EMBO J, 1998 Nov 16, 17(22), 6508 - 15 C- to N-terminal translocation of preproteins into mitochondria; Folsch H et al.; Nuclear-encoded mitochondrial matrix proteins in most cases contain N-terminal targeting signals and are imported in a linear N- to C-terminal (N-->C) fashion . We asked whether import can also occur in a C- to N-terminal direction (C-->N) . We placed targeting signals at the C-terminus of passenger proteins . Import did occur in this 'backwards' fashion . It paralleled that of the 'normal' N-->C mechanism in terms of efficiency, rate, energetic requirements and ability to mediate unfolding and refolding during and following import of protein containing a folded domain . Furthermore, this reaction was mediated by the TIM17-23 machinery . The import pathway taken by certain inner-membrane proteins contains elements of such a C-->N translocation pathway, as they are targeted to mitochondria by internal targeting signals . These internal targeting signals appear to form loop structures together with neighbouring transmembrane segments, and penetrate the inner membrane in a membrane-potential-dependent manner . The dimeric TIM17-23 complex, together with mt-Hsp70, acts on both sides of the loop structure to facilitate their translocation into the matrix . On one side of the loop import occurs in the common N-->C direction, whereas the translocation of the other side involves the novel C-->N import direction . We conclude therefore that the mitochondrial import machinery displays no preference for the directionality of the import process. EMBO J, 1998 Nov 16, 17(22), 6477 - 86 Tim9p, an essential partner subunit of Tim10p for the import of mitochondrial carrier proteins; Koehler CM et al.; Tim10p, a protein of the yeast mitochondrial intermembrane space, was shown previously to be essential for the import of multispanning carrier proteins from the cytoplasm into the inner membrane . We now identify Tim9p, another essential component of this import pathway . Most of Tim9p is associated with Tim10p in a soluble 70 kDa complex . Tim9p and Tim10p co-purify in successive chromatographic fractionations and co-immunoprecipitated with each other . Tim9p can be cross-linked to a partly translocated carrier protein . A small fraction of Tim9p is bound to the outer face of the inner membrane in a 300 kDa complex whose other subunits include Tim54p, Tim22p, Tim12p and Tim10p . The sequence of Tim9p is 25% identical to that of Tim10p and Tim12p . A Ser67-->Cys67 mutation in Tim9p suppresses the temperature-sensitive growth defect of tim10-1 and tim12-1 mutants . Tim9p is a new subunit of the TIM machinery that guides hydrophobic inner membrane proteins across the aqueous intermembrane space. EMBO J, 1998 Nov 16, 17(22), 6449 - 64 A novel complex of membrane proteins required for formation of a spherical nucleus; Siniossoglou S et al.; Two membrane proteins were identified through their genetic interaction with the nucleoporin Nup84p and shown to participate in nuclear envelope morphogenesis in yeast . One component is a known sporulation factor Spo7p, and the other, Nem1p, a novel protein whose C-terminal domain is conserved during eukaryotic evolution . Spo7p and Nem1p localize to the nuclear/ER membrane and behave biochemically as integral membrane proteins . Nem1p binds to Spo7p via its conserved C-terminal domain . Although cells without Spo7p or Nem1p are viable, they exhibit a drastically altered nuclear morphology with long, pore-containing double nuclear membrane extensions . These protrusions emanate from a core nucleus which contains the DNA, and penetrate deeply into the cytoplasm . Interestingly, not only Spo7(-) and Nem1(-), but also several nucleoporin mutants are defective in sporulation . Thus, Spo7p and Nem1p, which exhibit a strong genetic link to nucleoporins of the Nup84p complex, fulfil an essential role in formation of a spherical nucleus and meiotic division. Curr Biol, 1998 Nov 19, 8(23), 1259 - 67 The 14-3-3 proteins positively regulate rapamycin-sensitive signaling; Bertram PG et al.; BACKGROUND: The kinase Tor is the target of the immunosuppressive drug rapamycin and is a member of the phosphatidylinositol kinase (PIK)-related kinase family . It plays an essential role in progression through the G1 phase of the cell cycle . The molecular details of Tor signaling remain obscure, however . RESULTS: We isolated two Saccharomyces cerevisiae genes, BMH1 and BMH2, as multicopy suppressors of the growth-inhibitory phenotype caused by rapamycin in budding yeast . BMH1 and BMH2 encode homologs of the 14-3-3 signal transduction proteins . Deletion of one or both BMH genes caused hypersensitivity to rapamycin in a manner that was dependent on gene dosage . In addition, alterations in the phosphopeptide-binding pocket of the 14-3-3 proteins had dramatically different effects on their ability to relieve the growth-arresting rapamycin phenotype . Mutations that prevented 14-3-3 from binding to a phosphoserine motif abolished its ability to confer rapamycin resistance . In contrast, substitution of two residues in 14-3-3 that surround these phosphoserine-binding sites conferred a dominant rapamycin-resistant phenotype . CONCLUSIONS: Our studies reveal 14-3-3 as an important component in rapamycin-sensitive signaling and provide significant new insights into the structure and function of 14-3-3 proteins. Curr Biol, 1998 Nov 19, 8(23), R832 - 5 Meiosis: Avoiding inappropriate relationships; Haber JE; Meiosis is distinguished from mitosis by the way double-strand breaks are made and by the synapsis and segregation of homologous chromosomes . Recent studies with the yeast Saccharomyces cerevisiae have identified some of the key players that link homologous recombination to synaptonemal complex formation. Bioessays, 1998 Oct, 20(10), 808 - 18 Repression by the Mad(Mxi1)-Sin3 complex; Schreiber-Agus N et al.; The functions of Myc in transformation and transactivation are countered by the suppressive actions of the Mad(Mxi1) family . Mad(Mxi1) proteins not only compete with Myc for dimerization to Max and binding to Myc/Max consensus sites but also recruit powerful repressors of gene expression . A prediction of the yin-yang relationship between Myc and Mad(Mxi1) families would be that the latter constitutes a new class of tumor suppressors . Here, we review the current literature on the Mad(Mxi1) family, with particular attention paid to the molecular mechanisms by which these proteins antagonize the actions of Myc in normal and neoplastic cells. Bioessays, 1998 Sep, 20(9), 771 - 80 Chromatin remodeling: a marriage between two families? Pollard KJ, Peterson CL. The compaction of the eukaryotic genome into a highly folded chromatin structure necessitates cellular mechanisms for allowing access of regulatory proteins to the DNA template . Recent advances in the fields of gene silencing, transcription, recombination, and DNA repair have led to the identification of two distinct families of chromatin remodeling enzymes--nuclear histone acetyltransferases and multisubunit complexes that harbor a SWI2/SNF2 ATPase family member . This paper reviews the current notion of how these enzymes function in remodeling chromatin; we then discuss some tantalizing lines of evidence that lead to the hypothesis that members of both families may actually function in concert to facilitate cellular processes in the context of chromatin. FEBS Lett, 1998 Oct 30, 438(1-2), 41 - 8 Cleavage of translation initiation factor 4G (eIF4G) during anti-Fas IgM-induced apoptosis does not require signalling through the p38 mitogen-activated protein (MAP) kinase; Morley SJ et al.; Initiation factor (eIF) 4G plays a key role in the regulation of translation, acting as a bridge between eIF4E and eIF3, to allow an mRNA molecule to associate with the 40S ribosomal subunit . In this study, we show that activation of the Fas/CD95 receptor complex in Jurkat cells induces the degradation of eIF4G, the inhibition of total protein synthesis and cell death . These responses were prevented by the caspase inhibitors, zVAD.FMK and zDEVD.FMK . We also show that, in contrast to Saccharomyces cerevisiae, although rapamycin caused a modest inhibition of protein synthesis it did not induce apoptosis or the cleavage of eIF4G . Studies with the specific inhibitor, SB203580, have shown that signalling through the p38 MAP kinase pathway is not required for either the Fas/CD95-induced cleavage of eIF4G or cell death . These data suggest that the cleavage of eIF4G and the inhibition of translation play an integral role in Fas/CD95-induced cell death in Jurkat cells. Biochem J, 1998 Dec 1, 336 ( Pt 2), 471 - 81 Human carbon catabolite repressor protein (CCR4)-associative factor 1: cloning, expression and characterization of its interaction with the B-cell translocation protein BTG1; Bogdan JA et al.; The human BTG1 protein is thought to be a potential tumour suppressor because its overexpression inhibits NIH 3T3 cell proliferation . However, little is known about how BTG1 exerts its anti-proliferative activity . In this study, we used the yeast 'two-hybrid' system to screen for interacting protein partners and identified human carbon catabolite repressor protein (CCR4)-associative factor 1 (hCAF-1), a homologue of mouse CAF-1 (mCAF-1) and Saccharomyces cerevisiae yCAF-1/POP2 . In vitro the hCAF-1/BTG1 complex formation was dependent on the phosphorylation of a putative p34cdc2 kinase site on BTG1 (Ser-159) . In yeast, the Ala-159 mutant did not interact with hCAF-1 . In addition, phosphorylation of Ser-159 in vitro showed specificity for the cell cycle kinases p34CDK2/cyclin E and p34CDK2/cyclin A, but not for p34CDK4/cyclin D1 or p34cdc2/cyclin B . Cell synchrony experiments with primary cultures of rat aortic smooth-muscle cells (RSMCs) demonstrated that message and protein levels of rat CAF-1 (rCAF-1) were up-regulated under conditions of cell contact, as previously reported for BTG1 {Wilcox, Scott, Subramanian, Ross, Adams-Burton, Stoltenborg and Corjay (1995) Circulation 92, I34-I35} . Western blot and immunohistochemical analysis showed that rCAF-1 localizes to the nucleus of contact-inhibited RSMCs, where it was physically associated with BTG1, as determined by co-immunoprecipitation with anti-hCAF-1 antisera . Overexpression of hCAF-1 in NIH 3T3 and osteosarcoma (U-2-OS) cells was itself anti-proliferative with colony formation reduced by 67% and 90% respectively . Taken together, these results indicate that formation of the hCAF-1/BTG1 complex is driven by phosphorylation at BTG1 (Ser-159) and implicates this complex in the signalling events of cell division that lead to changes in cellular proliferation associated with cell-cell contact. Biochem J, 1998 Dec 1, 336 ( Pt 2), 271 - 82 Molecular aspects of the endocytic pathway; Clague MJ; Observation of the flow of material along the endocytic pathway has lead to the description of the basic architecture of the pathway and provided insight into the relationship between compartments . Significant advances have been made in the study of endocytic transport steps at the molecular level, of which studies of cargo selection, vesicle budding and membrane fusion events comprise the major part . Progress in this area has been driven by two approaches, yeast genetics and in vitro or cell-free assays, which reconstitute particular transport steps and allow biochemical manipulation . The complex protein machineries that control vesicle budding and fusion are significantly conserved between the secretory and endocytic pathways such that proteins that regulate particular steps are often part of a larger family of proteins which exercise a conserved function at other locations within the cell . Well characterized examples include vesicle coat proteins, rabs (small GTPases) and soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment protein (SNAP) receptors (SNAREs) . Intracompartmental pH, lipid composition and cytoskeletal organization have also been identified as important determinants of the orderly flow of material within the endocytic pathway. Trends Genet, 1998 Oct, 14(10), 403 - 9 Putting the genome on the map; Bridger JM et al.; The first complete genomic sequence of a eukaryote (Saccharomyces cerevisiae) has already been accomplished . It is estimated that the sequence of the human genome will be known early in the next millennium . Yet it is already apparent that, despite their immense length, these linear primary sequence maps will be inadequate descriptions of the eukaryotic genome, be it of a budding yeast or a human . To reflect our growing awareness of the importance of spatial context in chromosome function and in gene expression we argue that a more complete map of the genome should seek to embody the richness of information that we expect of the maps we use to navigate our way around the outside world. Mol Biotechnol, 1998 Oct, 10(2), 103 - 6 A simple program to calculate codon bias index; Wang TT et al.; A computer program (PCBI) was developed to quickly calculate codon bias index (CBI) . PCBI can analyze a gene containing introns . The 22 preferred codons defined from Saccharomyces cerevisiae were used in PCBI as the standard to measure the CBI values . However, users can modify the preferred codons to suit each organism . The data PCBI provides include DNA sequence of open reading frame without introns, amino acid sequence of gene product, a table of amino acid composition, a table of codon usage and (G + C) content, parameters for calculating CBI, and the value of CBI . PCBI runs on a DOS or Windows environment, but results can be saved in ASCII text format. Mol Cell Biol, 1998 Dec, 18(12), 7444 - 54 Distinct subclasses of small GTPases interact with guanine nucleotide exchange factors in a similar manner; Day GJ et al.; The Ras-related GTPases are small, 20- to 25-kDa proteins which cycle between an inactive GDP-bound form and an active GTP-bound state . The Ras superfamily includes the Ras, Rho, Ran, Arf, and Rab/YPT1 families, each of which controls distinct cellular functions . The crystal structures of Ras, Rac, Arf, and Ran reveal a nearly superimposible structure surrounding the GTP-binding pocket, and it is generally presumed that the Rab/YPT1 family shares this core structure . The Ras, Rac, Ran, Arf, and Rab/YPT1 families are activated by interaction with family-specific guanine nucleotide exchange factors (GEFs) . The structural determinants of GTPases required for interaction with family-specific GEFs have begun to emerge . We sought to determine the sites on YPT1 which interact with GEFs . We found that mutations of YPT1 at position 42, 43, or 49 (effector loop; switch I), position 69, 71, 73, or 75 (switch II), and position 107, 109, or 115 (alpha-helix 3-loop 7 {alpha3-L7}) are intragenic suppressors of dominant interfering YPT1 mutant N22 (YPT1-N22), suggesting these mutations prevent YPT1-N22 from binding to and sequestering an endogenous GEF . Mutations at these positions prevent interaction with the DSS4 GEF in vitro . Mutations in the switch II and alpha3-L7 regions do not prevent downstream signaling in yeast when combined with a GTPase-defective (activating) mutation . Together, these results show that the YPT1 GTPase interacts with GEFs in a manner reminiscent of that for Ras and Arf in that these GTPases use divergent sequences corresponding to the switch I and II regions and alpha3-L7 of Ras to interact with family-specific GEFs . This finding suggests that GTPases of the Ras superfamily each may share common features of GEF-mediated guanine nucleotide exchange even though the GEFs for each of the Ras subfamilies appear evolutionarily unrelated. Mol Cell Biol, 1998 Dec, 18(12), 7304 - 16 Inhibition of double-stranded RNA-dependent protein kinase PKR by vaccinia virus E3: role of complex formation and the E3 N-terminal domain; Romano PR et al.; The human double-stranded RNA (dsRNA)-dependent protein kinase PKR inhibits protein synthesis by phosphorylating translation initiation factor 2alpha (eIF2alpha) . Vaccinia virus E3L encodes a dsRNA binding protein that inhibits PKR in virus-infected cells, presumably by sequestering dsRNA activators . Expression of PKR in Saccharomyces cerevisiae inhibits protein synthesis by phosphorylation of eIF2alpha, dependent on its two dsRNA binding motifs (DRBMs) . We found that expression of E3 in yeast overcomes the lethal effect of PKR in a manner requiring key residues (Lys-167 and Arg-168) needed for dsRNA binding by E3 in vitro . Unexpectedly, the N-terminal half of E3, and residue Trp-66 in particular, also is required for anti-PKR function . Because the E3 N-terminal region does not contribute to dsRNA binding in vitro, it appears that sequestering dsRNA is not the sole function of E3 needed for inhibition of PKR . This conclusion was supported by the fact that E3 activity was antagonized, not augmented, by overexpressing the catalytically defective PKR-K296R protein containing functional DRBMs . Coimmunoprecipitation experiments showed that a majority of PKR in yeast extracts was in a complex with E3, whose formation was completely dependent on the dsRNA binding activity of E3 and enhanced by the N-terminal half of E3 . In yeast two-hybrid assays and in vitro protein binding experiments, segments of E3 and PKR containing their respective DRBMs interacted in a manner requiring E3 residues Lys-167 and Arg-168 . We also detected interactions between PKR and the N-terminal half of E3 in the yeast two-hybrid and lambda repressor dimerization assays . In the latter case, the N-terminal half of E3 interacted with the kinase domain of PKR, dependent on E3 residue Trp-66 . We propose that effective inhibition of PKR in yeast requires formation of an E3-PKR-dsRNA complex, in which the N-terminal half of E3 physically interacts with the protein kinase domain of PKR. Mol Cell Biol, 1998 Dec, 18(12), 7278 - 87 Identification of a family of sorting nexin molecules and characterization of their association with receptors; Haft CR et al.; Sorting nexin 1 (SNX1) is a protein that binds to the epidermal growth factor (EGF) receptor and is proposed to play a role in directing EGF receptors to lysosomes for degradation (R . C . Kurten, D . L . Cadena, and G . N . Gill, Science 272:1008-1010, 1996) . We have obtained full-length cDNAs and deduced the amino acid sequences of three novel homologous proteins, which were denoted human sorting nexins (SNX2, SNX3, and SNX4) . In addition, we identified a presumed splice variant isoform of SNX1 (SNX1A) . These molecules contain a conserved domain of approximately 100 amino acids, which was termed the phox homology (PX) domain . Human SNX1 (522 amino acids), SNX1A (457 amino acids), SNX2 (519 amino acids), SNX3 (162 amino acids), and SNX4 (450 amino acids) are part of a larger family of hydrophilic molecules including proteins identified in Caenorhabditis elegans and Saccharomyces cerevisiae . Despite their hydrophilic nature, the sorting nexins are found partially associated with cellular membranes . They are widely expressed, although the tissue distribution of each sorting nexin mRNA varies . When expressed in COS7 cells, epitope-tagged sorting nexins SNX1, SNX1A, SNX2, and SNX4 coimmunoprecipitated with receptor tyrosine kinases for EGF, platelet-derived growth factor, and insulin . These sorting nexins also associated with the long isoform of the leptin receptor but not with the short and medium isoforms . Interestingly, endogenous COS7 transferrin receptors associated exclusively with SNX1 and SNX1A, while SNX3 was not found to associate with any of the receptors studied . Our demonstration of a large conserved family of sorting nexins that interact with a variety of receptor types suggests that these proteins may be involved in several stages of intracellular trafficking in mammalian cells. Mol Cell Biol, 1998 Dec, 18(12), 7205 - 15 Identification of a polar region in transmembrane domain 6 that regulates the function of the G protein-coupled alpha-factor receptor; Dube P et al.; The alpha-factor pheromone receptor (Ste2p) of the yeast Saccharomyces cerevisiae belongs to the family of G protein-coupled receptors that contain seven transmembrane domains (TMDs) . Because polar residues can influence receptor structure by forming intramolecular contacts between TMDs, we tested the role of the five polar amino acids in TMD6 of the alpha-factor receptor by mutating these residues to nonpolar leucine . Interestingly, a subset of these mutants showed increased affinity for ligand and constitutive receptor activity . The mutation of the most polar residue, Q253L, resulted in 25-fold increased affinity and a 5-fold-higher basal level of signaling that was equal to about 19% of the alpha-factor induced maximum signal . Mutation of the adjacent residue, S254L, caused weaker constitutive activity and a 5-fold increase in affinity . Comparison of nine different mutations affecting Ser254 showed that an S254F mutation caused higher constitutive activity, suggesting that a large hydrophobic amino acid residue at position 254 alters transmembrane helix packing . Thus, these studies indicate that Gln253 and Ser254 are likely to be involved in intramolecular interactions with other TMDs . Furthermore, Gln253 and Ser254 fall on one side of the transmembrane helix that is on the opposite side from residues that do not cause constitutive activity when mutated . These results suggest that Gln253 and Ser254 face inward toward the other TMDs and thus provide the first experimental evidence to suggest the orientation of a TMD in this receptor . Consistent with this, we identified two residues in TMD7 (Ser288 and Ser292) that are potential contact residues for Gln253 because mutations affecting these residues also cause constitutive activity . Altogether, these results identify a new domain of the alpha-factor receptor that regulates its ability to enter the activated conformation. Mol Cell Biol, 1998 Dec, 18(12), 7176 - 84 ETO, a target of t(8;21) in acute leukemia, interacts with the N-CoR and mSin3 corepressors; Lutterbach B et al.; t(8;21) is one of the most frequent translocations associated with acute myeloid leukemia . It produces a chimeric protein, acute myeloid leukemia-1 (AML-1)-eight-twenty-one (ETO), that contains the amino-terminal DNA binding domain of the AML-1 transcriptional regulator fused to nearly all of ETO . Here we demonstrate that ETO interacts with the nuclear receptor corepressor N-CoR, the mSin3 corepressors, and histone deacetylases . Endogenous ETO also cosediments on sucrose gradients with mSin3A, N-CoR, and histone deacetylases, suggesting that it is a component of one or more corepressor complexes . Deletion mutagenesis indicates that ETO interacts with mSin3A independently of its association with N-CoR . Single amino acid mutations that impair the ability of ETO to interact with the central portion of N-CoR affect the ability of the t(8;21) fusion protein to repress transcription . Finally, AML-1/ETO associates with histone deacetylase activity and a histone deacetylase inhibitor impairs the ability of the fusion protein to repress transcription . Thus, t(8;21) fuses a component of a corepressor complex to AML-1 to repress transcription. J Cell Sci, 1998 Dec 18, 111 ( Pt 24), 3655 - 61 An essential function of Grr1 for the degradation of Cln2 is to act as a binding core that links Cln2 to Skp1; Kishi T et al.; In budding yeast, SCF complexes, composed of Skp1, Cdc53 and one of the F-box proteins, have been implicated in Cdc34-dependent ubiquitination . Grr1, which is required for degradation of G1 cyclins Cln1 and Cln2 as well as for regulation of glucose repression, is an F-box protein and interacts with Skp1 through the F-box motif . Grr1 also interacts in vitro with phosphorylated Cln1 and Cln2 . However, ubiquitination of Cln1 has not been successful in an in vitro reconstituted system . In this study, domain analysis was performed to understand the role of Grr1 in the degradation of Cln2 . Grr1 has another motif, leucine-rich repeats (LRR), in addition to the F-box . We found that the LRR is a domain for Cln2 binding . A deletion of half of the LRR abolished the interaction of Grr1 with phosphorylated Cln2 but not with Skp1 in vivo, and a deletion of the F-box abolished the interaction of Grr1 with Skp1 but not with phosphorylated Cln2 in vivo . Based on these results, we constructed grr1 mutants that are defective in association with either Skp1 or Cln2 . Cln2 was highly stabilized and accumulated in the phosphorylated forms in the mutant cells . Furthermore, Skp1 associated in vivo with phosphorylated Cln2 in a Grr1-dependent manner . These data suggest that Grr1 is required for degradation of Cln2 through linking phosphorylated Cln2 to Skp1 in a SCFGrr1 complex. Biochemistry, 1998 Nov 17, 37(46), 16349 - 59 Conformational transitions of an unmodified tRNA: implications for RNA folding; Maglott EJ et al.; Unmodified tRNAs are powerful systems to study the effects of posttranscriptional modifications and site-directed mutations on both the structure and function of these ribonucleic acids . To define the general limitations of synthetic constructs as models for native tRNAs, it is necessary to elucidate the conformational states of unmodified tRNAs as a function of solution conditions . Here we report the conformational properties of unmodified yeast tRNAPhe as a function of ionic strength, {Mg2+}, and temperature using a combination of spectroscopic measurements along with chemical and enzymatic probes . We find that in low {Na+} buffer at low temperature, native yeast tRNAPhe adopts tertiary structure in the absence of Mg2+ . By contrast, tertiary folding of unmodified yeast tRNAPhe has an absolute requirement for Mg2+ . Below the melting temperature of the cloverleaf, unmodified yeast tRNAPhe exists in a Mg2+-dependent equilibrium between secondary and tertiary structure . Taken together, our findings suggest that although the tertiary structures of tRNAs are broadly comparable, the intrinsic stability of the tertiary fold, the conformational properties of intermediate states, and the stability of intermediate states can differ significantly between tRNA sequences . Thus, the use of unmodified tRNAs as models for native constructs can have significant limitations . Broad conclusions regarding "tRNA folding" as a whole must be viewed cautiously, particularly in cases where structural changes occur, such as during protein synthesis. Hum Mol Genet, 1998 Dec, 7(13), 2157 - 66 Cloning and characterization of Krct, a member of a novel subfamily of serine/threonine kinases; Stairs DB et al.; Protein kinases frequently play key roles in the normal regulation of growth and development in eukaryotic organisms . As a consequence, aberrant expression or mutations in this family of molecules frequently result in transformation . Previously, we have conducted a screen to identify protein kinases that are expressed in the mouse during mammary gland development and in breast cancer cell lines . We now describe the molecular cloning, characterization and expression of Krct, a novel serine/threonine protein kinase unrelated to previously defined families of protein kinases . At the mRNA level, Krct is widely expressed throughout murine development and in adult tissues . Despite its ubiquitous expression, Krct is expressed preferentially within specific cellular compartments in multiple tissues, in particular within the testis and gastrointestinal tract . At the amino acid level, Krct is most closely related to four previously undescribed kinases in Saccharomyces cerevisiae, Arabidopsis thaliana and Caenorhabditis elegans . Together, these kinases appear to define a novel subfamily of serine/threonine protein kinases . Krct possesses an unusually long 5'-untranslated region containing multiple upstream initiation codons and, in this regard, is similar to many proto-oncogenes that regulate normal growth and differentiation . In addition, Krct is located on mouse chromosome 11 closely linked to the epidermal growth factor receptor and, therefore, is likely to be co-amplified in a variety of human tumors. J Cell Biol, 1998 Nov 16, 143(4), 1041 - 52 When overexpressed, a novel centrosomal protein, RanBPM, causes ectopic microtubule nucleation similar to gamma-tubulin; Nakamura M et al.; A novel human protein with a molecular mass of 55 kD, designated RanBPM, was isolated with the two-hybrid method using Ran as a bait . Mouse and hamster RanBPM possessed a polypeptide identical to the human one . Furthermore, Saccharomyces cerevisiae was found to have a gene, YGL227w, the COOH-terminal half of which is 30% identical to RanBPM . Anti-RanBPM antibodies revealed that RanBPM was localized within the centrosome throughout the cell cycle . Overexpression of RanBPM produced multiple spots which were colocalized with gamma-tubulin and acted as ectopic microtubule nucleation sites, resulting in a reorganization of microtubule network . RanBPM cosedimented with the centrosomal fractions by sucrose- density gradient centrifugation . The formation of microtubule asters was inhibited not only by anti- RanBPM antibodies, but also by nonhydrolyzable GTP-Ran . Indeed, RanBPM specifically interacted with GTP-Ran in two-hybrid assay . The central part of asters stained by anti-RanBPM antibodies or by the mAb to gamma-tubulin was faded by the addition of GTPgammaS-Ran, but not by the addition of anti-RanBPM anti- bodies . These results provide evidence that the Ran-binding protein, RanBPM, is involved in microtubule nucleation, thereby suggesting that Ran regulates the centrosome through RanBPM. AIDS, 1998 Oct 22, 12(15), 1957 - 64 Recruitment of the TATA-binding protein to the HIV-1 promoter is a limiting step for Tat transactivation; Majello B et al.; OBJECTIVES: To examine the functional interaction between HIV-1 Tat protein and the TATA-binding protein (TBP) . DESIGN: HIV long terminal repeat reporter plasmids were cotransfected into mammalian and Drosophila insect cells with expression vectors encoding Tat and vectors encoding TBP either alone or linked to an heterologous DNA-binding domain . METHODS: The activity of the different reporters was compared in the presence or absence of Tat or TBP, or both, upon cotransfections into human and Drosophila insect cell lines . RESULTS: Tat protein is unable to transactivate enhancerless HIV-1 minimal promoter bearing only the TATA box and TAR . Artificial recruitment of human TBP (hTBP) to the enhancerless HIV minimal promoter was found to trigger gene expression and coexpression of Tat resulted in a marked synergy . Tat protein cooperated with DNA-bound hTBP by inducing high levels of processive viral transcripts . Synergy between Tat and hTBP was also observed when both factors were targeted to a promoter DNA template . The functional cooperation between TBP and Tat was further demonstrated using the Drosophila Schneider SL2 cells . In these cells Tat protein alone was ineffective; however, coexpression of Drosophila TBP and Tat resulted in a trans-activating response region-dependent synergistic transactivation of basal transcription . CONCLUSION: The strong synergy between TBP and Tat in the absence of any DNA-bound activator suggests that Tat stimulates transcription in an activator-independent manner most likely by a functional interaction with general transcription factors that occurs after TBP recruitment . Thus, efficient recruitment of TBP represents a limiting step for Tat transactivation. RNA, 1998 Nov, 4(11), 1357 - 72 Synthetic lethal interactions with conditional poly(A) polymerase alleles identify LCP5, a gene involved in 18S rRNA maturation; Wiederkehr T et al.; To identify new genes involved in 3'-end formation of mRNAs in Saccharomyces cerevisiae, we carried out a screen for synthetic lethal mutants with the conditional poly(A) polymerase allele, pap1-7 . Five independent temperature-sensitive mutations called Icp1 to Icp5 (for lethal with conditional pap1 allele) were isolated . Here, we describe the characterization of the essential gene LCP5 which codes for a protein with a calculated molecular mass of 40.8 kD . Unexpectedly, we found that mutations in LCP5 caused defects in pre-ribosomal RNA (pre-rRNA) processing, whereas mRNA 3'-end formation in vitro was comparable to wild-type . Early cleavage steps (denoted A0 to A2) that lead to the production of mature 18S rRNA were impaired . In vivo depletion of Lcp5p also inhibited pre-rRNA processing . As a consequence, mutant and depleted cells showed decreased levels of polysomes compared to wild-type cells . Indirect immunofluorescence indicated a predominant localization of Lcp5p in the nucleolus . In addition, antibodies directed against Lcp5p specifically immunoprecipitated the yeast U3 snoRNA snR17, suggesting that the protein is directly involved in pre-rRNA processing. J Cell Biol, 1998 Nov 2, 143(3), 709 - 17 The septins are required for the mitosis-specific activation of the Gin4 kinase; Carroll CW et al.; In budding yeast, a protein kinase called Gin4 is specifically activated during mitosis and functions in a pathway initiated by the Clb2 cyclin to control bud growth . We have used genetics and biochemistry to identify additional proteins that function with Gin4 in this pathway, and both of these approaches have identified members of the septin family . Loss of septin function produces a phenotype that is very similar to the phenotype caused by loss of Gin4 function, and the septins are required early in mitosis to activate Gin4 kinase activity . Furthermore, septin mutants display a prolonged mitotic delay at the short spindle stage, consistent with a role for the septins in the control of mitotic events . Members of the septin family bind directly to Gin4, demonstrating that the functions of Gin4 and the septins must be closely linked within the cell . These results demonstrate that the septins in budding yeast play an integral role in the mitosis-specific regulation of the Gin4 kinase and that they carry out functions early in mitosis. J Cell Biol, 1998 Nov 2, 143(3), 589 - 99 Reconstitution of retrograde transport from the Golgi to the ER in vitro; Spang A et al.; Retrograde transport from the Golgi to the ER is an essential process . Resident ER proteins that escape the ER and proteins that cycle between the Golgi and the ER must be retrieved . The interdependence of anterograde and retrograde vesicle trafficking makes the dissection of both processes difficult in vivo . We have developed an in vitro system that measures the retrieval of a soluble reporter protein, the precursor of the yeast pheromone alpha-factor fused to a retrieval signal (HDEL) at its COOH terminus (Dean, N., and H.R.B Pelham . 1990 . J . Cell Biol . 111:369-377) . Retrieval depends on the HDEL sequence; the alpha-factor precursor, naturally lacking this sequence, is not retrieved . A full cycle of anterograde and retrograde transport requires a simple set of purified cytosolic proteins, including Sec18p, the Lma1p complex, Uso1p, coatomer, and Arf1p . Among the membrane-bound v-SNAP receptor (v-SNARE) proteins, Bos1p is required only for forward transport, Sec22p only for retrograde trafficking, and Bet1p is implicated in both avenues of transport . Putative retrograde carriers (COPI vesicles) generated from Golgi-enriched membranes contain v-SNAREs as well as Emp47p as cargo. J Biol Chem, 1998 Nov 20, 273(47), 31068 - 74 A model for Ku heterodimer assembly and interaction with DNA . Implications for the function of Ku antigen; Wang J et al.; Ku autoantigen, a heterodimer of 70- and 80-kDa subunits, is a DNA end-binding factor critical for DNA repair . Two domains of p70 mediate DNA binding, one on the C-terminal and one on the N-terminal portion . The latter must dimerize with p80 in order to bind DNA, whereas the former is p80-independent . Both must be intact for end binding activity in gel shift assays . To evaluate the role of p80 in DNA binding, deletion mutants were co-expressed with full-length p70 using recombinant baculoviruses . We show by several criteria that amino acids 371-510 of p80 interact with p70 . Both of the p70 dimerization domains bind to the same region of p80, but apparently to separate sites within that region . In DNA immunoprecipitation assays, amino acids 179-510 of p80 were required for p80-dependent DNA binding of p70, whereas in gel shift assays, amino acids 179-732 were necessary . Interestingly, both the p80-dependent and the p80-independent DNA binding sites preferentially bound to DNA ends, suggesting a model in which a single Ku heterodimer may juxtapose two broken DNA ends physically, facilitating their rejoining by DNA ligases. J Biol Chem, 1998 Nov 20, 273(47), 31032 - 9 The isolated complex of the translocase of the outer membrane of mitochondria . Characterization of the cation-selective and voltage-gated preprotein-conducting pore; Kunkele KP et al.; The complex of the translocase mitochondrial outer membrane (TOM), mediates recognition, unfolding, and translocation of preproteins . We have used a combination of biochemical and electrophysiological methods to study the properties of the preprotein-conducting pore of the purified TOM complex . The pore is cation-selective and voltage-gated . It shows three main conductance levels with characteristic slow and fast kinetics transitions to states of lower conductance following application of transmembrane voltages . These electrical properties distinguish it from the mitochondrial voltage-dependent anion channel (porin) and are identical to those of the previously described peptide-sensitive channel . Binding of antibodies to the C terminus of Tom40 on the intermembrane space side of the outer membrane modifies the channel properties and allows determination of the orientation of the channel within the lipid bilayer . Mitochondrial presequence peptides specifically interact with the pore and decrease the ion flow through the channel in a voltage-dependent manner . We propose that the presequence-induced closures of the pore are related to structural alterations of the TOM complex observed during the various stages of preprotein movement across the mitochondrial outer membrane. J Biol Chem, 1998 Nov 20, 273(47), 30995 - 1001 Cytoprotection by Bcl-2 requires the pore-forming alpha5 and alpha6 helices; Matsuyama S et al.; We explored whether the putative channel-forming fifth and sixth alpha-helices of Bcl-2 and Bax account for Bcl-2-mediated cell survival and Bax-induced cell death in mammalian cells and in the yeast Saccharomyces cerevisiae . When alpha5-alpha6 were either deleted or swapped with each other, the Bcl-2Deltaalpha5alpha6 deletion mutant and Bcl-2-Bax(alpha5alpha6) chimeric protein failed to block apoptosis induced by either Bax or staurosporine in human cells and were unable to prevent Bax-induced cell death in yeast, implying that the alpha5-alpha6 region of Bcl-2 is essential for its cytoprotective function . Additional experiments indicated that, although alpha5-alpha6 is necessary, it is also insufficient for the anti-apoptotic activity of Bcl-2 . In contrast, deletion or substitution of alpha5-alpha6 in Bax reduced but did not abrogate apoptosis induction in human cells, whereas it did completely nullify cytotoxic activity in yeast, implying that the pore-forming segments of Bax are critical for conferring a lethal phenotype in yeast but not necessarily in human cells . BaxDeltaalpha5alpha6 and Bax-Bcl-2(alpha5alpha6) also retained the ability to dimerize with Bcl-2 . Bax therefore may have redundant mechanisms for inducing apoptosis in mammalian cells, based on its ability to form alpha5-alpha6-dependent channels in membranes and to dimerize with and antagonize anti-apoptotic proteins such as Bcl-2. J Biol Chem, 1998 Nov 20, 273(47), 30847 - 50 SRC-1 and GRIP1 coactivate transcription with hepatocyte nuclear factor 4; Wang JC et al.; Hepatocyte nuclear factor-4 (HNF4), a member of the nuclear receptor superfamily, plays an important role in tissue-specific gene expression, including genes involved in hepatic glucose metabolism . In this study, we show that SRC-1 and GRIP1, which act as coactivators for various nuclear receptors, associate with HNF4 in vivo and enhance its transactivation potential . The AF-2 domain of HNF4 is required for this interaction and for the potentiation of transcriptional activity by these coactivators . p300 can also serve as a coactivator with HNF4, and it synergizes with SRC-1 to further augment the activity of HNF4 . HNF4 is also a key regulator of the expression of hepatocyte nuclear factor-1 (HNF1) . The overexpression of SRC-1 or GRIP1 enhances expression from a HNF1 gene promoter-reporter in HepG2 hepatoma cells, and this requires an intact HNF4-binding site in the HNF1 gene promoter . Type 1 maturity onset diabetes of young (MODY), which is characterized by abnormal glucose-mediated insulin secretion, is caused by mutations of the HNF4 gene . A mutation of the HNF4-binding site in the HNF1 gene promoter has also been associated with MODY . Thus, HNF4 is involved in the regulation of glucose homeostasis at several levels and along with the SRC-1, GRIP1, and p300 may play an important role in the pathophysiology of non-insulin-dependent diabetes mellitus. Proc Natl Acad Sci U S A, 1998 Nov 10, 95(23), 13905 - 10 Hybrid Ty1/HIV-1 elements used to detect inhibitors and monitor the activity of HIV-1 reverse transcriptase; Nissley DV et al.; We previously demonstrated that hybrid retrotransposons composed of the yeast Ty1 element and the reverse transcriptase (RT) of HIV-1 are active in the yeast Saccharomyces cerevisiae . The RT activity of these hybrid Ty1/HIV-1 (his3AI/AIDS RT; HART) elements can be monitored by using a simple genetic assay . HART element reverse transcription depends on both the polymerase and RNase H domains of HIV-1 RT . Here we demonstrate that the HART assay is sensitive to inhibitors of HIV-1 RT . (-)-(S)-8-Chloro-4,5,6, 7-tetrahydro-5-methyl-6-(3-methyl-2-butenyl)imidazo{4,5,1-jk}{1, 4}-benzodiazepin-2(1H)-thione monohydrochloride (8 Cl-TIBO), a well characterized non-nucleoside RT inhibitor (NNRTI) of HIV-1 RT, blocks propagation of HART elements . HART elements that express NNRTI-resistant RT variants of HIV-1 are insensitive to 8 Cl-TIBO, demonstrating the specificity of inhibition in this assay . HART elements carrying NNRTI-resistant variants of HIV-1 RT can be used to identify compounds that are active against drug-resistant viruses. Proc Natl Acad Sci U S A, 1998 Nov 10, 95(23), 13543 - 8 A transcriptional activating region with two contrasting modes of protein interaction; Ansari AZ et al.; A C-terminal segment of the yeast activator Gal4 manifests two functions: When tethered to DNA, it elicits gene activation, and it binds the inhibitor Gal80 . Here we examine the effects on these two functions of cysteine and proline substitutions . We find that, although certain cysteine substitutions diminish interaction with Gal80, those substitutions have little effect on the activating function in vivo and interaction with TATA box-binding protein (TBP) in vitro . Proline substitutions introduced near residues critical for Gal80 binding abolish that interaction but once again have no effect on the activating function . Crosslinking experiments show that a defined position in the activating peptide is in close proximity to TBP and Gal80 in the two separate reactions and show that binding of the inhibitor blocks binding to TBP . Thus, the same stretch of amino acids are involved in two quite different protein-protein interactions: binding to Gal80, which depends on a precise sequence and the formation of a defined secondary structure, or interactions with the transcriptional machinery in vivo, which are not impaired by perturbations of either sequence or structure. Proc Natl Acad Sci U S A, 1998 Nov 10, 95(23), 13501 - 6 Long-distance transcriptional enhancement by the histone acetyltransferase PCAF; Krumm A et al.; Enhancers are defined by their ability to stimulate gene activity from remote sites and their requirement for promoter-proximal upstream activators to activate transcription . Here we demonstrate that recruitment of the p300/CBP-associated factor PCAF to a reporter gene is sufficient to stimulate promoter activity . The PCAF-mediated stimulation of transcription from either a distant or promoter-proximal position depends on the presence of an upstream activator (Sp1) . These data suggest that acetyltransferase activity may be a primary component of enhancer function, and that recruitment of polymerase and enhancement of transcription are separable . Transcriptional activation by PCAF requires both its acetyltransferase activity and an additional activity within its N terminus . We also show that the simian virus 40 enhancer and PCAF itself are sufficient to counteract Mad-mediated repression . These results are compatible with recent models in which gene activity is regulated by the competition between deacetylase-mediated repression and enhancer-mediated recruitment of acetyltransferases. Proc Natl Acad Sci U S A, 1998 Nov 10, 95(23), 13419 - 24 An autonomous folding unit mediates the assembly of two-stranded coiled coils; Kammerer RA et al.; Subunit oligomerization of many proteins is mediated by coiled-coil domains . Although the basic features contributing to the thermodynamic stability of coiled coils are well understood, the mechanistic details of their assembly have not yet been dissected . Here we report a 13-residue sequence pattern that occurs with limited sequence variations in many two-stranded coiled coils and that is absolutely required for the assembly of the Dictyostelium discoideum actin-bundling protein cortexillin I and the yeast transcriptional activator GCN4 . The functional relationship between coiled-coil "trigger" sequences was manifested by replacing the intrinsic trigger motif of GCN4 with the related sequence from cortexillin I . We demonstrate that these trigger sequences represent autonomous helical folding units that, in contrast to arbitrarily chosen heptad repeats, can mediate coiled-coil formation . Aside from being of general interest for protein folding, trigger motifs should be of particular importance in the protein de novo design. J Virol, 1998 Dec, 72(12), 10020 - 8 RNA 5'-triphosphatase, nucleoside triphosphatase, and guanylyltransferase activities of baculovirus LEF-4 protein; Gross CH et al.; Autographa californica nuclear polyhedrosis virus late and very late mRNAs are transcribed by an RNA polymerase consisting of four virus-encoded polypeptides: LEF-8, LEF-9, LEF-4, and p47 . The 464-amino-acid LEF-4 subunit contains the signature motifs of GTP:RNA guanylyltransferases (capping enzymes) . Here, we show that the purified recombinant LEF-4 protein catalyzes two reactions involved in RNA cap formation . LEF-4 is an RNA 5'-triphosphatase that hydrolyzes the gamma phosphate of triphosphate-terminated RNA and a guanylyltransferase that reacts with GTP to form a covalent protein-guanylate adduct . The RNA triphosphatase activity depends absolutely on a divalent cation; the cofactor requirement is satisfied by either magnesium or manganese . LEF-4 also hydrolyzes ATP to ADP and Pi (Km = 43 microM ATP; Vmax = 30 s-1) and GTP to GDP and Pi . The LEF-4 nucleoside triphosphatase (NTPase) is activated by manganese or cobalt but not by magnesium . The RNA triphosphatase and NTPase activities of baculovirus LEF-4 resemble those of the vaccinia virus and Saccharomyces cerevisiae mRNA capping enzymes . We suggest that these proteins comprise a novel family of metal-dependent triphosphatases. J Virol, 1998 Dec, 72(12), 9827 - 34 Zeta chain of the T-cell receptor interacts with nef of simian immunodeficiency virus and human immunodeficiency virus type 2; Howe AY et al.; A truncated version of the nef gene of simian immunodeficiency virus SIVmac239 capable of encoding amino acids 98 to 263 was used as bait to screen a cDNA library from activated lymphocytes in a yeast two-hybrid system . The zeta chain of the T-cell receptor (TCRzeta) was found to interact specifically not only with truncated SIV nef in yeast cells but also with full-length glutathione S-transferase (GST)-SIVnef fusion protein in vitro . Coimmunoprecipitation of TCRzeta with full-length SIV nef was demonstrated in transfected Jurkat cells and in Cos 18 cells which express the cytoplasmic domain of TCRzeta fused to the external domain of CD8 via the CD8 transmembrane domain . Using a series of nef deletion mutants, we have mapped the binding site within the central core domain of nef (amino acids 98 to 235) . Binding of TCRzeta was specific for nef isolated from SIVmac239, SIVsmH4, and human immunodeficiency virus (HIV)-2ST and was not detected with nef from five different HIV-1 isolates . An active tyrosine kinase was coprecipitated with nef-TCRzeta complexes from Jurkat cells but not from J.CAM1.6 cells which lack a functional Lck tyrosine kinase . These results demonstrate a specific association of SIV and HIV-2 nef, but not HIV-1 nef, with TCRzeta. Curr Biol, 1998 Nov 5, 8(22), 1211 - 4 The Rho1 effector Pkc1, but not Bni1, mediates signalling from Tor2 to the actin cytoskeleton; Helliwell SB et al.; In Saccharomyces cerevisiae, the phosphatidylinositol kinase homologue Tor2 controls the cell-cycle-dependent organisation of the actin cytoskeleton by activating the small GTPase Rho1 via the exchange factor Rom2 {1,2} . Four Rho1 effectors are known, protein kinase C 1 (Pkc1), the formin-family protein Bni1, the glucan synthase Fks and the signalling protein Skn7 {2,3} . Rho1 has been suggested to signal to the actin cytoskeleton via Bni1 and Pkc1; rho1 mutants have never been shown to have defects in actin organisation, however {2,4} . We have further investigated the role of Rho1 in controlling actin organisation and have analysed which of the Rho1 effectors mediates Tor2 signalling to the actin cytoskeleton . We show that some, but not all, rho1 temperature-sensitive (rho1ts) mutants arrest growth with a disorganised actin cytoskeleton . Both the growth defect and the actin organisation defect of the rho1-2ts mutant were suppressed by upregulation of Pkc1 but not by upregulation of Bni1, Fks or Skn7 . Overexpression of Pkc1, but not overexpression of Bni1, Fks or Skn7, also rescued a tor2ts mutant, and deletion of BNI1 or SKN7 did not prevent the suppression of the tor2ts mutation by overexpressed Rom2 . Furthermore, overexpression of the Pkc1-controlled mitogen-activated protein (MAP) kinase Mpk1 suppressed the actin defect of tor2ts and rho1-2ts mutants . Thus, Tor2 signals to the actin cytoskeleton via Rho1, Pkc1 and the cell integrity MAP kinase cascade. Curr Biol, 1998 Nov 5, 8(22), 1207 - 10 Activation of the human anaphase-promoting complex by proteins of the CDC20/Fizzy family; Kramer ER et al.; The initiation of anaphase and exit from mitosis depend on the activation of the cyclosome/anaphase-promoting complex (APC) that ubiquitinates regulatory proteins such as anaphase inhibitors and mitotic cyclins {1-4} . Genetic experiments have demonstrated that two related WD40-repeat proteins--called Cdc20p and Hct1p/Cdh1p in budding yeast and Fizzy and Fizzy-related in Drosophila--are essential for APC--dependent proteolysis {5-11} . Human orthologs of these proteins--hCDC20/p55CDC {12} and hCDH1--have recently been found to associate with APC in a cell-cycle-dependent manner {13,14} . Here, we show that the amount of hCDC20 and hCDH1 bound to APC correlates with a high ubiquitination activity of APC and that binding of recombinant hCDC20 and hCDH1 can activate APC in vitro . Our results suggest that the association between hCDH1 and APC is regulated by post-translational mechanisms, whereas the amount of hCDC20 bound to APC may in addition be controlled by hCDC20 synthesis and destruction {15} . The temporally distinct association of hCDC20 and hCDH1 with APC suggests that these proteins are, respectively, mitosis-specific and G1-specific activating subunits of APC. J Cell Sci, 1998 Dec, 111 ( Pt 23), 3459 - 70 Alpha-COP can discriminate between distinct, functional di-lysine signals in vitro and regulates access into retrograde transport; Schroder-Kohne S et al.; Emp47p is a yeast Golgi transmembrane protein with a retrograde, Golgi to ER transport di-lysine signal in its cytoplasmic tail . Emp47p has previously been shown to recycle between the Golgi complex and the ER and to require its di-lysine signal for Golgi localization . In contrast to other proteins with di-lysine signals, the Golgi-localization of Emp47p has been shown to be preserved in ret1-1 cells expressing a mutant alpha-COP subunit of coatomer . Here we demonstrate by sucrose gradient fractionation and immunofluorescence analysis that recycling of Emp47p was unimpaired in ret1-1 . Furthermore we have characterized three new alleles of ret1 and showed that Golgi localization of Emp47p was intact in cells with those mutant alleles . We could correlate the ongoing recycling of Emp47p in ret1-1 with preserved in vitro binding of coatomer from ret1-1 cells to immobilized GST-Emp47p-tail fusion protein . As previously reported, the di-lysine signal of Wbp1p was not recognized by ret1-1 mutant coatomer, suggesting a possible role for alpha-COP in the differential binding to distinct di-lysine signals . In contrast to results with alpha-COP mutants, we found that Emp47p was mislocalised to the vacuole in mutants affecting beta'-, gamma-, delta-, and zeta-COP subunits of coatomer and that the mutant coatomer bound neither to the Emp47p nor to the Wbp1p di-lysine signal in vitro . Therefore, the retrograde transport of Emp47p displayed a differential requirement for individual coatomer subunits and a special role of alpha-COP for a particular transport step in vivo. Oncogene, 1998 Oct 29, 17(17), 2187 - 93 hpttg, a human homologue of rat pttg, is overexpressed in hematopoietic neoplasms . Evidence for a transcriptional activation function of hPTTG; Dominguez A et al.; We have isolated a human cDNA clone encoding a novel protein of 22 kDa that is a human counterpart of the rat oncoprotein PTTG . We show that the corresponding gene (hpttg) is overexpressed in Jurkat cells (a human T lymphoma cell line) and in samples from patients with different kinds of hematopoietic malignancies . Analysis of the sequence showed that hPTTG has an amino-terminal basic domain and a carboxyl-terminal acidic domain, and that it is a proline-rich protein with several putative SH3-binding sites . Subcellular fractionation studies show that, although hPTTG is mainly a cytosolic protein, it is partially localized in the nucleus . In addition we demonstrate that the acidic carboxyl-terminal region of hPTTG acts as a transactivation domain when fused to a heterologous DNA binding domain, both in yeast and in mammalian cells. Nat Med, 1998 Nov, 4(11), 1293 - 301 Drug target validation and identification of secondary drug target effects using DNA microarrays; Marton MJ et al.; We describe here a method for drug target validation and identification of secondary drug target effects based on genome-wide gene expression patterns . The method is demonstrated by several experiments, including treatment of yeast mutant strains defective in calcineurin, immunophilins or other genes with the immunosuppressants cyclosporin A or FK506 . Presence or absence of the characteristic drug 'signature' pattern of altered gene expression in drug-treated cells with a mutation in the gene encoding a putative target established whether that target was required to generate the drug signature . Drug dependent effects were seen in 'targetless' cells, showing that FK506 affects additional pathways independent of calcineurin and the immunophilins . The described method permits the direct confirmation of drug targets and recognition of drug-dependent changes in gene expression that are modulated through pathways distinct from the drug's intended target . Such a method may prove useful in improving the efficiency of drug development programs. Cancer Res, 1998 Nov 1, 58(21), 4811 - 6 Multinuclearity and increased ploidy caused by overexpression of the aurora- and Ipl1-like midbody-associated protein mitotic kinase in human cancer cells; Tatsuka M et al.; Aurora- and Ipl1-like midbody-associated protein (AIM-1) is a serine/ threonine kinase that is structurally related to Drosophila aurora and Saccharomyces cerevisiae Ipl1, both of which are required for chromosome segregation . A kinase-negative form of AIM-1 inhibits the formation of cleavage furrow without affecting nuclear division, indicating that the gene controls entry into cytokinesis during M phase in mammalian cells . A human gene that encodes the protein AIM-1 was overexpressed in colorectal and other tumor cell lines . The regulation of AIM-1 expression was cell cycle dependent in normal and tumor cells, and the maximum accumulation was observed at G2-M . Exogenous overexpression of wild-type AIM-1 produced multinuclearity in human cells, suggesting that the excess amount of AIM-1 had a dominant-negative effect on the overexpressing cells . In long-term culture of AIM-1-overexpressing cells, multiple nuclei in a cell were occasionally fused, and then an increased ploidy and aneuploidy were induced . Thus, the overexpression of AIM-1 in colorectal tumor cell lines is thought to have a causal relationship with multinuclearity and increased ploidy . Cytokinesis error caused by AIM-1 overexpression is a major factor in the predisposition of tumor cells to the perturbation of chromosomal integrity that is commonly observed in human neoplasia . Thus, defects of pathways essential for mitotic regulation are important during human cancer development. FEMS Microbiol Lett, 1998 Oct 15, 167(2), 171 - 7 Characterization and expression of the co-transcribed cyc1 and cyc2 genes encoding the cytochrome c4 (c552) and a high-molecular-mass cytochrome c from Thiobacillus ferrooxidans ATCC 33020; Appia-Ayme C et al.; The sequence of the cyc1 gene encoding the Thiobacillus ferrooxidans ATCC 33020 c552 cytochrome, shows that this cytochrome is a 21-kDa periplasmic c4-type cytochrome containing two similar monohaem domains . The kinetics of reduction and the fact that cytochromes c4 are considered to be physiological electron donors of cytochrome oxidases suggest that the last steps of the iron respiratory chain are: rusticyanin-->cytochrome c4-->cytochrome oxidase . In Thiobacillus ferrooxidans, cyc1 is co-transcribed with the cyc2 gene, encoding a high-molecular-mass monohaem cytochrome c . This suggests that the cytochromes encoded by these genes belong to the same electron transfer chain. Mol Cell, 1998 Oct, 2(4), 485 - 93 Formation of a novel four-helix bundle and molecular recognition sites by dimerization of a response regulator phosphotransferase; Varughese KI et al.; A basis for understanding specificity of molecular recognition between phosphorelay proteins has been deduced from the 2.6 A structure of the Spo0B phosphotransferase of the phosphorelay regulating sporulation initiation . Spo0B consists of two domains: an N-terminal alpha-helical hairpin domain and a C-terminal alpha/beta domain . Two subunits of Spo0B dimerize by a parallel association of helical hairpins to form a novel four-helix bundle from which the active histidine protrudes . Docking studies show that both the monomers interact with a Spo0F molecule at the region surrounding the active site aspartate to position it for phosphotransfer . It is apparent that different surfaces of response regulators may be involved in recognition of the protein partners to which they are paired. Int J Cancer, 1998 Nov 23, 78(5), 576 - 80 Mutations of the human MUT S homologue 6 gene in ampullary carcinoma and gastric cancer; Imai Y et al.; MSH6 has been implicated in repair of single base mispairs and single-base deletion/insertion mutations . Established MSH6-null mice present a frequent occurrence of gastrointestinal tumors without microsatellite instability (MI), suggesting the possibility of the APC gene being a mutational target . Because human ampullary carcinomas and gastric cancers manifest frequent missense or I-base deletion mutations in cancer-related genes such as p53 and TGFbeta-RII, we suspected that the hMSH6 gene mutation might play a role in the carcinogenesis process . Out of the whole coding sequences, hMSH6 (C)8 (codons 1085-1087) and hMSH3 (A)8 repeats (codons 381-383) have been shown to be hotspots for frameshift mutations in a certain group of cancers, contributing to an increased genomic instability . We therefore investigated mutations of hMSH6 (C)8 and hMSH3 (A)8 in association with microsatellite mutator phenotype (MMP) in 18 ampullary carcinomas and 30 gastric cancers . In addition, overexpression of the P53 protein and mutational status of APC (AG)5 (codons 1462-1465) and (A)6 (codons 1554-1556) repeats were also investigated as a potential target of genetic instability secondary to MSH6 dysfunction . Mutation of the hMSH6 gene was not found in ampullary carcinomas and was irrelevant to TGFbeta-RII gene mutation . Mutation of the hMSH6 gene was observed in a subset of gastric cancers (4/30, 13.3%), but was not associated with P53 overexpression or APC gene mutation . In contrast to MSH6-null mice that do not show MI, hMSH6 gene mutation in human gastric cancers was closely correlated with MMP (3/10 MMP vs . 1/20 non-MMP) . In conclusion, hMSH6 mutation appears only in association with MMP and may underlie augmented MI, resulting in missense or I-base frameshift mutations in other genes in human gastric cancers. Plant Physiol, 1998 Nov, 118(3), 867 - 74 A nitrilase-like protein interacts with GCC box DNA-binding proteins involved in ethylene and defense responses; Xu P et al.; Ethylene-responsive element-binding proteins (EREBPs) of tobacco (Nicotiana tabacum L.) bind to the GCC box of many pathogenesis-related (PR) gene promoters, including osmotin (PR-5) . The two GCC boxes on the osmotin promoter are known to be required, but not sufficient, for maximal ethylene responsiveness . EREBPs participate in the signal transduction pathway leading from exogenous ethylene application and pathogen infection to PR gene induction . In this study EREBP3 was used as bait in a yeast two-hybrid interaction trap with a tobacco cDNA library as prey to isolate signal transduction pathway intermediates that interact with EREBPs . One of the strongest interactors was found to encode a nitrilase-like protein (NLP) . Nitrilase is an enzyme involved in auxin biosynthesis . NLP interacted with other EREBP family members, namely tobacco EREBP2 and tomato (Lycopersicon esculentum L.) Pti4/5/6 . The EREBP2-EREBP3 interaction with NLP required part of the DNA-binding domain . The specificity of interaction was further confirmed by protein-binding studies in solution . We propose that the EREBP-NLP interaction serves to regulate PR gene expression by sequestration of EREBPs in the cytoplasm. Plant Physiol, 1998 Nov, 118(3), 817 - 25 A high-affinity Ca2+ pump, ECA1, from the endoplasmic reticulum is inhibited by cyclopiazonic acid but not by thapsigargin; Liang F et al.; To identify and characterize individual Ca2+ pumps, we have expressed an Arabidopsis ECA1 gene encoding an endoplasmic reticulum-type Ca2+-ATPase homolog in the yeast (Saccharomyces cerevisiae) mutant K616 . The mutant (pmc1pmr1cnb1) lacks a Golgi and a vacuolar membrane Ca2+ pump and grows very poorly on Ca2+-depleted medium . Membranes isolated from the mutant showed high H+/Ca2+-antiport but no Ca2+-pump activity . Expression of ECA1 in endomembranes increased mutant growth by 10- to 20-fold in Ca2+-depleted medium . 45Ca2+ pumping into vesicles from ECA1 transformants was detected after the H+/Ca2+-antiport activity was eliminated with bafilomycin A1 and gramicidin D . The pump had a high affinity for Ca2+ (Km = 30 nM) and displayed two affinities for ATP (Km of 20 and 235 microM) . Cyclopiazonic acid, a specific blocker of animal sarcoplasmic/endoplasmic reticulum Ca2+-ATPase, inhibited Ca2+ transport (50% inhibition dose = 3 nmol/mg protein), but thapsigargin (3 microM) did not . Transport was insensitive to calmodulin . These results suggest that this endoplasmic reticulum-type Ca2+-ATPase could support cell growth in plants as in yeast by maintaining submicromolar levels of cytosolic Ca2+ and replenishing Ca2+ in endomembrane compartments . This study demonstrates that the yeast K616 mutant provides a powerful expression system to study the structure/function relationships of Ca2+ pumps from eukaryotes. J Nutr, 1998 Nov, 128(11), 1841 - 4 Recently identified molecular aspects of intestinal iron absorption; Wood RJ et al.; Gene mapping techniques to identify gene mutations in humans and animals with phenotypic abnormalities in iron metabolism are providing important insights into the probable molecular mediators of intestinal iron absorption . Positional gene cloning in humans with hereditary hemochromatosis has identified a mutation in a novel major histocompatibility complex (MHC) gene called HFE that is likely to be involved in regulating intestinal iron absorption . In addition, recent observations based on positional cloning strategies in the mk/mk mouse and the Belgrade (b/b) rat rodent models of hypochromic, microcytic anemia have shown that the phenotypic abnormality in iron metabolism is associated with a mutation in the Nramp2 gene . Functional cloning studies in Xenopus oocytes have characterized DCT1 (Nramp2) as an iron-regulated proton-coupled divalent cation transporter . Nramp2 is likely to be the membrane transporter that functions in controlling iron entry across the apical membrane and in the export of iron out of endosomal vesicles . The observation that the expression of both HFE and Nramp2 mRNAs are reciprocally regulated by cellular iron status in Caco-2 cells, a human intestinal cell line, lends additional credence to the notion that these proteins may work in concert to regulate intestinal iron absorption. Genes Dev, 1998 Nov 1, 12(21), 3442 - 51 Not just scaffolding: plectin regulates actin dynamics in cultured cells; Andra K et al.; Plectin, a major linker and scaffolding protein of the cytoskeleton, has been shown to be essential for the mechanical integrity of skin, skeletal muscle, and heart . Studying fibroblast and astroglial cell cultures derived from plectin (-/-) mice, we found that their actin cytoskeleton, including focal adhesion contacts, was developed more extensively than in wild-type cells . Also it failed to show characteristic short-term rearrangments in response to extracellular stimuli activating the Rho/Rac/Cdc42 signaling cascades . As a consequence, cell motility, adherence, and shear stress resistance were altered, and morphogenic processes were delayed . Furthermore, we show that plectin interacts with G-actin in vitro in a phosphatidylinositol-4,5-biphosphate-dependent manner and associates with actin stress fibers in living cells . The actin stress fiber phenotype of plectin-deficient fibroblasts could be reversed to a large degree by transient transfection of full-length plectin or plectin fragments containing the amino-terminal actin-binding domain (ABD) . These results reveal a novel role of plectin as regulator of cellular processes involving actin filament dynamics that goes beyond its proposed role in scaffolding and mechanical stabilization of cells. Plant J, 1998 Oct, 16(1), 41 - 52 The bifactorial endosperm box of gamma-zein gene: characterisation and function of the Pb3 and GZM cis-acting elements; Marzabal P et al.; The proximal region of the gamma-zein promoter (gamma Z) has a functional bifactorial prolamin box element containing two cis-acting elements, a prolamin-box motif (Pb3) and a GCN4-like motif (GZM) . By particle bombardment of maize endosperms with 5' deletions and internal deletions of gamma Z fused to the GUS gene, we have shown that a 135 bp region containing the bifactorial element is involved in the transcriptional activation of the gamma Z promoter . However, the 135 bp region was unable to activate the gamma Z promoter in the absence of a 84 bp downstream sequence . Using in vivo footprinting and gel mobility shift assays with 15 DAP endosperm nuclear extracts, we have demonstrated the presence of trans-acting factors that interact with Pb3 and GZM target sites . Base-substitution mutations within Pb3 and GZM decreased transcription activity of the gamma Z promoter suggesting a co-ordinated function between the two cis-acting elements . Two additional cis-motifs upstream of the bifactorial prolamin element have been identified: a motif with high homology to the AACA elements of rice glutelin genes and an AZM motif containing an ACGT core which binds nuclear proteins other than the Opaque 2 (O2). Plant J, 1998 Sep, 15(6), 755 - 64 FUSCA3 encodes a protein with a conserved VP1/AB13-like B3 domain which is of functional importance for the regulation of seed maturation in Arabidopsis thaliana; Luerssen H et al.; Conditionally lethal mutant alleles of the FUSCA3 (FUS3) gene of Arabidopsis thaliana are specifically defective in the gene expression program responsible for seed maturation . FUS3 was isolated by map-based cloning and expression of the FUS3 cDNA resulted in complementation of the Fus3- phenotype . In the predicted FUS3 gene product, a continuous stretch of more than 100 amino acids shows significant sequence similarity to the B3 domains of the polypeptides encoded by ABI3 (Arabidopsis) and VP1 (maize) . FUS3 transcription was detected mainly in siliques and was found to be developmentally regulated during embryogenesis . Transcripts of abnormal sizes were observed in fus3 mutants due to aberrant splicing caused by point mutations at intron termini . Sequence analysis of mutant and wild-type FUS3 alleles, as well as sequencing of fus3 cDNAs, revealed small inframe deletions at two different sites of the coding region . While a deletion between B3 and the C-terminus of the predicted polypeptide was found in conjunction with normal FUS3 function, another deletion located within the conserved B3 domain (as well as truncations therein) were associated with the Fus3- phenotype . It is apparent, therefore, that an intact B3 domain is essential for the regulation of seed maturation by FUS3. Mutat Res, 1998 Oct 21, 409(1), 11 - 6 Genomic organization of a potential human DNA-crosslink repair gene, KIAA0086; Demuth I et al.; In the present study, we describe the genomic structure of the KIAA0086 gene and the 5'-flanking sequence . The analysis is based on the alignment of the KIAA0086 cDNA and a corresponding genomic BAC sequence which was identified in a basic BLAST similarity search using the cDNA sequence as a template . The gene contains nine exons spanning approximately 20 kb . All splice sites conform to the GT-AG rule . Analysis of the upstream untranscribed region identified one GC box but no TATA box, suggesting that the KIAA0086 gene is a housekeeping gene . The promoter region contains putative recognition sites for several transcription factors, e.g., AP1, Sp1 and NFkappaB . The homology of the KIAA0086 gene to the yeast SNM1 gene, which is involved in the cellular response to DNA-interstrand crosslinks, is discussed with respect to a possible role of the KIAA0086 gene in the human disorder, Fanconi anemia. Clin Chem Lab Med, 1998 Aug, 36(8), 659 - 62 Genetic basis of congenital hypothyroidism: abnormalities in the TSHbeta gene, the PIT1 gene, and the NIS gene; Tatsumi K et al.; We have elucidated the molecular pathology of three types of congenital hypothyroidism . Thyrotropin (TSH) is the major regulator of thyroid function . In cases of isolated congenital TSH deficiency, we found that they are caused by a missense mutation in the conserved CAGYC region of the TSHbeta gene . Pit-1/GHF-1 is a pituitary specific POU-domain DNA binding factor, which transactivates the growth hormone (GH), prolactin (PRL), TSHbeta genes, and the PIT1 gene itself . In cases of combined deficiency of GH, PRL, and TSH, we found that they are caused by abnormalities in the PIT1 gene, either recessively or dominantly . Sodium dependent iodide symporter (NIS) actively transports iodide into the thyroid cells to produce thyroid hormones . In cases of iodide transport defect, we elucidated that a missense mutation in the transmembrane region of the NIS gene caused them. Mutat Res, 1998 Sep 11, 408(3), 171 - 82 A new UV-sensitive mutant that suggests a second excision repair pathway in Neurospora crassa; Ishii C et al.; In an attempt to understand the relationship between photorepair and dark repair in Neurospora crassa, a new mutant was isolated, which showed defects in both repair processes . The new mutant, mus-38, is moderately sensitive to UV and shows imperfect photoreactivation following UV irradiation . DNA was purified from this mutant and the other UV-sensitive mutants, and analyzed for the removal of cyclobutane pyrimidine dimers (CPDs) . UV-specific endonuclease-sensitive sites (ESS) completely disappeared with 1 h of photoreactivation in mus-38 DNA, although the survival recovery with photoreactivation was greatly reduced in this mutant . This suggests that the insufficient survival recovery with photoreactivation in mus-38 does not result from a failure of photo-reversal of CPDs . Removal of ESS during liquid holding (dark repair) was slower in mus-38 compared to wild type . To test the possibility that this mutant was involved in excision repair, the double mutant was made between mus-38 and mus-18, which encodes a UV-damage-specific endonuclease . CPD excision in the mus-18 null mutant was severely affected but not completely inhibited . The double mutant showed a complete loss of the excision activity and was super sensitive to UV . These results indicate that mus-38 participates in an excision pathway that is different from the mus-18 pathway . The mus-38 mutant was sensitive not only to UV but also to some chemical mutagens which make adducts on DNA . Thus, mus-38 is possibly involved in an excision-repair pathway that is related to the Saccharomyces cerevisiae RAD3 pathway. Carcinogenesis, 1998 Oct, 19(10), 1847 - 53 Metabolism of benzo{a}pyrene and benzo{a}pyrene-7,8-diol by human cytochrome P450 1B1; Kim JH et al.; Benzo{a}pyrene (B{a}P), a ubiquitous environmental, tobacco and dietary carcinogen, has been implicated in human cancer etiology . The role of human cytochrome P450 1B1 in the metabolism of B{a}P is poorly understood . Using microsomal preparations of human P450 1A1, 1A2 and 1B1 expressed in baculovirus-infected insect cells, as well as human and rat P450 1B1 expressed in yeast, we have determined the metabolism of B{a}P, with and without the addition of exogenous epoxide hydrolase, and B{a}P-7,8-dihydrodiol (7,8-diol), each substrate at a concentration of 10 microM . HPLC analysis detected eight major metabolites of B{a}P and four metabolites of the 7,8-diol . The results of these studies indicate that cytochrome P450 1B1 carries out metabolism of B{a}P along the pathway to the postulated ultimate carcinogen, the diol epoxide 2, at rates much higher than P450 1A2 but less than P450 1A1 . The rates of formation of the 7,8-diol metabolite in incubations with epoxide hydrolase are 0.17 and 0.38 nmol/min/nmol P450 for human P450 1B1 and 1A1, respectively, and undetectable for 1A2 . The rates of total tetrol metabolite formation from the 7,8-diol, which are indicative of diol epoxide formation, are 0.60, 0.43 and 2.58 nmol/min/nmol P450 for 1B1, 1A2 and 1A1 respectively . In agreement with other reports of rat P450 1B1 activity, our data show this rat enzyme to be very active for B{a}P and 7,8-diol, with rates higher than human P450 1B1 . In addition to the established role of P450 1A1 in B{a}P metabolism, P450 1B1 may significantly contribute to B{a}P and 7,8-diol metabolism and carcinogenesis in rodent tumor models and in humans. J Biol Chem, 1998 Nov 13, 273(46), 30524 - 9 Identification of Lys-403 in the PI-SceI homing endonuclease as part of a symmetric catalytic center; Gimble FS et al.; Superposition of the PI-SceI and I-CreI homing endonuclease three-dimensional x-ray structures indicates general similarity between the I-CreI homodimer and the PI-SceI endonuclease domain . Saddle-shaped structures are present in each protein that are proposed to bind DNA . At the putative endonucleolytic active sites, the superposition reveals that two lysine (Lys-301 and Lys-403 in PI-SceI and Lys-98 and Lys-98' in I-CreI) and two aspartic acid residues (Asp-218 and Asp-326 in PI-SceI and Asp-20 and Asp-20' in I-CreI) are related by 2-fold symmetry . The critical role of Lys-301, Asp-218, and Asp-326 in the PI-SceI reaction pathway was reported previously . Here, we demonstrate the significance of the active-site symmetry by showing that alanine substitution at Lys-403 reduces cleavage activity by greater than 50-fold but has little effect on the DNA binding activity of the mutant enzyme . Substitution of Lys-403 with arginine, which maintains the positive charge, has only a modest effect on activity . Interestingly, even though the Lys-301 and Lys-403 residues display pseudosymmetry, PI-SceI mutant proteins with substitutions at these positions have different behaviors . The presence of similar basic and acidic residues in many LAGLIDADG homing endonucleases suggests that these enzymes use a common reaction mechanism to cleave double-stranded DNA. J Biol Chem, 1998 Nov 13, 273(46), 30104 - 9 Reactions of hydrogen peroxide with familial amyotrophic lateral sclerosis mutant human copper-zinc superoxide dismutases studied by pulse radiolysis; Goto JJ et al.; Mutations in copper-zinc superoxide dismutase (CuZn-SOD) have been implicated in the familial form of the motor neuron disease amyotrophic lateral sclerosis (Lou Gehrig's disease) . We have expressed and purified recombinant human wild type (hWT) and G93A (hG93A) CuZn-SOD, and we have used pulse radiolysis to measure their superoxide dismutase activities and their rates of deactivation upon exposure to hydrogen peroxide or heat . Both hG93A and hWT CuZn-SOD were found to have high SOD activities in their copper and zinc containing as-isolated forms as well as when remetallated entirely with copper (CuCu) . Rates of deactivation by hydrogen peroxide of the as-isolated hWT and hG93A enzymes were determined and were found to be similar, suggesting that the FALS mutant enzyme is not inactivated at a higher rate than wild type by generation of and subsequent reaction with hydroxyl radical, .OH, when it is in the CuZn form . However, rates of deactivation by hydrogen peroxide of the CuCu derivatives of both hWT and hG93A were significantly greater than those of the copper and zinc containing as-isolated enzymes . Rates of thermal deactivation were also similar for the mutant and hWT as-isolated CuZn forms but were greater for the CuCu derivatives of both enzymes . Reactions of hydrogen peroxide with the Cu(II)Cu(II) derivative of the WT enzyme demonstrate that the copper ion in the copper site is reduced much more rapidly than the copper in the zinc site, leading to the conclusion that reaction of hydrogen peroxide with Cu(I) in the copper site is the source of deactivation in the CuCu as well as the CuZn enzymes. Mol Pharmacol, 1998 Nov, 54(5), 864 - 73 A linear hexapeptide somatostatin antagonist blocks somatostatin activity in vitro and influences growth hormone release in rats; Baumbach WR et al.; Somatostatin (SRIF) is the main inhibitory peptide regulating growth hormone (GH) secretion . It has been difficult to establish the role of endogenous SRIF release in the absence of pure SRIF antagonists . Although several SRIF antagonists have recently been described, none have been shown to possess in vivo activity in the absence of added SRIF . Here, an SRIF antagonist with no detectable agonist activity has been identified from a synthetic combinatorial hexapeptide library containing 6.4 x 10(7) unique peptides . Each peptide in the library is amino-terminally acetylated and carboxyl-terminally amidated and consists entirely of D-amino acids . A SRIF-responsive yeast growth assay was used as a primary screening tool, and cAMP accumulation, competitive binding, and microphysiometry also were used to confirm and further characterize SRIF antagonist activity . The hexapeptide library was screened in stepwise iterative fashion to identify AC-178,335, a pure SRIF antagonist of the sequence Ac-hfirwf-NH2 . This D-hexapeptide bound SRIF receptor type 2 with an affinity constant (Ki) of 172 +/- 12 nM, blocked SRIF inhibition of adenylate cyclase in vitro (IC50 = 5.1 +/- 1.4 microM), and induced GH release when given alone (50 micrograms intravenously) to anesthetized rats with or without pretreatment with a long-acting SRIF agonist. Science, 1998 Nov 6, 282(5391), 1126 - 32 Chromosome 2 sequence of the human malaria parasite Plasmodium falciparum; Gardner MJ et al.; Chromosome 2 of Plasmodium falciparum was sequenced; this sequence contains 947,103 base pairs and encodes 210 predicted genes . In comparison with the Saccharomyces cerevisiae genome, chromosome 2 has a lower gene density, introns are more frequent, and proteins are markedly enriched in nonglobular domains . A family of surface proteins, rifins, that may play a role in antigenic variation was identified . The complete sequencing of chromosome 2 has shown that sequencing of the A+T-rich P . falciparum genome is technically feasible. J Invest Dermatol, 1998 Nov, 111(5), 722 - 6 Expression and regulation of mRNA for putative fatty acid transport related proteins and fatty acyl CoA synthase in murine epidermis and cultured human keratinocytes; Harris IR et al.; The epidermis has a requirement for fatty acids in order to synthesize cellular membranes and the extracellular lipid lamellar membranes in the stratum corneum . Despite high endogenous production of fatty acids the transport of exogenous essential fatty acids into the epidermis is an absolute requirement . Fatty acid uptake by keratinocytes has been shown to be mediated by a transport system . In this study we determined in murine epidermis and human cultured keratinocytes the expression of three putative fatty acid transport related proteins and fatty acyl CoA synthase, an enzyme that facilitates the uptake of fatty acids by promoting their metabolism . In cultured human keratinocytes, mRNA for fatty acid transport protein (FATP), plasma membrane fatty acid binding protein (FABP-pm), and fatty acyl CoA synthase (FACS) were detectable . Differentiation, induced by high calcium, did not affect FATP mRNA levels, but resulted in an approximately 50% increase in FACS mRNA, while decreasing FABP-pm mRNA by 50% . Fatty acid translocase (FAT) mRNA was not detected in cultured human keratinocytes . In murine epidermis, FATP, FABP-pm, FACS, and FAT mRNA were all present . Barrier disruption by either tape stripping or acetone treatment increased FAT mRNA levels by approximately 2-fold without affecting FATP, FABP-pm, or FACS . Occlusion with an impermeable membrane immediately after barrier disruption completely blocked the increase in FAT mRNA levels, indicating that this increase is related to barrier disruption rather than a nonspecific injury effect . In summary, this study demonstrates that several putative fatty acid transport related proteins as well as fatty acyl CoA synthase are expressed in keratinocytes and epidermis, and that the expression of these proteins may be regulated by differentiation and/ or barrier disruption. FEBS Lett, 1998 Oct 16, 437(1-2), 142 - 4 A sterol biosynthetic pathway in Mycobacterium; Lamb DC et al.; The genome sequence of Mycobacterium tuberculosis (and also M . leprae) revealed a significant number of homologies to Saccharomyces cerevisiae sterol biosynthetic enzymes . We addressed the hypothesis of a potential sterol biosynthetic pathway existing in Mycobacterium using cultures of Mycobacterum smegmatis . Non-saponifiable lipid extracts subjected to analysis by gas chromatography-mass spectrometry (GC-MS) showed cholesterol was present . Sterol synthesis by M . smegmatis was confirmed using 14C-radiolabelled mevalonic acid and incorporation into C4-desmethyl sterol co-migrating with authentic cholesterol on TLC . The sterol biosynthetic pathway has provided a rich source of targets for commercially important bioactive molecules and such agents represent new opportunities for Mycobacteria chemotherapy. Mol Biochem Parasitol, 1998 Sep 15, 95(2), 229 - 40 Characterisation of the Cdc2-related kinase 2 gene from Plasmodium knowlesi and P . berghei; Vinkenoog R et al.; The complete sequence of the cdc2-related kinase 2 (CRK2) gene from Plasmodium knowlesi and from P . berghei was determined . In both species, the CRK2 gene is closely linked to an elongation factor 1 alpha gene . The two CRK2 proteins are highly homologous to the P . falciparum PfPK5 protein . The CRK2 gene of both species is expressed at a low level during the asexual cell-cycle within the host erythrocytes . The P . berghei CRK2 mRNA is also present in gametocytes and in stages during development in the mosquito, suggesting a role of this protein in different parts of the life cycle . A conserved sequence located in the 5' untranslated region immediately upstream of the initiator ATG has the potential to form a stem-loop structure . Although the CRK2 protein possesses most of the domains that are conserved among cdc2-proteins, neither a recombinant P . knowlesi CRK2 protein nor a recombinant P . berghei protein was able to complement a yeast cdc28ts mutant . Furthermore and in contrast to the P . falciparum PfPK5 protein, a recombinant monomeric P . knowlesi CRK2 protein showed no kinase activity. DNA Res, 1998 Aug 31, 5(4), 241 - 6 Cloning and characterization of human Sep1 (hSEP1) gene and cytoplasmic localization of its product; Sato Y et al.; We isolated and sequenced a human cDNA (designated as hSEP1) encoding both a homologue of mouse Dhm2 and budding yeast SEP1 . The gene was shown to be located on the long arm of chromosome 3 (3q25-26.1) . The putative hSEP1 product (hSEP1p) consisted of 1694 amino acid residues with a molecular mass of about 190 kDa . Northern blot analysis showed a major 10-kb mRNA expressed ubiquitously in various organs as well as a minor 5.5-kb mRNA expressed relatively highly in the testis and placenta . hSEP1p is localized in the cytoplasm as examined by cytochemical and Western blot analyses of fractionated cellular extracts . The biological function of hSEP1p was discussed in correlation with its cytoplasmic localization. FEBS Lett, 1998 Oct 9, 436(3), 318 - 22 Interaction between the Arabidopsis thaliana heat shock transcription factor HSF1 and the TATA binding protein TBP; Reindl A et al.; The heat shock factor (HSF1) is the central regulator of the heat stress (hs) response and is required for stimulating the transcription of the hs genes and consequently the expression of heat shock proteins . To promote the polymerase II-dependent transcription of the hs genes, HSF has to communicate with the basal transcription machinery . Here, we report that the Arabidopsis thaliana HSF1 interacts directly with TBP, the general TATA box binding transcription factor, as shown by affinity chromatography and electrophoretic mobility shift analyses in vitro . An in vivo interaction between AtHSF1 and AtTBP1 was suggested by results employing the yeast two-hybrid system. Philos Trans R Soc Lond B Biol Sci, 1998 Sep 29, 353(1374), 1439 - 44 Signalling of abscisic acid to regulate plant growth; Himmelbach A et al.; Abscisic acid (ABA) mediated growth control is a fundamental response of plants to adverse environmental cues . The linkage between ABA perception and growth control is currently being unravelled by using different experimental approaches such as mutant analysis and microinjection experiments . So far, two protein phosphatases, ABI1 and ABI2, cADPR, pH, and Ca2+ have been identified as main components of the ABA signalling pathway . Here, the ABA signal transduction pathway is compared to signalling cascades from yeast and mammalian cells . A model for a bifurcated ABA signal transduction pathway exerting a positive and negative control mechanism is proposed. J Lipid Res, 1998 Nov, 39(11), 2161 - 71 Localization of nervonic acid beta-oxidation in human and rodent peroxisomes: impaired oxidation in Zellweger syndrome and X-linked adrenoleukodystrophy; Sandhir R et al.; Studies with purified subcellular organelles from rat liver indicate that nervonic acid (C24:1) is beta-oxidized preferentially in peroxisomes . Lack of effect by etomoxir, inhibitor of mitochondrial beta-oxidation, on beta-oxidation of lignoceric acid (C24:0), a peroxisomal function, and that of nervonic acid (24:1) compared to the inhibition of palmitic acid (16:0) oxidation, a mitochondrial function, supports the conclusion that nervonic acid is oxidized in peroxisomes . Moreover, the oxidation of nervonic and lignoceric acids was deficient in fibroblasts from patients with defects in peroxisomal beta-oxidation {Zellweger syndrome (ZS) and X-linked adrenoleukodystrophy (X-ALD)} . Similar to lignoceric acid, the activation and beta-oxidation of nervonic acid was deficient in peroxisomes isolated from X-ALD fibroblasts . Transfection of X-ALD fibroblasts with human cDNA encoding for ALDP (X-ALD gene product) restored the oxidation of both nervonic and lignoceric acids, demonstrating that the same molecular defect may be responsible for the abnormality in the oxidation of nervonic as well as lignoceric acid . Moreover, immunoprecipitation of activities for acyl-CoA ligase for both lignoceric acid and nervonic acid indicate that saturated and monoenoic very long chain (VLC) fatty acids may be activated by the same enzyme . These results clearly demonstrate that similar to saturated VLC fatty acids (e.g., lignoceric acid), VLC monounsaturated fatty acids (e.g., nervonic acid) are oxidized preferentially in peroxisomes and that this activity is impaired in X-ALD . In view of the fact that the oxidation of unsaturated VLC fatty acids is defective in X-ALD patients, the efficacy of dietary monoene therapy, "Lorenzo's oil," in X-ALD needs to be evaluated. Curr Biol, 1998 Oct 22, 8(21), R771 - 3 Replication conrol: choreographing replication origins; Diffley JF; Budding yeast replication origins are activated during S phase according to a predetermined temporal programme . Two recent studies indicate that this programme is executed, at least in part, by the S-phase-promoting cyclins that act to assemble a pre-initiation complex which includes the Cdc45 protein. Arch Biochem Biophys, 1998 Nov 1, 359(1), 128 - 32 The alpha 1-helix in the extra-membranous domain contributes to the stability of the iron-sulfur protein of the cytochrome bc1 complex; Obungu VH et al.; Previously, we reported that several charged amino acids located in the alpha1-beta4 loop of the iron-sulfur protein are required to maintain the stability of the protein and its assembly into the cytochrome bc1 complex . The current study extends this analysis to several amino acids localized in the single alpha-helix present in the extra-membranous domain of the protein . Three charged and two uncharged residues in the alpha1-helix were mutated and used to transform yeast cells (JPJ1) lacking the iron-sulfur protein gene . Mutants V132L and H124L grew at half the rate of the wild type in medium containing glycerol/ethanol, while E125Q grew more slowly than the wild type . The rates of growth of T122A and E128Q were identical to that of the wild-type cells . Activity of the cytochrome bc1 complex was decreased 70, 40, and 80% in mutants H124L, E125Q, and V132L, respectively, while the activity of T122A and E128Q was decreased 30 and 20% relative to the wild type . Western blotting experiments revealed that the content of the iron-sulfur protein was decreased in mutants H124L and V132L; however, no decrease in the content of the iron-sulfur protein was observed in the other mutants . These results suggest that several amino acids located in the alpha1-helix of the protein are important in maintaining the stability and proper assembly of the iron-sulfur protein in the bc1 complex . Dev Genes Evol, 1998 Nov, 208(9), 531 - 6 The Drosophila Sin3 gene encodes a widely distributed transcription factor essential for embryonic viability; Pennetta G et al.; Expression of many mammalian genes is activated by the binding of heterodimers of the Myc and Max proteins to specific DNA sequences called the E-boxes . Transcription of the same genes is repressed upon binding to the same sequences of complexes composed of Max, Mad/Mxi1, the co-repressors Sin3 and N-CoR, and the histone deacetylase Rpd3 . Max-Mad/Mxi1 heterodimers, which bind to E-boxes in absence of co-repressors, do not inhibit gene expression simply by competition with Myc-Max heterodimers, but require Sin3 and Rpd3 for efficient repression of transcription . We have cloned a Drosophila homolog of Sin3 (dSin3) and found it to be ubiquitously expressed during embryonic development . Yeast, mouse and Drosophila proteins share six blocks of strong homologies, including four potential paired amphipathic helix domains . In addition, the domain of binding to the histone deacetylase Rpd3 is strongly conserved . Null mutations cause recessive embryonic lethality. Dev Genes Evol, 1998 Oct, 208(8), 440 - 6 Specific interactions between vestigial and scalloped are required to promote wing tissue proliferation in Drosophila melanogaster; Paumard-Rigal S et al.; The two genes vestigial (vg) and scalloped (sd) are required for wing development in Drosophila melanogaster . They present similar patterns of expression in second and third instar wing discs and similar wing mutant phenotypes . vg encodes a nuclear protein without any recognized nucleic acid-binding motif . Sd is a transcription factor homologous to the human TEF-1 factor whose promoter activity depends on cell-specific cofactors . We postulate that Vg could be a cofactor of Sd in the wing morphogenetic process and that, together, they could constitute a functional transcription complex . We investigated genetic interactions between the two genes . We show here that vg and sd co-operate in vivo in a manner dependent on the structure of the Vg protein . We ectopically expressed vg in the patch (ptc) domains . We show evidence that wing-like outgrowths induced by ectopic expression of vg are severely reduced in vg or sd mutant backgrounds . Accordingly, we demonstrate that ptc-GAL4-driven expression of vg induces both expressions of the endogenous vg and sd genes and that the two Vg and Sd proteins have to be produced together to promote wing proliferation . Furthermore, we show an interaction between the two proteins by double hybrid experiments in yeast . Our results therefore support the hypothesis that Sd and Vg directly interact in vivo to form a complex regulating the proliferation of wing tissue. Curr Genet, 1998 Oct, 34(4), 269 - 79 Gene-conversion tract directionality is influenced by the chromosome environment; Cho JW et al.; Spontaneous and double-strand break (DSB)-induced gene conversion in Saccharomyces cerevisiae was assayed using non-tandem chromosomal direct repeat crosses and plasmid x chromosome crosses . Each cross involved identical ura3 alleles marked with phenotypically silent restriction fragment length polymorphic (RFLP) mutations at approximately 100-bp intervals . DSBs introduced in vivo at HO sites in one allele stimulated recombination to Ura+ by more than two orders of magnitude . Spontaneous gene-conversion products were isolated from a related strain lacking a functional HO nuclease gene . The multiple markers did not appear to influence the frequency of direct repeat deletions for spontaneous or DSB-induced events . DSB-induced conversion reflected efficient mismatch repair of heteroduplex DNA . Conversion frequencies of equidistant markers on opposites sides of the DSB were similar in the direct repeat cross . In contrast, markers 5' of the DSB (promoter-proximal) converted more often than 3' markers in plasmid x chromosome crosses, a possible consequence of crossing-over associated with long conversion tracts . With direct repeats, bidirectional tracts (extending 5' and 3' of the DSB) occurred twice as often as in a plasmid x chromosome cross in which DSBs were introduced into the plasmid-borne allele . A key difference between the direct-repeat and plasmidxchromosome crosses is that the ends of a broken plasmid are linked, whereas the ends of a broken chromosome are unlinked . We tested whether linkage of ends influenced tract directionality using a second plasmid x chromosome cross in which DSBs were introduced into the chromosomal allele and found few bidirectional tracts . Thus, chromosome environment, but not linkage of ends, influences tract directionality . The similar tract spectra of the two plasmid x chromosome crosses suggest that similar mechanisms are involved whether recombination is initiated by DSBs in plasmid or chromosomal alleles. Genetics, 1998 Nov, 150(3), 1037 - 47 Chromosome break-induced DNA replication leads to nonreciprocal translocations and telomere capture; Bosco G et al.; In yeast, broken chromosomes can be repaired by recombination, resulting in nonreciprocal translocations . In haploid cells suffering an HO endonuclease-induced, double-strand break (DSB), nearly 2% of the broken chromosome ends recombined with a sequence near the opposite chromosome end, which shares only 72 bp of homology with the cut sequence . This produced a repaired chromosome with the same 20-kb sequence at each end . Diploid strains were constructed in which the broken chromosome shared homology with the unbroken chromosome only on the centromere-proximal side of the DSB . More than half of these cells repaired the DSB by copying sequences distal to the break from the unbroken template chromosome . All these events were RAD52 dependent . Pedigree analysis established that DSBs occurring in G1 were repaired by a replicative mechanism, producing two identical daughter cells . We discuss the implications of these data in understanding telomerase-independent replication of telomeres, gene amplification, and the evolution of chromosomal ends. EMBO J, 1998 Nov 2, 17(21), 6200 - 9 Genetic and morphological analyses reveal a critical interaction between the C-termini of two SNARE proteins and a parallel four helical arrangement for the exocytic SNARE complex; Katz L et al.; In a screen for suppressors of a temperature-sensitive mutation in the yeast SNAP-25 homolog, Sec9, we have identified a gain-of-function mutation in the yeast synaptobrevin homolog, Snc2 . The genetic properties of this suppression point to a specific interaction between the C-termini of Sec9 and Snc2 within the SNARE complex . Biochemical analysis of interactions between the wild-type and mutant proteins confirms this prediction, demonstrating specific effects of these mutations on interactions between the SNAREs . The location of the mutations suggests that the C-terminal H2 helical domain of Sec9 is likely to be aligned in parallel with Snc2 in the SNARE complex . To test this prediction, we examined the structure of the yeast exocytic SNARE complex by deep-etch electron microscopy . Like the neuronal SNARE complex, it is a rod approximately 14 nm long . Using epitope tags, antibodies and maltose-binding protein markers, we find that the helical domains of Sso, Snc and both halves of Sec9 are all aligned in parallel within the SNARE complex, suggesting that the yeast exocytic SNARE complex consists of a parallel four helix bundle . Finally, we find a similar arrangement for SNAP-25 in the neuronal SNARE complex . This provides strong evidence that the exocytic SNARE complex is a highly conserved structure composed of four parallel helical domains whose C-termini must converge in order to bring about membrane fusion. Thromb Haemost, 1998 Oct, 80(4), 585 - 7 Activity of secreted Kunitz domain 1 variants of tissue factor pathway inhibitor; Johnson K et al.; Tissue factor pathway inhibitors (TFPI and TFPI-2) are Kunitz domain-type serine protease inhibitors which inhibit factor VIIa/tissue factor (VIIa/TF) complexes in a factor Xa-dependent manner . The VIIa/TF and Xa inhibitory activity has been localized to the first two Kunitz domains, respectively . Unlike TFPI, TFPI-2 has been reported to exhibit significant Xa-independent VIIa/TF inhibitory activity, perhaps due to an arginine at the P1 residue in the first Kunitz domain of TFPI-2 as opposed to a lysine at the comparable residue in TFPI . Two domain TFPI variants, differing in the first Kunitz domain but containing the second Kunitz domain of TFPI, were constructed and secreted by Saccharomyces cerevisiae in order to test the possibility that a TFPI first Kunitz domain with a P1 lysine to arginine change or a hybrid containing the TFPI-2 first Kunitz domain may represent more potent VIIa/TF inhibitors . When yeast supernatants were analyzed for specific activity in the Xa-dependent inhibition of VIIa/TF, neither variant was as active as the truncated TFPI. Int J Radiat Biol, 1998 Oct, 74(4), 481 - 9 Expression, localization and functional interactions of Ku70 subunit of DNA-PK in peripheral lymphocytes and Nalm-19 cells after irradiation; Kumaravel TS et al.; PURPOSE: To investigate the changes in expression, localization and functional interaction of Ku70 subunit of DNA-PK after irradiation . MATERIALS AND METHODS: Protein expression by Western blotting, cellular localization by immunofluorescence and functional interactions by immunoprecipitation were investigated after gamma-irradiation in two different cell systems: (1) quiescent human peripheral lymphocytes (0, 2 and 4 Gy) stimulated to proliferate with PHA; and (2) in a proliferating Nalm-19 cell line (0 and 2 Gy) . Cell cycle analysis was performed by FACS . Immunofluorescence on extended chromatin fibres was also performed to show close association of Ku70 with the chromatin fibre . RESULTS: Gamma-irradiation induced dose-dependent expression of Ku70 in both cell systems . Confocal microscopy on immunostained cells and cell cycle analysis showed that Ku70 protein translocates to the cytoplasm after the G1 phase . There was a delay in the cytoplasmic shift of Ku70 in the irradiated group, corresponding with the G1 delay . The cytoplasmic localization was supported by ultracentrifugation studies . Immunofluorescence with Ku70 antibody on an extended chromatin fibre showed that Ku70 is closely associated with the chromatin fibre, and irradiation produced many spots of intense fluorescence on it . Ku70 coprecipitated with c-ABL and p21 after irradiation . The c-ABL was coprecipitated throughout the time course of observation, whereas p21 was transiently (0.2 h) associated with Ku70 and only in the lower dose groups . CONCLUSIONS: These studies have demonstrated new biological characteristics of Ku70 in the cellular response to irradiation. J Mol Evol, 1998 Nov, 47(5), 531 - 43 Identification of the first fungal annexin: analysis of annexin gene duplications and implications for eukaryotic evolution; Braun EL et al.; Annexin homologues have been found in animals, plants, and distinct protist lineages . We report the identification of the first fungal annexin, encoded by the anx14 gene of the filamentous ascomycete Neurospora crassa . Annexins have a complex evolutionary history and exhibit a large number of gene duplications and gene losses in various taxa, including the complete loss of annexin sequences from another ascomycete, the budding yeast Saccharomyces cerevisiae . Surprisingly, the N . crassa annexin homologue is most closely related to the annexin homologue of the slime mold Dictyostelium discoideum, suggesting a phylogenetic link between cellular slime molds and true fungi . Both of these annexin homologues are closely related to the family of annexin homologues present in animals, an observation consistent with the existence of the animal-fungal clade . These data further suggest that the gene duplications that generated the family of annexin sequences present in animals, fungi, and slime molds began prior to the divergence of these taxa. Appl Environ Microbiol, 1998 Nov, 64(11), 4489 - 94 Cloning and high-level expression of alpha-galactosidase cDNA from Penicillium purpurogenum; Shibuya H et al.; The cDNA coding for Penicillium purpurogenum alpha-galactosidase (alphaGal) was cloned and sequenced . The deduced amino acid sequence of the alpha-Gal cDNA showed that the mature enzyme consisted of 419 amino acid residues with a molecular mass of 46,334 Da . The derived amino acid sequence of the enzyme showed similarity to eukaryotic alphaGals from plants, animals, yeasts, and filamentous fungi . The highest similarity observed (57% identity) was to Trichoderma reesei AGLI . The cDNA was expressed in Saccharomyces cerevisiae under the control of the yeast GAL10 promoter . Almost all of the enzyme produced was secreted into the culture medium, and the expression level reached was approximately 0.2 g/liter . The recombinant enzyme purified to homogeneity was highly glycosylated, showed slightly higher specific activity, and exhibited properties almost identical to those of the native enzyme from P . purpurogenum in terms of the N-terminal amino acid sequence, thermoactivity, pH profile, and mode of action on galacto-oligosaccharides. Protein Eng, 1998 Sep, 11(9), 783 - 8 Stabilization of Aspergillus awamori glucoamylase by proline substitution and combining stabilizing mutations; Allen MJ et al.; To stabilize Aspergillus awamori glucoamylase (GA), three proline substitution mutations were constructed . When expressed in Saccharomyces cerevisiae, Ser30-->Pro (S30P) stabilized the enzyme without decreased activity, whereas Asp345-->Pro (D345P) did not significantly alter and Glu408-->Pro (E408P) greatly decreased enzyme thermostability . The S30P mutation was combined with two previously identified stabilizing mutations: Gly137-->Ala, and Asn20-->Cys/Ala27-->Cys (which creates a disulfide bond between positions 20 and 27) . The combined mutants demonstrated cumulative stabilization as shown by decreased irreversible thermoinactivation rates between 65 and 80 degrees C . Additionally, two of the combined mutants outperformed wild-type GA in high-temperature (65 degrees C) saccharifications of DE 10 maltodextrin and were more active than the wild-type enzyme when assayed using maltose as substrate. Eur J Pharm Biopharm, 1998 Sep, 46(2), 233 - 6 Preliminary investigation of some polysaccharides as a carrier for cell entrapment; Sriamornsak P; Entrapment of cells within spheres of polysaccharide gel has become the most widely used technique for immobilizing living cells . Polysaccharide pectin, formed gel with calcium ions, was investigated as a precursor of spherical calcium polysaccharide gel beads . The type of pectin sample was found to be important in the formation of the beads . Partially deesterified pectin with a lower degree of esterification provided spherical beads and was chosen for immobilization of the yeast cells, Sacchararomyces cerevisiae, and compared to those with alginate . The effect of storage condition of the beads on the viability of the entrapped cells was also studied . After storage at 4 degreesC or -40 degreesC for 1 month, even lyophilization before storage, the beads with entrapped cells were sufficiently stable when compared to suspension of non-entrapped yeast cells. Biochem J, 1998 Nov 1, 335 ( Pt 3), 567 - 72 Specificity of interaction between adaptor-complex medium chains and the tyrosine-based sorting motifs of TGN38 and lgp120; Stephens DJ et al.; Multiple sorting steps within eukaryotic cells are mediated by tyrosine-based sorting motifs . Motifs conforming to the consensus -YXXO- (where O indicates a bulky hydrophobic residue) have been shown to specify high-efficiency internalization from the plasma membrane, targeting from the plasma membrane to the trans-Golgi network and targeting to lysosomal compartments as well as being involved in basolateral sorting in polarized cells . These motifs are recognized by the medium-chain subunits of heterotetrameric adaptor complexes . Whereas these motifs have been shown to be sufficient to mediate interaction with the mu-chains, we and others have shown that their context is important in determining the affinity of interaction . In this study we have investigated the interaction between the tyrosine motifs of the type-1 integral membrane proteins TGN38 and lgp120 with medium-chain subunits using the yeast two-hybrid system . Whereas the wild-type version of the cytosolic domain of TGN38 interacts with highest affinity with mu2, we show that the cytosolic domain of lgp120 interacts almost exclusively with mu3A . The specificity of binding of tyrosine-based sorting motifs to mu-chains is shown to be highly sensitive to the context in which the motif lies . For example, the -YQTI- motif of lgp120 is effectively non-functional with regard to mu-chain binding when placed in the context of the TGN38 cytosolic domain . Deletion of four amino acids (NLKL) at the extreme C-terminus of TGN38, leaving the YXXO motif as the C-terminus, greatly enhances the affinity of interaction with mu2 . Furthermore, addition of these same residues to the extreme C-terminus of lgp120 effectively abolishes the interaction of the cytosolic domain of lgp120 with mu-chains . We also show that the newly identified mu-adaptin-related protein 2 (mu4) only interacts weakly with tyrosine-based sorting motifs. Endocrinology, 1998 Nov, 139(11), 4455 - 65 Transcriptional activation of the ovine follicle-stimulating hormone beta-subunit gene by gonadotropin-releasing hormone: involvement of two activating protein-1-binding sites and protein kinase C; Strahl BD et al.; FSH is an alpha/beta heterodimeric glycoprotein, the formation of which is regulated primarily by expression of its beta-subunit . Recent studies on transcriptional regulation of the ovine FSH beta-subunit gene (oFSHbeta) have defined two functional activating protein-1 (AP-1) enhancers in the proximal promoter (located at -120 and -83 bp) that are probably physiologically important for FSHbeta expression . As GnRH is a major regulator of FSHbeta expression and is also known to stimulate the synthesis of Jun and Fos family members (AP-1), we investigated the possibility that oFSHbeta transcription may be regulated by GnRH through AP-1 . Here we report the use of an in vitro cell system involving transient transfection of GnRH receptors (GnRHR) into HeLa cells to define regulatory elements involved in GnRH-mediated induction of oFSHbeta . This system was used to show that expression of luciferase constructs containing either the -4741/+759 region of the oFSHbeta gene (-4741oFSHbeta-Luc) or the -846/+44 region of the human alpha gene (alpha-Luc; a positive control) was stimulated 3.1 +/- 0.3- and 7.7 +/- 1.9-fold, respectively, by 100 nM GnRH . Another luciferase expression plasmid containing the Rous sarcoma virus promoter (a negative control) showed no response to GnRH . Similar results with these constructs were obtained in COS-7 cells . Studies with progressive 5'-deletion constructs and site-specific mutations demonstrated that this stimulation was dependent on each AP-1 site in the proximal promoter of oFSHbeta . Gel shift assays demonstrated the ability of GnRHR in HeLa cells to increase AP-1 binding activity . Responses in the HeLa cell system were dependent on GnRH (ED50 = 0.5 nM) and GnRHR, which was identified by photoaffinity labeling . In addition, GnRHR-expressing HeLa cells exhibited a normal GnRH-dependent mobilization of intracellular calcium . Finally, as protein kinase C (PKC) is a known target of GnRH action in gonadotropes, the role of PKC in transcriptional regulation of oFSHbeta and alpha-subunit genes by GnRH in HeLa cells was investigated . Although 12-O-tetradecanoyl 13-acetate induction of alpha-Luc and -215oFSHbeta-Luc could be completely blocked in a dose-dependent manner by the specific PKC inhibitor bisindolylmaleimide I, only 57-65% of the GnRH-mediated stimulation of these promoters was blocked, demonstrating the involvement of PKC as well as other signaling systems in GnRH induction . These data define a molecular action of GnRH on oFSHbeta gene transcription that involves two proximal AP-1 enhancer elements and PKC activation . Furthermore, these studies establish the usefulness of HeLa and COS-7 cells to investigate specific aspects of GnRH action on gonadotropin subunit gene expression, as similar signaling pathways and transcription factors that are activated by GnRH in gonadotropes (such as PKC, mitogen-activated protein kinase, Ca2+, and AP-1) exist in these cells. J Immunol, 1998 Nov 1, 161(9), 4572 - 82 Adenovirus-mediated expression of a dominant negative mutant of p65/RelA inhibits proinflammatory gene expression in endothelial cells without sensitizing to apoptosis; Soares MP et al.; We hypothesized that blocking the induction of proinflammatory genes associated with endothelial cell (EC) activation, by inhibiting the transcription factor nuclear factor kappaB (NF-kappaB), would prolong survival of vascularized xenografts . Our previous studies have shown that inhibition of NF-kappaB by adenovirus-mediated overexpression of I kappaB alpha suppresses the induction of proinflammatory genes in EC . However, I kappaB alpha sensitizes EC to TNF-alpha-mediated apoptosis, presumably by suppressing the induction of the NF-kappaB-dependent anti-apoptotic genes A20, A1, manganese superoxide dismutase (MnSOD), and cellular inhibitor of apoptosis 2 . We report here that adenovirus mediated expression of a dominant negative C-terminal truncation mutant of p65/RelA (p65RHD) inhibits the induction of proinflammatory genes, such as E-selectin, ICAM-1, VCAM-1, IL-8, and inducible nitric oxide synthase, in EC as efficiently as does I kappaB alpha . However, contrary to I kappaB alpha, p65RHD does not sensitize EC to TNF-alpha-mediated apoptosis although both inhibitors suppressed the induction of the anti-apoptotic genes A20, A1, and MnSOD equally well . We present evidence that this difference in sensitization of EC to apoptos