the original UCP from hamster . With both {14C}GTP and dansyl-GTP, the KD in wt increased 16-19-fold from pH 6.0 to 7.5, while in E190Q this increase was only 2.5-2.9-fold . As a result, at pH 7.5, both {14C}GTP and dansyl-GTP bind 6-fold tighter to E190Q than to wt . The binding rate of GTP decreased 10-fold from pH 6.0 to 7.5 in wt and only 4-fold in E190Q . Woodward reagent K (WRK) known to interact specifically with E190 {Winkler, E., Wachter, E., and Klingenberg, M . (1997) Biochemistry 36, 148-155} abolished {14C}GTP and dansyl-GTP binding to wt UCP, whereas binding to E190Q was fully resistant to WRK . H+ and Cl- transport activity in reconstituted vesicles were the same with wt and E190Q . At pH 7.5, 5 microM GTP is unable to inhibit H+ and Cl- transport in wt but inhibits in E190Q to maximum level . The different sensitivity toward GTP versus GDP found in wt is absent in E190Q . Thus, the mutation E190Q results in the predicted gain of function in binding and proves the role of the intrahelical E190 as a pH sensor for nucleotide binding but excludes a role in transport. Neuroreport, 1997 Jul 7, 8(9-10), 2143 - 8 Long-term morphine treatment increases Ku protein DNA end-binding activity; Bakalkin G et al.; Human neuroblastoma SH-SY5Y and small-cell lung carcinoma U1690 cells of neuroendocrine origin were exposed to morphine for 1 h, 3 h or 5 days . These treatments did not alter activities of AP-1, NF-kappa B and YY1 transcription factors in SH-SY5Y cells or NF-kappa B and YY1 in U1690 cells . Five-day morphine treatment, however, caused a twofold increase in the activity of a sequence-non-specific, spermidine-activated DNA-binding factor in U1690 cells . The morphine effect was prevented by the antagonist naloxone . The DNA-binding factor bound preferentially to double-stranded DNA ends . This fact and data on subunit composition, molecular masses of subunits, and supershift/inhibition by specific antibodies in a band shift assay, show the spermidine-activated factor to be identical with the Ku protein, the DNA-binding subunit of DNA-dependent protein kinase . The effect observed may be one of the mechanisms through which opioids influence gene regulation. J Biotechnol, 1997 Jul 4, 55(3), 171 - 9 Bioaffinity layering: a novel strategy for the immobilization of large quantities of glycoenzymes; Farooqi M et al.; A simple strategy for increasing considerably the quantities of glycoenzymes immobilized on insoluble supports is described . The strategy that we call bioaffinity layering makes use of the multivalent nature of concanavalin A (Con A) and the multiple oligosaccharide chains of most glycoenzymes to build alternating lectin and glycoenzyme layers on a Sepharose matrix with precoupled Con A . Using this procedure, it was possible to increase the amounts of several glycoenzymes immobilized on Sepharose and 19.0 mg glucose oxidase could be associated with one ml Sepharose matrix after seven Con A/glucose oxidase incubation cycles . Bioaffinity layered preparations of glycoenzymes exhibited high activities as indicated by very high effectiveness factor (eta) values and those of glucose oxidase and invertase exhibited a layer-by-layer increase in thermostability . The sensitivity of a flow-through glucose monitoring cartridge integrated into a flow injection analysis (FIA) system was enhanced significantly by increasing the amount of immobilized glucose oxidase via bioaffinity layering . A cartridge bearing six layers of glucose oxidase on Sepharose support was used effectively and repeatedly for analysis of medium glucose concentration during a fed-batch cultivation of the yeast Saccharomyces cerevisiae. J Biol Chem, 1997 Jul 4, 272(27), 17145 - 53 P11, a unique member of the S100 family of calcium-binding proteins, interacts with and inhibits the activity of the 85-kDa cytosolic phospholipase A2; Wu T et al.; Using a two hybrid system screen of a human cDNA library, we have found that p11, a unique member of the S100 family of calcium-binding proteins, interacts with the carboxyl region of the 85-kDa cytosolic phospholipase A2 (cPLA2) . p11 synthesized in a cell-free system interacts with cPLA2 in vitro . The p11-cPLA2 complex is detectable from a human bronchial epithelial cell line (BEAS 2B) . Furthermore, p11 inhibits cPLA2 activity in vitro . Selective inhibition of p11 expression in the BEAS 2B cells by antisense RNA results in an increased PLA2 activity as well as an increased release of prelabeled arachidonic acid . This study demonstrates a novel mechanism for the regulation of cPLA2 by an S100 protein. J Biol Chem, 1997 Jul 4, 272(27), 17134 - 8 The synaptobrevin-related domains of Bos1p and Sec22p bind to the syntaxin-like region of Sed5p; Sacher M et al.; SNAREs (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors) are cytoplasmically oriented membrane proteins that reside on vesicular carriers (v-SNARE) and target organelles (t-SNARE) . The pairing of a stage-specific v-SNARE with its cognate t-SNARE may mediate the specificity of membrane traffic . In the yeast Saccharomyces cerevisiae transport between the endoplasmic reticulum and Golgi complex employs two v-SNAREs, Bos1p and Sec22p, each containing a domain that is related to the neuronal v-SNARE synaptobrevin . Sed5p, which is homologous to syntaxin, is the t-SNARE that functions at this stage of the secretory pathway . Here we report that regions of Bos1p and Sec22p, which are homologous to synaptobrevin, bind to the syntaxin-like domain of Sed5p . Furthermore, we demonstrate that efficient v-SNARE/t-SNARE interactions require the participation of both v-SNAREs, indicating that, unlike post-Golgi membrane traffic, the active form of the endoplasmic reticulum to Golgi v-SNARE is a heteromeric complex. Nature, 1997 Jul 3, 388(6637), 78 - 82 Cofilin promotes rapid actin filament turnover in vivo; Lappalainen P et al.; The ability of actin filaments to function in cell morphogenesis and motility is coupled to their capacity for rapid assembly and disassembly . Because disassembly in vitro is much slower than in vivo, cellular factors that stimulate disassembly have long been assumed to exist . Although numerous proteins can affect actin dynamics in vitro, demonstration of in vivo relevance of these effects has not been achieved . We have used genetics and an actin-inhibitor in yeast to demonstrate that rapid cycles of actin assembly and disassembly depend on the small actin-binding protein cofilin, and that cofilin stimulates filament disassembly . These results may explain why cofilin is ubiquitous in eukaryotes and is essential for viability in every organism in which its function has been tested genetically . Magnitudes of disassembly defects in cofilin mutants in vivo were found to be correlated closely with the magnitudes of disassembly defects observed in vitro, supporting our conclusions . Furthermore, these cofilin mutants provided an opportunity to distinguish in living cells those actin functions that depend specifically on filament turnover (endocytosis) from those that do not (cortical actin patch motility). Nature, 1997 Jul 3, 388(6637), 75 - 8 p47 is a cofactor for p97-mediated membrane fusion; Kondo H et al.; At least two distinct ATPases, NSF and p97, are known to be involved in the heterotypic fusion of transport vesicles with their target membranes and the homotypic fusion of membrane compartments . The NSF-mediated fusion pathway is the best characterized, many of the components having been identified and their functions analysed . In contrast, none of the accessory proteins for the p97-mediated fusion pathway has been identified . Now we have identified the first such component, a protein of relative molecular mass 47,000 (p47), which forms a tight, stoichiometric complex with cytosolic p97 (one trimer of p47 per hexamer of p97) . It is essential for the p97-mediated regrowth of Golgi cisternae from mitotic Golgi fragments, a process restricted to animal cells . As a homologue of p47 exists in budding yeast, this indicates that it might also be involved in other membrane fusion reactions catalysed by p97, such as karyogamy. Biol Chem, 1997 Jul, 378(7), 591 - 7 Telomere length regulation: getting the measure of chromosome ends; Shore D; Telomeres, the protein-DNA complexes that comprise the ends of linear eukaryotic chromosomes, serve to protect the chromosome ends and allow their complete replication . Telomeres also appear to play an essential role in chromosome segregation . In most organisms telomeric DNA consists of a series of short repeats that are variable in length, but regulated at a fixed average value in the germline . The possible involvement of telomere repeat shortening in aging and carcinogenesis has recently attracted attention to the more basic question of how telomere length is sensed and regulated by the cell . Telomere length in the budding yeast Saccharomyces cerevisiae has been known for over a decade now to be under complex genetic control, and this organism has provided a useful model system to address basic mechanistic questions . This review focuses on recent studies in yeast which indicate that the double-strand telomere-repeat binding protein Rap1 may play an important role in a negative-feedback mechanism that senses and controls the length of the telomere repeats . Although the same carboxy-terminal domain of Rap1p is involved in both telomere length regulation and telomeric silencing (telomere position effect), it appears that these two functions are mediated by separate sets of Rap1p-interacting proteins . Results from other systems suggest that negative regulation of telomere elongation by a double-stranded telomere-repeat binding protein may be a highly conserved strategy for telomere length control. J Biochem (Tokyo), 1997 Jul, 122(1), 212 - 6 Alternative translation initiation generates acyl-CoA synthetase 3 isoforms with heterogeneous amino termini; Fujino T et al.; ACS3 is a recently identified acyl-CoA synthetase (ACS) isozyme that preferentially utilizes laurate, myristate, arachidonate, and eicosapentaenoate among saturated and unsaturated long chain fatty acids . The ACS3 purified from COS cells transfected with the ACS3 cDNA was separated by SDS-PAGE into two major forms of 79 and 80 kDa . We report here that alternative translation initiation from ACS3 mRNA gives rise to these two isoforms of ACS3 . In vitro mutagenesis of the ACS3 cDNA revealed that the translation of the 80-kDa and 79-kDa isoforms started from the first and second in-frame AUGs, respectively . The two isoforms of ACS3 expressed in COS cells exhibited similar levels of ACS activities toward palmitate and myristate . Immunocytochemistry of intact COS cells transfected with various ACS3 expression vectors suggested that the two forms are localized in the extranuclear compartment, where they exhibit a reticular pattern . In rat cerebrum, the 80-kDa isoform of ACS3 was detected mainly in the microsomal fraction . Only a trace amount of the 79-kDa isoform was detected in rat cerebrum, whereas both forms were detected in rat glioma cell line KEG1 cells. Mol Gen Genet, 1997 Jul, 255(4), 429 - 37 Isolation and characterization of the krev-1 gene, a novel member of ras superfamily in Neurospora crassa: involvement in sexual cycle progression; Ito S et al.; Genes belonging to the ras superfamily encode low-molecular-weight GTP/GDP-binding proteins that are highly conserved in wide variety of organisms . We used the polymerase chain reaction (PCR) to isolate a novel member of the ras superfamily from the filamentous fungus Neurospora crassa and obtained a mammalian Krev-1 homolog . We named the gene krev-1 and analyzed its structure and function . The krev-1 gene encodes a polypeptide of 225 amino acids, which is nearly 60% homologous to the mammalian Krev-1 p21 . The krev-1 gene product (KREV1) is functionally analogous to mammalian Krev-1 p21 and Rsr1p/Bud1p, a Krev-1 homolog from the yeast Saccharomyces cerevisiae . GAL1-driven expression of KREV1 in a wild-type yeast strain resulted in a random budding pattern, as did its mammalian counterpart Krev-1 p21 . We disrupted the krev-1 gene by RIP (repeat-induced point mutation), but the krev-1 disruptants showed no abnormalities . By in vitro mutagenesis, we constructed several mutant krev-1 genes (G21V, A68T, and D128A) which mimic constitutively active mutants of Ha-ras, and the krev-1 (K25N) mutant which is analogous to a dominant-negative mutant of Ha-ras . Each mutant gene was introduced into the wild-type strain and the phenotypes were analyzed . We could not observe any difference in vegetative growth between these transformants . When each strain was used as the female in mating tests, the development of perithecia from protoperithecia was inhibited in all cases . The results indicate that the krev-1 gene may be involved in sexual cycle progression. Plant J, 1997 Jul, 12(1), 121 - 31 Very-long-chain fatty acid biosynthesis is controlled through the expression and specificity of the condensing enzyme; Millar AA et al.; The Arabidopsis FATTY ACID ELONGATION1 (FAE1) gene encodes a putative seed-specific condensing enzyme . It is the first of four enzyme activities that comprise the microsomal fatty acid elongase (FAE) involved in the biosynthesis of very-long-chain fatty acids (VLCFAs) . FAE1 has been expressed in yeast and in tissues of Arabidopsis and tobacco, where significant quantities of VLCFAs are not found . The introduction of FAE1 alone in these systems is sufficient for the production of VLCFAs, for wherever FAE1 was expressed, VLCFAs accumulated . These results indicate that FAE1 is the rate-limiting enzyme for VLCFA biosynthesis in Arabidopsis seed, because introduction of extra copies of FAE1 resulted in higher levels of the VLCFAs . Furthermore, the condensing enzyme is the activity of the elongase that determines the acyl chain length of the VLCFAs produced . In contrast, it appears that the other three enzyme activities of the elongase are found ubiquitously throughout the plant, are not rate-limiting and play no role in the control of VLCFA synthesis . The ability of yeast containing FAE1 to synthesize VLCFAs suggests that the expression and the acyl chain length specificity of the condensing enzyme, along with the apparent broad specificities of the other three FAE activities, may be a universal eukaryotic mechanism for regulating the amounts and acyl chain length of VLCFAs synthesized. Domest Anim Endocrinol, 1997 Jul, 14(4), 241 - 9 Somatotroph function in the neonatal pig; Matteri RL et al.; The objective of this study was to evaluate developmental changes in somatotroph function and related gene expression in neonatal pigs . Male piglets were sacrificed at 1, 7, 14, 21, 28, 35, and 42 d of age (8/age group) for the collection of tissue and blood . Serum concentrations of GH were determined . Quantitations of mRNA were performed for pituitary Pit-1, GH, and GHRH receptor . Cultures of pituitary cells from each pig were stimulated with 0, 0.1, 1, or 10 nM GHRH; 2 mM 8-Br-cAMP; or 100 nM phorbol myristate acetate . Elevated serum concentrations of GH were observed at 1 d of age, followed by a pronounced decrease to basal levels thereafter (P < 0.0001) . A mild transient increase in circulating GH occurred at Day 28 . In vitro GH secretion was significantly stimulated by secretagogue treatments (P < 0.0001) . Age-related declines in in vitro GH secretion were observed regardless of if the cells were stimulated by GHRH or by secretagogues that bypass the GHRH receptor (P < 0.001) . Similarly, cellular GH content varied with age (P = 0.01) . Levels of pituitary GH mRNA (P = 0.01) and GHRH receptor mRNA (P = 0.0002) decreased with age . The quantity of GHRH receptor mRNA was correlated with GH mRNA levels (r = 0.55, P = 0.02), serum GH concentrations (r = 0.55, P = 0.02), and in vitro GH secretion (r = 0.66, P = 0.001) . Pituitary Pit-1 mRNA levels at 7 and 14 d of age were significantly elevated relative to all other sampling times (P = 0.0002) . Levels of Pit-1 and GH mRNAs were significantly correlated (r = 0.64, P = 0.003) . These results demonstrate a strong developmental regulation of somatotrophic function and related gene expression during the early neonatal period of the pig . Age-related decreases in secretory function may be mediated by concurrent mechanisms relating to the expression of the GHRH receptor and of GH. Mech Dev, 1997 Jul, 65(1-2), 3 - 17 Miz1, a novel zinc finger transcription factor that interacts with Msx2 and enhances its affinity for DNA; Wu L et al.; Msx2 is a homeobox gene with a regulatory role in inductive tissue interactions, including those that pattern the skull . We demonstrated previously that individuals affected with an autosomal dominant disorder of skull morphogenesis (craniosynostosis, Boston type) bear a mutated form of Msx2 in which a histidine is substituted for a highly conserved proline in position 7 of the N-terminal arm of the homeodomain (p148h) . The mutation behaves as a dominant positive in transgenic mice . The location of the mutation in the N-terminal arm of the homeodomain, a region which in other homeodomain proteins plays a key part in protein-protein interactions, prompted us to undertake a yeast two hybrid screen for Msx2-interacting proteins . Here we present a functional analysis of one such protein, designated Miz1 (Msx-interacting-zinc finger) . Miz1 is a zinc finger-containing protein whose amino acid sequence closely resembles that of the yeast protein, Nfi-1 . Together these proteins define a new, highly conserved protein family . Analysis of Miz1 expression by Northern blot and in situ hybridization revealed a spatiotemporal pattern that overlaps that of Msx2 . Further, Miz1 is a sequence specific DNA binding protein, and it can function as a positive-acting transcription factor . Miz1 interacts directly with Msx2 in vitro and enhances the DNA binding affinity of Msx2 for a functionally important element in the rat osteocalcin promoter . The p148h mutation in Msx2 augments the Miz1 effect on Msx2 DNA binding, suggesting a reason why this mutation behaves in vivo as a dominant positive, and providing a potential explanation of the craniosynostosis phenotype. J Mol Med, 1997 Jul, 75(7), 467 - 9 The breast cancer gene product TSG101: a regulator of ubiquitination? Ponting CP, Cai YD, Bork P. Sequence analysis is a powerful tool to obtain structural and functional information about genes and their products . Here we show that TSG101, a gene subjected to somatic mutations in breast cancer, contains an amino terminal domain that is a homologue of ubiquitin conjugating enzymes (UBCs) and not, as previously proposed, DNA-binding domains . As the UBC active site residue is replaced in the TSG101 sequence in a similar manner to several other members of the UBC family, we propose a role for TSG101 in regulating the ubiquitination of short-lived gene products. Am J Physiol, 1997 Jul, 273(1 Pt 1), E37 - 45 Cholate inhibits high-fat diet-induced hyperglycemia and obesity with acyl-CoA synthetase mRNA decrease; Ikemoto S et al.; The effects of sodium cholate on high-fat diet-induced hyperglycemia and obesity were investigated . Insulin resistance was estimated by measuring 2-deoxyglucose uptake in epitrochlearis muscles incubated in vitro . Addition of 0.5% cholate to high-safflower oil diet completely prevented high fat-induced hyperglycemia and obesity in C57BL/6J mice with a slight decrease of energy intake but with no inhibition of fat absorption . Furthermore, the addition of cholate decreased blood insulin levels and prevented high-fat diet-induced decrease of glucose uptake in epitrochlearis . However, there was no change in the unsaturation index of fatty acids in skeletal muscles and in GLUT-4 levels by cholate . In liver, cholate addition resulted in cholesterol accumulation and completely prevented high-fat diet-induced triglyceride accumulation . The changes of triglyceride level in the liver were paralleled to the changes of acyl-CoA synthetase (ACS) mRNA . ACS catalyzes the formation of acyl-CoA from fatty acid, and acyl-CoA is utilized for triglyceride formation in liver . ACS has a sterol-responsive element 1 in its promoter region . These data indicate that the favorable effects of cholate could be partly the result of downregulation of ACS mRNA. Immunity, 1997 Jul, 7(1), 37 - 47 V(D)J recombination in Ku86-deficient mice: distinct effects on coding, signal, and hybrid joint formation; Bogue MA et al.; Ku, a heterodimer of 70 and 86 kDa subunits, plays a critical but poorly understood role in V(D)J recombination . Although Ku86-deficient mice are defective in coding and signal joint formation, rare recombination products have been detected by PCR . Here, we report nucleotide sequences of 99 junctions from Ku86-deficient mice . Over 90% of the coding joints, but not signal or hybrid joints, exhibit short sequence homologies, indicating that homology is required to join coding ends in the absence of Ku86 . Our results suggest that Ku86 may normally have distinct functions in the formation of these different types of junctions . Furthermore, Ku86(-/-) joints are unexpectedly devoid of N-region diversity, suggesting a novel role for Ku in the addition of N nucleotides by terminal deoxynucleotidyl transferase. Plant Mol Biol, 1997 Jul, 34(4), 693 - 700 Molecular genetic and biochemical analysis of Brassica napus proliferating cell nuclear antigen function; Markley NA et al.; A cDNA encoding the proliferating cell nuclear antigen (PCNA) from Brassica napus (oilseed rape) was shown to complement the lethal deletion mutation in the PCNA gene (delta POL30) of Saccharomyces cerevisiae . We provide unequivocal evidence that the B . napus PCNA can perform all the essential functions of the yeast PCNA in DNA replication, although some species-specific differences may exist . In addition, the B . napus PCNA expressed as a fusion polypeptide with glutathione S-transferase (GST) was shown to stimulate the activity and processivity of two delta-like DNA polymerases from wheat in vitro . These experiments provide direct biochemical evidence that the B . napus PCNA may function as an auxiliary factor in plant cell DNA replication. J Cell Sci, 1997 Jul, 110 ( Pt 14), 1647 - 54 A genetic screen reveals a role for the late G1-specific transcription factor Swi4p in diverse cellular functions including cytokinesis; Igual JC et al.; The transcription factor Swi4p plays a crucial role in the control of the initiation of the cell cycle in budding yeast . To further understand Swi4p function, we set up a synthetic lethal screen for genes interacting with SWI4 . Fourteen conditional mutations which resulted in lethality only in the absence of SWI4 have been isolated . Only two of them were suppressed by ectopic expression of CLN2, indicating that Swi4p is involved in diverse cellular processes in addition to its requirement for CLN1,2 regulation . In most of the mutants a cell cycle phenotype was observed, including defects in G1 progression, budding, the G2/M transition and cytokinesis . In addition, four of the mutations resulted in massive cell lysis at the restrictive temperature, indicating that Swi4p is involved in the maintenance of cell integrity . One of the mutants, rsf1 swi4delta, was characterized in detail and it is defective in cytokinesis at the restrictive temperature . Staining with Calcofluor revealed that the rsf1 swi4delta mutant is impaired in chitin biosynthesis . rsf1 is allelic to the AGM1 gene, coding for N-acetylglucosamine-phosphate mutase, an enzyme involved in the biosynthesis of chitin . A single copy of SWI4 suppressed the cytokinesis defect . The above data suggest that Swi4p has a role in cytokinesis and becomes essential in this process when chitin biosynthesis is compromised . As overexpression or ectopic expression of CLN did not suppress the rsf1 swi4delta mutant phenotype, Swi4p must control some other gene(s) involved in cytokinesis. Chem Pharm Bull (Tokyo), 1997 Jul, 45(7), 1169 - 76 Synthesis and antifungal activity of novel thiazole-containing triazole antifungals; Tsuruoka A et al.; A new series of thiazole-containing triazole antifungals was synthesized and evaluated for antifungal activity against a variety of clinically isolated pathogenic fungi in vitro and against systemic candidosis in vivo . Among these compounds, (+/-)-1-(2,4-difluorophenyl)-1-{4-(2,4-difluorophenyl) thiazol-2-yl}-2-(1H-1,2,4-triazol-1-yl)ethanol (ER24161) showed the most potent and well-balanced in vitro activities and excellent in vivo efficacy . We also achieved an enantioselective synthesis of the more potent enantiomer of ER-24161. J Protein Chem, 1997 Jul, 16(5), 513 - 22 Structure and sites of phosphorylation of 14-3-3 protein: role in coordinating signal transduction pathways; Dubois T et al.; The 14-3-3 family are homo- and heterodimeric proteins whose biological role has been unclear for some time, although they are now gaining acceptance as a novel type of 'adaptor' protein that modulates interactions between components of signal transduction pathways, rather than by direct activation or inhibition . It is becoming apparent that phosphorylation of the binding partner and possibly also the 14-3-3 proteins may regulate these interactions . 14-3-3 isoforms interact with a novel phosphoserine (Sp) motif on many proteins, RSX1,2SpXP . The two isoforms that interact with Raf-1 are phosphorylated in vivo on Ser185 in a consensus sequence motif for proline-directed kinases . The crystal structure of 14-3-3 indicates that this phosphorylation could regulate interaction of 14-3-3 with its target proteins . We have now identified a number of additional phosphorylation sites on distinct mammalian and yeast isoforms. Mol Biol Cell, 1997 Jul, 8(7), 1305 - 16 Mss4 does not function as an exchange factor for Rab in endoplasmic reticulum to Golgi transport; Nuoffer C et al.; Mss4 and its yeast homologue, Dss4, have been proposed to function as guanine nucleotide exchange factors (GEFs) for a subset of Rab proteins in the secretory pathway . We have previously shown that Rab1A mutants defective in GTP-binding potently inhibit endoplasmic reticulum to Golgi transport, presumably by sequestering an unknown GEF regulating its function . We now demonstrate that these mutants stably associate with Mss4 both in vivo and in vitro and that Mss4 effectively neutralizes the inhibitory activity of the Rab1A mutants . An equivalent Rab3A mutant (Rab3A{N135I}), a Rab protein specifically involved in regulated secretion at the cell surface, associates with Mss4 as efficiently as the Rab1A{N124I} mutant . Although Rab3A{N135I} prevents the ability of Mss4 to neutralize the inhibitory effects of Rab1A mutants on transport, it has no effect on Rab1 function or endoplasmic reticulum to Golgi transport . Furthermore, quantitative immunodepletion of Mss4 fails to inhibit transport in vitro . We conclude that Mss4 and its yeast homologue, Dss4, are not GEFs mediating activation of Rab, but rather, they interact with the transient guanine nucleotide-free state, defining a new class of Ras-superfamily GTPase effectors that function as guanine nucleotide-free chaperones (GFCs). Mol Biol Cell, 1997 Jul, 8(7), 1175 - 81 Bet1p activates the v-SNARE Bos1p; Stone S et al.; Bet1p is a type II membrane protein that is required for vesicular transport between the endoplasmic reticulum and Golgi complex in the yeast Saccharomyces cerevisiae . A domain of Bet1p, that shows potential to be involved in a coiled-coil interaction, is homologous to a region of the neuronal protein SNAP-25 . Here, we used in vitro binding studies to demonstrate that Bet1p plays a role in potentiating soluble NSF attachment protein receptor (SNARE) interactions . Mutational analysis points to the coiled-coil region as necessary for Bet1p function, and circular dichroism experiments support this theory . In vitro binding studies were also used to demonstrate that a direct interaction between Bet1p and Bos1p is required for the efficient interaction of the vesicle SNARE with its SNARE target . Genetic studies suggest that the interactions of Bet1p with Bos1p are regulated by the small GTP-binding protein Ypt1p. Eur J Cell Biol, 1997 Jul, 73(3), 240 - 51 Conservation of mitochondrial targeting sequence function in mitochondrial and hydrogenosomal proteins from the early-branching eukaryotes Crithidia, Trypanosoma and Trichomonas; Hausler T et al.; Kinetoplastid protozoa are the earliest-branching eukaryotes to possess a true mitochondrion . This organelle is host to a variety of intriguing and unique features, including RNA editing . We examined the characteristics of protein import into mitochondria of Trypanosoma brucei . Dihydrofolate reductase (DHFR) carrying a yeast mitochondrial targeting signal was correctly translocated into trypanosome mitochondria in vivo, as were DHFR fusion proteins bearing two unusually short (7-9 amino acids) presequences from trypanosomatids . The short trypanosomal targeting signals were functional in Saccharomyces cerevisiae as well, but their targeting efficiency was lower and processing was absent . Trichomonads branched even earlier than kinetoplastids in eukaryotic evolution and contain energy-generating organelles called hydrogenosomes . The origin of hydrogenosomes has been controversial, but most evidence suggests that they are related to mitochondria . Putative hydrogenosomal targeting signals from Trichomonas vaginalis are short (5-12 amino acids) . Three such sequences were capable of targeting a passenger protein to mitochondria both in yeast and in trypanosomes, and one of the hydrogenosomal presequences was efficiently processed in both organisms . These findings suggest a resemblance between the import machineries of mitochondria and hydrogenosomes. Methods, 1997 Jul, 12(3), 264 - 75 Assaying CTD kinases in vitro and phosphorylation-modulated properties of RNA polymerase II in vivo; Morris DP et al.; The functional properties of RNA polymerase II are modulated by hyperphosphorylation of its unique C-terminal repeat domain (CTD) . A number of enzymes with CTD kinase activity have been identified, and correlations between CTD phosphorylation and RNA polymerase II function have been made . Here we describe methods for assaying CTD kinases and for characterizing them enzymologically . In addition we present approaches for studying phosphorylation-mediated behavior of chromosome-associated RNA polymerase II by using CTD-directed, phosphorylation state-sensitive antibodies and in situ localization techniques . The methods described here should, in conjunction with genetic approaches, contribute to elucidating the physiological roles of CTD kinases. Yeast, 1997 Jul, 13(9), 809 - 17 Characterization of two new genes down-regulated by alpha-factor; Seidel J et al.; To detect genes directly down-regulated by alpha-factor, 55,000 plaque-forming units of a Saccharomyces cerevisiae lambda gt10 gene bank were differentially screened with cDNA of cells treated with alpha-factor for 20 min . Two new genes were detected in this way, called alpha0.5 and alpha0.6 . The former is transiently down-regulated by alpha-factor; it is very highly transcribed in late exponential-phase cells . The gene, located on the right arm of chromosome XIII, codes for a 59 amino-acid protein with a signal peptide . The protein has been shown with an antibody to be present in the membrane fraction . The gene has also been cloned as HOR7 (hyperosmolarity-responsive protein; Hirayama et al., 1995) . No other homologous sequences have been detected in the yeast genome . alpha0.6, located on the right arm of chromosome XII, corresponds to the open reading frame YLR110c; it codes for a 133 amino-acid protein containing a signal peptide . Its derived amino-acid sequence is homologous to the N-terminal half of the SED1 gene product . SED1, when overexpressed, is able to suppress a defect in the HDEL receptor coded for by the ERD2 gene (Hardwick and Pelham, 1994); however, alpha0.6 is not able to do so . The disruption of alpha0.5 or alpha0.6 does not lead to a special phenotype. EMBO J, 1997 Jul 1, 16(13), 4092 - 106 An evolutionarily conserved U5 snRNP-specific protein is a GTP-binding factor closely related to the ribosomal translocase EF-2; Fabrizio P et al.; The driving forces behind the many RNA conformational changes occurring in the spliceosome are not well understood . Here we characterize an evolutionarily conserved human U5 small nuclear ribonucleoprotein (snRNP) protein (U