S. cerevisiae

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 Cdc28p/cyclin complexes through an unknown mechanism . We have identified mammalian p50Cdc37 as a protein kinase-targeting subunit of the molecular chaperone Hsp90 . Previously, p50 was observed in complexes with pp60v-src and Raf-1, but its identity and function have remained elusive . In mouse fibroblasts, a primary target of Cdc37 is Cdk4 . This kinase is activated by D-type cyclins and functions in passage through G1 . In insect cells, Cdc37 is sufficient to target Hsp90 to Cdk4 and both in vitro and in vivo, Cdc37/Hsp90 associates preferentially with the fraction of Cdk4 not bound to D-type cyclins . Cdc37 is coexpressed with cyclin Dl in cells undergoing programmed proliferation in vivo, consistent with a positive role in cell cycle progression . Pharmacological inactivation of Cdc37/Hsp90 function decreases the half-life of newly synthesized Cdk4, indicating a role for Cdc37/Hsp90 in Cdk4 stabilization . This study suggests a general role for p50Cdc37 in signaling pathways dependent on intrinsically unstable protein kinases and reveals a previously unrecognized chaperone-dependent step in the production of Cdk4/cyclin D complexes.

Genes Dev, 1996 Jun 15, 10(12), 1479 - 90
Activator-dependent regulation of transcriptional pausing on nucleosomal templates; Brown SA et al.; Promoter-proximal pausing during transcriptional elongation is an important way of regulating many diverse genes, including human c-myc and c-fos, some HIV genes, and the Drosophila heat shock loci . To characterize the mechanisms that regulate pausing, we have established an in vitro system using the human hsp7O gene . We demonstrate that nucleosome formation increases by >100-fold the duration of a transcriptional pause on the human hsp7O gene in vitro at the same location as pausing is observed in vivo . Readthrough of this pause is increased by an activator that contains the human heat shock factor 1 (HSF1) transcriptional activation domains . Maximal effect of the activator requires that the system be supplemented with fractions that have hSWI/SNF activity, which has been shown previously to alter nucleosome structure . No significant readthrough is observed in the absence of activator, and neither the activator nor the hSWI/SNF fraction affected elongation on naked DNA; therefore, these results suggest that an activator can cause increased readthrough of promoter-proximal pausing by decreasing the inhibitory effect of nucleosomes on transcriptional elongation.

FEMS Microbiol Lett, 1996 Jun 15, 140(1), 77 - 83
Trehalose-6-phosphate synthase A affects citrate accumulation by Aspergillus niger under conditions of high glycolytic flux; Arisan-Atac I et al.; Accumulation of citric acid by Aspergillus niger depends on a high flux through glycolysis . We have investigated the possibility of control of this flux by trehalose 6-phosphate, an inhibitor of hexokinase of Saccharomyces cerevisiae and other eukaryotes (Blasquez et al., FEBS Lett . (1993) 329, 51-54) . Hexokinase of A . niger was shown in vitro to be only weakly inhibited by trehalose 6-phosphate (KI 1.5-2 mM) . To investigate the in vivo relevance of this inhibition, we used isogenic strains of A . niger, carrying either a disruption or an amplification of the trehalose-6-phosphate synthase A (T6PSA)-encoding gene (ggsA) and exhibiting corresponding differences in T6PSA activity . These strains produced citric acid at comparable rates and with similar yields on 1 or 2.5% (w/v) sucrose . At 5-14% (w/v) sucrose, the ggsA disrupted strain initiated citric acid accumulation earlier, whereas the multicopy strain showed the reverse effect . When sucrose was replaced by lactose, which enabled only low rates of catabolism irrespective of its concentration (1-8%), no differences in the initiation or rate of citric acid accumulation were observed between the three strains . The possible mechanisms by which ggsA controls glycolytic flux in A . niger in the presence of high sugar concentrations are discussed.

Cell Immunol, 1996 Jun 15, 170(2), 202 - 11
Changes in superoxide anion production and phagocytosis by circulating neutrophils during tumor progression in a rat model; Szucs S et al.; The functional state of circulating neutrophils was monitored in a rat model of mesoblastic nephroma during tumor progression . Superoxide anion (O2.-) production in response to PMA and phagocytosis of yeast particles (Saccharomyces cerevisiae) were measured every second day after tumor cell implantation . Both phagocytosis and PMA-induced 02.- generation were found to be enhanced in the first period (on Days 6, 8, and 10), while they became significantly reduced in the advanced stage of cancer (on Days 12, 14, 16, and 18) . The suppression of PMNL functions was accompanied with tumor progression and an increased number of neutrophils in the peripheral blood . Studies were also carried out on PMNLs isolated from normal rats and the cells were treated with plasma samples obtained from tumor-bearing animals at different stages of nephroma . Incubation of the normal cells with plasmas separated on the 2nd and 8th days of tumor growth influenced neither the 02.- generation nor the phagocytosis . In contrast, plasma preparations obtained on the 14th day significantly inhibited both 02.- production and phagocytosis by normal neutrophils . The alterations in 02'- generation and phagocytosis by PMNLs were observed in close association with tumor growth, thus they could be considered as indicators of tumor progression . However, further studies are required to see whether the granulocyte dysfunctions observed in our animal model could provide additional prognostic information in the case of human malignancies as well as to clarify the origin of inhibitory factor(s) present in the blood of tumorous animals.

CMAJ, 1996 Jun 15, 154(12), 1871 - 3
The Ku autoantigen: cast in a new light; Robinson A; With his associates at the University of Ottawa, Dr . Robert Hache has demonstrated that the Ku autoantigen, a protein that plays a role in DNA repair and in immunoglobulin gene recombination, also modifies the action of steroid hormones . This finding has potential implications for the treatment of tumours whose growth is influenced by steroid hormones . The researchers suggest that Ku could be involved in the control of gene expression through a mechanism involving protein phosphorylation . They hope that a better understanding of the role Ku plays in many cellular processes may improve our understanding of autoimmune diseases, diseases of immune deficiency and cancer.

Cell, 1996 Jun 14, 85(6), 875 - 85
The YTA10-12 complex, an AAA protease with chaperone-like activity in the inner membrane of mitochondria; Arlt H et al.; The mitochondrial members of the highly conserved AAA family, Yta10p and Yta12p, constitute a membrane-embedded complex of about 850 kDa . As an ATP dependent metallopeptidase (AAA protease), the YTA10-12 complex mediates the degradation of nonassembled inner membrane proteins . In contrast to nucleotide-dependent complex formation and substrate binding, proteolysis of bound polypeptides depends on the hydrolysis of ATP and the metallopeptidase activity of both subunits . Independent of its proteolytic function, the chaperone-like activity of the YTA10-12 complex is required for assembly of the membrane-associated ATP synthase . We propose that proteolytic and chaperone-like activities in the YTA10-12 complex mediate assembly and degradation processes of membrane protein complexes and thereby exert key functions in the maintenance of membrane integrity.

J Biol Chem, 1996 Jun 14, 271(24), 13935 - 8
Expression cloning of a novel suppressor of the Lec15 and Lec35 glycosylation mutations of Chinese hamster ovary cells; Ware FE et al.; Lec15 and Lec35 are recessive Chinese hamster ovary (CHO) cell glycosylation mutations characterized by inefficient synthesis and utilization, respectively, of mannose-P-dolichol (MPD) . Consequently, Lec15 and Lec35 cells accumulate Man5GlcNAc2-P-P-dolichol and glucosaminyl-acylphosphatidylinositol . This report describes the cloning of a suppressor (termed SL15) of the Lec15 and Lec35 mutations from a CHO cDNA library by functional expression in Lec15 cells, employing phytohemagglutinin/swainsonine selection . The SL15 protein has a predicted molecular weight of 26,693 with two potential membrane spanning regions and a likely C-terminal endoplasmic reticulum retention signal (Lys-Lys-Glu-Gln) . Lec15 cells transfected with SL15 have normal levels of MPD synthase activity in vitro and convert Man5GlcNAc2-P-P-dolichol to Glc0-3Man9GlcNAc2-P-P-dolichol in vivo . Surprisingly, SL15 also corrects the defective mannosylation in Lec35 cells . The SL15 protein bears no apparent similarity to Saccharomyces cerevisiae MPD synthase (the DPM1 protein), but is highly similar to the hypothetical F38E1.9 protein encoded on Caenorhabditis elegans chromosome 5 . These results indicate a novel function for the SL15 protein and suggest that MPD synthesis is more complex than previously suspected.

J Biol Chem, 1996 Jun 14, 271(24), 14098 - 104
Characterization of a Ku86 variant protein that results in altered DNA binding and diminished DNA-dependent protein kinase activity; Han Z et al.; Three proteins known to play a critical role in mammalian DNA double-strand break repair and lymphoid V(D)J recombination are the autoantigens Ku86 and Ku70 and a 465-kDa serine/threonine protein kinase catalytic subunit (DNA-PKcs) . These proteins physically associate to form a complex (DNA.PK) with DNA-dependent protein kinase activity . In this study, we demonstrate using electrophoretic mobility shift assays (EMSAs) that the nuclear DNA end-binding activity of Ku is altered in the human promyelocytic leukemic HL-60 cell line . Western blot and EMSA supershift analyses revealed that HL-60 cells expressed both full-length and variant Ku86 proteins . However, a combined EMSA and immunoanalysis revealed that the Ku heterodimers complexed with DNA in HL-60 cells contained only the variant Ku86 proteins . Finally, UV cross-linking experiments and DNA.PK assays demonstrated that the Ku complexes containing variant Ku86 had a greatly reduced ability to interact with DNA-PKcs and that consequently HL-60 cells had severely diminished DNA.K activity . These data provide important insights into the interaction between Ku and DNA-PKcs and into the role of DNA.PK in DNA double-strand break repair.

J Biol Chem, 1996 Jun 14, 271(24), 14430 - 7
Cloning and characterization of a novel serine/threonine protein kinase expressed in early Xenopus embryos; Su JY et al.; We have cloned from a Xenopus ovary cDNA library a novel protein kinase gene whose expression peaks in the oocyte and unfertilized egg, begins to decrease gradually after fertilization, and disappears during the gastrulation stage of embryogenesis . The cloned gene, termed XEEK1 (for Xenopus egg and embryo kinase), encodes a protein with a predicted molecular mass of 49 kDa . Bacterially expressed XEEK1 migrates at 57 kDa upon polyacrylamide gel electrophoresis analysis, and a XEEK1-specific antibody recognizes a protein of 57 kDa in Xenopus oocyte and egg extracts . The XEEK1 kinase domain shares 35% identity (approximately 65% similarity) with the yeast SNF1 kinase and related kinases . However, expression of XEEK1 does not complement a snf1 deletion mutation in yeast, which suggests that it is probably not a Xenopus homolog of SNF1 . Recombinant XEEK1 protein autophosphorylates on threonine residues in vitro in a reaction that prefers Mn2+ to Mg2+ ions . Site-directed mutagenesis of the conserved lysine residue (Lys-81) within the kinase domain to isoleucine totally abolishes kinase activity, and threonine 192 has been identified as the autophosphorylation site . This site is distinct from the conserved threonine (Thr-215 in XEEK1) present in the protein kinase activation loop that is the site of autophosphorylation for many protein kinases . XEEK1 is a substrate for the cyclic AMP-dependent protein kinase both in vitro and in vivo, suggesting a possible mode of regulation of XEEK1 . An immunoprecipitate of oocyte/egg extracts with anti-XEEK1 serum contains a protein of approximately 155 kDa that may be a substrate and/or a regulatory component of the kinase.

Science, 1996 Jun 14, 272(5268), 1662 - 5
Activation of Gal4p by galactose-dependent interaction of galactokinase and Gal80p; Zenke FT et al.; Yeast galactokinase (Gal1p) is an enzyme and a regulator of transcription . In addition to phosphorylating galactose, Gal1p activates Gal4p, the activator of GAL genes, but the mechanism of this regulation has been unclear . Here, biochemical and genetic evidence is presented to show that Gal1p activates Gal4p by direct interaction with the Gal4p inhibitor Gal80p . Interaction requires galactose, adenosine triphosphate, and the regulatory function of Gal1p . These data indicate that Gal1p-Gal80p complex formation results in the inactivation of Gal80p, thereby transmitting the galactose signal to Gal4p.

Gene, 1996 Jun 12, 172(1), 25 - 31
Heterodimerization between two classes of homeodomain proteins in the mushroom Coprinus cinereus brings together potential DNA-binding and activation domains; Asante-Owusu RN et al.; The A mating type-genes of the mushroom, Coprinus cinereus, encode two classes of homeodomain-containing proteins distinguished as HD1 and HD2 on the basis of conserved, but distinctly different motifs . Compatible mating partners bring together versions of the proteins that can heterodimerize, thereby generating an active transcription factor complex that commits mated cells to sexual development . We have previously described a rare mutation in which an HD2::HD1 gene fusion generates a 'fused dimer' lacking much of HD1 including the homeodomain yet capable of constitutively promoting development {Kues et al., EMBO J . 13 (1994b) 4054-4059} . Here, we exploit this mutation to help identify contributions made by each protein class to normal heterodimer function . We show that the HD2 homeodomain is essential; deletion within the HD1 homeodomain can be tolerated in a normal heterodimer, as well as in the mutant fusion protein, but not substitution of a critical amino acid . We define, by deletion analysis, an essential C-terminal region of the HD1 and demonstrate its potential activation function by the ability to activate transcription in yeast when fused to the GAL4 DNA-binding domain . We also identify a potential role in transcriptional repression for the predicted C-terminal helix of HD1 proteins.

Gene, 1996 Jun 12, 172(1), 143 - 7
Missense mutations at the FKBP12-rapamycin-binding site of TOR1; Freeman K et al.; The TOR genes were first identified in Saccharomyces cerevisiae by the isolation of mutants which exhibit dominant resistance to the immunosuppressive and antifungal drug rapamycin (Rm) . The originally characterized Rm-resistant (RmR) TOR1-1 and TOR2-1 alleles contain an Arg in place of a conserved Ser residue, which lies adjacent to the phosphatidylinositol (PI) kinase-related domain of TOR (Ser1972 in TOR1; Ser1975 in TOR2) . Additional spontaneous RmR mutants containing Lys, Ile or Asn substitutions were subsequently isolated . As this Ser is a potential site for protein kinase C phosphorylation, we were interested in determining whether the observed RmR is due to steric hindrance of the FKBP12-Rm-TOR interaction or whether phosphorylation at this site is required to mediate the interaction . Using site-directed mutagenesis, we replaced the Ser1972 residue of TOR1 with either a conservative residue, Ala, an alternative potential phosphorylation site, Thr, or Asp to mimic phosphorylation . The TOR1 (S1972A) mutant protein retained Rm sensitivity (RmS), whereas both the Thr and Asp substitutions conferred RmR . RmS correlated with the ability to interact with FKBP12-Rm in a two-hybrid assay: both wild-type TOR1 and the S1972A mutant retained the ability to interact with FKBP12-Rm, whereas the S1972T, S1972D and S1972R mutants failed to interact . All mutant TOR1 proteins were able to complement the growth defect of tor1 null alleles, suggesting that the Ser1972 residue may not be required for TOR1 function in cycling cells . Since a TOR1(S1972A) mutant protein confers a RmS phenotype, interacts with FKBP12-Rm in a two-hybrid assay, and functions in vivo, we conclude that phosphorylation at Ser1972 is not necessary for the interaction between TOR1 and FKBP12-Rm.

Biochemistry, 1996 Jun 11, 35(23), 7459 - 65
Simultaneous binding and bending of promoter DNA by the TATA binding protein: real time kinetic measurements; Parkhurst KM et al.; The binding and bending of tetramethylrhodamine-5'-(GGGCTATAAAAGGG) duplex-3'-fluorescein by native Saccharomyces cerevisiae TATA binding protein (TBP) have been investigated using fluorescence resonance energy transfer . Probability distributions derived from fluorescein emission lifetime measurements show a decrease in the mean 3'-fluorescein-5'- rhodamine distance from 56.5 to 46.8 A upon binding of the oligomer to TBP, consistent with the DNA bend observed by X-ray crystallography . The kinetics, monitored in real time using stopped flow fluorimetry, demonstrate simultaneous binding and bending of a TATA box by TBP with a single second-order rate constant of (2.4 +/- 0.3) x 10(6) M-1 s-1 at 30 degrees C.

Biochemistry, 1996 Jun 11, 35(23), 7422 - 8
Unusual effects of an engineered disulfide on global and local protein stability; Betz SF et al.; The global and local stabilities of a eukaryotic ferricytochrome c variant with an engineered disulfide are examined . The disulfide connects position 20, which is usually a valine, to position 102, which is usually a threonine . The cross-linked variant is approximately 1.2 kcal mol-1 less stable than the wild-type protein at 298 K, pH 4.6, in H2O and D2O . Circular dichroism studies show that the decreased stability results from structure-induced stabilization of the denatured state {Betz, S . F., & Pielak, G . J . (1992) Biochemistry 31, 12337-12344} . Here, we use proton chemical shift, paramagnetic shift, and amide proton exchange data to obtain atomic level structural and energetic information . Chemical and paramagnetic shift data indicate only minor native state structural changes . Local stability is obtained from amide proton-deuterium exchange data, using model peptide intrinsic exchange rates . As expected, the exchange data indicate that cross-link incorporation decreases the majority of local stabilities . Near the cross-link, however, local stability seems to increase despite the overall global stability decrease . Furthermore, local stability changes for hydrophobic core residues seem to be greater than the global stability change . We interpret these observations as cross-link-induced changes in exchange competent states and relate them to changes in the denatured state.

Biochemistry, 1996 Jun 11, 35(23), 7403 - 11
Changing the transition state for protein (Un) folding; Doyle DF et al.; (Un)folding transition states of Saccharomyces cerevisiae iso-1-ferri- and ferrocytochromes c were studied using equilibrium and kinetic denaturation experiments . The wild-type protein and the global suppressor variant, N52I (isoleucine replaces asparagine 52), were examined . Denaturation was induced by guanidinium chloride (GdmCI) and monitored by circular dichroism (CD) spectropolarimetry without stopped-flow devices . Soret CD spectra indicate that thermal and GdmCl denatured states are different, and heat is the more effective denaturant . Equilibrium data show that the high stability of ferrocytochrome c can be rationalized as a requirement to bury the oxidation-induced positive charge and remain folded under physiological conditions . Kinetic data are monoexponential and permit characterization of the rate-limiting transition state for unfolding as a function of {GdmCl} . For the oxidized wild-type protein, the transition state solvent accessibility is nearly the same as that of the denatured state . Three perturbations, reducing the wild-type protein, reducing the N52I variant, and substituting position 52 in the oxidized protein, change the free energy and solvent accessibility of the transition state . In contrast, substituting position 52 in the reduced protein apparently does not change the transition state solvent accessibility, allowing more detailed characterization . In the reduced proteins' transition states at 4.3 M GdmCl, the position 52 side chain is in a denatured environment, even though transition state solvent accessibility is only one-third that of the denatured state (relative to the native state).

Biochemistry, 1996 Jun 11, 35(23), 7299 - 307
Influence of the carbohydrate moiety on the stability of glycoproteins; Wang C et al.; To study the role of oligosaccharides on the properties of glycoproteins, five glycoproteins (yeast external invertase, bovine serum fetuin, glucoamylase from Aspergillus niger, and chicken egg white ovotransferrin and avidin) of previously established glycan patterns were purified to homogeneity and deglycosylated with endo- and exo-glycosidases in native conditions . Thermal stability and conformational changes were measured by high-resolution differential scanning microcalorimetry and circular dicroism spectroscopy before and after they were deglycosylated . It was found that deglycosylation decreases protein thermal stability, as judged by the decrease in denaturation temperature and denaturation enthalpy, while it does not affect substantially the conformation as indicated by the CD spectra in the far UV range . The destabilization effect of deglycosylation seems to depend on the carbohydrate content, i.e., the maximum effect was observed for the most heavily glycosylated protein, irrespective of the types (N-linked or O-linked) or patterns (mono- or multi-branched) of the covalently attached carbohydrate chains . In addition, studies of the reversibility to heat denaturation revealed that deglycosylated proteins have a poorer thermal reversibility in calorimetric scans than their native counterparts and tend to aggregate during thermal inactivation at acidic pH . These results suggest that carbohydrate moieties, in addition to the apparent stabilizing effect, may prevent the unfolded or partially folded protein molecules from aggregation . Our results support the hypothesis that the general function of protein glycosylation is to aid in folding of the nascent polypeptide chain and in stabilization of the conformation of the mature glycoprotein.

Mol Cell Biochem, 1996 Jun 7, 159(1), 1 - 6
Long-chain fatty Acyl-CoA synthetase enzymatic activity in rat liver cell nuclei; Ves-Losada A et al.; A long-chain fatty acyl-CoA synthetase that catalyzes the activation of long-chain fatty acids as thioesters of CoA, was described in rat liver nuclei . This is the first step for further metabolization of fatty acids in the cell . Up to now, it has been shown that long-chain fatty acyl-CoA synthetase is located in the endoplasmic reticulum, in plasma membrane, in mitochondria and in peroxisomes . The nuclear long-chain fatty acyl-CoA synthetase was assayed using palmitic (16:0), linoleic (18:2n-6) and 8,11,14-eicosatrienoic (20:3n-6) acids as substrates and was stimulated linearly with nuclear protein concentration and with incubation time The higher enzymatic activity was observed with 18:2n-6 and 20:3n-6 acids as substrates . The synthesis of palmitoyl-CoA, linoleyl-CoA and 8,11,14-eicosatrienoyl-CoA followed normal Michaelis-Menten kinetics with respect to the corresponding substrate concentrations . The acyl-CoA synthetase seems to be saturated at a substrate concentration of 12.8 microM for all the acids tested . The apparent Km values decreased in the following order 20:3n-6 > 18:2n-6 > 16:0 . The lowest apparent Km for palmitic acid indicates a preference for acylation of this acid in the cell nucleus.

J Biol Chem, 1996 Jun 7, 271(23), 13861 - 7
Human Ku autoantigen binds cisplatin-damaged DNA but fails to stimulate human DNA-activated protein kinase; Turchi JJ et al.; We have identified a series of proteins based on an affinity for cisplatin-damaged DNA . One protein termed DRP-1 has been purified to homogeneity and was isolated as two distinct complexes . The first complex is a heterodimer of 83- and 68-kDa subunits, while the second complex is a heterotrimer of 350-, 83-, and 68-kDa subunits in a 1:1:1 ratio . The 83- and 68-kDa subunits in each complex are identical . The 83-kDa subunit of DRP-1 was identified as the p80 subunit of Ku autoantigen by N-terminal protein sequence analysis and reactivity with a monoclonal antibody directed against human Ku p80 subunit . The 68-kDa subunit of DRP-1 cross-reacted with monoclonal antisera raised against the Ku autoantigen p70 subunit . The 350-kDa subunit was identified as DNA-PKcs, the catalytic subunit of the human DNA-activated protein kinase, DNA-PK . DRP-1/Ku DNA binding was assessed in mobility shift assays and competition binding assays using cisplatin-damaged DNA . Results indicate that DNA binding was essentially unaffected by cisplatin-DNA adducts in the presence or absence of DNA-PKcs . DNA-PK activity was only stimulated with undamaged DNA, despite the ability of Ku to bind to cisplatin-damaged DNA . The lack of DNA-PK stimulation by cisplatin-damaged DNA correlated with the extent of cisplatin-DNA adduct formation . These results demonstrate that Ku can bind cisplatin-damaged DNA but fails to activate DNA-PK . These results are discussed with respect to the repair of cisplatin-DNA adducts and the role of DNA-PK in coordinating DNA repair processes.

Science, 1996 Jun 7, 272(5267), 1495 - 7
A subfamily of P-type ATPases with aminophospholipid transporting activity; Tang X et al.; The appearance of phosphatidylserine on the surface of animal cells triggers phagocytosis and blood coagulation . Normally, phosphatidylserine is confined to the inner leaflet of the plasma membrane by an aminophospholipid translocase, which has now been cloned and sequenced . The bovine enzyme is a member of a previously unrecognized subfamily of P-type adenosine triphosphatases (ATPases) that may have diverged from the primordial enzyme before the separation of the known families of ion-translocating ATPases . Studies in Saccharomyces cerevisiae suggest that aminophospholipid translocation is a general function of members of this family.

Science, 1996 Jun 7, 272(5267), 1489 - 92
Synergistic activation of estrogen receptor with combinations of environmental chemicals; Arnold SF et al.; Certain chemicals in the environment are estrogenic . The low potencies of these compounds, when studied singly, suggest that they may have little effect on biological systems . The estrogenic potencies of combinations of such chemicals were screened in a simple yeast estrogen system (YES) containing human estrogen receptor (hER) . Combinations of two weak environmental estrogens, such as dieldrin, endosulfan, or toxaphene, were 1000 times as potent in hER-mediated transactivation as any chemical alone . Hydroxylated polychlorinated biphenyls shown previously to synergistically alter sexual development in turtles also synergized in the YES . The synergistic interaction of chemical mixtures with the estrogen receptor may have profound environmental implications . These results may represent a previously uncharacterized level of regulation of estrogen-associated responses.

Science, 1996 Jun 7, 272(5267), 1473 - 6
Evidence that Spt6p controls chromatin structure by a direct interaction with histones; Bortvin A et al.; Genetic analysis has implicated SPT6, an essential gene of Saccharomyces cerevisiae, in the control of chromatin structure . Mutations in SPT6 and particular mutations in histone genes are able to overcome transcriptional defects in strains lacking the Snf/Swi protein complex . Here it is shown that an spt6 mutation causes changes in chromatin structure in vivo . In addition, both in vivo and in vitro experiments provide evidence that Spt6p interacts directly with histones and primarily with histone H3 . Consistent with these findings, Spt6p is capable of nucleosome assembly in vitro.

Oncogene, 1996 Jun 6, 12(11), 2331 - 42
Transcriptional repression by the proto-oncogene BCL-6; Seyfert VL et al.; In up to 45% of reported cases of the non-Hodgkin's lymphoma, diffuse large cell lymphoma, there are translocations of the BCL-6 gene, which are presumed to deregulate its expression . The BCL-6 protein, which is unmutated in these lymphomas, contains six Kruppel-like zinc fingers at its carboxy terminus and a 121 amino acid domain at its amino terminus, termed the POZ domain, which bears homology with amino terminal domains in a subset zinc finger transcription factors . In this study, we tested whether BCL-6 regulates transcription and if the POZ domain has a role in this function . The BCL-6 POZ domain, when fused to the GAL4 DNA binding domain, strongly repressed transcriptional activation initiated from several different promoters including the SV40 enhancer/promoter . Repression was also observed when the fusion protein was bound at a distance of 200 bp 5' of the promoter . When the GAL4/BCL6 POZ domain fusion protein was expressed in yeast, it was able to homodimerize in the nucleus . Nevertheless, in contrast with mammalian cells, the fusion protein did not repress transcription . To test the ability of the full length BC1-6 protein to repress transcription when bound to DNA through its zinc finger DNA binding domain, high affinity BCL-6 binding sites were selected from a pool of random oligonucleotides . Full length BCL-6 was able to strongly repress transcription when bound to its cognate site cloned upstream of the thymidine kinase promoter . This repression was mediated, in large measure, by the POZ domain, although a variant of BCL-6 lacking the POZ domain was able to repress transcription modestly . The ability of BCL-6 to function as a transcriptional repressor may contribute to its ability to transform B lymphocytes in diffuse large cell lymphoma.

Biochem Biophys Res Commun, 1996 Jun 5, 223(1), 48 - 53
Tissue specific expression of testis angiotensin converting enzyme is not determined by the -32 nonconsensus TATA motif; Zhou Y et al.; Testis ACE is an isozyme of angiotensin-converting enzyme (ACE) made by male germ cells . These cells recognize a small intragenic promoter which contains a positive regulatory element, TCTTAT, at position -32 . A probe containing this element was gel shifted to an identical location by rat testis and rat liver nuclear extracts; formation of the complex was blocked by anti-TATA binding protein antibody . Recombinant TATA binding protein recognized the testis ACE TCTTAT motif as well as a probe containing the consensus TATA motif . Mice transgenic for testis ACE promoter constructs containing either the wild type testis ACE motif or a consensus TATA sequence expressed a reporter gene at high levels only within the testis . These data suggest that the testis ACE motif TCTTAT is a non-consensus TATA, but is not responsible for the highly restricted pattern of testis ACE gene expression.

Biochemistry, 1996 Jun 4, 35(22), 7131 - 41
Studies on the hydrolytic properties of (serine) carboxypeptidase Y; Stennicke HR et al.; The activity of serine carboxypeptidases is dependent on a catalytic triad, an oxyanion hole, and a binding site equivalent to those found in the serine endopeptidases . The action of carboxypeptidase Y on substrates containing amino acids, alcohols, and amines as leaving groups is described . It is demonstrated that the features common to serine endopeptidases and carboxypeptidases are sufficient for hydrolysis of ester bonds . However, rapid hydrolysis of amide bonds is dependent on interactions between the C-terminal carboxylate group of the substrate and the C-terminal recognition site of the enzyme . Furthermore, on the basis of the pH dependencies of wild-type and mutant enzyme, combined with the ability of the enzyme to utilize binding energy to promote catalysis, alternative models for the high activity of carboxypeptidase Y at low pH are discussed . They describe how the catalytically essential histidine is maintained in its active deprotonated state through perturbation of its pKa value in the enzyme-substrate complex.

EMBO J, 1996 Jun 3, 15(11), 2810 - 9
Utilizing the GCN4 leader region to investigate the role of the sequence determinants in nonsense-mediated mRNA decay; Ruiz-Echevarria MJ et al.; In the yeast Saccharomyces cerevisiae, premature translation termination promotes rapid degradation of mRNAs . Accelerated decay requires the presence of specific cis-acting sequences which have been defined as downstream elements . It has been proposed that the role of the downstream element may be to promote translational reinitiation or ribosomal pausing . The GCN4 gene produces an mRNA that contains four short upstream open reading frames (uORFs) preceding the GCN4 protein-coding region in which translational initiation and reinitiation events occur . It was anticipated that these uORFs would function in a manner analogous to nonsense codons, promoting rapid degradation of the mRNA . However, the GCN4 transcript was not degraded by the nonsense-mediated mRNA decay pathway . We have investigated the role of the leader region of the GCN4 transcript in an effort to identify possible sequence elements that inactivate this decay pathway . We show that the GCN4 leader region does not harbor a downstream element needed to promote mRNA decay . In addition, using hybrid GCN4-PGK1 transcripts, we demonstrate that if a translational reinitiation signal precedes a downstream element, the mRNA will no longer be sensitive to nonsense-mediated decay . Furthermore, we demonstrate that the downstream element is functional only after a translational initiation and termination cycle has been completed but is unable to promote nonsense-mediated mRNA decay if it is situated 5' of a translational initiation site . Based on these results, the role of the downstream element will be discussed.

EMBO J, 1996 Jun 3, 15(11), 2668 - 77
Differential requirement for the mitochondrial Hsp70-Tim44 complex in unfolding and translocation of preproteins; Voos W et al.; The mitochondrial heat shock protein Hsp70 is essential for import of nuclear-encoded proteins, involved in both unfolding and membrane translocation of preproteins . mtHsp70 interacts reversibly with Tim44 of the mitochondrial inner membrane, yet the role of this interaction is unknown . We analysed this role by using two yeast mutants of mtHsp70 that differentially influenced its interaction with Tim44 . One mutant mtHsp70 (Ssc1-2p) efficiently bound preproteins, but did not show a detectable complex formation with Tim44; the mitochondria imported loosely folded preproteins with wild-type kinetics, yet were impaired in unfolding of preproteins . The other mutant Hsp70 (Ssc1-3p') bound both Tim44 and preproteins, but the mitochondria did not import folded polypeptides and were impaired in import of unfolded preproteins; Ssc1-3p' was defective in its ATPase domain and did not undergo a nucleotide-dependent conformational change, resulting in permanent binding to Tim44 . The following conclusions are suggested . (i) The import of loosely folded polypeptides (translocase function of mtHsp70) does not depend on formation of a detectable Hsp70-Tim44 complex . Two explanations are possible: a trapping mechanism by soluble mtHsp70, or a weak/very transient interaction of Ssc1-2p with Tim44 that leads to a weak force generation sufficient for import of loosely folded, but not folded, polypeptides . (ii) Import of folded preproteins (unfoldase function of mtHsp70) involves a reversible nucleotide-dependent interaction of mtHsp70 with Tim44, including a conformational change in mtHsp70 . This is consistent with a model that the dynamic interaction of mtHsp70 with Tim44 generates a pulling force on preproteins which supports unfolding during translocation.

J Pharm Biomed Anal, 1996 Jun, 14(8-10), 1007 - 13
Toxicity order of cholanic acids using an immobilised cell biosensor; Campanella L et al.; There is considerable published evidence of the use of cells of various species to evaluate the toxicity of numerous compounds, many of pharmaceutical interest . The coupling of cell colonies with a suitable transduction device has led to the development in recent years of toxicity biosensors based on the alteration of a process or a cell metabolic function by the toxic substance under examination . A biosensor based on immobilised yeast cells (Saccharomyces cerevisiae) has been developed recently in this department for the purpose of performing a rapid toxicity test in aqueous environmental matrices . This biosensor has now been used in the toxicity screening of a number of sodium salts of conjugated and free cholanic acids . The "toxicity degree" scale, which was found by placing in decreasing order the values of the slopes of the straight lines obtained by quantifying changes in the behaviour of the respirometric curve, plotted before and after incubation, using known concentrations of cholanic acid sodium salts, was: deoxycholic acid > chenodeoxycholic acid > ursodeoxycholic acid > cholic acid, for free cholanic acids; and glycodeoxycholic acid > glycochenodeoxycholic acid > glycocholic acid, for glycocholanic acids . These values are in good agreement with published toxicity data obtained in vitro . This sensor can thus be considered to provide a valid instrument for the preliminary evaluation of the toxicity of organic compounds or drugs.

Mol Biol Cell, 1996 Jun, 7(6), 917 - 34
Pleiotropic nuclear defects associated with a conditional allele of the novel nucleoporin Rat9p/Nup85p; Goldstein AL et al.; In a screen for mutants defective in nucleocytoplasmic export of mRNA, we have identified a new component of the Saccharomyces cerevisiae nuclear pore complex (NPC) . The RAT9/NUP85 (ribonucleic acid trafficking) gene encodes an 84.9-kDa protein that we have localized to NPCs by tagging the RAT9/NUP85 gene with the in vivo molecular marker Green Fluorescent Protein . In cells containing either the rat9-1 allele or a complete deletion of the RAT9/NUP85 gene, poly(A)+ RNA accumulates rapidly in nuclei after a shift from 23 degrees C to 37 degrees C . Under these same conditions, rapid fragmentation of the nucleolus was also observed . At the permissive growth temperature in rat9-1 or RAT9 deletion strains, the nuclear envelope (NE) becomes detached from the main body of the nucleus, forming long thin double sheets of NE . NPCs within these sheets are clustered and some appear to be locked together between opposing sheets of NE such that their nucleoplasmic faces are in contact . The Rat9/Nup85 protein could not be detected in cells carrying a mutation of RAT2/NUP120, suggesting that Rat9p/Nup85p cannot be assembled into NPCs in the absence of Rat2p/Nup120p . In contrast,Rat9/ Nup85 protein was readily incorporated into NPCs in strains carrying mutant alleles of other nucleoporin genes . The possible role of Rat9p/Nup85p in NE integrity and the loss of nucleoporins when another nucleoporin is mutant or absent are discussed.

Mol Immunol, 1996 Jun, 33(9), 787 - 96
Ku is a general inhibitor of DNA-protein complex formation and transcription; Ono M et al.; Ku is a ubiquitous and abundant DNA binding protein . Recently, it has been shown that Ku plays a crucial role in double stranded-DNA (dsDNA) break repair such as occurs during the V(D)J recombination of Ig genes . Ku has also been found to provide DNA binding activity to the catalytic domain of DNA-PK which is known to phosphorylate several transcription factors, suggesting that Ku is a multifunctional protein that participates as a component of several functional DNA-protein complexes . Here, we examined the interaction of Ku with several DNA binding proteins . Firstly, the DNA binding interaction between Ku and well-characterized transcription factors (OTF-1, Sp-1, AP-1) was analysed by EMSA . Although sequence non-specific, Ku was strongly competitive with these sequence specific transcription factors on compatible DNA elements, displacing them because of its high affinity association with DNA ends . Secondly, to determine whether this competitive effect was functionally relevant, we tested Ku in an in vitro transcription system with the adenovirus major late promoter . We found that Ku inhibited transcription from linear, but not from circular template DNA . These results suggest that Ku inhibits transcription when it is able to bind to template DNA and that the inhibition is the result of Ku displacing specific transcription factors from DNA.

J Med Vet Mycol, 1996 Jun-Jul, 34(3), 163 - 9
Isolation and characterization of a calmodulin-encoding cDNA from the pathogenic fungus Histoplasma capsulatum; el-Rady J et al.; We describe in this paper the isolation and complete sequence of a calmodulin (CaM) encoding cDNA from the dimorphic pathogenic fungus Histoplasma capsulatum (GenBank accession U12505) . The deduced amino acid sequence was identical to the CaM of Aspergillus nidulans and had only one amino acid difference from the CaM of Neurospora crassa . Saccharomyces cerevisiae CaM, however, has only about 60% amino acid identity compared with H . capsulatum . These data further support the close relationship of Histoplasma to the filamentous ascomycetes . Histoplasma CaM was expressed as a single major transcript of approximately 1200 nt . in both the yeast and mould growth forms . CaM mRNA levels were approximately two-fold greater in the yeast than in the mould form of the organism . Southern blot analysis indicated that the H . capsulatum CaM gene is present in a single copy.

J Cell Sci, 1996 Jun, 109 ( Pt 6), 1575 - 83
The SAC3 gene encodes a nuclear protein required for normal progression of mitosis; Bauer A et al.; The SAC3 gene of Saccharomyces serevisiae has been implicated in actin function by genetic experiments showing that a temperature sensitive mutation in the essential actin gene (actl-1) can be suppressed by mutations in SAC3 . An involvement of SAC3 in actin function is further suggested by the observation that the actin cytoskeleton is altered in SAC3 mutants . Our fractionation experiments, however, point to a nuclear localization of Sac3p . On sucrose density gradients Sac3p co-fractionated with the nuclear organelle markers examined . Furthermore, Sac3p was enriched 10-fold in a nuclei preparation along with the nuclear protein Nop1p . In this report we further show that SAC3 function is required for normal progression of mitosis . SAC3 mutants showed a higher fraction of large-budded cells in culture, indicative of a cell cycle delay . The predominant population among the large-budded sac3 cells were cells with a single nucleus at the bud-neck and a short intranuclear spindle . This suggests that a cell cycle delay occurs in mitosis prior to anaphase . The observation that SAC3 mutants lose chromosomes with higher frequency than wild-type is another indication for a mitotic defect in SAC3 mutants . We further noticed that SAC3 mutants are more resistant against the microtubule destabilizing drug benomyl . This finding suggests that SAC3 is involved, directly or indirectly, in microtubule function . In summary, our data indicate that SAC3 is involved in a process which affects both the actin cytoskeleton and mitosis.

J Cell Sci, 1996 Jun, 109 ( Pt 6), 1297 - 310
Mutations which block the binding of calmodulin to Spc110p cause multiple mitotic defects; Stirling DA et al.; We have generated three temperature-sensitive alleles of SPC110, which encodes the 110 kDa component of the yeast spindle pole body (SPB) . Each of these alleles carries point mutations within the calmodulin (CaM) binding site of Spc110p which affect CaM binding in vitro; two of the mutant proteins fail to bind CaM detectably (spc110-111, spc110-118) while binding to the third (spc110-124) is temperature-sensitive . All three alleles are suppressed to a greater or lesser extent by elevated dosage of the CaM gene (CMD1), suggesting that disruption of CaM binding is the primary defect in each instance . To determine the consequences on Spc110p function of loss of effective CaM binding, we have therefore examined in detail the progression of synchronous cultures through the cell division cycle at the restrictive temperature . In each case, cells replicate their DNA but then lose viability . In spc110-124, most cells duplicate and partially separate the SPBs but fail to generate a functional mitotic spindle, a phenotype which we term 'abnormal metaphase' . Conversely, spc110-111 cells initially produce nuclear microtubules which appear well-organised but on entry into mitosis accumulate cells with 'broken spindles', where one SPB has become completely detached from the nuclear DNA . In both cases, the bulk of the cells suffer a lethal failure to segregate the DNA.

J Cell Sci, 1996 Jun, 109 ( Pt 6), 1241 - 51
The organization of ribosomal RNA processing correlates with the distribution of nucleolar snRNAs; Beven AF et al.; We have analyzed the organization of pre-rRNA processing by confocal microscopy in pea root cell nucleoli using a variety of probes for fluorescence in situ hybridization and immunofluorescence . Our results show that transcript processing within the nucleolus is spatially highly organized . Probes to the 5' external transcribed spacer (ETS) and first internal transcribed spacer (ITS1) showed that the excision of the ETS occurred in a sub-region of the dense fibrillar component (DFC), whereas the excision of ITS1 occurred in the surrounding region, broadly corresponding to the granular component . In situ labelling with probes to the snoRNAs U3 and U14, and immunofluorescence labelling with antibodies to fibrillarin and SSB1 showed a high degree of coincidence with the ETS pattern, confirming that ETS cleavage and 18 S rRNA production occur in the DFC . ETS, U14, fibrillarin and SSB1 showed a fine substructure within the DFC comprising closely packed small foci, whereas U3 appeared more diffuse throughout the DFC . A third snoRNA, 7-2/MRP, was localised to the region surrounding the ETS, in agreement with its suggested role in ITS1 cleavage . All three snoRNAs were also frequently observed in numerous small foci in the nucleolar vacuoles, but none was detectable in coiled bodies . Antibodies to fibrillarin and SSB1 labelled coiled bodies strongly, though neither protein was detected in the nucleolar vacuoles . During mitosis, all the components analyzed, including pre-rRNA, were dispersed through the cell at metaphase, then became concentrated around the periphery of all the chromosomes at anaphase, before being localized to the developing nucleoli at late telophase . Pre-rRNA (ETS and ITS1 probes), U3 and U14 were also concentrated into small bodies, presumed to be pre-nucleolar bodies at anaphase.

Curr Biol, 1996 Jun 1, 6(6), 730 - 8
ARF and PITP restore GTP gamma S-stimulated protein secretion from cytosol-depleted HL60 cells by promoting PIP2 synthesis; Fensome A et al.; BACKGROUND: In many cell types, including neutrophils and HL60 cells, there is an absolute requirement for a GTP-dependent step to elicit Ca(2+)-regulated secretion . Neutrophils and HL60 cells secrete lysosomal enzymes from azurophilic granules; this secretion is inhibited by 1% ethanol, indicating that phosphatidate (PA) produced by phospholipase D (PLD) activity may be involved . PLD can use primary alcohols in preference to water during the hydrolytic step, generating the corresponding phosphatidylalcohol instead of PA, its normal product . As ARF (ADP-ribosylation factor) proteins regulate PLD activity and are implicated in constitutive vesicular traffic, we have investigated whether ARF is also required for GTP-dependent secretion in HL60 cells . RESULTS: We have used a cell-permeabilization protocol that allows HL60 cells to become refractory to stimulation with GTP gamma S plus 10 microM Ca2+ with regard to secretion and PLD activity . Permeabilization with streptolysin O for 10 minutes permitted the loss of freely diffusable cytosolic proteins, including ARF proteins . Fractions derived from brain cytosol, enriched in ARF proteins, restored secretory function and PLD activity . The major contaminating protein present in these ARF-enriched fractions was identified as phosphatidylinositol transfer protein (PITP) . Unexpectedly, PITP was also found to restore GTP gamma S-dependent secretion . Restoration of secretory function was characterized using recombinant proteins, rARF1 and rPITP alpha and rPITP beta . The rARF1 protein restored both secretory function and PLD activity, whereas PITP only restored secretory function . However, both ARF and PITP were capable of stimulating phosphatidylinositol bis phosphate (PIP2) synthesis . CONCLUSIONS: ARF and PITP restore secretory function in cytosol-depleted cells when stimulated with GTP gamma S plus Ca2+ . We have previously shown that PITP participates in the synthesis of PIP2 . In comparison, ARF1 activates PLD, producing PA, which is a known activator of phosphatidylinositol-4-phosphate 5 kinase, the enzyme responsible for PIP2 synthesis . We propose that ARF and PITP both restore exocytosis by a common mechanism-promoting PIP2 synthesis.

Curr Opin Genet Dev, 1996 Jun, 6(3), 366 - 70
Genetic analysis of mechanisms of aging; Rose MR et al.; A wide range of genetic models with postponed aging are now available, from selected mice and Drosophilia to mutant Caenorhabditis elegans and Saccharomyces cerevisiae . These systems allow efficient testing of alternative mechanistic hypotheses for aging . Genetic analysis is forging stronger connections between particular alleles and susceptibility to particular 'diseases of aging'; for example, two different genes for Alzheimer disease have been identified.

Appl Environ Microbiol, 1996 Jun, 62(6), 1951 - 7
Analysis of heterologous protein production in defined recombinant Aspergillus awamori strains; Gouka RJ et al.; A study was carried out to obtain more insight into the parameters that determine the secretion of heterologous proteins from filamentous fungi . A strategy was chosen in which the mRNA levels and protein levels of a number of heterologous genes of different origins were compared . All genes were under control of the Aspergillus awamori 1,4-beta-endoxylanase A (exlA) expression signals and were integrated in a single copy at the A . awamori pyrG locus . A Northern (RNA) analysis showed that large differences occurred in the steady-state mRNA levels obtained with the various genes; those levels varied from high values for genes of fungal origin (A . awamori 1,4-beta-endoxylanase A, Aspergillus niger glucoamylase, and Thermomyces lanuginosa lipase) to low values for genes of nonfungal origin (human interleukin 6 and Cyamopsis tetragonoloba {guar} alpha-galactosidase) . With the C . tetragonoloba alpha-galactosidase wild-type gene full-length mRNA was even undetectable . Surprisingly, small amounts of full-length mRNA could be detected when a C . tetragonoloba alpha-galactosidase gene with an optimized Saccharomyces cerevisiae codon preference was expressed . In all cases except human interleukin 6, the protein levels corresponded to the amounts expected on basis of the mRNA levels . For human interleukin 6, very low protein levels were observed, whereas relatively high steady-state mRNA levels were obtained . Our data suggest that intracellular protein degradation is the most likely explanation for the low levels of secreted human interleukin 6.

J Mol Endocrinol, 1996 Jun, 16(3), 277 - 85
Mutational analysis of the estrogen receptor ligand-binding domain: influence of ligand structure and stereochemistry on transactivation; Kohno H et al.; The mouse estrogen receptor (mER) exhibits ligand stereochemical specificity for indenestrol A (IA), a stilbestrol estrogen . IA has a chiral C3 methyl group, and the mER preferentially binds the S-enantiomer (IA-S), resulting in elevated biological activity when compared with the IA-R enantiomer . To elucidate the mechanisms for this stereochemical recognition, we have constructed a series of mERs with individual amino acid substitutions at Met521, His528, Met532, and Val537 . The abilities of yeast-expressed wild-type and mutant mERs to transactivate an estrogen-responsive reporter gene construct were measured in the presence of diethylstilbestrol (DES) and IA enantiomers . The concentration of IA-S required to induce half-maximal transactivation by wild-type mER was 10-fold lower than IA-R, which is attributed to the 15-fold greater binding affinity for IA-S . Wild-type mER displayed similar dose-response curves for IA-R and demethyl IA, which lacks a C3 methyl group, demonstrating that the presence and correct orientation of the C3 methyl group on the IA compound is required for high-affinity ligand binding and transcriptional activity . Each mutant exhibited a reduced preference for IA-S enantiomer with respect to transactivation, suggesting that this region of the mER functions in ligand stereochemical recognition and activation . A mutation at Met532 diminished DES- and IA-S-induced transactivation by 7.5-fold and 40-fold respectively, with minimal change on their binding affinity . These data suggest that Met532 is required for transactivation induced by the potent agonist, IA-S, and the M532G mutation effectively uncouples IA-S ligand binding from transactivation . Use of these stereochemically different ligands in combination with mutagenesis of the mER demonstrates that ligand structure could influence transactivation by specifically altering the conformation of the mER AF-2 region.

Am J Physiol, 1996 Jun, 270(6 Pt 1), E995 - 1002
Rapid enhancement of acyl-CoA synthetase, LPL, and GLUT-4 mRNAs in adipose tissue of VMH rats; Shimomura I et al.; Obesity is frequently accompanied by metabolic and cardiovascular complications . The accumulation of intra-abdominal visceral fat has been shown to be more closely related to various complications of obesity than that of subcutaneous fat . To elucidate the metabolic characteristics of visceral fat during fat accumulation, we examined the changes of acyl-CoA synthetase (ACS) mRNA abundance and its activity, glucose transporter (GLUT)-4, lipoprotein lipase (LPL), and very-low-density lipoprotein (VLDL) receptor mRNA abundances in mesenteric and subcutaneous fat in early stages of ventromedial hypothalamus (VMH)-lesioned rats . ACS activity increased 4.9-fold in the mesenteric fat on the 1st day, remaining unchanged in the subcutaneous fat . ACS, GLUT-4, and LPL mRNA levels were all increased in both fat tissues of VMH rats . The relative increase of mRNAs in VMH day 1 was greater in the mesenteric fat, suggesting that mesenteric fat shows rapid response during fat accumulation . VLDL receptor mRNA levels showed no significant change in either fat tissue . We conclude that ACS, GLUT-4, and LPL may contribute to fat accumulation at the gene expression level from a very early stage during the development of obesity.

Sci China C Life Sci, 1996 Jun, 39(3), 243 - 50
Effect of nucleotides 37 and 38 on cleavage of tRNAPhe precursors; Liu J et al.; Splicing is required for tRNA maturation when the precursors contain the introns . In order to determine whether nucleotides 37 and 38 affect splicing, yeast tRNAPhe precursors with different nucleotides 37 and 38 were prepared by in vitro mutagenesis and cleaved by the purified yeast tRNA-splicing endonuclease . The precursors with purine nucleotides at N37 and N38 were found to be the best substrates for the enzyme . When N37 and N38 were replaced by pyrimidine nucleotides, few precursors could be cleaved by the endonuclease . If one is pyrimidine nucleotide, the other one is purine nucleotide at these positions, the cleavage efficiencies are between the two groups of precursors stated above . The pyrimidine nucleotides at these positions might affect the fine structures of the precursors or the distance between the splicing sites, so that the precursors can not be fixed or anchored on the enzyme well, leading to the poor cutting.

Curr Opin Cell Biol, 1996 Jun, 8(3), 369 - 73
Nucleosome assembly: the CAF and the HAT; Kaufman PD; Recent data argue strongly that a protein complex termed chromatin assembly factor-1 (CAF-I) plays a major role in de novo nucleosome assembly during DNA replication . Human CAF-I deposits newly synthesized, acetylated histones onto replicated DNA in vitro and localizes to sites of DNA replication in S-phase cells . Specific lysines of the histones used for nucleosome assembly are acetylated; in the past year the first gene encoding a histone acetyltransferase was cloned . However, mechanistic links between histone acetylation and nucleosome assembly have not been established in vivo or in vitro.

Curr Opin Cell Biol, 1996 Jun, 8(3), 354 - 7
Influences of the cell cycle on silencing; Fox CA et al.; Silencing in Saccharomyces cerevisiae is a form of transcriptional repression that involves the assembly of a specialized and heritable structure of chromatin . The HML and HMR loci, which contain copies of the genes found at the yeast mating-type locus, are silenced, as are telomeres . These examples share several features which are also found in position-effect variegation in flies and X-chromosome inactivation and genomic imprinting in mammals . Silenced chromatin is confined to a few special domains of the yeast genome, and active genes inserted into these domains become silenced . Molecular and genetic evidence has suggested that the establishment of silenced chromatin requires some S phase specific function . Recent experiments indicate that the assembly and maintenance of silenced chromatin can also be influenced at other phases of the cell cycle.

Genetics, 1996 Jun, 143(2), 755 - 67
Homologous and homeologous intermolecular gene conversion are not differentially affected by mutations in the DNA damage or the mismatch repair genes RAD1, RAD50, RAD51, RAD52, RAD54, PMS1 and MSH2; Porter G et al.; Mismatch repair (MMR) genes or genes involved in both DNA damage repair and homologous recombination might affect homeologous vs . homologous recombination differentially . Spontaneous mitotic gene conversion between a chromosome and a homologous or homeologous donor sequence (14% diverged) on a single copy plasmid was examined in wild-type Saccharomyces cerevisiae strains and in MMR or DNA damage repair mutants . Homologous recombination in rad51, rad52 and rad54 mutants was considerably reduced, while there was little effect of rad1, rad50, pms1 and msh2 null mutations . DNA divergence resulted in no differential effect on recombination rates in the wild type or the mutants; there was only a five to 10-fold reduction in homeologous relative to homologous recombination regardless of background . Since DNA divergence is known to affect recombination in some systems, we propose that differences in the role of MMR depends on the mode of recombination and/or the level of divergence . Based on analysis of the recombination breakpoints, there is a minimum of three homologous bases required at a recombination junction . A comparison of Rad+ vs . rad52 strains revealed that while all conversion tracts are continuous, elimination of RAD52 leads to the appearance of a novel class of very short conversion tracts.

RNA, 1996 Jun, 2(6), 535 - 50
Essential domains of the PRP21 splicing factor are implicated in the binding to PRP9 and PRP11 proteins and are conserved through evolution; Rain JC et al.; The yeast Prp9p, Prp11p, Prp21p proteins form a multimolecular complex identified as the SF3a splicing factor in higher eukaryotes . This factor is required for the assembly of the prespliceosome . Prp21p interacts with both Prp9p and Prp11p, but the molecular basis of these interactions is unknown . Prp21p, its human homologue, and the so-called SWAP proteins share a tandemly repeated motif, the surp module . Given the evolutionary conservation and the role of SWAP proteins as splicing regulators, it has been proposed that surp motifs are essential for interactions between Prp21p and other splicing factors . In order to characterize functional domains of Prp21p and to identify potential additional functions of this protein, we isolated a series of heat-sensitive prp21 mutants . Our results indicate that prp21 heat-sensitive mutations are associated with defects in the interaction with Prp9p, but not with Prp11p . Interestingly, most heat-sensitive point mutants associate a strong splicing defect with a pre-mRNA nuclear export phenotype, as does the prp9-1 heat-sensitive mutant . Deletion analyses led to the definition of domains required for viability . These domains are responsible for the interaction with Prp9p and Prp11p and are conserved through evolution . They do not include the most conserved surp1 module, suggesting that the conservation of this motif in two families of proteins may reflect a still unknown function dispensable in yeast under standard conditions.

Biochem J, 1996 Jun 1, 316 ( Pt 2), 647 - 54
Influence of amino acid residue 374 of cytochrome P-450 2D6 (CYP2D6) on the regio- and enantio-selective metabolism of metoprolol; Ellis SW et al.; Cytochrome P-450 2D6 (CYP2D6) is an important human drug-metabolizing enzyme responsible for the oxidation of more than 30 widely used therapeutic agents . The enzymes encoded by the published genomic {Kimura, Umeno, Skoda, Meyer and Gonzalez (1989) Am . J . Hum . Genet . 45, 889-904} and cDNA {Gonzalez, Skoda, Kimura, Umeno, Zanger, Nebert, Gelboin, Hardwick and Meyer (1988) Nature 331, 442-446} sequences of CYP2D6, and presumed to represent wild-type sequences, differ at residue 374 and encode valine (CYP2D6-Val) and methionine (CYP2D6-Met) respectively . The influence of this amino acid difference on cytochrome P-450 expression, ligand binding, catalysis and stereoselective oxidation of metoprolol was investigated by the heterologous expression of the corresponding cDNAs in the yeast Saccharomyces cerevisiae . The level of expression of apo- and holo-protein was similar with each form of CYP2D6 cDNA, and the binding affinities of a series of ligands to CYP2D6-Val and CYP2D6-Met were identical . The enantioselective O-demethylation and alpha-hydroxylation of metoprolol were also similar with each form of CYP2D6, O-demethylation being R-(+)- enantioselective (CYP2D6-Val: R/S, 1.6; CYP2D6-Met: R/S, 1.4), whereas alpha-hydroxylation showed a preference for S-(-)-metoprolol (CYP2D6-Val: R/S, 0.7; CYP2D6-Met: R/S, 0.8) . However, although the favoured regiomer overall was O-demethylmetoprolol (ODM), the regioselectivity for O-demethylation of each metoprolol enantiomer was significantly greater for CYP2D6-Val {R-(+)-: ODM/alpha-hydroxymetoprolol (alpha OH), 5.9; S-(-)-: ODM/alpha OH, 2.5) than that observed for CYP2D6-Met {R-(+)-: ODM/alpha OH, 2.2; S-(-)-: ODM/alpha OH, 1.4} . The stereoselective properties of CYP2D6-Val were consistent with those observed for CYP2D6 in human liver microsomes . The difference in the stereoselective properties of CYP2D6-Val and CYP2D6-Met were rationalized with respect to a homology model of the active site of CYP2D6 based on an alignment with the crystal structure of the bacterial cytochrome P-450BM-3' CYP102.

J Cell Biol, 1996 Jun, 133(6), 1163 - 76
Dynamic localization of the nuclear import receptor and its interactions with transport factors; Koepp DM et al.; Characterization of the interactions between soluble factors required for nuclear transport is key to understanding the process of nuclear trafficking . Using a synthetic lethal screen with the rna1-1 strain, we have identified a genetic interaction between Rna1p, a GTPase activating protein required for nuclear transport, and yeast importin-beta, a component of the nuclear localization signal receptor . By the use of fusion proteins, we demonstrate that Rna1p physically interacts with importin-beta . Mutants in importin-beta exhibit in vivo nuclear protein import defects, and importin-beta localizes to the nuclear envelope along with other proteins associated with the nuclear pore complex . In addition, we present evidence that importin-alpha, but not importin-beta, mislocalizes to the nucleus in cells where the GTPase Ran is likely to be in the GDP-bound state . We suggest a model of nuclear transport in which Ran-mediated hydrolysis of GTP is necessary for the import of importin-alpha and the nuclear localization signal-bearing substrate into the nucleus, while exchange of GDP for GTP on Ran is required for the export of both mRNA and importin-alpha from the nucleus.

J Cell Biol, 1996 Jun, 133(6), 1141 - 52
Nic96p is required for nuclear pore formation and functionally interacts with a novel nucleoporin, Nup188p; Zabel U et al.; The amino-terminal domain of Nic96p physically interacts with the Nsp1p complex which is involved in nucleocytoplasmic transport . Here we show that thermosensitive mutations mapping in the central domain of Nic96p inhibit nuclear pore formation at the nonpermissive temperature . Furthermore, the carboxyterminal domain of Nic96p functionally interacts with a novel nucleoporin Nup188p in an allele-specific fashion, and when ProtA-Nup188p was affinity purified, a fraction of Nic96p was found in physical interaction . Although NUP188 is not essential for viability, a null mutant exhibits striking abnormalities in nuclear envelope and nuclear pore morphology . We propose that Nic96p is a multivalent protein of the nuclear pore complex linked to several nuclear pore proteins via its different domains.

Nucleic Acids Res, 1996 Jun 1, 24(11), 2112 - 8
Transcriptional activation by Oct-3: evidence for a specific role of the POU-specific domain in mediating functional interaction with Oct-1; Vigano MA et al.; Oct-3, a member of the POU family of transcription factors, is expressed in pluripotent cells of early mammalian embryos and in undifferentiated embryonal carcinoma cell lines . Using a variety of Oct-3 mutants, we have identified two different domains of Oct-3 which activate transcription in transfected mammalian cells . One of these domains, located in the C-terminal part of the protein, plays a major role in transcriptional activation when Oct-3 is bound to its cognate site, the octamer motif . An Oct-3 mutant containing a single amino acid substitution in the POU homeodomain is unable to bind the octamer target in vitro, yet is still able to activate transcription in an octamer-dependent manner . We provide evidence that transactivation by this mutant involves protein-protein interactions with the ubiquitous octamer binding factor Oct-1 . This interaction requires the POU-specific domain of Oct-3 and allows recruitment of Oct-3 to the target promoter even in the absence of Oct-3 DNA binding.

Nucleic Acids Res, 1996 Jun 1, 24(11), 2053 - 8
Extrachromosomal recombination occurs efficiently in cells defective in various DNA repair systems; Morrison C et al.; A series of different frameshift mutations of a firefly luciferase reporter plasmid was created so that no activity was obtained when they were transfected into mammalian cells . Co-transfection of these constructs with short fragments of the original sequence resulted in luciferase activity in different cell lines (A-549, NIH 3T3 and Jurkat) . The level of this activity was dependent on the length of the fragment, regardless of cell line examined . Two different transfection techniques (lipofection and adenovirus-enhanced gene transfer) gave similar results . It was shown by polymerase chain reaction that expression of detectable luciferase required recombination of the transfected molecules . Cells with defined defects in DNA repair pathways were examined for their ability to perform this extrachromosomal recombination . Cells lacking normal Ku p80, (ADP-ribosyl)transferase, MLH1 or XP-C were all capable of restoring expression to the frameshifted constructs . Given the pivotal roles of the above molecules in the pathways of DNA repair, it seems that this recombination derives from a different activity.

J Immunol, 1996 Jun 1, 156(11), 4484 - 91
Calreticulin binds hYRNA and the 52-kDa polypeptide component of the Ro/SS-A ribonucleoprotein autoantigen; Cheng ST et al.; Calreticulin (CR) is a multifunctional, calcium-binding protein that has recently been shown to bind to and promote the replication of the rubella virus genome in mammalian cells . While CR is now widely recognized as a new human autoantigen, the relationship between CR and the Ro/SS-A ribonucleo-protein (RNP) autoantigen has been somewhat controversial . In this work, we demonstrate that unphosphorylated human rCR binds specifically and distinctly to in vitro transcribed forms of hYRNA, the RNA backbone of the Ro/SS-A RNP particle . This interaction appears to be mediated by binding through the N- and C-terminal domains of CR, but not by the central proline-rich domain . Furthermore, our studies indicate that CR can facilitate the binding of the 60-kDa polypeptide component of the Ro/SS-A RNP (Ro60) to hYRNA . In addition, CR and the 52-kDa Ro/SS-A polypeptide (Ro52) appear to be capable of interacting through direct protein-protein binding . These studies confirm that CR is an hYRNA-binding protein, and provide for the first time a molecular mechanism by which Ro52 can be linked physically to hYRNA . Through these molecular interactions and its known functional role as a chaperone, it is suggested that CR plays a supportive role in the formation of the Ro/SS-A RNP complex . The capacity of CR to interact with RNA viruses such as rubella provides an additional argument for an infectious trigger for autoantibody production against self RNP particles such as Ro/SS-A.

FASEB J, 1996 Jun, 10(8), 849 - 58
Biochemistry, molecular biology, and genetics of the oligosaccharyltransferase; Silberstein S et al.; Asparagine-linked glycosylation is a highly conserved protein modification reaction that occurs in all eukaryotes . The initial stage in the biosynthesis of N-linked glycoproteins, catalyzed by the enzyme oligosaccharyltransferase (OST), involves the transfer of a preassembled high-mannose oligosaccharide from a dolichol-linked oligosaccharide donor onto asparagine acceptor sites in nascent proteins in the lumen of the rough endoplasmic reticulum . Biochemical, molecular biological, and genetic studies conducted during the past 5 years have resulted in an explosive growth in our knowledge concerning the OST . Although the basic biochemical properties of the enzyme were determined more than a decade ago using intact microsomal membranes, recent studies provide novel insight into the catalytic mechanism of the enzyme . The OST was recently purified as a large heteroligomeric membrane protein complex; the sequences of many of the subunits have been determined from both fungal and vertebrate sources . Consistent with the evolutionary conservation of N-linked glycosylation, protein sequence comparisons reveal significant homologies between vertebrate, invertebrate, plant, and fungal OST subunits . Yeast molecular genetic methods have been instrumental in the functional characterization of the OST subunits, and have proven to be powerful tools for the identification of novel gene products that influence oligosaccharide transfer in vivo.

Curr Genet, 1996 Jun, 30(1), 62 - 7
Molecular cloning of thi-4, a gene necessary for the biosynthesis of thiamine in Neurospora crassa; Akiyama M et al.; The thiamine-4 (thi-4) gene was cloned by functional complementation of a thi-4 mutant of Neurospora crassa . The product of this gene is believed to be involved in the condensation of pyrimidine and thiazole precursors, which is necessary for the synthesis of thiamine . The thi-4 gene has ten introns which vary in length from 57 to 200 bp and the junction and internal (lariat) sequences are in good agreement with the consensus sequences for the splicing of introns in N . crassa . The thi-4 gene encodes a protein of 538 amino acids which is similar in terms of amino-acid sequence to proteins encoded by Saccharomyces cerevisiae whose function is unknown . The expression of the thi-4 gene in N . crassa was not repressed by thiamine.

Genomics, 1996 Jun 1, 34(2), 166 - 72
Cloning and characterization of a putative human holocytochrome c-type synthetase gene (HCCS) isolated from the critical region for microphthalmia with linear skin defects (MLS); Schaefer L et al.; Microphthalmia with linear skin defects syndrome (MLS) is an X-linked male-lethal disorder associated with X chromosomal rearrangements resulting in monosomy from Xpter to Xp22 . Features include micro- phthalmia, sclerocornea, linear skin defects, and agenesis of the corpus callosum . Using a cross-species conservation strategy, an expressed sequence from the 450- to the 550-kb MLS critical region on Xp22 was identified by screening a human embryo cDNA library . Northern analysis revealed a transcript of approximately 2.6 kb in all tissues examined, with weaker expression of approximately 1.2- and approximately 5.2-kb transcripts . The strongest expression was observed in heart and skeletal muscle . Sequence analysis of a 3-kb cDNA contig revealed an 807-bp open reading frame encoding a putative 268-amino-acid protein . Comparison of the sequence with sequences in the databases revealed homology with holocytochrome c-type synthetases, which catalyze the covalent addition of a heme group onto c-type cytochromes in the mitochondria . The c-type cytochromes are required for proper functioning of the electron transport pathway . The human gene (HGMW-approved symbol HCCS) and the corresponding murine gene characterized in this paper are the first mammalian holocytochrome c-type synthetases to be described in the literature . Because of the lack of a neuromuscular phenotype in MLS, it is uncertain whether the deletion of a mitochondrial holocytochrome synthetase would contribute to the phenotype seen in MLS . The expression pattern of this gene and knowledge about the function of holocytochrome synthetases, however, suggest that it is a good candidate for X-linked encephalomyopathies typically associated with mitochondrial dysfunction.

J Bacteriol, 1996 Jun, 178(11), 3406 - 9
The tamA gene of Aspergillus nidulans contains a putative zinc cluster motif which is not required for gene function; Davis MA et al.; Expression of many nitrogen catabolic enzymes is controlled by nitrogen metabolite repression in Aspergillus nidulans . Although the phenotypes of tamA mutants have implicated this gene in nitrogen regulation, its function is unknown . We have cloned the tamA gene by complementation of a new tamA allele . The tamA sequence shares significant homology with the UGA35/DAL81/DURL gene of Saccharomyces cerevisiae . In vitro mutagenesis of sequences encoding a putative zinc cluster DNA binding domain indicated that this motif is not required for in vivo TamA function.

Mol Cell Biol, 1996 Jun, 16(6), 3169 - 78
An SH3 domain-containing GTPase-activating protein for Rho and Cdc42 associates with focal adhesion kinase; Hildebrand JD et al.; The integrin family of cell surface receptors mediates cell adhesion to components of the extracellular matrix (ECM) . Integrin engagement with the ECM initiates signaling cascades that regulate the organization of the actin-cytoskeleton and changes in gene expression . The Rho subfamily of Ras-related low-molecular-weight GTP-binding proteins and several protein tyrosine kinases have been implicated in mediating various aspects of integrin-dependent alterations in cell homeostasis . Focal adhesion kinase (FAK or pp125FAK) is one of the tyrosine kinases predicted to be a critical component of integrin signaling . To elucidate the mechanisms by which FAK participates in integrin-mediated signaling, we have used expression cloning to identify cDNAs that encode potential FAK-binding proteins . We report here the identification of a cDNA that encodes a new member of the GTPase-activating protein (GAP) family of GTPase regulators . This GAP, termed Graf (for GTPase regulator associated with FAK), binds to the C-terminal domain of FAK in an SH3 domain-dependent manner and preferentially stimulates the GTPase activity of the GTP-binding proteins RhoA and Cdc42 . Subcellular localization studies using Graf-transfected chicken embryo cells indicates that Graf colocalizes with actin stress fibers, cortical actin structures, and focal adhesions . Graf mRNA is expressed in a variety of avian tissues and is particularly abundant in embryonic brain and liver . Graf represents the first example of a regulator of the Rho family of small GTP-binding proteins that exhibits binding to a protein tyrosine kinase . We suggest that Graf may function to mediate cross talk between the tyrosine kinases such as FAK and the Rho family GTPase that control steps in integrin-initiated signaling events.

Mol Cell Biol, 1996 Jun, 16(6), 3066 - 73
Interaction of Vav with ENX-1, a putative transcriptional regulator of homeobox gene expression; Hobert O et al.; The proto-oncogene product Vav plays a critical role in hematopoietic signal transduction . By using the yeast two-hybrid system, we identified a novel human protein, ENX-1, which interacts specifically with Vav both in vitro and in vivo . ENX-1 represents the human homolog of the Drosophila Enhancer of zeste gene, a member of the Polycomb group of genes, which are transcriptional regulators of homeobox gene expression . Interaction with ENX-1 suggests that Vav functions as an upstream element in the transcriptional regulation of homeobox genes, known to be important effectors in the hematopoietic system.

Mol Cell Biol, 1996 Jun, 16(6), 2951 - 7
Effects of terminal nonhomology and homeology on double-strand-break-induced gene conversion tract directionality; Nelson HH et al.; Double-strand breaks (DSBs) greatly enhance gene conversion in the yeast Saccharomyces cerevisiae . In prior plasmid x chromosome crosses, conversion tracts were often short ( < 53 bp) and usually extended in only one direction from a DSB in an HO recognition sequence inserted into ura3 . To allow fine-structure analysis of short and unidirectional tracts, phenotypically silent markers were introduced at 3- and 6-bp intervals flanking the HO site . These markers, which created a 70-bp homeologous region (71% homology), greatly increased the proportion of bidirectional tracts . Among products with short or unidirectional tracts, 85% were highly directional, converting markers on only one side (the nearest marker being 6 bp from the HO site) . A DSB in an HO site insertion creates terminal nonhomologies . The high degree of directionality is a likely consequence of the precise cleavage at homology/nonhomology borders in hybrid DNA by Rad1/10 endonuclease . In contrast, terminal homeology alone yielded mostly unidirectional tracts . Thus, nonhomology flanked by homeology yields primarily bidirectional tracts, but terminal homeology or nonhomology alone yields primarily unidirectional tracts . These results are inconsistent with uni- and bidirectional tracts arising from one- and two-ended invasion mechanisms, respectively, as reduced homology would be expected to favor one-ended events . Tract spectra with terminal homeology alone with similar in RAD1 and rad1 cells, indicating that the high proportion of bidirectional tracts seen with homeology flanking nonhomology is not a consequence of Rad1/10 cleavage at homology/homeology boundaries . Instead, tract directionality appears to reflect the influence of the degree of broken-end homology on mismatch repair.

Mol Cell Biol, 1996 Jun, 16(6), 2865 - 9
Accessibility of alpha 2-repressed promoters to the activator Gal4; Redd MJ et al.; It has been proposed that eukaryotic repressors of transcription can act by organizing chromatin, thereby preventing the accessibility of nearby DNA to activator proteins required for transcription initiation . In this study, we test this idea for the yeast alpha 2 repressor using a simple, artificial promoter that contains a single binding site for the activator protein Gal4 and a single binding site for the repressor alpha 2 . When both the repressor and the activator are expressed in the same cell, the artificial promoter is efficiently repressed . In vivo footprinting experiments demonstrate that Gal4 can occupy its binding site even when the promoter is repressed . This result indicates that alpha 2-directed repression must result from interference with some stage in transcription initiation other than activator binding to DNA.

Mol Cell Biol, 1996 Jun, 16(6), 2830 - 7
Ste12 and Mcm1 regulate cell cycle-dependent transcription of FAR1; Oehlen LJ et al.; The transcripts of many genes involved in Saccharomyces cerevisiae mating were found to fluctuate during the cell cycle . In the absence of a functional Ste12 transcription factor, both the levels and the cell cycle pattern of expression of these genes were affected . FUS1 and AGA1 levels, which are maximally expressed only in G1-phase cells, were strongly reduced in ste12- cells . The cell cycle transcription pattern for FAR1 was changed in ste12- cells: the gene was still significantly expressed in G2/M, but transcript levels were strongly reduced in G1 phase, resulting in a lack of Far1 protein accumulation . G2/M transcription of FAR1 was dependent on the transcription factor Mcm1, and expression of a gene with Mcm1 fused to a strong transcriptional activation domain resulted in increased levels of FAR1 transcription . The pattern of cell cycle-regulated transcription of FAR1 could involve combinatorial control of Ste12 and Mcm1 . Forced G1 expression of FAR1 from the GAL1 promoter resorted the ability to arrest in response to pheromone in ste12-cells . This indicates that transcription of FAR1 in the G1 phase is essential for accumulation of the protein and for pheromone-induced cell cycle arrest.

Mol Cell Biol, 1996 Jun, 16(6), 2756 - 63
A mouse Fas-associated protein with homology to the human Mort1/FADD protein is essential for Fas-induced apoptosis; Zhang J et al.; The Fas cell surface receptor belongs to the tumor necrosis factor receptor family and can initiate apoptosis in a variety of cell types . Using the Fas cytoplasmic domain as bait in a yeast two-hybrid screening, we isolated a mouse cDNA encoding a 205-amino-acid protein . Its predicted protein sequence shows 68% identity and 80% similarity with the sequence of recently described human Mort/FADD . This protein, most likely the mouse homolog of human FADD, associates with Fas in vivo only upon the induction of cell death . A fraction of this protein is highly phosphorylated at serine/threonine residues, with both phosphorylated and unphosphorylated forms being capable of binding to FAS . Stable expression of a truncated form of the Mort/FADD protein protects cells from Fas-mediated apoptosis by interfering with the wild-type protein-Fas interaction . Thus, mouse Mort/FADD is an essential downstream component that mediates Fas-induced apoptosis.

Mol Cell Biol, 1996 Jun, 16(6), 2744 - 55
The SAP, a new family of proteins, associate and function positively with the SIT4 phosphatase; Luke MM et al.; SIT4 is the catalytic subunit of a type 2A-related protein phosphatase in Saccharomyces cerevisiae that is required for G1 cyclin transcription and for bud formation . SIT4 associates with several high-molecular-mass proteins in a cell cycle-dependent fashion . We purified two SIT4-associated proteins, SAP155 and SAP190, and cloned the corresponding genes . By sequence homology, we isolated two additional SAP genes, SAP185 and SAP4 . Through such an association is not yet proven for SAP4, each of SAP155, SAP185, and SAP190 physically associates with SIT4 in separate complexes . The SAPs function positively with SIT4, and by several criteria, the loss of all four SAPs is equivalent to the loss of SIT4 . The data suggest that the SAPs are not functional in the absence of SIT4 and likewise that SIT4 is not functional in the absence of the SAPs . The SAPs are hyperphoshorylated in cells lacking SIT4, raising the possibility that the SAPs are substrates of SIT4 . By sequence similarity, the SAPs fall into two groups, the SAP4/SAP155 group and the SAP185/SAP190 group . Overexpression of a SAP from one group does not suppress the defects due to the loss of the other group . These findings and others indicate that the SAPs have distinct functions.

Mol Cell Biol, 1996 Jun, 16(6), 2678 - 88
Repression by HoxA7 is mediated by the homeodomain and the modulatory action of its N-terminal-arm residues; Schnabel CA et al.; Hox genes encode homeodomain-containing proteins that are presumed to control spatial patterning during murine embryogenesis through their actions as transcriptional regulatory proteins . In this study, we have investigated the transcriptional function of a prototypic member of this family, HoxA7 . We demonstrate that HoxA7 function as a potent transcriptional repressor and that its action as such requires several domains, including both activator and repressor regions . The repressor regions are contained within the homeodomain and a C-terminal acidic region, both of which are well conserved among members of the Hox family . Accordingly, we show that two other members of this family also function as repressors, although they vary in their relative repressor potency . Finally, we explore the novel observation that the homeodomain of HoxA7 functions as a transcriptional repressor domain . We show that the homeodomain compared with two other DNA-binding domains, is unique in its ability to function as a repressor domain and that repression requires conserved residues, in helix III . We further show that residues in the N-terminal arm of the homeodomain contribute to the differential repressor actions of various Hox proteins . These findings demonstrate that the transcriptional function of HoxA7 and possibility of Hox proteins in general is determined by their unique combination of conserved and nonconserved regions as well as through the complex actions of their homeodomains.

J Virol, 1996 Jun, 70(6), 3440 - 8
Mutations in the Ty3 major homology region affect multiple steps in Ty3 retrotransposition; Orlinsky KJ et al.; The Saccharomyces cerevisiae retroviruslike element Ty3 encodes the major structural proteins capsid (CA) and nucleocapsid in the GAG3 open reading frame . The Ty3 CA protein contains a sequence (QGX2EX5FX3LX3H, where H is a hydrophobic residue) which has not been observed in other retrotransposons but which is similar to the major homology region (MHR) described for retrovirus CA . In this study the effects of mutations in the Ty3 MHR on particle formation, processing, DNA synthesis, and transposition were examined . Each of the mutations tested resulted in severe defects in transposition, with disruption occurring prior to or at particle formation, subsequent to particle formation and prior to completion of DNA synthesis, and subsequent to DNA synthesis . Changing the Q in the motif to R had relatively little effect on particle formation but decreased transposition to about 13% of that of a wild-type element . Changing G to A or V almost completely eliminated the formation of intracellular particles, possibly by disruption of CA-CA interactions . Changes introduced at the position of E resulted in blocked processing, blocked DNA synthesis, or a block at some post-reverse transcription step, depending on the nature of the mutation introduced . These results showed that the integrity of the Ty3 MHR is required for multiple aspects of Ty3 replication involving CA . These functions are independent of extracellular budding and of infection, aspects of the retroviral life cycle which are not recapitulated in replication of the Ty3 retrotransposon.

Nat Struct Biol, 1996 Jun, 3(6), 510 - 5
An engineered allosteric switch in leucine-zipper oligomerization; Gonzalez L Jr et al.; Controversy remains about the role of core side-chain packing in specifying protein structure . To investigate the influence of core packing on the oligomeric structure of a coiled coil, we engineered a GCN4 leucine zipper mutant that switches from two to three strands upon binding the hydrophobic ligands cyclohexane and benzene . In solution these ligands increased the apparent thermal stability and the oligomerization order of the mutant leucine zipper . The crystal structure of the peptide-benzene complex shows a single benzene molecule bound at the engineered site in the core of the trimer . These results indicate that coiled coils are well-suited to function as molecular switches and emphasize that core packing is an important determinant of oligomerization specificity.

Nat Genet, 1996 Jun, 13(2), 230 - 2
Sex reversal by loss of the C-terminal transactivation domain of human SOX9; Sudbeck P et al.; Haploinsufficiency for SOX9 has recently been identified as the cause for both campomelic dysplasia (CD), a human skeletal malformation syndrome, and the associated autosomal XY sex reversal . SOX9 contains a putative DNA-binding motif known as the high-mobility group (HMG) domain characterizing a whole class of transcription factors . We show in cell transfection experiments that SOX9 can transactivate transcription from a reporter plasmid through the motif AACAAAG, a sequence recognized by other HMG domain transcription factors . By fusing all or part of SOX9 to the DNA-binding domain of yeast GAL4, the transactivating function was mapped to a transcription activation (TA) domain at the C terminus of SOX9 . This non-acidic TA domain is evolutionarily conserved and rich in proline, glutamine and serine . With one exception, all SOX9 nonsense and frame shift mutations described so far in CD/sex reversal patients lead to truncation of the TA domain, suggesting that impairment of gonadal and skeletal development in these cases results, at least in part, from loss of transactivation of genes downstream of SOX9.

J Biol Chem, 1996 May 31, 271(22), 12691 - 4
Mitogenic signaling by Ret/ptc2 requires association with enigma via a LIM domain; Durick K et al.; The ret/ptc2 papillary thyroid cancer oncogene, an oncogenic form of the c-Ret receptor tyrosine kinase, is the product of a somatic crossover event fusing the dimerization domain of the type Ialpha regulatory subunit of cyclic AMP-dependent protein kinase (RI) with the tyrosine kinase domain of c-Ret . Mitogenic activity of Ret/ptc2 required dimerization via the N terminus of RI and a tyrosine residue located C-terminal to the kinase core of Ret, Tyr-586 (Durick, K., Yao, V . J., Borrello, M . G., Bongarzone, I., Pierotti, M . A . and Taylor, S . S . (1995) J . Biol . Chem . 270, 24642-24645) . Using the yeast two-hybrid system, Ret/ptc2 binding proteins were identified, and the sites of interaction with Ret/ptc2 were mapped . The SH2 domains of phospholipase Cgamma and Grb10 were both identified, and binding depended on phosphorylation of Tyr-539 and Tyr-429, respectively . These interactions, however, were not required for mitogenic signaling . The second of the three LIM domains in Enigma (Wu, R . Y., and Gill, G . N . (1994) J . Biol . Chem . 269, 25085-25090) was also identified as a Ret/ptc2 binding domain . Enigma, a 455-residue protein, was discovered based on its interaction with the insulin receptor through the C-terminal LIM domain . Although the association with Enigma required Tyr-586 of Ret/ptc2, the interaction was phosphorylation-independent . In contrast to the SH2 interactions, disruption of the interaction with Enigma abolished Ret/ptc2 mitogenic signaling, suggesting that LIM domain recognition of an unphosphorylated tyrosine-based motif is required for Ret signal transduction.

J Biol Chem, 1996 May 31, 271(22), 12852 - 8
CD30 contains two binding sites with different specificities for members of the tumor necrosis factor receptor-associated factor family of signal transducing proteins; Gedrich RW et al.; CD30 is a member of the tumor necrosis factor (TNF) receptor family of proteins . CD30 can regulate proliferation of lymphocytes and may also play an important role in human immunodeficiency virus replication . However, little is known about CD30 signal transduction . We performed a yeast two-hybrid library screen with the cytoplasmic domain of CD30 and isolated multiple independent cDNAs encoding human tumor necrosis factor receptor-associated factor (TRAF) 1, TRAF2, and CRAF1 (TRAF3) . The ability of TRAF1, TRAF2, and CRAF1 to associate with CD30 was confirmed using an in vitro coprecipitation assay, further demonstrating that the interaction was specific and direct . The TRAF-binding domain of CD30 was mapped to the COOH-terminal 36 amino acid residues, which contained two independent binding sites . CRAF1 bound only a single site, which contained the sequence PEQET, whereas TRAF1 and TRAF2 were capable of binding to either the PEQET site or an additional downstream domain . These data indicate that the TRAF protein binding pattern of CD30 differs from other TNF receptor family members and suggest that signaling specificity through TNF receptor family proteins may be achieved through differences in their abilities to bind TRAF proteins.

Proc Natl Acad Sci U S A, 1996 May 28, 93(11), 5663 - 7
Constitutively active human Notch1 binds to the transcription factor CBF1 and stimulates transcription through a promoter containing a CBF1-responsive element; Lu FM et al.; Notch is a transmembrane receptor that plays a critical role in cell fate determination . In Drosophila, Notch binds to and signals through Suppressor of Hairless . A mammalian homologue of Suppressor of Hairless, named CBF1 (or RBPJk), is a ubiquitous transcription factor whose function in mammalian Notch signaling is unknown . To determine whether mammalian Notch can stimulate transcription through a CBF1-responsive element (RE), we cotransfected a CBF1-RE-containing chloramphenicol acetyltransferase reporter and N1(deltaEC), a constitutively active form of human Notch1 lacking the extracellular domain, into DG75, COS-1, HeLa, and 293T cells, which all contain endogenous CBF1 . N1(deltaEC) dramatically increased chloramphenicol acetyltransferase activity in these cells, indicating functional coupling of Notch1 and CBF1 . The activity was comparable to that produced by the Epstein-Barr virus protein EBNA2, a well-characterized, potent transactivator of CBF1 . To test whether CBF1 and Notch1 interact physically, we tagged CBF1 with an epitope from the influenza virus hemagglutinin or with the N-terminal domain of gal4, and transfected the tagged CBF1 plus N1(deltaEC) into COS-1 cells . Cell lysates were immunoprecipitated and immunoblotted with several anti-Notch1 antibodies {to detect N1(deltaEC)} or with antibodies to hemagglutinin or gal4 (to detect CBF1) . Each immunoprecipitate contained a complex of N1(deltaEC) and CBF1 . In summary, we find that the truncated, active form of human Notch1, N1(deltaEC), binds CBF1 and activates transcription through a CBF1-RE-containing promoter . We conclude that CBF1 is a critical downstream protein in the human Notch1 signaling pathway.

Proc Natl Acad Sci U S A, 1996 May 28, 93(11), 5615 - 8
Direct interaction of the Wiskott-Aldrich syndrome protein with the GTPase Cdc42; Kolluri R et al.; Wiskott-Aldrich syndrome (WAS) is an X-linked immunodeficiency disorder with the most severe pathology in the T lymphocytes and platelets . The disease arises from mutations in the gene encoding the WAS protein . T lymphocytes of affected males with WAS exhibit a severe disturbance of the actin cytoskeleton, suggesting that the WAS protein could regulate its organization . We show here that WAS protein interacts with a member of the Rho family of GTPases, Cdc42 . This interaction, which is guanosine 5'-triphosphate (GTP)-dependent, was detected in cell lysates, in transient transfections and with purified recombinant proteins . A weaker interaction was also detected with Rac1 using WAS protein from cell lysates . It was also found that different mutant WAS proteins from three affected males retained their ability to interact with Cdc42 and that the level of expression of the WAS protein in these mutants was only 2-5% of normal . Taken together these data suggest that the WAS protein might function as a signal transduction adaptor downstream of Cdc42, and in affected males, the cytoskeletal abnormalities may result from a defect in Cdc42 signaling.

Proc Natl Acad Sci U S A, 1996 May 28, 93(11), 5455 - 9
Mechanism for transcriptional gain of function resulting from chromosomal translocation in alveolar rhabdomyosarcoma; Bennicelli JL et al.; The t(2;13) translocation of alveolar rhabdomyosarcoma results in tumor-specific expression of a chimeric transcription factor containing the N-terminal DNA-binding domain of PAX3 and the C-terminal transactivation domain of FKHR . Here we have tested the hypothesis that PAX3-FKHR gains function relative to PAX3 as a consequence of switching PAX3 and FKHR transactivation domains, which were previously shown to have similar potency but distinct structural motifs . In transient cotransfection assays with human expression constructs, we have demonstrated the increased ability of PAX3-FKHR to activate transcription of a reporter gene located downstream of multimerized e5, PRS-9, or CD19 DNA-binding sites in three cell lines . For example, PAX3-FKHR was 100-fold more potent than PAX3 as an activator binding to e5 sites in NIH 3T3 cells . To compare transactivation potency independent of PAX3-specific DNA binding, we tested GAL4 fusions of full-length PAX3 and PAX3-FKHR or their respective C-terminal transactivation domains on a reporter with GAL4 DNA-binding sites . In this context, full-length PAX3-FKHR was also much more potent than PAX3 . Additionally, the activity of each full-length protein was decreased relative to its C-terminal domain, demonstrating that N-terminal sequences are inhibitory . By deletion analysis, we mapped a bipartite cis-acting inhibitory domain to the same subregions within the DNA-binding domains of both PAX3 and PAX3-FKHR . We have shown, however, that the structurally distinct transactivation domains of PAX3 and PAX3-FKHR differ 10- to 100-fold in their susceptibility to inhibition, thus elucidating a mechanism by which PAX3 gains enhanced function during oncogenesis.

Proc Natl Acad Sci U S A, 1996 May 28, 93(11), 5419 - 24
Recombinational repair of gaps in DNA is asymmetric in Ustilago maydis and can be explained by a migrating D-loop model; Ferguson DO et al.; Recombinational repair of double-stranded DNA gaps was investigated in Ustilago maydis . The experimental system was designed for analysis of repair of an autonomously replicating plasmid containing a cloned gene disabled by an internal deletion . It was discovered that crossing over rarely accompanied gap repair . The strong bias against crossing over was observed in three different genes regardless of gap size . These results indicate that gap repair in U . maydis is unlikely to proceed by the mechanism envisioned in the double-stranded break repair model of recombination, which was developed to account for recombination in Saccharomyces cerevisiae . Experiments aimed at exploring processing of DNA ends were performed to gain understanding of the mechanism responsible for the observed bias . A heterologous insert placed within a gap in the coding sequence of two different marker genes strongly inhibited repair if the DNA was cleaved at the promoter-proximal junction joining the insert and coding sequence but had little effect on repair if the DNA was cleaved at the promoter-distal junction . Gene conversion of plasmid restriction fragment length polymorphism markers engineered in sequences flanking both sides of a gap accompanied repair but was directionally biased . These results are interpreted to mean that the DNA ends flanking a gap are subject to different types of processing . A model featuring a single migrating D-loop is proposed to explain the bias in gap repair outcome based on the observed asymmetry in processing the DNA ends.

Biochemistry, 1996 May 28, 35(21), 6706 - 14
Human bleomycin hydrolase: molecular cloning, sequencing, functional expression, and enzymatic characterization; Bromme D et al.; We have cloned the cDNA of human bleomycin hydrolase (hBH), a protease which is thought to be involved in the metabolic inactivation of the antineoplastic drug bleomycin . The open reading frame consists of 1365 base pairs and is predicted to encode a 52 kDa protein . The protein shares 40% identity with yeast bleomycin hydrolase and contains the conserved active site residues (Cys, His, Asn) characteristic for cysteine proteases of the papain superfamily . Human bleomycin hydrolase has been functionally expressed in Spodoptera frugiperda Sf9 cells using the Autographa californica nuclear polyhedrosis virus . The 52 kDa recombinant protein forms a hexamer of 310 kDa and acts strictly as an aminopeptidase with a broad substrate specificity . The lack of a leader sequence and its pH optimum at 7.2 suggest a cytosolic/nuclear localization . Human bleomycin hydrolase was detected at low to moderate expression levels in most of the human organs tested . Significantly higher RNA levels have been observed in a variety of tumor cell lines . The human enzyme effectively degrades both forms of bleomycin (A2 and B2) in vitro and could indeed be responsible for the resistance of various tumors to this widely used anticancer drug.

Gene, 1996 May 24, 171(1), 123 - 7
Isolation and characterization of a cDNA from Trichoderma harzianum P1 encoding a 14-3-3 protein homolog; Klemsdal SS et al.; A full-length cDNA close, Th1433, (GenBank accession No . U24158), was isolated and characterized from the filamentous fungus, Trichoderma harzianum . The deduced amino acid (aa) sequence showed an acidic 30-kDa protein homologous to the 14-3-3 proteins, a family of putative kinase regulators originally characterized in mammalian brain tissue . The greatest homology, 71% identical aa, was found to BMH1, the corresponding protein from Saccharomyces cerevisiae and to the epsilon isoform from sheep brain . Southern analysis of genomic DNA indicated that Th1433 is a member of a small genomic family . At least two genes encoding 14-3-3-like proteins exist in T . harzianum . Northern analysis showed the highest level of expression during the first day after inoculation of the culture with conidial spores.

Science, 1996 May 24, 272(5265), 1179 - 82
TAB1: an activator of the TAK1 MAPKKK in TGF-beta signal transduction; Shibuya H et al.; Transforming growth factor-beta (TGF-beta) regulates many aspects of cellular function . A member of the mitogen-activated protein kinase kinase kinase (MAPKKK) family, TAK1, was previously identified as a mediator in the signaling pathway of TGF-beta superfamily members . The yeast two-hybrid system has now revealed two human proteins, termed TAB1 and TAB2 (for TAK1 binding protein), that interact with TAK1 . TAB1 and TAK1 were co-immunoprecipitated from mammalian cells . Overproduction of TAB1 enhanced activity of the plasminogen activator inhibitor 1 gene promoter, which is regulated by TGF-beta, and increased the kinase activity of TAK1 . TAB1 may function as an activator of the TAK1 MAPKKK in TGF-beta signal transduction.

Nature, 1996 May 23, 381(6580), 332 - 5
Efficient integration of an intron RNA into double-stranded DNA by reverse splicing; Yang J et al.; Some group II introns are mobile elements as well as catalytic RNAs . Introns aI1 and aI2 found in the gene COX1 in yeast mitochondria encode reverse transcriptases which promote site-specific insertion of the intron into intronless alleles ('homing') . For aI2 this predominantly occurs by reverse transcription of unspliced precursor RNA at a break in double-strand DNA made by an endonuclease encoded by the intron . The aI2 endonuclease involves both the excised intron RNA, which cleaves the DNA's sense strand by partial reverse splicing; and the intron-encoded reverse transcriptase which cleaves the anti-sense strand . Here we show that aI1 encodes an analogous endonuclease specific for a different target site compatible with the different exon-binding sequences of the intron RNA . Over half of aI1 undergoes complete reverse splicing in vitro, thus integrating linear intron RNA directly into the DNA . This unprecedented reaction has implications for both intron mobility and evolution, and potential genetic engineering applications.

Nature, 1996 May 23, 381(6580), 328 - 31
Effects on NF-kappa B1/p105 processing of the interaction between the HTLV-1 transactivator Tax and the proteasome; Rousset R et al.; The viral Tax protein, which is encoded by human T-cell leukaemia virus HTLV-I, activates nuclear translocation of the NF-kappa B/Rel transcription factors and relieves cytoplasmic sequestration of RelA and Rel by heterodimerization with NF-kappa B1/p1O5 (refs 1,2) . Proteolytic maturation of this precursor protein is performed by the proteasome complex . Here we show that Tax binds specifically to two subunits of the 20S proteasome, HsN3 and HC9 . This interaction is weakened with HsN3 and lost for HC9 when a mutant of Tax is substituted that is selectively defective for NF-kappa B activation . Immunoprecipitation shows that p1O5 binds weakly to HC9 and that this interaction is reinforced by Tax . No bridging function of Tax between p1O5 and HsN3 was observed . From these results, we propose that Tax accelerates the proteolytic maturation of P105 by favouring its anchorage to the proteasome.

Biochemistry, 1996 May 21, 35(20), 6345 - 50
New insights into the catalytic cycle of flavocytochrome b2; Daff S et al.; Flavocytochrome b2 from Saccharomyces cerevisiae couples L-lactate dehydrogenation to cytochrome c reduction in the mitochondrial intermembrane space . The catalytic cycle for this process can be described in terms of five consecutive electron-transfer events . L-Lactate dehydrogenation results in the two-electron reduction of FMN . The two electrons are individually passed to b2-heme (intramolecular electron transfer) and then onto cytochrome c (intermolecular electron transfer) . At 25 degrees C, I 0.10, in the presence of saturating concentrations of ferricytochrome c and L-lactate, the catalytic cycle progresses with rate constant 104 (+/- 5) s-1 {per L-lactate oxidized; Miles, C . S., Rouviere-Fourmy, N., Lederer, F., Mathews, F . S., Reid, G . A., & Chapman, S . K . (1992) Biochem . J . 285, 187-192} . Stopped-flow spectrophotometry has been used to show that the major rate-limiting step in the catalytic cycle is electron transfer from flavin semiquinone to b2-heme . This conclusion is based on the observation that pre-steady-state flavin oxidation by ferricytochrome c takes place at 120 s-1 . Although flavin oxidation involves several other electron transfer steps, these are considered too fast to contribute significantly to the rate constant . It was also shown that the reaction product, pyruvate, is able to inhibit pre-steady-state flavin oxidation (Ki = 40 +/- 17 mM) consistent with reports that it acts as a noncompetitive inhibitor in the steady state at high concentrations {Ki = 30 mM; Lederer, F . (1978) Eur . J . Biochem, 88, 425-431} . This novel way of measuring the electron transfer rate constant is directly applicable to the catalytic cycle and has enabled us to derive a self-consistent model for it, based also on data collected for enzyme reduction {Miles, C . S., Rouviere-Fourmy, N., Lederer, F., Mathews, F . S., Reid, G . A., & Chapman, S . K . (1992) Biochem . J . 285, 187-192} and its interaction with cytochrome c {Daff, S., Sharp, R . E., Short, D . M., Bell, C., White, P., Manson, F . D . C., Reid, G . A., & Chapman, S . K . (1996) Biochemistry 35, 6351-6357} . Rapid-freezing quenched-flow EPR has been used to confirm the model by demonstrating that during steady-state turnover of the enzyme approximately 75% of the flavin is in the semiquinone oxidation state.

FEBS Lett, 1996 May 20, 386(2-3), 201 - 4
Limulus kexin: a new type of Kex2-like endoprotease specifically expressed in hemocytes of the horseshoe crab; Kawabata S et al.; A Kex2-like protease was identified in hemocytes of the horseshoe crab (Tachypleus tridentatus), named limulus kexin, and a full-length cDNA was obtained from a hemocyte cDNA library . The deduced amino acid sequence contains 752 residues, composed of five domains with a signal sequence, a propeptide, a catalytic domain, a Ser/Thr-rich domain, and a transmembrane domain . The domain organization is very similar to that of the yeast Kex2 except that limulus kexin does not have a cytoplasmic tail . The catalytic domain exhibits striking sequence identities with those of furins, especially Drosophila furin1 (79%) . Northern blotting showed specific expression of limulus kexin in hemocytes, suggesting the involvement in proteolytic processing of the granule components of hemocytes.

J Chromatogr B Biomed Appl, 1996 May 17, 680(1-2), 213 - 9
RNA partitioning accompanied by adsorption: high-molecular-mass RNA adsorbed at the interface like a particle; Kimura K et al.; In a potassium phosphate-poly(ethylene glycol) (PEG) system . RNA partitioning was accompanied by adsorption at the interface, depending on the molecular mass . Low-molecular-mass RNA showed the typical partition behavior of a soluble substance . Conversely, high-molecular-mass RNA was significantly adsorbed at the interface in the potassium phosphate-PEG1500 system . The adsorbed amount was proportional to the added amount, regardless of the phase volume ratio . The partitioning of high-molecular-mass RNA showed an amount-ratio-dependent distribution like that of a particle.

J Chromatogr B Biomed Appl, 1996 May 17, 680(1-2), 131 - 6
Enzyme purification with aqueous two-phase systems: comparison between systems composed of pure polymers and systems composed of crude polymers; Venancio A et al.; The main drawback when using aqueous two-phase systems for macromolecule purification is the high cost of most polymers used . The purification of an enzyme, alcohol dehydrogenase, from a crude extract of Saccharomyces cerevisiae was tested in systems composed of poly(ethylene glycol) and a crude hydroxypropyl starch or Reppal PES 100, a purified fraction of hydroxypropyl starch . Purification factors measured for the enzyme were very similar in both systems (between 0.8 and 1.4 for both systems in the upper phase) . However, systems composed of Reppal PES present a greater recovery of enzyme, between 77% and 100% versus 60% and 100%, while systems composed of crude hydroxypropyl starch exhibit a larger delta log K for the tested ligand, 1.26 versus 0.81.

J Chromatogr B Biomed Appl, 1996 May 17, 680(1-2), 81 - 9
Impact of cell disruption and polymer recycling upon aqueous two-phase processes for protein recovery; Rito-Palomares M et al.; A practical study is presented of the influence of cell debris and polymer recycling upon the operation of two-stage aqueous two-phase systems (ATPS) for the recovery of yeast bulk protein, pyruvate kinase and fumarase . Brewers' yeast was disrupted using one of two types of high-pressure homogenisers or a bead mill . The different cell debris suspensions were partitioned in a single PEG-phosphate ATPS extraction and the efficiency of solid-liquid separation was examined . A continuously operated two-stage ATPS process, using spray columns, is presented and practical problems of polymer recycling are discussed . Conclusions are drawn concerning the generic implementation and operational stability of ATPS in practical protein recoveries.

Science, 1996 May 17, 272(5264), 1030 - 3
DNA replication fork pause sites dependent on transcription; Deshpande AM et al.; Replication fork pause (RFP) sites transiently arresting replication fork movement were mapped to transfer RNA (tRNA) genes of Saccharomyces cerevisiae in vivo . RFP sites are polar, stalling replication forks only when they oppose the direction of tRNA transcription . Mutant tRNA genes defective in assembly of transcription initiation complexes and a temperature-sensitive RNA polymerase III mutant (rpc160-41) defective in initiation of transcription do not stall replication forks, suggesting that transcription is required for RFP activity.

Science, 1996 May 17, 272(5264), 1004 - 7
Specification of pituitary cell lineages by the LIM homeobox gene Lhx3; Sheng HZ et al.; During pituitary organogenesis, the progressive differentiation of distinct pituitary-specific cell lineages from a common primordium involves a series of developmental decisions and inductive interactions . Targeted gene disruption in mice showed that Lhx3, a LIM homeobox gene expressed in the pituitary throughout development, is essential for differentiation and proliferation of pituitary cell lineages . In mice homozygous for the Lhx3 mutation, Rathke's pouch formed but failed to grow and differentiate; such mice lacked both the anterior and intermediate lobes of the pituitary . The determination of all pituitary cell lineages, except the corticotrophs, was affected, suggesting that a distinct, Lhx3-independent ontogenetic pathway exists for the initial specification of this lineage.

Nature, 1996 May 16, 381(6579), 251 - 3
Role of interactions between the origin recognition complex and SIR1 in transcriptional silencing; Triolo T et al.; Transcriptional silencing of the HM mating-type loci in the yeast Saccharomyces cerevisiae is caused by the localized formation of an altered chromatin structure, analogous to heterochromatin in higher eukaryotes . Silencing depends on cis-acting sequences, termed silencers, as well as several trans-acting factors, including histones H4 and H3, proteins RAP1 and ABF1, and the four SIR proteins (SIR1-4) . Each of the four HM silencers contains an autonomously replicating sequence (ARS) to which the origin replication complex (ORC) binds . This six-protein complex is required for initiation of DNA replication, as well as for silencing . Efficient establishment of the silenced state requires both passage through the S phase of the cell cycle and SIR1 protein . Previous experiments suggested that SIR1 might be localized to the silencers by binding to ORC and/or RAP1 . Here we report that SIR1 can bind directly to ORC1, the largest of the ORC subunits, and that targeting of SIR1 to ORC1 at a silencer is sufficient to establish a silenced state.

Genes Dev, 1996 May 15, 10(10), 1260 - 70
Promoter specificity mediates the independent regulation of neighboring genes; Merli C et al.; Although enhancers can exert their influence over great distances, their effect is generally limited to a single gene . To discern the mechanism by which this constraint can he mediated, we have studied three neighboring Drosophila genes: decapentaplegic (dpp), SLY1 homologous (Slh) and out at first (oaf) . Several dpp enhancers are positioned close to Slh and oaf, and yet these genes are unaffected by the dpp elements . However, when a transposon is located within the oaf gene, the dpp enhancers activate the more distant transposon promoters while still ignoring the closer Slh and oaf start sites . To test whether this promoter specificity accounts for the regulatory autonomy normally found for the three genes, we used in vivo gene targeting to replace the oaf promoter with a dpp-compatible one in an otherwise normal chromosome . Strikingly, this chimeric gene is now activated by the dpp enhancers . Thus, the properties of the promoters themselves are sufficient to mediate the autonomous regulation of genes in this region.

Genes Dev, 1996 May 15, 10(10), 1233 - 46
A protein that shuttles between the nucleus and the cytoplasm is an important mediator of RNA export; Lee MS et al.; The connection between RNA and protein export from the nucleus was examined in the budding yeast Saccharomyces cerevisiae . NPL3 encodes an RNA-binding protein that shuttles in and out of the nucleus . Export of poly(A)+ RNA has been shown previously to be blocked in np13-1 mutants . To understand the role of Np13p in RNA export, we have developed a novel assay that effectively uncouples nuclear protein export from reimport . With this assay, we show that Np13p satisfies several of the predicted requirements for a protein carrier for mRNA export . Temperature-sensitive mutations in the RNA recognition motifs of Np13p result in nuclear accumulation of poly(A)+ RNA . One such mutation prevents nuclear export of Np13p . Moreover, Np13p export depends on ongoing RNA polymerase II transcription . Export ceases in either the presence of the RNA synthesis inhibitor thiolutin or in a temperature-sensitive RNA polymerase (rpb1) mutant . Together, these findings support a model in which Np13p exits the nucleus in association with poly(A)+ RNA, deposits the RNA in the cytoplasm, and is rapidly reimported for another cycle of export.

Biochem Biophys Res Commun, 1996 May 15, 222(2), 541 - 6
Cloning of Xenopus TFIIS and its expression in oocytes and early embryos; Kugawa F et al.; The transcriptional factor TFIIS has been cloned from Xenopus laevis . The length of the cDNA is 1668bp and contains the complete open reading frame of 303 amino acids . Xenopus TFIIS has high homologies to its human and mouse counterparts . In Northern blot analyses, TFIIS mRNAs that consisted of four different sizes were expressed relatively highly from the early stages of Xenopus oogenesis . During oocyte maturation, the pattern of Xenopus TFIIS messages showed a transient peak of expression . TFIIS mRNA occurred maternally and its level increased in later stage embryos . These data suggest that TFIIS mRNA is expressed in a developmentally regulated way in Xenopus laevis.

Eur J Biochem, 1996 May 15, 238(1), 104 - 11
Two distinct long-chain-acyl-CoA synthetases in guinea pig Harderian gland; Kono M et al.; Two distinct long-chain-acyl-CoA synthetases which have different kinetic properties were identified in the guinea pig Harderian gland . One was localized in the microsomes and the other in the mitochondria . The relative V(max) values of the microsomal enzyme were 8.1, 1.7 and 1 and the apparent Km values were 66.7, 12.0 and 30.0 microM for palmitic, linoleic and arachidonic acids, respectively . The relative V(max) values of the mitochondrial enzyme were 2.7, 3.5 and 1 and the apparent Km values were 33.3, 29.9 and 30.0 microM for palmitic, linoleic and arachidonic acids, respectively . The relative V(max) values for the liver microsomal enzyme were 2.0, 2.5 and 1, while those of the liver mitochondrial enzyme were 4.1, 3.9 and 1 with palmitic, linoleic and arachidonic acids, respectively . There were no difference between the microsomal and the mitochondrial enzymes in the liver, regarding apparent Km values; these were 38.4, 29.9 and 22.0 microM for palmitic, linoleic and arachidonic acids, respectively . Thus, the substrate specificity and catalytic rate of the mitochondrial enzyme in Harderian gland for palmitic, linoleic and arachidonic acids were similar to the liver enzyme, but not to the microsomal enzyme in Harderian gland . On the other hand, the antiserum raised against the rat liver enzyme immune-titrated and immuno-blotted the enzymes from Harderian gland microsomes and liver, but not so the enzyme from Harderian gland mitochondria . Thus, the microsomal enzyme in Harderian gland had a common immunogenic epitope(s) with the liver enzyme, but the mitochondrial enzyme did not . The Harderian gland mitochondrial enzyme was a distinct protein from liver enzymes . The catalytic and immunogenic characteristics suggest that the enzyme proteins in the Harderian gland are unique, that is, different from that in the liver . The large V(max) value of the Harderian gland microsomal enzyme for palmitic acid suggests that it contributes to the synthesis of a large amount of the secretory lipid and the high Km value to maintenance of cellular lipid in this organ . The evidence that long-chain-acyl-CoA synthetase in the mitochondria is distinct from that in the microsomes was first found in guinea pig Harderian gland.

EMBO J, 1996 May 15, 15(10), 2508 - 18
Acetylation of histone H4 plays a primary role in enhancing transcription factor binding to nucleosomal DNA in vitro; Vettese-Dadey M et al.; Core histones isolated from normal and butyrate-treated HeLa cells have been reconstituted into nucleosome cores in order to analyze the role of histone acetylation in enhancing transcription factor binding to recognition sites in nucleosomal DNA . Moderate stimulation of nucleosome binding was observed for the basic helix-loop-helix factor USF and the Zn cluster DNA binding domain factor GAL4-AH using heterogeneously acetylated histones . However, by coupling novel immunoblotting techniques to a gel retardation assay, we observed that nucleosome cores containing the most highly acetylated forms of histone H4 have the highest affinity for these two transcription factors . Western analysis of gel-purified USF-nucleosome and GAL4-AH-nucleosome complexes demonstrated the predominant presence of acetylated histone H4 relative to acetylated histone H3 . Immunoprecipitation of USF-nucleosome complexes with anti-USF antibodies also demonstrated that these complexes were enriched preferentially in acetylated histone H4 . These data show that USF and GAL4-AH preferentially interact with nucleosome cores containing highly acetylated histone H4 . Acetylation of histone H4 thus appears to play a primary role in the structural changes that mediate enhanced binding of transcription factors to their recognition sites within nucleosomes.

EMBO J, 1996 May 15, 15(10), 2371 - 80
WW domains of Nedd4 bind to the proline-rich PY motifs in the epithelial Na+ channel deleted in Liddle's syndrome; Staub O et al.; The amiloride-sensitive epithelial sodium channel (ENaC) plays a major role in sodium transport in kidney and other epithelia, and in regulating blood pressure . The channel is composed of three subunits (alphabetagamma) each containing two proline-rich sequences (P1 and P2) at its C-terminus . The P2 regions in human beta and gammaENaC, identical to the rat betagammarENaC, were recently shown to be deleted in patients with Liddle's syndrome (a hereditary form of hypertension), leading to hyperactivation of the channel . Using a yeast two-hybrid screen, we have now identified the rat homologue of Nedd4 (rNedd4) as the binding partner for the P2 regions of beta and gammarENaC . rNedd4 contains a Ca2+ lipid binding (CaLB or C2) domain, three WW domains and a ubiquitin ligase (Hect) domain . Our yeast two-hybrid and in vitro binding studies revealed that the rNedd4-WW domains mediate this association by binding to the P2 regions, which include the PY motifs (XPPXY) of either betarENaC (PPPNY) or gammarENaC (PPPRY) . SH3 domains were unable to bind these sequences . Moreover, mutations to Ala of Pro616 or Tyr618 within the betarENaC P2 sequence (to PPANY or PPPNA, respectively), recently described in Liddle's patients, led to abrogation of rNedd4-WW binding . Nedd4-WW domains also bound to the proline-rich C-terminus (containing the sequence PPPAY) of alpharENaC, and endogenous Nedd4 co-immunoprecipitated with alpharENaC expressed in MDCK cells . These results demonstrate that the WW domains of rNedd4 bind to the PY motifs deleted from beta or gammaENaC in Liddle's syndrome patients, and suggest that Nedd4 may be a regulator (suppressor) of the epithelial Na+ channel.

Ann N Y Acad Sci, 1996 May 15, 782, 462 - 77
The use of antibodies for characterization and quantification of a recombinant protein; Sabater M et al.; In this study, we characterized proteinase A secreted by recombinant Saccharomyces cerevisiae bearing a multicopy plasmid containing the encoding gene (PEP4) . Polyclonal and monoclonal antibodies were raised to study the product heterogeneity . Characterization of proteinase A was performed by immunoelectrophoresis and immunoblotting techniques . None of the monoclonal antibodies raised against proteinase A was found to react with the glycosyl side chains; thus cross-reaction with other glycosylated proteins (e.g . carboxypeptidase Y) was very low . This study allowed us to develop an ELISA method for the quantification of proteinase A in culture supernatants as well as the evaluation of monoclonal antibodies for their use in immunoaffinity chromatography.

Nucleic Acids Res, 1996 May 15, 24(10), 1971 - 8
The transactivation potential of a c-Myc N-terminal region (residues 92-143) is regulated by growth factor/Ras signaling; Colman MS et al.; The colony stimulating factor-1 receptor (CSF-1R) affects mitogenic growth and gene expression in NIH 3T3 cells through signaling pathways that require the products of the c-ras and c-myc proto-oncogenes . In this work we tested the hypothesis that there is direct communication between the Ras and Myc pathways . In transient transfection assays Ras increased by 5-fold transcriptional transactivation by chimeric c-Myc-Gal4 proteins . A constitutive active form of the CSF-1R also stimulated this activity and co-expression of a dominant negative ras gene ablated receptor stimulation . Deletion analysis of the c-Myc N-terminal region demonstrated that amino acid residues between positions 92 and 143 are the targets for Ras action . Transactivation by chimeric Myc proteins that were stably expressed could be transiently enhanced by either CSF-1 or serum, with peak activity occurring 2 h after mitogen stimulation . The steady-state levels of the chimeric c-Myc transactivators were increased following stimulation with CSF-1 or serum, but this increase in steady-state protein level did not strictly correlate with the increase in transactivation activity . Thus, Ras signaling may directly affect the activity of the c-Myc N-terminal region.

Mutat Res, 1996 May 15, 363(1), 43 - 56
Restoration of X-ray and etoposide resistance, Ku-end binding activity and V(D) J recombination to the Chinese hamster sxi-3 mutant by a hamster Ku86 cDNA; He DM et al.; Ku is a heterodimeric protein composed of 86 and 70 kDa subunits that binds preferentially to the double-stranded ends of DNA . Recent molecular characterization of ionizing-radiation sensitive (IRs) mutants belonging to the XRCC5 complementation group demonstrated the involvement of Ku in DNA double-strand break (DSB) repair and lymphoid V(D)J recombination . Here, we describe the isolation of a full-length hamster cDNA encoding the large subunit of the Ku heterodimer and demonstrate that the stable expression of this cDNA can functionally restore IR, Ku DNA end-binding activity and V(D)J recombination proficiency in the Chinese hamster IRs sxi-3 mutant . Moreover, we also demonstrate that sxi-3 cells are hypersensitive to etoposide, a DNA topoisomerase II inhibitor, and that resistance to this drug was restored by the Ku86 cDNA . These experiments suggest that a defect in the large subunit of the heterodimeric Ku protein is the sole factor responsible for the known defects of sxi-3 cells and our data of further support the role of Ku in DNA DSB repair and V(D)J recombination.

Proc Natl Acad Sci U S A, 1996 May 14, 93(10), 4974 - 8
Cloning and expression of apoptosis inhibitory protein homologs that function to inhibit apoptosis and/or bind tumor necrosis factor receptor-associated factors; Uren AG et al.; Baculovirus inhibitors of apoptosis (IAPs) act in insect cells to prevent cell death . Here we describe three mammalian homologs of IAP, MIHA, MIHB, and MIHC, and a Drosophila IAP homolog, DIHA . Each protein bears three baculovirus IAP repeats and an N-terminal ring finger motif . Apoptosis mediated by interleukin 1beta converting enzyme (ICE), which can be inhibited by Orgyia pseudotsugata nuclear polyhedrosis virus IAP (OpIAP) and cowpox virus crmA, was also inhibited by MIHA and MIHB . As MIHB and MIHC were able to bind to the tumor necrosis factor receptor-associated factors TRAF1 and TRAF2 in yeast two-hybrid assays, these results suggest that IAP proteins that inhibit apoptosis may do so by regulating signals required for activation of ICE-like proteases.

Proc Natl Acad Sci U S A, 1996 May 14, 93(10), 4572 - 6
Functional interaction and colocalization of the herpes simplex virus 1 major regulatory protein ICP4 with EAP, a nucleolar-ribosomal protein; Leopardi R et al.; The herpes simplex virus 1 infected cell protein 4 (ICP4) binds to DNA and regulates gene expression both positively and negatively . EAP (Epstein-Barr virus-encoded small nuclear RNA-associated protein) binds to small nonpolyadenylylated nuclear RNAs and is found in nucleoli and in ribosomes, where it is also known as L22 . We report that EAP interacts with a domain of ICP4 that is known to bind viral DNA response elements and transcriptional factors . In a gel-shift assay, a glutathione S-transferase (GST)-EAP fusion protein disrupted the binding of ICP4 to its cognate site on DNA in a dose-dependent manner . This effect appeared to be specifically due to EAP binding to ICP4 because (i) GST alone did not alter the binding of ICP4 to DNA, (ii) GST-EAP did not bind to the probe DNA, and (iii) GST-EAP did not influence the binding of the alpha gene trans-inducing factor (alphaTIF or VP16) to its DNA cognate site . Early in infection, ICP4 was dispersed throughout the nucleoplasm, whereas EAP was localized to the nucleoli . Late in infection, EAP was translocated from nucleoli and colocalized with ICP4 in small, dense nuclear structures . The formation of dense structures and the colocalization of EAP and ICP4 did not occur if virus DNA synthesis and late gene expression were prevented by the infection of cells at the nonpermissive temperature with a mutant virus defective in DNA synthesis, or in cells infected and maintained in the presence of phosphonoacetate, which is an inhibitor of viral DNA synthesis . These results suggest that the translocation of EAP from the nucleolus to the nucleoplasm is a viral function and that EAP plays a role in the regulatory functions expressed by ICP4.

FEBS Lett, 1996 May 13, 386(1), 75 - 81
Purification and characterization of the cytoplasmic histone acetyltransferase B of maize embryos; Eberharter A et al.; From a soluble cellular fraction of maize embryos we purified to apparent homogeneity a cytoplasmic histone acetyltransferase, which matches all criteria for a B-type enzyme . Using 8 chromatographic steps, we achieved a 6700-fold purification of an enzymatically active protein with a molecular weight of approximately 90 kDa . Under denaturing conditions the protein split into 2 components which migrated at 45 and 50 kDa in SDS-PAGE, suggesting that the native enzyme is a heterodimer . The purified enzyme was characterized in terms of physicochemical and kinetic properties, and substrate specificity . It was specific for histone H4, leading to acetylation of non-acetylated H4 subspecies into the di-acetylated state in vitro . Its activity was coincident with the intensity of DNA replication in meristematic cells during embryo germination . We established an electrophoretic system under non-denaturing conditions for detection of enzyme activity within the gel matrix; in combination with second dimension SDS-PAGE the procedure allowed the unambiguous identification of histone acetyltransferase, even in crude enzyme preparations.

J Biol Chem, 1996 May 10, 271(19), 11563 - 70
Substrate recognition by recombinant serine collagenase 1 from Uca pugilator; Tsu CA et al.; Uca pugilator serine collagenase 1 was cloned and sequenced from a fiddler crab hepatopancreas cDNA library . A full-length sequence encodes a 270-amino acid pre-pro-enzyme highly identical in structure to the chymotrypsin family of serine proteases . The zymogen form of the enzyme was expressed in Saccharomyces cerevisiae as a fusion with the alpha-factor signal sequence under control of the alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase promoter . Upon activation with trypsin, the recombinant collagenase possesses collagenolytic properties identical to those of the enzyme isolated from the crab hepatopancreas . The collagenase substrate binding pocket recognizes a wide range of basic, hydrophobic, and neutral polar residues . beta-Branched and acidic amino acids are poor substrates . Acylation is rate-limiting for collagenase versus peptidyl amides, rather than deacylation, as for trypsin and chymotrypsin . Correlations relating substrate volume and hydrophobicity to catalysis were found for collagenase and compared to those for chymotrypsin and elastase . Relative enzyme efficiencies on single amino acid versus tetrapeptide amide substrates show that collagenase derives less catalytic efficiency from binding of the primary substrate residue than trypsin or chymotrypsin, but compensates in binding of the extended peptidyl residues . Serine collagenase 1 is a novel member of the chymotrypsin protease family, by virtue of its amino acid sequence and multifunctional active site.

J Biol Chem, 1996 May 10, 271(19), 11182 - 90
Transcription factor IIA mutations show activator-specific defects and reveal a IIA function distinct from stimulation of TBP-DNA binding; Ozer J et al.; The general transcription factor IIA (TFIIA) binds to the TATA binding protein (TBP) and mediates transcriptional activation by distinct classes of activators . To elucidate the function of TFIIA in transcriptional activation, point mutants were created in the human TFIIA-gamma subunit at positions conserved with the yeast homologue . We have identified a class of TFIIA mutants that stimulate TBP-DNA binding (T-A complex) but fail to support transcriptional activation by several different activators, suggesting that these mutants are defective in their ability to facilitate an activation step subsequent to TBP promoter binding . Point mutations of the hydrophobic core of conserved residues from 65 to 74 resulted in various activation-defective phenotypes . These residues were found to be important for TFIIA gamma-gamma interactions, suggesting that gamma-gamma interactions are critical for TFIIA function as a coactivator . A subset of these TFIIA-gamma mutations disrupted transcriptional activation by all activators tested, except for the Epstein-Barr virus-encoded Zta protein . The gamma Y65F, gamma W72A, and gamma W72F mutants mediate Zta activation, but not GAL4-AH, AP-1, GAL4-CTF, or GAL4-VP16 activation . The gamma W72A mutant failed to stimulate TFIID-DNA binding (D-A complex) but was able to form a complex with TFIID and DNA in the presence of Zta (Z-D-A complex) . Thus, the ability of Zta to activate transcription with gamma W72A appears to result from a unique ability to form the stable Z-D-A complex with this mutant . Our results show that different activators utilize the general factor TFIIA in unique ways and that TFIIA contributes transcription activation functions in addition to the facilitation of TBP-DNA binding.

Biochemistry, 1996 May 7, 35(18), 5764 - 77
DNA replication machinery: functional characterization of a complex containing DNA polymerase alpha, DNA polymerase delta, and replication factor C suggests an asymmetric DNA polymerase dimer; Maga G et al.; By using a complementation assay for a replication factor C dependent DNA polymerase activity on a singly-primed M13 DNA template, we have isolated from calf thymus a multiprotein complex active in DNA replication . For this, the inclusion of ATP during the entire isolation procedure was essential, since the complex decayed after omission of ATP . This complex contains at least DNA polymerase alpha/primase, DNA polymerase delta, and replication factor C as shown by gel-filtration and coimmunoprecipitation experiments . It is functionally active in replication of primed and unprimed single-stranded M13 DNA templates . Furthermore, in the presence of proliferating cell nuclear antigen and ATP, it forms an isolatable holoenzyme/template-primer complex . Replication factor C apparently mediates the interaction of DNA polymerase delta in the complex with proliferating cell nuclear antigen, through an ATP-dependent mechanism . This interaction appears to stabilize the binding of the complex to a template-primer and to coordinate the activity of DNA polymerase alpha/primase and DNA polymerase delta during replication of a single-stranded DNA template . Our data suggest the existence of an asymmetric DNA polymerase complex in mammalian cells.

FEBS Lett, 1996 May 6, 385(3), 229 - 32
An SH3 domain-mediated interaction between the phagocyte NADPH oxidase factors p40phox and p47phox; Ito T et al.; The phagocyte NADPH oxidase is activated during phagocytosis to produce superoxide, following assembly of a membrane-integrated cytochrome b558 with cytosolic proteins, p47phox, p67phox and p40phox, each containing Src homology 3 (SH3) domains . While both p47phox and p67phox are indispensable for the oxidase activity, role of p40phox remains obscure . Here we study interaction between p40phox and p47phox by two independent methods, a two-hybrid system in the yeast and an in vitro binding assay using purified proteins . The present results show that the interaction is mediated via binding of the SH3 domain of p40phox to a C-terminal proline-rich region of p47phox . This proline-rich region is also the target for binding of p67phox, and the SH3 domain of p40phox can inhibit the binding of the C-terminal one of p67phox to p47phox.

FEBS Lett, 1996 May 6, 385(3), 221 - 4
Characterization of the interaction between RhoA and the amino-terminal region of PKN; Shibata H et al.; The yeast two-hybrid system and in vitro binding assay were carried out to characterize the interaction between PKN and a small GTP-binding protein, RhoA . It was revealed that the region corresponding to the amino acid residues 33-111 in the amino-terminal region of PKN was sufficient to confer the ability to associate with RhoA . Each synthetic peptide fragment corresponding to the amino acid residues 74-93 and 94-113 of PKN inhibited the interaction between PKN and RhoA in the in vitro binding assay, suggesting that this region is important in the association with RhoA . The endogenous and the GAP-stimulated GTPase activity of RhoA was inhibited by the interaction with PKN, suggesting the presence of a regulatory mechanism that sustains the GTP-bound active form of RhoA.

J Biol Chem, 1996 May 3, 271(18), 10910 - 6
Vitamin D receptors repress basal transcription and exert dominant negative activity on triiodothyronine-mediated transcriptional activity; Yen PM et al.; We have examined vitamin D receptor (VDR), thyroid hormone receptor (TR), and retinoid X receptor beta (RXR beta) binding to vitamin D response elements (VDREs), two thyroid hormone response elements (TREs) (DR4 and F2), and a retinoic acid response element (DR5) . VDR/RXR bound well to the VDREs and to DR4 and DR5 using the electrophoretic mobility shift assay . Surprisingly, VDR/RXR also bound well to F2, which contains half-sites arranged as an inverted palindrome . In co-transfection experiments using CV-1 cells, we observed that VDR repressed basal transcription in the absence of ligand on DR3 and osteopontin VDREs and F2, but had no effect on DR4 or DR5 . VDR selectively mediated ligand-dependent transcription on only VDREs . VDR also exhibited dominant negative activity as it blocked triiodothyronine (T3)-mediated transcriptional activity on DR4 and F2 . These results demonstrate that VDR/RXR heterodimers can bind promiscuously to a wide range of hormone response elements, including inverted palindromes . Moreover, they show that unliganded VDRs, similar to TRs and retinoic acid receptors, can repress basal transcription . Last, they also suggest a novel repressor function of VDR on T3-mediated transcription which may be significant in tissues where VDR and TR are co-expressed.

J Biol Chem, 1996 May 3, 271(18), 10904 - 9
Desmosomal cadherin binding domains of plakoglobin; Witcher LL et al.; Plakoglobin is a major component of both desmosomes and adherens junctions . At these sites it binds to the cytoplasmic domains of cadherin cell-cell adhesion proteins and regulates their adhesive and cytoskeletal binding functions . Plakoglobin also forms distinct cytosolic protein complexes that function in pathways of tumor suppression and cell fate determination . Recent studies in Xenopus suggest that cadherins inhibit the signaling functions of plakoglobin presumably by sequestering this protein at the membrane and depleting its cytosolic pool . To understand the reciprocal regulation between desmosomal cadherins (desmoglein and desmocollin) and plakoglobin, we have sought to identify the binding domains involved in the formation of these protein complexes . Plakoglobin comprises 13 central repeats flanked by amino-terminal and carboxyl-terminal domains . Our results show that repeats 1-4 are involved in binding desmoglein-1 . In contrast, the interaction of plakoglobin with desmocollin-1a is sensitive to deletion of either end of the central repeat domain . The binding sites for two adherens junction components, alpha-catenin and classical cadherins, overlap these sites . Competition among these proteins for binding sites on plakoglobin may therefore account for the distinct composition of adherens junctions and desmosomes.

Cell, 1996 May 3, 85(3), 423 - 33
Evidence for a new step in telomere maintenance; Wellinger RJ et al.; The strand of telomeric DNA that runs 5'-3' toward a chromosome end is typically G rich . Telomerase-generated G tails are expected at one end of individual DNA molecules . Saccharomyces telomeres acquire TG1-3 tails late in S phase . Moreover, the telomeres of linear plasmids can interact when the TG1-3 tails are present . Molecules that mimic the structures predicted for telomere replication intermediates were generated in vitro . These in vitro generated molecules formed telomere-telomere interactions similar to those on molecules isolated from yeast, but only if both ends that interacted had a TG1-3 tail . Moreover, TG1-3 tails were generated in vivo in cells lacking telomerase . These data suggest a new step in telomere maintenance, cell cycle-regulated degradation of the C1-3A strand, which can generate a potential substrate for telomerase and telomere-binding proteins at every telomere.

Cell, 1996 May 3, 85(3), 369 - 78
The cotranslational integration of membrane proteins into the phospholipid bilayer is a multistep process; Do H et al.; During the cotranslational integration of a nascent protein into the endoplasmic reticulum membrane, the transmembrane (TM) sequence moves out of an aqueous pore formed by Sec61alpha, TRAM, and other proteins and into the nonpolar lipid bilayer . Photocross-linking reveals that this movement involves the sequential passage of the TM domain through three different proteinaceous environments: one adjacent to Sec61alpha and TRAM and two adjacent to TRAM that place different restrictions on TM domain movement . In addition, the TM sequence is not allowed to diffuse into the bilayer from the final TRAM-proximal site until translation terminates . Cotranslational integration is therefore linked to translation and occurs via an ordered multistep pathway at an endoplasmic reticulum site that is multilayered both structurally and functionally.

Oncogene, 1996 May 2, 12(9), 1921 - 9
Identification of ArgBP1, an Arg protein tyrosine kinase binding protein that is the human homologue of a CNS-specific Xenopus gene; Wang B et al.; Arg and c-Abl represent the mammalian members of the Abelson family of nonreceptor protein-tyrosine kinases . To gain insight into the biological role of Arg we used the two-hybrid approach to identify interacting proteins . Using a C-terminal segment of Arg we identified a novel protein, ArgBP1 (Arg binding protein 1) . ArgBP1 contains a C-terminal SH3 domain, several PEST sequences, a serine rich domain and an SH3 binding site . ArgBP1 is ubiquitously expressed as two transcripts of approximately 2.2 kb and approximately 8 kb with highest levels in brain, heart and testis . The association of ArgBP1 with Arg in living cells was confirmed by coimmunoprecipitation in cotransfected COS cells . Analysis of the mechanism of association indicated that the ArgBP1 SH3 domain binds to a C-terminal Arg SH3-binding site, and that an N-terminal ArgBP1 proline-rich sequence binds to the Arg SH3 domain . Immunostaining indicated that the subcellular localization of ArgBP1 is cytoplasmic . The similarity of the ArgBP1 expression pattern and subcellular localization to those of Arg and the potential for a highly specific and potentially strong association mediated by two pairs of SH3 domain/proline-rich motif interactions, suggest that ArgBP1 is likely to be a regulator and/or effector of Arg function.

Biochim Biophys Acta, 1996 May 2, 1294(1), 15 - 24
Hydrostatic pressure induces conformational and catalytic changes on two alcohol dehydrogenases but no oligomeric dissociation; Dallet S et al.; A comparison between the pressure effects on the catalysis of Thermoanaerobium brockii alcohol dehydrogenase (TBADH: a thermostable tetrameric enzyme) and yeast alcohol dehydrogenase (YADH: a mesostable tetrameric enzyme) revealed a different behaviour . YADH activity is continuously inhibited by an increase of pressure, whereas YADH affinity seems less sensitive to pressure . TBADH activity is enhanced by pressure up to 100 MPa . TBADH affinity for alcoholic substrates increases if pressure increases, was TBADH affinity for NADP decreases when pressure increases . Hypothesis has been raised concerning the dissociation of oligomeric enzymes under high hydrostatic pressure ( < 200 MPa) {1} . But in the case of these two enzymes, unless the oligomers reassociate very quickly (< 1 min), the activity inhibition of YADH at all pressures and TBADH for pressures above 100 MPa is not correlated to subunit dissociation . Hence we suggest that enzymes under pressure encounter a molecular rearrangement which can either have a positive or a negative effect on activity . Finally, we have observed that the catalytic behaviour of alcohol dehydrogenases under pressure is connected to their thermostability.

Genes Cells, 1996 May, 1(5), 475 - 89
Communication between homologous chromosomes: genetic alterations at a nuclease-hypersensitive site can alter mitotic chromatin structure at that site both in cis and in trans; Keeney S et al.; BACKGROUND: In vegetatively growing diploid strains of the yeast Saccharomyces cerevisiae, homologous chromosomes appear to be paired via multiple interstitial interactions, likely as a regular feature of the diploid lifestyle . We have previously suggested that this pairing is guided by direct physical interactions between intact DNA duplexes in nuclease-hypersensitive regions and that homology is sensed directly at the DNA level . RESULTS: As a first test of this idea we have examined the level of DNase I sensitivity at a prominent nuclease-hypersensitive site in mitotic chromatin in strains that are either homozygous or heterozygous for a pair of alleles at this site . We find that the degree of nuclease sensitivity at this site on a given (maternal or paternal) chromosome can vary depending upon whether the homologue carries the same allele or the different allele . The data are suggestive that nuclease sensitivity is higher in the former case than in the latter, as though nuclease hypersensitivity might be increased when the two alleles match as compared to when they do not . CONCLUSIONS: Formally, these observations suggest that homologous chromosomes can communicate via a mechanism that senses the status of the assayed nuclease-hypersensitive site with resultant changes in chromatin structure at that site . The observed pattern of effects is fully compatible with direct physical interactions between homologues at nuclease-hypersensitive regions, but alternative scenarios also can be envisioned . Since DNase I hypersensitive sites occur in many important regions of chromosomes, homology-dependent interactions involving such regions could potentially affect diverse processes including gene expression (e.g . transvection), chromosome organization, domain structure, and/or DNA replication patterns.

Am J Physiol, 1996 May, 270(5 Pt 1), E873 - 81
Physiological concentrations of insulin and T3 stimulate 3T3-L1 adipocyte acyl-CoA synthetase gene transcription; Kansara MS et al.; Acyl-CoAsynthetase (ACS) is a key gene for cellular utilization of long-chain fatty acids . We characterized its regulation by physiological concentrations of insulin that acutely regulate metabolism . Our results demonstrate that subnanomolar insulin rapidly and maximally stimulates ACS gene transcription in the absence of protein synthesis; 0.5 nM insulin produced a 2.3 +/- 0.1-fold increase in ACS mRNA levels and induced ACS gene transcription 2.4 +/- 0.3-fold . The insulin sensitivity of ACS was compared with lipoprotein lipase (LPL) and stearoyl-CoA desaturase-1 (SCD-1), which were both less sensitive to insulin . Physiological triiodothyronine (10 nm) also induced ACS mRNA 2.4 +/- 0.1-fold and gene transcription 2.8 +/- 0.3-fold and coordinately induced LPL and SCD-1 mRNA and gene transcription . Because insulin and adenosine 3',5'-cyclic monophosphate often regulate genes involved in lipid and carbohydrate metabolism in a reciprocal manner, we evaluated effects of 1-methyl-3-isobutylxanthine (MIX).ACS mRNA levels were strongly downregulated by MIX in a dose-dependent manner, and ACS gene transcription inhibited in a coordinate manner with LPL and SCD-1 . These data demonstrate a uniquely sensitive pattern of stimulation of ACS gene transcription by insulin with reciprocal regulation by MIX, and they suggest a significant role for ACS as a tightly regulated "gatekeeper" gene participating in the control of adipocyte metabolism.

Dev Comp Immunol, 1996 May-Jun, 20(3), 207 - 16
In vitro studies on the regulation of rainbow trout (Oncorhynchus mykiss) macrophage respiratory burst activity; Novoa B et al.; Modulation of the respiratory burst activity of head kidney macrophages isolated from rainbow trout (Oncorhynchus mykiss) was observed following treatment with several biologically active substances . Macrophage-activating factor (MAF) induced the highest increment if respiratory burst activity relative to treatment with lipopolysaccharide (LPS), tumor necrosis factor alpha (TNF alpha) or beta-glucans from Saccharomyces cerevisiae . Increased responses were more evident when these molecules were combined in pairs . Negative regulation of respiratory burst activity was observed when diMePGE2 was added to the macrophages, with maximal inhibition seen using a concentration of 2.6 microM . Inhibition was also seen using stimulated macrophages, either by co-incubation of stimuli and diMePGE2 or by adding diMePGE2 to previously stimulated cells . The inhibitory effect on macrophages was detectable with 3 h of incubation with diMePGE2 and by 24 h the level of the response was even lower than that from unstimulated (control) macrophages . Of significance was the finding that the inhibitory effect of prostaglandin on macrophage function could be overcome by co-incubation with stimulatory molecules or by pre-treatment with MAF and LPS or MAF and TNF alpha Thus, the regulation of macrophage activation in fish is likely to be as complex as in mammals.

Hum Gene Ther, 1996 May 1, 7(7), 809 - 20
TAXI/UAS: A molecular switch to control expression of genes in vivo; Delort JP et al.; Numerous therapies and biological questions could be addressed in mammals by the application of a molecular switch that would allow physicians and/or investigators to turn individual genes on or off during the lifetime of the organism . We have constructed such a switch, composed of three elements: (i) an inducible promoter that is normally absent from mammalian genomes; (ii) a receptor that, when it is bound to an inducer drug, specifically activates transcription from the inducible promoter; and (iii) inducer drugs, such as RU486, whose pharmacological properties in humans and several mammalian species including mouse have been well studied . The molecular switch is functional in transiently and stably transfected cells . Importantly, both the total output and the induction levels of the reporter gene can be finely tuned, with induction levels of over 100-fold being readily attained . Finally, we demonstrate that the molecular switch can be used to regulate a mouse transgene using a gene therapy paradigm . The specificity of the system suggests that it should be useful in the analysis of gene function in transgenic animals and in the design of strategies for human gene therapy.

J Struct Biol, 1996 May-Jun, 116(3), 356 - 65
Uso1 protein is a dimer with two globular heads and a long coiled-coil tail; Yamakawa H et al.; USO1 is one of the essential genes in Saccharomyces cerevisiae whose gene products participate in protein transport from the endoplasmic reticulum to the Golgi apparatus . This product was purified to homogeneity . Electron microscopic study revealed that it has a single or double globular domain with a long tail and that the molecule is a dimer . A peak position of the distribution of rod length was 154.5 nm, in agreement with the secondary structure prediction that it has a long alpha-helix at the carboxyl terminus . Probability of coiled-coil formation was also predicted from the primary structure of the product, which asserts that it has a long alpha-helical coiled-coil at the carboxyl-terminal region with some interruptions . Certainly, the electron microscopic image of this molecule had some hinges within the rod region . The distance was measured between the globular domain and the hinges . Two peaks of the distribution of the hinge position exist at 23.1 and 85.5 nm from the globular domain . This is consistent with the predicted positions of interruption . These results give new experimental evidence that Uso1 protein is a dimer and has an alpha-helical coiled-coil tail with two globular heads.

Curr Biol, 1996 May 1, 6(5), 598 - 605
Human Ste20 homologue hPAK1 links GTPases to the JNK MAP kinase pathway; Brown JL et al.; BACKGROUND . The Rho-related GTP-binding proteins Cdc42 and Rac1 have been shown to regulate signaling pathways involved in cytoskeletal reorganization and stress-responsive JNK (Jun N-terminal kinase) activation . However, to date, the GTPase targets that mediate these effects have not been identified . PAK defines a growing family of mammalian kinases that are related to yeast Ste20 and are activated in vitro through binding to Cdc42 and Rac1 (PAK: p21 Cdc42-/Rac-activated kinase) . Clues to PAK function have come from studies of Ste20, which controls the activity of the yeast mating mitogen-activated protein (MAP) kinase cascade, in response to a heterotrimeric G protein and Cdc42 . RESULTS . To initiate studies of mammalian Ste20-related kinases, we identified a novel human PAK isoform, hPAK1 . When expressed in yeast, hPAK1 was able to replace Ste20 in the pheromone response pathway . Chemical mutagenesis of a plasmid encoding hPAK1, followed by transformation into yeast, led to the identification of a potent constitutively active hPAK1 with a substitution of a highly conserved amino-acid residue (L107F) in the Cdc42-binding domain . Expression of the hPAK1(L107F) allele in mammalian cells led to specific activation of the Jun N-terminal kinase MAP kinase pathway, but not the mechanistically related extracellular signal-regulated MAP kinase pathway . CONCLUSIONS . These results demonstrate that hPAK1 is a GTPase effector controlling a downstream MAP kinase pathway in mammalian cells, as Ste20 does in yeast . Thus, PAK and Ste20 kinases play key parts in linking extracellular signals from membrane components, such as receptor-associated G proteins and Rho-related GTPases, to nuclear responses, such as transcriptional activation.

Curr Biol, 1996 May 1, 6(5), 500 - 3
The complete code for a eukaryotic cell . Genome sequencing; Johnston M; The complete sequencing of the genome of a simple eukaryotic organism - the budding yeast Saccharomyces cerevisiae - is a milestone for biology, and sets the stage for a complete understanding of how a eukaryotic cell functions.

Curr Biol, 1996 May 1, 6(5), 551 - 4
Kinase connections on the cellular intranet . Signalling pathways; Pelech SL; The yeast mating factor-activated protein kinase pathway is a paradigm for understanding related pathways that transduce diverse signals . New studies of multicellular organisms, however, indicate higher levels of integration of these pathways within networks.

Tissue Antigens, 1996 May, 47(5), 382 - 90
Molecular and functional phenotypes of melanoma cells with abnormalities in HLA class I antigen expression; Wang Z et al.; Analysis of melanoma cell lines with abnormalities in HLA Class I antigen expression has identified two serological phenotypes caused by distinct molecular defects . One is characterized by lack of HLA Class I antigen expression which is not induced by IFN-gamma or by incubation at 25 degrees C for 24 hrs . This phenotype reflects structural changes in the beta(2)m gene which interfere with its transcription and/or translation or result in the synthesis of a defective beta(2)-mu polypeptide unable to associate with HLA Class I heavy chains . The other phenotype manifests very low HLA Class I antigen expression which is enhanced by IFN-gamma or by incubation at 25 degrees C for 24 hrs . This phenotype reflects abnormalities in TAP heterodimer expression, which cause defects in stable assembly and intracellular transport of the HLA Class I antigen trimolecular complex . Loss of HLA Class I antigens renders melanoma cells resistant to lysis by HLA Class I antigen-restricted cytotoxic T cells which specifically recognize melanoma associated antigens . Therefore, abnormalities in HLA Class I antigen expression may have a negative impact on the outcome of T cell based immunotherapy . Characterization of the molecular defects underlying loss of HLA Class I antigens may suggest approaches to restore their expression . Inclusion of these approaches in the protocols of T cell based immunotherapy may improve its efficacy.

Yeast, 1996 May, 12(6), 577 - 82
PetCR46, a gene which is essential for respiration and integrity of the mitochondrial genome; Coppee JY et al.; In the frame of the European Pilot Project for the functional analysis of newly discovered open reading frames (ORFs) from Saccharomyces cerevisiae chromosome III, we have deleted entirely the YCR46C ORF by a one-step polymerase chain reaction method and replaced it by the HIS3 marker in the strain W303 . The deletion has been checked by meiotic segregation and Southern blot analyses . Characterization of the deleted strain indicates that YCR46C is essential for respiration and maintenance of the mitochondrial genome since its deletion leads to the appearance of 100% of cytoplasmic petites . Hybridization with molecular probes from mtDNA of individual clones of such petites showed that about 50% did hybridize (rho- clones) while others did not (possibly rho degrees clones) . The wild-type gene has been cloned and shown to complement the deletion . The gene, which probably codes for a mitochondrial ribosomal protein, has been called petCR46.

Hum Mol Genet, 1996 May, 5(5), 705 - 8
Localisation of a gene for central areolar choroidal dystrophy to chromosome 17p; Lotery AJ et al.; Central areolar choroidal dystrophy (CACD) is a rare inherited retinal disease which causes progressive profound loss of vision in patients during their 4th decade . We have identified a Northern Irish family with 19 affected individuals in three living generations . We have performed a total genome search and established linkage of CACD in this family to chromosome 17p (multipoint Zmax = 5.65 at D17S938) . The genes for phosphatidylinositol transfer protein (PITPN), retinal guanylate cyclase (GUC2D), beta-arrestin 2 (ARRB2), pigment epithelium-derived factor (PEDF) and recoverin (RCV1) map to this region and are candidate genes for retinal disease . Analysis of the coding region of the PITPN gene failed to reveal any mutation in this family.

Genetics, 1996 May, 143(1), 345 - 51
Elimination of introns at the Drosophila suppressor-of-forked locus by P-element-mediated gene conversion shows that an RNA lacking a stop codon is dispensable; Williams CJ et al.; The suppressor of forked {su(f)} locus affects the phenotype of mutations caused by transposable element insertions at unlinked loci . It encodes a putative 84-kD protein with homology to two proteins involved in mRNA 3' end processing; the product of the yeast RNA14 gene and the 77-kD subunit of human cleavage stimulation factor . Three su(f) mRNAs are produced by alternative polyadenylation . The 2.6- and 2.9-kb mRNAs encode the same 84-kD protein while a 1.3-kb RNA, which terminates within the fourth intron, is unusual in having no stop codon . Using P-element-mediated gene replacement we have copied sequences from a transformation construct into the su(f) gene creating a su(f) allele at the normal genomic location that lacks the first five introns . This allele is viable and appears wild type for su(f) function, demonstrating that the 1.3-kb RNA and the sequences contained within the deleted introns are dispensable for su(f) function . Compared with studies on gene replacement at the white locus, chromosomal breaks at su(f) appear to be less efficiently repaired from ectopic sites, perhaps because of the location of su(f) at the euchromatin/heterochromatin boundary on the X chromosome.

Genetics, 1996 May, 143(1), 147 - 54
The vacuolar ATPase of Neurospora crassa is indispensable: inactivation of the vma-1 gene by repeat-induced point mutation; Ferea TL et al.; To analyze the phenotype of cells lacking the vacuolar ATPase, we inactivated the vma-1 gene, which encodes the catalytic subunit of the enzyme . Because preliminary experiments suggested the vma-1 gene was essential, we developed a method of simultaneously inactivating the gene and complementing it with a functional copy . We call this method repeat-induced point mutation (RIP) & Rescue . Two strains, both of which contained an extra copy of the vma-1 gene, were mated . Progeny that had inherited a functional copy of the gene at an ectopic site in the genome were selected . In some of these progeny the endogenous vma-1 gene had been altered by the RIP process . Sequencing showed the endogenous vma-1 gene had been inactivated by multiple point mutations . Progeny from strains with an inactive endogenous vma-1 gene were inviable unless a functional copy of the gene cosegregated, indicating that the vacuolar ATPase is essential in Neurospora crassa.

Genetics, 1996 May, 143(1), 95 - 102
Establishing genetic interactions by a synthetic dosage lethality phenotype; Kroll ES et al.; We have devised a genetic screen, termed synthetic dosage lethality, in which a cloned "reference" gene is inducibly overexpressed in a set of mutant strains carrying potential "target" mutations . To test the specificity of the method, two reference genes, CTF13, encoding a centromere binding protein, and ORC6, encoding a subunit of the origin of replication binding complex, were overexpressed in a large collection of mutants defective in either chromosome segregation or replication . CTF13 overexpression caused synthetic dosage lethality in combination with ctf14-42 (cbf2, ndc10), ctf17-61 (chl4), ctf19-58 and ctf19-26 . ORC6 overexpression caused synthetic dosage lethality in combination with cdc2-1, cdc6-1, cdc14-1, cdc16-1 and cdc46-1 . These relationships reflect specific interactions, as overexpression of CTF13 caused lethality in kinetochore mutants and overexpression of ORC6 caused lethality in replication mutants . In contrast, only one case of dosage suppression was observed . We suggest that synthetic dosage lethality identifies a broad spectrum of interacting mutations and is of general utility in detecting specific genetic interactions using a cloned wild-type gene as a starting point . Furthermore, synthetic dosage lethality is easily adapted to the study of cloned genes in other organisms.

Appl Biochem Biotechnol, 1996 May, 59(2), 167 - 75
Selectivity of methyl-fructoside synthesis with beta-fructofuranosidase; Rodriguez M et al.; Enzyme synthesis of methyl fructoside was studied using beta-fructofuranosidase from Sacharomyces cerevisiae and sucrose and methanol as substrates . Taking into account the inhibition and deactivation effects of methanol on the enzyme, a system with 4.9M (20%, v/v) methanol was selected . At this alcohol level, 35% of sucrose is converted to fructoside at low or high substrate concentrations . The effect of enzyme concentration, pH, and temperature on both the synthesis and the hydrolysis of the fructoside was investigated . It was found that if the reaction proceeds at pH 6.0, 4 degree C and/or 0.014 mg/mL (3 U/mL) of beta-fructofuranosidase at varying sucrose concentrations, methyl fructoside may be obtained with a minimum loss of the fructoside at the end of the reaction.

Kaohsiung J Med Sci, 1996 May, 12(5), 301 - 5
Elimination of false negative results in the two-hybrid system in the phagocyte NADPH oxidase; Hong YR et al.; The yeast two-hybrid system is finding increased use in the study of interactions between proteins . In this method, two polypeptides are expressed in yeast as fusion proteins to a transcriptional activator DNA-binding domain (bd) and activating domain (ad), respectively . Interaction between the two polypeptides reconstitutes function of a transactivator which controls expression of reporters . The phagocyte NADPH oxidase is a complex of membrane cytochrome b558 (comprised of subunits p22-phox and gp91-phox) and three cytosol proteins (p47-phox, p67-phox, and p21rac) that translocate to membrane and bind to cytochrome b558 . This is the first report to demonstrate that two of cytosolic components of cytochrome b558, p47-phox binding to p67-phox each other . We encountered several methodological problems in the two-hybrid system which are the focus of this report.

Nat Genet, 1996 May, 13(1), 35 - 42
A gene (RPGR) with homology to the RCC1 guanine nucleotide exchange factor is mutated in X-linked retinitis pigmentosa (RP3); Meindl A et al.; X-linked retinitis pigmentosa (xlRP) is a severe progressive retinal degeneration which affects about 1 in 25,000 of the population . The most common form of xlRP, RP3, has been localised to the interval between CYBB and OTC in Xp21.1 by linkage analysis and deletion mapping . Identification of microdeletions within this region has now led to the positional cloning of a gene, RPGR, that spans 60 kg of genomic DNA and is ubiquitously expressed . The predicted 90 kD protein contains in its N-terminal half a tandem repeat structure highly similar to RCC1 (regulator of chromosome condensation), suggesting an interaction with a small GTPase . The C-terminal half contains a domain, rich in acidic residues, and ends in a potential isoprenylation anchorage site . The two intragenic deletions, two nonsense and three missense mutations within conserved domains provide evidence that RPGR (retinitis pigmentosa GTPase regulator) is the RP3 gene.

Plant Cell, 1996 May, 8(5), 899 - 913
High-level transgene expression in plant cells: effects of a strong scaffold attachment region from tobacco; Allen GC et al.; We have previously shown that yeast scaffold attachment regions (SARs) flanking a chimeric beta-glucuronidase (GUS) reporter gene increased per-copy expression levels by 24-fold in tobacco suspension cell lines stably transformed by microprojectile bombardment . In this study, we examined the effect of a DNA fragment originally identified in a tobacco genomic clone by its activity in an in vitro binding assay . The tobacco SAR has much greater scaffold binding affinity than does the yeast SAR, and tobacco cell lines stably transformed with constructs containing the tobacco SAR accumulated greater than fivefold more GUS enzyme activity than did lines transformed with the yeast SAR construct . Relative to the control construct, flanking the GUS gene with plant SARs increased overall expression per transgene copy by almost 140-fold . In transient expression assays, the same construct increased expression only approximately threefold relative to a control without SARs, indicating that the full SAR effect requires integration into chromosomal DNA . GUS activity in individual stable transformants was not simply proportional to transgene copy number, and the SAR effect was maximal in cell lines with fewer than approximately 10 transgene copies per tobacco genome . Lines with significantly higher copy numbers showed greatly greatly reduced expression relative to the low-copy-number lines . Our results indicate that strong SARs flanking a transgene greatly increases expression without eliminating variation between transformants . We propose that SARs dramatically reduce the severity or likelihood of homology-dependent gene silencing in cells with small numbers of transgenes but do not prevent silencing of transgenes present in many copies.

J Cell Biol, 1996 May, 133(4), 761 - 75
Association of a dynamin-like protein with the Golgi apparatus in mammalian cells; Henley JR et al.; Dynamins are a family of 100-kD GTPases comprised of at least three distinct gene products and multiple alternatively spliced variants . Homologies with the shibire gene product in Drosophila melanogaster and with Vps1p and Dnm1p in Saccharomyces cerevisiae suggest that dynamins play an important role in vesicular transport . Morphological studies have localized brain dynamin to coated pits and tubular invaginations at the plasma membrane, where it is believed to facilitate the formation of endocytic vesicles . Because similar membrane-budding events occur at the Golgi apparatus and multiple dynamin isoforms exist, we have studied the distribution of dynamins in mammalian cells . To this end, we generated and characterized peptide-specific antibodies directed against conserved regions of the dynamin family . By immunoblot analysis, these antibodies reacted specifically with a 100-kD protein in fibroblasts that sedimented with membranes and microtubules in vitro in a manner similar to brain dynamin . By immunofluorescence microscopy, these antibodies strongly labeled the Golgi complex in cultured fibroblasts and melanocytes, as confirmed by double labeling with a Golgi-specific antibody . Furthermore, Western blot analysis showed significant enrichment of a 100-kD dynamin band in Golgi fractions isolated from the liver . To substantiate these findings, we use a specific antidynamin antibody to immunoisolate Golgi membranes from subcellular Golgi fractions, as determined by EM and immunoblot analysis . This study provides the first morphological and biochemical evidence that a dynamin-like protein associates with the Golgi apparatus in mammalian cells, and suggests that dynamin-related proteins may have multiple cytoplasmic distributions . The potential contributions of dynamin to the secretory and endocytic pathways are discussed.

RNA, 1996 May, 2(5), 492 - 505
RNA sequences upstream of the 3' splice site repress splicing of mutantyeast ACT1 introns; Kivens W et al.; A yeast ACT1 intron in which both the first and last intron nucleotides are mutated, the /a-c/ intron, splices 10% as well as wild type . We selected for additional cis-acting mutations that improve the splicing of /a-c/ introns and recovered small deletions upstream of the 3' splice site . For example, deletion of nucleotides -9 and -10 upstream of the 3' splice site increased the splicing activity of the /a-c/ intron to 30% that of the wild-type ACT1 intron . To determine if the increased /a-c/ splicing was due to changes in intron spacing or sequence, we made mutations that mimicked the local sequence of the delta-9, -10 deletion without deleting any nucleotides . These mutants also increased /a-c/ splicing, indicating that the increased splicing activity was due to changes in intron sequence . The delta-9, -10 deletion was not allele specific to the /a-c/ intron, and improved the splicing efficiency of many mutant introns with step II splicing defects . To further define the sequences required for improved splicing of mutant introns, we randomized the region upstream of the ACT1 3' splice site . We found that almost all sequence alterations improved the splicing of the /a-c/ intron . We postulate that this sequence near the 3' end of the intron represses the splicing of mutant introns, perhaps by serving as the binding site for a negative splicing factor.

J Mol Evol, 1996 May, 42(5), 552 - 9
18S rRNA suggests that Entoprocta are protostomes, unrelated to Ectoprocta; Mackey LY et al.; The Ento- and Ectoprocta are sometimes placed together in the Bryozoa, which have variously been regarded as proto- or deuterostomes . However, Entoprocta have also been allied to the pseudocoelomates, while Ectoprocta are often united with the Brachiopoda and Phoronida in the (super)phylum Lophophorata . Hence, the phylogenetic relationships of these taxa are still much debated . We determined complete 18S rRNA sequences of two entoprocts, an ectoproct, an inarticulate brachiopod, a phoronid, two annelids, and a platyhelminth . Phylogenetic analyses of these data show that (1) entoprocts and lophophorates have spiralian, protostomous affinities, (2) Ento- and Ectoprocta are not sister taxa, (3) phoronids and brachiopods form a monophyletic clade, and (4) neither Ectoprocta or Annelida appear to be monophyletic . Both deuterostomous and pseudocoelomate features may have arisen at least two times in evolutionary history . These results advocate a Spiralia-Radialia-based classification rather than one based on the Protostomia-Deuterostomia concept.

Hum Genet, 1996 May, 97(5), 620 - 4
The human lanosterol synthase gene maps to chromosome 21q22.3; Young M et al.; In order to contribute to the development of the transcriptional map of human chromosome 21 (HC21) we have used exon trapping to identify portions of HC21 genes . Using pools of random HC21-specific cosmids from the LL21NC02-Q library and cosmids from 21q22.3 we have identified five different coding regions with strong homology to the lanosterol synthase genes of rat and yeast . This enzyme catalyzes the cyclization of squalene-2,3-epoxide lanosterol, which is the parental compound of all steroids in mammals . Using somatic cell hybrids and HC21 yeast artificial chromosomes (YACS) and cosmids, we mapped the human lanosterol synthase cDNA gene to 2lq22.3 between markers D21S25 and 21qter . Cosmid Q7G8 from the LL21NC02-Q library and YAC 145D8 from the CEPH HC21 contig contain this human gene . We cloned a portion of the human lanosterol synthase cDNA (almost 85% of the coding region) from a brain cDNA library and determined its nucleotide sequence . The predicted human protein shows 83% identity to its rat and 40% to its yeast homolog . No obvious candidate human disease exists for lanosterol synthase deficiency and the role (if any) of triplication of this gene in the various phenotypes of trisomy 21 is unknown.

Nucleic Acids Res, 1996 May 1, 24(9), 1616 - 24
On the use of double FLP recognition targets (FRTs) in the LTR of retroviruses for the construction of high producer cell lines; Karreman S et al.; A pilot experiment for the construction of a hamster derived, high producer cell line using site specific recombination is described . In the experiment chromosomal loci with intrinsic high expression characteristics were sought via infection with a retroviral construct, containing double FRT sites and subsequent screening for overproduction of an encoded markergene . These sites were then targeted with a second vector, that recombined via the FLP/FRT system from Saccharomyces cerevisiae yielding cells that had the second construct at exactly the same position as the first . By using retroviral vectors with double and single FRT sites, respectively, stable clones can be created that can no longer be excised with FLP.

Biochem J, 1996 May 1, 315 ( Pt 3), 909 - 16
Probing the high-affinity site of beef heart cytochrome c oxidase by cross-linking; Malatesta F et al.; A covalent complex between cytochrome c oxidase and Saccharomyces cerevisiae iso-1-cytochrome c (called caa3) has been prepared at low ionic strength . Subunit III Cys-115 of beef heart cytochrome c oxidase cross-links by disulphide bond formation to thionitrobenzoate-modified yeast cytochrome c, a derivative shown to bind into the high-affinity site for substrate {Fuller, Darley-Usmar and Capaldi (1981) Biochemistry 20, 7046-7053} . Stopped-flow experiments show that (1) covalently bound yeast cytochrome c cannot donate electrons to cytochrome oxidase, whereas oxidation of exogenously added cytochrome c and electron transfer to cytochrome a are only slightly affected; (2) the steady-state reduction levels of cytochrome c and cytochrome a in the covalent complex caa3 are higher than those found in the native aa3 enzyme . However, (3) K(m) and Vmax values obtained from the non-linear Eadie-Hofstee plots are very similar in both caa3 and aa3 . The results imply that cytochrome c bound to the high-affinity site is not in a configuration optimal for electron transfer.

EMBO J, 1996 May 1, 15(9), 2125 - 37
Tom7 modulates the dynamics of the mitochondrial outer membrane translocase and plays a pathway-related role in protein import; Honlinger A et al.; The preprotein translocase of the outer mitochondrial membrane is a multi-subunit complex with receptors and a general import pore . We report the molecular identification of Tom7, a small subunit of the translocase that behaves as an integral membrane protein . The deletion of TOM7 inhibited the mitochondrial import of the outer membrane protein porin, whereas the import of preproteins destined for the mitochondrial interior was impaired only slightly . However, protein import into the mitochondrial interior was strongly inhibited when it occurred in two steps: preprotein accumulation at the outer membrane in the absence of a membrane potential and subsequent further import after the re-establishment of a membrane potential . The delay of protein import into tom7delta mitochondria seemed to occur after the binding of preproteins to the outer membrane receptor sites . A lack of Tom7 stabilized the interaction between the receptors Tom20 and Tom22 and the import pore component Tom40 . This indicated that Tom7 exerts a destabilizing effect on part of the outer membrane translocase, whereas Tom6 stabilizes the interaction between the receptors and the import pore . Synthetic growth defects of the double mutants tom7delta tom20delta and tom7delta tom6delta provided genetic evidence for the functional relationship of Tom7 with Tom20 and Tom6 . These results suggest that (i) Tom7 plays a role in sorting and accumulation of the preproteins at the outer membrane, and (ii) Tom7 and Tom6 perform complementary functions in modulating the dynamics of the outer membrane translocase.

EMBO J, 1996 May 1, 15(9), 2115 - 24
A mutation in protein phosphatase 2A regulatory subunit A affects auxin transport in Arabidopsis; Garbers C et al.; The phytohormone auxin controls processes such as cell elongation, root hair development and root branching . Tropisms, growth curvatures triggered by gravity, light and touch, are also auxin-mediated responses . Auxin is synthesized in the shoot apex and transported through the stem, but the molecular mechanism of auxin transport is not well understood . Naphthylphthalamic acid (NPA) and other inhibitors of auxin transport block tropic curvature responses and inhibit root and shoot elongation . We have isolated a novel Arabidopsis thaliana mutant designated roots curl in NPA (rcn1) . Mutant seedlings exhibit altered responses to NPA in root curling and hypocotyl elongation . Auxin efflux in mutant seedlings displays increased sensitivity to NPA . The rcn1 mutation was transferred-DNA (T-DNA) tagged and sequences flanking the T-DNA insert were cloned . Analysis of the RCN1 cDNA reveals that the T-DNA insertion disrupts a gene for the regulatory A subunit of protein phosphatase 2A (PP2A-A) . The RCN1 gene rescues the rcn1 mutant phenotype and also complements the temperature-sensitive phenotype of the Saccharomyces cerevisiae PP2A-A mutation, tpd3-1 . These data implicate protein phosphatase 2A in the regulation of auxin transport in Arabidopsis.

Bioessays, 1996 May, 18(5), 411 - 9
Meiotic recombination: a mechanism for tracking and eliminating mutations?
McKee BD.
The function of meiotic recombination has remained controversial, despite recent inroads into mechanisms . Ideas concerning a possible role of recombination in the elimination or efficient incorporation of mutations have been backed by theoretical studies but have lacked empirical support . Recent investigations into the basis for local variations in recombination frequency in yeast have uncovered a strong association between recombination initiation sites and transcriptional regulatory sequences . Other recent studies indicate a strong correlation between transcription and mutation rates in yeast genes . Taken together, these data imply that distributions of recombination and mutation frequencies may be strongly correlated . This suggests that recombination may be targeted to genomic sites of high mutation frequency; such a 'mutation-tracking' function would clearly aid in the shuffling of mutations to break up unfavorable and create favorable allelic combinations . Moreover, recent insights into the mechanism of gene conversion in yeast reveal a very strong inherent bias in favor of alleles on the non-initiating homolog . Combined with mutation tracking, these findings suggest a novel and general mechanism by which allelic gene conversion may act to eliminate mutations.

J Cell Biol, 1996 May, 133(3), 605 - 16
A purified Drosophila septin complex forms filaments and exhibits GTPase activity; Field CM et al.; Septin proteins are necessary for cytokinesis in budding yeast and Drosophila and are thought to be the subunits of the yeast neck filaments . To test whether septins actually form filaments, an immunoaffinity approach was used to isolate a septin complex from Drosophila embryos . The purified complex is comprised of the three previously identified septin polypeptides Pnut, Sep2, and Sep1 . Hydrodynamic and sequence data suggest that the complex is composed of a heterotrimer of homodimers . The complex copurifies with one molecule of bound guanine nucleotide per septin polypeptide . It binds and hydrolyzes exogenously added GTP . These observations together with conserved sequence motifs identify the septins as members of the GTPase superfamily . We discuss a model of filament structure and speculate as to how the filaments are organized within cells.

FASEB J, 1996 May, 10(7), 799 - 801
Functional interaction of hexokinase with ATP requires participation by both small and large lobes of the enzyme: implications for other proteins using the actin fold as a nucleotide binding motif; Wilson JE et al.; The actin fold is a structural motif involved in binding of ATP to an otherwise diverse family of proteins that includes hexokinase, actin, and the 70 kDa heat shock protein . Previous analyses have focused attention on a hydrophobic pocket within the large lobe of hexokinase (and analogous regions in other proteins possessing the actin fold motif) as the-site for binding of the adenine moiety . However, there is reason to believe that binding of the adenine moiety also involves a beta-sheet region located in the small lobe of hexokinase . It is postulated that functional interaction with ATP, required for catalysis, involves interactions with both regions . The expected structural consequences provide a basis for explaining the kinetic mechanism suggested for this enzyme, i.e., although both substrates can bind independently, the preferred kinetic pathway is not random but ordered, with glucose binding first . The structural features postulated to be involved in binding of ATP to hexokinase are also found in other proteins possessing the actin fold motif . Interaction of the nucleotide with the beta-sheet region, analogous to that found in the "small lobe" of hexokinase, may induce functionally important conformational changes in other proteins employing the actin fold as a nucleotide binding motif.

Mol Cell Biol, 1996 May, 16(5), 2394 - 401
Helix-loop-helix proteins LYL1 and E2a form heterodimeric complexes with distinctive DNA-binding properties in hematolymphoid cells; Miyamoto A et al.; LYL1 is a basic helix-loop-helix (HLH) protein that was originally discovered because of its translocation into the beta T-cell receptor locus in an acute lymphoblastic leukemia . LYL1 is expressed in many hematolymphoid cells, with the notable exceptions of thymocytes and T cells . Using the yeast two-hybrid system to screen a cDNA library constructed from B cells, we identified the E-box-binding proteins E12 and E47 as potential lymphoid dimerization partners for LYL1 . The interaction of LYL1 with E2a proteins was further characterized in vitro and shown to require the HLH motifs of both proteins . Immunoprecipitation analyses showed that in T-ALL and other cell lines, endogenous LYL1 exists in a complex with E2a proteins . A preferred DNA-binding sequence, 5'-AACAGATG(T/g)T-3', for the LYL1-E2a heterodimer was determined by PCR-assisted site selection . Endogenous protein complexes containing both LYL1 and E2a bound this sequence in various LYL1-expressing cell lines and could distinguish between the LYL1 consensus and muE2 sites . These data demonstrate that E2a proteins serve as dimerization partners for the basic HLH protein LYL1 to form complexes with distinctive DNA-binding properties and support the hypothesis that the leukemic properties of the LYL1 and TAL subfamily of HLH proteins could be mediated by recognition of a common set of target genes as heterodimeric complexes with class I HLH proteins.

Mol Cell Biol, 1996 May, 16(5), 2274 - 82
A unique transactivation sequence motif is found in the carboxyl-terminal domain of the single-strand-binding protein FBP; Duncan R et al.; The far-upstream element-binding protein (FBP) is one of several recently described factors which bind to a single strand of DNA in the 5' region of the c-myc gene . Although cotransfection of FBP increases expression from a far-upstream element-bearing c-myc promoter reporter, the mechanism of this stimulation is heretofore unknown . Can a single-strand-binding protein function as a classical transactivator, or are these proteins restricted to stabilizing or altering the conformation of DNA in an architectural role? Using chimeric GAL4-FBP fusion proteins we have shown that the carboxyl-terminal region (residues 448 to 644) is a potent transcriptional activation domain . This region contains three copies of a unique amino acid sequence motif containing tyrosine diads . Analysis of deletion mutants demonstrated that a single tyrosine motif alone (residues 609 to 644) was capable of activating transcription . The activation property of the C-terminal domain is repressed by the N-terminal 107 amino acids of FBP . These results show that FBP contains a transactivation domain which can function alone, suggesting that FBP contributes directly to c-myc transcription while bound to a single-strand site . Furthermore, activation is mediated by a new motif which can be negatively regulated by a repression domain of FBP.

Mol Cell Biol, 1996 May, 16(5), 2101 - 9
Adenovirus E1A proteins inhibit activation of transcription by p53; Steegenga WT et al.; p53 stimulates the transcription of a number of genes, such as MDM2, Waf1, and GADD45 . We and others have shown previously that this activity of p53 can be inhibited by adenovirus type 2 or 12 large E1B proteins . Here we show that the adenovirus E1A proteins also can repress the stimulation of transcription by p53, both in transient transfections and in stably transfected cell lines . The inhibition by E1A occurs without a significant effect on the DNA-binding capacity of p53 . Furthermore, the activity of a fusion protein containing the N-terminal part of p53 linked to the GAL4 DNA-binding domain can be suppressed by E1A . This indicates that E1A affects the transcription activation domain of p53, although tryptic phosphopeptide mapping revealed that the level of phosphorylation of this domain does not change significantly in E1A-expressing cell lines . Gel filtration studies, however, showed p53 to be present in complexes of increased molecular weight as a result of E1A expression . Apparently, E1A can cause increased homo- or hetero-oligomerization of p53, which might result in the inactivation of the transcription activation domain of p53 . Additionally, we found that transfectants stably expressing E1A have lost the ability to arrest in G1 after DNA damage, indicating that E1A can abolish the normal biological function of p53.

Mol Cell Biol, 1996 May, 16(5), 2044 - 55
Three functional classes of transcriptional activation domain; Blau J et al.; We have studied the abilities of different transactivation domains to stimulate the initiation and elongation (postinitiation) steps of RNA polymerase II transcription in vivo . Nuclear run-on and RNase protection analyses revealed three classes of activation domains: Sp1 and CTF stimulated initiation (type I); human immunodeficiency virus type 1 Tat fused to a DNA binding domain stimulated predominantly elongation (type IIA); and VP16, p53, and E2F1 stimulated both initiation and elongation (type IIB) . A quadruple point mutation of VP16 converted it from a type IIB to a type I activator . Type I and type IIA activators synergized with one another but not with type IIB activators . This observation implies that synergy can result from the concerted action of factors stimulating two different steps in transcription: initiation and elongation . The functional differences between activators may be explained by the different contacts they make with general transcription factors . In support of this idea, we found a correlation between the abilities of activators, including Tat, to stimulate elongation and their abilities to bind TFIIH.

J Virol, 1996 May, 70(5), 3176 - 88
PEST-dependent cytoplasmic retention of v-Rel by I(kappa)B-alpha: evidence that I(kappa)B-alpha regulates cellular localization of c-Rel and v-Rel by distinct mechanisms; Rottjakob EM et al.; Association of c-Rel with the inhibitor of kappaB-alpha (IkappaB-alpha) protein regulates both cellular localization and DNA binding . The ability of v-Rel, the oncogenic viral counterpart of avian c-Rel, to evade regulation by p40, the avian IkappaB-alpha protein, contributes to v-Rel-mediated oncogenesis . The yeast two-hybrid system was utilized to dissect Rel:IkappaB-alpha interactions in vivo . We find that distinct domains in c-Rel and v-Rel are required for association with p40 . Furthermore, while the ankyrin repeat domain of p40 is sufficient for association with c-Rel, both the ankyrin repeat domain and the PEST domain are required for association with v-Rel . Two amino acid differences between c-Rel and v-Rel that are principally responsible for PEST-dependent association of v-Rel with p40 were identified . These same amino acids were principally responsible for PEST-dependent cytoplasmic retention of v-Rel by p40 . The presence of mutations in c-Rel that were sufficient to confer PEST-dependent association of the mutant c-Rel protein with p40 did not increase the weak oncogenicity of c-Rel . However, the introduction of these two c-Rel-derived amino acids into v-Rel markedly reduced the oncogenicity of v-Rel . Deletion of the NLS of either c-Rel or v-Rel did not abolish association with p40, but did confer PEST-dependent association of c-Rel with p40 . Surprisingly, deletion of the nuclear localization signal in v-Rel did not affect oncogenicity by v-Rel . Analysis of several mutant c-Rel and v-Rel proteins demonstrated that association of Rel proteins with p40 is necessary but not sufficient for cytoplasmic retention . These results are not consistent with the hypothesis that p40 regulates cellular localization of v-Rel and c-Rel by the same mechanism . Rather, these results support the hypothesis that p40 regulates cellular localization of v-Rel and c-Rel by distinct mechanisms.

Mol Pharmacol, 1996 May, 49(5), 772 - 80
Inhibition of DNA topoisomerase II by imidazoacridinones, new antineoplastic agents with strong activity against solid tumors; Skladanowski A et al.; Imidazoacridinones are new antitumor compounds that exhibit strong antitumor effect against solid tumors such as human colon and breast carcinomas . The mechanism of action of imidazoacridinones is unknown, although a similarity in the chemical structure between active imidazoacridinones and mitoxantrone suggests common cellular targets . We show that imidazoacridinones inhibit the catalytic activity of purified topoisomerase II as determined by both relaxation and decatenation assays . All biologically active compounds stimulated the formation of cleavable complexes in vitro, whereas inactive compounds did not . The pattern of DNA cleavage in SV40 DNA was similar to that obtained for 4'-(9-acridinylamino)methane-sulfon-m-aniside, particularly within the matrix-associated region . Significant levels of DNA complexes were observed when DC-3F fibrosarcoma cells were treated with active compounds, whereas negligible amounts of these complexes were induced by inactive analogues . DC-3F/9-OHE cells, which are resistant to other topoisomerase II inhibitors, are 30-125-fold cross-resistant to active imidazoacridinones . The resistance is associated with a reduction in the formation of DNA/protein complexes and is highest for compounds that are potent topoisomerase II inhibitors in vitro . Interestingly, the two most active derivatives, C-1310 and C-1311, were equally cytotoxic toward fast-growing monolayer cultures and cells growing in three dimensions as multicellular spheroids, which have a slower growth fraction . In contrast, 4'-(9-acridinylamino)methanesulfon-m-aniside, mitoxantrone, and doxorubicin were more cytotoxic toward monolayer cultures . Taken together, the results suggest that DNA topoisomerase II is a major cellular target of biologically active imidazoacridinones and that these drugs show both similarities and dissimilarities compared with classic topoisomerase II inhibitors.

Cancer Res, 1996 May 1, 56(9), 1997 - 2002
Identification of two candidate tumor suppressor genes on chromosome 17p13.3; Schultz DC et al.; A second tumor suppressor locus on 17p that is distinct from TP53 has been identified in brain, breast, lung, and ovarian tumors . Using allelic loss mapping and positional cloning methods, we have recently identified two novel genes, which we refer to as OVCA1 and OVCA2, that map to 17p13.3 . The two genes are ubiquitously expressed and encode proteins of 443 and 227 amino acids, respectively, with no known functional motifs . Sequence comparison of OVCA1 and OVCA2 revealed extensive sequence identity and similarity to hypothetical proteins from Saccharomyces cerevisiae, Caenorhabditis elegans, and Rattus species . Northern blot analysis reveals that OVCA1 and OVCA2 mRNA were expressed in normal surface epithelial cells of the ovary, but the level of this transcript is significantly reduced or is undetectable in 92% (11/12) of the ovarian tumors and tumor cell lines analyzed . The location, high degree of amino acid conservation, and reduced expression in ovarian tumors and tumor cell lines suggest that decreased expression of these two genes contributes to ovarian tumorigenesis and should be considered candidate tumor suppressor genes.

Proc Natl Acad Sci U S A, 1996 Apr 30, 93(9), 4057 - 62
DNA transport by a type II topoisomerase: direct evidence for a two-gate mechanism; Roca J et al.; Recent biochemical and crystallographic results suggest that a type II DNA topoisomerase acts as an ATP-modulated clamp with two sets of jaws at opposite ends: a DNA-bound enzyme can admit a second DNA through one set of jaws; upon binding ATP, this DNA is passed through an enzyme-mediated opening in the first DNA and expelled from the enzyme through the other set of jaws . Experiments based on the introduction of reversible disulfide links across one dimer interface of yeast DNA topoisomerase II have confirmed this mechanism . The second DNA is found to enter the enzyme through the gate formed by the N-terminal parts of the enzyme and leave it through the gate close to the C termini.

Philos Trans R Soc Lond B Biol Sci, 1996 Apr 29, 351(1339), 569 - 78
Ligand-dependent interaction of nuclear receptors with potential transcriptional intermediary factors (mediators); Le Douarin B et al.; The activity of the ligand-inducible activation function 2 (AF-2) contained in the ligand binding domain (LBD) of nuclear receptors (NRs) is thought to be mediated by transcriptional intermediary factors (TIFs) . We have recently reported the isolation and characterization of two novel mouse proteins, designated TIF1 and mSUG1, that interact in a ligand-dependent fashion with the LBD (region E) of several NRs in vivo as well as in vitro . Remarkably, these interactions require the conserved core motif of the AF-2 activating domain (AF-2 AD) and can be blocked by AF-2 antagonists . TIF1 and mSUG1 might therefore represent TIFs/mediators for the ligand-dependent AF-2 of NRs . By comparing the interaction properties of these two putative TIFs with different NRs including the oestrogen (ER), thyroid hormone (TR), vitamin D3 (VDR), retinoic acid (RAR alpha) and retinoid X (RXR) receptors, we demonstrate that: (i) RXR alpha efficiently interacts with TIF1, but not with mSUG1, whereas TR alpha interacts much more efficiently with mSUG1 than with TIF1, and RAR alpha, VDR and ER efficiently interact with both TIF1 and mSUG1; (ii) the amphipathic alpha helix core of AF-2 AD is differentially involved in the interactions of RAR alpha with TIF1 and mSUG1; and (iii) the AF-2 AD cores of RAR alpha and ER are similarly involved in their interaction with TIF1, but not with mSUG1 . Thus the interaction interfaces between the various NRs and either TIF1 or mSUG1 may vary depending on the nature of both the receptor and the putative mediator of its AF-2 function . We discuss the possible roles of TIF1 and mSUG1 as mediators of the transcriptional activity of the AF-2 of NRs.

Philos Trans R Soc Lond B Biol Sci, 1996 Apr 29, 351(1339), 517 - 26
Mechanisms of transcriptional activation and repression can both involve TFIID; Manley JL et al.; Regulation of transcription involves the activities of activators and repressors . Recent experiments have provided evidence that the function of both types of regulators can involve interactions with one or more component of the basal transcription machinery . A principal target appears to be TFIID, which consists of the TATA binding protein (TBP) and associated factors (TAFs) . Here we describe experiments that provide added support for the idea that interactions affecting TFIID can play important roles in both activation and repression . We show, using transfection assays in Drosophila Schneider cells, that recruitment of TBP to a promoter as a GAL4-TBP fusion protein can provide a substantial activation of transcription . The conserved core of TBP is necessary and sufficient for this effect, which was observed with both TATA-containing and TATA-lacking basal promoters . These findings extend experiments performed in yeast, and strengthen the idea that recruitment of TBP (TFIID) can be an important mechanism of activation . We also provide further support for the idea that TBP can be a target for a transcriptional repressor, the Drosophila Even-skipped protein (Eve) . We present evidence that the homeodomain, which is necessary for binding TBP in vitro, can also be required for repression in vivo, independent of its role in DNA binding . On the other hand, deletion of the alanine/proline-rich region that is essential for repression in vivo and TBP binding in vitro does not significantly affect DNA binding by the purified protein . These results strengthen the view that TBP, either directly or indirectly as a component of TFIID, can be a target of both activators and repressors.

J Biol Chem, 1996 Apr 26, 271(17), 10282 - 90
Characterization of AMP-activated protein kinase beta and gamma subunits . Assembly of the heterotrimeric complex in vitro; Woods A et al.; There is growing evidence that mammalian AMP-activated protein kinase (AMPK) plays a role in protecting cells from stresses that cause ATP depletion by switching off ATP-consuming biosynthetic pathways . The active form of AMPK from rat liver exists as a heterotrimeric complex and we have previously shown that the catalytic subunit is structurally and functionally related to the SNF1 protein kinase from Saccharomyces cerevisiae . Here we describe the isolation and characterization of the two other polypeptides, termed AMPKbeta and AMPKgamma, that together with the catalytic subunit (AMPKalpha) form the active kinase complex in mammalian liver . Sequence analysis of cDNA clones encoding these subunits reveals that they are related to yeast proteins that interact with SNF1, providing further evidence that the regulation and function of AMPK and SNF1 have been conserved throughout evolution . The amino acid sequence of the beta subunit is most closely related to SIP2 (35% identity), while the amino acid sequence of the gamma subunit is 35% identical with SNF4 . We show that both AMPKbeta and AMPKgamma mRNA and protein are expressed widely in rat tissues . We show that AMPKbeta interacts with both AMPKalpha and AMPKgamma in vitro, whereas AMPKalpha does not interact with AMPKgamma under the same conditions . These results suggest that AMPKbeta mediates the association of the heterotrimeric AMPK complex in vitro, and will facilitate future studies aimed at investigating the regulation of AMPK in vivo.

J Biol Chem, 1996 Apr 26, 271(17), 10010 - 6
Computer simulation of the triosephosphate isomerase catalyzed reaction; Aqvist J et al.; A major challenge for theoretical simulation methods is the calculation of enzymic reaction rates directly from the three-dimensional protein structure together with some idea of the chemical reaction mechanism . Here, we report the evaluation of a complete free energy profile for all the elementary steps of the triosephosphate isomerase catalyzed reaction using such an approach . The results are compatible with available experimental data and also suggest which of the possible reaction intermediates is kinetically observable . In addition to previously identified catalytic residues, the simulations show that a crystallographically observed active site water molecule plays an important role during catalysis and an intersubunit interaction that could explain the low activity of the monomeric enzyme is also observed . The calculations clearly demonstrate the important catalytic effects associated with stabilization of charged high energy intermediates and reduction of reorganization energy, which are likely to be general principles of enzyme catalyzed charge transfer and separation reactions.

Science, 1996 Apr 26, 272(5261), 560 - 2
Linkage of replication to start by the Cdk inhibitor Sic1; Schneider BL et al.; In Saccharomyces cerevisiae, three G1 cyclins (Clns) are important for Start, the event committing cells to division . Sic1, an inhibitor of C1b-Cdc28 kinases, became phosphorylated at Start, and this phosphorylation depended on the activity of Clns . Sic1 was subsequently lost, which depended on the activity of Clns and the ubiquitin-conjugating enzyme Cdc34 . Inactivation of Sic1 was the only nonredundant essential function of Clns, because a sic1 deletion rescued the inviability of the cln1 cln2 cln3 triple mutant . In sic1 mutants, DNA replication became uncoupled from budding . Thus, Sic1 may be a substrate of Cln-Cdc28 complexes, and phosphorylation and proteolysis of Sic1 may regulate commitment to replication at Start.

J Mol Biol, 1996 Apr 19, 257(5), 1019 - 30
Expression of a continuous open reading frame encoding subunits 1 and 2 of cytochrome c oxidase in the mitochondrial DNA of Acanthamoeba castellanii; Lonergan KM et al.; We have investigated the expression of a continuous open reading frame (ORF) present in the mitochondrial genome of Acanthamoeba castellanii and specifying the two largest subunits (COX1 and COX2) of the cytochrome c oxidase complex . Northern hybridization and primer extension analysis demonstrated that this ORF (cox1/2, 873 codons) is transcribed as part of a 4.7 kb RNA that also includes the upstream small subunit rRNA sequence . Between the cox1 and cox2 portions of the transcript, RNA sequence exactly matches gene sequence, excluding the possibility that a standard cox1 termination codon is created by post-transcriptional RNA processing or editing . Western analysis revealed an A . castellanii COX2 protein with a mobility matching that of mature COX2 from yeast (Saccharomyces cerevisiae) mitochondria . These observations indicate that although A . castellanii COX1 and COX2 are apparently translated from the same ORF, they do not exist in mature form as a COX1-COX2 "fusion" protein . Whereas translation of COX2 could potentially be initiated from an internal AUG codon in the cox1/2 ORF, COX1 must be generated either through an unusual translation termination mechanism acting between the cox1 and cox2 coding regions of the cox1/2 mRNA, or by co-translational or post-translational proteolytic processing of a translation product whose synthesis continues into the cox2 coding region . Because the cox2 nucleotide sequence predicts a COX2 protein considerably larger than that observed by Western analysis, A . castellanii COX2 may undergo additional post-translational processing to its final form.

Cell, 1996 Apr 19, 85(2), 205 - 15
SNARE-mediated retrograde traffic from the Golgi complex to the endoplasmic reticulum; Lewis MJ et al.; Operation of the secretory pathway in eukaryotic cells requires the selective docking and fusion of transport vesicles with the appropriate target organelle . This is mediated in part by integral membrane proteins termed v-SNAREs (on vesicles) and t-SNAREs (on the target membranes) . We describe a novel yeast t-SNARE that resides on the endoplasmic reticulum and mediates retrograde traffic from the Golgi complex . Mutation of this protein prevents both the HDEL receptor and a membrane protein bearing a dibasic retrieval signal from recycling to the endoplasmic reticulum . Forward traffic is also blocked, but only indirectly . Comparison with other yeast mutants indicates that Sec21p (gamma-COP) and Sec20p (an endoplasmic reticulum membrane protein) are also involved primarily, if not exclusively, in retrograde transport.

Oncogene, 1996 Apr 18, 12(8), 1821 - 6
Development of thyroid papillary carcinomas secondary to tissue-specific expression of the RET/PTC1 oncogene in transgenic mice; Santoro M et al.; Gene rearrangements activating the RET proto-oncogene are frequently associated with human thyroid carcinomas belonging to the papillary subtype . These arrangements cause the fusion of the tyrosine-kinase domain of RET to the 5'-terminal region of different genes creating the RET/PTC chimeric oncogenes . Here we report the generation of transgenic mice lines expressing the RET/PTC1 oncogene under the control of the thyroid-specific rat thyroglobulin promoter . RET/PTC1-transgenic mice developed thyroid tumors displaying the histological aspect of papillary carcinomas . These tumors were slowly progressive and did not cause premature death of the animals . Two additional mice developed areas of thyroid hyperplasia . Immunohistochemical and reverse-transcriptase polymerase chain reaction analyses confirmed the thyroid-specific expression of the transgene . Given the frequency of activating rearrangements of RET in human papillary thyroid carcinomas we conclude that this animal system could be a good model for studying the neoplastic progression of thyroid carcinomas.

Nature, 1996 Apr 18, 380(6575), 642 - 6
Control of cAMP-regulated enhancers by the viral transactivator Tax through CREB and the co-activator CBP; Kwok RP et al.; The Tax protein of human T-lymphotropic virus (HTLV)-1 activates expression of the HTLV-1 long terminal repeat through a DNA element that resembles the cellular cyclic AMP-regulated enhancer (CRE) . Tax contains a transcriptional activation domain, but its ability to activate gene expression depends on interactions with cellular CRE-binding proteins such as CREB . Whether Tax can activate the expression of cellular CRE-containing genes has been controversial . Here we show that Tax can activate both the HTLV-1 and consensus cellular CREs, and propose that this activation may occur through mechanisms that are differentially dependent on CREB phosphorylation . Tax not only increases the binding of CREB to the viral CRE but also recruits the transcriptional co-activator CBP in a manner independent of CREB phosphorylation . In contrast, association of Tax with the cellular CRE occurs through CBP which, in turn, is recruited only in the presence of phosphorylated CREB.

Gene, 1996 Apr 17, 170(1), 119 - 23
An additional copy of the adenylate cyclase-encoding gene relieves developmental defects produced by a mutation in a vegetative incompatibility-controlling gene in Podospora anserina; Loubradou G et al.; To identify cellular functions involved in vegetative incompatibility in filamentous fungi, we have initiated the cloning of Podospora anserina (Pa) mod genes . These genes interfere with the lethal reaction triggered by interaction between incompatible het genes . A gene (Pa AC) has been cloned by complementation of developmental defects caused by a mutation in the mod-D gene . This gene encodes a protein of 2145 amino acids (aa)that exhibits strong similarities with many adenylate cyclases (AC) . About 65% aa identity has been found between the sequence of the polypeptide encoded by this Pa AC gene and the AC of Neurospora crassa . The organization of peptidic domains in the polypeptide encoded by Pa AC is closely related to that of Saccharomyces cerevisiae CYR1 . Restriction-fragment-length polymorphism (RFLP) and genetic analysis have shown that Pa AC and mod-D are distinct genes.

Gene, 1996 Apr 17, 170(1), 1 - 8
GCN4 eukaryotic transcription factor/FokI endonuclease-mediated 'Achilles' heel cleavage': quantitative study of protein-DNA interaction; Skowron PM et al.; Three proteins, yeast transcription regulatory protein GCN4, M.FokI DNA methyltransferase and R.FokI restriction endonuclease (ENase) were used to attain specific cleavage of DNA at the 18-20-bp GCN4 recognition site . This is a novel version of the 'Achilles' heel cleavage' (AC) technique {Koob et al., Science 241 (1988) 1084-1086} . Since the method employs a class-IIS ENase (R.FokI), which cleaves the DNA outside of its recognition sequence, it leaves the overlapping GCN4-binding intact . Thus, the same GCN4 site can be used in consecutive cleavage reactions . This novel GCN4-IIS-AC technique was applied to study the protein-DNA interaction . Quantitative analysis of the effect of temperature, reaction time, and GCN4 and M.FokI concentrations allowed determination of the GCN4-DNA complex half-life, which was found to be 7 h at 30 degrees C, 18 h at 22 degrees C and over 24 h at 10 degrees C . In addition, conditions for controlled, partial GCN4-IIS-AC digestion of DNA were determined, and applied to the physical mapping of large genomes.

Proc Natl Acad Sci U S A, 1996 Apr 16, 93(8), 3248 - 52
Fos and Jun do not bend the AP-1 recognition site; Sitlani A et al.; We have used a solution-based DNA cyclization assay and a gel-phasing method to show that contrary to previous reports {Kerppola, T . K . & Curran, T . (1991) Cell 66, 317-326}, basic region leucine zipper proteins Fos and Jun do not significantly bend their AP-1 recognition site . We have constructed two sets of DNA constructs that contain the 7-bp 5'-TGACTCA-3' AP-1 binding site, from either the yeast or the human collagenase gene, which is well separated from and phased by 3-4 helical turns against an A tract-directed bend . The cyclization probabilities of DNAs with altered phasings are not significantly affected by Fos-Jun binding . Similarly, Fos-Jun and Jun-Jun bound to differently phased DNA constructs show insignificant variations in gel mobilities . Both these methods independently indicate that Fos and Jun bend their AP-1 target site by <5 degrees, an observation that has important implications in understanding their mechanism of transcriptional regulation.

Structure, 1996 Apr 15, 4(4), 463 - 73
The evolution of an allosteric site in phosphorylase; Rath VL et al.; BACKGROUND: Glycogen phosphorylases consist of a conserved catalytic core onto which different regulatory sites are added . By comparing the structures of isozymes, we hope to understand the structural principles of allosteric regulation in this family of enzymes . Here, we focus on the differences in the glucose 6-phosphate (Glc-6-P) binding sites of two isozymes . RESULTS: We have refined the structure of Glc-6-P inhibited yeast phosphorylase b to 2.6 A and compared it with known structures of muscle phosphorylase . Glc-6-P binds in a novel way, interacting with a distinct set of secondary elements . Structural links connecting the Glc-6-P binding sites and catalytic sites are conserved, although the specific contacts are not . CONCLUSIONS: Our comparison reveals that the Glc-6-P binding site was modified over the course of evolution from yeast to vertebrates to become a bi-functional switch . The additional ability of muscle phosphorylase to be activated by AMP required the recruitment of structural elements into the binding site and sequence changes to create a binding subsite for adenine, whilst maintaining links to the catalytic site.

Arch Biochem Biophys, 1996 Apr 15, 328(2), 245 - 54
Characterization of the n-alkane and fatty acid hydroxylating cytochrome P450 forms 52A3 and 52A4; Scheller U et al.; Two enzymes, P450 52A3 (P450Cm1) and 52A4 (P450Cm2), the genes of which belong to the CYP52 multigene family occurring in the alkane-assimilating yeast Candida maltosa, have been characterized biochemically and compared in terms of their substrate specificities . For this purpose, both the p450 proteins and the corresponding C . maltosa NADPH-cytochrome P450 reductase were separately produced by expressing their cDNAs in Saccharomyces cerevisiae, purified, and reconstituted to active monooxygenase systems . Starting from microsomal fractions with a specific content of 0.75 nmol P450Cm1, 0.34 nmol P450Cm2, and 10.5 units reductase per milligram of protein, respectively, each individual recombinant protein was purified to homogeneity . P450 substrate difference spectra indicated strong type I spectral changes and high-affinity binding of n-hexadecane (Ks= 26 micron) and n-octadecane (Ks = 27 microM) to P450Cm1, whereas preferential binding to P450Cm2 was observed using lauric acid (Ks = 127 microM) and myristic acid (Ks = 134 microM) as substrates . These substrate selectivities were further substantiated by kinetic parameters, determined for n-alkane and fatty acid hydroxylation in a reconstituted system, which was composed of the purified components and phospholipid, as well as in microsomes obtained after coexpressing each of the P450 proteins with the reductase . The highest catalytic activities within the reconstituted system were measured for n-hexadecane hydroxylation to 1-hexadecanol by P450Cm1 (Vmax = 27 microM x min-1, Km = 54 microM) and oxidation of lauric acid to 16-hydroxylauric acid by P450Cm2 (Vmax = 30 microM x min-1, Km = 61 microM) . We conclude that P450Cm1 and P450Cm2 exhibit overlapping but distinct substrate specificities due to different chain-length dependencies and preferences for either n-alkanes or fatty acids.

EMBO J, 1996 Apr 15, 15(8), 1950 - 60
The human papillomavirus type 16 E7 gene product interacts with and trans-activates the AP1 family of transcription factors; Antinore MJ et al.; The E7 gene product of human papillomavirus type 16 (HPV16) binds to the retinoblastoma gene product (pRb) and dissociates pRb-E2F complexes . However, the observation that the ability of E7 to bind pRb is not required for the HPV16-induced immortalization of primary keratinocytes prompted a search for other cellular factors bound by E7 . Using a glutathione-S-transferase (GST) fusion protein system, we show that E7 complexes with AP1 transcription factors including c-Jun, JunB, JunD and c-Fos . The ability of E7 to complex with c-Jun in vivo is demonstrated by co-immunoprecipitation and the yeast two-hybrid system . An analysis of E7 point mutants in the GST system indicates that the E7 zinc-finger motif, but not the pRb binding domain, is involved in these interactions . Using c-Jun deletion mutants, E7 binding maps between amino acids 224 and 286 of c-Jun . E7 trans-activates c-Jun-induced transcription from a Jun responsive promoter, and this activity correlates with the ability of E7 mutants to bind Jun proteins . Finally, a transcriptionally inactive c-Jun deletion, which can bind E7, interferes with the E7-induced transformation of rat embryo fibroblasts in cooperation with an activated ras, indicating that the Jun-E7 interaction is physiologically relevant and that Jun factors may be targeted in the E7 transformation pathway.

EMBO J, 1996 Apr 15, 15(8), 1792 - 8
Delta- and zeta-COP, two coatomer subunits homologous to clathrin-associated proteins, are involved in ER retrieval; Cosson P et al.; Two new thermosensitive yeast mutants defective in retrieval of dilysine-tagged proteins from the Golgi back to the endoplasmic reticulum (ER) were characterized . While both ret2-1 and ret3-1 were defective for ER retrieval, only ret2-1 exhibited a defect in forward ER-to-Golgi transport at the non-permissive temperature . Coatomer (COPI) from both mutants could efficiently bind dilysine motifs in vitro . The corresponding RET2 and RET3 genes were cloned by complementation and found of encode the delta and zeta subunits of coatomer respectively . Both proteins show significant homology to clathrin adaptor subunits . These results emphasize the role of coatomer in retrieval of dilysine-tagged proteins back to the ER, and the similarity between clathrin and coatomer coats.

Biochem J, 1996 Apr 15, 315 ( Pt 2), 429 - 34
Molecular cloning and functional expression of human deoxyhypusine synthase cDNA based on expressed sequence tag information; Yan YP et al.; Deoxyhypusine synthase is an NAD(+)-dependent enzyme that catalyses the formation of a deoxyhypusine residue on the eukaryotic initiation factor 5A (eIF-5A) precursor by transferring an aminobutyl moiety from spermidine to the epsilon-amino group of a unique lysine residue . We have recently cloned and characterized the Neurospora crassa deoxyhypusine synthase cDNA using a reverse genetics approach . A GenBank search showed that a stretch of the deduced amino acid sequence (96 amino acids) of Neurospora deoxyhypusine synthase matches a short human expressed sequence tag (EST), Z25337, with greater than 70% amino acid identity . Gene-specific primers based on this EST were used together with universal primers to obtain 1219 bp and 1078 bp cDNAs from a human cDNA library . The 1219 bp and 1078 bp sequences, each containing an open reading frame, encode polypeptides of respectively 368 and 321 amino acids . The short sequence is identical to the long one except that it is missing a stretch of 47 amino acids spanning residues 261-307 . The 368-amino-acid sequence of human deoxyhypusine synthase shares a high degree of identity ( > 50%) and similarity ( > 60%) with that of the Neurospora and yeast deoxyhypusine synthases . After cloning into an expression vector, the 368-amino-acid recombinant protein exhibits high deoxyhypusine synthase activity . In contrast, the 321-amino-acid recombinant protein shows no detectable activity.

Genes Dev, 1996 Apr 15, 10(8), 921 - 33
Drosophila IRBP/Ku p70 corresponds to the mutagen-sensitive mus309 gene and is involved in P-element excision in vivo; Beall EL et al.; The P family of transposable elements in Drosophila transpose by a cut-and-paste mechanism involving double-strand gap repair . We report here that a Drosophila mutagen-sensitive mutant, mus3O9, contains a mutation in IRBP (inverted repeat binding protein), the Drosophila homolog of the mammalian Ku p70 gene . We show that the repair of double-strand DNA breaks after P-element excision is severely reduced in mus3O9 mutants using an in vivo assay for P-element transposase activity . In addition, excision products recovered from mus3O9 mutant embryos by use of a plasmid-based P-element mobility assay contain large deletions, suggesting that IRBP is involved in the repair of double-strand DNA breaks . Our findings provide the first demonstration that a mutation in the IRBP gene affects double-strand DNA break repair and suggest that DNA repair functions are conserved between Drosophila and mammals.

Genes Dev, 1996 Apr 15, 10(8), 905 - 20
Repression and activation by multiprotein complexes that alter chromatin structure; Kingston RE et al.; Recent studies have provided strong evidence that macromolecular complexes are used in the cell to remodel chromatin structure during activation and to create an inaccessible structure during repression, Although there is not yet any rigorous demonstration that modification of chromatin structure plays a direct, causal role in either activation or repression, there is sufficient smoke to indicate the presence of a blazing inferno nearby . It is clear that complexes that remodel chromatin are tractable in vitro; hopefully this will allow the establishment of systems that provide a direct analysis of the role that remodeling might play in activation . These studies indicate that establishment of functional systems to corroborate the elegant genetic studies on repression might also be tractable . As the mechanistic effects of these complexes are sorted out, it will become important to understand how the complexes are regulated . In many of the instances discussed above, the genes whose products make up these complexes were identified in genetic screens for effects on developmental processes . This implies a regulation of the activity of these complexes in response to developmental cues and further implies that the work to fully understand these complexes will occupy a generation of scientists.

Blood, 1996 Apr 15, 87(8), 3089 - 96
Cloning of human Bcl-2 homologue: inflammatory cytokines induce human A1 in cultured endothelial cells; Karsan A et al.; Bcl-2 is an intracellular membrane-associated protein that functions to block programmed cell death . Despite recurrent exposure to cellular toxins from the circulation and tissue, endothelial cells are remarkably resistant to cell death . Because Bcl-2 protein levels are low or undetectable in endothelial cells, we postulated that other members of the growing Bcl-2 family would be present in endothelial cells to provide protection against apoptosis . Degenerate primers to two conserved regions of the Bcl-2 family were used to amplify potential homologues in endothelial cells . This strategy resulted in the isolation of a human Bcl-2 homologue related to murine Al, a recently identified member of this family . We show here that, in endothelial cells, human Al is rapidly inducible by phorbol ester and the inflammatory cytokines, tumor necrosis factor-alpha and interleukin-1beta, but not by the growth factors, basic fibroblast growth factor or vascular endothelial growth factor . Al is the only known Bcl-2 family member that is inducible by inflammatory cytokines, suggesting that it may play a protective role during inflammation . Additionally, vascular smooth muscle cells and various nonhematopoietic tissues express human Al, indicating that human Al is a widely expressed Bcl-2 homologue.

J Biol Chem, 1996 Apr 12, 271(15), 8936 - 41
The DNA-dependent protein kinase is inactivated by autophosphorylation of the catalytic subunit; Chan DW et al.; The DNA-dependent protein kinase (DNA-PK) requires for activity free ends or other discontinuities in the structure of double strand DNA . In vitro, DNA-PK phosphorylates several transcription factors and other DNA-binding proteins and is thought to function in DNA damage recognition or repair and/or transcription . Here we show that in vitro DNA-PK undergoes autophosphorylation of all three protein subunits (DNA-PKcs, Ku p70 and Ku p80) and that phosphorylation correlates with inactivation of the serine/threonine kinase activity of DNA-PK . Significantly, activity is restored by the addition of purified native DNA-PKcs but not Ku, suggesting that inactivation is due to autophosphorylation of DNA-PKcs . Our data also suggest that autophosphorylation results in dissociation of DNA-PKcs from the Ku-DNA complex . We suggest that autophosphorylation is an important mechanism for the regulation of DNA-PK activity.

J Biol Chem, 1996 Apr 12, 271(15), 8887 - 94
The Hansenula polymorpha PER9 gene encodes a peroxisomal membrane protein essential for peroxisome assembly and integrity; Baerends RJ et al.; We have cloned and characterized the Hansenula polymorpha PER9 gene by functional complementation of the per9-1 mutant of H . polymorpha, which is defective in peroxisome biogenesis . The predicted product, Per9p, is a polypeptide of 52 kDa with sequence similarity to Pas3p, a protein involved in peroxisome biogenesis in Saccharomyces cerevisiae . In a per9 disruption strain (Deltaper9), peroxisomal matrix and membrane proteins are present at wild-type levels . The matrix proteins accumulated in the cytoplasm . However, the location of the membrane proteins remained obscure; fully induced Deltaper9 cells lacked residual peroxisomal vesicles ("ghosts") . Analysis of the activity of the PER9 promoter revealed that PER9 expression was low in cells grown on glucose, but was enhanced during growth of cells on peroxisome-inducing substrates . The highest expression levels were observed in cells grown on methanol . Localization studies revealed that Per9p is an integral membrane protein of the peroxisome . Targeting studies suggested that Per9p may be sorted to the peroxisome via the endoplasmic reticulum . Overexpression of PER9 induced a significant increase in the number of peroxisomes per cell, a result that suggests that Per9p may be involved in peroxisome proliferation and/or membrane biosynthesis . When PER9 expression was placed under the control of a strongly regulatable promoter and switched off, peroxisomes were observed to disintegrate over time in a manner that suggested that Per9p may be required for maintenance of the peroxisomal membrane.

J Biol Chem, 1996 Apr 12, 271(15), 8725 - 30
Characterization and analysis of conserved motifs in a peroxisomal ATP-binding cassette transporter; Shani N et al.; The adrenoleukodystrophy protein (ALDP) and the 70-kDa peroxisomal membrane protein are half ATP-binding cassette (ABC) transporters in the human peroxisome membrane . Both are implicated in genetic disorders of peroxisome biogenesis and function . Proteins homologous to ALDP and the 70-kDa peroxisomal membrane protein have been discovered in other eukaryotic organisms and form a growing group of peroxisomal half ABC transporters . Amino acid sequence alignment of these and other ABC transporters reveals several protein motifs that are highly conserved both in sequence and location . Here we characterize two of these, designated the EAA-like and the loop1 motifs . We study them by introducing missense mutations in Pxa1p, a Saccharomyces cerevisiae ortholog of ALDP, and show that both motifs are important for Pxa1p function . Interestingly, missense mutations in corresponding amino acids in ALDP cause adrenoleukodystrophy in humans . We conclude that these motifs are important for ABC transporter function and that the yeast protein Pxa1p is a useful system for understanding the molecular basis of adrenoleukodystrophy.

J Biol Chem, 1996 Apr 12, 271(15), 8661 - 6
Phosphorylation of the DNA polymerase alpha-primase B subunit is dependent on its association with the p180 polypeptide; Ferrari M et al.; The B subunit of the DNA polymerase (pol) alpha-primase complex executes an essential role at the initial stage of DNA replication in Saccharomyces cerevisiae and is phosphorylated in a cell cycle-dependent manner . In this report, we show that the four subunits of the yeast DNA polymerase alpha-primase complex are assembled throughout the cell cycle, and physical association between newly synthesized pol alpha (p180) and unphosphorylated B subunit (p86) occurs very rapidly . Therefore, B subunit phosphorylation does not appear to modulate p180.p86 interaction . Conversely, by depletion experiments and by using a yeast mutant strain, which produces a low and constitutive level of the p180 polypeptide, we found that formation of the p180.p86 subcomplex is required for B subunit phosphorylation.

J Biol Chem, 1996 Apr 12, 271(15), 8633 - 45
Loop replacement and random mutagenesis of omega-loop D, residues 70-84, in iso-1-cytochrome c; Mulligan-Pullyblank P et al.; To study the role of omega loop D, residues 70-84, in the structure and function of yeast iso-1-cytochrome c, this loop was replaced with homologous and heterologous loops . A novel method was developed for rapid insertion of these mutations into the yeast chromosome at the CYC1 locus . The strains containing these loop replacement cytochromes cannot grow on nonfermentable carbon sources, indicating that the proteins are nonfunctional . Whole cell difference spectroscopy shows that no holocytochrome c is present; however, apoprotein is found by immunoblot analysis . Thus, apoprotein is present in these mutant strains, but it cannot bind heme and cannot compete with wild type apoprotein conversion to holoprotein . This is a unique example of a set of loop replacements that do not produce folded protein, and these results suggest that the loop D amino acid sequence in iso-1-cytochrome c plays a significant role in cytochrome c biosynthesis in vivo . To identify the significant amino acids in loop D, random mutagenesis of six highly conserved loop residues, Tyr-74, Ile-75, Pro-76, Gly-77, Thr-78, and Lys-79, was accomplished . Sequencing of the random mutants shows that strict conservation of none of these residues is required to produce a minimally functional cytochrome c . Preferences are found for small, hydrophilic or aromatic residues at position 74, hydrophobic residues at position 75, glycine and arginine at positions 76 and 77, and beta-branched amino acids at position 78 . Implications for the role of loop D in the structure and function of iso-1-cytochrome c are discussed.

J Biol Chem, 1996 Apr 12, 271(15), 8593 - 8
The interaction between Ku antigen and REF1 protein mediates negative gene regulation by extracellular calcium; Chung U et al.; Through the specific binding of a negative calcium-responsive element to its binding protein in response to extracellular Ca (Ca2+e), negative calcium-responsive element-bearing genes, such as the human parathyroid hormone gene, are negatively regulated by Ca2+e . The Ku antigen mediated negative gene regulation by Ca2+e by interacting with a redox factor protein, REF1 . Although sequence-nonspecific DNA binding activity of the Ku antigen has been well characterized, the mechanism of its sequence-specific DNA binding remained obscure . Here, we report that the specific binding of the Ku antigen to another protein, REF1, leads to DNA-protein complex formation with a novel sequence specificity and thereby regulates gene expression.

Science, 1996 Apr 12, 272(5259), 268 - 70
Accurate processing of a eukaryotic precursor ribosomal RNA by ribonuclease MRP in vitro; Lygerou Z et al.; Very few of the enzymes required for eukaryotic precursor ribosomal RNA (pre-rRNA) processing have been identified . Ribonuclease (RNase) MRP was characterized as a nuclease that cleaves mitochondrial replication primers, but it is predominantly nucleolar . Previous genetic evidence revealed that this ribonucleoprotein is required, directly or indirectly, for cleavage of the yeast pre-rRNA in vivo at site A3 . Here, an in vitro processing system that accurately reproduces this cleavage is described . Biochemical purification and the use of extracts depleted of the MRP RNA demonstrate that endonucleolytic cleavage of the pre-rRNA is directly mediated by RNase MRP . This establishes a role for RNase MRP in the nucleolus.

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