Proc Natl Acad Sci U S A, 1996 Jul 23, 93(15), 7594 - 9 DP-2, a heterodimeric partner of E2F: identification and characterization of DP-2 proteins expressed in vivo; Rogers KT et al.; E2F is a heterodimeric transcription factor that regulates the expression of genes at the G1/S boundary and is composed of two related but distinct families of proteins, E2F and DP . E2F/DP heterodimers form complexes with the retinoblastoma (Rb) protein, the Rb-related proteins p107 and p130, and cyclins/cdks in a cell cycle-dependent fashion in vivo . E2F is encoded by at least five closely related genes, E2F-1 through -5 . Here we report studies of DP-2, the second member of the DP family of genes . Our results indicate that (i) DP-2 encodes at least five distinct mRNAs, (ii) a site of alternative splicing occurs within the 5' untranslated region of DP-2 mRNA, (iii) at least three DP-2-related proteins (of 55, 48, and 43 kDa) are expressed in vivo, (iv) each of these proteins is phosphorylated, and (v) one DP-2 protein (43 kDa) carries a truncated amino terminus . Our data also strongly suggest that the 55-kDa DP-2-related protein is a novel DP-2 isoform that results from alternative splicing . Thus, we conclude that DP-2 encodes a set of structurally, and perhaps functionally, distinct proteins in vivo. Proc Natl Acad Sci U S A, 1996 Jul 23, 93(15), 7567 - 71 SMRT isoforms mediate repression and anti-repression of nuclear receptor heterodimers; Chen JD et al.; Transcriptional repression represents an important component in the regulation of cell differentiation and oncogenesis mediated by nuclear hormone receptors . Hormones act to relieve repression, thus allowing receptors to function as transcriptional activators . The transcriptional corepressor SMRT was identified as a silencing mediator for retinoid and thyroid hormone receptors . SMRT is highly related to another corepressor, N-CoR, suggesting the existence of a new family of receptor-interacting proteins . We demonstrate that SMRT is a ubiquitous nuclear protein that interacts with unliganded receptor heterodimers in mammalian cells . Furthermore, expression of the receptor-interacting domain of SMRT acts as an antirepressor, suggesting the potential importance of splicing variants as modulators of thyroid hormone and retinoic acid signaling. FEBS Lett, 1996 Jul 22, 390(2), 226 - 8 Probing the higher order structure of RNA with peroxonitrous acid; Gotte M et al.; Potassium peroxonitrite (ONOOK) and {Fe(EDTA)}2- were used to analyze the influence of chemically entirely different hydroxyl radical sources on tRNA cleavage profiles . {Fe(EDTA)}2- gives rise to hydroxyl radicals via a Fenton-like reaction during the oxidation of chelated Fe2+, while ONOOK generates hydroxyl radicals via its conjugate acid (ONOOH) when adding a stable alkaline solution of ONOOK in samples buffered at neutral pH . {Fe(EDTA)}2- is known to induce oxidative strand scission at sugar moieties thought to be solvent accessible, while those residues located in the 'inside' of structured RNAs are protected . Although ONOOH is neutral and significantly smaller than the metal complex, both reagents generate the same protection pattern on tRNAs, suggesting that access of the commonly formed hydroxyl radical, rather than access of its source, is the determining factor when probing the higher order structure of RNA . Strong difference in reactivity is only seen at the modified 2-thiouridine S34 of tRNA(Lys3) which shows hyperreactivity towards ONOOK treatment . This particular reaction may require interaction between the peroxonitrite anion and the thiocarbonyl group of the base, since hyperreactivity is not observed when probing the dethiolated tRNA(Lys3). J Biol Chem, 1996 Jul 19, 271(29), 17183 - 9 Tmp21 and p24A, two type I proteins enriched in pancreatic microsomal membranes, are members of a protein family involved in vesicular trafficking; Blum R et al.; We report here on the isolation, cloning, and expression of two Mr 21,000 proteins from rat pancreatic acinar cells, the rat-Tmp21 (transmembrane protein, Mr 21,000) and the rat-p24A . Both proteins are transmembrane proteins with type I topology and share weak but significant homology to one another (23% identity) . We further show the cloning and characterization of the human homologs, hum-Tmp21, which is expressed in two variants (Tmp21-I and Tmp21-II), and hum-p24A . Tmp21 proteins and p24A have highly conserved COOH-terminal tails, which contain motifs related to the endoplasmic reticulum retention and retrieval consensus sequence KKXX . The rat-p24 sequence is identical to the hamster CHOp24, a recently characterized component of coatomer-coated transport vesicles, which defines a family of proteins (called the p24 family) proposed to be involved in vesicular transport processes (Stamnes, M . A., Craighead, M . W., Hoe, M . H., Lampen, N., Geromanos, S., Tempst, P., and Rothman, J . E.(1995) Proc . Natl . Acad . Sci . U . S . A . 92, 8011-8015) . Sequence alignment and structural features identify the Tmp21 protein as a new member of this p24 family . Northern analysis of various tissues indicates that the Tmp21 proteins and the p24A protein are ubiquitously expressed . The integral membrane components Tmp21 and p24A are localized in microsomal membranes, zymogen granule membranes, and the plasma membrane and are absent from the cytosol . Both p24A and Tmp21 show weak homology to the yeast protein Emp24p, which recently has been shown to be involved in secretory protein transport from the endoplasmic reticulum to the Golgi apparatus . This leads us to conclude that the receptor-like Tmp21 and p24A are involved in vesicular targeting and protein transport. J Biol Chem, 1996 Jul 19, 271(29), 17152 - 6 An FK506-sensitive transporter selectively decreases intracellular levels and potency of steroid hormones; Kralli A et al.; Steroid hormones bind and activate intracellular receptors that are ligand-regulated transcription factors . Mammalian steroid receptors can confer hormone-dependent transcriptional enhancement when expressed in yeast, thereby enabling the genetic identification of nonreceptor proteins that function in the hormone signal transduction pathway . Pdr5p (Lem1/Sts1/Ydr1p), a yeast ATP-binding cassette transporter, selectively decreases the intracellular levels of particular steroid hormones, indicating that active processes can affect the passage of steroids across biological membranes . In yeast, the immunosuppressive drug FK506 inhibited Pdr5p, thereby potentiating activation of the glucocorticoid receptor by dexamethasone, a ligand that is exported by Pdr5p . In mammalian L929 cells but not in HeLa cells, FK506 potentiated dexamethasone responsiveness and increased dexamethasone accumulation, without altering the hormone-binding properties of the glucocorticoid receptor . We suggest that an FK506-sensitive transporter in L929 cells selectively decreases intracellular hormone levels and, consequently, the potency of particular steroids . Thus, steroid transporters may modulate, in a cell-specific manner, an initial step in signaling, the availability of hormone to the receptor. Oncogene, 1996 Jul 18, 13(2), 353 - 62 RalGDS-like factor (Rlf) is a novel Ras and Rap 1A-associating protein; Wolthuis RM et al.; The small GTPase Rap 1A is a close relative of Ras that, when overexpressed, is able to revert oncogenic transformation induced by active Ras . We screened a mouse embryonic cDNA library using the yeast two-hybrid system and isolated the cDNA of a novel Rap 1A-interacting protein . The open reading frame encodes for an 84 kDa protein with a Cdc25-homology domain which shares approximately 30% identity with Ral guanine nucleotide dissociation stimulator (RalGDS) and RalGDS-like (Rg1) . The C-terminal region reveals a striking conservation of sequences with the Ras-binding domain of RalGDS . We designated this protein Rlf, for RalGDS-like factor . In the yeast system, Rlf interacts with Rap 1A, H-Ras and R-Ras, but not with Rac and Rho . In addition, we found that Rlf interacts with Rap 1Aval12 but not with Rap 1AAsn17 . In vitro binding studies revealed that a C-terminally located 91 amino acid region of Rlf is sufficient for direct association with the GTP-bound form of Ras and Rap 1A . The observed dissociation constants are 0.6 microM and 0.4 microM, respectively . No significant association with Ras-GDP or Rap 1A-GDP could be detected . These binding characteristics indicate that Rlf is a putative effector for Ras and Rap 1A. Biochim Biophys Acta, 1996 Jul 17, 1307(3), 254 - 8 RNA editing in the cox3 mRNA of Magnolia is more extensive than in other dicot or monocot plants; Perrotta G et al.; The Magnoliaceae are discussed as one of the key species at the root of the flowering plants . To obtain molecular information for one of these phylogenetically interesting plant species, we determined genomic and cDNA sequences of the mitochondrial cox3 gene in Magnolia grandiflora . Twenty-two RNA editing events are identified to alter cytidines in the mRNA to uridines, all but one of which change the encoded amino acid identity . RNA editing in the cox3 coding region is thus more frequent in Magnolia than in other dicot or monocot plants investigated and almost as predominant as in some gymnosperms . The cox3 RNA editing frequency in Magnolia thus occupies an intermediate position between angiosperms and gymnosperms consistent with the phylogenetic position of the Magnoliales. Biochem Biophys Res Commun, 1996 Jul 16, 224(2), 457 - 61 Specific targeting of ISP6 to mitochondria is mediated by sequences other than its amino terminus; Cao W et al.; Most proteins synthesized in cytoplasm target to mitochondria through sequences at their amino termini . However, a previous study suggests that the native carboxyl terminus of ISP6 might be critical of its specific delivery . Here we investigated the sequence directing ISP6 to yeast mitochondrial outer membrane . Unlike mitochondrial presequences, a region at the amino terminus of ISP6 is dispensable for importing the rest of the protein . The carboxyl-terminal end and the nearby transmembrane region of ISP6 are essential to direct the protein exclusively to its correct membrane destination . ISP6 thus may be directed to mitochondria by an unusual sequence. J Neurosci Res, 1996 Jul 15, 45(2), 89 - 95 The role of Sec1p-related proteins in vesicle trafficking in the nerve terminal; Pevsner J; Vesicle trafficking at multiple stages of the secretory pathway depends on a family of soluble proteins related to yeast Sec1p . In yeast, this family consists of four members: the late-acting Sec1p that is required for vesicular transport between the Golgi apparatus and the cell surface; Vps33p and Vps45p which are required for trafficking between the Golgi complex and the lysosome-like vacuole; and Sly1p that is essential for trafficking between the endoplasmic reticulum and the Golgi apparatus . In mammalian systems, homologues of these proteins have been identified . In particular, a neural-specific Sec1p homologue (n-sec1/Munc-18) binds the plasma membrane protein syntaxin and may regulate synaptic vesicle docking . The Sec1p family of proteins is essential for vesicle trafficking in both regulated and constitutive trafficking pathways, and n-sec1 is critical in the regulated release of neurotransmitter from the nerve terminal. Biochem J, 1996 Jul 15, 317 ( Pt 2), 425 - 31 Interaction of dibutyltin-3-hydroxyflavone bromide with the 16 kDa proteolipid indicates the disposition of proton translocation sites of the vacuolar ATPase; Hughes G et al.; The organotin complex dibutyltin-3-hydroxyflavone bromide {Bu2Sn(of)Br} has been shown to bind to the 16 kDa proteolipid of Nephrops norvegicus, either in the form of the native protein or after heterologous expression in Saccharomyces and assembly into a hybrid vacuolar H(+)-ATPase . Titration of Bu2Sn(of)Br against the 16 kDa proteolipid results in a marked fluorescence enhancement, consistent with binding to a single affinity site on the protein . Vacuolar ATPase-dependent ATP hydrolysis was also inhibited by Bu2Sn(of)Br, with the inhibition constant correlating well with dissociation constants determined for binding of Bu2Sn(of)Br complex to the proteolipid . The fluorescence enhancement produced by interaction of probe with proteolipid can be back-titrated by dicyclohexylcarbodiimide (DCCD), which covalently modifies Glu140 on helix-4 of the polypeptide . Expression of a mutant proteolipid in which Glu140 was changed to a glycine resulted in assembly of a vacuolar ATPase which was inactive in proton pumping and which had reduced ATPase activity . Co-expression studies with this mutant and wild-type proteolipids suggest that proton pumping can only occur in a vacuolar ATPase containing exclusively wild-type proteolipid . The fluorescent enhancement of affinity of Bu2Sn(of)Br for the mutant proteolipid was not significantly altered, with the organotin complex having no effect on residual ATPase activity . Interaction of the probe with mutant proteolipid was unaffected by DCCD . These data suggest an overlap in the binding sites of organotin and DCCD, and have implications for the organization and structure of proton-translocating pathways in the facuolar H(+)-ATPase. Eur J Biochem, 1996 Jul 15, 239(2), 460 - 8 Isolation and expression of a cDNA clone encoding human kynureninase; Alberati-Giani D et al.; Kynureninase (L-kynurenine hydrolase), a pyridoxal-5'-phosphate-(pyridoxal-P)-dependent enzyme, catalyses the cleavage of L-kynurenine and L-3-hydroxykynurenine into anthranilic and 3-hydroxyanthranilic acids, respectively . In this report, we describe the isolation of a cDNA clone encoding human kynureninase . Degenerate oligonucleotides designed from the amino acid sequences of peptides from rat liver kynureninase, were used as primers for reverse-transcription PCR of rat kidney RNA . The resulting rat cDNA product was then used to screen a human hepatoma cell line (Hep G2) cDNA library . Analysis of a positive cDNA clone showed the presence of an insert of 1651 nucleotides containing an open reading frame coding for a protein of 456 amino acids (theoretical molecular mass = 52357 Da) . The predicted amino acid sequence of human kynureninase displayed high similarity to that reported for the rat enzyme and to a Saccharomyces cerevisiae gene product putatively ascribed to kynureninase . Profile analysis of kynureninase primary structure indicated the presence of a pyridoxal-P-binding site consensus sequence assigned to class-V aminotransferases, with Lys276 being the residue binding the cofactor . RNA blot analysis of human tissues, including brain, showed the presence of an approximately 2.0-kb mRNA species in all tissues tested . A second mRNA species (approximately 2.6 kb) was also detected in some tissues . After transfection of HEK-293 cells with the cDNA coding for kynureninase, the K(m) values of L-kynurenine and DL-3-hydroxykynurenine for the recombinant enzyme were 671 +/- 37 microM and 13.2 +/- 2.0 microM, respectively. Eur J Biochem, 1996 Jul 15, 239(2), 445 - 50 Activation of the uncoupling protein by fatty acids is modulated by mutations in the C-terminal region of the protein; Gonzalez-Barroso MM et al.; The transport properties of the uncoupling protein (UCP) from brown adipose tissue have been studied in mutants where Cys304 has been replaced by either Gly, Ala, Ser, Thr, Ile or Trp . This position is only two residues away from the C-terminus of the protein, a region that faces the cytosolic side of the mitochondrial inner membrane . Mutant proteins have been expressed in Saccharomyces cerevisiae and their activity determined in situ by comparing yeast growth rates in the presence and absence of 2-bromopalmitate . Their bioenergetic properties have been studied in isolated mitochondria by determining the effects of fatty acids and nucleotides on the proton permeability and NADH oxidation rate . It is revealed that substitution of Cys304 by non-charged residues alters the response of UCP to fatty acids . The most effective substitution is Cys for Gly since it greatly enhances the sensitivity to palmitate, decreasing threefold the concentration required for half-maximal stimulation of respiration . The opposite extreme is the substitution by Ala which increases twofold the half-maximal concentration . We conclude that the C-terminal region participates in the fatty acid regulation of UCP activity . The observed correlation between yeast growth rates in the presence of bromopalmitate and the calculated activation constants for respiration in isolated mitochondria validates growth analysis as a method to screen the in situ activity of UCP mutants. Eur J Biochem, 1996 Jul 15, 239(2), 362 - 8 Characterization of the interaction of the monomeric GTP-binding protein Rab3a with geranylgeranyl transferase II; Johannes L et al.; The monomeric GTP-binding protein Rab3a controls exocytosis in neuroendocrine and neuronal cells . Like other members of the Rab family, Rab3a is posttranslationally modified by the addition of hydrophobic geranylgeranyl groups to its C-terminus . The geranylgeranylation reaction is catalysed by the heterotrimeric geranylgeranyl transferase II . We describe the cDNA cloning of the beta-subunit of human geranylgeranyl transferase II by means of the yeast two-hybrid system . The human enzyme, which is 49% and 96% similar to yeast and rat isoforms, respectively, can complement the beta-subunit deficiency in the yeast strain ANY119 . Furthermore, by means of the two-hybrid system and in vitro geranylgeranylation reactions with purified recombinant rat geranylgeranyl transferase II, we have characterized Rab3a domains implicated in the interaction with geranylgeranyl transferase II . We find that the N-terminus, the effector loop, the hypervariable region of the C-terminus, and the geranylgeranyl-acceptor cysteines have roles in this interaction . The GDP-bound form of Rab3a is the preferred substrate of geranylgeranyl transferase II. Eur J Biochem, 1996 Jul 15, 239(2), 302 - 9 Rat pristanoyl-CoA oxidase . cDNA cloning and recognition of its C-terminal (SQL) by the peroxisomal-targeting signal 1 receptor; Vanhooren JC et al.; The composite pristanoyl-CoA oxidase cDNA sequence, derived from two overlapping clones from a rat liver cDNA library and a 5'-RACE (rapid amplification of cDNA ends) PCR fragment, consisted of 2600 bases and contained an open reading frame of 2100 bases, encoding a protein of 700 amino acids with a calculated molecular mass of 78445 Da . This value is somewhat larger than the reported molecular mass of 70 kDa as determined earlier by SDS-gel electrophoresis . The amino acid identity with rat palmitoyl-CoA oxidase was rather low (28%) and barely higher than that with the yeast acyl-CoA oxidases (20%), suggesting that the palmitoyl-CoA oxidase/pristanoyl-CoA oxidase duplication occurred early in evolution . The carboxy-terminal tripeptide of pristanoyl-CoA oxidase was SQL . In vitro studies with the bacterially expressed human peroxisomal-targeting signal-1 import receptor indicated that SQL functions as a peroxisome-targeting signal . Northern analysis of tissues from control and clofibrate treated rats demonstrated that the pristanoyl-CoA oxidase gene is transcribed in liver and extrahepatic tissues and that transcription is not enhanced by treatment of rats with peroxisome proliferators . No mRNA could be detected by northern analysis of human tissues, suggesting that the human pristanoyl-CoA oxidase gene, if present, is only poorly or not transcribed. Genomics, 1996 Jul 15, 35(2), 321 - 7 Cloning the cDNA of human PWP2, which encodes a protein with WD repeats and maps to 21q22.3; Lalioti MD et al.; We have used exon trapping to contribute to the development of the transcription map of chromosome 21q22.3 and to clone the genes responsible for disorders that map in the 21q22.3 region . Polypeptides deduced from three trapped sequences that map near PFKL showed homology to the yeast PWP2 gene . The full-length coding region of a human homologue of this yeast gene was subsequently cloned from human infant brain and fetal kidney cDNA libraries . The 919-codon open reading frame of human PWP2 belongs to the family of genes that contain tryptophan-aspartate (WD) repeats; other than its yeast counterpart, PWP2 is most closely homologous to the beta subunits of the trimeric G-protein family and may putatively be involved in signal transduction . Northern blot analysis revealed that the PWP2 gene is expressed in all fetal and adult human tissues examined (3.4 kb mRNA species) . This single-copy gene maps approximately 200 kb proximal to PFKL in chromosome 21q22.3 between markers EHOC-1 and D21S25. J Biol Chem, 1996 Jul 12, 271(28), 16934 - 8 Characterization of multiple mRNAs that encode mammalian translation initiation factor 5 (eIF-5); Si K et al.; Eukaryotic translation initiation factor 5 (eIF-5) interacts with the 40 S initiation complex (40S.mRNA.MettRNAf.eIF-2.GTP) to promote the hydrolysis of bound GTP with the concomitant joining of the 60 S ribosomal subunit to the 40 S initiation complex to form a functional 80 S initiation complex . In this paper, the multiple mRNAs that encode mammalian eIF-5 have been characterized . In rat tissues, three major eIF-5 mRNAs of 3.5, 2.8, and 2.2 kilobases in length are detected . All major eIF-5 mRNAs are initiated from a single transcription initiation site, contain identical 5'-untranslated and coding regions, but differ from one another only in the length of their 3'-untranslated regions . The different lengths of the 3'-untranslated region of eIF-5 mRNAs are generated by the use of alternative polyadenylation signals . Additionally, we demonstrate tissue-specific variations in eIF-5 mRNA expression as well as preference for polyadenylation sites . These results should lead to increased understanding of the regulation of eIF-5 gene expression. J Biol Chem, 1996 Jul 12, 271(28), 16477 - 84 CAAT/enhancer-binding proteins are involved in beta-globin gene expression and are differentially expressed in murine erythroleukemia and K562 cells; Wall L et al.; Acting in cis with the beta-globin locus control region, the CAAT box of the beta-globin gene promoter stimulates transcription 10-fold in murine erythroleukemia (MEL) cells but is without effect in K562 cells . Our previous studies suggested that of four proteins from MEL cells that bind to this CAAT box region (CP1, GATA-1, and two factors that were denoted DSFr and DSF1) DSFr is involved in the up-regulation of transcription . In the present report, the DSFr protein in MEL cells was identified as C/EBPgamma through expression cloning and antibody studies . C/EBPgamma DNA binding activity could not be detected in K562 cells . However, K562 cells, but not MEL cells, were found to express LIP, which is a truncated form of C/EBPbeta and is an inhibitor of transcription . Thus, the differential expression of C/EBP members could account for the ability of the beta-globin CAAT box to stimulate transcription in MEL cells, but not function in K562 cells . Juxtaposing a specific C/EBP binding sequence next to the beta-globin promoter, in constructs in which the CAAT box had been rendered inactive by mutation or deletion, restored full promoter activity in MEL cells only if CP1 still bound to the promoter . In conjunction with previous mutation analyses, these results suggest that C/EBPgamma may collaborate with CP1 to enhance transcription through the beta-globin CAAT box. J Biol Chem, 1996 Jul 12, 271(28), 16748 - 52 Molecular characterization and expression of rat acyl-CoA synthetase 3; Fujino T et al.; Isolation and characterization of a rat brain cDNA identified a third acyl-CoA synthetase (ACS) designated ACS3 . The deduced amino acid sequence of the cDNA revealed that ACS3 consists of 720 amino acids and exhibits a structural architecture common to ACSs from various origins . ACS3 expressed in COS cells was purified to near homogeneity . The purified ACS3 resolved by SDS-polyacrylamide gel electrophoresis into two major proteins of 79 and 80 kDa . Cell-free translation of a synthetic mRNA encoding the entire region of ACS3 revealed that the two isoforms were derived from the same mRNA . The purified ACS3 utilizes laurate and myristate most efficiently among C8-C22 saturated fatty acids and arachidonate and eicosapentaenoate among C16-C20 unsaturated fatty acids . Northern blot analysis revealed that ACS3 mRNA is most abundant in brain and, to a much lesser extent, in lung, adrenal gland, kidney, and small intestine . During the development of the rat brain, expression of ACS3 mRNA reached a maximum level at 15 days after birth and then declined gradually to 10% of the maximum in the adult brain. J Biol Chem, 1996 Jul 12, 271(28), 16827 - 32 DNA binding specificities of YPF1, a Drosophila homolog to the DNA binding subunit of human DNA-dependent protein kinase, Ku; Jacoby DB et al.; YPF1, a heterodimeric protein from Drosophila melanogaster, is a homolog to Ku, the DNA binding subunit of human DNA-dependent protein kinase . This kinase is crucial in transcriptional activation, V(D)J recombination, double-strand break repair, and both topoisomerase and helicase activities . To investigate functional homology between YPF1 and Ku, we examined DNA binding properties of YPF1 . Like Ku, at 100 mM KCl, YPF1 binding has no detectable DNA sequence specificity, requires a DNA terminus, and has a concentration-dependent stoichiometry consistent with subsequent translocation along DNA . YPF1 differs from Ku by having a 10(5)-fold higher affinity . At 400 mM KCl, YPF1 still prefers DNA termini but shows binding specificities not observed previously with Ku . In descending order of affinity, YPF1 binds to: specific DNA sequences with a specific polarity and spacing relative to DNA termini; nonspecific linear DNA; and circular DNA . At this higher ionic strength, binding stoichiometry is concentration independent, indicating that YPF1 remains bound to ends . These results demonstrate a strong functional homology between YPF1 and Ku at physiological ionic strength . The strong binding of YPF1 has also allowed us to detect underlying binding specificities that may be specific to YPF1 and its function. Exp Cell Res, 1996 Jul 10, 226(1), 154 - 63 Complementation of Ah receptor deficiency in hepatoma cells: negative feedback regulation and cell cycle control by the Ah receptor; Weiss C et al.; The Ah receptor (AhR) is a ligand-dependent transcription factor subunit that heterodimerizes with the AhR nuclear translocator (Arnt) and mediates the predominant biological effects of 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) . TCDD activates target genes in xenobiotica metabolism in many cell lines and, more specifically, delays G1-S progression of 5L hepatoma cells . Here we describe transient and stable AhR-expression analysis in AhR-deficient subclones of the TCDD-sensitive 5L cells . We tested the integrity of the AhR-signaling system beyond the lack of the receptor in the variant subclone and analyzed the role of AhR in cell cycle regulation . Transiently expressed AhR has a high basal activity on promoters containing AhR-binding sites, so-called XREs, when transfected into receptor-deficient variant cells compared to wild-type cells . Single- and double-hybrid analysis dissociates AhR ligand responsiveness, transactivation, and heterodimerization with Arnt from receptor binding to an XRE . Hybrid receptors also show the high basal activity in the absence of exogenous TCDD in AhR-deficient variant cells, indicating that the endogenous AhR-activating signal acts directly on the receptor rather than XRE-dependent promoters or DNA binding of the receptor . Stable expression of AhR in variant cell clones by retroviral infection fully reconstitutes TCDD responsiveness, including target-gene induction and delay of cell cycle progression . These AhR-reconstituted cells, like AhR-containing wild-type cells, show low basal activity of the transiently expressed AhR hybrid . Thus, the increased basal activity in AhR-deficient cells suggests a negative feedback control of AhR activity . In vitro ligand-binding assays are compatible with the idea that the increased basal activity is due to the accumulation of an AhR-binding endogenous ligand . In conclusion, AhR is causally responsible for TCDD-dependent cell cycle regulation and feedback control of AhR activity. Proc Natl Acad Sci U S A, 1996 Jul 9, 93(14), 7125 - 30 Mini-chromosomes derived from the human Y chromosome by telomere directed chromosome breakage; Heller R et al.; We have used telomeric DNA to break two acrocentric derivatives of the human Y chromosome into mini-chromosomes that are small enough to be size- fractionated by pulsed-field gel electrophoresis . One of the mini-chromosomes is about 7 Mb in size and sequence-tagged site analysis of this molecule suggests that it corresponds to a simple truncation of the short arm of the Y chromosome . Five of the mini-chromosomes are derived from the long arm, are all rearranged by more than a simple truncation, and range in size from 4.0 Mb to 9 Mb . We have studied the mitotic stabilities of these mini-chromosomes and shown that they are stably maintained by cells proliferating in culture for about 100 cell divisions. Proc Natl Acad Sci U S A, 1996 Jul 9, 93(14), 7108 - 13 Selection for genes encoding secreted proteins and receptors; Klein RD et al.; Extracellular proteins play an essential role in the formation, differentiation, and maintenance of multicellular organisms . Despite that, the systematic identification of genes encoding these proteins has not been possible . We describe here a highly efficient method to isolate genes encoding secreted and membrane-bound proteins by using a single-step selection in yeast . Application of this method, termed signal peptide selection, to various tissues yielded 559 clones that appear to encode known or novel extracellular proteins . These include members of the transforming growth factor and epidermal growth factor protein families, endocrine hormones, tyrosine kinase receptors, serine/threonine kinase receptors, seven transmembrane receptors, cell adhesion molecules, extracellular matrix proteins, plasma proteins, and ion channels . The eventual identification of most, or all, extracellular signaling molecules will advance our understanding of fundamental biological processes and our ability to intervene in disease states. Proc Natl Acad Sci U S A, 1996 Jul 9, 93(14), 7063 - 8 Bcl-2 interacting protein, BAG-1, binds to and activates the kinase Raf-1; Wang HG et al.; The Bcl-2 protein blocks programmed cell death (apoptosis) through an unknown mechanism . Previously we identified a Bcl-2 interacting protein BAG-1 that enhances the anti-apoptotic effects of Bcl-2 . Like BAG-1, the serine/threonine protein kinase Raf-1 also can functionally cooperate with Bcl-2 in suppressing apoptosis . Here we show that Raf-1 and BAG-1 specifically interact in vitro and in yeast two-hybrid assays . Raf-1 and BAG-1 can also be coimmunoprecipitated from mammalian cells and from insect cells infected with recombinant baculoviruses encoding these proteins . Furthermore, bacterially-produced BAG-1 protein can increase the kinase activity of Raf-1 in vitro . BAG-1 also activates this mammalian kinase in yeast . These observations suggest that the Bcl-2 binding protein BAG-1 joins Ras and 14-3-3 proteins as potential activators of the kinase Raf-1. Proc Natl Acad Sci U S A, 1996 Jul 9, 93(14), 6975 - 80 The C-terminal domain of the largest subunit of RNA polymerase II interacts with a novel set of serine/arginine-rich proteins; Yuryev A et al.; Although transcription and pre-mRNA processing are colocalized in eukaryotic nuclei, molecules linking these processes have not previously been described . We have identified four novel rat proteins by their ability to interact with the repetitive C-terminal domain (CTD) of RNA polymerase II in a yeast two-hybrid assay . A yeast homolog of one of the rat proteins has also been shown to interact with the CTD . These CTD-binding proteins are all similar to the SR (serine/arginine-rich) family of proteins that have been shown to be involved in constitutive and regulated splicing . In addition to alternating Ser-Arg domains, these proteins each contain discrete N-terminal or C-terminal CTD-binding domains . We have identified SR-related proteins in a complex that can be immunoprecipitated from nuclear extracts with antibodies directed against RNA polymerase II . In addition, in vitro splicing is inhibited either by an antibody directed against the CTD or by wild-type but not mutant CTD peptides . Thus, these results suggest that the CTD and a set of CTD-binding proteins may act to physically and functionally link transcription and pre-mRNA processing. Proc Natl Acad Sci U S A, 1996 Jul 9, 93(14), 6964 - 9 Molecular characterization of a FKBP-type immunophilin from higher plants; Luan S et al.; Immunophilins are intracellular receptors for the immunosuppressants cyclosporin A, FK506, and rapamycin . In addition to their use in organ transplantation, these natural products have been used to investigate signaling pathways in yeast, plant, and mammalian cells . We have recently described the identification of an immunosuppressant-sensitive signaling pathway in and the purification of several immunophilins from Vicia faba plants . We now report the molecular characterization of a 15 kDa FK506- and rapamycin-binding protein from V . faba (VfFKBP15) . The amino acid sequence deduced from the cDNA starts with a signal peptide of 22 hydrophobic amino acids . The core region of VfFKBP15 is most similar to yeast and mammalian FKBP13 localized in the endoplasmic reticulum (ER) . In addition, VfFKBP15 has a carboxyl-terminal sequence that is ended with SSEL, a putative ER retention signal . These findings suggest that VfFKBP15 is a functional homolog of FKBP13 from other organisms . Interestingly, two distinct cDNAs corresponding to two isoforms of FKBP15 have been cloned from Arabidopsis and also identified from rice data base, suggesting that pFKBP15 (plant FKBP15) is encoded by a small gene family in plants . This adds to the diversity of plant FKBP members even with the same subcellular localization and is in contrast with the situation in mammalian and yeast systems in which only one FKBP13 gene has been found . Like the mammalian and yeast FKBP13, the recombinant VfFKBP15 protein has rotamase activity that is inhibited by both FK506 and rapamycin with a Ki value of 30 nM and 0.9 nM, respectively, illustrating that VfFKBP15 binds rapamycin in preference over FK506 . The mRNA of VfFKBP15 is ubiquitously expressed in various plant tissues including leaves, stems, and roots, consistent with the ER localization of the protein . Levels of VfFKBP15 mRNA are elevated by heat shock, suggesting a possible role for this FKBP member under stress conditions. Biochem Biophys Res Commun, 1996 Jul 5, 224(1), 206 - 11 Expressed sequence tags identify human isologs of the ARF-dependent phospholipase D; Ribbes G et al.; By searching into Expressed Sequence Tags databases (dbEST) using Blast X algorithm software and a plant phospholipase D as template, we have identified a cDNA from human brain (Z45777) which encodes for a protein similar to the amino acid region 743-929 of the human phospholipase D1 (PLD1), and a cDNA from human liver (R93485) which encodes for a protein similar to region 815-932 of PLD1 . Sequence comparison between cloned phospholipases showed the presence of 3 conserved amino acid sequences: AFVGGIDLAYGRWD (box A), IIGSANINDRS (box B), and YIYIENQFFI (box C) . Phylogenic analysis indicated that the cDNA from brain and liver encoded for human isologs of PLD1. J Biol Chem, 1996 Jul 5, 271(27), 15866 - 9 Mammalian Sly1 regulates syntaxin 5 function in endoplasmic reticulum to Golgi transport; Dascher C et al.; Members of the syntaxin gene family are components of protein complexes which regulate vesicle docking and/or fusion during transport of cargo through the secretory pathway of eukaryotic cells . We have previously demonstrated that syntaxin 5 is specifically required for endoplasmic reticulum to Golgi transport (Dascher, C., Matteson, J., and Balch, W . E.(1994) J . Biol . Chem . 269, 29363-29366) . To extend these observations we have now cloned a protein from rat liver membranes which forms a native complex with syntaxin 5 . We demonstrate that this protein is the mammalian homologue to yeast Sly1p, previously identified as a protein which genetically and biochemically interacts with the small GTPase Ypt1p and Sed5p, proteins involved in docking/fusion in the early secretory pathway of yeast . Using transient expression we find that overexpression of rat liver Sly1 (rSly1) can neutralize the dominant negative effects of excess syntaxin 5 on endoplasmic reticulum to Golgi transport . These results suggest that rSly1 functions to positively regulate syntaxin 5 function. J Biol Chem, 1996 Jul 5, 271(27), 15934 - 41 Specificity of LIM domain interactions with receptor tyrosine kinases; Wu R et al.; LIM domains, Cys-rich motifs containing approximately 50 amino acids found in a variety of proteins, are proposed to direct protein*protein interactions . To identify structural targets recognized by LIM domains, we have utilized random peptide library selection, the yeast two-hybrid system, and glutathione S-transferase fusions . Enigma contains three LIM domains within its carboxyl terminus and LIM3 of Enigma specifically recognizes active but not mutant endocytic codes of the insulin receptor (InsR) (Wu, R . Y., and Gill, G . N . (1994) J . Biol . Chem . 269, 25085-25090) . Interaction of two random peptide libraries with glutathione S-transferase-LIM3 of Enigma indicated specific binding to Gly-Pro-Hyd-Gly-Pro-Hyd-Tyr-Ala corresponding to the major endocytic code of InsR . Peptide competition demonstrated that both Pro and Tyr residues were required for specific interaction of InsR with Enigma . In contrast to LIM3 of Enigma binding to InsR, LIM2 of Enigma associated specifically with the receptor tyrosine kinase, Ret . Ret was specific for LIM2 of Enigma and did not bind other LIM domains tested . Mutational analysis indicated that the residues responsible for binding to Enigma were localized to the carboxyl-terminal 61 amino acids of Ret . A peptide corresponding to the carboxyl-terminal 20 amino acids of Ret dissociated Enigma and Ret complexes, while a mutant that changed Asn-Lys-Leu-Tyr in the peptide to Ala-Lys-Leu-Ala or a peptide corresponding to exon16 of InsR failed to disrupt the complexes, indicating the Asn-Lys-Leu-Tyr sequence of Ret is essential to the recognition motif for LIM2 of Enigma . We conclude that LIM domains of Enigma recognize tyrosine-containing motifs with specificity residing in both the LIM domains and in the target structures. J Biol Chem, 1996 Jul 5, 271(27), 16393 - 8 Alternatively spliced transcripts from the Drosophila eIF4E gene produce two different Cap-binding proteins; Lavoie CA et al.; Eukaryotic initiation factor 4E (eIF4E) is the subunit of eIF4F that binds to the cap structure at the 5' end of messenger RNA and is a critical component for the regulation of translation initiation . Using 7-methyl-GTP-Sepharose affinity chromatography, two distinct cap-binding proteins that migrate on SDS-polyacrylamide gel electrophoresis at approximately 35 kDa were purified from Drosophila adults . Peptide microsequence analysis indicated that these two proteins differ at their amino termini . Analysis of a set of cDNA clones encoding eIF4E led to the conclusion that the two different protein isoforms, which we term eIF4EI and eIF4EII, result from three alternatively spliced transcripts from a single eIF4E gene, which maps to region 67A8-B2 on polytene chromosomes . The three eIF4E transcripts also vary greatly in the lengths of their 5'-UTRs, suggesting the possibility of complex translational control of expression of the two eIF4E isoforms. Science, 1996 Jul 5, 273(5271), 54 - 9 Longevity, genes, and aging; Jazwinski SM; Until recently, biogerontology was a backwater of biology, but progress in the qualitative and quantitative genetic analysis of longevity has led to a revolution in aging research . This research has revealed that extended longevity is frequently associated with enhanced metabolic capacity and response to stress . Moreover, it suggests that there are multiple mechanisms of aging . Because of its complexity, the aging process takes us into the realm of integrative biology, and thus, biogerontology should prove instrumental in deciphering the functional and regulatory circuitry of the sequenced genome. Mol Biol Cell, 1996 Jul, 7(7), 1043 - 58 Genes that control the fidelity of endoplasmic reticulum to Golgi transport identified as suppressors of vesicle budding mutations; Elrod-Erickson MJ et al.; Although convergent evidence suggests that proteins destined for export from the endoplasmic reticulum (ER) are separated from resident ER proteins and are concentrated into transport vesicles, the proteins that regulate this process have remained largely unknown . In a screen for suppressors of mutations in the essential COPII gene SEC13, we identified three genes (BST1, BST2/EMP24, and BST3) that negatively regulate COPII vesicle formation, preventing the production of vesicles with defective or missing subunits . Mutations in these genes slow the secretion of some secretory proteins and cause the resident ER proteins Kar2p and Pdi1p to leak more rapidly from the ER, indicating that these genes are also required for proper discrimination between resident ER proteins and Golgi-bound cargo molecules . The BST1 and BST2/EMP24 genes code for integral membrane proteins that reside predominantly in the ER . Our data suggest that the BST gene products represent a novel class of ER proteins that link the regulation of vesicle coat assembly to cargo sorting. Yeast, 1996 Jul, 12(9), 849 - 57 Identification and characterization of cytosolic Hansenula polymorpha proteins belonging to the Hsp70 protein family; Titorenko VI et al.; We have isolated two members of the Hsp70 protein family from the yeast Hansenula polymorpha using affinity chromatography . Both proteins were located in the cytoplasm . One of these, designated Hsp72, was inducible in nature (e.g . by heat shock) . The second protein (designated Hsc74) was constitutively present . Peptides derived from both Hsp72 and Hsc74 showed sequence homology to the cytosolic Saccharomyces cerevisiae Hsp70s, Ssa1p and Ssa2p . The gene encoding Hsp72 (designated HSA1) was cloned, sequenced and used to construct HSA1 disruption and HSA1 overexpression strains . Comparison of the stress tolerances of these strains with those of wild-type H . polymorpha revealed that HSA1 overexpression negatively affected the tolerance of the cells to killing effects of temperature or ethanol, but enhanced the tolerance to copper and cadmium . The tolerance for other chemicals (arsenite, arsenate, H2O2) or to high osmolarity was unaffected by either deletion or overexpression of HSA1. J Cell Sci, 1996 Jul, 109 ( Pt 7), 1937 - 46 Intracellular redistribution of Ku immunoreactivity in response to cell-cell contact and growth modulating components in the medium; Fewell JW et al.; Ku is a heterodimeric protein first recognized as a human autoantigen but now known to be widely distributed in mammalian cells . Analysis of repair-deficient mutant cells has shown that Ku is required for DNA repair, and roles in DNA replication and transcription have also been suggested on the basis of in vitro observations . Ku is generally regarded as a nuclear component . However, in the present paper, we show that a quantitatively significant fraction (half or more) of Ku is located in the cytoplasm of cultured primate cells, and that major changes in epitope accessibility of both nuclear and cytoplasmic Ku components are associated with the transition from sparse to confluent cell densities . The same changes in immunoreactivity were seen in HeLa, 293, CV-1 (monkey) and HPV-transformed keratinocyte cell lines, and in primary cultures of human keratinocytes . The immunostaining pattern of sparsely grown cells could be converted to the 'confluent' configuration by re-plating them at the same low density on a monolayer of mouse 3T3 cells . The confluent antigen pattern could also be induced in sparse cells within 15-30 minutes by exposure of the cells to serum- or Ca(2+)-free medium or overnight with 2 mM hydroxyurea . Somatostatin at 0.12 mM blocked the effects of serum/Ca2+ deprivation of Ku p70 antigen distribution in sparse CV-1 cells, and in confluent cultures reversed the usual nuclear concentration of p70 immunoreactivity . However, somatostatin did not alter the expected immunostaining patterns of p86 . Preliminary studies indicate that sparse CV-1 cells, but not HeLa cells, respond to as little as 1 pM of TGF-beta 1 in the culture medium by the rapid appearance of nuclear immunoreactivity . TGF-alpha had no apparent effect . These findings are consistent with the participation of Ku in a signal transduction system responsive to the inhibitory effect of cell-cell contact on the one hand and to cytokines and growth-supportive components of the culture medium on the other. J Cell Sci, 1996 Jul, 109 ( Pt 7), 1677 - 87 Mutational analysis of Hsp90 alpha dimerization and subcellular localization: dimer disruption does not impede "in vivo' interaction with estrogen receptor; Meng X et al.; The molecular chaperone Hsp90 has been found ubiquitously as a predominantly cytoplasmic dimer . By interacting with cytoplasmic or nuclear proteins such as pp60v-src or steroid receptors, Hsp90 helps its targets to become competent for full biological activity . Mutational deletion analysis of some properties of chicken Hsp90 alpha was undertaken after transient transfection of the constructs in COS7 cells . First, Hsp90 mutants were analyzed for their ability to behave as cytosolic dimers . We confirmed that the C-terminal Hsp90 region (amino acids 446-728) was sufficient for dimerization, and found that deletion of three small subregions in the 200 C-terminal residues precluded Hsp90 dimer formation . Moreover, we demonstrated that the N-terminal region of the protein (1-442) was not involved in dimerization . Second, the subcellular localization of the wild-type (WT) protein and mutants was analyzed by specific immunodetection and confocal microscopy . Most of the mutants were cytoplasmic like Hsp90WT, a nuclear localization being barely detectable in the WT protein or in mutants with a C-terminal truncation equal to or shorter than 286 residues . Surprisingly a mutant encoding the N-terminal region (1-285) was nuclear localized . In addition, the in vivo interaction between the cytoplasmic Hsp90 and the nuclear ER was documented after coexpression of both proteins in the same cells: some Hsp90 was shifted into the nucleus via its interaction with ER . From an analysis of dimeric or monomeric cytoplasmic Hsp90 mutants, we found that disruption of Hsp90 dimer did not systematically impede its interaction with ER . Finally, Hsp90WT and cytoplasmic mutants were tested for their ability to rescue from lethality a yeast strain deleted of both Hsp90 genes . Interestingly, the delta 661-677 mutant that showed an impaired dimerization but interacted with ER was able to confer viability, while the mutant deleted of the 30 C-terminal residues (NC6) was monomeric, did not confer viability and did not interact with ER . We therefore suggest that Hsp90 properties analyzed here are not necessarily interdependent. Plant Mol Biol, 1996 Jul, 31(4), 761 - 70 Characterization of pyruvate decarboxylase genes from rice; Hossain MA et al.; The pdc1 gene encoding pyruvate decarboxylase has been isolated and sequenced from an IR54 rice genomic library . In contrast to a previously isolated intron-less rice genomic pdc, pRgpdc3, this gene contains five intervening introns in the coding region and corresponds to a cDNA clone, pRcpdc1, isolated from an IR54-cDNA library constructed from anaerobically-induced mRNAs . Comparison of the deduced amino acid sequence of this gene with that of the rice pdc2 and pdc3 showed 88% and 89% similarity, and 78% and 79% identity, respectively . Southern blots indicated that more than three genes constitute the pdc gene family in rice . pdc1 is highly inducible under anaerobic conditions . Rice pdc2 is also inducible by anoxia but to a much lesser extent than pdc1. Biophys J, 1996 Jul, 71(1), 451 - 65 Forces on chromosomal DNA during anaphase; Jannink G et al.; In the course of anaphase, the chromosomal DNA is submitted to the traction of the spindle . Several physical problems are associated with this action . In particular, the sister chromatids are generally topologically intertwined at the onset of anaphase, and the removal of the intertwinings results from a coupling between the enzymatic action of type II DNA topoisomerases and the force exerted by the spindle . We propose a physical analysis of some of these problems: 1) We compare the maximum force the spindle can produce with the force required to break a DNA molecule, and define the conditions compatible with biological safety during anaphase . 2) We show that the behavior of the sister chromatids in the absence of type II DNA topoisomerases can be described by two distinct models: a chain pullout model accounts for the experimental observations made in the budding yeast, and a model of the mechanical rupture of rubbers accounts for the nondisjunction in standard cases . 3) Using the fluctuation-dissipation theorem, we introduce an effective protein friction associated with the strand-passing activity of type II DNA topoisomerases . We show that this friction can be used to describe the situation in which one chromosome passes entirely through another one . Possible experiments that could test these theoretical analyses are discussed. Genome Res, 1996 Jul, 6(7), 639 - 45 A DNA microarray system for analyzing complex DNA samples using two-color fluorescent probe hybridization; Shalon D et al.; Detecting and determining the relative abundance of diverse individual sequences in complex DNA samples is a recurring experimental challenge in analyzing genomes . We describe a general experimental approach to this problem, using microscopic arrays of DNA fragments on glass substrates for differential hybridization analysis of fluorescently labeled DNA samples . To test the system, 864 physically mapped lambda clones of yeast genomic DNA, together representing >75% of the yeast genome, were arranged into 1.8-cm x 1.8-cm arrays, each containing a total of 1744 elements . The microarrays were characterized by simultaneous hybridization of two different sets of isolated yeast chromosomes labeled with two different fluorophores . A laser fluorescent scanner was used to detect the hybridization signals from the two fluorophores . The results demonstrate the utility of DNA microarrays in the analysis of complex DNA samples . This system should find numerous applications in genome-wide genetic mapping, physical mapping, and gene expression studies. Endocrinology, 1996 Jul, 137(7), 3144 - 7 Leptin is a metabolic signal to the reproductive system; Barash IA et al.; Leptin, a newly-discovered hormonal product of the obese (ob) gene, is expressed by adipocytes and thought to play a role in the regulation of food intake and metabolism . We tested the hypothesis that leptin signals metabolic information to the reproductive system by examining its effects on the reproductive system of ob/ob mice, which have a congenital deficiency in leptin and are infertile . We treated pair-fed males and females with leptin (50 microg twice daily, ip) or vehicle (n=10/group) for 14 days, after which the animals were bled and killed . Leptin-treated females had significantly elevated serum levels of LH, increased ovarian and uterine weights, and stimulated aspects of ovarian and uterine histology compared to controls . Leptin-treated males had significantly elevated serum levels of FSH, increased testicular and seminal vesicle weights, greater seminal vesicle epithelial cell height, and elevated sperm counts compared to controls . These results demonstrate that leptin stimulates the reproductive endocrine system in both sexes of ob/ob mice and suggest that leptin may serve as a permissive signal to the reproductive system of normal animals. FEBS Lett, 1996 Jul 1, 389(2), 219 - 23 Species differences in the intracellular distribution of ciprofibroyl-CoA hydrolase . Implications for peroxisome proliferation; Urrea R et al.; Peroxisomal proliferators (HPP), such as ciprofibrate and clofibric acid, are species-specific drugs . Since HPP-coenzyme A derivatives might be involved in their action, we studied the subcellular distribution of liver ciprofibroyl-CoA hydrolase in rat and in two HPP-unresponsive species, humans and guinea pig . Total activity was similar in the three species and was not induced by clofibric acid treatment . In guinea pig, as in humans, the enzyme is localized in the mitochondrial and soluble fractions and no changes are observed after drug treatment . In the rat, the enzyme has a microsomal localization, but upon clofibric acid treatment it changes to a mitochondrial and soluble distribution, as in unresponsive species . These results raise the possibility that drug-induced hydrolases in rats might be normally expressed in humans and guinea pigs. Bioessays, 1996 Jul, 18(7), 567 - 77 Protein kinase cascades activated by stress and inflammatory cytokines; Kyriakis JM et al.; Signal transduction pathways constructed around a core module of three consecutive protein kinases, the most distal being a member of the extracellular signal-regulated kinase (ERK) family, are ubiquitous among eukaryotes . Recent work has defined two cascades activated preferentially by the inflammatory cytokines TNF-alpha and IL-1-beta, as well as by a wide variety of cellular stresses such as UV and ionizing radiation, hyperosmolarity, heat stress, oxidative stress, etc . One pathway converges on the ERK subfamily known as the "stress activated' protein kinases (SAPKs, also termed Jun N-terminal kinases, JNKs), whereas the second pathway recruits the p38 kinases . Upstream inputs are diverse, and include small GTPases (primarily Rac and Cdc42; secondarily Ras) acting through mammalian homologs of the yeast Ste20 kinase, other kinase subfamilies (e.g . GC kinase) and ceramide, a putative second messenger for certain TNF-alpha actions . These two cascades signal cell cycle delay, cellular repair or apoptosis in most cells, as well as activation of immune and reticuloendothelial cells. Microbiology, 1996 Jul, 142 ( Pt 7), 1757 - 63 A cb-type cytochrome-c oxidase terminates the respiratory chain in Helicobacter pylori; Nagata K et al.; A Helicobacter pylori membrane fraction oxidized yeast and equine cytochrome c, and N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) . When ascorbate was used as reductant, the Vmax and apparent Km values were 612 nmol electron min-1 (mg protein)-1 and 14 microM for yeast, and 419 nmol electron min-1 (mg protein)-1 and 19 microM for equine cytochrome c, respectively . For TMPD oxidation, the Vmax and Km values were 640 nmol electron min-1 (mg protein)-1 and 182 microM, respectively . These oxidase activities showed a high affinity for oxygen . Inhibition of both cytochrome-c and TMPD oxidase activities by 50% was caused by about 4 microM cyanide and about 0.5 mM azide . Redox difference spectra of the membrane solubilized with Triton X-100 showed b- or c-type cytochromes but not aa3-type cytochromes . c-type and a part of some b-type cytochromes were reduced with ascorbate plus TMPD . A CO difference spectrum revealed that protohaem, but not an aa3-type cytochrome, may be interacting with CO/oxygen . Only protohaem was detected in the haem fraction extracted from the membrane . Three polypeptides (60, 38 and 29 kDa) were found to be bearing haem c after SDS-PAGE of the membrane . From these results, it was suggested that the cbb3-type cytochrome-c oxidase, having a haem-copper binuclear centre like the cytochrome aa3-type oxidase, but differing in a few other properties, functions as a terminal oxidase in the respiratory chain of H . pylori. Microbiology, 1996 Jul, 142 ( Pt 7), 1591 - 6 The localization of chitin synthase in membranous vesicles (chitosomes) in Neurospora crassa; Sietsma JH et al.; Polyclonal anti-chitin synthase antibodies raised against the Saccharomyces cerevisiae CHS2 gene product were used to identify and localize chitin synthase in the filamentous ascomycete Neurospora crassa . A single band of approximately 110 kDa was observed in Western blots of total protein extracts of N . crassa, probed with these antibodies . However, several additional bands were labelled when membrane fraction proteins (microsomes) were probed . Histo-immunochemical localization of chitin synthase confirmed that the polypeptide is compartmentalized in membranous vesicles (chitosomes), which are abundant in the vicinity of the hyphal tip . TEM analysis did not reveal chitin synthase in the plasma membrane . However, dense labelling of membrane-associated chitin synthase was observed by light-microscopic analysis of N . crassa protoplasts and at young hyphal tips. Appl Biochem Biotechnol, 1996 Jul, 60(1), 33 - 9 Stability of invertase in reverse micelles; Subramani S et al.; The stability of invertase was studied under various conditions, including at 75 degrees C, in presence of stabilizers (sorbitol and glycerol) at 75 degrees C, and in the presence of denaturants (urea and trichloroacetic acid) at 37 degrees C in reverse micelles . Stability of the invertase in reverse micelles was found to be improved over that of the enzyme in bulk aqueous solution . Sorbitol could enhance enzyme stability as it does in the bulk aqueous system . The stabilizing effect of glycerol was reduced in reverse micelles . The denaturation pattern of urea remains unaltered . However, the denaturation effect of trichloroacetic acid has been reduced in reverse micelles. Trends Biochem Sci, 1996 Jul, 21(7), 247 - 50 The biochemistry of polyadenylation; Wahle E et al.; During the synthesis of mRNA in the nucleus, 3'-ends are generated by endonucleolytic cleavage followed by polyadenylation . The machinery responsible for this simple reaction is surprisingly complex . In vitro reconstitution of 3'-end processing has demonstrated the importance of cooperative interactions in RNA recognition and catalysis . However, the inventory of processing factors is still incomplete and important mechanistic questions have not yet been answered. J Cell Biol, 1996 Jul, 134(2), 307 - 13 The human mitochondrial import receptor, hTom20p, prevents a cryptic matrix targeting sequence from gaining access to the protein translocation machinery; McBride HM et al.; Yeast Mas70p and NADH cytochrome b5 reductase are bitopic integral proteins of the mitochondrial outer membrane and are inserted into the lipid-bilayer in an Nin-Ccyto orientation via an NH2-terminal signal-anchor sequence . The signal anchor of both proteins is comprised of a short, positively charged domain followed by the predicted transmembrane segment . The positively charged domain is capable of functioning independently as a matrix-targeting signal in yeast mitochondria in vitro but does not support import into mammalian mitochondria (rat or human) . Rather, this domain represents a cryptic signal that can direct import into mammalian mitochondria only if proximal components of the outer membrane import machinery are removed . This can be accomplished either by treating the surface of the intact mitochondria with trypsin or by generating mitoplasts . The import receptor Tom20p (Mas20p/MOM19) is responsible for excluding the cryptic matrix-targeting signal from mammalian mitochondria since replacement of yeast Tom20p with the human receptor confers this property to the yeast organelle while at the same time maintaining import of other proteins . In addition to contributing to positive recognition of precursor proteins, therefore, the results suggest that hTom20p may also have the ability to screen potential matrix-targeting sequences and exclude certain proteins that would otherwise be recognized and imported by distal components of the outer and inner membrane protein-translocation machinery . These findings also indicate, however, that cryptic signals, if they exist within otherwise native precursor proteins, may remain topogenically silent until the precursor successfully clears hTom20p, at which time the activity of the cryptic signal is manifested and can contribute to subsequent translocation and sorting of the polypeptide. J Cell Biol, 1996 Jul, 134(2), 279 - 93 Endoplasmic reticulum localization of Sec12p is achieved by two mechanisms: Rer1p-dependent retrieval that requires the transmembrane domain and Rer1p-independent retention that involves the cytoplasmic domain; Sato M et al.; Yeast Sec12p is a type II transmembrane protein in the ER, which is essential for the formation of transport vesicles . From biochemical and morphological lines of evidence, we have proposed that Sec12p is localized to the ER by two mechanisms: static retention in the ER and dynamic retrieval from the early Golgi compartment . We have also shown that Rer1p, a membrane protein in the Golgi, is required for correct localization of Sec12p . In the present study, we have performed a systematic analysis to determine the ER localization signals in Sec12p corresponding to these two mechanisms . Both the transmembrane domain (TMD) and the NH2-terminal cytoplasmic domain of Sec12p show the ability to localize the protein to the ER . The effect of the TMD is potent and sufficient by itself for the ER localization and is strongly dependent on Rer1p . On the other hand, the cytoplasmic domain shows a moderate ER-localization capability which is independent of Rer1p . The rate of mannosyl modification has been measured to distinguish between retention and retrieval . The cytoplasmic domain significantly delays the transport from the ER to the cis-Golgi . In contrast, the TMD shows only a subtle retardation in the transport from the ER to the cis-Golgi but strictly prevents the transport beyond there . From these observations, we conclude that the TMD mainly acts as the retrieval signal and the cytoplasmic domain contains the retention signal . This study not only supports the two-mechanisms hypothesis but also provides powerful tools to dissect the two. J Cell Biol, 1996 Jul, 134(2), 269 - 78 Signal sequences specify the targeting route to the endoplasmic reticulum membrane; Ng DT et al.; In the yeast Saccharomyces cerevisiae, only a subset of preproteins that are translocated across the ER membrane require the function of the signal recognition particle (SRP), suggesting that an alternative, SRP-independent pathway must exist (Hann, B.C., and P . Walter . 1991 . Cell . 67:131-144) . We have established that the two targeting pathways function in parallel . Mutant alleles of SEC62 and SEC63 were isolated that specifically impaired the translocation of SRP-independent preproteins in vivo and in vitro, whereas SRP-dependent preproteins were unaffected . Based on this analysis, preproteins fall into three distinct classes: SRP dependent, SRP independent, and those that can use both pathways . Pathway specificity is conferred by the hydrophobic core of signal sequences . Our studies show a previously unrecognized diversity in ER-directed signal sequences, that carry structural information that serves to identify the route taken. J Leukoc Biol, 1996 Jul, 60(1), 58 - 68 Platelets enhance Fc(gamma) receptor-mediated phagocytosis and respiratory burst in neutrophils: the role of purinergic modulation and actin polymerization; Zalavary S et al.; The interaction of platelets with neutrophil granulocytes is considered to play an important role in the inflammatory process, and the present study was focused on platelet-induced modulation of Fcgamma receptor-mediated functions in neutrophils . We found that phagocytosis and the respiratory burst (measured as luminol-enhanced chemiluminescence), triggered in neutrophils by immunoglobulin G (IgG)-opsonized yeast particles, were potentiated by platelets and that maximal enhancement was achieved at a physiological neutrophil/platelet ratio of about 1:50 to 1:100 . Platelets both increased the intra- and extracellular generation of oxygen radicals as well as the release of myeloperoxidase from stimulated neutrophils . The presence of platelets also induced a cortical actin polymerization in neutrophils, which might explain the increased phagocytic capacity . Platelets appear to affect neutrophil function in a contact-independent manner that most likely involves ATP, indicated by the following: (1) platelet supernatants, but not fixed platelets, affected neutrophil function in the same way as viable platelets; (2) platelets raised the extracellular ATP level four- to fivefold; (3) exogenous ATP mimicked the effects of platelets on actin polymerization, phagocytosis, and the respiratory burst in neutrophils; (4) hydrolysis of extracellular ATP with apyrase or blocking of ATP receptors with suramin reversed the platelet-induced enhancement of neutrophil function . An increased accumulation of extracellular adenosine, induced by inhibiting endogenous adenosine deaminase or adding exogenous adenosine, reversed the effects of platelets . The platelet-induced potentiation of the respiratory burst was inhibited by the tyrosine kinase inhibitor genistein, suggesting that tyrosine phosphorylation is involved . However, platelets did not significantly affect the Fcgamma receptor-triggered calcium response in neutrophils . In conclusion, we show that platelets, through an ATP-dependent mechanism, potentiate IgG-mediated ingestion and production of oxygen metabolites in neutrophils. Nucleic Acids Res, 1996 Jul 1, 24(13), 2483 - 7 A new class of genome rare cutters; Veselkov AG et al.; Although significant efforts have been directed at developing efficient techniques for rare and super rare genome cutting, only limited success has been achieved . Here we propose a new approach to solve this problem . We demonstrate that peptide nucleic acid 'clamps' (bis-PNAs) bind strongly and sequence specifically to short homopyrimidine sites on lambda and yeast genomic DNAs . Such binding efficiently shields methylation/restriction sites which overlap with the bis-PNA binding sites from enzymatic methylation . After removing the bis-PNA, the genomic DNAs are quantitatively cleaved by restriction enzymes into a limited number of pieces of lengths from several hundred kbp to several Mbp . By combining various bis-PNAs with different methylation/restriction enzyme pairs, a huge new class of genome rare cutters can be created . These cutters cover the range of recognition specificities where very few, if any, cutters are now available. Lab Invest, 1996 Jul, 75(1), 97 - 107 AAMP, a conserved protein with immunoglobulin and WD40 domains, regulates endothelial tube formation in vitro; Beckner ME et al.; Angio-associated migratory cell protein (AAMP) is a newly discovered protein that is widely distributed with strong expression in endothelial cells and others with migratory potential (cytotrophoblasts, carcinoma cells, etc) . AAMP is 52 kd with an isoelectric point of 5.2 . Its sequence contains immunoglobulin type domains, WD40 repeats, a large acidic region with an acid box, a potential transmembrane region, potential serine/threonine phosphorylation sites, and a positively charged amino-terminal region with strong heparin binding potential (Kd = 14 pmol) . Human umbilical vein endothelial cells cultured on Matrigel, a basement membrane material, form endothelial tubes (capillary-like structures) . Anti-recombinant AAMP (anti-rAAMP) (1 to 10 microg/ml) inhibits this process under conditions that favor cross-linking of its ligand (AAMP) . Immunofluorescent staining has shown that AAMP is distributed both intracellularly and extracellularly in cultures of endothelial cells and tubes . Molecular analysis of AAMP's protein sequence shows a striking evolutionary relationship with the YCR072c protein in Saccharomyces cerevisiae . Both the human and yeast proteins show an unusual and almost identical arrangement of immunoglobulin type domains, WD40 repeats, a protein kinase C phosphorylation consensus site in the carboxyl region, and a positively charged amino-terminal region that in AAMP has heparin binding potential . Detection of YCR072c's immunoglobulin type domains is new . Thus, AAMP is a protein that has been highly conserved in evolution and may function in the regulation of endothelial tube formation. J Virol, 1996 Jul, 70(7), 4737 - 47 Ty3 integrase mutants defective in reverse transcription or 3'-end processing of extrachromosomal Ty3 DNA; Kirchner J et al.; Ty3, a retroviruslike element in Saccharomyces cerevisiae, encodes an integrase (IN) which is essential for position-specific transposition . The Ty3 integrase contains the highly conserved His-Xaa(3-7)-His-Xaa(23-32)-Cys-Xaa(2)-Cys and Asp, Asp-Xaa(35)-Glu {D,D(35)E} motifs found in retroviral integrases . Mutations were introduced into the coding region for the Ty3 integrase to determine the effects in vivo of changes in conserved residues of the putative catalytic triad D,D(35)E and the nonconserved carboxyl-terminal region . Ty3 viruslike particles were found to be associated with significant amounts of linear DNA of the approximate size expected for a full-length reverse transcription product and with plus-strand strong-stop DNA . The full-length, preintegrative DNA has at each 3' end 2 bp that are removed prior to or during integration . Such 3'-end processing has not been observed for other retroviruslike elements . A mutation at either D-225 or E-261 of the Ty3 integrase blocked transposition and prevented processing of the 3' ends of Ty3 DNA in vivo, suggesting that the D,D(35)E region is part of the catalytic domain of Ty3 IN . Carboxyl-terminal deletions of integrase caused a dramatic reduction in the amount of Ty3 DNA in vivo and a decrease in reverse transcriptase activity in vitro but did not affect the apparent size or amount of the 55-kDa reverse transcriptase in viruslike particles . The 115-kDa viruslike particle protein, previously shown to react with antibodies to Ty3 integrase, was shown to be a reverse transcriptase-IN fusion protein . These results are consistent with a role for the integrase domain either in proper folding of reverse transcriptase or as part of a heterodimeric reverse transcriptase molecule. Nat Genet, 1996 Jul, 13(3), 303 - 8 Identification of the murine beige gene by YAC complementation and positional cloning; Perou CM et al.; The beige mutation is a murine autosomal recessive disorder, resulting in hypopigmentation, bleeding and immune cell dysfunction . The gene defective in beige is thought to be a homologue of the gene for the human disorder Chediak-Higashi syndrome . We have identified the murine beige gene by in vitro complementation and positional cloning, and confirmed its identification by defining mutations in two independent mutant alleles . The sequence of the beige gene message shows strong nucleotide homology to multiple human ESTs, one or more of which may be associated with the Chediak-Higashi syndrome gene . The amino acid sequence of the Beige protein revealed a novel protein with significant amino acid homology to orphan proteins identified in Saccharomyces cerevisiae, Caenorhabditis elegans and humans. Curr Eye Res, 1996 Jul, 15(7), 774 - 81 Comparison of leucine aminopeptidase and aminopeptidase III activities in lens; Sharma KK et al.; PURPOSE . To evaluate the relative contribution of leucine aminopeptidase and aminopeptidase III activities to the total aminopeptidase activity in bovine and human lenses under in vivo pH conditions . METHODS . Bovine and human lens extracts were fractionated on a Sephadex G-200 column at pH 6.9 and 8.5 and all the fractions were assayed with Leu-pNA and Arg-pNA as substrates at in vivo lens pH (6.9) and optimum pH for leucine aminopeptidase, (8 . 5) . The major peptidases were purified and their activities compared with that of LAP and AP III isolated from bovine lens . The ability of bovine and human lens extracts and purified bovine lens LAP and AP III to hydrolyze various peptide bonds in synthetic peptides, VHLPTVEK, bradykinin and Ile-Ser-bradykinin was determined by amino acid analysis of the reaction products . RESULTS . Sephadex G-200 gel chromatography and assay of all the fractions at pH 6.9 showed that the elution volume for the predominant aminopeptidase present in bovine lens extract is the same as that of purified AP III from the same lenses . However, when the assays were done at pH 8.5, the major activity eluting from the Sephadex G-200 column was found in fractions having LAP . A similar study of human lens extracts at pH 6 . 9 and 8.5 showed one major peak with elution volume corresponding to that of purified bovine lens AP III . The human lens extracts displayed a very low level of LAP activity . The hydrolysis pattern of peptide substrates by AP III paralleled that of bovine and human lens extract at pH 6.9 . The X-Pro bond resistant to LAP in peptide substrate, VHLTPVEK was hydrolyzed by AP III as well as lens extracts . CONCLUSION . Both bovine and human lenses have very low LAP activity compared to AP III activity at in vivo pH 6.9 . AP III, by its higher activity, broad specificity and its ability to cleave peptide bonds that are resistant to LAP, is likely to play a major role in lens during epithelial cell differentiation into fiber cells and complete hydrolysis of peptides generated in vivo. Mol Cell Biol, 1996 Jul, 16(7), 3799 - 806 Modulation of thermal induction of hsp70 expression by Ku autoantigen or its individual subunits; Yang SH et al.; Previously, we proposed a dual control mechanism for the regulation of the heat shock response in mammalian cells: a positive control mediated by the heat shock transcription factor HSF1 and a negative control mediated by the constitutive heat shock element-binding factor (CHBF) . To study the physiological role of CHBF in the regulation of heat shock response, we purified CHBF to apparent homogeneity and showed it to be identical to the Ku autoantigen, a heterodimer consisting of 70-kDa (Ku-70) and 86-kDa (Ku-80) polypeptides . To study further the functional significance of Ku/CHBF in the cellular response to heat shock, we established rodent cell lines that stably and constitutively overexpressed one or both subunits of the human Ku protein, and examined the thermal induction of hsp70 and other heat shock proteins in these Ku-overexpressing ing cells . We show that expression of the human Ku-70 and Ku-80 subunits jointly or of the Ku-70 subunit alone specifically inhibits heat-induced hsp70 expression . Conversely, expression of human Ku-80 alone does not have this effect . Thermal induction of other heat shock proteins in all of the Ku-overexpressing cell lines appears not to be significantly affected, nor is the state of phosphorylation or the DNA-binding ability of HSF1 affected . These findings support a model in which hsp70 expression is controlled by a second regulatory factor in addition to the positive activation of HSF1 . The Ku protein, specifically the Ku-70 subunit, is involved in the regulation of hsp70 gene expression. Mol Cell Biol, 1996 Jul, 16(7), 3773 - 80 DNA sequence preferences of GAL4 and PPR1: how a subset of Zn2 Cys6 binuclear cluster proteins recognizes DNA; Liang SD et al.; Biophysical and genetic experiments have defined how the Saccharomyces cerevisiae protein GAL4 and a subset of related proteins recognize specific DNA sequences . We assessed DNA sequence preferences of GAL4 and a related protein, PPR1, in an in vitro DNA binding assay . For GAL4, the palindromic CGG triplets at the ends of the 17-bp recognition site are essential for tight binding, whereas the identities of the internal 11 bp are much less important, results consistent with the GAL4-DNA crystal structure . Small reductions in affinity due to mutations at the center-most 5 bp are consistent with the idea that an observed constriction in the minor groove in the crystalline GAL4-DNA complex is sequence dependent . The crystal structure suggests that this sequence dependence is due to phosphate contacts mediated by arginine 51, as part of a network of hydrogen bonds . Here we show that the mutant protein GAL4(1-100)R51A fails to discriminate sites with alterations in the center of the site from the wild-type site . PPR1, a relative of GAL4, also recognizes palindromic CGG triplets at the ends of its 12-bp recognition sequence . The identities of the internal 6 bp do not influence the binding of PPR1 . We also show that the PPR1 site consists of a 12-bp duplex rather than 16 bp as reported previously: the two T residues immediately 5' to the CGG sequence in each half site, although highly conserved, are not important for binding by PPR1 . Thus, GAL4 and PPR1 share common CGG half sites, but they prefer DNA sequences with the palindromic CGG separated by the appropriate number of base pairs, 11 for GAL4 and 6 for PPR1. Mol Cell Biol, 1996 Jul, 16(7), 3707 - 13 Actions of Rho family small G proteins and p21-activated protein kinases on mitogen-activated protein kinase family members; Frost JA et al.; The mitogen-activated protein (MAP) kinases are a family of serine/threonine kinases that are regulated by distinct extracellular stimuli . The currently known members include extracellular signal-regulated protein kinase 1 (ERK1), ERK2, the c-Jun N-terminal kinase/stress-activated protein kinases (JNK/SAPKs), and p38 MAP kinases . We find that overexpression of the Ste20-related enzymes p21-activated kinase 1 (PAK1) and PAK2 in 293 cells is sufficient to activate JNK/SAPK and to a lesser extent p38 MAP kinase but not ERK2 . Rat MAP/ERK kinase kinase 1 can stimulate the activity of each of these MAP kinases . Although neither activated Rac nor the PAKs stimulate ERK2 activity, overexpression of either dominant negative Rac2 or the N-terminal regulatory domain of PAK1 inhibits Ras-mediated activation of ERK2, suggesting a permissive role for Rac in the control of the ERK pathway . Furthermore, constitutively active Rac2, Cdc42hs, and RhoA synergize with an activated form of Raf to increase ERK2 activity . These findings reveal a previously unrecognized connection between Rho family small G proteins and the ERK pathway. Mol Cell Biol, 1996 Jul, 16(7), 3679 - 84 The molecular chaperone Ydj1 is required for the p34CDC28-dependent phosphorylation of the cyclin Cln3 that signals its degradation; Yaglom JA et al.; The G1 cyclin Cln3 of the yeast Saccharomyces cerevisiae is rapidly degraded by the ubiquitin-proteasome pathway . This process is triggered by p34CDC28-dependent phosphorylation of Cln3 . Here we demonstrate that the molecular chaperone Ydj1, a DnaJ homolog, is required for this phosphorylation . In a ydj1 mutant at the nonpermissive temperature, both phosphorylation and degradation of Cln3 were deficient . No change was seen upon inactivation of Sis1, another DnaJ homolog . The phosphorylation defect in the ydj1 mutant was specific to Cln3, because no reduction in the phosphorylation of Cln2 or histone H1, which also requires p34CDC28, was observed . Ydj1 was required for Cln3 phosphorylation and degradation rather than for the proper folding of this cyclin, since Cln3 produced in the ydj1 mutant was fully active in the stimulation of p34CDC28 histone kinase activity . Moreover, Ydj1 directly associates with Cln3 in close proximity to the segment that is phosphorylated and signals degradation . Thus, binding of Ydj1 to this domain of Cln3 seems to be essential for the phosphorylation and breakdown of this cyclin . In a cell-free system, purified Ydj1 stimulated the p34CDC28-dependent phosphorylation of the C-terminal segment of Cln3 and did not affect phosphorylation of Cln2 (as was found in vivo) . The reconstitution of this process with pure components provides evidence of a direct role for the chaperone in the phosphorylation of Cln3. Mol Cell Biol, 1996 Jul, 16(7), 3446 - 53 Molecular genetic analysis of volatile-anesthetic action; Keil RL et al.; The mechanism(s) and site(s) of action of volatile inhaled anesthetics are unknown in spite of the clinical use of these agents for more than 150 years . In the present study, the model eukaryote Saccharomyces cerevisiae was used to investigate the action of anesthetic agents because of its powerful molecular genetics . It was found that growth of yeast cells is inhibited by the five common volatile anesthetics tested (isoflurane, halothane, enflurane, sevoflurane, and methoxyflurane) . Growth inhibition by the agents is relatively rapid and reversible . The potency of these compounds as yeast growth inhibitors directly correlates with their lipophilicity as is predicted by the Meyer-Overton relationship, which directly correlates anesthetic potency of agents and their lipophilicity . The effects of isoflurane on yeast cells were characterized in the most detail . Yeast cells survive at least 48 h in a concentration of isoflurane that inhibits colony formation . Mutants resistant to the growth-inhibitory effects of isoflurane are readily selected . The gene identified by one of these mutations, zzz4-1, has been cloned and characterized . The predicted ZZZ4 gene product has extensive homology to phospholipase A2-activating protein, a GO effector protein of mice . Both zzz4-1 and a deletion of ZZZ4 confer resistance to all five of the agents tested, suggesting that signal transduction may be involved in the response of these cells to volatile anesthetics. Mol Cell Biol, 1996 Jul, 16(7), 3264 - 74 EGT2 gene transcription is induced predominantly by Swi5 in early G1; Kovacech B et al.; In a screen for cell cycle-regulated genes in the yeast Saccharomyces cerevisiae, we have identified a gene, EGT2, which is involved in cell separation in the G1 stage of the cell cycle . Transcription of EGT2 is tightly regulated in a cell cycle-dependent manner . Transcriptional levels peak at the boundary of mitosis and early G1 The transcription factors responsible for EGT2 expression in early G1 are Swi5 and, to a lesser extent, Ace2 . Swi5 is involved in the transcriptional activation of the HO gene during late G1 and early S phase, and Ace2 induces CTS1 transcription during early and late G1 We show that Swi5 activates EGT2 transcription as soon as it enters the nucleus at the end of mitosis in a concentration-dependent manner . Since Swi5 is unstable in the nucleus, its level drops rapidly, causing termination of EGT2 transcription before cells are committed to the next cell cycle . However, Swi5 is still able to activate transcription of HO in late G1 in conjunction with additional activators such as Swi4 and Swi6. Genomics, 1996 Jul 1, 35(1), 55 - 65 Structure and methylation-associated silencing of a gene within a homozygously deleted region of human chromosome band 8p22; MacGrogan D et al.; The structure and expression pattern of a human gene located within a homozygously deleted region of a metastatic prostate cancer have been characterized . Multiple cDNA fragments of this gene were isolated by hybrid capture with yeast artificial chromosome clones covering the deletion region . Eleven coding exons spanned 205-220 kb of the 730- to 970-kb deletion . The predicted amino acid sequence was 43% identical to that of an anonymous Caenorhabditis elegans gene and 20% identical to an accessory or regulatory subunit of the oligosaccharyltransferase enzyme complex in Saccharomyces cerevisiae . Hydrophobicity profiles of all three gene products were similar and showed four putative membrane-spanning domains in the molecules' C-terminal halves, suggesting a general conservation of function . The gene was expressed as an approximately 1.5-kb mRNA in most nonlymphoid human cells/tissues including prostate, lung, liver, and colon . Expression was detected in many epithelial tumor cell lines, but was undetectable by Northern blot or RT-PCR in 14 of 15 colorectal, 1 of 8 lung, and 1 of 4 liver cancer cell lines . Lack of expression in tumor cell lines was highly correlated with hypermethylation of a CpG island located at the gene's 5' end . These findings form a basis for further work on this candidate tumor suppressor gene. Arch Biochem Biophys, 1996 Jul 1, 331(1), 134 - 40 Active-site topologies of human CYP2D6 and its aspartate-301 --> glutamate, asparagine, and glycine mutants; Mackman R et al.; Cytochrome P450 2D6 (CYP2D6) catalyzes the oxidation of substrates with a positively charged nitrogen atom 5-7 angstroms from the site of the oxidation . The active-site topology of CYP2D6 is examined here with phenyl-, 2-naphthyl-, and p-biphenyldiazene, which react with P450 enzymes to form sigma-bonded aryl-iron (Fe-Ar) complexes . Ferricyanide-mediated migration of the aryl group from the iron to the porphyrin nitrogens produces the N-arylprotoporphyrin IX regioisomers (NB:NA:NC:ND, in which the aryl group is bound to the nitrogen of pyrrole rings B, A, C, and D, respectively) in the following ratios (zero means <5%): phenyl, 10:90:00:00; 2-naphthyl, 09:91:00:00; and p-biphenyl, 16:84:00:00 . These results suggest that the CYP2D6 active site is open above pyrrole ring A and to a small extent above pyrrole ring B but is closed above pyrrole rings C and D . This geometry differs from those determined by the same method for P450s for which crystal structures are available . Replacement of Asp-301 by a Glu, which preserves the carboxylate side chain, causes no detectable change in the N-aryl porphyrin regioisomer patterns and only minor changes in the catalytic activity . Replacement of Asp-301 by an Asn or Gly, which eliminates the negatively charged side chain, suppresses migration of the aryl groups to pyrrole ring B without impairing migration to pyrrole ring A and virtually abolishes catalytic activity . These results provide a refined model of the active site of CYP2D6 . They confirm, furthermore, that the loss of activity observed when Asp-301 is replaced by a neutral residue is due to loss of the charge-pairing interaction with the substrate positive charge and/or subtle structural effects in the vicinity of pyrrole ring B, but not to major structural reorganization of the active site. Arch Biochem Biophys, 1996 Jul 1, 331(1), 1 - 8 Purification and characterization of protein phosphatase 2C in rat parotid acinar cells: two forms of Mg(2+)-activated histone phosphatase and phosphorylation by cAMP-dependent protein kinase; Yokoyama N et al.; Two forms of Mg(2+)-activated histone phosphatase activities were partially purified from rat parotid acinar cells using Mono Q and gel filtration chromatography . Both enzymes activities were dependent on the presence of Mg2+, showing little activity in the presence of EDTA . The activities fractionated on the Mono Q column into two peaks: the first was a minor peak of histone phosphatase activity; the second was a major peak . These two peaks eluted at distinct positions on the gel filtration column . The molecular masses of the two peak fractions corresponded to 46 and 55 kDa, respectively on SDS-gels . The first 46-kDa peak immunoreacted with anti-PP2Calpha phosphatase antibody and like PP2Calpha phosphatase could be phosphorylated by cAMP-dependent protein kinase . The second 55-kDa peak showed neither reactivity with anti-PP2Calpha phosphatase antibody nor phosphorylability by cAMP-dependent protein kinase, but retained a Mg2+ or Mn2+ dependence for its histone phosphatase activity . Ca2+ showed a strong inhibition on this activity . On the basis of these observations, we have identified the first peak enzyme as PP2Calpha phosphatase and the second peak as a novel PP2C-like phosphatase. J Biol Chem, 1996 Jun 28, 271(26), 15315 - 21 Removal of hydrogen peroxide by thiol-specific antioxidant enzyme (TSA) is involved with its antioxidant properties . TSA possesses thiol peroxidase activity; LES Netto et al.; The thiol-specific antioxidant protein (TSA) protects glutamine synthetase from inactivation by a metal-catalyzed oxidation (MCO) system comprised of dithiothreitol (DTT)/Fe3+/O2 but not by the ascorbate/Fe3+/O2 MCO system . The removal of sulfur-centered radicals or H2O2 has been proposed as the protective mechanism of TSA . Like catalase, TSA prevents the initiation of the rapid O2 uptake phase during MCO of DTT but causes only partial inhibition when added after the reaction is well into the propagation phase . Stoichiometric studies showed that the antioxidant property of TSA is, at least in part, due to its ability to catalyze the destruction of H2O2 by the overall reaction 2 RSH + H2O2 --> RSSR + H2O . Results of kinetic studies demonstrate that the removal of H2O2 by TSA correlates with its ability to protect glutamine synthetase from inactivation . In the presence of thioredoxin, TSA is more active, whereas C170S (an active mutant of TSA in which cysteine 170 was replaced by a serine) and open reading frame 6 (a human antioxidant protein homologous to TSA with only one conserved cysteine residue) are only slightly affected . The thiol specificity of the protective activity of TSA derives from the fact that the oxidized form of TSA can be converted back to its sulfhydryl form by treatment with thiols but not by ascorbate. Gene, 1996 Jun 26, 172(2), 299 - 302 Isolation and sequencing of the cDNA encoding phosphatidylinositol transfer protein from rabbit lung; Tsao FH et al.; The cDNA clones encoding rabbit lung phosphatidylinositol transfer protein (PI-TP) were isolated and sequenced . The putative polypeptide consisted of 270 amino acid (aa) residues, the same as human PI-TP, but one aa residue less than the PI-TP of rat and mouse . PI-TP RNA expression in various tissues of a pregnant rabbit was analyzed by Northern blot . Brain, placenta and fallopian tube had the highest PI-TP RNA expression . PI-TP RNA expression in alveolar epithelial type-II cells isolated from rabbit lung markedly increased after a 24-h culture, suggesting that PI-TP RNA expression in type-II cells can be modified by ambient factors. Proc Natl Acad Sci U S A, 1996 Jun 25, 93(13), 6764 - 9 Mutation of Pro-258 in transmembrane domain 6 constitutively activates the G protein-coupled alpha-factor receptor; Konopka JB et al.; The alpha-factor pheromone receptor stimulates MATa yeast cells to undergo conjugation . The receptor contains seven transmembrane domains that function in ligand binding and in transducing a signal to the cytoplasmic receptor sequences to mediate G protein activation . A genetic screen was used to isolate receptor mutations that constitutively signal in the absence of alpha-factor . The Pro-258-->Leu (P258L) mutation caused constitutive receptor signaling that was equivalent to about 45% of the maximum level observed in wild-type cells stimulated with alpha-factor . Mutations of both Pro-258 and the adjacent Ser-259 to Leu increased constitutive signaling to > or = 90% of the maximum level . Since Pro-258 occurs in the central portion of transmembrane domain 6, and since proline residues are expected to cause a kink in alpha-helical domains, the P258L mutation is predicted to alter the structure of transmembrane domain 6 . The P258L mutation did not result in a global distortion of receptor structure because alpha-factor bound to the mutant receptors with high affinity and induced even higher levels of signaling . These results suggest that sequences surrounding Pro-258 may be involved in ligand activation of the receptor . Conformational changes in transmembrane domain 6 may effect a change in the adjacent sequences in the third intracellular loop that are thought to function in G protein activation . Greater than 90% of all G protein-coupled receptors contain a proline residue at a similar position in transmembrane domain 6, suggesting that this aspect of receptor activation may be conserved in other receptors. Proc Natl Acad Sci U S A, 1996 Jun 25, 93(13), 6521 - 6 In vitro reconstitution of human replication factor C from its five subunits; Uhlmann F et al.; Replication factor C (RFC, also called Activator I) is part of the processive eukaryotic DNA polymerase holoenzymes . The processive elongation of DNA chains requires that DNA polymerases are tethered to template DNA at primer ends . In eukaryotes the ring-shaped homotrimeric protein, proliferating cell nuclear antigen (PCNA), ensures tight template-polymerase interaction by encircling the DNA strand . Proliferating cell nuclear antigen is loaded onto DNA through the action of RFC in an ATP-dependent reaction . Human RFC is a protein complex consisting of five distinct subunits that migrate through SDS/polyacrylamide gels as protein bands of 140, 40, 38, 37, and 36 kDa . All five genes encoding the RFC subunits have been cloned and sequenced . A functionally identical RFC complex has been isolated from Saccharomyces cerevisiae and the deduced amino acid sequences among the corresponding human and yeast subunits are homologous . Here we report the expression of the five cloned human genes using an in vitro coupled transcription/translation system and show that the gene products form a complex resembling native RFC that is active in supporting an RFC-dependent replication reaction . Studies on the interactions between the five subunits suggest a cooperative mechanism in the assembly of the RFC complex . A three-subunit core complex, consisting of p36, p37, and p40, was identified and evidence is presented that p38 is essential for the interaction between this core complex and the large p140 subunit. Mol Gen Genet, 1996 Jun 24, 251(4), 412 - 21 The hapC gene of Aspergillus nidulans is involved in the expression of CCAAT-containing promoters; Papagiannopoulos P et al.; The 5' regulatory region of the amdS gene of Aspergillus nidulans, which encodes an acetamidase required for growth on acetamide as a carbon and nitrogen source, contains a CCAAT sequence which is required for setting the basal level of amdS expression . Mobility shift studies have identified a factor in A . nidulans nuclear extracts which binds to this CCAAT sequence . In Saccharomyces cerevisiae the HAP3 gene encodes one component of a multisubunit complex that binds CCAAT sequences . A search of the EMBL and SwissProt databases has revealed an A . nidulans sequence with significant homology to the HAP3 gene adjacent to the previously cloned regulatory gene amdR . Sequencing of the remainder of this region has confirmed the presence of a gene, designated hapC, with extensive homology to HAP3 . The predicted amino acid sequence of HapC shows extensive identity to HAP3 in the central conserved domain, but shows little conservation in the flanking sequences . A haploid carrying a hapC deletion has been created and is viable, but grows poorly on all media tested . This null mutant grows especially slowly on acetamide as a sole carbon and nitrogen source, indicating that hapC plays a role in amdS expression . In agreement with this notion, it has been shown that the hapC deletion results in reduced levels of expression of an amdS::lacZ reporter gene and this effect is particularly evident under conditions of carbon limitation . Nuclear extracts prepared from the hapC deletion mutant show no CCAAT binding activity to the amdS or gatA promoters, indicating that hapC may encode a component of the complex binding at this sequence. J Mol Biol, 1996 Jun 21, 259(4), 645 - 54 Functional base-pairing interaction between highly conserved elements of U3 small nucleolar RNA and the small ribosomal subunit RNA; Hughes JM; The U3 nucleolar RNA has a remarkably wide phyletic distribution extending from the Eukarya to the Archaea . It functions in maturation of the small subunit (SSU) rRNA through a mechanism which is as yet unknown but which involves base-pairing with pre-rRNA . The most conserved part of U3 is within 30 nucleotides of the 5' end, but as yet no function for this domain has been proposed . Elements within this domain are complementary to highly conserved sequences in the SSU rRNA which, in the mature form, fold into a universally conserved pseudoknot . The nature of the complementarity suggests a novel mechanism for U3 function whereby U3 facilitates correct folding of the pseudoknot . Wide phylogenetic comparison provides compelling evidence in support of the interaction in that significant complementary changes have taken place, particularly in the archaeon Sulfolobus, which maintain the base-pairing . Base-substitution mutations in yeast U3 designed to disrupt the base-pairing indicate that the interaction is probably essential . These include cold-sensitivity mutations which exhibit phenotypes similar to U3-depletion, but without impairment of the AO processing step, which occurs within the 5' ETS . These phenotypes are consistent with the destabilization of SSU precursors and partial impairment of the processing steps A1, at the 5' ETS/18 S boundary, and A2, within the ITS1. J Biol Chem, 1996 Jun 21, 271(25), 14653 - 6 Interaction between an integral protein of the nuclear envelope inner membrane and human chromodomain proteins homologous to Drosophila HP1; Ye Q et al.; At the nuclear envelope in higher eukaryotic cells, the nuclear lamina and the heterochromatin are adjacent to the inner nuclear membrane, and their attachment is presumably mediated by integral membrane proteins . In a yeast two-hybrid screen, the nucleoplasmic domain of lamin B receptor (LBR), an integral protein of the inner nuclear membrane, associated with two human polypeptides homologous to Drosophila HP1, a heterochromatin protein involved in position-effect variegation . LBR fusion proteins bound to HP1 proteins synthesized by in vitro translation and present in cell lysates . Antibodies against LBR also co-immunoprecipitated HP1 proteins from cell extracts . LBR can interact with chromodomain proteins that are highly conserved in eukaryotic species and may function in the attachment of heterochromatin to the inner nuclear membrane in cells. Oncogene, 1996 Jun 20, 12(12), 2631 - 40 Cyclin C/CDK8 is a novel CTD kinase associated with RNA polymerase II; Rickert P et al.; A number of cyclin/kinase complexes have been identified in mammalian cells that are essential for controlled cell proliferation . Cyclin C was isolated by virtue of its ability to rescue the triple CLN mutation in yeast; however, until now its function has remained unclear . Cyclin C associates with a novel cyclin dependent kinase, CDK8, and we demonstrate that this complex is associated with kinase activity towards the carboxy-terminal domain (CTD) of RNA polymerase II . We have identified at least two distinct cyclin C/CDK8 containing complexes within the cell, a larger complex over 500 kD in size, that also contains the largest subunit of RNA polymerase II, and a smaller 170 kD species . Both of these cyclin C complexes retain potent CTD kinase activity . We further demonstrate that the cyclin C/CDK8 complex associates with the large subunit of RNA polymerase II in vivo, implicating a potential role for cyclin C/CDK8 in regulating its activities. Nature, 1996 Jun 20, 381(6584), 709 - 13 An RNA-dependent ATPase associated with U2/U6 snRNAs in pre-mRNA splicing; Xu D et al.; The hydrolysis of ATP by a group of RNA-dependent ATPases (DEAD/H proteins) is required for spliceosome assembly, but not for the subsequent transesterification reactions . Little is known about the function of these ATPases in relation to the RNA conformational changes that occur in formation of active structures, in which U2/U6 small nuclear RNA (snRNA) interactions are essential for splicing to take place . Using a synthetic lethal genetic screen, we have isolated four yeast splicing factors involved in U2/U6 snRNA interactions (D.X . et al., manuscript in preparation) . The RNA-dependent ATPase activity associated with one such factor, the Slt22 protein, is stimulated preferentially by annealed U2/U6 snRNAs . Both mutant slt22-1 and U2 snRNA cause a reduction in stimulation . The slt22-1 mutation blocks splicing at or before the first step, resulting in the accumulation of an unusual complex which lacks U5 snRNA . Our results indicate that the U2/U6 snRNA interactions facilitated by Slt22 are also involved in the interaction of U5 snRNA with the spliceosome. Biochemistry, 1996 Jun 18, 35(24), 8058 - 67 Iterative optimization of high-affinity protease inhibitors using phage display . 2 . Plasma kallikrein and thrombin; Markland W et al.; As discussed in the accompanying paper {Markland, W., Ley, A . C., & Ladner, R . C . (1996) Biochemistry 35, 8045-8057}, we generated libraries from the first Kunitz domain of human lipoprotein-associated coagulation inhibitor (LACI-D1) using multivalent M13 III display and derived potent inhibitors of human plasmin (PLA) by iterative variegation and selection . Here, we show that high-affinity, high-specificity binders to human plasma kallikrein (pKAL) and human thrombin (THBN) can be obtained starting from the identical library and employing the same iterative variegation procedures used to obtain PLA inhibitors . Lib#1 (allowing 31 200 variants involving five positions near the P1 residue of LACI-D1) and its pKAL-biased derivative, Lib#4 (allowing an additional 1600 variants at residues 31, 32, 34, and 39), were screened against pKAL, yielding potent inhibitors . One of these, EPI-K401, has Ki = 284 pM, very high specificity, and excellent stability . We used information from Lib#4 selectants to design Lib#5 (allowing 1.5 x 10(6) amino-acid sequences involving nine varied positions) from which we obtained an inhibitor (EPI-K503) having high affinity for pKAL (Ki = 40 pM) and retaining the high specificity of EPI-K401 . When we screened Lib#1 and its THBN-tailored derivative, Lib#6, against THBN, we obtained a different and very homogeneous population of selected molecules . The purified proteins derived from Lib#6 selectants bound to THBN-agarose beads but did not inhibit proteolytic activity of THBN, suggesting that these selectants bind to a site on THBN other than the catalytic site . Thus, a single large combinatorial library can serve as a source to obtain highly specific, high-affinity binding molecules for each of several targets . Furthermore, the results with THBN show that the binding of Kunitz domains to other proteins is not limited to the catalytic sites of trypsin-homologous proteases. Biochemistry, 1996 Jun 18, 35(24), 7890 - 4 Mutation of the conserved domains of two inositol polyphosphate 5-phosphatases; Jefferson AB et al.; Two short amino acid motifs, WXGDXNXR and PXWCDRXL, define a large family of inositol polyphosphate 5-phosphatases . We tested the importance of seven of these conserved amino acids to substrate binding and catalysis by mutating each to alanine in the platelet 75 kDa inositol polyphosphate 5-phosphatase II (5-phosphatase II) . Native and mutant forms of 5-phosphatase II were expressed in baculovirus-infected Sf9 cells, and the recombinant proteins were purified by Mono Q chromatography and studied for enzyme activity . Mutants D476A, N478A, D553A, and R554A had no detectable activity using all four known substrates for this enzyme . Mutants R480A, W551A, and I555A showed greatly reduced hydrolysis of Ins(1,4,5)P3 when compared to native enzyme {Km = 75 microM, Vm = 8300 nmol of Ins(1,4,5)P3 hydrolyzed min-1 (mg of protein)-1} . Mutants W551A and I555A had a Km for Ins(1,4,5)P3 hydrolysis similar to that of the native enzyme (35 microM and 81 microM, respectively), suggesting that these amino acids do not play a role in binding substrate . By contrast, mutant R480A had both increased Km (634 microM) and decreased Vm {855 nmol of Ins(1,4,5)P3 hydrolyzed min-1 (mg of protein)-1} . As judged by measurement of Km, mutant R480A retained normal binding of Ins(1,3,4,5)P4, suggesting that the arginine in motif 2 has a greater role in Ins(1,4,5)P3 binding than in Ins(1,3,4,5)P4 binding . Mutant I555A bound Ins(1,3,4,5)P4 with 8-fold reduced affinity . These mutations markedly reduced 5-phosphatase II hydrolysis of the three other substrates, Ins(1,3,4,5)P4, PtdIns(4,5)P2, and PtdIns(3,4,5)P3 . We also tested a mutation comparable to D553A, D460A, in the 110 kDa form of the signaling inositol polyphosphate 5-phosphatase (5SIP110) . 5SIP110 D460A had no detectable enzyme activity but retained the ability to bind GRB2 . These results are consistent with a role for these conserved amino acids in substrate binding and catalysis. Biochemistry, 1996 Jun 18, 35(24), 7705 - 14 Modulation of cyclobutane pyrimidine dimer formation in a positioned nucleosome containing poly(dA.dT) tracts; Schieferstein U et al.; We have used a defined-sequence nucleosome to concomitantly investigate the generation and location of DNA lesions in nucleosomes and their influence on nucleosome positioning (translational and rotational setting) . A 134 bp HISAT sequence from the yeast DED1 promoter, containing a polypyrimidine region (40 bp) with a T6-tract, two T5-tracts, and a T9-tract, was reconstituted in nucleosomes with a defined rotational setting . T-tracts adopt unusually rigid DNA structures in solution ("T-tract structure") and are hot spots of cyclobutane pyrimidine dimer (CPD) formation by UV light (254 nm) . DNA was irradiated with UV light before or after reconstitution . The CPD yields and distribution were analyzed by cleavage with T4 endonuclease V . The rotational setting of nucleosomal DNA was characterized by DNase I digestion . With the exception of one T5-tract (1T5), the T6-, the 2T5-, and the T9-tracts formed T-tract structure in solution . T-tract structure was lost upon folding in nucleosomes, demonstrating a dominant constraint of DNA folding in nucleosomes over that of T-tract structure . CPD formation was strongly modulated by the nucleosome structure, but the CPD distribution differed from that reported for mixed-sequence DNA . CPD formation in the nucleosome had no effect on the rotational setting of nucleosomal DNA, but the rotational setting was affected when nucleosomes were assembled on damaged DNA . The toleration of DNA distortions imposed by CPDs in nucleosomes may have important implications for the recognition and repair of these damages in chromatin. FEBS Lett, 1996 Jun 17, 388(2-3), 185 - 8 MBA1 encodes a mitochondrial membrane-associated protein required for biogenesis of the respiratory chain; Rep M et al.; The yeast MBA 1 gene (Multi-copy Bypass of AFG3) is one of three genes whose overexpression suppresses afg3-null and rca1-null mutations . Bypass of AFG3 and RCA1, whose products are essential for assembly of mitochondrial inner membrane enzyme complexes, suggests a related role for MBA1 . The predicted translation product is a 30 kDa hydrophilic protein with a putative mitochondrial targeting sequence and no homology to any sequence in protein or EST databases . Gene disruption leads to a partial respiratory growth defect, which is more pronounced at temperatures above 30 degrees C . Concomitantly, amounts of cytochromes b and aa3 are reduced . A C-terminal c-myc-tagged MBA1 gene product is functional and is found associated with the mitochondrial inner membrane, from which it can he extracted by carbonate, but not by high salt . These observations give further support to a role of MBA1 in assembly of the respiratory chain. EMBO J, 1996 Jun 17, 15(12), 3135 - 43 RNA-DNA hybrid formation at the human mitochondrial heavy-strand origin ceases at replication start sites: an implication for RNA-DNA hybrids serving as primers; Xu B et al.; Critical elements of a mammalian mitochondrial DNA heavy-strand replication origin include a promoter and three downstream conserved sequence blocks (CSBIII, CSBII and CSBI) . We found recently that a stable and persistent RNA-DNA hybrid forms during in vitro transcription at Saccharomyces cerevisiae mitochondrial origins; hybrid formation was dependent on the conserved CSBII element . We report here that during in vitro transcription with human mitochondrial RNA polymerase, stable and persistent RNA-DNA hybrid formation is also evident at the human mitochondrial heavy-strand origin . As predicted, hybrid formation was dependent on the GC-rich CSBII element . The human RNA-DNA hybrids terminate within or downstream of CSBI at locations implicated in initiation of mitochondrial DNA replication . Interestingly, efficient hybrid formation in the human system is influenced by sequence 5' to the RNA-DNA hybrid, including the CSBIII element . These results suggest that the RNA-DNA hybrids formed during transcription across the mitochondrial DNA heavy-strand origin provide RNA primers for initiation of mitochondrial DNA replication. EMBO J, 1996 Jun 17, 15(12), 3028 - 39 Oligomerization and phosphorylation of the Ire1p kinase during intracellular signaling from the endoplasmic reticulum to the nucleus; Shamu CE et al.; The transmembrane kinase Ire1p is required for activation of the unfolded protein response (UPR), the increase in transcription of genes encoding endoplasmic reticulum (ER) resident proteins that occurs in response to the accumulation of unfolded proteins in the ER . Ire1p spans the ER membrane (or the nuclear membrane with which the ER is continuous), with its kinase domain localized in the cytoplasm or in the nucleus . Consistent with this arrangement, it has been proposed that Ire1p senses the accumulation of unfolded proteins in the ER and transmits the signal across the membrane toward the transcription machinery, possibly by phosphorylating downstream components of the UPR pathway . Molecular genetic and biochemical studies described here suggest that, as in the case of growth factor receptors of higher eukaryotic cells, Ire1p oligomerizes in response to the accumulation of unfolded proteins in the ER and is phosphorylated in trans by other Ire1p molecules as a result of oligomerization . In addition to its kinase domain, a C-terminal tail domain of Ire1p is required for induction of the UPR . The role of the tail is probably to bind other proteins that transmit the unfolded protein signal to the nucleus. Genomics, 1996 Jun 15, 34(3), 328 - 33 Identification and analysis of the human and murine putative chromatin structure regulator SUPT6H and Supt6h; Chiang PW et al.; We have isolated and sequenced SUPT6H and Supt6h, the human and murine homologues of the Saccharomyces cerevisiae and Caenorhabditis elegans genes SPT6 (P using 1603 aa = 6.7 e-95) and emb-5 (P using 1603 aa = 7.0 e-288), respectively . The human and murine SPT6 homologues are virtually identical, as they share >98% identity and >99% similarity at the protein level . The derived amino acid sequences of these two genes predict a 1603-aa protein (human) and a 1726-bp protein (mouse), respectively . There were several known features, including a highly acidic 5'-region, a degenerate SH2 domain, and a leucine zipper . These features are consistent with a nuclear protein that regulates transcription, whose extreme conservation underscores the likely importance of this gene in mammalian development . Expression of human and murine SPT6 homologues was analyzed by Northern blotting, which revealed a 7 . 0-kb transcript that was expressed constitutively . The SPT6 homologue was mapped to chromosome 17q11.2 in human by somatic cell hybrid analysis and in situ hybridization . These data indicate that SUPT6H and Supt6h are functionally analogous to SPT6 and emb-5 and may therefore regulate transcription through establishment or maintenance of chromatin structure. Nucleic Acids Res, 1996 Jun 15, 24(12), 2416 - 21 CTF5--a new transcriptional activator of the NFI/CTF family; Wenzelides S et al.; NFI/CTF is a family of polypeptides involved in stimulating the initiation of adenovirus DNA replication and the activation of transcription driven by RNA polymerase II . Several naturally occurring NFI/CTF variants display distinctive transactivation activities in vivo . To define more precisely the role of the NFI/CTF family in regulating gene expression, we cloned the splice variant CTF5, analyzed transcriptional activation patterns in a yeast transcription assay, and compared it with other CTF proteins . CTF5, which lacks exons 9 and 10 including a CTD-like motif essential for transcriptional activation by full-length CTF1, enhances transcription to a greater extent than CTF1 . In addition, CTF5 is even more active than CTF7, which lacks exons 7-9 . These findings indicate that CTF proteins formed by differential splicing display a much broader range of transcriptional activities as observed previously. Nucleic Acids Res, 1996 Jun 15, 24(12), 2324 - 30 Characterization of the interaction between the acidic activation domain of VP16 and the RNA polymerase II initiation factor TFIIB; Gupta R et al.; Contact between a transcriptional activator and one or more components of the RNA polymerase II transcription initiation machinery is generally believed important for activators to function . Several different molecular targets have been suggested for direct contact by herpes simplex virus virion protein VP16, including the general initiation factor TFIIB . In this report we have used several strategies to critically assess this interaction between VP16 and TFIIB . Affinity columns of VP16 bound TFIIB activity from HeLa cell extracts and the binding was reduced by mutations in the activation domain of VP16 . In assays of direct binding, VP16 bound recombinant human TFIIB but not Drosophila or yeast TFIIB . Unlike binding from an extract, however, we found that the interaction between VP16 and recombinant human TFIIB was not affected by mutations in VP16 that reduce transactivation . Point mutations within human TFIIB that reduce transactivation by VP16 have been shown to reduce VP16 binding, but we show here that these same mutations critically affect both the important TBP-TFIIB interaction and the ability of TFIIB to support activator-independent basal transcription in vitro . Taken together our results suggest more evidence is needed to support the notion that TFIIB is a functionally important target for the activator VP16. Genes Dev, 1996 Jun 15, 10(12), 1491 - 502 Mammalian p50Cdc37 is a protein kinase-targeting subunit of Hsp90 that binds and stabilizes Cdk4; Stepanova L et al.; CDC37, an essential gene in Saccharomyces cerevisiae, interacts genetically with multiple protein kinases and is required for production of