S. cerevisiae

Proc Natl Acad Sci U S A, 1994 Dec 20, 91(26), 12980 - 4
Interaction mating reveals binary and ternary connections between Drosophila cell cycle regulators; Finley RL Jr et al.; We characterized interactions between Drosophila melanogaster cell cycle regulatory proteins by a yeast interaction-mating technique . The results were displayed as two-dimensional matrices that revealed individual binary interactions between proteins . Each protein (Cdi, cyclin-dependent kinase interactor) interacted with a distinct spectrum of cyclin-dependent kinases (Cdk) from Drosophila and other organisms . Some Cdis interacted with other Cdis, indicating that these proteins may form trimeric complexes that include Cdks . Similar analysis of interaction matrices may be generally useful in detecting other multiprotein complexes and in establishing connectivity between individual complex members . Moreover, such analysis may also help assign function to newly identified proteins, identify domains involved in protein-protein interactions, and aid the dissection of genetic regulatory networks.

Proc Natl Acad Sci U S A, 1994 Dec 20, 91(26), 12681 - 5
A Drosophila protein homologous to the human p70 Ku autoimmune antigen interacts with the P transposable element inverted repeats; Beall EL et al.; P transposable elements in Drosophila are mobilized via a cut-and-paste mechanism . This mode of transposition requires repair of both a double-strand break at the donor DNA site and gapped DNA at the target site . Biochemical studies have identified a cellular non-P element-encoded DNA binding protein, termed the inverted repeat binding protein (IRBP), that specifically interacts with the outer half of the 31-bp terminal inverted repeats . Protein sequence information was used to isolate cDNA clones encoding IRBP . Sequence analysis shows that IRBP is related to the 70-kDa subunit of the human Ku autoimmune antigen . The mammalian Ku antigen binds free DNA termini and has been implicated in immunoglobulin VDJ recombination, DNA repair, and transcription . In addition, Ku is the DNA binding subunit of the double-strand DNA-dependent protein kinase . Cytogenetic mapping indicates that the IRBP gene maps to chromosomal position 86E on the right arm of the third chromosome.

Cell, 1994 Dec 16, 79(6), 1103 - 9
Relationship of CDK-activating kinase and RNA polymerase II CTD kinase TFIIH/TFIIK; Feaver WJ et al.; KIN28, a member of the p34cdc2/CDC28 family of protein kinases, is identified as a subunit of yeast RNA polymerase transcription factor IIH (TFIIH) on the basis of sequence determination, immunological reactivity, and copurification . KIN28 is, moreover, one of three subunits of TFIIK, a subassembly of TFIIH with protein kinase activity directed toward the C-terminal repeat domain (CTD) of the largest subunit of RNA polymerase II . Itself a phosphoprotein, KIN28 interacts specifically with the two largest subunits of RNA polymerase II . Previous work of others points to two further associations: KIN28 interacts in vivo with the cyclin CCL1, and KIN28 and CCL1 are homologous to human MO15 and cyclin H, which form the cyclin-dependent kinase-activating kinase (CAK) . We show that human CAK possesses the CTD kinase activity characteristic of TFIIH.

J Biol Chem, 1994 Dec 16, 269(50), 31962 - 8
Incorporation of 5-fluorouracil into U2 and U6 snRNA inhibits mRNA precursor splicing; Lenz HJ et al.; The splicing activities of 5-fluorouracil (FUra)-substituted U2 and U6 small nuclear RNAs (snRNAs) were examined in an in vitro splicing system . Yeast splicing extracts were specifically depleted of endogenous U2 and U6 snRNAs by antisense oligonucleotide-directed RNase H hydrolysis . Splicing activity was recovered when the extracts were reconstituted with synthetic U2 and U6 snRNAs . However, U2 snRNA with all uracils substituted with FUra (FU2) did not restore any splicing activity . Nondenaturing gel electrophoresis showed that FU2 failed to promote the assembly of spliceosome complexes . The ability of U2 snRNA to restore splicing in U2-depleted extracts increased as FUra content decreased but was still only 60% of control activity at 25% substitution of uracils with FUra . Addition of FU2 to nondepleted extracts caused strong inhibition of splicing accompanied by increased degradation of the pre-mRNA, suggesting that FU2 forms an inactive complex with a protein splicing factor that normally binds to the pre-mRNA . FU6 restored full splicing activity to U6-depleted extracts, but at a 5-fold higher concentration than U6 snRNA . These results demonstrate that the incorporation of FUra can impair the functions of catalytic RNA molecules.

J Biol Chem, 1994 Dec 16, 269(50), 31630 - 4
Inhibition of the human methylmalonyl-CoA mutase by various CoA-esters; Taoka S et al.; Human methylmalonyl-CoA mutase is inhibited by ethylmalonyl-CoA, cyclopropylcarbonyl-CoA carboxylate, and methylenecyclopropylacetyl-CoA, which are substrate, intermediate, and product analogs, respectively . The mode of inhibition by each analog is reversible and mixed with respect to the substrate, methylmalonyl-CoA . This implies that the inhibitors are able to bind to both free enzyme and to the enzyme-substrate complex, although with affinities that are 4.5- to 10-fold different for the two species . The Ki1 for the cyclopropylcarbonyl-CoA carboxylate (0.26 +/- 0.07 mM), is 4-fold greater than the Km(app) measured for the substrate, methylmalonyl-CoA . Additionally, ethylmalonyl-CoA functions as an alternate substrate and is metabolized to methylsuccinyl-CoA . The human mutase is a homodimer that binds 1 mol of cobalamin per subunit . So, the observed mixed inhibition kinetics by substrate analogs is curious . Our finding that methylenecyclopropylacetyl-CoA, the causative agent of Jamaican "vomiting sickness," inhibits methylmalonyl-CoA mutase, while interesting, is probably not physiologically important because of the relatively high inhibition constants (Ki1 = 0.47 +/- 0.12 mM and Ki2 = 2 +/- 0.34 mM) observed with this compound.

Genes Dev, 1994 Dec 15, 8(24), 2939 - 52
Growth suppression by p18, a p16INK4/MTS1- and p14INK4B/MTS2-related CDK6 inhibitor, correlates with wild-type pRb function; Guan KL et al.; The D-type cyclin-dependent kinases CDK4 and CDK6 are complexed with many small cellular proteins (p14, p15, p16, p18, and p20) . We have isolated cDNA sequences corresponding to the MTS2 genomic fragment that encodes the CDK4- and CDK6-associated p14 protein . By use of a yeast interaction screen to search for CDK6-interacting proteins, we have also identified an 18-kD human protein, p18, that is a homolog of the cyclin D-CDK4 inhibitors p16 (INK4A/MTS1) and p14 (MTS2/INK4B) . Both in vivo and in vitro, p18 interacts strongly with CDK6, weakly with CDK4, and exhibits no detectable interaction with the other known CDKs . Recombinant p18 inhibits the kinase activity of cyclin D-CDK6 . Distinct from the p21/p27 family of CDK inhibitors that form ternary complexes with cyclin-CDKs, only binary complexes of p14, p16, and p18 were found in association with CDK4 and/or CDK6 . Ectopic expression of p18 or p16 suppresses cell growth with a correlated dependence on endogenous wild-type pRb.

Nature, 1994 Dec 15, 372(6507), 698 - 701
Ypt1p implicated in v-SNARE activation; Lian JP et al.; Synaptobrevin-like membrane proteins that reside on transport vesicles, called the vesicle SNARE (v-SNARE), play a key role in ensuring that a vesicle targets and fuses with its correct acceptor compartment . Here we show that Bos1p, the v-SNARE of yeast endoplasmic reticulum-to-Golgi transport vesicles, pairs with another integral membrane protein of similar topology (Sec22p) on vesicles . This pairing, which appears to require functional Ypt1p (Rab in mammalian cells), may aid the activity of Bos1p on this compartment . These findings suggest that Rabs regulate the specificity of membrane fusion by selectively activating the v-SNARE on carrier vesicles . Because the v-SNARE resides on more than one membrane, such a regulated activation step may be necessary to prevent the premature fusion of donor and acceptor compartments.

Mol Gen Genet, 1994 Dec 15, 245(6), 781 - 6
The identification of a Caenorhabditis elegans homolog of p34cdc2 kinase; Mori H et al.; We have identified a Caenorhabditis elegans homolog of p34cdc2 kinase . The C . elegans homolog, ncc-1, is approximately 60% identical to p34cdc2 of Homo sapiens . When expressed from a constitutive yeast promoter, ncc-1 is capable of complementing a conditional lethal mutation in the CDC28 gene of Saccharomyces cerevisiae, indicating that this C . elegans homolog can properly regulate the cell cycle.

Eur J Biochem, 1994 Dec 15, 226(3), 899 - 907
The phosphotyrosyl phosphatase activator of protein phosphatase 2A . A novel purification method, immunological and enzymic characterization; Van Hoof C et al.; A simple, improved procedure for the isolation of the phosphotyrosyl phosphatase activator (PTPA) from rabbit skeletal muscle has been developed . The majority of the protein phosphatase 2A (PP2A) was separated from PTPA at an early stage in the procedure . The procedure yields approximately 1 mg essentially pure PTPA/kg rabbit skeletal muscle; it was also applied to porcine brain and the yeast Saccharomyces cerevisiae . The physico-chemical properties of PTPA obtained from all sources are very similar . The pure rabbit skeletal muscle protein was used to raise polyclonal goat antibodies and to affinity purify these antibodies . Immunological studies revealed the presence of PTPA in all mammalian tissues and cell lines examined with differences in tissue distribution, brain showing the highest concentration . PTPA could only be detected in cytosolic fractions . Using a semi-quantitative immunological assay (Western blot), the in vivo concentration could be estimated to be micromolar, which is in the same range as the PP2A target . The purified Xenopus oocyte PTPA showed only a weak cross reactivity, whereas yeast PTPA was not recognised by the antibody indicating some evolutionary diversity of the protein . In a PTPA-affinity column chromatography, the weak interaction with PP2A was independent of the presence of ATP.Mg, a necessary cofactor in the activation process . Interaction of PTPA with PP2A in a 1:1 ratio induces a low (kcat = 3 min-1) ATPase activity that is inhibited by okadaic acid, ADP and non-hydrolysable ATP analogues.

EMBO J, 1994 Dec 15, 13(24), 6087 - 98
Human cyclin F; Bai C et al.; Cyclins are important regulators of cell cycle transitions through their ability to bind and activate cyclin-dependent protein kinases . In mammals several classes of cyclins exist which are thought to co-ordinate the timing of different events necessary for cell cycle progression . Here we describe the identification of a novel human cyclin, cyclin F, isolated as a suppressor of the G1/S deficiency of a Saccharomyces cerevisiae cdc4 mutant . Cyclin F is the largest cyclin, with a molecular weight of 87 kDa, and migrates as a 100-110 kDa protein . It contains an extensive PEST-rich C-terminus and a cyclin box region that is most closely related to cyclins A and B . Cyclin F mRNA is ubiquitiously expressed in human tissues . It fluctuates dramatically through the cell cycle, peaking in G2 like cyclin A and decreasing prior to decline of cyclin B mRNA . Cyclin F protein accumulates in interphase and is destroyed at mitosis at a time distinct from cyclin B . Cyclin F shows regulated subcellular localization, being localized in the nucleus in most cells, with a significant percentage of cells displaying only perinuclear staining . Overexpression of cyclin F, or a mutant lacking the PEST region, in human cells resulted in a significant increase in the G2 population, implicating cyclin F in the regulation of cell cycle transitions . The ubiquitous expression and phylogentic conservation of cyclin F suggests that it is likely to coordinate essential cell cycle events distinct from those regulated by other cyclins.

EMBO J, 1994 Dec 15, 13(24), 6062 - 75
A novel nuclear pore protein Nup133p with distinct roles in poly(A)+ RNA transport and nuclear pore distribution; Doye V et al.; Temperature-sensitive nucleoporin nup49-316 mutant cells accumulate poly(A)+ RNA inside the nucleus when shifted to restrictive temperature . We performed a synthetic lethal screen with this mutant allele to identify further components of the mRNA export machinery . A synthetic lethal mutant slv21 was isolated, which exhibited a ts phenotype and showed nuclear accumulation of poly(A)+ RNA at 37 degrees C . The wild-type gene complementing slv21 was cloned and sequenced . It encodes a novel protein Nup133p which is located at the nuclear pore complex . NUP133 is not an essential gene, but cells in which NUP133 is disrupted grow slowly at permissive temperatures and stop growing at 37 degrees C . Concomitant with the growth inhibition, nup133- cells accumulate poly(A)+ RNA inside the nucleus whereas nuclear import of a karyophilic reporter protein is not altered . Strikingly, nup133- cells display extensive clustering of nuclear pore complexes at a few sites on the nuclear envelope . However, the nuclear pore clustering phenotype and intranuclear accumulation of poly(A)+ RNA are not obligatorily linked, since an amino-terminally truncated Nup133p allows normal poly(A)+ RNA export, but does not complement the clustering phenotype of nup133- cells.

EMBO J, 1994 Dec 15, 13(24), 6031 - 40
Differential repression of transcription factor binding by histone H1 is regulated by the core histone amino termini; Juan LJ et al.; In order to investigate the interrelated roles of nucleosome cores and histone H1 in transcription repression, we have employed a purified system to analyze the function of H1 in the repression of transcription factor binding to nucleosomes . H1 binding to nucleosome cores resulted in the repression of USF binding to nucleosomes . By contrast, H1 only slightly inhibited the binding of GAL4-AH, indicating that H1 differentially represses the binding of factors with different DNA-binding domains . H1-mediated repression of factor binding was dependent on the core histone amino-terminal tails . Removal of these domains alleviated H1-mediated repression and increased acetylation of these domains partly alleviated repression by H1 . H1 binding assays suggest a less stable interaction of histone H1 with the core particle in the absence of the amino termini.

EMBO J, 1994 Dec 15, 13(24), 6021 - 30
Regulated degradation of the transcription factor Gcn4; Kornitzer D et al.; We report that Gcn4, a yeast transcriptional activator of the bZIP family involved in the regulation of the biosynthesis of amino acids and purines, is rapidly turned over . This degradation is inhibited under conditions of starvation for amino acids . Degradation is also inhibited by single amino acid alterations in a region adjacent to the Gcn4 activation domain . Furthermore, we show that degradation of Gcn4 proceeds through the ubiquitin pathway, a major proteolytic system for cytoplasmic proteins, and is dependent on two specific ubiquitin conjugating enzymes, Cdc34 (Ubc3) and Rad6 (Ubc2) . As a first step towards reconstituting the Gcn4 degradation pathway in vitro, we show that purified Cdc34 and Rad6 proteins are able to direct the specific ubiquitination of Gcn4.

Biochem Biophys Res Commun, 1994 Dec 15, 205(2), 1201 - 5
WWP, a new amino acid motif present in single or multiple copies in various proteins including dystrophin and the SH3-binding Yes-associated protein YAP65; Andre B et al.; A new repeating amino acid motif, which we called WWP, was found in several proteins of yeast, nematod or vertebrate origin . Among these are dystrophin, the product of the Duchenne muscular dystrophy locus, a protein (YAP65) which associates in vitro with the Src homology domain 3 (SH3) of the Yes proto-oncogene product, and a human putative GTPase-activating protein . As is the case for proteins which contain the SH2, SH3 and PH domains, at least some of the WWP-containing proteins appear to be signaling or cytoskeletal proteins, associated with plasma or organellar membranes, and specific protein-protein contacts are likely to be crucial to their function.

Biochemistry, 1994 Dec 13, 33(49), 14887 - 95
Subcellular location of enzymes involved in core histone acetylation; Grabher A et al.; Multiple enzyme forms of histone deacetylase and histone acetyltransferase exist in germinating maize embryos . We analyzed the association of the different enzymes to chromatin by ion exchange chromatography of subcellular fractions from different time points of embryo germination . The vast majority of histone deacetylase HD-1A was not bound to chromatin, since it was solubilized during chromatin isolation, regardless of its phosphorylation state and the phase of embryo germination . In contrast, HD-2 was chromatin bound during the entire germination pathway . Histone deacetylase HD-1B was present in a chromatin-bound and a soluble form; the ratio between these two forms changed during germination . Both nuclear histone acetyltransferases, HAT-A1 and HAT-A2, were tightly chromatin-bound and could only be released from chromatin by salt extraction . To test whether histone acetyltransferases or deacetylases are associated with the nuclear matrix, we analyzed nuclear matrix preparations from yeast, Physarum, and maize step by step for both enzyme activities . This analysis confirmed that part of the activity is chromatin bound, but no significant enzyme activity could be found in the final nuclear matrix, regardless of the preparation protocol . This result was further substantiated by detailed analysis of histone deacetylases and acetyltransferases during cellular fractionation and nuclear matrix preparation of chicken erythrocytes . Altogether our results suggest that the participation of these enzymes in different nuclear processes may partly be regulated by a distinct location to intranuclear components.

FEBS Lett, 1994 Dec 12, 356(1), 94 - 100
Autoantigen Ku protein is involved in DNA binding proteins which recognize the U5 repressive element of human T-cell leukemia virus type I long terminal repeat; Okumura K et al.; We have identified and analyzed a 27-nucleotide sequence (U5 repressive element, designated as U5RE) at the U5 region of the human T-cell leukemia virus type I (HTLV-I) long terminal repeat (LTR) which is required for HTLV-I basal transcriptional repression . The basal promoter strength of constructs that contained deletions in the U5 region of the LTR was analyzed by chloramphenicol acetyltransferase (CAT) assays following transfection of HeLa cells or Jurkat T-cells in the presence or absence of viral transactivator tax protein . We consistently observed a 2- to 5-fold increase in basal promoter activity when sequences between +277 to +306 were deleted . In vivo competition experiments suggested that the U5 DNA fragment from +269 to +295 contains a functional repressive element (U5RE) . Using gel mobility shift assays, we have purified a highly enriched fraction that could specifically bind U5RE . This DNA affinity column fraction contained three major detectable proteins on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with silver staining: 110-, 80- and 70-kDa proteins . The 110-kDa protein appeared to be a novel DNA-binding protein whose characteristics are still obscure, while the 70- and 80-kDa proteins were shown to be related to the human autoantigen Ku, the Ku (p70/p80) complex, as demonstrated by amino acid sequencing and immunological analyses . As Ku is known to be involved in transcriptional regulation, the specific interaction of Ku with U5RE raises intriguing possibilities for its function in HTLV-I basal transcriptional repression.

FEBS Lett, 1994 Dec 12, 356(1), 56 - 9
Blockage of the urokinase receptor on the cell surface: construction and characterization of a hybrid protein consisting of the N-terminal fragment of human urokinase and human albumin; Lu H et al.; Receptor-bound urokinase is likely to be a crucial determinant in both tumor invasion and angiogenesis . We report here that a yeast-derived genetic conjugate between human serum albumin and the 1-135 N-terminal residues of urokinase (u-PA) competitively inhibits the binding of exogenous and endogenous u-PA to its cell-anchored receptor (u-PAR) . This hybrid molecule (ATF-HSA) also inhibits in vitro pro-urokinase-dependent plasminogen activation in the presence of u-PAR bearing cells . These effects are probably responsible for the observed in vitro inhibition of tumor cell invasion in a reconstituted basement membrane extract (Matrigel).

Nature, 1994 Dec 8, 372(6506), 563 - 6
Activation of the kinase Pelle by Tube in the dorsoventral signal transduction pathway of Drosophila embryo; Grosshans J et al.; The concentration of Dorsal protein in the nucleus determines cell fate along the dorsoventral axis of the Drosophila embryo . The dorsal-group genes and the cactus gene are required for production and transmission of a localized signal on the ventral side of the embryo which determines the position of the highest nuclear concentration of Dorsal protein . The ventralizing signal produced in somatic cells is transmitted through the perivitelline space to the integral membrane protein Toll . Inside the embryo it leads to dissociation of the cytoplasmic Dorsal-Cactus complex and subsequent nuclear localization of Dorsal protein . Two components are known to mediate the signal transduction between Toll and Dorsal-Cactus: Pelle, a serine/threonine protein kinase, and Tube, a protein with an unknown biochemical activity . Here we construct gain-of-function alleles of pelle and tube and show that pelle functions downstream of tube . In addition, Pelle and Tube interact directly with one another . We propose that Tube is a direct activator of the protein kinase Pelle.

Proc Natl Acad Sci U S A, 1994 Dec 6, 91(25), 11938 - 42
Directing transcription of an RNA polymerase III gene via GAL4 sites; Marsolier MC et al.; A yeast chimeric RNA polymerase III transcription system was constructed to explore the ordered, multistep process of gene activation in vivo . A promoter-deficient U6 RNA gene harboring GAL4-binding sites could be reactivated by fusing the GAL4 DNA-binding domain to components of the general transcription factor TFIIIC (tau) or TFIIIB . Expression of chimeric tau 138 or tau 131 (but not tau 95) subunits activated transcription from GAL4-binding sites located at various positions, including upstream of or within the gene . The function(s) of the B block binding domain of TFIIIC was provided by the fused GAL4-(1-147) domain . The GAL4-(1-147)-TFIIIB70 fusion protein acted at a distance like an activator of transcription . In contrast, none of the 10 different GAL4-(1-147)-polymerase subunit fusions was able to induce transcription, suggesting that RNA polymerase recruitment is not sufficient to initiate transcription.

Proc Natl Acad Sci U S A, 1994 Dec 6, 91(25), 11934 - 7
Initiation of meiotic recombination is independent of interhomologue interactions; Gilbertson LA et al.; In yeast meiosis, crossing-over between homologues is dependent upon double-strand breaks . We demonstrate that the occurrence of these breaks is independent of pairing between homologues by showing that they occur with normal frequency, timing, and position in the absence of a homologue . This observation supports models that view double-strand breaks as initiating events and crossing-over as a consequence of repair of these breaks.

Proc Natl Acad Sci U S A, 1994 Dec 6, 91(25), 11802 - 6
Identification of a 50-kDa systemin-binding protein in tomato plasma membranes having Kex2p-like properties; Schaller A et al.; A protein of 50-kDa (SBP50) was identified in plasma membranes of tomato leaves which resembles proteases of the family of Kex2p-like prohormone convertases . To our knowledge, proteases of this class have not been reported in plants previously . A biotinylated derivative of systemin, the 18-aa polypeptide inducer of proteinase inhibitors in tomato and potato leaves, was bound by SBP50 with high specificity . When a systemin derivative was labeled with biotin at residue 8 and with {35S}methionine at position 15, the biotin moiety but not the radioactive label was bound by SBP50 . At least 4 aa from the C terminus that included {35S}methionine were missing, indicating that proteolytic cleavage had occurred . Whereas residues in systemin most important for binding SBP50 appear to be located in the N-terminal half of the molecule, amino acids crucial for proteinase inhibitor induction are located within the C terminus . The residues important for binding include a cleavage site for furin, a member of the family of Kex2p-like prohormone-processing enzymes . Processing of systemin at the predicted furin cleavage site was confirmed in vitro . An antiserum against a Kex2p-like protease from Drosophila inhibited binding of biotinylsystemin to SBP50 and recognized a protein of about 60 kDa in Western blot analyses of tomato plasma membrane proteins . The data suggest a possible role for a membrane bound, furin-like protease in the mechanism of defense gene signaling by systemin.

Biochemistry, 1994 Dec 6, 33(48), 14426 - 33
Sequence 18-29 on actin: antibody and spectroscopic probing of conformational changes; Adams SB et al.; Experimental evidence for the involvement of the 18-29 site within actin subdomain-1 in the actomyosin weak binding interface includes the inhibition of actomyosin ATPase activity by specific peptide antibodies {Adams, S., & Reisler, E . (1993) Biochemistry 32, 5051-5056} and by the Dictyostelium actin mutant D24H/D25H {Johara, M., et al . (1993) Proc . Natl . Acad . Sci . U.S.A . 90, 2127-2131} . In this work, the effect of the 18-29 peptide antibodies on the polymerization and conformation of actin has been characterized . Binding of antibody to the 18-29 site strongly inhibited the MgCl2-induced polymerization of G-actin, had a much weaker impact on the CaCl2 polymerization of actin, and showed very little effect on the NaCl polymerization of G-actin . These observations were linked to the binding of the 18-29 antibody to the different forms of actin . In sedimentation assays, the (18-29) IgG bound more strongly to Mg-F- and Mg-G-actins than to Ca-F- and Ca-G-actins, respectively . The binding of IgG to F-actin decreased sharply with an increase in ionic strength . Antibody binding to the 18-29 site induced conformational changes within the nucleotide cleft, both slowing the rate of nucleotide exchange and increasing the fluorescence intensity of actin-bound epsilon ATP . The increased fluorescence of a dansyl probe attached to Gln-41 and a pyrene probe attached to Cys-374 demonstrated that antibody binding also caused local perturbations in the DNase I loop of subdomain-2 and at the C-terminus of actin . These results are discussed in terms of actin plasticity and its implications for actomyosin interactions.

Proc Natl Acad Sci U S A, 1994 Dec 6, 91(25), 12317 - 21
cDNA cloning of the Sm proteins D2 and D3 from human small nuclear ribonucleoproteins: evidence for a direct D1-D2 interaction; Lehmeier T et al.; The major small nuclear ribonucleoproteins (snRNPs) U1, U2, U4/U6, and U5 share a set of common proteins denoted B/B', D1, D2, D3, E, F, and G which play an important part in the biogenesis of the snRNPs . In addition, there is a link between the common proteins and autoimmunity; the three D proteins, together with B/B', are the major autoantigens for the so-called anti-Sm antibodies often produced by patients suffering from systemic lupus erythematosus . Here we describe the characterization of the human proteins D2 and D3 by cDNA cloning and immunological methods . D2 and D3 are encoded by distinct genes and are 118 and 126 amino acids in length, respectively . Both proteins prepared by in vitro translation exhibit Sm epitopes and can be precipitated by anti-Sm autoantibodies . They react differently with various patient sera, in a manner consistent with the reaction pattern on immunoblots of the D proteins isolated from HeLa cells . D1 and D2 synthesized in vitro form specific complexes, a result that is significant for the assembly pathway of the various core proteins into an snRNP's core ribonucleoprotein structure . The D3 protein is homologous to the human D1 protein, showing an overall amino acid sequence identity of 29%, including two regions with over 60% identity . D2 has less than 15% sequence identity with D1 and D3 . A data bank search revealed a striking similarity (with more than 40% sequence identity) between human D3 and a Saccharomyces cerevisiae gene, previously published as the 5' flanking gene of yeast pep3 {Preston, R.A., Manolson, M., Becherer, K., Weidenhammer, E., Kirkpatrick, D., Wright, R . & Jones, E . (1991) Mol . Cell . Biol . 11, 5801-5812}, suggesting that this gene encodes the yeast homologue of the human D3 protein.

J Biol Chem, 1994 Dec 2, 269(48), 30154 - 7
Activation of the Eck receptor protein tyrosine kinase stimulates phosphatidylinositol 3-kinase activity; Pandey A et al.; The Eph/Eck subfamily of receptor protein tyrosine kinases is currently the largest subfamily of receptor protein tyrosine kinases with a dozen members (Van der Geer, P., Hunter, T., and Lindberg, R . A . (1994) Annu . Rev . Cell Biol . 10, 251-337) . Using the cytoplasmic domain of Eck as bait in a yeast two-hybrid screen of mouse embryonic and T-cell cDNA libraries, it was discovered that the p85 subunit of phosphatidylinositol 3-kinase bound Eck . Further, using glutathione S-transferase fusion proteins, it was found that the C-terminal src homology 2 domain of the p85 subunit specifically interacted with Eck . Additionally, Eck coimmunoprecipitated with p85 in ligand activated cells confirming their interaction in vivo . In keeping with the above observations, activation of Eck by its ligand, B61, increased phosphatidylinositol 3-kinase activity . This is the first description of a signal transduction pathway initiated by any member of the Eph/Eck family.

J Biol Chem, 1994 Dec 2, 269(48), 30101 - 4
Initiation of transcription by RNA polymerase II is limited by melting of the promoter DNA in the region immediately upstream of the initiation site; Pan G et al.; To further elucidate the mechanism of transcriptional initiation, we used synthetic oligonucleotides to prepare templates containing heteroduplex regions of varying size and location along the DNA of the adenovirus major late promoter . Unlike closed, linear DNA, or DNA with a downstream mismatch, DNA with a mismatch upstream of the initiation site only required the general factors TATA box-binding protein and transcription factor (TF) IIB to direct specific and accurate initiation in vitro by calf thymus RNA polymerase II . In the presence of TFIIF, initiation was possible on closed, linear DNA, but an upstream mismatch region still stimulated transcriptional initiation by more than 100-fold, leading to production of approximately 0.5 transcript/template in the absence of TFIIE, TFIIH, or ATP . The presence of a DNA mismatch was most effective in the -9 to -1 region; furthermore, stimulation by a mismatch did not require that the initiation site be included in the heteroduplex region . Efficient initiation at the immunoglobulin heavy chain promoter in the presence of TATA box-binding protein and TFIIB was also achieved when a mismatch region was introduced from -9 to +3 . Our results suggest that initiation by RNA polymerase II in the absence of transcriptional activation is limited by melting of the promoter DNA upstream of the initiation site.

J Mol Biol, 1994 Dec 2, 244(3), 255 - 8
Fungal virus capsids, cytoplasmic compartments for the replication of double-stranded RNA, formed as icosahedral shells of asymmetric Gag dimers; Cheng RH et al.; The primary functions of most virus capsids are to protect the viral genome in the extra-cellular milieu and deliver it to the host . In contrast, the capsids of fungal viruses, like the cores of all other known double stranded RNA viruses, are not involved in host recognition but do shield their genomes, and they also carry out transcription and replication . Nascent (+) strands are extruded from transcribing virions . The capsids of the yeast virus L-A are composed of Gag (capsid protein; 76 kDa), with a few molecules of Gag-Pol (170 kDa) . Analysis of these 420 A diameter shells and those of the fungal P4 virus by cryo-electron microscopy and image reconstruction shows that they share the same novel icosahedral structure . Both capsids consist of 60 equivalent Gag dimers, whose two subunits occupy non-equivalent bonding environments . Stoichiometry data on other double-stranded RNA viruses indicate that the 120-subunit structure is widespread, implying that this molecular architecture has features that are particularly favorable to the design of a capsid that is also a biosynthetic compartment.

J Biol Chem, 1994 Dec 2, 269(48), 30069 - 72
A novel RING finger protein interacts with the cytoplasmic domain of CD40; Hu HM et al.; CD40 is a member of the tumor necrosis factor receptor family and, like other members, it appears to possess no intrinsic signaling capacity (e.g . kinase activity), suggesting that signal transduction is likely mediated by associating molecules . To identify such molecules, we have utilized the yeast two-hybrid system to clone cDNAs encoding proteins that bind the CD40 cytoplasmic domain . One such interacting protein, designated CD40-binding protein, has a N-terminal RING finger motif that is found in a number of DNA-binding proteins, including the V(D)J recombination activating gene RAG1 . In addition, it contains a prominent central coiled-coil segment that may allow homo- or hetero-oligomerization . The C terminus possesses substantial homology to the tumor necrosis factor receptor-associated factor (TRAF) domain that is found in two proteins (TRAF1 and TRAF2) that associate with the cytoplasmic domain of the related 75-kDa tumor necrosis factor receptor . This is the first identification of a molecule that interacts with CD40 and whose sequence suggests a potential role in signaling.

Eur J Biochem, 1994 Dec 1, 226(2), 649 - 56
A method for increasing the concentration of a specific internal metabolite in steady-state systems; Small JR et al.; An approach is described by which it is possible to increase the concentration of any internal metabolite without affecting the concentrations of other metabolites and fluxes in the organism . This approach requires the manipulation of only a limited number of enzyme activities . The method shows which enzymes to manipulate and the extent of the manipulation required to achieve a given increase in a chosen metabolite . A case study involving tryptophan overproduction in Saccharomyces cerevisae is given as a practical example of how this method could be used.

Mol Carcinog, 1994 Dec, 11(4), 227 - 35
DNA recombination induced by aflatoxin B1 activated by cytochrome P450 1A enzymes; Sengstag C et al.; Mutations in tumor suppressor genes are intricately associated with the etiology of neoplasia . Often, such mutations are followed by the loss of the second, functional alleles of tumor suppressor genes, a phenomenon known as loss of heterozygosity . Loss of heterozygosity may occur by different molecular mechanisms, including mitotic recombination, and it is conceivable that these molecular events are influenced by endogenous as well as exogenous factors . To test whether mitotic recombination is induced by certain carcinogens, we genetically engineered a Saccharomyces cerevisiae tester strain so that it metabolizes two important classes of carcinogens, polycyclic aromatic hydrocarbons and heterocyclic arylamines . This was accomplished by expressing human cDNA's coding for the cytochrome P450 (CYP) enzymes CYP1A1 or CYP1A2 in combination with NADPH-CYP oxidoreductase in a strain heterozygous for two mutations in the trp5 gene . Microsomes isolated from the transformed yeast strains activated various xenobiotics to powerful mutagens that were detected in the Ames test . Of these, the mycotoxin aflatoxin B1, when activated intracellularly in the strains containing either human CYP enzyme, significantly induced mitotic recombination . These results are discussed in light of possible mechanisms that are involved in aflatoxin B1-mediated hepatocarcinogenesis . Similarly, benzo{a}pyrene-trans-7,8-dihydrodiol and 3-amino-1-methyl-5H-pyrido{4,3-b}indole were activated to recombinagenic products, whereas benzo{a}pyrene and 2-amino-3,8-dimethylimidazo{4,5-f}quinoxaline were negative in this assay . Our results argue that the constructed yeast strains may be a valuable tool for the investigation of drug-induced mitotic recombination.

Genes Dev, 1994 Dec 1, 8(23), 2868 - 78
TFIIF-TAF-RNA polymerase II connection; Henry NL et al.; RNA polymerase transcription factor IIF (TFIIF) is required for initiation at most, if not all, polymerase II promoters . We report here the cloning and sequencing of genes for a yeast protein that is the homolog of mammalian TFIIF . This yeast protein, previously designated factor g, contains two subunits, Tfg1 and Tfg2, both of which are required for transcription, essential for yeast cell viability, and whose sequences exhibit significant similarity to those of the mammalian factor . The yeast protein also contains a third subunit, Tfg3, which is less tightly associated and at most stimulatory to transcription, dispensable for cell viability, and has no known counterpart in mammalian TFIIF . Remarkably, the TFG3 gene encodes yeast TAF30, and furthermore, is identical to ANC1, a gene implicated in actin cytoskeletal function in vivo (Welch and Drubin 1994) . Tfg3 is also a component of the recently described mediator complex (Kim et al . 1994), whose interaction with the carboxy-terminal repeat domain of RNA polymerase II enables transcriptional activation . Deletion of TFG3 results in diminished transcription in vivo.

Genes Dev, 1994 Dec 1, 8(23), 2842 - 56
Influence of a steroid receptor DNA-binding domain on transcriptional regulatory functions; Lefstin JA et al.; We have isolated two independent mutations in the DNA-binding domain of the rat glucocorticoid receptor, P493R and S459A, that implicate DNA binding in the control of attached transcriptional activation domains, either that of the receptor itself or of VP16 . The mutants are capable of activating transcription normally, but unlike wild-type receptors, they interfere with particular transcriptional activators in yeast and mammalian cells, and inhibit growth when overexpressed in yeast . The mutant residues reside at positions within the three-dimensional structure of the receptor that could, in principle, transduce structural changes from the DNA-binding surface of the receptor to other functional domains . These findings, together with the salt dependence of specific and nonspecific DNA binding by these receptors, suggest that specific DNA acts as an allosteric effector that directs the functional interaction of the receptor with targets of transcriptional activation and that the P493R and S459A mutants mimic the allosteric effect of specific DNA, allowing the receptor to interact with regulatory targets even in the absence of specific DNA binding.

J Clin Invest, 1994 Dec, 94(6), 2475 - 80
Estrogen levels in childhood determined by an ultrasensitive recombinant cell bioassay; Klein KO et al.; We hypothesized that estradiol levels are higher in prepubertal girls than in prepubertal boys and that this greater secretion of estradiol might drive the more rapid epiphyseal development and earlier puberty in girls . Since previous estradiol assays have lacked adequate sensitivity to test the hypothesis of higher estradiol levels in girls, we developed a new ultrasensitive assay to measure estrogen levels . The assay uses a strain of Saccharomyces cerevisiae genetically engineered for extreme sensitivity to estrogen . Yeast were transformed with plasmids encoding the human estrogen receptor and an estrogen-responsive promoter fused to the structural gene for beta-galactosidase . Ether extracts of 0.8 ml of serum were incubated with yeast for 8 h and the beta-galactosidase response was used to determine estrogen bioactivity relative to estradiol standards prepared in charcoal-stripped plasma . The assay was highly specific for estradiol with < 3% cross-reactivity with estrone, estriol, or estradiol metabolites . The detection limit was < 0.02 pg/ml estradiol equivalents (100-fold lower than existing assays) . Using this assay, we measured estrogen levels in 23 prepubertal boys (9.4 +/- 2.0 yr) and 21 prepubertal girls (7.7 +/- 1.9 {SD} yr) . The estrogen level in girls, 0.6 +/- 0.6 pg/ml estradiol equivalents, was significantly greater than the level in boys, 0.08 +/- 0.2 pg/ml estradiol equivalents (P < 0.05) . We conclude that the ultrasensitive recombinant cell bioassay for estrogen is approximately 100-fold more sensitive than previous estradiol assays, that estrogen levels are much lower prepubertally, in both sexes, than reported previously, and that prepubertal girls have 8-fold higher estrogen levels than prepubertal boys.

Nature, 1994 Dec 1, 372(6505), 471 - 4
A polyadenylation factor subunit is the human homologue of the Drosophila suppressor of forked protein; Takagaki Y et al.; Polyadenylation of messenger RNA precursors is a complex process that requires multiple protein factors (for reviews, see refs 1, 2) . Cleavage stimulation factor (CstF) is one of these, functioning together with cleavage-polyadenylation specificity factor, two cleavage factors, and poly(A)+ polymerase . CstF is composed of three subunits of M(r) 77, 64 and 50K . The 64K and 50K subunits contain, respectively, an RNP-type RNA-binding domain that contacts the pre-mRNA and transducin repeats characteristic of G-protein beta-subunits . Here we report the cloning and characterization of the 77K subunit of human CstF (referred to as 77K) . We show that the 77K subunit is required for formation of active CstF and bridges the 64K and 50K subunits . Sequence analyses indicate that the 77K subunit is the homologue of the protein encoded by the Drosophila melanogaster suppressor of forked (su(f)) gene . Mutations in su(f) can enhance or suppress the effects of transposable element insertions, and our data indicate that this is due to changes in polyadenylation . Both the 77K subunit and the su(f) protein share homology with Saccharomyces cerevisiae RNA14, previously shown to be involved in mRNA metabolism . Our results thus also indicate that components of the complex polyadenylation machinery are conserved from yeast to man.

Mol Cell Biol, 1994 Dec, 14(12), 8376 - 84
Signal transduction by tumor necrosis factor mediated by JNK protein kinases; Sluss HK et al.; JNK protein kinases are distantly related to mitogen-activated protein kinases (ERKs) and are activated by dual phosphorylation on Tyr and Thr . The JNK protein kinase group includes the 46-kDa isoform JNK1 . Here we describe the molecular cloning of a second member of the JNK group, the 55-kDa protein kinase JNK2 . The activities of both JNK isoforms are markedly increased by exposure of cells to UV radiation . Furthermore, JNK protein kinase activation is observed in cells treated with tumor necrosis factor . Although both JNK isoforms phosphorylate the NH2-terminal activation domain of the transcription factor c-Jun, the activity of JNK2 was approximately 10-fold greater than that of JNK1 . This difference in c-Jun phosphorylation correlates with increased binding of c-Jun to JNK2 compared with JNK1 . The distinct in vitro biochemical properties of these JNK isoforms suggest that they may have different functions in vivo . Evidence in favor of this hypothesis was obtained from the observation that JNK1, but not JNK2, complements a defect in the expression of the mitogen-activated protein kinase HOG1 in the yeast Saccharomyces cerevisiae . Together, these data indicate a role for the JNK group of protein kinases in the signal transduction pathway initiated by proinflammatory cytokines and UV radiation.

Mol Cell Biol, 1994 Dec, 14(12), 8315 - 21
Distinct residues of human p53 implicated in binding to DNA, simian virus 40 large T antigen, 53BP1, and 53BP2; Thukral SK et al.; We identified a minimal domain of human p53 required for the transactivation of a p53 response element in Saccharomyces cerevisiae . This domain contains the central region of p53 sufficient for specific DNA binding, which colocalizes with the region responsible for binding simian virus 40 large T antigen, 53BP1, and 53BP2 . Thirty amino acid positions, including natural mutational hot spots (R175, R213, R248, R249, and R273), in the minimal DNA-binding domain were mutated by alanine substitution . Alanine substitutions at positions R213, R248, R249, D281, R282, R283, E286, and N288 affected transactivation but allowed binding to at least one of the three interacting proteins; these amino acids may be involved in amino acid-base pair contacts . Surprisingly, alanine substitution at the mutational hot spot R175 did not affect DNA binding, transactivation, or T-antigen binding, although it nearly eliminated binding to 53BP1 and 53BP2 . Mutation of H168 significantly affected only T-antigen binding, and mutation of E285 affected only 53BP1 binding . Thus, we implicate specific residues of p53 in different DNA and protein interactions.

Mol Cell Biol, 1994 Dec, 14(12), 8117 - 22
Amino acid residues in the CDC25 guanine nucleotide exchange factor critical for interaction with Ras; Park W et al.; Previously we found that negatively charged residues at positions 62, 63, and 69 of H-Ras are involved in binding to the CDC25 guanine nucleotide exchange factor (GEF) . Using site-directed mutagenesis, we have changed conserved, positively charged residues of CDC25GEF to glutamic acid . We find the nonfunctional CDC25R1374E mutant and the nonfunctional H-RasE63K mutant cooperate in suppression of the loss of CDC25 function in Saccharomyces cerevisiae . Also, peptides corresponding to residues 1364 to 1383 of CDC25GEF inhibit interaction between GEFs and H-Ras . We propose that residues 1374 of CDC25GEF and 63 of H-Ras form an ion pair and that when this ion pair is reversed, functional interaction can still occur.

Mol Cell Biol, 1994 Dec, 14(12), 8107 - 16
Special peptidyl-tRNA molecules can promote translational frameshifting without slippage; Vimaladithan A et al.; Recently we described an unusual programmed +1 frameshift event in yeast retrotransposon Ty3 . Frameshifting depends on the presence of peptidyl-tRNA(AlaCGC) on the GCG codon in the ribosomal P site and on a translational pause stimulated by the slowly decoded AGU codon . Frameshifting occurs on the sequence GCG-AGU-U by out-of-frame binding of a valyl-tRNA to GUU without slippage of peptidyl-tRNA(AlaCGC) . This mechanism challenges the conventional understanding that frameshift efficiency must correlate with the ability of mRNA-bound tRNA to slip between cognate or near-cognate codons . Though frameshifting does not require slippery tRNAs, it does require special peptidyl-tRNAs . We show that overproducing a second isoacceptor whose anticodon had been changed to CGC eliminated frameshifting; peptidyl-tRNA(AlaCGC) must have a special capacity to induce +1 frameshifting in the adjacent ribosomal A site . In order to identify other special peptidyl-tRNAs, we tested the ability of each of the other 63 codons to replace GCG in the P site . We found no correlation between the ability to stimulate +1 frameshifting and the ability of the cognate tRNA to slip on the mRNA--several codons predicted to slip efficiently do not stimulate frameshifting, while several predicted not to slip do stimulate frameshifting . By inducing a severe translational pause, we identified eight tRNAs capable of inducing measurable +1 frameshifting, only four of which are predicted to slip on the mRNA . We conclude that in Saccharomyces cerevisiae, special peptidyl-tRNAs can induce frameshifting dependent on some characteristic(s) other than the ability to slip on the mRNA.

Mol Cell Biol, 1994 Dec, 14(12), 7884 - 90
Replication factor A is required in vivo for DNA replication, repair, and recombination; Longhese MP et al.; Replication factor A (RF-A) is a heterotrimeric single-stranded-DNA-binding protein which is conserved in all eukaryotes . Since the availability of conditional mutants is an essential step to define functions and interactions of RF-A in vivo, we have produced and characterized mutations in the RFA1 gene, encoding the p70 subunit of the complex in Saccharomyces cerevisiae . This analysis provides the first in vivo evidence that RF-A function is critical not only for DNA replication but also for efficient DNA repair and recombination . Moreover, genetic evidence indicate that p70 interacts both with the DNA polymerase alpha-primase complex and with DNA polymerase delta.

J Med Microbiol, 1994 Dec, 41(6), 384 - 8
Interaction of Candida krusei with human neutrophils in vitro; Richardson MD et al.; In human neutrophil monolayer assays the percentage phagocytosis of Candida krusei by neutrophils was found to be significantly lower (9%) than that for C . albicans (37%) . Both organisms required opsonisation with complement products for ingestion . The number of competent neutrophils phagocytosing C . krusei was increased with: antisera specific to C . albicans (49%); the neutrophil chemo-attractant formyl-methionyl-leucyl-phenylalanine (fmlp) (49%); mannan extracted from the cell wall of C . albicans (72%); and a crude cell extract from C . krusei (61%) . In the case of C . albicans all but one of these methods increased the proportion of phagocytosing neutrophils per slide . The data provide evidence for differences in quantitative phagocytosis of C . krusei and C . albicans and suggest that C . krusei is resistant to phagocytosis in vitro.

J Cell Biol, 1994 Dec, 127(5), 1381 - 94
Control of cellular morphogenesis by the Ip12/Bem2 GTPase-activating protein: possible role of protein phosphorylation; Kim YJ et al.; The IPL2 gene is known to be required for normal polarized cell growth in the budding yeast Saccharomyces cerevisiae . We now show that IPL2 is identical to the previously identified BEM2 gene . bem2 mutants are defective in bud site selection at 26 degrees C and localized cell surface growth and organization of the actin cytoskeleton at 37 degrees C . BEM2 encodes a protein with a COOH-terminal domain homologous to sequences found in several GTPase-activating proteins, including human Bcr . The GTPase-activating protein-domain from the Bem2 protein (Bem2p) or human Bcr can functionally substitute for Bem2p . The Rho1 and Rho2 GTPases are the likely in vivo targets of Bem2p because bem2 mutant phenotypes can be partially suppressed by increasing the gene dosage of RHO1 or RHO2 . CDC55 encodes the putative regulatory B subunit of protein phosphatase 2A, and mutations in BEM2 have previously been identified as suppressors of the cdc55-1 mutation . We show here that mutations in the previously identified GRR1 gene can suppress bem2 mutations . grr1 and cdc55 mutants are both elongated in shape and cold-sensitive for growth, and cells lacking both GRR1 and CDC55 exhibit a synthetic lethal phenotype . bem2 mutant phenotypes also can be suppressed by the SSD1-vl (also known as SRK1) mutation, which was shown previously to suppress mutations in the protein phosphatase-encoding SIT4 gene . Cells lacking both BEM2 and SIT4 exhibit a synthetic lethal phenotype even in the presence of the SSD1-v1 suppressor . These genetic interactions together suggest that protein phosphorylation and dephosphorylation play an important role in the BEM2-mediated process of polarized cell growth.

J Cell Biol, 1994 Dec, 127(5), 1245 - 57
An oligomeric protein is imported into peroxisomes in vivo; McNew JA et al.; The mechanism of translocation of peroxisomal proteins from the cytoplasm into the matrix is largely unknown . We have been studying this problem in yeast . We show that the peroxisomal targeting sequences SKL or AKL, with or without a spacer of nine glycines (G9), are sufficient to target chloramphenicol acetyltransferase (CAT) to peroxisomes of Saccharomyces cerevisiae in vivo . The mature form of CAT is a homotrimer, and complete trimerization of CAT was found to occur within a few minutes of synthesis . In contrast, import, measured by immunoelectron microscopy and organellar fractionation, occurred over several hours . To confirm that import of preassembled CAT trimers was occurring, we co-expressed CAT-G9-AKL with CAT lacking a peroxisomal targeting sequence but containing a hemagglutinin-derived epitope tag (HA-CAT) . We found that HA-CAT was not imported unless it was co-expressed with CAT-G9-AKL . Both proteins were released from the organelles under mild conditions (pH 8.5) that released other matrix proteins, indicating that import had occurred . These results strongly suggested that HA-CAT was imported as a heterotrimer with CAT-G9-AKL . The process of oligomeric import also occurs in animal cells . When HA-CAT was co-expressed with CAT-G9-AKL in CV-1 cells, HA-CAT co-localized with peroxisomes but was cytoplasmic when expressed alone . It is not clear whether the import of globular proteins into peroxisomes occurs through peroxisomal membrane pores or involves membrane internalization . Both possibilities are discussed.

Curr Opin Cell Biol, 1994 Dec, 6(6), 847 - 52
Cdk inhibitors: on the threshold of checkpoints and development; Elledge SJ et al.; The regulation of cyclin-dependent kinases is at the heart of cell cycle control and, by inference, the control of cell proliferation . Recent advances in regulation of these kinases have uncovered a group of small proteins that bind to and inhibit them, thus preventing cell cycle progression . Linking these proteins to tumor suppressor functions has provided a much sought after connection between cancer and cell cycle control.

Trends Genet, 1994 Dec, 10(12), 436 - 40
GAPs for rho-related GTPases; Lamarche N et al.; Ras-related GTP-binding proteins (GTPases) of the rho subfamily play important roles in regulating the organization of the actin cytoskeleton . A large number of multifunctional proteins that can stimulate their intrinsic GTPase activity have been identified . Here, we discuss the nature of such GTPase-activating proteins (GAPs) and their potential importance for cell signalling.

Development, 1994 Dec, 120(12), 3367 - 77
Diaphanous is required for cytokinesis in Drosophila and shares domains of similarity with the products of the limb deformity gene; Castrillon DH et al.; We show that the Drosophila gene diaphanous is required for cytokinesis . Males homozygous for the dia1 mutation are sterile due to a defect in cytokinesis in the germline . Females trans-heterozygous for dia1 and a deficiency are sterile and lay eggs with defective eggshells; failure of cytokinesis is observed in the follicle cell layer . Null alleles are lethal . Death occurs at the onset of pupation due to the absence of imaginal discs . Mitotic figures in larval neuroblasts were found to be polyploid, apparently due to a defect in cytokinesis . The predicted 123 x 10(3) M(r) protein contains two domains shared by the formin proteins, encoded by the limb deformity gene in the mouse . These formin homology domains, which we have termed FH1 and FH2, are also found in Bni1p, the product of a Saccharomyces cerevisiae gene required for normal cytokinesis in diploid yeast cells.

Mol Gen Genet, 1994 Dec 1, 245(5), 616 - 22
Cytosolic and plastid forms of 5-enolpyruvylshikimate-3-phosphate synthase in Euglena gracilis are differentially expressed during light-induced chloroplast development; Reinbothe C et al.; The enzyme 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase (EC 2.5.1.19), the target of the herbicide glyphosate {N-(phosphonomethyl)glycine}, exists in two molecular forms in Euglena gracilis . One form has previously been characterized as a monofunctional 59 kDa protein . The other form constitutes a single domain of the multifunctional 165 kDa arom protein . The two enzyme forms are inversely regulated at the protein and mRNA levels during light-induced chloroplast development, as demonstrated by the determination of their enzyme activities after non-denaturing polyacrylamide gel electrophoresis and Northern hybridization analysis with a Saccharomyces cerevisiae ARO1 gene probe . The arom protein and its mRNA predominate in dark-grown cells, and the levels of both decline upon illumination . In contrast, the monofunctional EPSP synthase and its mRNA are induced by light, the increase in mRNA abundance preceding accumulation of the protein . The two enzymes are localized in different subcellular compartments, as demonstrated by comparing total protein patterns with those of isolated organelles . Glyphosate-adapted wild-type cells and glyphosate-tolerant cells of a plastid-free mutant of E . gracilis, W10BSmL, were used for organelle isolation and protein extraction, as these cell lines overproduce EPSP synthase and the arom protein, respectively . Evidence was obtained for the cytosolic localization of the arom protein and the plastid compartmentalization of the monofunctional EPSP synthase . These conclusions are further supported by the observation that EPSP synthase precursor, produced by in vitro translation of the hybrid-selected mRNA, was efficiently taken up and processed to mature size by isolated chloroplasts from photoautotrophic wild-type E . gracilis cells, while the in vitro-synthesized arom protein was not sequestered by isolated Euglena plastids.

J Cell Biol, 1994 Dec, 127(6 Pt 1), 1547 - 56
Mitochondrial protein import: biochemical and genetic evidence for interaction of matrix hsp70 and the inner membrane protein MIM44; Rassow J et al.; The import of preproteins into mitochondria involves translocation of the polypeptide chains through putative channels in the outer and inner membranes . Preprotein-binding proteins are needed to drive the unidirectional translocation of the precursor polypeptides . Two of these preprotein-binding proteins are the peripheral inner membrane protein MIM44 and the matrix heat shock protein hsp70 . We report here that MIM44 is mainly exposed on the matrix side, and a fraction of mt-hsp70 is reversibly bound to the inner membrane . Mt-hsp70 binds to MIM44 in a 1:1 ratio, suggesting that mt-hsp70 is localizing to the membrane via its interaction with MIM44 . Formation of the complex requires a functional ATPase domain of mt-hsp70 . Addition of Mg-ATP leads to dissociation of the complex . Overexpression of mt-hsp70 rescues the protein import defect of mutants in MIM44; conversely, overexpression of MIM44 rescues protein import defects of mt-hsp70 mutants . In addition, yeast strains with conditional mutations in both MIM44 and mt-hsp70 are barely viable, showing a synthetic growth defect compared to strains carrying single mutations . We propose that MIM44 and mt-hsp70 cooperate in translocation of preproteins . By binding to MIM44, mt-hsp70 is recruited at the protein import sites of the inner membrane, and preproteins arriving at MIM44 may be directly handed over to mt-hsp70.

Curr Opin Biotechnol, 1994 Dec, 5(6), 599 - 603
cDNA sequencing: a means of understanding cellular physiology; Weinstock KG et al.; High-throughput automated sequencing has enabled researchers to examine large numbers of clones from a cDNA library as a measure of the steady-state levels of mRNA species . The past year has witnessed many new applications of this technique to allow the qualitative and quantitative comparison of the changes in transcript levels from multiple genes.

Protein Sci, 1994 Dec, 3(12), 2435 - 46
High-sensitivity sequencing of large proteins: partial structure of the rapamycin-FKBP12 target; Erdjument-Bromage H et al.; We report on studies leading to refinements of various steps of the protein internal sequencing process . Specifically, the developments comprise (1) higher-sensitivity chemical sequencing through background reduction; (2) improved peptide recovery from rapid in situ digests of nanogram amount, nitrocellulose-bound proteins; and (3) accurate UV spectroscopic identification of Trp- and Cys-containing peptides . In addition, we describe strategies for 2-dimensional liquid chromatographic peptide isolation from complex mixtures and a multi-analytical approach to peptide sequence analysis (Edman sequencing, matrix-assisted laser desorption mass spectrometry, and UV spectroscopy) . Both strategies were applied in tandem to the primary structural analysis of a gel-purified, 250-kDa protein (mammalian target of rapamycin-FKBP12 complex), available in low picomolar quantities only . More than 300-amino acids worth of sequence was obtained in mostly uninterrupted stretches, several containing Trp, Cys, His, and Ser . That information has allowed the matching of a biological function of a mammalian protein to a yeast gene product with a well-characterized mutant phenotype . The results also demonstrate that extended chemical sequencing analysis (e.g., 26 successive amino acids) is now feasible, starting with initial yields well below 1 pmol.

Yeast, 1994 Dec, 10(12), 1613 - 20
Effect of site-directed mutagenesis of conserved lysine residues upon Pas1 protein function in peroxisome biogenesis; Krause T et al.; The Pas1 protein (Pas1p) is required for peroxisome biogenesis in Saccharomyces cerevisiae and contains two putative ATP-binding sites, each within a domain which is conserved among members of the recently characterized AAA-family . To elucidate whether both putative ATP-binding sites are essential for Pas1p function, lysine 467 of the first and lysine 744 of the second putative ATP-binding site were each changed to glutamate by site-directed mutagenesis . While replacement of lysine 744 abolished the function of the Pas1 protein in peroxisome biogenesis, replacement of lysine 467 had no obvious effect.

Chromosoma, 1994 Dec, 103(7), 441 - 9
Histone acetylation: facts and questions; Loidl P; The DNA of eukaryotic cells is organized in a complex with proteins, either as interphase chromatin or mitotic chromosomes . Nucleosomes, the structural subunits of chromatin, have long been considered as static structures, incompatible with processes occurring in chromatin . During the past few years it has become evident that the histone part of the nucleosome has important regulatory functions . Some of these functions are mediated by the N-terminal core histone domains which contain sites for posttranslational modifications, among them lysine residues for reversible acetylation . Recent results indicate that acetylation and deacetylation of N-terminal lysines of nucleosomal core histones represent a means of molecular communication between chromatin and the cellular signal transduction network, resulting in heritable epigenetic information . Data on enzymes involved in acetylation and the pattern of acetylated lysine sites on chromosomes, as well as genetic data on yeast transcriptional repression, suggest that acetylation may lead to structural transitions as well as specific signalling within distinct chromatin domains.

Semin Cell Biol, 1994 Dec, 5(6), 409 - 16
Roles of the Snf1/Rkin1/AMP-activated protein kinase family in the response to environmental and nutritional stress; Hardie DG et al.; The AMP-activated protein kinase (AMPK) inhibits several biosynthetic pathways in mammals, and is activated in response to stresses which cause ATP depletion, e.g . heat shock . This system may therefore protect cells against environmental stress by switching off biosynthesis (i.e . growth) to conserve ATP . Recent biochemical and molecular genetic studies have shown that AMPK is closely related to the SNF1 gene product from Saccharomyces cerevisiae, and its homologues in higher plants . SNF1 is required for the response to starvation for glucose . Thus the novel function of providing protection against environmental stress may have evolved from a more ancient response to nutritional stress.

J Cell Sci, 1994 Dec, 107 ( Pt 12), 3449 - 59
Mutations in the VPS45 gene, a SEC1 homologue, result in vacuolar protein sorting defects and accumulation of membrane vesicles; Cowles CR et al.; Genetic analyses of vacuolar protein sorting in Saccharomyces cerevisiae have uncovered a large number of mutants (vps) that missort and secrete vacuolar hydrolases . A small subset of vps mutants exhibit a temperature-conditional growth phenotype and show a severe defect in the localization of soluble vacuolar proteins, yet maintain a near-normal vacuole structure . Here, we report on the cloning and characterization of the gene affected in one of these mutants, VPS45, which has been found to encode a member of a protein family that includes the yeast proteins Sec1p, Sly1p and Vps33p, as well as n-Sec1, UNC18 and Rop from other eukaryotic organisms . These proteins are thought to participate in vesicle-mediated protein transport events . Polyclonal antiserum raised against a TrpE-Vps45 fusion protein specifically detects a stable 67 kDa protein in labeled yeast cell extracts . Subcellular fractionation studies demonstrate that the majority of Vps45p is associated with a high-speed membrane pellet fraction that includes Golgi, transport vesicles and, potentially, endosomal membranes . Significantly, this fraction lacks ER, vacuole and plasma membranes . Overexpression of Vps45p saturates the sites with which Vps45p associates . A vps45 null mutant accumulates vesicles, many of which were found to be present in large clusters . This accumulation of potential transport vesicles indicates that Vps45p may facilitate the targeting and/or fusion of these vesicles in the vacuolar protein sorting pathway.

Curr Biol, 1994 Dec 1, 4(12), 1149 - 51
Recombination and repair . Ku starts at the end; Troelstra C et al.; Genetic evidence suggests that the Ku DNA-end-binding protein complex is central to the recombination-based repair of double-strand breaks that protects DNA from radiation and underlies antibody gene rearrangement.

Curr Biol, 1994 Dec 1, 4(12), 1118 - 21
MAP kinase pathways . Straight and narrow or tortuous and intersecting?
Cooper JA.
Stress and mitogens stimulate overlapping sets of MAP kinases in mammalian cells; MAP kinase pathways appear more distinct in yeast, but differences between the two systems may be less than is presently evident.

Biochemistry, 1994 Nov 29, 33(47), 14221 - 6
Topology of an amphiphilic mitochondrial signal sequence in the membrane-inserted state: a spin labeling study; Yu YG et al.; To investigate the interaction of the presequence of the precursor of yeast cytochrome C oxidase subunit IV (COX IV) with phospholipid membranes, a series of single- and double-cysteine-substituted peptide variants derived from the 25-residue NH2-terminal presequence has been synthesized and modified with nitroxide spin labels . The immersion depth, orientation, and secondary structure of the peptide in the POPC bilayer containing 10 mol % POPG were determined using electron paramagnetic resonance (EPR) spectroscopy . EPR saturation analysis of singly labeled variants reveals that the nitroxides attached to the NH2-terminal region of the peptide insert into the acyl chain region of the bilayer, approximately 13 A deep from the membrane surface . EPR line shape analysis of doubly labeled variants indicates that the peptide predominantly exists as an extended conformation, with little secondary structure . The experimental results, together with the energetic consideration of peptide-bilayer interactions, suggest that the presequence is located near the interface between the head group region and the acyl chain region, such that the hydrophobic side chains are solvated by the acyl chains and the charged side chains extended toward the polar environment at the bilayer surface.

FEBS Lett, 1994 Nov 28, 355(2), 109 - 13
A genetic approach to elucidating eukaryotic iron metabolism; Klausner RD et al.; Studies of mutants of the yeast Saccharomyces cerevisiae have led to the identification of genes required for high affinity iron uptake . Reduction of iron (III) outside the cell is accomplished by means of reductases encoded by FRE1 and FRE2, homologues of the gp91-phox component of the oxygen reductase of human granulocytes . High affinity iron (II) transport from the exterior to the interior of the cell occurs by means of a transport system that has not been molecularly characterized . However, the transport process requires the activity of a copper-containing oxidase encoded by FET3 . The amino acid sequence of this protein resembles other multi-copper oxidases, including mammalian ceruloplasmin . High affinity copper uptake mediated by the copper transport protein encoded by CTR1 is required to provide the FET3 protein with copper, and thus copper uptake is indirectly required for ferrous iron uptake . These genetic elements of yeast and their relationships may be conserved in complex eukaryotic organisms.

Science, 1994 Nov 25, 266(5189), 1388 - 91
Cell cycle control by a complex of the cyclin HCS26 (PCL1) and the kinase PHO85; Espinoza FH et al.; The events of the eukaryotic cell cycle are governed by cyclin-dependent kinases (cdk's), whose activation requires association with cyclin regulatory subunits expressed at specific cell cycle stages . In the budding yeast Saccharomyces cerevisiae, the cell cycle is thought to be controlled by a single cdk, CDC28 . Passage through the G1 phase of the cell cycle is regulated by complexes of CDC28 and G1 cyclins (CLN1, CLN2, and CLN3) . A putative G1 cyclin, HCS26, has recently been identified . In a/alpha diploid cells lacking CLN1 and CLN2, HCS26 is required for passage through G1 . HCS26 does not associate with CDC28, but instead associates with PHO85, a closely related protein kinase . Thus, budding yeast, like higher eukaryotes, use multiple cdk's in the regulation of cell cycle progression.

Science, 1994 Nov 25, 266(5189), 1383 - 7
Catalytic site components common to both splicing steps of a group II intron; Chanfreau G et al.; The splicing of group II introns occurs in two steps involving substrates with different chemical configurations . The question of whether these two steps are catalyzed by a single or two separate active sites is a matter of debate . Here, certain bases and phosphate oxygen atoms at conserved positions in domain V of a group II self-splicing intron are shown to be required for catalysis of both splicing steps . These results show that the active sites catalyzing the two steps must, at least, share common components, ruling out the existence of two completely distinct active sites in group II introns.

Nucleic Acids Res, 1994 Nov 25, 22(23), 4932 - 6
Role of a small RNA pol II subunit in TATA to transcription start site spacing; Furter-Graves EM et al.; The yeast shi mutation affects the spacing between the TATA promoter element and transcription initiation sites; for the H2B and ADH1 genes, a series of start sites located approximately 50-80 bp downstream of TATA is used in addition to the wild-type initiation sites located at around 100 bp from TATA (1) . Here, the yeast SHI wild-type gene has been isolated by complementation and shown to be identical to RPB9, the gene encoding a small subunit of RNA polymerase II . A point mutation in the shi gene, changing a cysteine residue in a putative zinc ribbon motif into a phenylalanine residue, was demonstrated to permit the observed usage of upstream initiation sites . Deletion of the non-essential SHI gene also results in usage of upstream initiation sites and causes conditional growth defects.

Proc Natl Acad Sci U S A, 1994 Nov 22, 91(24), 11398 - 402
AtVPS34, a phosphatidylinositol 3-kinase of Arabidopsis thaliana, is an essential protein with homology to a calcium-dependent lipid binding domain; Welters P et al.; The cDNA encoding phosphatidylinositol (PI) 3-kinase was cloned from Arabidopsis thaliana, and the derived amino acid sequence (AtVPS34) has a significantly higher homology to yeast PI 3-kinase (VPS34) than to the mammalian (p110) . The protein has two conserved domains: a catalytic site with the ATP-binding site near the C terminus and a calcium-dependent lipid-binding domain near the N terminus . The plant cDNA does not rescue a yeast vps34 deletion mutant, but a chimeric gene in which the coding sequence for the C-terminal third of VPS34 is replaced by the corresponding sequence from the plant gene does rescue the yeast mutant . PI 3-kinase activity is detectable in extracts from plants that overexpress the plant PI 3-kinase . Expression of antisense constructs gives rise to second-generation transformed plants severely inhibited in growth and development.

J Biol Chem, 1994 Nov 18, 269(46), 28878 - 84
Cloning and characterization of a human phosphatidylinositol 4-kinase; Wong K et al.; Phosphatidylinositol (PtdIns) 4-kinase catalyzes the first committed step in the biosynthesis of phosphatidylinositol 4,5-bisphosphate . Here we report the first mammalian cDNA clone of a PtdIns 4-kinase (named PI4K alpha) . The 2.6-kb cDNA encodes a protein of 854 amino acids that is highly homologous to the recently cloned yeast PtdIns 4-kinase STT4 and is also homologous to a second yeast PtdIns 4-kinase, PIK1 . PI4K alpha has more distant sequence homology to the catalytic domains of mammalian and yeast PtdIns 3-kinases and to the yeast Tor family of proteins . It also has a region of similarity to pleckstrin homology domains and a potential ankyrin repeat . Cross-hybridizing messages were detected in all human tissues investigated . The enzymatic properties of the protein expressed in insect cells are characteristic of type II PtdIns 4-kinases (activated by detergent and inhibited by adenosine), and PI4K alpha is recognized by an antibody specific for type II PtdIns 4-kinases.

Gene, 1994 Nov 18, 149(2), 345 - 50
Characterization and chromosomal localization of the human homologue of a rat AMP-activated protein kinase-encoding gene: a major regulator of lipid metabolism in mammals; Aguan K et al.; AMP-activated protein kinase (AMPK) phosphorylates and inactivates acetyl-CoA carboxylase and beta-hydroxy beta-methylglutaryl-coenzyme A (HMG-CoA) reductase which are the major enzymes involved in fatty acid and lipid biosyntheses . The AMPK gene from rat (rAMPK) has recently been cloned {Carling et al., J . Biol . Chem . 269 (1994) 11442-11448} . In order to study the structure and function of the human AMPK gene (hAMPK), we have cloned the gene, and report in this communication its nucleotide (nt) sequence, tissue distribution and chromosomal location . Our results show that the ORF of hAMPK encodes 552 amino acids (aa) (62.250 kDa) and is highly conserved with rAMPK with identities of 97.3 and 90% at the aa and nt levels, respectively . The hAMPK gene bears homology to a yeast protein kinase-encoding gene (snf1) that regulates carbohydrate metabolism, and also with three other genes encoding SNF1-like kinases from different plant species, namely Arabidopsis thaliana, Hordeum vulgare and Secale cereale . As determined by fluorescent in situ hybridization of a human metaphase chromosome spread, hAMPK maps to chromosome 1p31 . The size of the hAMPK transcript is 8.5 kb and the transcription start point (tsp) is located approx . 46 bp upstream from the ATG codon . While 10-15% of AMPK is alternatively spliced in most tissues of the rat, our RT-PCR analyses of the hAMPK mRNA did not reveal the presence of any alternatively spliced form of the gene in human tissues . An interesting aspect of AMPK is that its expression, unlike in rat liver, could not be detected in human liver, and thus the purported role of the gene in controlling fatty-acid synthesis in the human liver remains to be determined.

Gene, 1994 Nov 18, 149(2), 311 - 4
Conservation in human and mouse Pur alpha of a motif common to several proteins involved in initiation of DNA replication; Ma ZW et al.; Pur alpha is a sequence-specific single-stranded DNA-binding protein with affinity for an element present in several eukaryotic origins of DNA replication (ori) and gene regulatory regions . We report here the cDNA sequence for mouse pur alpha and an extraordinary degree of conservation between human and mouse Pur alpha (hPurA and mPurA, respectively) . There are only two single-amino-acid (aa) changes between hPurA (322 aa) and mPurA (321 aa) . One PurA region of 22 aa, termed the 'psycho' motif, possesses significant homology to a counterpart in the SV40 large T-antigen, to several other transforming proteins of DNA tumor viruses, and to certain cellular proteins in yeast and human cells that may also be involved in the initiation of DNA replication.

Cell, 1994 Nov 18, 79(4), 639 - 48
Solution structure of a specific DNA complex of the Myb DNA-binding domain with cooperative recognition helices; Ogata K et al.; The DNA-binding region of Myb consists of three imperfect tandem repeats (R1, R2, and R3) . We have determined the solution structure of a specific DNA complex of the minimum DNA-binding domain (R2R3) by heteronuclear multidimensional NMR . Both R2 and R3 contain three helices, and the third helix in each is found to be a recognition helix . R2 and R3 are closely packed in the major groove, so that the two recognition helices contact each other directly to bind to the specific base sequence, AACNG cooperatively; this is a significant arrangement of recognition helices . The three key base pairs in this sequence are specifically recognized by Asn-183 (R3), Lys-182 (R3), and Lys-128 (R2) . In contrast, R1 has no specific interactions with DNA from our NMR study of the DNA complex of the full DNA-binding domain (R1R2R3).

Biochim Biophys Acta, 1994 Nov 17, 1215(1-2), 87 - 92
Activation of alkylthioacrylic acids in subcellular fractions of rat tissues: a new spectrophotometric method for assay of acyl-CoA synthetase; Wu P et al.; Alkylthioacrylic acids are activated by the long-chain acyl-CoA synthetase in mitochondria and microsomes from rat liver, heart and kidney . The activation of a corresponding dicarboxylic acid was also demonstrated . The highest rate of activation was found in liver microsomes . The activation rate of the long-chain alkylthioacrylic acids is about one third of that of corresponding normal long-chain saturated fatty acids . The short-chain (methyl-, butyl-) thioacrylic acids showed no detectable activation in intact mitochondria or mitochondrial extracts . The cofactor requirement, chain length specificity and mutual inhibition of normal fatty acids and alkylthioacrylic acids in activation indicate that the long-chain fatty acid activating enzyme (long-chain acyl-CoA synthetase) of microsomes and mitochondria accepts alkylthioacrylic acids as substrates, while the short- and medium-chain fatty acid activating enzymes of the mitochondrial matrix do not . The UV absorption at 312 nm of the coenzyme A esters of alkylthioacrylic acid with a high molar extinction coefficient (22 mM-1 cm-1) makes a specific spectrophotometric assay of the long-chain acyl-CoA synthetase possible in spite of a slower reaction rate than with normal fatty acids.

EMBO J, 1994 Nov 15, 13(22), 5410 - 20
The transcription factor, the Cdk, its cyclin and their regulator: directing the transcriptional response to a nutritional signal; Hirst K et al.; The Pho80-Pho85 cyclin-cdk complex prevents transcription of PHO5 by inhibiting the ability of the basic-helix-loop-helix transcription factor Pho4 to activate transcription in response to high phosphate conditions . In low phosphate the Pho80-Pho85 complex is inactivated and Pho4 is then able to activate the acid phosphatase gene PHO5 . We show here that Pho4 and the homeobox protein Pho2 interact in vivo and act cooperatively to activate the PHO5 UAS, with interaction being regulated by the phosphate switch . In addition, we also demonstrate that an additional factor, Pho81, interacts in high phosphate with both the Pho80 cyclin and with Pho4 . In low phosphate, Pho80 and Pho81 dissociate from Pho4, but retain the ability to interact with each other . The evidence presented here supports the idea that Pho81 acts as a phosphate-sensitive trigger that regulates the ability of the Pho80-Pho85 cyclin-cdk complex to bind Pho4, while DNA binding by Pho4 is dependent on the phosphate-sensitive interaction with Pho2.

EMBO J, 1994 Nov 15, 13(22), 5253 - 61
A Sec62p-related component of the secretory protein translocon from Drosophila displays developmentally complex behavior; Noel P et al.; The isolation and characterization of a Drosophila melanogaster gene (Dtrp1) that encodes a protein displaying the properties of both a structural and functional homolog of the yeast endoplasmic reticulum membrane-bound translocation protein Sec62p is reported . We show that Dtrp1 can not only rescue the lethality associated with a SEC62 gene knockout in yeast, but also complement the sec62-associated defective transport of a precursor polypeptide from the cytoplasm into the lumen of the endoplasmic reticulum . Expression of the Dtrp1 gene throughout Drosophila development is characterized by peaks in mid-embryo-genesis and mid-pupation, followed by a sustained period of mRNA accumulation in adults . The examination of male reproductive tissues showed a very high level of preferential expression of a 1.6 kb message, while a 2.2 kb message was confined almost exclusively to the non-reproductive tissues . Within the reproductive tract itself the 1.6 kb message was expressed in testes, ejaculatory duct and particularly strongly in the paragonial glands . Since these latter organs are specialized secretory tissues we suggest that the 1.6 kb message may encode a protein isoform that performs a unique, tissue-specific role in the protein translocation pathway . Such observations may indicate a hitherto unexpected diversity in components of the protein translocation pathway in respect to stage, tissue and, potentially, substrate specificity.

FEBS Lett, 1994 Nov 14, 354(3), 245 - 7
A leucine motif in the amino acid sequence of subunit 9 of the mitochondrial ATPase, and other hydrophobic membrane proteins, that is highly conserved by editing; Konstantinov YM et al.; Subunit 9 of the mitochondrial ATPase, but also other hydrophobic mitochondrially encoded proteins, contains a high frequency of the leucine motif, -Leu-X9-Leu-, which is highly conserved through RNA editing . The leucine motif may provide specific recognition sites between membrane-spanning domains of the F0-ATPase and between other hydrophobic subunits during the assembly of multienzyme complexes in the inner mitochondrial membrane.

Nucleic Acids Res, 1994 Nov 11, 22(22), 4712 - 8
GAGA factor and TBF1 bind DNA elements that direct ubiquitous transcription of the Drosophila alpha 1-tubulin gene; O'Donnell KH et al.; Three DNA regions (TE1, TE2 and the intron) regulate the ubiquitous expression of the alpha 1-tubulin gene of Drosophila melanogaster . In this report, we identify two proteins that bind these DNA regions . One is the previously characterized GAGA transcription factor and the other is a newly identified 62 kDa polypeptide, TBF1 (TE1-binding factor 1) . Purified GAGA factor binds three sites in TE2 and at least three in the intron . TBF1 was purified from embryos and binds to both TE1 and TE2 . Together, the two proteins produce the same DNase I footprints in TE1 and TE2 as does a nuclear extract that transcribes the gene accurately . These footprints cover most of the TE1 and TE2 DNA . Moreover, one binding site for each protein coincides with a site that activates transcription in vitro . The characteristics of the GAGA factor and the genes it regulates suggest roles these two proteins are likely to play in regulating ubiquitous expression.

J Biol Chem, 1994 Nov 11, 269(45), 28460 - 4
Rupture of the mitochondrial outer membrane impairs porin assembly; Smith M et al.; Outer membranes isolated from yeast mitochondria were capable mediating the in vitro insertion of porin . As with the outer membrane of intact mitochondria, the insertion was ATP-dependent, and the inserted porin was resistant to trypsin treatment after detergent solubilization . However, the extent of porin insertion into isolated outer membranes was much less per mg of outer membrane protein than with intact mitochondria . The greater efficiency of intact mitochondria was not due to contact site-mediated translocation as isolated contact sites were less able to insert porin than isolated outer membranes, and blockade of the contact site channel in intact mitochondria did not affect porin insertion . However, mitochondria that had been subjected to osmotic shock sufficient to rupture the outer membrane and deplete the contents of the intermembrane space (i.e . mitoplasts) lost most of their ability to insert porin . Since outer membranes are isolated from mitoplasts, the low insertion activity of mitoplasts explains the low efficiency of insertion into isolated outer membranes . These results also indicate that, unlike proteins that are imported to the inner membrane and matrix of the mitochondria, porin's assembly is severely reduced by breaching the outer membrane and depletion of the intermembrane space contents.

FEBS Lett, 1994 Nov 7, 354(2), 183 - 6
Troponin T is capable of binding dystrophin via a leucine zipper; Pearlman JA et al.; Using genetic and physical assays for protein-protein interactions, we identified a fast isoform of troponin T that binds to dystrophin . Troponin T specifically bound to the first of two highly conserved leucine zipper motifs in the carboxy terminus of dystrophin {1,2} . Single amino acid changes in the zipper predicted to disrupt alpha-helix formation or cause steric hindrance abolished this binding . These data support the hypothesis that dystrophin couples the contractile apparatus to the sarcolemma and indicate that leucine zipper mediated protein-protein interactions are functionally important in the cytoskeleton as well as the nucleus.

Cell, 1994 Nov 4, 79(3), 535 - 46
Rit1, a tRNA backbone-modifying enzyme that mediates initiator and elongator tRNA discrimination; Astrom SU et al.; Using a genetic screen in yeast aimed at identifying cellular factors involved in initiator and elongator methionine tRNA discrimination in the translational process, we have identified a mutation that abolish the requirement for elongator methionine tRNA . The gene affected, which we call the ribosylation of the initiator tRNA gene or RIT1, encodes a 2'-O-ribosyl phosphate transferase . This enzyme modifies exclusively the initiator tRNA in position 64 using 5'-phosphoribosyl-1'-pyrophosphate as the modification donor . As the initiator tRNA participates both in the initiation and elongation of translation in a rit1 strain, we conclude that the 2'-O-ribosyl phosphate modification discriminates the initiator tRNAs from the elongator tRNAs during protein synthesis . The modification enzyme was shown to recognize the stem-loop IV region that is unique in eukaryotic cytoplasmic initiator tRNAs.

Cell, 1994 Nov 4, 79(3), 459 - 74
DPY-27:a chromosome condensation protein homolog that regulates C . elegans dosage compensation through association with the X chromosome; Chuang PT et al.; dpy-27 is an essential dosage compensation gene that acts to reduce expression of both hermaphrodite X chromosomes . The DPY-27 protein becomes specifically localized to the X chromosomes of wild-type XX embryos, but remains diffusely distributed throughout the nuclei of male (XO) embryos . In xol-1 mutant XO embryos that activate the XX mode of dosage compensation and die from inappropriately low X chromosome transcript levels, DPY-27 becomes localized to X . Therefore, sex specificity of the dosage compensation process is regulated at the step of DPY-27 X chromosome localization . DPY-27 exhibits striking similarity to proteins required for assembly and structural maintenance of Xenopus chromosomes in vitro and for segregation of yeast chromosomes in vivo . These findings suggest a link between global regulation of gene expression and higher order chromosome structure . We propose that DPY-27 implements dosage compensation by condensing the chromatin structure of X in a manner that causes general reduction of X chromosome expression.

J Mol Biol, 1994 Nov 4, 243(4), 696 - 718
Protein three-dimensional structure determination and sequence-specific assignment of 13C and 15N-separated NOE data . A novel real-space ab initio approach; Kraulis PJ; The sequence-specific assignment of resonances is considered to be a requirement for the determination of the three-dimensional (3D) structure of a protein in solution by nuclear magnetic resonance methods . The main source of structural information is the nuclear Overhauser effect spectroscopy (NOESY) spectrum, which contains information about spatially close pairs of protons . Currently, various J-correlated spectra must be recorded in order to obtain the sequence-specific assignments necessary to interpret the NOESY spectra . In this work, a novel procedure to determine the 3D structure and the sequence-specific assignments of a protein using only data from 13C and 15N-separated multidimensional NOESY spectra is described . No information from J-correlated spectra is required . The algorithm is called ANSRS (Assignment of NOESY Spectra in Real Space) and is based on an inversion of the traditional strategy . A 3D real-space structure of detected, but unassigned, 1H spins is calculated from the nuclear Overhauser effect (NOE) distance restraints using a dynamical simulated annealing procedure . The sequence-specific assignments are then determined by searching among the 1H spins in the 3D real-space structure for plausible residue assignments . The search uses a Monte Carlo simulated annealing algorithm based on assignment probabilities derived from the 1H, 15N and 13C chemical shifts, various spatial constraints, and the known sequence of the protein . The procedure has been tested on semi-synthetic data sets comprising published experimental chemical shifts and NOE distance restraints derived from the known 3D structures of the two proteins GAL4 (residues 9 to 41) and bovine pancreatic trypsin inhibitor . The ANSRS procedure was able to determine the sequence-specific assignments for more than 95% of the spins, and was fairly robust with respect to missing NOE data . The potential of the ANSRS approach with respect to automated assignment, reduction of the number of NMR spectra required for a structure determination, assignment of homologous and mutant proteins, and the possibility of analysing spectra recorded at high pH is discussed.

Biophys Chem, 1994 Nov, 52(3), 191 - 218
Effect of ethidium binding and superhelix density on the supercoiling free energy and torsion and bending constants of p30 delta DNA; Clendenning JB et al.; Topoisomer distributions created by the action of topoisomerase I on p30 delta DNA in the presence of various concentrations of ethidium are measured and analyzed using recently developed theory to obtain the twist energy parameter (ET) that governs the free energy of supercoiling in each case . Competitive dialysis experiments to investigate the relative affinity of ethidium for linear and supercoiled DNAs at different binding ratios are assayed fluorometrically and the results are analyzed using related theory . The topoisomer distributions and fluorescence intensity ratios agree well with the theory, which is based on the assumption that the supercoiling free energy varies quadratically with the effective linking difference, regardless of ethidium binding or superhelix density . The topoisomer distribution experiments alone yield an average best-fit value, ET = 950 +/- 80, independent of ethidium binding ratio from r = 0 to 0.082, while the combined topoisomer distribution and ethidium binding experiments yield an average best-fit value, ET = 1030 +/- 90, which is essentially independent of ethidium binding ratio from r = 0 to 0.082 and superhelix density from sigma = 0 to (-)0.053 . One may conclude that the supercoiling free-energy-varies quadratically with effective linking difference over the entire range of observed ethidium binding ratios and superhelix densities . The independently measured torsion constant (alpha) of p30 delta DNA is likewise essentially independent of superhelix density and ethidium binding ratio . The observed invariance of ET and alpha implies that the bending constant kappa beta is similarly invariant to superhelix density and ethidium binding ratio . The apparently ideal behavior displayed by p30 delta DNA is not exhibited by pBR322 DNA, which is discussed in the following companion paper.

J Cell Biol, 1994 Nov, 127(4), 893 - 902
Mitochondrial heat shock protein 70, a molecular chaperone for proteins encoded by mitochondrial DNA; Herrmann JM et al.; Mitochondrial heat shock protein 70 (mt-Hsp70) has been shown to play an important role in facilitating import into, as well as folding and assembly of nuclear-encoded proteins in the mitochondrial matrix . Here, we describe a role for mt-Hsp70 in chaperoning proteins encoded by mitochondrial DNA and synthesized within mitochondria . The availability of mt-Hsp70 function influences the pattern of proteins synthesized in mitochondria of yeast both in vivo and in vitro . In particular, we show that mt-Hsp70 acts in maintaining the var1 protein, the only mitochondrially encoded subunit of mitochondrial ribosomes, in an assembly competent state, especially under heat stress conditions . Furthermore, mt-Hsp70 helps to facilitate assembly of mitochondrially encoded subunits of the ATP synthase complex . By interacting with the ATP-ase 9 oligomer, mt-Hsp70 promotes assembly of ATP-ase 6, and thereby protects the latter protein from proteolytic degradation . Thus mt-Hsp70 by acting as a chaperone for proteins encoded by the mitochondrial DNA, has a critical role in the assembly of supra-molecular complexes.

Invest Ophthalmol Vis Sci, 1994 Nov, 35(12), 4023 - 30
Isolation of cDNA clones encoding the 80-kd subunit protein of the human autoantigen Ku (p70/p80) by antisera raised against ciliary processes of human eye donors; Hong TJ et al.; PURPOSE . To isolate and characterize human nonpigmented ocular ciliary epithelial cDNA clones by screening a cDNA library constructed from the established human nonpigmented ciliary epithelial cell line (ODM-2) with sera raised against ciliary processes of human eye donors (cadavers) . METHODS . An ODM-2 cDNA expression library in lambda Uni-ZAP-XR vector was screened with sera of mouse anti-human ciliary processes . The cDNA inserts from plaque-purified clones were sequenced by the dideoxy-chain termination method with T3 and T7 polymerase . Sera were assayed by Western blot analysis and indirect immunofluorescence and were then compared with sera, enriched in anti-Ku antibodies, collected from patients with scleroderma polymyositis overlap syndrome . RESULTS . Immunoscreening of 2.5 x 10(5) independent clones with sera induced experimentally in total tissue homogenates of human ciliary processes led to the isolation of four cDNA-positive clones . Three clones, designated A1, D1, and D3 and containing inserts measuring 1.4 kb to 1.8 kb, were found to be identical in DNA sequence analysis to that of the 80-kd subunit protein of the human autoantigen Ku (p70/p80) . The fourth clone, designated D2, failed to show any significantly similarity with previously known genes . Cell extracts from human ciliary processes and ODM-2 cells were analyzed by immunoblotting with anti-human ciliary process sera . The pattern of proteins immunodetected was compared with the profile of proteins immunodetected with human anti-Ku sera from a patient with scleroderma polymyositis overlap syndrome . These results demonstrated that sera from anti-human ciliary processes recognize a variety of antigens in ODM-2 cells and are strikingly rich in antibodies to the 80-kd subunit protein of Ku (p70/p80) . Furthermore, indirect immunofluorescence analysis supported the observation that anti-human ciliary processes sera labeled nuclear antigens in a human ciliary epithelium cell line and cross species in bovine ciliary epithelial cell, thus demonstrating the nuclear localization of Ku antigen . The mRNA in the human eye was found to be widely expressed in all tested ocular tissues and in ODM-2 cells . CONCLUSIONS . The ocular ciliary epithelium induces a species-specific immune response in xenogenic animals . In particular, the human ciliary epithelium elicits a strong immune recognition of the 80-kd subunit protein of the human autoantigen Ku (p70/p80) in ODM-2 cells.

Exp Cell Res, 1994 Nov, 215(1), 109 - 13
A novel acidic form of the phosphatidylinositol transfer protein is preferentially retained in permeabilized Swiss mouse 3T3 fibroblasts; de Vries KJ et al.; By use of indirect immunofluorescence it was shown that phophatidylinositol transfer protein (PI-TP) remains associated with the Golgi system of Swiss mouse 3T3 fibroblasts after permeabilization with streptolysin O . By Western blot analysis it was demonstrated that intact cells contain the phosphatidylinositol-bound form of PI-TP (pI 5.5) and a novel more acidic form of PI-TP (pI 5.4) in approximately equal amounts . After permeabilization, about 50% of the PI-TP was retained in the cells with an enrichment of the pH 5.4 form relative to the pH 5.5 form; the opposite was observed for the PI-TP released into the medium . Subfractionation of cell homogenates by centrifugation provided evidence that a distinct amount of PI-TP is strongly bound to the membrane fraction with the pH 5.4 form more prominently present than the pH 5.5 form.

EMBO J, 1994 Nov 1, 13(21), 5135 - 45
Molecular chaperones cooperate with PIM1 protease in the degradation of misfolded proteins in mitochondria; Wagner I et al.; ATP dependent proteolytic degradation of misfolded proteins in the mitochondrial matrix is mediated by the PIM1 protease and depends on the molecular chaperone proteins mt-hsp70 and Mdj1p . Chaperone function is essential to maintain misfolded proteins in a soluble state, a prerequisite for their degradation by PIM1 protease . In the absence of functional mt-hsp70 or Mdj1p misfolded proteins either remain associated with mt-hsp70 or form aggregates and thereby are no longer substrates for PIM1 protease . Mdj1p is shown to regulate the ATP dependent association of an unfolded polypeptide chain with mt-hsp70 affecting binding to as well as release from mt-hsp70 . These findings establish a central role of molecular chaperone proteins in the degradation of misfolded proteins by PIM1 protease and thereby demonstrate a functional interrelation between components of the folding machinery and the proteolytic system within mitochondria.

Biochemistry, 1994 Nov 1, 33(43), 12787 - 94
Eight histidine residues are catalytically essential in a membrane-associated iron enzyme, stearoyl-CoA desaturase, and are conserved in alkane hydroxylase and xylene monooxygenase; Shanklin J et al.; The eukaryotic fatty acid desaturases are iron-containing enzymes that catalyze the NAD-(P)H- and O2-dependent introduction of double bonds into methylene-interrupted fatty acyl chains . Examination of deduced amino acid sequences for the membrane desaturases from mammals, fungi, insects, higher plants, and cyanobacteria has revealed three regions of conserved primary sequence containing HX(3 or 4)H,HX(2 or 3)HH, and HX(2 or 3)HH . This motif is also present in the bacterial membrane enzymes alkane hydroxylase (omega-hydroxylase) and xylene monooxygenase . Hydropathy analyses indicate that these enzymes contain up to three long hydrophobic domains which would be long enough to span the membrane bilayer twice . The conserved His-containing regions have a consistent positioning with respect to these potential membrane spanning domains . Taken together, these observations suggest that the membrane fatty acid desaturases and hydrocarbon hydroxylases have a related protein fold, possibly arising from a common ancestral origin . In order to examine the functional role of these conserved His residues, we have made use of the ability of the rat delta 9 desaturase gene to complement a yeast strain deficient in the delta 9 desaturase gene function (ole1) . By site-directed mutagenesis, eight conserved His residues in the rat delta 9 desaturase were individually converted to Ala . Each His-->Ala mutation failed to complement the yeast ole1 mutant . In contrast, mutation of three nonconserved flanking His residues or a partially conserved Arg residue within the conserved motif to Ala allowed for complementation of the ole1 phenotype.(ABSTRACT TRUNCATED AT 250 WORDS)

Mol Cell Biol, 1994 Nov, 14(11), 7652 - 9
Domain B of ARS307 contains two functional elements and contributes to chromosomal replication origin function; Theis JF et al.; ARS307 is highly active as a replication origin in its native location on chromosome III of Saccharomyces cerevisiae . Its ability to confer autonomous replication activity on plasmids requires the presence of an 11-bp autonomously replicating sequence (ARS) consensus sequence (ACS), which is also required for chromosomal origin function, as well as approximately 100 bp of sequence flanking the ACS called domain B . To further define the sequences required for ARS function, a linker substitution mutagenesis of domain B was carried out . The mutations defined two sequences, B1 and B2, that contribute to ARS activity . Therefore, like ARS1, domain B of ARS307 is composed of functional subdomains . Constructs carrying mutations in the B1 element were used to replace the chromosomal copy of ARS307 . These mutations caused a reduction in chromosomal origin activity, demonstrating that the B1 element is required for efficient chromosomal origin function.

Mol Cell Biol, 1994 Nov, 14(11), 7507 - 16
A highly conserved domain of RNA polymerase II shares a functional element with acidic activation domains of upstream transcription factors; Xiao H et al.; We report here that the largest subunit of yeast RNA polymerase II contains an acidic domain that is similar to acidic activators of transcription . This domain includes the highly conserved homology box H . A hybrid protein containing this acidic domain fused to the DNA-binding domain of GAL4 is a potent activator of transcription in the yeast Saccharomyces cerevisiae . Interestingly, mutations that reduce the upstream activating activity of this acidic domain also abolish the normal function of RNA polymerase II . Such functional defects can be rescued by the acidic activation domains of VP16 and GAL4 when inserted into the mutant derivatives of RNA polymerase II . We further show that this acidic domain of RNA polymerase II interacts directly with two general transcription factors, the TATA-binding protein and TFIIB, and that the acidic activation domain of VP16 can compete specifically with the acidic domain of the RNA polymerase for these interactions . We discuss the implications of this finding for the mechanisms of transcriptional activation in eucaryotes.

Mol Cell Biol, 1994 Nov, 14(11), 7492 - 8
Active-site assembly and mode of DNA cleavage by Flp recombinase during full-site recombination; Whang I et al.; A combination of half-site substrates and step arrest mutants of Flp, a site-specific recombinase of the integrase family, had earlier revealed the following features of the half-site recombination reaction . (i) The Flp active site is assembled by sharing of catalytic residues from at least two monomers of the protein . (ii) A Flp monomer does not cleave the half site to which it is bound (DNA cleavage in cis); rather, it cleaves a half site bound by a second Flp monomer (DNA cleavage in trans) . For the lambda integrase (Int protein), the prototype member of the Int family, catalytic complementation between two active-site mutants has been observed in reactions with a suicide attL substrate . By analogy with Flp, this observation is strongly suggestive of a shared active site and of trans DNA cleavage . However, reactions with linear suicide attB substrates and synthetic Holliday junctions are more compatible with cis than with trans DNA cleavage . These Int results either argue against a common mode of active-site assembly within the Int family or challenge the validity of Flp half sites as mimics of the normal full-site substrates . We devised a strategy to assay catalytic complementation between Flp monomers in full sites . We found that the full-site reaction follows the shared active-site paradigm and the trans mode of DNA cleavage . These results suggest that within the Int family, a unitary chemical mechanism of recombination is achieved by more than one mode of physical interaction among the recombinase monomers.

Mol Cell Biol, 1994 Nov, 14(11), 7483 - 91
ralGDS family members interact with the effector loop of ras p21; Kikuchi A et al.; Using a yeast two-hybrid system, we identified a novel protein which interacts with ras p21 . This protein shares 69% amino acid homology with ral guanine nucleotide dissociation stimulator (ralGDS), a GDP/GTP exchange protein for ral p24 . We designated this protein RGL, for ralGDS-like . Using the yeast two-hybrid system, we found that an effector loop mutant of ras p21 was defective in interacting with the ras p21-interacting domain of RGL, suggesting that this domain binds to ras p21 through the effector loop of ras p21 . Since ralGDS contained a region highly homologous with the ras p21-interacting domain of RGL, we examined whether ralGDS could interact with ras p21 . In the yeast two-hybrid system, ralGDS failed to interact with an effector loop mutant of ras p21 . In insect cells, ralGDS made a complex with v-ras p21 but not with a dominant negative mutant of ras p21 . ralGDS interacted with the GTP-bound form of ras p21 but not with the GDP-bound form in vitro . ralGDS inhibited both the GTPase-activating activity of the neurofibromatosis gene product (NF1) for ras p21 and the interaction of Raf with ras p21 in vitro . These results demonstrate that ralGDS specifically interacts with the active form of ras p21 and that ralGDS can compete with NF1 and Raf for binding to the effector loop of ras p21 . Therefore, ralGDS family members may be effector proteins of ras p21 or may inhibit interactions between ras p21 and its effectors.

J Virol, 1994 Nov, 68(11), 7275 - 83
Serum response factor has functional roles both in indirect binding to the CArG box and in the transcriptional activation function of human T-cell leukemia virus type I Tax; Fujii M et al.; We previously reported that Tax1 of human T-cell leukemia virus type I interacts directly with serum response factor (SRF) and that Tax1 activates the transcription of several cellular immediate-early genes through the SRF binding site (CArG box) . This activation required the transcriptional activation function of Tax1, identified as an activity of GALTax (a chimeric Tax1 with the yeast transcription factor GAL4) at the GAL4-binding site . In this study, we examined whether SRF plays a role in the transcriptional activation function of Tax1 . Expression of Tax1 suppressed the GALTax activity at the GAL4 site as a result of squelching, and the suppressed activity was recovered by the overexpression of SRF, suggesting that SRF is a factor that is required for GALTax activity and that is inhibited by competition with Tax1 . The expression of antisense SRF RNA specifically inhibited GALTax activity to less than 20% . Deletion of the Tax1 interaction domain of SRF at its C terminus converted SRF from an activator of GALTax to an inhibitor . These results suggest that SRF is an essential component of the transcriptional activation of Tax1 in addition to a mediator of CArG box binding.

Curr Biol, 1994 Nov 1, 4(11), 973 - 82
Use of an oriented peptide library to determine the optimal substrates of protein kinases; Songyang Z et al.; BACKGROUND: Phosphorylation by protein kinases is an important general mechanism for controlling intracellular processes, and plays an essential part in the signal transduction pathways that regulate cell growth in response to extracellular signals . A great number of protein kinases have been discovered, and the identification of their biological targets is still a very active research area . Protein kinases must have the appropriate substrate specificity to ensure that signals are transmitted correctly . Previous studies have demonstrated the importance of primary sequences within substrate proteins in determining protein kinase specificity, but efficient ways of identifying these sequences are lacking . RESULTS: We have developed a new technique for determining the substrate specificity of protein kinases, using an oriented library of more than 2.5 billion peptide substrates . In this approach, the consensus sequence of optimal substrates is determined by sequencing the mixture of products generated during a brief reaction with the kinase of interest . The optimal substrate predicted for cAMP-dependent protein kinase (PKA) by this technique is consistent with the sequences of known PKA substrates . The optimal sequences predicted for cyclin-dependent kinases (CDKs) cyclin B-Cdc2 and cyclin A-CDK2 also agree well with sites thought to be phosphorylated in vivo by these kinases . In addition, we determined the optimal substrate for SLK1, a homologue of the STE20 protein serine kinase of hitherto unknown substrate specificity . We also discuss a model incorporating the optimal cyclin B-Cdc2 substrate into the known crystal structure of this kinase . CONCLUSIONS: Using the new technique we have developed, the sequence specificity of protein kinases can rapidly be predicted and, from this information, potential targets of the kinases can be identified.

Curr Biol, 1994 Nov 1, 4(11), 1019 - 22
Vacuolar sorting . Tracking down an elusive receptor; Chapman RE; The receptor that sorts carboxypeptidase Y for transport to the vacuole in yeast has recently been identified; how vacuolar sorting is regulated is far from understood, but an intriguing model has been proposed.

Trends Biochem Sci, 1994 Nov, 19(11), 470 - 3
MAPKs: new JNK expands the group; Davis RJ; Mitogen-activated protein kinases (MAPKs) are activated by dual phosphorylation on threonine and tyrosine in response to a wide array of extracellular stimuli . In the yeast Saccharomyces cerevisiae, a series of extracellular stimuli . In the yeast Saccharomyces cerevisiae, a series of MAPK signal transduction pathways have been demonstrated to control many cellular functions . By contrast, mammalian MAPKs are more poorly understood . However, recent studies have established important roles for three separate groups of mammalian MAPKs, which are characterized by distinct dual phosphorylation motifs . Together, these protein kinases mediate signal transduction in mammalian tissues and control many aspects of cellular physiology.

Bioessays, 1994 Nov, 16(11), 809 - 15
Mitogenesis and protein synthesis: a role for ribosomal protein S6 phosphorylation?
Stewart MJ, Thomas G.
It has been known for 20 years that the ribosomal protein S6 is rapidly phosphorylated when cells are stimulated to grow or divide . Furthermore, numerous studies have documented that there is a strong correlation between increases in S6 phosphorylation and protein synthesis, leading to the idea that S6 phosphorylation is involved in up-regulating translation . In an attempt to define a mechanism by which S6 phosphorylation exerts translational control, other studies have focused on characterizing the sites of phosphorylation of this protein and its location within the ribosome . Recent data show that S6 is a protein which may have diverse cellular functions and is essential for normal development, and that it may be involved in the translational regulation of a specific class of messages.

Plant Physiol, 1994 Nov, 106(3), 1157 - 62
Arabidopsis thaliana expresses three divergent Srp54 genes; Chu B et al.; The Arabidopsis thaliana Srp54 gene family was determined to consist of three genes, all of which were cloned and sequenced . In addition, cDNAs corresponding to two of the genes were obtained . To our knowledge this is the first description of multiple Srp54 genes within an organism . In contrast to the situation in mammals, where there are only three amino acid differences between the mouse and canine sequences, there was significant amino acid sequence diversity among the genes, particularly in the methionine-rich region of the protein, which is the region responsible for binding to the 7S RNA of the signal recognition particle and to the signal sequence of newly synthesized proteins . The amino acid sequences of the GTP-binding domains of the three clones were 86% identical, whereas the methionine-rich domains were only 65% identical . RNA gel blots of various tissues and developmental stages hybridized with gene-specific probes revealed that all three genes were expressed in all the tissues investigated . There were, however, quantitative differences in expression levels.

Bioorg Med Chem, 1994 Nov, 2(11), 1243 - 50
The chemoenzymatic synthesis of neoglycolipids and lipid-linked oligosaccharides using glycosyltransferases; Flitsch SL et al.; The application of glycosyltransferases to the chemoenzymatic synthesis of neoglycosphingolipids and lipid-linked oligosaccharides allows the regio- and stereoselective formation of glycosidic bonds . In our laboratory galactosyl-, sialyl-, and fucosyltransferases have been used to assemble oligosaccharide headgroups directly on a sphingosine derivative without the need for any protection group strategies, including the Lewisx antigen . In complementary studies on N-linked oligosaccharide biosynthesis, chemically phosphorylated dolichol analogues have been tested as substrates for Dol-P-Man synthetase . Also, the substrate recognition of the core beta-1,4-mannosyltransferase from yeast has been investigated using a range of chitobiose derivatives as potential substrates.

Bioorg Med Chem, 1994 Nov, 2(11), 1133 - 41
A comparison of proteins and peptides as substrates for microsomal and solubilized oligosaccharyltransferase; Liu YL et al.; A chemoenzymatic synthesis of homogeneous neoglycoproteins and glycopeptides was explored using oligosaccharyltransferase isolated from yeast . Neither the microsomal form nor the solubilized form of the enzyme catalyzed the transfer of the core Glc3Man9(GlcNAc)2 oligosaccharide to chemically modified ribonuclease A or alpha-lactalbumin . Similarly, no transfer was observed to the 32-amino acid peptide hormone, calcitonin, by either the membrane-bound or soluble form of oligosaccharyltransferase . However, a 17-amino acid fragment of yeast invertase with the unusual sequence containing two overlapping glycosylation sequons proved to be a good substrate, slightly less effective than the well studied tripeptide, Bz-Asn-Leu-Thr-NH2 . Product analysis using gel permeation chromatography showed that the expected glycopeptides were formed and endo H-catalyzed cleavage of the oligosaccharide portion from the glycopeptides demonstrated that the glycopeptides contained the same carbohydrate moiety.

Biochem Mol Biol Int, 1994 Nov, 34(5), 883 - 95
Enhanced protein degradation by photoilluminated riboflavin in the presence of Cu(II); Jazzar MM et al.; Riboflavin is known to generate superoxide anion upon photoillumination and in the presence of Cu(II) causes fragmentation of DNA . In the present study we examined the effect of riboflavin and Cu(II) on bovine serum albumin, invertase and lysozyme . Using fluorescence quenching experiments, it is shown that riboflavin binds to protein and causes fragmentation which in the presence of Cu(II) is enhanced . Using neocuproine as the Cu(I) sequestering reagent, it has also been shown that Cu(I) is an essential intermediate in the protein fragmentation reaction . Results obtained with various scavengers of active oxygen species strongly suggest that the species predominantly responsible for protein breakage is hydroxyl radical.

J Cell Sci, 1994 Nov, 107 ( Pt 11), 3223 - 33
Assembly and DNA binding of recombinant Ku (p70/p80) autoantigen defined by a novel monoclonal antibody specific for p70/p80 heterodimers; Wang J et al.; The Ku autoantigen is a heterodimer of 70 kDa (p70) and -80 kDa (p80) subunits that is the DNA-binding component of a DNA-dependent protein kinase (DNA-PK) . The 350 kDa (p350) catalytic subunit of DNA-PK phosphorylates Sp-1, Oct-1, p53 and RNA polymerase II in vitro, but the precise cellular role of DNA-PK remains unclear . In the present studies, the assembly of p70/p80 heterodimers and the interaction of Ku with DNA was investigated using recombinant vaccinia viruses directing the synthesis of human p70 (p70-vacc) and p80 (p80-vacc), and monoclonal antibodies (mAbs) . Expression of human Ku antigens in rabbit kidney (RK13) cells could be demonstrated by immunofluorescent staining because this cell line contains little endogenous Ku . A novel mAb designated 162 stained the nuclei of RK13 cells coinfected with p70-vacc and p80-vacc, but not cells that were infected with either virus alone, suggesting that it recognized the p70/p80 heterodimer but not monomeric p70 or p80 . In agreement with the immunofluorescence data, 162 immunoprecipitated both p70 and p80 from extracts of coinfected cells, but did not immunoprecipitate either subunit by itself from extracts of cells infected with p70-vacc or p80-vacc, respectively . Conversely, the binding of 162 to Ku isolated from human K562 cells stabilized the p70/p80 heterodimer under conditions that normally dissociate p70 from p80 . The nuclei of cells infected with p70-vacc alone could be stained with mAb N3H10 (anti-p70) and cells infected with p80-vacc alone could be stained with mAb 111 (anti-p80), indicating that the formation of p70/p80 heterodimers was not required for nuclear transport . Finally, free recombinant and cellular p70 both bound to DNA efficiently in vitro, suggesting that free p70, like the p70/p80 heterodimer, serves as a DNA-binding factor . Moreover, free human p70 could be released from the nuclei of p70-vacc-infected RK13 cells by deoxyribonuclease I treatment, suggesting that it was associated with chromatin in vivo . The nuclear transport of free p70 and the association of free p70 with chromatin in vivo raise the possibility that newly synthesized cellular p70 might undergo nuclear transport and DNA-binding prior to dimerization with p80 or assembly with p350.

FEBS Lett, 1994 Oct 31, 354(1), 23 - 9
Specificities of the two center N inhibitors of mitochondial bc1 complex, antimycin and funiculosin: strong involvement of cytochrome b-asparagine-208 in funiculosin binding; Brasseur G et al.; Funiculosin, a center N inhibitor of the bc1 complex, induces a blue-shift in the cytochrome b spectrum . A thermosensitive revertant {Coppee, J.Y . et al., J . Biol . Chem . 269 (1994) 4221-4226} isolated from a cytochrome b respiratory-deficient mutant, exhibits a red-shift instead of the blue-shift retained in the original mutant and shows resistance to this inhibitor . Replacing cytochrome b-Asparagine-208 by Lysine in this revertant, keeping the original mutation S206L, leads, when mitochondria are incubated at non-permissive temperature, to complete loss of bc1 complex activity and funiculosin-binding, while the antimycin-binding is conserved . These data suggest some inhibitor site specificity and close proximity between the funiculosin-binding site and the catalytic center N domain (QN).

Nature, 1994 Oct 27, 371(6500), 768 - 74
Mitochondrial Hsp70/MIM44 complex facilitates protein import; Schneider HC et al.; Protein translocation into mitochondria requires the mitochondrial protein Hsp70 . This molecular chaperone of the mitochondrial matrix is recruited to the protein import machinery by MIM44, a component associated with the inner membrane of the mitochondria . Formation of the mt-Hsp70/MIM44 complex is regulated by ATP . MIM44 and mt-Hsp 70 interact in a sequential manner with incoming segments of unfolded preproteins and thereby facilitate stepwise vectorial translocation of proteins across the mitochondrial membranes . The complex appears to act as a molecular ratchet which is energetically driven by the hydrolysis of ATP.

Nucleic Acids Res, 1994 Oct 25, 22(21), 4510 - 9
SWAP pre-mRNA splicing regulators are a novel, ancient protein family sharing a highly conserved sequence motif with the prp21 family of constitutive splicing proteins; Spikes DA et al.; Regulators responsible for the pervasive, nonsex-specific alternative pre-mRNA splicing characteristic of metazoans are almost entirely unknown or uncertain . We describe here a novel family of splicing regulators present throughout metazoans . Specifically, we analyze two nematode (Caenorhabditis elegans) genes . One, CeSWAP, is a cognate of the suppressor-of-white-apricot (DmSWAP) splicing regulator from the arthropod Drosophila . Our results define the ancient, conserved SWAP protein family whose members share a colinearly arrayed series of novel sequence motifs . Further, we describe evidence that the CeSWAP protein autoregulates its levels by feedback control of splicing of its own pre-mRNA analogously to the DmSWAP protein and as expected of a splicing regulator . The second nematode gene, Ceprp21, encodes an abundant nuclear cognate of the constitutive yeast splicing protein, prp21, on the basis of several lines of evidence . Our analysis defines prp21 as a second novel, ancient protein family . One of the motifs conserved in prp21 proteins--designated surp--is shared with SWAP proteins . Several lines of evidence indicate that both new families of surp-containing proteins act at the same (or very similar) step in early prespliceosome assembly . We discuss implications of our results for regulated metazoan pre-mRNA splicing.

Nucleic Acids Res, 1994 Oct 25, 22(21), 4405 - 13
Exon2 of HIV-2 Tat contributes to transactivation of the HIV-2 LTR by increasing binding affinity to HIV-2 TAR RNA; Rhim H et al.; Human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) express related Tat proteins that are encoded in two exons . Tat proteins bind directly to the TAR RNA element contained in the 5' ends of viral transcripts and thereby stimulate transcription through an as yet unidentified mechanism . We have investigated the functional significance of exon2 of the HIV-2 Tat protein by examining properties of proteins consisting of exon1 alone or exon1 + 2 . In transactivation assays in vivo, exon2 modestly increased HIV-2 Tat stimulation of transcription from the HIV-2 long terminal repeat (LTR) but had no effect on transcription from the HIV-1 LTR . In HeLa cells, exon2 increased transactivation of the HIV-2 LTR by approximately three-fold, while in COS and Jurkat cells this value was less than two-fold . In binding assays in vitro, exon2 increased the binding affinity of the HIV-2 Tat protein to HIV-2 TAR RNA . Results with GAL4 fusion proteins and a synthetic promoter containing GAL4 DNA binding sites indicated that exon2 does not contribute to the HIV-2 Tat activation domain . These observations suggest that exon2 of HIV-2 Tat contributes to transactivation of the HIV-2 LTR by increasing the binding affinity to HIV-2 TAR RNA.

Nucleic Acids Res, 1994 Oct 25, 22(21), 4386 - 94
SARs stimulate but do not confer position independent gene expression; Poljak L et al.; Two minimal scaffold-associated regions (SARs) from Drosophila were tested in stably transformed cells for their effects on the expression of reporter genes . The expression of genes bounded by two SARs is consistently stimulated by about 20- to 40-fold, if the average of a pool of cell transformants is analyzed . However, analysis of individual, stable cell transformants demonstrates that flanking SAR elements do not confer position-independent expression on the reporter gene and that the extent of position-dependent variegation is similarly large with or without the flanking SAR elements . The SAR stimulation of expression is observed in stable but not in transiently transfected cell lines . The Drosophila scs and scs' boundary elements, which do not bind to the nuclear matrix in vitro, are only about one-tenth as active as SARs in stimulating expression in stable transformants . Interestingly, the SAR stimulatory effect can be blocked by a fragment containing CpG islands (approximately 70% GC), if positioned between the SAR and the enhancer . In contrast, when inserted in the same position, control fragments, such as the scs/scs' elements, do not interfere with SAR function.

Proc Natl Acad Sci U S A, 1994 Oct 25, 91(22), 10581 - 5
Cloning of a cDNA for the FAD-linked glycerol-3-phosphate dehydrogenase from rat liver and its regulation by thyroid hormones; Muller S et al.; A full-length 2.4-kb cDNA for the FAD-linked glycerol-3-phosphate dehydrogenase (EC 1.1.99.5) was cloned from rat liver using PCR techniques . The cloned gene encodes a protein of 727 amino acids . The calculated molecular mass of 80,898 Da is higher than the apparent molecular mass observed by SDS/PAGE (74,000 Da) of the purified enzyme . This result indicates that the enzyme is synthesized as a precursor with a putative mitochondrial signal sequence . mRNA for this gene was detected in liver, heart, muscle, brain, testes, and pancreas . With the exception of testes, basal expression levels were very low in all tissues examined . However, application of thyroid hormones led to a 10- to 15-fold increase in liver glycerol-3-phosphate dehydrogenase mRNA, whereas hypothyroidism further decreased the mRNA level.

Proc Natl Acad Sci U S A, 1994 Oct 25, 91(22), 10340 - 4
Split ubiquitin as a sensor of protein interactions in vivo; Johnsson N et al.; We describe an assay for in vivo protein interactions . Protein fusions containing ubiquitin, a 76-residue, single-domain protein, are rapidly cleaved in vivo by ubiquitin-specific proteases, which recognize the folded conformation of ubiquitin . When a C-terminal fragment of ubiquitin (C(ub)) is expressed as a fusion to a reporter protein, the fusion is cleaved only if an N-terminal fragment of ubiquitin (Nub) is also expressed in the same cell . This reconstitution of native ubiquitin from its fragments, detectable by the in vivo cleavage assay, is not observed with a mutationally altered Nub . However, if C(ub) and the altered Nub are each linked to polypeptides that interact in vivo, the cleavage of the fusion containing C(ub) is restored, yielding a generally applicable assay for kinetic and equilibrium aspects of in vivo protein interactions . This method, termed USPS (ubiquitin-based split-protein sensor), makes it possible to monitor a protein-protein interaction as a function of time, at the natural sites of this interaction in a living cell.

Gene, 1994 Oct 21, 148(2), 327 - 30
The peroxisome proliferator-activated receptor interacts with the retinoid X receptor in vivo; Miyata KS et al.; The peroxisome proliferator-activated receptor (PPAR) binds cooperatively to cognate peroxisome proliferator-responsive elements (PPRE) in vitro through heterodimerization with retinoid X receptors (RXR) . We used the yeast two-hybrid system to determine whether these two nuclear receptors physically interact in vivo . Mouse (m) PPAR and human (h) RXR alpha were synthesized as fusion proteins to either the DNA-binding domain (GBD) or the transactivation domain (GAD) of the yeast GAL4 transcription-activator protein, and were tested for their ability to activate expression of a GAL1::lacZ reporter gene . Strong activation was observed only in yeast transformed with combinations of GBD::mPPAR and GAD::hRXR alpha or with GAD::mPPAR and GBD::hRXR alpha . Homodimeric interaction by mPPAR was not detected . These results provide evidence for the interaction of PPAR and RXR alpha in vivo in the absence of a PPRE target site or exogenously added ligands.

Cell, 1994 Oct 21, 79(2), 283 - 92
Crossover interference is abolished in the absence of a synaptonemal complex protein; Sym M et al.; In the zip1 mutant, meiotic chromosomes fail to synapse, owing to the absence of a structural component of the synaptonemal complex (SC) . This mutant has been analyzed for the ability to carry out several functions that have been proposed for the SC . The data presented show that the zip1 mutation does not affect chiasma function and confers only modest defects in meiotic recombination and sister chromatid cohesion . In contrast, crossover interference is completely abolished in the absence of Zip1 . These data are the first to establish a molecular link between cytological observations of the SC and the genetic phenomenon of interference.

J Mol Biol, 1994 Oct 21, 243(2), 199 - 207
Resolution of immobile chi structures by the FLP recombinase of 2 microns plasmid; Dixon JE et al.; FLP is a conservative site-specific recombinase that is encoded by the 2 microns plasmid of the yeast, Saccharomyces cerevisiae . FLP is member of the integrase family of recombinases that mediate the recombination reaction through a Holliday intermediate . The FLP recognition target (FRT) sites lie within two 599 bp inverted repeats of the 2 microns plasmid . The minimal target contains two inverted FLP binding sites (13 bp) that surround an 8 bp core region . FLP nicks the top and the bottom strands of the FRT site at the margins of the core and these nicks are thought to be the sites of strand exchange . Hence, recombination generates heteroduplex DNA in the core region . It is known that heterology between the core regions of two FRT sites inhibits their ability to engage in recombination . It is possible that two homologous cores are required to allow the junction of the Holliday intermediate to branch migrate through the core during resolution . If so, an immobile Holliday junction point should inhibit the recombination activity of FLP in the same manner as a heterology between the cores of two double-stranded FRT sites . In order to test this prediction, we generated synthetic Holliday structures specific for FLP that had the junction immobilised at representative points within the FRT core . We used either sequence heterologies or nicked strands in order to immobilise the junction . We found that immobilisation of a Holliday junction within the core region did not inhibit resolution of the Holliday structure by FLP . Hence, homology is not required for the resolution of the Holliday intermediate but rather, for an earlier step in the reaction.

J Biol Chem, 1994 Oct 21, 269(42), 26402 - 10
Biochemical, molecular, and functional characterization of porin isoforms from potato mitochondria; Heins L et al.; The mitochondrial outer membrane of eukaryotic cells contains a voltage-dependent anion channel termed porin . In the organisms studied so far only one type of porin has been identified at the protein level . Here we present a biochemical and molecular genetic analysis of two different porin polypeptides of M(r) 34,000 and 36,000 from the outer membranes of potato mitochondria (termed POM 34 and POM 36, respectively) . N-terminal sequencing and the use of labeled oligonucleotide mixtures derived from these amino acid sequences allowed the isolation of cDNA clones encoding the 34- and 36-kDa proteins . They have similar steady state protein levels and share about 75% identical amino acids suggesting that they represent isoforms . In addition, a third cDNA clone coding for a slightly different isoform of the 36-kDa protein was characterized . The polypeptides encoded by the three cDNA clones share the highest degree of sequence identity with mitochondrial porins from fungi and mammals . Tentative models of the secondary structure of the 34- and 36-kDa proteins suggest the occurrence of a 16-stranded beta-barrel typical for bacterial and mitochondrial porins . Purification of the 34-kDa protein by hydroxyapatite chromatography allowed conductance measurements in artificial bilayers . The 34-kDa protein is a voltage-dependent, channel-forming component with single channel conductances of 3.5 and 2.0 nanosiemens in 1 M KCl . In spite of the striking functional similarities to mitochondrial porins from other organisms neither the 34- nor the 36-kDa proteins are able to complement the respiratory defect of a yeast por- mutant.

Biochim Biophys Acta, 1994 Oct 18, 1219(2), 328 - 34
Primary structure and expression of a human CTP:phosphocholine cytidylyltransferase; Kalmar GB et al.; Human CTP:phosphocholine cytidylyltransferase (CT) cDNAs were isolated by PCR amplification of a human erythroleukemic K562 cell library . Initially two degenerate oligonucleotide primers derived from the sequence of the rat liver CT cDNA were used to amplify a centrally located 230 bp fragment . Subsequently overlapping 5' and 3' fragments were amplified, each using one human CT primer and one vector-specific primer . Two cDNAs encoding the entire translated domain were also amplified . The human CT (HCT) has close homology at the nucleotide and amino acid level with other mammalian CTs (from rat liver, mouse testis or mouse B6SutA hemopoietic cells and Chinese hamster ovary) . The region which deviates most from the rat liver CT sequence is near the C-terminus, where 7 changes are clustered within 34 residues (345-359), of the putative phosphorylation domain . The region of the proposed catalytic domain (residues 75-235) is 100% identical with the rat liver sequence . Significant homology was observed between the proposed catalytic domain of CT and the Saccharomyces cerevisiae MUQ1 gene product, and between the proposed amphipathic alpha-helical membrane binding domains of CT and soybean oleosin, a phospholipid-binding protein . There are several shared characteristics of these amphipathic helices . An approx . 42,000 Da protein was over-expressed in COS cells using a pAX142 expression vector containing one of the full-length HCT cDNA clones . The specific activity of the HCT in COS cell homogenates was the same as that of analogously expressed rat liver CT . The activity of HCT was lipid dependent . The soluble form was activated 3 to 4-fold by anionic phospholipids and by oleic acid or diacylglycerol-containing PC vesicles.

EMBO J, 1994 Oct 17, 13(20), 4856 - 62
The transactivation domain of Pho4 is required for nucleosome disruption at the PHO5 promoter; Svaren J et al.; The chromatin structure of the PHO5 promoter is disrupted when the promoter is derepressed by phosphate starvation . The transactivator, Pho4, is primarily responsible for this change . We have used deletion mutations of Pho4 in order to determine which protein domains are involved in nucleosome dissolution . Our results show that the DNA binding domain by itself is not sufficient to trigger chromatin disruption, even when overexpressed . In vivo footprinting reveals that Pho4 derivatives lacking the N-terminal activation domain can bind to UASp1, which resides in a constitutively nucleosome-free region, but not to UASp2, which lies within a nucleosome in the repressed PHO5 promoter . The acidic activation domain of Pho4 appears to be involved in nucleosome disruption . Substitution of the native transactivation domain of Pho4 with that from VP16 results in substantial chromatin disruption . In every case, the ability of the Pho4 mutants to activate transcription correlates with their ability to disrupt nucleosome structure in the PHO5 promoter . Therefore, we conclude that the Pho4 activation domain has at least two roles: (i) to trigger disruption of nucleosome structure over the promoter, thereby facilitating the binding of transcription factors, and (ii) to interact with the transcriptional apparatus at the proximal promoter.

EMBO J, 1994 Oct 17, 13(20), 4848 - 55
A nucleosome precludes binding of the transcription factor Pho4 in vivo to a critical target site in the PHO5 promoter; Venter U et al.; Activation of the Saccharomyces cerevisiae PHO5 gene by phosphate starvation is accompanied by the disappearance of two pairs of positioned nucleosomes that flank a short hypersensitive region in the promoter . The transcription factor Pho4 is the key regulator of this transition . By in vitro footprinting it was previously shown that there is a low affinity site (UASp1) which is contained in the short hypersensitive region in the inactive promoter, and a high affinity site (UASp2) which is located in the adjacent nucleosome . To investigate the interplay between nucleosomes and Pho4, we have performed in vivo footprinting experiments with dimethylsulfate . Pho4 was found to bind to both sites in the active promoter . In contrast, it binds to neither site in the repressed promoter . Lack of binding under repressing conditions is largely due to the low affinity of Pho4 for its binding sites under these conditions . Despite the increased affinity of Pho4 for its target sites under activating conditions, binding to UASp2 is prevented by the presence of the nucleosome and can only occur after prior disruption of this nucleosome in a process that requires UASp1 . Protection of the PHO5 UASp2 by the nucleosome is not absolute, however, since overexpression of Pho4 can disrupt this nucleosome even when UASp1 is deleted . Also under these conditions, with only UASp2 present, all four nucleosomes at the PHO5 promoter are disrupted, whereas no chromatin change at all is observed when both UAS elements are destroyed.

EMBO J, 1994 Oct 17, 13(20), 4807 - 15
Functional similarity and physical association between GCN5 and ADA2: putative transcriptional adaptors; Marcus GA et al.; A selection for yeast mutants resistant to GAL4-VP16-induced toxicity previously identified two genes, ADA2 and ADA3, which may function as adaptors for some transcriptional activation domains and thereby facilitate activation . Here we identify two new genes by the same selection, one of which is identical to GCN5 . We show that gcn5 mutants share properties with ada mutants, including slow growth, temperature sensitivity and reduced activation by the VP16 and GCN4 activation domains . Double mutant studies suggest that ADA2 and GCN5 function together in a complex or pathway . Moreover, we demonstrate that GCN5 binds to ADA2 both by the two-hybrid assay in vivo and by co-immunoprecipitation in vitro . This suggests that ADA2 and GCN5 are part of a heteromeric complex that mediates transcriptional activation . Finally, we demonstrate the functional importance of the bromodomain of GCN5, a sequence found in other global transcription factors such as the SWI/SNF complex and the TATA binding protein-associated factors . This domain is not required for the interaction between GCN5 and ADA2 and thus may mediate a more general activity of transcription factors.

FEBS Lett, 1994 Oct 17, 353(2), 201 - 6
Yta10p is required for the ATP-dependent degradation of polypeptides in the inner membrane of mitochondria; Pajic A et al.; Incompletely synthesized polypeptides in the mitochondrial inner membrane are subject to rapid proteolysis . We demonstrate that Yta10p, a mitochondrial homologue of a conserved family of putative ATPases in Saccharomyces cerevisiae, is essential for this proteolytic process . Yta10p-dependent degradation requires divalent metal ions and the hydrolysis of ATP . Yta10p is an integral protein of the inner mitochondrial membrane exposing the carboxy terminus to the mitochondrial matrix space . Based on the presence of consensus binding sites for ATP, and for divalent metal ions found in a number of metal dependent endopeptidases, a direct role of Yta10p in the proteolytic breakdown of membrane-associated polypeptides in mitochondria is suggested.

Genes Dev, 1994 Oct 15, 8(20), 2504 - 12
Crystal structure of a PPR1-DNA complex: DNA recognition by proteins containing a Zn2Cys6 binuclear cluster; Marmorstein R et al.; PPR1 is a yeast transcription factor that contains a six-cysteine, two-zinc (Zn) domain, homologous to a similar structure in GAL4 . Like GAL4, it binds to DNA sites with conserved CGG triplets symmetrically placed near each end . Whereas the GAL4 site has 11 intervening base pairs, the PPR1 site has 6 . The crystal structure of a 95-residue fragment of PPR1 in specific complex with DNA shows that the protein binds to a symmetrical 14-bp recognition site as a nonsymmetrical homodimer . An amino-terminal Zn domain interacts with a conserved CGG triplet near each end of the site through major groove contacts, and the carboxy-terminal residues mediate dimerization through a coiled-coil element and an extended strand . A linker region, connecting the Zn domain and the coiled-coil, folds into a beta-hairpin . This hairpin packs differently on the two subunits and leads to a striking asymmetry, which is largely restricted to the dimerization and linker regions of the protein . Comparison with the GAL4-DNA structure shows that their specificities for sites of different length are determined by the preferred folds of their respective linker segments and by residues at the amino-terminal ends of their coiled-coils . None of these residues contact DNA in PPR1, and they contact only the sugar phosphate backbone in GAL4 . We propose that this novel mode of DNA site selection is employed by other proteins that contain a Zn2Cys6 binuclear cluster.

Genes Dev, 1994 Oct 15, 8(20), 2491 - 503
Inhibition of the DNA-binding activity of Drosophila suppressor of hairless and of its human homolog, KBF2/RBP-J kappa, by direct protein-protein interaction with Drosophila hairless; Brou C et al.; We have purified the sequence-specific DNA-binding protein KBF2 and cloned the corresponding cDNA, which is derived from the previously described RBP-J kappa gene, the human homolog of the Drosophila Suppressor of Hairless {Su(H)} gene . Deletion studies of the RBP-J kappa and Su(H) proteins allowed us to define a DNA-binding domain conserved during evolution . Because Su(H) mutant alleles exhibit dose-sensitive interactions with Hairless (H) loss-of-function mutations, we have investigated whether the RBP-J kappa or Su(H) proteins directly interact with the H protein in vitro . We show here that H can inhibit the DNA binding of both Su(H) and RBP-J kappa through direct protein-protein interactions . Consistent with this in vitro inhibitory effect, transcriptional activation driven by Su(H) in transfected Drosophila S2 cells is inhibited by H . These results support a model in which H acts, at least in part, as a negative regulator of Su(H) activity . This model offers a molecular view to the antagonistic activities encoded by the H and Su(H) genes for the control of sensory organ cell fates in Drosophila . We further propose that a similar mechanism might occur in mammals.

J Immunol, 1994 Oct 15, 153(8), 3574 - 83
The CD39 lymphoid cell activation antigen . Molecular cloning and structural characterization; Maliszewski CR et al.; CD39, a 70- to 100-kDa molecule expressed primarily on activated lymphoid cells, was previously shown to mediate B cell homotypic adhesion when ligated with a subset of anti-CD39 mAbs . In the present study, we describe the cloning and molecular characterization of human and murine CD39 . The nucleotide sequence of human CD39 includes an open reading frame encoding a putative 510 amino acid protein with six potential N-linked glycosylation sites, 11 Cys residues, and two potential transmembrane regions . Murine CD39 shares 75% amino acid sequence identity with human CD39 but fails to cross-react with anti-human CD39 mAbs . Although there were no significant similarities with other mammalian genes, considerable homology was found between CD39 and a guanosine diphosphatase from yeast . A series of mouse-human hybrid molecules was constructed to determine the general topology of CD39 and the location of a biologically functional epitope . These findings and supporting evidence from an anti-CD39 mAb-selected phage peptide display library indicate a likely model wherein a short intracellular N-terminus is followed by a large extracellular loop containing the epitope recognized by stimulatory anti-CD39 mAbs, and a short intracellular C terminus . The results demonstrate that CD39 is a novel cell surface glycoprotein with unusual structural characteristics.

Biochem Biophys Res Commun, 1994 Oct 14, 204(1), 224 - 9
Chinifur, a selective inhibitor and "subversive substrate" for Trypanosoma congolense trypanothione reductase; Cenas N et al.; Nitrofurans with aromatic and heterocyclic substituents inhibit Trypanosoma congolense trypanothione reductase (TR) and yeast glutathione reductase (GR), acting as uncompetitive inhibitors vs . NADPH and noncompetitive or uncompetitive inhibitors vs . disulfide substrate . Many of these compounds inhibited trypanothione reductase more efficiently than glutathione reductase . Chinifur (2-{5'-nitro(furo-2'-yl)-ethene-1-yl}-4(N,N-diethylamino)-1-methyl-but-1 -yl - aminocarbonyl-4-quinoline) was the most selective inhibitor of, and free radical-generating substrate for, trypanothione reductase (Ki = 4.5 microns, TN = 3 s-1, TN/Km = 3.2 x 10(4) M-1 s-1), only weakly inhibiting glutathione reductase (Ki = 100 microns) . These findings point to the importance of hydrophobic interactions in the design of redox active heteroaromatic compounds acting as selective inhibitors of, and "subversive substrates" for, trypanothione reductase.

Science, 1994 Oct 14, 266(5183), 288 - 91
Restoration of X-ray resistance and V(D)J recombination in mutant cells by Ku cDNA; Smider V et al.; Three genetic complementation groups of rodent cells are defective for both repair of x-ray-induced double-strand breaks and V(D)J recombination . Cells from one group lack a DNA end-binding activity that is biochemically and antigenically similar to the Ku autoantigen . Transfection of complementary DNA (cDNA) that encoded the 86-kilodalton subunit of Ku rescued these mutant cells for DNA end-binding activity, x-ray resistance, and V(D)J recombination activity . These results establish a role for Ku in DNA repair and recombination . Furthermore, as a component of a DNA-dependent protein kinase, Ku may initiate a signaling pathway induced by DNA damage.

Science, 1994 Oct 14, 266(5183), 280 - 2
Increased recruitment of TATA-binding protein to the promoter by transcriptional activation domains in vivo; Klein C et al.; The rate at which the TATA-binding protein (TBP) interacts with the TATA element and promotes transcription by RNA polymerase II was determined in yeast cells . A TBP derivative with altered TATA-element specificity was rapidly induced, and transcription from promoters with appropriately mutated TATA elements was measured . Without a functional activator protein, basal transcription was observed only after a lag of several hours . In contrast, GCN4-activated transcription occurred rapidly upon induction of the TBP derivative . These results suggest that accessibility of TBP to the chromatin template in vivo is rate limiting and that activation domains increase recruitment of TBP to the promoter.

J Biol Chem, 1994 Oct 14, 269(41), 25483 - 93
L-type voltage-sensitive calcium channel activation stimulates gene expression by a serum response factor-dependent pathway; Misra RP et al.; A mechanism by which calcium-induced signals are transduced to the nucleus to activate transcription of the c-fos proto-oncogene has been characterized . The serum response element (SRE), a region of the c-fos gene which controls growth factor-induced transcription, is now shown to mediate c-fos transcription in response to activation of L-type voltage-sensitive calcium channels . Calcium-dependent transcriptional activation through the SRE is mediated by the serum response factor (SRF) . Membrane depolarization induces phosphorylation of SRF at Ser-103, an event shown to enhance the ability of SRF to bind the SRE . Ca(2+)-induced SRF phosphorylation occurs via a pathway that may involve Ca2+/calmodulin-dependent kinases.

J Biol Chem, 1994 Oct 14, 269(41), 25243 - 6
Activators of protein kinase C stimulate association of Shc and the PEST tyrosine phosphatase; Habib T et al.; Using the yeast two-hybrid system, complementary DNA clones were isolated from a HeLa cell library encoding proteins that interacted with p52shc . One of these clones encoded the non-catalytic, COOH-terminal half of the cytosolic protein tyrosine phosphatase PTP-PEST . Expression of truncated forms of p52shc in the two-hybrid system revealed that the amino-terminal half of p52shc was sufficient for interaction with PTP-PEST . The p52 and p66 forms of Shc, but not the p46 form, bound to a glutathione S-transferase fusion protein containing the region of PTP-PEST isolated from the two-hybrid screen . Similarly, when HeLa cell lysates were immunoprecipitated with PTP-PEST antiserum, p52shc and p66shc proteins, but not p46shc, co-precipitated . Shc-PTP-PEST complex formation was stimulated 6-8-fold by the protein kinase C activator phorbol 12-myristate 13-acetate, while epidermal growth factor and serum had no effect . Phorbol 12-myristate 13-acetate also stimulated phosphorylation of p52shc and p66shc . The muscarinic agonist carbachol (also an activator of protein kinase C) stimulated complex formation 3-5-fold in SH-SY5Y neuroblastoma cells . These results suggest a role for PTP-PEST in G protein receptor signaling and in cross-talk between G protein receptor and tyrosine kinase receptor pathways.

Nature, 1994 Oct 13, 371(6498), 612 - 4
Activation of Raf-1 by 14-3-3 proteins; Fantl WJ et al.; The protein Raf-1, a key mediator of mitogenesis and differentiation, associates with p21ras (refs 1-3) . However, the regulation of the serine/threonine kinase activity of Raf-1 is still not understood . Using the yeast two-hybrid system, we identified two structurally related proteins that interact with the aminoterminal region of Raf-1 . These proteins, 14-3-3 zeta (PLA2) and 14-3-3 beta (HS1), are members of the 14-3-3 family of proteins . Expression of 14-3-3 proteins in Xenopus oocytes enhanced Raf-1 activity and promoted Raf-1-dependent oocyte maturation . A dominant negative mutant of Raf-1 blocked the effects of 14-3-3 protein.

Proc Natl Acad Sci U S A, 1994 Oct 11, 91(21), 9847 - 51
The primer tRNA sequence is not inherited during Ty1 retrotransposition; Lauermann V et al.; Yeast retrotransposon Ty1 transposes through an RNA intermediate by a mechanism resembling retroviral replication . Long terminal repeat retroelements require primers for initiation of reverse transcription . The primer for minus-strand DNA synthesis is the 3' end of a cellular tRNA that base pairs to the complementary region of genomic RNA (the primer binding site) . The genomic RNA of retroviruses and retrotransposons is shorter than its proviral DNA counterpart, lacking complete long terminal repeats . A variety of models have been proposed to describe how complete long terminal repeats are regenerated during reverse transcription . A common feature of these models is the requirement that the 3' portion of the primer tRNA be reverse-transcribed and then utilized in a strand-transfer reaction . We introduced a silent mutation into the Ty1 primer binding site and followed its fate during a single cycle of reverse transcription to directly test this aspect of the reverse transcription model . We demonstrate that the tRNA sequence is not inherited by progeny Ty1 elements during reverse transcription.

Proc Natl Acad Sci U S A, 1994 Oct 11, 91(21), 10212 - 6
Purification of a Ran-interacting protein that is required for protein import into the nucleus; Moore MS et al.; Previously we reported the isolation of two cytosolic fractions (A and B) from Xenopus ovary that are required sequentially to support protein import into the nuclei of digitonin-permeabilized cells . Fraction A is required for recognition of the nuclear localization sequence and targeting to the nuclear envelope, whereas fraction B is required for the subsequent translocation of the bound substrate into the nucleus . The first protein required for fraction B activity to be purified was the small GTPase Ran (ras-related nuclear protein) . Here we report the purification of the second (and final) protein required for fraction B activity . By SDS/PAGE, the purified protein appeared as a single band with an apparent molecular mass of 10 kDa, but the native protein fractionated upon gel filtration chromatography with an apparent size of 30 kDa . Peptide sequence analysis indicated that the purified protein was highly homologous to a previously identified human protein of unknown function called placental protein 15 (pp15) and to the predicted protein product of a yeast open reading frame from Saccharomyces cerevisiae.

Nucleic Acids Res, 1994 Oct 11, 22(20), 4250 - 8
Functional differences between the Oct2 transactivation domains determine the transactivation potential of individual Oct2 isoforms; Annweiler A et al.; The lymphocyte specific transcription factor Oct2 is involved in mediating the B-cell specific transcriptional activity of the octamer motif . Mutational analyses in the context of the complete Oct2 protein had indicated that Oct2 contains two transactivation domains . These two domains appeared to be redundant for activation from a promoter proximal position, whereas stimulation from a remote enhancer position specifically required the C-terminal transactivation domain and an additional B-cell restricted activity . We have generated fusion proteins between the DNA binding domain of the yeast Gal4 transcription factor and individual Oct2 protein domains to analyze their transactivation potential separately . We show that both N- and C-terminal domains can stimulate transcription from a promoter proximal position independently . However, only the C-terminal transactivation domain activates from a distance and it can only do so in B-cells . The C-terminal transactivation domain represents a composite transactivation domain . Whereas removal of just 9 aminoacids from the extreme C-terminus lead to a complete inactivation of this domain deletions from the other side resulted in a gradual loss of activity . We also characterized the transactivation potential of different N-terminal regions of Oct2 generated by alternative splicing . We show that the N-terminus of one of the isoforms, Oct2.3, contains a negative regulatory domain (NRD), which can inactivate the neighbouring glutamine-rich transactivation in cis . The presence of this NRD affects the overall phosphorylation state of the Oct2 protein . This result suggests that the mechanism of inactivation might involve differential protein phosphorylation.

Nucleic Acids Res, 1994 Oct 11, 22(20), 4031 - 8
Strong transcriptional activators isolated from viral DNA by the 'activator trap', a novel selection system in mammalian cells; Gstaiger M et al.; Transcription factors often contain activation domains that interact with the basic transcription machinery . We have developed a functional screening strategy in mammalian cells to selectively isolate activation domains from a library of random DNA inserts . For this, sonicated DNA fragments are cloned next to the DNA binding domain of GAL4 factor in a plasmid that also contains the SV40 origin of replication . Pools of fusion protein clones are transfected into CV-1-5GT monkey cells containing an SV40 T antigen gene under the control of a promoter with GAL4 binding sites . Plasmids that express functional transactivating fusion proteins activate the T antigen gene, thus promoting selective amplification of the plasmid in the mammalian host cell line . Using this method, we were able to select strong enhancer-type activation domains from the immediate early regions of two herpesviruses, namely pseudorabies virus and bovine herpesvirus 1 . In both cases, the activation domains selected were homologues of the ICP4 regulatory protein of herpes simplex virus . The activation domain from pseudorabies virus is four times stronger than the activation domain of herpes simplex virus protein VP16 (Vmw65), making it the strongest activation domain characterized so far . This activator trap method should be useful for precisely localizing activation domain(s) in known factors, or to identify mammalian transcriptional adaptors that do not bind DNA and which may escape conventional detection methods.

Biochem Pharmacol, 1994 Oct 7, 48(7), 1363 - 9
Metabolic interactions of methoxsalen and coumarin in humans and mice; Maenpaa J et al.; Methoxsalen (8-methoxypsoralen) is a very potent inhibitor of human cytochrome P450 2A6 (CYP2A6) and mouse Cyp2a-5-mediated coumarin 7-hydroxylation in vitro . To determine the effect of methoxsalen on coumarin 7-hydroxylation in humans in vivo, five subjects were given 45 mg of methoxsalen and 5 mg of coumarin . Methoxsalen inhibited in vivo coumarin metabolism by 47 +/- 9.2% (mean +/- SEM) . Methoxsalen was metabolized in human liver microsomes at the rate of 50-100 pmol/mg protein/min (approx . 30% of the activity in mouse liver microsomes) . Metabolism was not inhibited by the anti-Cyp2a-5 antibody in human liver microsomes . NIH 3T3 cells stably expressing catalytically active CYP2A6 enzyme did not metabolize methoxsalen, indicating that CYP2A6 does not accept methoxsalen as a substrate . In pyrazole-induced mouse liver microsomes, methoxsalen metabolism was inhibited by the anti-Cyp2a-5 antibody . Cyp2a-5 protein expressed in the yeast Saccharomyces cerevisiae was capable of metabolizing methoxsalen, indicating that methoxsalen is a substrate of Cyp2a-5 . Although kinetic studies indicated that the inhibition of coumarin 7-hydroxylation by methoxsalen is competitive in human liver microsomes, methoxsalen does not appear to be a substrate for CYP2A6 . Methoxsalen and coumarin have the potential of strong metabolic interactions in man.

Science, 1994 Oct 7, 266(5182), 122 - 6
Phosphate-regulated inactivation of the kinase PHO80-PHO85 by the CDK inhibitor PHO81; Schneider KR et al.; A complex consisting of the cyclin-dependent kinase (CDK) PHO85 and the cyclin PHO80 phosphorylates and is thought to inactivate the transcription factor PHO4 when yeast cells are grown in medium containing high concentrations of phosphate . The CDK inhibitor PHO81 inhibits the kinase activity of the PHO80-PHO85 complex when Saccharomyces cerevisiae cells are grown in medium depleted of phosphate . A region of PHO81 with similarity to the mammalian CDK inhibitor p16INK4 is sufficient for inhibition in vitro . These studies demonstrate that CDK inhibitors are used to regulate kinases involved in processes other than cell cycle control and suggest that the ankyrin repeat motif may be commonly used for interaction with cyclin-CDK complexes.

Biochim Biophys Acta, 1994 Oct 6, 1214(3), 288 - 94
Phytanic acid must be activated to phytanoyl-CoA prior to its alpha-oxidation in rat liver peroxisomes; Watkins PA et al.; alpha-Oxidation of the branched-chain fatty acid, phytanic acid, is defective in patients with Refsum's disease, the disorders of peroxisome biogenesis (e.g., Zellweger syndrome), and in rhizomelic chondrodysplasia punctata . 3H-Release from {2,3-3H}phytanic acid, which is impaired in cultured skin fibroblasts from these patients, was investigated in rat liver peroxisomes . Cofactors necessary for optimal 3H-release, ATP, Mg2+, and coenzyme A, were also necessary for optimal acyl-CoA synthetase activity, suggesting that the substrate for 3H-release might be phytanoyl-CoA . 5,8,11,14-Eicosatetraynoic acid (ETYA), an inhibitor of long-chain acyl-CoA synthetase activity, blocked phytanoyl-CoA synthesis as well as 3H-release from {2,3-3H}phytanic acid in a dose-dependent manner . However, this inhibitor had little effect on 3H-release from {2,3-3H}phytanoyl-CoA . Tetradecylglycidic acid (TDGA) inhibited 3H-release from {2,3-3H}phytanic acid in peroxisomal but not in mitochondrial fractions from rat liver . This agent inhibited 3H-release from {2,3-3H}phytanic acid and {2,3-3H}phytanoyl-CoA equally . In contrast to ETYA, which appeared to decrease 3H-release as a consequence of synthetase inhibition, TDGA appeared to act directly on the enzyme catalyzing 3H-release . This enzyme was partially purified from rat liver . The purified enzyme, which did not possess phytanoyl-CoA synthetase activity, catalyzed tritium release from {2,3-3H}phytanoyl-CoA . This enzyme catalyzed 3H-release from {2,3-3H}phytanic acid only if a source of phytanoyl-CoA synthetase was present . We conclude that in rat liver peroxisomes, phytanic acid must be activated to its coenzyme A derivative prior to subsequent alpha-oxidation.

Biochemistry, 1994 Oct 4, 33(39), 11833 - 41
Regulation of cytochrome c oxidase by interaction of ATP at two binding sites, one on subunit VIa; Taanman JW et al.; Cytochrome c oxidase isolated from a wild-type yeast strain and a mutant in which the gene for subunit VIa had been disrupted were used to study the interaction of adenine nucleotides with the enzyme complex . At low ionic strength (25 mM potassium phosphate), in the absence of nucleotides, the cytochrome c oxidase activity of the mutant enzyme lacking subunit VIa was higher than that of the wild-type enzyme . Increasing concentrations of ATP, in the physiological range, enhanced the cytochrome c oxidase activity of the mutant much more than the activity of the wild-type strain, whereas ADP, in the same concentration range, had no significant effect on the activity of the cytochrome c oxidase of either strain . These results indicate an interaction of ATP with subunit VIa in the wild-type enzyme that prevents the stimulation of the activity observed in the mutant enzyme . The stimulation of the mutant enzyme implies the presence of a second ATP binding site on the enzyme . Quantitative titrations with the fluorescent adenine nucleotide analogues 2'(or 3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate (TNP-ATP) and 2'(or 3')-O-(2,4,6-trinitrophenyl)adenosine 5'-diphosphate (TNP-ADP) confirmed the presence of two binding sites for adenine nucleotides per monomer of wild-type cytochrome c oxidase and one binding site per monomer of mutant enzyme . Covalent photolabeling of yeast cytochrome c oxidase with radioactive 2-azido-ATP further confirmed the presence of an ATP binding site on subunit VIa.(ABSTRACT TRUNCATED AT 250 WORDS)

Toxicol Lett, 1994 Oct, 74(1), 15 - 21
Enoximone inhibits hepatic mitochondrial long-chain acyl-CoA synthetase; Youssef J et al.; The phosphodiesterase inhibitor, enoximone, was previously shown to cause paradoxical effects on cardiac lipid metabolism . The present study was undertaken to elucidate the effects of enoximone on the hepatic mitochondrial pathway of fatty acid oxidation . Results presented here show that in isolated rat liver mitochondria, palmitate oxidation was inhibited progressively by increasing concentrations of enoximone . Maximum inhibition (35%) of mitochondrial oxygen uptake was attained at 250 microM enoximone . At this concentration, enoximone did not affect the oxidation of either palmitoyl-CoA or palmitoyl carnitine . Also, enoximone did not inhibit the oxidation of the short-chain fatty acid, hexanoate, neither did it affect the respiratory chain in the mitochondria . These data suggest that enoximone specifically inhibits long-chain acyl-CoA synthetase activity . This was confirmed experimentally when the activity of this enzyme was determined in the absence and presence of enoximone . Discovering inhibitors of specific steps in lipid metabolism should provide a useful tool to investigate mechanisms regulating this pathway.

Oncogene, 1994 Oct, 9(10), 3057 - 61
Evolutionary conservation of the EPS8 gene and its mapping to human chromosome 12q23-q24; Wong WT et al.; We have previously isolated the coding sequence for a novel substrate for tyrosine kinases, eps8, from NIH3T3 fibroblasts . Eps8 was phosphorylated in vivo by several receptor tyrosine kinases (RTKs) and, upon overexpression, was able to enhance EGFR-mediated mitogenic signaling in NIH3T3 cells . To gain understanding of eps8 function as well as its role in normal and neoplastic proliferation, we cloned the human eps8 coding sequence and studied expression of the human RNA and protein, evolutionary conservation, and chromosomal location . In addition to a previously identified SH3 domain, the predicted amino acid sequence of human eps8 revealed a non-random distribution of prolines, clustered in a way to suggest SH3-binding sites and a putative PH domain . Eps8 was expressed in all epithelial and fibroblastic lines examined and in some, but not all, hematopoietic cells . An essential function of eps8 in cell growth regulation was underscored by its conservation during evolution, where eps8-related sequences were detected as early as in Saccharomyces cerevisiae . Finally, the human EPS8 locus was mapped to chromosome 12q23-q24.

J Virol, 1994 Oct, 68(10), 6137 - 46
Visna virus Tat protein: a potent transcription factor with both activator and suppressor domains; Carruth LM et al.; Visna virus is a pathogenic lentivirus of sheep tat is distantly related to the primate lentiviruses, including human immunodeficiency virus type 1 . The visna virus genome encodes a small regulatory protein, Tat, which is necessary for efficient viral replication and enhanced viral transcription . To investigate the mechanism of action of the visna Tat protein and to localize the protein domain(s) responsible for transcriptional activation, chimeric proteins containing visna virus Tat sequences fused to the DNA binding domain of the yeast transactivation factor GAL4 (residues 1 to 147) were made . The GAL4-Tat fusion proteins were transfected into cells and tested for the ability to activate the adenovirus E1b promoter via upstream GAL4 DNA binding sites . Full-length GAL4-Tat fusion proteins were weak transactivators in this system, giving only a two- to fourfold increase in transcription in several cell types, including HeLa and sheep choroid plexus cells . In contrast, fusion of the N-terminal region of the Tat protein to GAL4 revealed a potent activation domain . Amino acids 13 to 38 appeared to be the most critical for activation . No other region of the protein showed any activation in the GAL4 system . This N-terminal region of the visna virus Tat protein has a large number of acidic and hydrophobic residues, suggesting that Tat has an acidic activation domain common to many transcriptional transactivators . Mutations in hydrophobic and bulky aromatic residues dramatically reduced the activity of the chimeric protein . Competition experiments suggest that mechanism of the visna virus Tat activation domain may closely resemble that of the herpesvirus activator VP16 and human immunodeficiency virus Tat, a related lentivirus activator, since both significantly reduce the level of visna virus Tat activation . Finally, a domain between residues 39 and 53 was identified in the Tat protein that, in the GAL4 system, negatively regulates activation by Tat.

Plant Cell, 1994 Oct, 6(10), 1467 - 75
Two classes of homeodomain proteins specify the multiple a mating types of the mushroom Coprinus cinereus; Kues U et al.; The A mating type locus of the mushroom Coprinus cinereus regulates essential steps in sexual development . The locus is complex and contains several functionally redundant, multiallelic genes that encode putative transcription factors . Here, we compare four genes from an A locus designated A42 . Overall, the DNA sequences are very different (approximately 50% homology), but two classes of genes can be distinguished on the basis of a conserved homeodomain motif in their predicted proteins (HD1 and HD2) . Development is postulated to be triggered by an HD1 and an HD2 gene from different A loci . Thus, proteins encoded by genes of the same locus must be distinguished from those encoded by another locus . Individual proteins of both classes recognize each other using the region N-terminal to the homeodomain . These N-terminal specificity regions (COP1 and COP2) are predicted to be helical and are potential dimerization interfaces . The amino acid composition of the C-terminal regions of HD1 proteins suggests a role in activation, and gene truncations indicate that this region is essential for function in vivo . A corresponding C-terminal region in HD2 proteins can be dispensed with in vivo . We will discuss these predicted structural features of the C . cinereus A proteins, their proposed interactions following a compatible cell fusion, and their similarities to the a1 and alpha 2 mating type proteins of the yeast Saccharomyces cerevisiae.

Nippon Rinsho, 1994 Oct, 52(10), 2544 - 9
{Analysis of the gene encoding human PC2, a prohormone processing enzyme}; Ohagi S et al.; Prohormone Convertase 2 (PC2) is a specific endoprotease responsible for the processing of proinsulin to insulin . PC2 is expressed in pancreatic islets, pituitary and brain but is very low or absent in most other tissues, such as liver, spleen and kidney . To evaluate the regulated expression of the human PC2 gene we have analyzed its structure and characterized its promoter . The gene spans > 130 kilobase pairs and consists of 12 exons . Comparison with the structure of the gene encoding human furin, revealed a high degree of conservation of exon-intron junctions . The hPC2 gene was localized to chromosome 20, band p11.2 . The promoter region of the PC2 gene is very G + C rich and contains six potential Sp1 binding sites but no TATA or CAAT box . Analysis of the level of chrolamphenicol acetyltransferase activity with several deletion mutants identified the region from -1100 to -539 from the translation start site as essential for the PC2 promoter activity.

Mol Pharmacol, 1994 Oct, 46(4), 773 - 7
Mutations in the gyrB domain of eukaryotic topoisomerase II can lead to partially dominant resistance to etoposide and amsacrine; Nitiss JL et al.; Anti-topoisomerase II agents represent a major class of anticancer therapeutic agents . Resistance to this class of agents can be mediated by several possible mechanisms . One mechanism may involve mutations in the structural gene(s) for topoisomerases, altering the drug sensitivity of the enzymes . Several mutations have been described in mammalian cell lines that were selected for resistance to topoisomerase II-targeting drugs such as Adriamycin, etoposide, or amsacrine . The difficulty of performing genetic analysis in mammalian cell lines has complicated the determination of whether the observed mutations are responsible for drug resistance . We have reconstructed, in the yeast topoisomerase II gene, the arginine to glutamine mutation at position 450 of human topoisomerase II alpha that was originally identified by Bugg et al . {Proc . Natl . Acad . Sci . USA 88:7654-7658 (1991)} . Mutation of Lys439, the equivalent amino acid in the yeast protein, to either glutamine or glutamic acid confers resistance to etoposide and amsacrine . Interestingly, in diploid yeast cells the heterozygous mutation can still confer partial drug resistance, compared with a diploid strain that is homozygous for wild-type topoisomerase II . Because mutations in the topoisomerase II gene that can confer dominant resistance to anti-topoisomerase II agents are relatively rare, mutations in the gyrB region may be important in the development of clinical drug resistance.

Genes Dev, 1994 Oct 1, 8(19), 2324 - 35
Molecular cloning of the small (gamma) subunit of human TFIIA reveals functions critical for activated transcription; Ozer J et al.; TFIIA is thought to play an important role in transcriptional regulation in higher eukaryotes, but its precise function is unclear . A human cDNA encoding a protein with 45% identity to the small subunit of yeast TFIIA has been isolated . TFIIA activity could be reconstituted by the mixing of recombinant large (alpha beta) and small (gamma) subunits . TFIIA-depleted HeLa nuclear extracts were used to demonstrate that TFIIA is essential for basal and activated transcription by several distinct classes of activators . Recombinant TFIIA functioned in transcriptional activation whether expressed as a dimer (alpha beta+gamma) or as a trimer (alpha+beta+gamma), which closely resembles the native form . Yeast TFIIA also functioned in transcriptional activation, and the human gamma subunit was functionally interchangeable with TOA2, its yeast homolog . Recombinant TFIIA mediated the stimulation of TFIID binding to the TATA region and downstream promoter sequences by the Zta transcriptional activator . Significantly, we found that TFIIA bound directly to Zta in an activation domain-dependent manner . One consequence of the TFIIA-mediated interaction between Zta and TFIID was the formation of a promoter-bound complex resistant to TATA oligonucleotide competition . These results demonstrate that TFIIA is an evolutionarily conserved general factor critical for activator-regulated transcription.

Plant Mol Biol, 1994 Oct, 26(1), 327 - 38
Characterisation of the S-adenosylmethionine decarboxylase (SAMDC) gene of potato; Mad Arif SA et al.; S-adenosylmethionine decarboxylase (SAMDC) is involved in the biosynthesis of the polyamines, spermidine and spermine . Recently, we reported the isolation of a putative cDNA clone of the SAMDC clone of potato (Plant Mol Biol 20; 641-651) . In order to confirm that the potato genes does encode SAMDC, a complementation experiment with a yeast strain that possesses a null mutation in the SAMDC gene was performed . The yeast strain contains a deletion-insertion mutation in the SAMDC gene and has an absolute requirement for the addition of exogenous spermidine for growth . When the full-length potato cDNA was expressed in the mutant yeast strain there was no longer a requirement for exogenous spermidine . Immunoblotting experiments suggest that the potato SAMDC gene product has an apparent molecular mass of 39 kDa . Expression of the SAMDC gene was high in the young and actively dividing tissues and low in the mature and non-dividing tissues of both vegetative and reproductive organs . Additionally, isolation and characterisation of the corresponding genomic clone is reported . The gene has one intron in its 5'-untranslated sequence but otherwise the transcribed portion is identical to the cDNA clone.

Mol Cell Biol, 1994 Oct, 14(10), 7013 - 24
Binding of basal transcription factor TFIIH to the acidic activation domains of VP16 and p53; Xiao H et al.; Acidic transcriptional activation domains function well in both yeast and mammalian cells, and some have been shown to bind the general transcription factors TFIID and TFIIB . We now show that two acidic transactivators, herpes simplex virus VP16 and human p53, directly interact with the multisubunit human general transcription factor TFIIH and its Saccharomyces cerevisiae counterpart, factor b . The VP16- and p53-binding domains in these factors lie in the p62 subunit of TFIIH and in the homologous subunit, TFB1, of factor b . Point mutations in VP16 that reduce its transactivation activity in both yeast and mammalian cells weaken its binding to both yeast and human TFIIH . This suggests that binding of activation domains to TFIIH is an important aspect of transcriptional activation.

Mol Cell Biol, 1994 Oct, 14(10), 6962 - 74
Topoisomerase II forms multimers in vitro: effects of metals, beta-glycerophosphate, and phosphorylation of its C-terminal domain; Vassetzky YS et al.; We present a novel assay for the study of protein-protein interactions involving DNA topoisomerase II . Under various conditions of incubation we observe that topoisomerase II forms complexes at least tetrameric in size, which can be sedimented by centrifugation through glycerol . The multimers are enzymatically active and can be visualized by electron microscopy . Dephosphorylation of topoisomerase II inhibits its multimerization, which can be restored at least partially by rephosphorylation of multiple sites within its 200 C-terminal amino acids by casein kinase II . Truncation of topoisomerase II just upstream of the major phosphoacceptor sites reduces its aggregation, rendering the truncated enzyme insensitive to either kinase treatments or phosphatase treatments . This is consistent with a model in which interactions involving the phosphorylated C-terminal domain of topoisomerase II aid either in chromosome segregation or in chromosome condensation.

Mol Cell Biol, 1994 Oct, 14(10), 6433 - 42
Characterization of an interaction between insulin receptor substrate 1 and the insulin receptor by using the two-hybrid system; O'Neill TJ et al.; Insulin receptor substrate 1 (IRS-1) is a major substrate of the insulin receptor and has been implicated in insulin signaling . Although IRS-1 is thought to interact with the insulin receptor, the nature of the interaction has not been defined . In this study, we used the two-hybrid assay of protein-protein interaction in the yeast Saccharomyces cerevisiae to study the interaction between human IRS-1 and the insulin receptor . We demonstrate that IRS-1 forms a specific complex with the cytoplasmic domain of the insulin receptor when both are expressed as hybrid proteins in yeast cells . We show that the interaction is strictly dependent upon receptor tyrosine kinase activity, since IRS-1 shows no interaction with a kinase-inactive receptor hybrid containing a mutated ATP-binding site . Furthermore, mutation of receptor tyrosine 960 to phenylalanine eliminates IRS-1 interaction in the two-hybrid assay . These data suggest that the interaction between IRS-1 and the receptor is direct and provide evidence that the juxtamembrane domain of the receptor is involved . Furthermore, we show that a 356-amino-acid region encompassed by amino acids 160 through 516 of IRS-1 is sufficient for interaction with the receptor in the two-hybrid assay . Lastly, in agreement with our findings for yeast cells, we show that the insulin receptor is unable to phosphorylate an IRS-1 protein containing a deletion of amino acids 45 to 516 when expressed in COS cells . The two-hybrid assay should provide a facile means by which to pursue a detailed understanding of this interaction.

J Cell Biol, 1994 Oct, 127(2), 357 - 71
Localization of Sed5, a putative vesicle targeting molecule, to the cis-Golgi network involves both its transmembrane and cytoplasmic domains; Banfield DK et al.; The yeast Sed5 protein, which is required for vesicular transport between ER and Golgi complex, is a membrane protein of the syntaxin family . These proteins are thought to provide the specific targets that are recognized by transport vesicles . We have investigated the mechanism by which Sed5 protein is itself localized . Expression of epitope-tagged versions of the yeast, Drosophila and rat Sed5 homologues in COS cells results in a perinuclear distribution; immuno-EM reveals that the majority of the protein is in a tubulo-vesicular compartment on the cis side of the Golgi apparatus . A similar distribution was obtained with a chimeric molecule consisting of a plasma membrane syntaxin with the Drosophila Sed5 transmembrane domain . This indicates that the membrane-spanning domain contains targeting information, as is the case with resident Golgi enzymes . However, alterations to the transmembrane domain of Drosophila Sed5 itself did not result in its mistargeting, implying that an additional targeting mechanism exists which involves only the cytoplasmic part of the protein . This was confirmed by modifying the transmembrane domain of the yeast Sed5 protein: substitution with the corresponding region from the Sso1 protein (a plasma membrane syntaxin homologue) did not affect yeast Sed5 function in vivo.

J Cell Biol, 1994 Oct, 127(2), 319 - 32
nup1 mutants exhibit pleiotropic defects in nuclear pore complex function; Bogerd AM et al.; The NUP1 gene of Saccharomyces cerevisiae encodes one member of a family of nuclear pore complex proteins (nucleoporins) conserved from yeast to vertebrates . We have used mutational analysis to investigate the function of Nup1p . Deletion of either the amino- or carboxy-terminal domain confers a lethal phenotype, but partial truncations at either end affect growth to varying extents . Amino-terminal truncation causes mislocalization and degradation of the mutant protein, suggesting that this domain is required for targeting Nup1p to the nuclear pore complex . Carboxy-terminal mutants are stable but do not have wild-type function, and confer a temperature sensitive phenotype . Both import of nuclear proteins and export of poly(A) RNA are defective at the nonpermissive temperature . In addition, nup1 mutant cells become multinucleate at all temperatures, a phenotype suggestive of a defect in nuclear migration . Tubulin staining revealed that the mitotic spindle appears to be oriented randomly with respect to the bud, in spite of the presence of apparently normal cytoplasmic microtubules connecting one spindle pole body to the bud tip . EM analysis showed that the nuclear envelope forms long projections extending into the cytoplasm, which appear to have detached from the bulk of the nucleus . Our results suggest that Nup1p may be required to retain the structural integrity between the nuclear envelope and an underlying nuclear scaffold, and that this connection is required to allow reorientation of the nucleus in response to cytoskeletal forces.

J Cell Biol, 1994 Oct, 127(2), 303 - 18
ScII: an abundant chromosome scaffold protein is a member of a family of putative ATPases with an unusual predicted tertiary structure; Saitoh N et al.; Here, we describe the cloning and characterization of ScII, the second most abundant protein after topoisomerase II, of the chromosome scaffold fraction to be identified . ScII is structurally related to a protein, Smc1p, previously found to be required for accurate chromosome segregation in Saccharomyces cerevisiae . ScII and the other members of the emerging family of SMC1-like proteins are likely to be novel ATPases, with NTP-binding A and B sites separated by two lengthy regions predicted to form an alpha-helical coiled-coil . Analysis of the ScII B site predicted that ScII might use ATP by a mechanism similar to the bacterial recN DNA repair and recombination enzyme . ScII is a mitosis-specific scaffold protein that colocalizes with topoisomerase II in mitotic chromosomes . However, ScII appears not to be associated with the interphase nuclear matrix . ScII might thus play a role in mitotic processes such as chromosome condensation or sister chromatid disjunction, both of which have been previously shown to involve topoisomerase II.

Hepatology, 1994 Oct, 20(4 Pt 1), 975 - 83
Heat shock response in the liver: expression and regulation of the hsp70 gene family and early response genes after in vivo hyperthermia; Schiaffonati L et al.; Heat shock response in cultured cells has been studied extensively; however few data are available on heat shock response in an intact organ of a living animal . In this study we analyzed the kinetics of expression of the heat shock protein 70 gene family (heat shock protein 70, heat shock cognate protein 73 and glucose-regulated protein 78) in the liver of the thermally stressed rat . New synthesis of heat shock protein 70 and heat shock cognate protein 73 was shown in liver slices pulse labeled in vitro with 35S-methionine . Accumulation of heat shock protein 70 and heat shock cognate protein 73 proteins was shown in total cellular extracts . 32P-labeled complementary DNA probes encoding heat shock protein 70, heat shock cognate protein 73 and glucose-regulated protein 78 were used to show that the levels of the corresponding messenger RNAs increase as a fraction of total RNA and in polysomes at different extents and with different kinetics . The induction of heat shock protein 70 and heat shock cognate protein 73 messenger RNAs reflected the increase in the synthesis of the corresponding proteins . Run-on transcription analysis indicated that the expression of heat shock protein 70 and heat shock cognate protein 73 genes was mainly regulated at the transcriptional level . On the contrary, both transcriptional and posttranscriptional regulatory mechanisms can explain the induction of the glucose-regulated protein 78 gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Eur J Biochem, 1994 Oct 1, 225(1), 305 - 10
ATPase strongly bound to higher eukaryotic ribosomes; Rodnina MV et al.; 80 S ribosomes from a number of higher eukaryotic organisms are able to hydrolyse ATP and GTP without the addition of soluble protein factors . ATPase seems to be an intrinsic activity of the ribosome, as indicated by the findings that ATPase activity is not diminished upon dissociation of ribosomes and reassociation of subunits, by washing with 0.66 M (KCl + NH4Cl) or 0.6 M LiCl treatment and ethanol precipitation; 1.5 M LiCl treatment removes only 40% ATPase activity . 80 S ribosomes are able to bind a variety of NTPs, NDPs and NTP analogues, with a preference for ATP . Effective inhibitors of the ribosomal ATPase are ammonium metavanadate and alcaloid emetine . The ATPase activity is present on both ribosomal subunits, which may reflect the existence of two catalytical sites for ATP on the 80 S ribosome . Ribosomal ATPase is stimulated by the occupancy of the A site, in particular with charged tRNA . The ATPase inhibitor adenylylimidodiphosphate almost completely prevents elongation-factor(EF)-1-dependent binding of Phe-tRNA(Phe) to the A site . The hydrolysis of ATP, therefore, is likely to be involved in the mechanism of tRNA binding to the A site of the 80 S ribosome . As far as wide substrate specificity and possible participation in tRNA interaction with the ribosome are concerned, the ribosomal ATPase seems to be similar to EF-3 found in fungi . A synergism in ATPase activities of yeast EF-3 and rabbit liver ribosomes at high ATP concentration and certain ribosome/EF-3 ratios have been observed . Rabbit liver ribosomes seem to stimulate the ATPase activity of yeast EF-3 similar to the mechanism in yeast ribosomes, though less efficiently.

J Biochem (Tokyo), 1994 Oct, 116(4), 752 - 8
Divalent metal ion-dependent mitochondrial degradation of unassembled subunits 2 and 3 of cytochrome c oxidase; Nakai T et al.; Intracellular protein degradation plays an important role in maintaining the stoichiometry of the different subunits of an oligomeric enzyme . In a Saccharomyces cerevisiae mutant defective in cytochrome c oxidase subunit 4 encoded in nuclear DNA, mitochondrial-encoded subunits 2 and 3 cannot assemble normally {Dowhan et al . (1985) EMBO J . 4, 179-184} . In this study, we show that those unassembled forms of subunits 2 and 3 in this strain are eliminated rapidly by degradation . Reduction of the intracellular ATP level by inhibiting the glycolytic pathway, or inhibition of the entry of ATP into mitochondria by bongkrekic acid, both of which are expected to reduce the intramitochondrial ATP level in respiratory-deficient cells such as WD1, significantly suppressed the degradation, suggesting that the degradation requires intramitochondrial ATP . The degradation was also inhibited by o-phenanthroline, a membrane-permeable metal chelator, and this inhibitory effect was suppressed by addition of an excess amount of Co2+, Mn2+, or Zn2+, but not by Ca2+ or Mg2+, suggesting a novel metal-dependence of the degradation of unassembled Cox II and Cox III which has not been reported previously for mitochondrial metabolic protein degradation systems . A potential advantage of using this strain for identifying the factor(s) involved by a genetical approach is discussed.

Mol Biol Cell, 1994 Oct, 5(10), 1129 - 43
Inhibition of endoplasmic reticulum (ER)-to-Golgi transport induces relocalization of binding protein (BiP) within the ER to form the BiP bodies; Nishikawa S et al.; Immunofluorescence staining of yeast cells with anti-binding protein (BiP) antibodies shows uniform staining of the endoplasmic reticulum (ER) . We have found that overproduction of Sec12p, an ER membrane protein, causes a change of BiP distribution within the cell . Upon induction of Sec12p by the GAL1 promoter, the staining pattern of BiP turns into bright dots scattering in the cell, whereas the staining of Sec12p remains to be the typical ER figure . Overproduction of other ER membrane proteins, HMG-CoA reductase or Sed4 protein, does not induce such relocalization of BiP . Pulse-chase experiments and electron microscopy have revealed that the overproduction of Sec12p inhibits protein transport from the ER to the Golgi apparatus . When the transport is arrested by one of the sec mutations that block the ER-to-Golgi step at the restrictive temperature, the BiP staining also changes into the punctate pattern . In contrast, the sec mutants that block later or earlier steps of the secretory pathway do not induce such change of BiP localization . These observations indicate that relocalization of BiP is caused by the inhibition of ER-to-Golgi transport . Using immunoelectron microscopy, we have found that the punctate staining is because of the accumulation of BiP in the restricted region of the ER, which we propose to call the "BiP body." This implicates existence of ER subdomains in yeast . A vacuolar protein, proteinase A, appears to colocalize in the BiP body when the ER-to-Golgi transport is blocked, suggesting that the BiP body may have a role as the site of accumulation of cargo molecules before exit from the ER.

Genomics, 1994 Oct, 23(3), 566 - 74
Molecular cloning of murine pig-a, a gene for GPI-anchor biosynthesis, and demonstration of interspecies conservation of its structure, function, and genetic locus; Kawagoe K et al.; Many membrane proteins are anchored to the cell membrane by glycosylphosphatidylinositol (GPI) . The core structure and biosynthesis of the GPI anchor are well conserved in eukaryote cells . We previously cloned a human PIGA gene that participates in GPI anchor biosynthesis . We have now cloned complementary and genomic DNA of Pig-a, the murine homologue of PIGA, and compared its function and gene structure with those of PIGA . The deduced amino acid sequence of mouse PIG-A is 88% identical with that of human PIG-A . Transfection of Pig-a cDNA complemented the defects of both a PIG-A-deficient murine cell line and a PIG-A-deficient human cell line, demonstrating that functions of mouse and human PIG-A are conserved . Like human PIGA, the chromosomal Pig-a gene has six exons and spans approximately 16 kb . Moreover, Pig-a was mapped to X-F3/4, which is syntenic to human Xp22.1, where PIGA is located . Thus, murine Pig-a provides a good animal model to study paroxysmal nocturnal hemoglobinuria, a disease caused by a somatic mutation of PIGA . Database analysis demonstrated that a yeast gene, SPT14, is homologous to Pig-a and PIGA and that these genes are members of a glycosyltransferase gene family.

Curr Biol, 1994 Oct 1, 4(10), 907 - 10
Cell division . Septins in common?
Sanders SL, Field CM.
Two apparently quite distinct processes, cytokinesis in animal cells and in budding yeast cells, have been shown to involve proteins of the same family, the septins, suggesting that the two may not be so different after all.

Hum Mol Genet, 1994 Oct, 3(10), 1783 - 8
Structural and mutational analysis of the xeroderma pigmentosum group D (XPD) gene; Frederick GD et al.; Individuals affected by the autosomal recessive disease xeroderma pigmentosum (XP) are acutely sensitive to sunlight and predisposed to skin cancer on exposed areas . Cells cultured from XP patients are both UV sensitive and defective in the nucleotide excision repair of damaged DNA . These cellular phenotypes are amenable to experimental strategies employing complementation, an approach previously used to demonstrate the correction of XP-D phenotypes following the introduction of the XPD (ERCC2) gene . In the present study, we have characterized the genomic organization of the XPD (ERCC2) gene and found it to be comprised of 23 exons . These data were helpful in evaluating the functional integrity of alleles in two XP-D cell lines . In cell line GM436 a C-->G transversion was found at nucleotide position 1411 in the XPD (ERCC2) cDNA, a change expected to result in a Leu461Val substitution . Cell line XP67MA carries a C-->T transition in genomic DNA at nucleotide position 2176 in exon 22, introducing the termination codon TAG at amino acid 726 . The latter would be expected to produce a protein truncated by 34 amino acids . Although expression of the normal XPD cDNA could be shown to correct the UV sensitivity phenotype in XP-D cells, cDNA constructs bearing either of the two mutations failed to yield complementation . These results confirm the role of ERCC2 in XP-D and illustrate the power of utilizing cellular phenotypes to evaluate the significance of single nucleotide substitutions.

Proteins, 1994 Oct, 20(2), 105 - 23
Helix packing in proteins: prediction and energetic analysis of dimeric, trimeric, and tetrameric GCN4 coiled coil structures; DeLano WL et al.; A simulated annealing method for atomic resolution structure prediction of alpha-helical coiled coil proteins is described which draws upon knowledge of the oligomerization state, the helix directionality, and the properties of heptad repeat sequences . Unknown structural parameters, such as the coiled coil twist angle and the side chain conformations, are heavily sampled while allowing for flexibility in the helix backbone geometry . Structures of the wild-type GCN4 dimer {O'Shea et al., Science 254:539-544, 1991} and a mutant tetramer {Harbury et al., Science 292:1401-1407, 1993} have been generated and compared with the X-ray crystal structures . The wild-type dimer model has a root mean square coordinate deviation from the crystal structure of 0.73 A for nonhydrogen atoms in the dimerization interface . Structures of a mutant dimer and a mutant trimer have been predicted . Packing energetics were analyzed for core leucine and isoleucine side chains in dimeric and tetrameric coiled coils . Strong packing preferences were found in the dimers but not in the tetramers . Thus, packing in the dimer may be responsible for the switch from a two-stranded to a four-stranded coiled coil caused by the GCN4 leucine zipper mutations.

Z Lebensm Unters Forsch, 1994 Oct, 199(4), 270 - 4
{The decomposition of Maillard reaction products by amylolytic enzymes . 1 . Reversible inhibition of alpha- and glucoamylase and alpha-glucosidase by oligosaccharide Amidori compounds}; Schumacher D et al.; The influence of Amadori-compounds (fructosyl-, maltulosyl- and maltotriulosylglycin) on the activity of the enzymes alpha-glucosidase (from Saccharomyces cerevisiae), glucoamylase (from Aspergillus niger) and alpha-amylase (from porcine pancreas) was studied . Fructosylglycin was not hydrolyzed by all three enzymes . alpha-Glucosidase hydrolyzes maltulosylglycin 10 times slower than maltotriulosylglycin . Glucoamylase and alpha-amylase catalyze only the cleavage of maltotriulosylglycin to form glucose and maltulosylglycin . The activities of alpha-glucosidase and glucoamylase are inhibited through the Amadori-compounds fructosyl- and maltulosylglycin . These Amadori-compounds don't influence the activity of alpha-amylase . Electronic effects or interactions between the secondary amino function of Amadori-compounds and the carboxyl- or carboxylate groups of active centres could be responsible for such an inhibition.

Mol Microbiol, 1994 Oct, 14(2), 323 - 34
Overexpression of flbA, an early regulator of Aspergillus asexual sporulation, leads to activation of brlA and premature initiation of development; Lee BN et al.; Aspergillus nidulans reproduces asexually by forming thousands of mitotically derived spores atop highly specialized multicellular organs termed conidiophores . We have identified a gene called flbA (for fluffy low brlA expression) that is required for initiation of A . nidulans conidiophore development . flbA mutants form abnormal colonies that have a distinct fluffy phenotype characterized by tightly interwoven aerial hyphae that autolyse as the colony matures . The requirement for flbA in conidiophore development precedes activation of brlA, a primary regulator of conidiophore development . The wild-type flbA gene was isolated and found to encode a 3.0 kb mRNA that is expressed throughout the A . nidulans asexual life cycle . Overexpression of flbA using an inducible promoter resulted in misscheduled expression of brlA in vegetative cells and caused hyphal tips to differentiate into spore-producing structures . Sequence analysis of a nearly full-length flbA cDNA clone showed that flbA is predicted to encode a 717-amino-acid polypeptide with 30% identity to the Saccharomyces cerevisiae SST2 protein . SST2 is required by yeast cells for resuming growth following prolonged exposure to yeast mating pheromone and for mating partner discrimination . We propose that flbA plays a related role in a signalling pathway for Aspergillus conidiophore development.

Mol Reprod Dev, 1994 Oct, 39(2), 215 - 25
Basal components of the transcription apparatus (RNA polymerase II, TATA-binding protein) contain activation domains: is the repetitive C-terminal domain (CTD) of RNA polymerase II a "portable enhancer domain"?
Seipel K, Georgiev O, Gerber HP, Schaffner W.
Regions rich in serine, threonine, and proline residues can be found in transcriptional activation domains, as well as in the N-terminal parts of mammalian TATA-binding proteins, where they are interrupted by polyglutamine stretches . Likewise, the C-terminal domain of the largest subunit of RNA polymerase II contains multiple repeats of the consensus heptapeptide sequence YSPTSPS . To test directly for possible activation functions, we fused the GAL4 DNA-binding domain to the N-terminal domain of human TBP or subdomains of it, and to the C-terminal domain (CTD) of mouse RNA polymerase II or synthetic polymers of a CTD consensus repeat . We found that these chimeric proteins were able to activate transcription when bound to a GAL4 site in front of the TATA box, a function characteristic of transcription factors . However, while subdomains of TBP functioned only from a position close to the TATA box ("promoter" position), multiple repeats of the CTD consensus sequence were also able to mediate transcriptional activation from a remote ("enhancer") position . Our findings suggest that a region of TBP that is unique to mammals functionally cooperates with "proximal" activation domains of promoter-bound transcription factors . They also imply that the C-terminal domain of RNA polymerase II includes a function that is otherwise confined to remote activation domains of enhancer-bound transcription factors . We suggest that the CTD of RNA polymerase II contains a "portable" remote activation domain that may also facilitate chromatin opening within the transcription unit.

Trends Biochem Sci, 1994 Oct, 19(10), 409 - 14
Translational control of GCN4: an in vivo barometer of initiation-factor activity; Hinnebusch AG; Phosphorylation of translation initiation factor-2 (eIF-2) is an adaptive mechanism for downregulating protein synthesis under conditions of starvation and stress . The yeast Saccharomyces has evolved a sophisticated means of increasing translation of GCN4 mRNA when eIF-2 is phosphorylated, allowing the induction of an important stress-response protein when expression of most other genes is decreasing . Because translation of GCN4 mRNA is so tightly coupled to eIF-2 activity, genetic analysis of this system has provided unexpected insights into the regulation of eIF-2 and its guanine nucleotide exchange factor, eIF-2B.

Biosci Biotechnol Biochem, 1994 Oct, 58(10), 1828 - 35
Isolation and characterization of two chitin synthase genes from Aspergillus nidulans; Yanai K et al.; Two chitin synthase genes, designated chsA and chsB, were isolated from Aspergillus nidulans with the Saccharomyces cerevisiae CHS2 gene as the hybridization probe . Nucleotide sequencing showed that chsA and chsB encoded polypeptides consisting of 1013 and 916 amino acid residues, respectively; the hydropathy profiles of the enzymes were similar to those of other fungal chitin synthases . Northern analysis indicated that both genes were transcribed, suggesting that cellular chitin in A . nidulans is synthesized by at least two chitin synthases . For examination of the roles of the chitin synthase genes in cell growth, gene disruption experiments were done . The chsA disruptant grew as well as the wild-type strain, but the chsB disruptant had severe growth defects that could not be overcome by the addition of 1.2M sorbitol as an osmotic stabilizer . These findings suggested that chsB but not chsA is essential for hyphal growth.

Curr Opin Biotechnol, 1994 Oct, 5(5), 511 - 5
Regulation of protein function through expression of chimaeric proteins; Picard D; The hormone-binding domain of steroid receptors can be used to subject heterologous protein functions to hormonal control in cis . Recent studies have established that these regulatory domains constitute a set of up to five different reversible molecular switches for the post-translational regulation of a wide variety of cytoplasmic and nuclear proteins, including kinases . This approach has been applied both in vertebrate cells and in budding yeast.

Nat Struct Biol, 1994 Oct, 1(10), 681 - 90
Parallel evolution in two homologues of phosphorylase; Rath VL et al.; The structure of the unphosphorylated, inactive form of yeast glycogen phosphorylase has been determined to a resolution of 2.6 A . The structure is similar to the phosphorylated, active form of muscle phosphorylase in the orientations of the subunits and catalytic residues, but resembles the inactive muscle enzyme in the closed, or substrate excluding, orientation of the two domains . Part of the unique yeast amino-terminal extension of 40 residues binds near the catalytic site of the second subunit in the homodimer, preventing the domain movement required for substrate access . Phosphorylation may displace the amino terminus from the active site, allowing the domains to separate.

Glycoconj J, 1994 Oct, 11(5), 469 - 76
Does an animal peptide: N-glycanase have the dual role as an enzyme and a carbohydrate-binding protein?
Suzuki T, Kitajima K, Inoue S, Inoue Y.
Recently, we have reported purification and characterization of a de-N-glycosylating enzyme, peptide: N-glycanase (PNGase) found in C3H mouse fibroblast L-929 cells, and designated L-929 PNGase {Suzuki T, Seko A, Kitajima K, Inoue Y, Inoue S (1994) J Biol Chem 269, 17611-18} . The unique properties of L-929 PNGase are that the enzyme had a high affinity to the substrate glycopeptide (e.g . Km = 114 microM for fetuin derived glycopentapeptide) and that the PNGase-catalysed reaction is strongly inhibited by the released free oligosaccharides but not by the free peptides formed, suggesting that L-929 PNGase is able to bind to a certain type of carbohydrate chain . In this study, we report the new findings of the mannan-binding property of L-929 PNGase: the de-N-glycosylating enzyme activity of L-929 PNGase was inhibited by yeast mannan and triomannose, Man alpha 1-->3(Man alpha 1-->6)Man, but not by mannose and alpha-methyl-D-mannoside . Furthermore, L-929 PNGase was revealed to bind to the glycan moiety of yeast mannan by using mannan-conjugated Sepharose 4B gel as a ligand, suggesting that L-929 PNGase could serve not only as an enzyme but also as a carbohydrate recognition protein in vivo . Such 'dual' properties found for animal-derived L-929 PNGase are unique and are not shared with other previously characterized plant- and bacterial-origin PNGases--PNGase A and PNGase F, respectively.

Mol Cell Biol, 1994 Oct, 14(10), 6540 - 51
Efficient homologous recombination of Ty1 element cDNA when integration is blocked; Sharon G et al.; Integration of the yeast retrotransposon Ty1 into the genome requires the self-encoded integrase (IN) protein and specific terminal nucleotides present on full-length Ty1 cDNA . Ty1 mutants with defects in IN, the conserved termini of Ty1 cDNA, or priming plus-strand DNA synthesis, however, were still able to efficiently insert into the genome when the elements were expressed from the GAL1 promoter present on a multicopy plasmid . As with normal transposition, formation of the exceptional insertions required an RNA intermediate, Ty1 reverse transcriptase, and Ty1 protease . In contrast to Ty1 transposition, at least 70% of the chromosomal insertions consisted of complex multimeric Ty1 elements . Ty1 cDNA was transferred to the inducing plasmid as well as to the genome, and transfer required the recombination and repair gene RAD52 . Furthermore, multimeric insertions occurred without altering the levels of total Ty1 RNA, virus-like particle-associated RNA or cDNA, Ty1 capsid proteins, or IN . These results suggest that Ty1 cDNA is utilized much more efficiently for homologous recombination when IN-mediated integration is blocked.

Mol Carcinog, 1994 Oct, 11(2), 59 - 64
Evidence for a p53-independent pathway for upregulation of SDI1/CIP1/WAF1/p21 RNA in human cells; Johnson M et al.; SDI1 is an inhibitor of DNA synthesis that we isolated by expression screening cDNAs prepared from senescent, terminally nondividing human cells . Other groups then cloned this gene as a cyclin-dependent kinase (cdk)-interacting protein (CIP1, p21) that inhibits cdks; the gene was also isolated by screening for genes transactivated by p53 (WAF1) . p53 levels are low in senescent and quiescent contact-inhibited or serum-deprived normal human cells, which we have found express high levels of SDI1 mRNA . This indicates that alternate pathways for upregulation of message level of this gene may exist . We therefore proceeded with the study presented here, treating human cells with a variety of growth-arrest-inducing agents, including some that damaged DNA, and found that RNA levels of SDI1 were increased in all cases that resulted in growth inhibition . More important, with the exception of gamma-radiation, most of these agents were able to elevate SDI1 message levels in cells lacking wild-type p53 . At least two distinct kinetic profiles for RNA induction were observed, one that implicated p53 transactivation and occurred early enough to cause arrest, and another that clearly was p53 independent and suggested a role for the SDI1 gene product in the maintenance rather than in the cause of inhibition of DNA synthesis.

Biochem Biophys Res Commun, 1994 Sep 30, 203(3), 1668 - 74
Glutathione peroxidase and glutathione reductase activities towards glutathione-derived antioxidants; Gaullier JM et al.; A new class of glutathione derivatives with antioxidant properties has been prepared by transformation of the NH2 group into a pyrrole ring with various substitutions at the 2 and 5 positions . Due to steric hindrance and/or hydrophobicity of the 2-5-disubstituted pyrrole ring, the reduced glutathione derivatives are poor substrates of the glutathione peroxidase and do not effectively compete with GSH . The oxidized glutathione derivatives are, in turn, relatively good substrates (Km = 1.5 mM) of the glutathione reductase as compared to natural oxidized glutathione (Km = 0.51 mM) but are not effective competitors of the enzyme . It can be considered that the new glutathione derivatives do not strongly interfere with the natural enzymatic defence against fatty acid hydroperoxides formed during an oxidative stress.

Biochem Biophys Res Commun, 1994 Sep 30, 203(3), 1589 - 98
Nuclear localization of p185neu tyrosine kinase and its association with transcriptional transactivation; Xie Y et al.; The rat neu protooncogene encodes a 185 kD transmembrane protein (p185neu), which is a member of the epidermal growth factor receptor (EGFr) family . In searching for the signaling transducer of p185neu by using a two-hybrid selection system, we found, surprisingly, that the cytoplasmic domain of p185neu, when fused to the DNA-binding domain of GAL4 (amino acids 1-147), functioned as a transcriptional activator . We subsequently observed nuclear localization of p185neu . Interestingly, nuclear p185neu has a much higher extent of tyrosine phosphorylation than its nonnuclear counterpart . Our results suggest that a transmembrane receptor tyrosine kinase may enter the nucleus and be involved in transcriptional activation . This novel finding unveils a clue in the understanding of the mechanism of receptor tyrosine kinase-mediated signal transduction.

Proc Natl Acad Sci U S A, 1994 Sep 27, 91(20), 9500 - 4
Cloning and characterization of a Neurospora crassa gene required for (1,3) beta-glucan synthase activity and cell wall formation; Enderlin CS et al.; The glucan synthase 1 gene (gs-1) is required for (1,3) beta-glucan synthase activity {E.C . 2.4.1.34; UDP glucose:1,3-beta-D-glucan 3-beta-D-glucosyltransferase} and for cell wall formation . The gs-1 gene was cloned by functional complementation of the cell-wall-less defect of the (1,3) beta-glucan synthase-deficient mutant, TM1, by using a genomic Neurospora crassa cosmid library . A 2568-nucleotide gs-1 cDNA sequence revealed a 532-amino acid open reading frame encoding a polypeptide of 59 kDa . The predicted gs-1 gene product has no obvious signal peptide cleavage sites or transmembrane domains . A gs-1 null mutant is defective for cell wall formation and (1,3) beta-glucan synthase activity . The predicted GS-1 protein is weakly homologous to a putative Saccharomyces cerevisiae transcriptional regulatory protein.

Biochim Biophys Acta, 1994 Sep 27, 1187(3), 289 - 95
Circular dichroism studies of the binding of mammalian and non-mammalian cytochromes c to cytochrome c oxidase, cytochrome c peroxidase, and polyanions; Garber EA et al.; The effects of binding of Candida krusei, Drosophila melanogaster, horse, human, and rat cytochromes c to beef cytochrome c oxidase (ferrocytochrome c: oxygen oxidoreductase, EC 1.9.3.1) and yeast cytochrome c peroxidase (ferricytochrome c: hydrogen-peroxide oxidoreductase, EC 1.11.1.5) on their circular dichroism spectra were determined . The binding to cytochrome oxidase results in a positive increase in the ellipticities of the positive and negative Cotton effects at 404 nm and 417 nm of cytochrome c . The horse, human, and rat cytochromes c display less of an increase in the ellipticity of the positive Cotton effect at 404 nm, but more of a positive change in the negative Cotton effect at 417 nm than the C . krusei or D . melanogaster proteins . Interaction with yeast cytochrome c peroxidase elicits only a positive change in the ellipticity of the positive Cotton effect at 404 nm . No significant change is observed in the negative Cotton effect at 417 nm . Rat cytochrome c variants with a phenylalanine in place of tyrosine-67 and/or an alanine in place of proline-30 all display circular dichroism spectral changes upon binding to cytochrome c oxidase or cytochrome c peroxidase identical to those of the unaltered protein . The increase in ellipticity at 404 nm upon binding occurs even though replacement of tyrosine-67 results in the loss of the positive Cotton effect at this position . Polyglutamate and phosvitin complexes of cytochrome c show changes in the circular dichroism spectrum similar to those observed with cytochrome c peroxidase . However, the magnitudes of the spectral changes were considerably less . A model is proposed in which the main cause of the circular dichroism spectral changes observed upon complexation arise from the exclusion of solvent from the exposed front heme edge . According to this model, the exclusion of solvent changes the relative asymmetry of the environment of the electronic transitions of the heme prosthetic group of cytochrome c, resulting in observed circular dichroic effects.

Nucleic Acids Res, 1994 Sep 25, 22(19), 3983 - 9
Plant activating sequences: positively charged peptides are functional as transcriptional activation domains; Estruch JJ et al.; Plant sequences that act as transcriptional activation domains in yeast as well as in plants have been isolated by genetic selection in yeast . The selection was based on the reconstitution of a functional GAL4 transcriptional activator . Since the peptides show no homology with reported activation domains, they represent a new class of activating sequences . The sequence P1, which is 10 amino acids long, is the shortest functional activation domain reported . A cDNA that encodes the P14 class (peptides P14-P18) activating sequence have been cloned . The protein exhibits strong homology (higher than 50% amino acid identity) with the BBC1-related sequences, a highly conserved family of basic proteins containing nuclear localization signals . The P14 and P15 peptides are the most effective plant activating sequences . The P14 and P15 peptides are highly hydrophilic, positively charged and mostly unstructured . These properties are at odds with the ones usually found in known activation domains.

Nucleic Acids Res, 1994 Sep 25, 22(19), 3918 - 24
Production and characterization of recombinant human Ku antigen; Ono M et al.; Ku is an ubiquitous nuclear heterodimeric protein consisting of p70 and p86 subunits that binds double-stranded DNA termini and associates with chromosomes in vivo . It was originally described as an autoantigen in patients with certain autoimmune diseases . The individual subunits of Ku have been difficult to isolate from human cells without denaturation and attempts to produce functional recombinant Ku have been largely unsuccessful . Here, we utilize two recombinant baculoviral vectors that carry p70 or p86 cDNA and express the Ku subunits individually as well as assemble them into the complete Ku heterodimer . In an electrophoretic mobility shift assay, recombinant Ku binds to linear double-stranded DNA but not to supercoiled, nicked circular, nor linear single-stranded DNA . Neither subunit binds DNA by itself indicating that heterodimerization is essential for function . We also describe a simple purification method for the isolation of highly purified recombinant Ku using a hexahistidine tag . The baculovirus expression system provides a stable and efficient source of not only the p70 and p86 subunits but also the functional Ku heterodimer.

Cell, 1994 Sep 23, 78(6), 949 - 61
Membrane targeting of the nucleotide exchange factor Sos is sufficient for activating the Ras signaling pathway; Aronheim A et al.; Activation of growth factor receptors results in tyrosine autophosphorylation and recruitment of SH2 domain-containing effectors, including Grb2 . Grb2 recruitment mediates activation of the Ras nucleotide exchanger Sos by an unknown mechanism . To examine the role of membrane recruitment, we prepared Sos derivatives containing either myristoylation or farnesylation signals . This resulted in plasma membrane targeting of Sos and stimulation of the Ras signaling pathway, including ERK and AP-1 activities leading to oncogenic transformation . Sos derivatives with nonfunctional myristoylation or farnesylation sequences were inactive . Farnesylation of Sos also activated Ras signaling in yeast . In both mammalian cells and yeast, membrane-targeted Sos derivatives lacking the C-terminal region were considerably more active . Therefore, targeting of Sos to the plasma membrane in the vicinity of Ras appears to be the primary mechanism leading to activation of the Ras pathway . A secondary mechanism could involve relief of the inhibitory effect of the Sos C-terminal region.

Cell, 1994 Sep 23, 78(6), 937 - 48
A rab protein is required for the assembly of SNARE complexes in the docking of transport vesicles; Sogaard M et al.; Rab proteins are generally required for transport vesicle docking . We have exploited yeast secretion mutants to demonstrate that a rab protein is required for v-SNAREs and t-SNAREs to assemble . The absence of the rab protein in the docking complex suggests that, in a broad sense, rab proteins participate in a reaction catalyzing SNARE complex assembly . In so doing, rab proteins could help impart an additional layer of specificity to vesicle docking . This mechanism likely involves the Sec1 homolog Sly1, which we identified in isolated docking complexes . We also report the identification of a novel v-SNARE (Ykt6p) component of the yeast ER-Golgi docking complex that has a CAAX box and is predicted to be lipid anchored . The surprising finding that docking complexes can contain many distinct species of SNAREs (Sed5p, Bos1p, Sec22p, Ykt6p, and likely Bet1p, p28, and p14) suggests that multimeric interactions are features of the fusion machinery, and may also improve the fidelity of vesicle targeting.

Biochim Biophys Acta, 1994 Sep 21, 1208(1), 127 - 35
Small molecule probes of glyoxalase I and glyoxalase II; Barnard JF et al.; A number of synthetic tropolones and hydrophobic S-blocked glutathione analogues were investigated as potential inhibitors of glyoxalase I from Saccharomyces cerevisiae and glyoxalase II from bovine liver . Several tropolones containing a free C-2 hydroxy group were found to be potent inhibitors of glyoxalase I, whereas the glutathione conjugates were found to be modest to poor inhibitors of this enzyme . Most tropolones and glutathione conjugates, except 5-p-tolylazotropolone and S-carbobenzoxy-L-glutathione, were found to be poor inhibitors of glyoxalase II . A recent report on an extremely active glyoxalase system from Plasmodium falciparum suggested that several of the more potent inhibitors may have antimalarial properties . A number of these compounds in fact, exhibited antimalarial activity in the low micromolar range . Further studies are required to fully elucidate the mechanism(s) of the antimalarial properties of these compounds.

Biochemistry, 1994 Sep 20, 33(37), 11150 - 7
Membrane location of spin-labeled apocytochrome c and cytochrome c determined by paramagnetic relaxation agents; Snel MM et al.; The mitochondrial precursor protein horse heart apocytochrome c was spin-labeled on the cysteine residue at position 14 or 17 in the N-terminal region, and the mature protein yeast cytochrome c was similarly labeled on the single free cysteine residue at position 102 at the C-terminal . The proteins were bound to negatively charged phospholipid bilayers, and the accessibility of the spin-labeled cysteine residues to lipid-soluble molecular oxygen and to the lipid-impermeant chromium oxalate anion was determined from the saturation properties of the ESR spectra . Binding of the protein was found to have a considerable effect on the local oxygen concentrations within the lipid bilayer . The accessibilities of the spin-labeled proteins relative to those obtained for phospholipids spin-labeled either in the headgroup or at positions in the sn-2 acyl chain, in the presence of unlabeled protein, identify the position of the spin-labeled cysteine residues in the phospholipid bilayer . The spin label on apocytochrome c bound to phosphatidylglycerol bilayers lies between the 5- and 14-C positions of the lipid acyl chain . Admixture of > or = 75 mol % phosphatidylcholine induces an additional surface-associated apocytochrome c population . The spin label on native and heat-denatured cytochrome c is located at the membrane surface . These different extents of membrane penetration correlate also with the reduction in local oxygen concentration experienced by spin-labeled phospholipids on binding of apo- and holocytochrome c . The possible biological implications of the data are discussed.

Science, 1994 Sep 16, 265(5179), 1713 - 6
Binding of 14-3-3 proteins to the protein kinase Raf and effects on its activation; Freed E et al.; To identify proteins that may participate in the activation of the protein kinase Raf, proteins that interact with Raf were selected in a two-hybrid screen . Two members of the 14-3-3 protein family were isolated that interacted with both the amino terminal regulatory regions of Raf and the kinase domain of Raf, but did not compete with the guanine nucleotide-binding protein Ras for binding to Raf . 14-3-3 proteins associated with Raf in mammalian cells and accompanied Raf to the membrane in the presence of activated Ras . In yeast cells expressing Raf and MEK, mammalian 14-3-3 beta or 14-3-3 zeta activated Raf to a similar extent as did expression of Ras . Therefore, 14-3-3 proteins may participate in or be required for the regulation of Raf function . These findings suggest a role for 14-3-3 proteins in Raf-mediated signal transduction.

Gene, 1994 Sep 15, 147(1), 107 - 10
A mutation in the effector region of Ras2 can be partially suppressed by alteration of a 'nonessential' region of Ras; Chen L et al.; Phenotypically normal revertants of budding yeast cells that contain the hyperactive RAS2Val19 allele often result from second-site mutations within the RAS2 locus itself . Several such intragenic revertants harboring a suppressed RAS2Val19 allele as their only RAS gene were analyzed . All such suppressors resulted from single amino acid substitutions that affected either: (i) the effector region of Ras2, (ii) the C-terminal CAAX box of Ras2, or (iii) residues known to be critical for GTP binding in Ras proteins . While these suppressor mutations completely suppressed the hyperactive phenotype induced by the Val19 substitution, they did not block the ability of Ras2 to promote growth at normal temperatures . These results suggest that in yeast, attenuation of Ras proteins can effectively block hyperactive phenotypes without completely blocking the growth-promoting function . A spontaneous intragenic mutation that restored function to an effector mutant was mapped to a 'nonessential' region of Ras proteins . Based on this genetic interaction with the effector region and the report that deletions of this region affect Ras/GAP interaction, we suggest that this region may have a functional role in Ras activation of target effectors.

Nature, 1994 Sep 15, 371(6494), 254 - 7
A cyclin associated with the CDK-activating kinase MO15; Makela TP et al.; The eukaryotic cell cycle is regulated by the sequential activation of cyclin-dependent kinases (CDKs) . CDK activation is dependent on cyclin binding and phosphorylation of a conserved threonine (T161 in Cdc2) mediated by the CDK-activating kinase CAK . A CDK-related kinase, MO15 (ref . 10), has been identified as the catalytic subunit of CAK (refs 11-13) . Here we use a yeast two-hybrid screen to show that a new human cyclin (cyclin H) is a MO15-associated protein . Cyclin H is a major MO15 partner in vivo and enhances the kinase activity of MO15 towards Cdk2/cyclin A . These findings demonstrate that a cyclin/kinase complex can function as a regulator of other cyclin/kinase complexes, and suggest that cyclin/kinase cascades may exist.

EMBO J, 1994 Sep 15, 13(18), 4321 - 8
Cdc16p, Cdc23p and Cdc27p form a complex essential for mitosis; Lamb JR et al.; Cdc16p, Cdc23p and Cdc27p are all essential proteins required for cell cycle progression through mitosis in Saccharomyces cerevisiae . All three proteins contain multiple tandemly repeated 34 amino acid tetratricopeptide repeats (TPRs) . Using two independent assays, two-hybrid analysis in vivo and co-immunoprecipitation in vitro, we demonstrate that Cdc16p, Cdc23p and Cdc27p self associate and interact with one another to form a macromolecular complex . A temperature sensitive mutation in the most highly conserved TPR domain of Cdc27p results in a greatly reduced ability to interact with Cdc23p, but has no effect on interactions with wild-type Cdc27p or Cdc16p . The specificity of this effect indicates that TPRs can mediate protein-protein interactions and that this mutation may define an essential interaction for cell cycle progression in yeast . The conservation of at least two of the three proteins from yeast to man suggests that this protein complex is essential for mitosis in a wide range of eukaryotes.

EMBO J, 1994 Sep 15, 13(18), 4278 - 90
A novel human protein serine/threonine phosphatase, which possesses four tetratricopeptide repeat motifs and localizes to the nucleus; Chen MX et al.; A novel human protein serine/threonine phosphatase, PP5, and a structurally related phosphatase in Saccharomyces cerevisiae, PPT1, have been identified from their cDNA and gene respectively . Their predicted molecular mass is 58 kDa and they comprise a C-terminal phosphatase catalytic domain and an N-terminal domain, which has four repeats of 34 amino acids, three of which are tandemly arranged . The phosphatase domain possesses all the invariant motifs of the PP1/PP2A/PP2B gene family, but is not closely related to any other known member (< or = 40% identity) . Thus PP5 and PPT1 comprise a new subfamily . The repeats in the N-terminal domain are similar to the tetratricopeptide repeat (TPR) motifs which have been found in several proteins that are required for mitosis, transcription and RNA splicing . Bacterially expressed PP5 is able to dephosphorylate serine residues in proteins and is more sensitive than PP1 to the tumour promoter okadaic acid . A 2.3 kb mRNA encoding PP5 is present in all human tissues examined . Investigation of the intracellular distribution of PP5 by immunofluorescence, using two different antibodies raised against the TPR and phosphatase domains, localizes PP5 predominantly to the nucleus . This suggests that, like other nuclear TPR-containing proteins, it may play a role in the regulation of RNA biogenesis and/or mitosis.

Proc Natl Acad Sci U S A, 1994 Sep 13, 91(19), 9101 - 5
Enhancer 1 binding factor (E1BF), a Ku-related protein, is a growth-regulated RNA polymerase I transcription factor: association of a repressor activity with purified E1BF from serum-deprived cells; Niu H et al.; Previous studies from this laboratory have demonstrated that the enhancer 1 binding factor (E1BF), a Ku-related protein, purified from the serum-enriched cells functions as a positive factor in an RNA polymerase (pol I) transcription system . We have now shown that E1BF purified from the serum-deprived cells (E1BFs) can inhibit rDNA transcription completely in a fractionated extract from the cells grown in serum-enriched medium . The suppression of transcription was overcome by the addition of control E1BF (E1BFc) . Immunoprecipitation of purified E1BFs by the anti-Ku monoclonal antibody and addition of the supernatant to the transcription reaction mixture prevented the inhibition significantly, whereas immunoprecipitation with the control mouse IgG did not restore the transcription . The transcriptional repressor activity associated with the final DNA affinity column fractions copurified with E1BF . Neither the amount of E1BF nor its promoter binding activity was altered following serum depletion . E1BFs selectively inhibited the initiation of rDNA transcription . The inhibitory activity of E1BFs was not due to a nonspecific RNase activity . These data suggest that E1BF is post-translationally modified following serum starvation of cells, and that the repressor activity of E1BFs is largely responsible for the down-regulation of pol I transcription in serum-deprived cells.

FEBS Lett, 1994 Sep 12, 351(3), 437 - 42
The crystal structure of the iron-free cytochrome c peroxidase and its implication for the enzymatic mechanism; Su XD et al.; We report the refined structure of an iron-free form of cytochrome c peroxidase (CcP) at 2.3 A resolution . The backbone comparison between native CcP and iron-free CcP shows that the two structures have the same protein fold within experimental error . The only difference noted is in the heme pocket where the distance between the proximal histidine and the center of the protoporphyrin has increased . The results show that the iron-free CcP should be a good substitute for native CcP in fluorescence studies and thus also validate previous studies using iron-free CcPs as efficient fluorescent probes in electron transfer studies.

J Biol Chem, 1994 Sep 9, 269(36), 22492 - 5
Aggregation of the intracellular domain of the type 1 tumor necrosis factor receptor defined by the two-hybrid system; Song HY et al.; The yeast-based two hybrid system has been used to determine whether oligomerization of the intracellular domain of the 55-kDa type 1 tumor necrosis factor (TNF) receptor may occur during TNF action . This assay depends upon reconstitution of the function of the GAL4 transcriptional activator through interaction of a protein fused to the GAL4 DNA binding domain with a protein fused to the transcriptional activation domain of GAL4 . Fusion of the type 1 TNF receptor intracellular domain with the DNA binding domain and the transactivation domain of GAL4 led to activation of the lacZ indicator gene, demonstrating interaction of the receptor intracellular domain with itself . A HeLa cell cDNA library was searched for proteins that interact with the intracellular domain of the type 1 TNF receptor . A protein corresponding to amino acids 329-426 in the type 1 TNF receptor intracellular domain was identified by this screen . The aggregation domain was further defined by testing the ability of deletion mutants of the type 1 TNF receptor intracellular region to interact with the complete intracellular domain . These experiments map the aggregation domain to a sequence of amino acids previously shown to be responsible for mediating TNF-induced cytotoxicity . These results suggest that aggregation of type 1 TNF receptor intracellular domains may be important in TNF signal transduction.

FEBS Lett, 1994 Sep 5, 351(2), 219 - 24
Similar DNA binding properties of free P70 (KU) subunit and P70/P80 heterodimer; Wang J et al.; The Ku antigen consists of 70 and 80 kDa protein subunits (p70 and p80, respectively) that form the DNA binding component of a DNA-dependent protein kinase (DNA-PK) . It is controversial whether the interaction of Ku with DNA is mediated by p70 alone or requires formation of p70/p80 dimers . In the present studies, the DNA binding properties of p70/p80 heterodimers and full-length human p70 expressed in the absence of p80 were investigated . The binding of free p70 and p70/p80 heterodimers to DNA showed similar sensitivity to high ionic strength buffers . Competitive DNA binding studies revealed that free p70, like the p70/p80 heterodimer, bound preferentially to linear double stranded DNA fragments, whereas tRNA and closed circular DNA molecules competed poorly with the radiolabeled linear DNA for binding to Ku . These studies suggest that free p70 and p70/p80 heterodimers have similar DNA binding properties, and that the interaction of Ku with DNA may depend primarily on the p70 subunit, possibly with implications for the assembly and function of DNA-PK.

FEBS Lett, 1994 Sep 5, 351(2), 155 - 8
The first hydrophobic segment of the ABC-transporter, Ste6, functions as a signal sequence; Kolling R et al.; The Ste6 protein of Saccharomyces cerevisiae is a member of the ABC-transporter family containing 12 putative membrane spanning segments . To test whether Ste6 is inserted into the endoplasmic reticulum (ER) membrane by a sequential insertion mechanism we constructed a Ste6-invertase fusion containing the first hydrophobic segment of Ste6 fused to invertase lacking its own signal sequence . The resulting protein became glycosylated demonstrating that it was translocated across the ER-membrane . The finding that the N-terminal hydrophobic segment of Ste6 is recognized by the ER-translocation machinery suggests that Ste6 is inserted sequentially into the ER-membrane . Furthermore, our experiments support the Nin orientation of Ste6 predicted from the Ste6 sequence . Several findings suggest that invertase is cleaved from the Ste6 membrane anchor: (i) the gel mobility of deglycosylated wild-type invertase and fusion protein derived invertase is the same; (ii) the periplasmic invertase activity is found in the cell wall fraction, i.e . it is not associated with the cell body; (iii) a signal peptide cleavage site is predicted in the Ste6 sequence . Although the membrane anchor appeared to be cleaved, most of the invertase was retained in the ER, probably due to aggregate formation.

Gene, 1994 Sep 2, 146(2), 239 - 44
Cloning, genomic organization and transcription of the Entamoeba histolytica alpha-tubulin-encoding gene; Sanchez MA et al.; We have isolated and characterized an Entamoeba histolytica alpha-tubulin (alpha Tub)-encoding gene (Eh alpha tub) . A 700-bp DNA fragment was amplified by PCR using primers derived from consensus alpha- and beta-Tub amino acid (aa) sequences from different organisms and E . histolytica DNA as the template . These PCR fragments were used to screen both genomic DNA and cDNA libraries in order to isolate an Eh alpha tub structural gene . Two overlapping clones containing the complete alpha tub ORF (1392 bp) were isolated from the genome and cDNA libraries . The deduced aa sequence of Eh alpha Tub has 55.5, 50 and 52% identity to Plasmodium falciparum alpha Tub 2, Saccharomyces cerevisiae alpha Tub 2 and human alpha Tub, respectively . Interestingly, the predicted Eh alpha Tub protein lacks a poly-acidic motif at its C terminus which is involved in Tub polymerization and microtubule-associated protein binding in other organisms . This fact may indicate a difference in tubule assembly in this organism and could provide a potential key for the development of therapeutic agents . According to Southern blot experiments and the sequences of several clones, at least two non-adjacent copies of alpha tub are present in the E . histolytica genome . A 1.5-kb transcript corresponding to the alpha tub mRNA was detected in mRNA from asynchronous E . histolytica trophozoites.

Gene, 1994 Sep 2, 146(2), 227 - 32
PCR-based construction of promoter/G-free templates for in vitro transcription analysis allows selection of plasmids with optimal activity in homologous extracts; Ruan Y et al.; In vitro transcription has been used for dissecting transcriptional controls in many eukaryotic systems . One modification which greatly reduces background non-specific transcription is the placement of a guanosine-free (G-free) region of DNA immediately downstream from a promoter {Sawadogo and Roeder, Proc . Natl . Acad . Sci . USA 82 (1985) 4394-4398}; transcription in the presence of RNase T1 and 3' O-Me-GTP eliminates non-specific transcripts, and produces the G-free transcripts initiated at the promoter . Restriction site-based fusion of a G-free cassette downstream from promoters is complicated by the requirement for G nucleotides to be excluded from the coding strand downstream from the transcription start points . We present an approach to add a G-free template onto a eukaryotic promoter by combining PCR-based termini construction and terminal deoxynucleotidyl transferase extension . The pisatin demethylase promoter (PDA1p) of the filamentous fungus Nectria haematococca was used as the test promoter . Three PDA1p/G- free constructs were tested in heterologous Drosophila melanogaster and HeLa and homologous N . haematococca transcription extracts . Each extract produced a PDA1p-specific transcript from each construct, but the relative level of transcription between constructs varied, particularly in the homologous extract . Since the choice of G-free sequence influences transcription differently among systems, this method for producing multiple G-free constructs should be useful for constructing and selecting optimal promoter/G-free templates for in vitro transcription in other homologous systems.

Gene, 1994 Sep 2, 146(2), 145 - 58
Evolution of transcription-regulating proteins by enzyme recruitment: molecular models for nitrogen metabolite repression and ethanol utilisation in eukaryotes; Hawkins AR et al.; Studies on the quinic acid utilisation gene (qut) cluster in Aspergillus nidulans showed that the genes encoding transcriptional activator and repressor proteins evolved by co-opting duplicated copies of genes encoding metabolic enzymes . In order to test the hypothesis that this was a general route for the genesis of regulatory proteins, the origins of the major control protein mediating nitrogen metabolite repression (an example of inter-pathway regulation) and ethanol utilisation (an example of intra-pathway regulation) in filamentous fungi were sought . The regulatory proteins mediating nitrogen metabolite repression were deduced to have originated in a duplication of genes encoding the anthranilate synthase complex which is active in the shikimate pathway . The major protein regulating ethanol utilisation was deduced to have its origin in the fusion of duplicated genes encoding the aldehyde and alcohol dehydrogenases (ALDA and ALCA) . These data strongly support the view that transcriptional regulatory proteins evolve by the recruitment of functional domains provided by metabolic enzymes.

Science, 1994 Sep 2, 265(5177), 1454 - 8
Degradation of G alpha by the N-end rule pathway; Madura K et al.; The N-end rule relates the in vivo half-life of a protein to the identity of its amino-terminal residue . Overexpression of targeting components of the N-end rule pathway in Saccharomyces cerevisiae inhibited the growth of haploid but not diploid cells . This ploidy-dependent toxicity was shown to result from enhanced degradation of Gpa1, the alpha subunit (G alpha) of a heterotrimeric guanine nucleotide-binding protein (G protein) that regulates cell differentiation in response to mating pheromones . Sst2, a protein whose absence renders cells hypersensitive to pheromone, was essential for degradation of G alpha but not other N-end rule substrates, suggesting the involvement of an indirect, or trans-, targeting mechanism . G alpha degradation by the N-end rule pathway adds another regulatory dimension to the multitude of signaling functions mediated by G proteins.

Science, 1994 Sep 2, 265(5177), 1442 - 5
Ku80: product of the XRCC5 gene and its role in DNA repair and V(D)J recombination; Taccioli GE et al.; The radiosensitive mutant xrs-6, derived from Chinese hamster ovary cells, is defective in DNA double-strand break repair and in ability to undergo V(D)J recombination . The human XRCC5 DNA repair gene, which complements this mutant, is shown here through genetic and biochemical evidence to be the 80-kilodalton subunit of the Ku protein . Ku binds to free double-stranded DNA ends and is the DNA-binding component of the DNA-dependent protein kinase . Thus, the Ku protein is involved in DNA repair and in V(D)J recombination, and these results may also indicate a role for the Ku-DNA-dependent protein kinase complex in those same processes.

J Biol Chem, 1994 Sep 2, 269(35), 22007 - 13
Refolding of a carboxypeptidase Y folding intermediate in vitro by low-affinity binding of the proregion; Winther JR et al.; Efficient folding of carboxypeptidase Y is dependent on the presence of the proregion . Thus, denatured procarboxypeptidase Y, in contrast to the mature enzyme, refolds efficiently in vitro in low ionic strength buffers . Under these conditions denatured mature carboxypeptidase Y forms an inactive, soluble folding intermediate, which has been characterized in the present study . The inactive intermediate can be folded into the active enzyme at a low efficiency (5-10%) by the addition of 0.9 M ammonium sulfate . The refolding is accompanied by pronounced structural changes . As seen for other protease zymogens the isolated proregion from carboxypeptidase Y was found to stimulate refolding without covalent linkage to the mature part . However, the added proregion does not form a stable complex with the native enzyme and requires the presence of 0.9 M ammonium sulfate to exhibit its function . The proregion increases the yield of correctly folded enzyme, and kinetic analysis suggests that this is due to a reduction of the rate of nonproductive folding or aggregation . In addition, the proregion stabilizes carboxypeptidase Y toward thermoinactivation.

J Cell Biol, 1994 Sep, 126(6), 1361 - 73
Regulation of mitochondrial morphology and inheritance by Mdm10p, a protein of the mitochondrial outer membrane; Sogo LF et al.; Yeast cells with the mdm10 mutation possess giant spherical mitochondria and are defective for mitochondrial inheritance . The giant mitochondria display classical features of mitochondrial ultrastructure, yet they appear incapable of movement or division . Genetic analysis indicated that the mutant phenotypes resulted from a single nuclear mutation, and the isolated MDM10 gene restored wild-type mitochondrial distribution and morphology when introduced into mutant cells . MDM10 encodes a protein of 56.2 kD located in the mitochondrial outer membrane . Depletion of Mdm10p from cells led to a condensation of normally extended, tubular mitochondria into giant spheres, and reexpression of the protein resulted in a rapid restoration of normal mitochondrial morphology . These results demonstrate that Mdm10p can control mitochondrial morphology, and that it plays a role in the inheritance of mitochondria.

Arch Biochem Biophys, 1994 Sep, 313(2), 235 - 40
Isolation of a novel soybean gene encoding a mitochondrial ATP synthase subunit; Smith MK et al.; A novel ATP synthase gene from soybean has been cloned and characterized . A subunit from the FA portion of the complex is encoded by two nuclear genes . The genomic clone(s) contain five exons encoding a protein of 179 amino acids . The amino terminal end contains many properties of a mitochondrial targeting sequence and preliminary in vitro import studies indicate that there is a cleavable precursor of approximately 30 amino acids . The predicted protein sequence shows high homology with the N-terminal sequence from an isolated subunit of ATP synthase complex from spinach (Hamsaur and Glaser (1992) Eur . J . Biochem . 205, 409-416) . The subunit was tentatively identified as the equivalent of subunit d in bovine and P18 in yeast based on structural identity.

Mol Gen Genet, 1994 Sep 1, 244(5), 548 - 56
Differential expression of several E2-type ubiquitin carrier protein genes at different developmental stages in Arabidopsis thaliana and Nicotiana sylvestris; Genschik P et al.; We characterized three genes encoding different E2-type ubiquitin carrier proteins involved in the ubiquitin-mediated proteolytic pathway: UbcAt3 shows homologies to the yeast CDC34 gene and Ub-cAt4a and UbcAt4b are two different genes homologous to the Ubc1/4/5 subfamily in yeast . Their accumulation was analysed and compared with that of the different families encoding polyubiquitins, as well as the monoubiquitin fusion protein, which is considered as a marker for cell division, during various developmental stages including G0/S transition and senescence of higher plant cells . Our results imply that these Ubc genes are under the control of complex mechanisms, and are differentially regulated, but not necessarily co-regulated with ubiquitin genes . Even the closely related UbcAt4a and UbcAt4b genes of the same multigene subfamily are controlled by distinct regulatory mechanisms.

Nature, 1994 Sep 1, 371(6492), 80 - 3
Crystal structure of an isoleucine-zipper trimer; Harbury PB et al.; Subunit oligomerization in many proteins is mediated by short coiled-coil motifs . These motifs share a characteristic seven-amino-acid repeat containing hydrophobic residues at the first (a) and fourth (d) positions . Despite this common pattern, different sequences form two-, three- and four-stranded helical ropes . We have investigated the basis for oligomer choice by characterizing variants of the GCN4 leucine-zipper dimerization domain that adopt trimeric or tetrameric structures in response to mutations at the a and d positions . We now report the high-resolution X-ray crystal structure of an isoleucine-containing mutant that folds into a parallel three-stranded, alpha-helical coiled coil . In contrast to the dimer and tetramer structures, the interior packing of the trimer can accommodate beta-branched residues in the most preferred rotamer at both hydrophobic positions . Compatibility of the shape of the core amino acids with the distinct packing spaces in the two-, three- and four-stranded conformations appears to determine the oligomerization state of the GCN4 leucine-zipper variants.

Mol Cell Biol, 1994 Sep, 14(9), 6046 - 55
The Oct-2 glutamine-rich and proline-rich activation domains can synergize with each other or duplicates of themselves to activate transcription; Tanaka M et al.; The B-cell POU homeodomain protein Oct-2 contains two transcriptional activation domains, one N terminal and the other C terminal of the central DNA-binding POU domain . The synergistic action of these two activation domains makes Oct-2 a more potent activator of mRNA promoters than the related broadly expressed octamer motif-binding protein Oct-1, which contains an N-terminal but not a C-terminal Oct-2-like activation domain . Both Oct-2 mRNA promoter activation domains were delineated by truncation analysis: the N-terminal Q domain is a 66-amino-acid region rich in glutamines, and the C-terminal P domain is a 42-amino-acid region rich in prolines . The Q and P domains synergized with each other or duplicates of themselves, independently of their N-terminal or C-terminal position relative to the POU domain . The C-terminal P domain, which differentiates Oct-2 from Oct-1, also activated transcription in conjunction with the heterologous GAL4 DNA-binding domain . Oct-2 thus contains three modular functional units, the DNA-binding POU domain and the two P and Q activation domains . An electrophoretic mobility shift assay with a variety of these Oct-2 activators revealed a distinct complex called QA that was dependent on the presence of an active glutamine-rich activation domain and migrated more slowly than the Oct-2-DNA complexes . Formation of the QA complex is consistent with interaction of the glutamine-rich activation domains with a regulatory protein important for the process of transcriptional activation.

Mol Cell Biol, 1994 Sep, 14(9), 5719 - 30
In vitro integration of retrotransposon Ty1: a direct physical assay; Braiterman LT et al.; Retrotransposon Ty1 of Saccharomyces cerevisiae inserts a double-stranded Ty1 cDNA into the yeast genome by a reaction analogous to the integration mechanism used by retroviruses . A quantitative in vitro integration assay that directly detects integrative recombination products was developed for Ty1 . Blunt-ended artificial radioactive substrates bearing Ty1 termini integrate into circular or linear target DNAs . The reaction is specific for native integrase isolated in the form of virus-like particles; virus-like particles prepared from integrase mutants were completely inactive in this assay . The products are radioactive, allowing direct detection after gel electrophoresis by autoradiography . Using this simple and amenable system, we characterized the biochemical requirements of the system and the structures of the major integration products . Two classes of products were detected: those that were the result of bona fide complete integration events (concerted reactions) and single-end joinings of substrate to target (half-reactions) . Additionally, we used a genetic selection scheme to identify and characterize target sites of complete integration events into a circular target plasmid; a 5-bp target site duplication flanking the inserted DNA resembling the duplication characteristic of in vivo integration was observed.

J Cell Biol, 1994 Sep, 126(5), 1133 - 48
Characteristics of endoplasmic reticulum-derived transport vesicles; Rexach MF et al.; We have isolated vesicles that mediate protein transport from the ER to Golgi membranes in perforated yeast . These vesicles, which form de novo during in vitro incubations, carry lumenal and membrane proteins that include core-glycosylated pro-alpha-factor, Bet1, Sec22, and Bos1, but not ER-resident Kar2 or Sec61 proteins . Thus, lumenal and membrane proteins in the ER are sorted prior to transport vesicle scission . Inhibition of Ypt1p-function, which prevents newly formed vesicles from docking to cis-Golgi membranes, was used to block transport . Vesicles that accumulate are competent for fusion with cis-Golgi membranes, but not with ER membranes, and thus are functionally committed to vectorial transport . A 900-fold enrichment was developed using differential centrifugation and a series of velocity and equilibrium density gradients . Electron microscopic analysis shows a uniform population of 60 nm vesicles that lack peripheral protein coats . Quantitative Western blot analysis indicates that protein markers of cytosol and cellular membranes are depleted throughout the purification, whereas the synaptobrevin-like Bet1, Sec22, and Bos1 proteins are highly enriched . Uncoated ER-derived transport vesicles (ERV) contain twelve major proteins that associate tightly with the membrane . The ERV proteins may represent abundant cargo and additional targeting molecules.

Oncogene, 1994 Sep, 9(9), 2633 - 8
Functional studies of E7 proteins from different HPV types; Ciccolini F et al.; We have performed comparative studies on the E7 proteins from malignant and non-malignant Human Papillomavirus types HPV 1, 6, 11, 16, 18, 33) . GST/E7 fusion proteins from all these HPV types associate with Rb1, p107 and the cyclin A/CDK2 complex . As has been shown for Rb1, the association with p107 and Cyclin A was weaker for the 'low risk' HPV6 and 11 E7 proteins as compared to 'high risk' HPV16, 18 and 33 E7 proteins . In contrast the E7 protein of the benign type HPV1 bound Rb1, p107 and cyclin A with the same affinity as the 'high risk' E7 proteins . The affinities of the E7/Rb1 interaction have been confirmed in vivo by the 'two hybrid' method in the yeast Saccharomyces cerevisiae . Although HPV1 E7 showed the same affinity in vitro and in vivo for Rb1 as the high risk HPV E7s, it did not have the ability to activate the E2F-1 transcription factor inhibited by Rb1, nor did it have any transforming activity when coexpressed with activated ras in primary rodent cells.

J Virol, 1994 Sep, 68(9), 6014 - 20
A cryptic RNA-binding domain in the Pol region of the L-A double-stranded RNA virus Gag-Pol fusion protein; Ribas JC et al.; The Pol region of the Gag-Pol fusion protein of the L-A double-stranded (ds) RNA virus of Saccharomyces cerevisiae has (i) a domain essential for packaging viral positive strands, (ii) consensus amino acid sequence patterns typical of RNA-dependent RNA polymerases, and (iii) two single-stranded RNA binding domains . We describe here a third single-stranded RNA binding domain (Pol residues 374 to 432), which is unique in being cryptic . Its activity is revealed only after deletion of an inhibitory region C terminal to the binding domain itself . This cryptic RNA binding domain is necessary for propagation of M1 satellite dsRNA, but it is not necessary for viral particle assembly or for packaging of viral positive-strand single-stranded RNA . The cryptic RNA binding domain includes a sequence pattern common among positive-strand single-stranded RNA and dsRNA viral RNA-dependent RNA polymerases, suggesting that it has a role in RNA polymerase activity.

J Virol, 1994 Sep, 68(9), 5375 - 83
EBNA-2 upregulation of Epstein-Barr virus latency promoters and the cellular CD23 promoter utilizes a common targeting intermediate, CBF1; Ling PD et al.; The EBNA-2 protein is essential for the establishment of a latent Epstein-Barr virus (EBV) infection and for B-cell immortalization . EBNA-2 functions as a transcriptional activator that modulates viral latency gene expression as well as the expression of cellular genes, including CD23 . We recently demonstrated that EBNA-2 transactivation of the EBV latency C promoter (Cp) is dependent on an interaction with a cellular DNA-binding protein, CBF1, for promoter targeting . To determine whether targeting via CBF1 is a common mechanism for EBNA-2-mediated transactivation, we have examined the requirements for activation of the cellular CD23 promoter . Binding of CBF1 to a 192-bp mapped EBNA-2-responsive region located at position -85 bp to -277 bp upstream of the CD23 promoter was detected in electrophoretic mobility shift assays . The identity of the bound protein as CBF1 was established by showing that the bound complex was competed for by the CBF1 binding site from the EBV Cp, that the bound protein could be supershifted with a bacterially expressed fusion protein' containing amino acids 252 to 425 of EBNA-2 but was unable to interact with a non-CBF1-binding EBNA-2 mutant (WW323SR), and that in UV cross-linking experiments, the Cp CBF1 binding site and the CD23 probe bound proteins of the same size . The requirement for interaction with CBF1 was demonstrated in a transient cotransfection assay in which the multimerized 192-bp CD23 response region was transactivated by wild-type EBNA-2 but not by the WW323SR mutant . Reporter constructions carrying multimerized copies of the 192-bp CD23 response region or multimers of the CBF1 binding site from the CD23 promoter were significantly less responsive to EBNA-2 transactivation than equivalent constructions carrying a multimerized region from the Cp or multimers of the CBF1 binding site from the Cp . Direct binding and competition assays using 30-mer oligonucleotide probes representing the individual CBF1 binding sites indicated that CBF1 bound less efficiently to the CD23 promoter and the EBV LMP-1 promoter sites than to the Cp site . To investigate the basis for this difference, we synthesized a series of oligonucleotides carrying mutations across the CBF1 binding site and used these as competitors in electrophoretic mobility shift assays . The competition experiments indicated that a central core sequence, GTGGGAA, common to all known EBNA-2-responsive elements, is crucial for CBF1 binding . Flanking sequences on either side of this core influence the affinity for CBF1.(ABSTRACT TRUNCATED AT 400 WORDS)

Int J Biochem, 1994 Sep, 26(9), 1095 - 101
Activation of 3-methyl-branched fatty acids in rat liver; Vanhooren JC et al.; 1 . Subcellular fractionation of rat liver revealed that 3-methylmargaric acid, a monobranched phytanic acid analogue, can be activated by mitochondria, endoplasmic reticulum and peroxisomes . 2 . Indirect data (effects of pyrophosphate and Triton X-100) suggested that the peroxisomal activation of 3-methylmargaric, 2-methylpalmitic and palmitic acid is catalyzed by different enzymes . 3 . Despite many attempts, column chromatography of solubilized peroxisomal membrane proteins so far did not provide more conclusive data . On various matrices, lignoceroyl-CoA synthetase clearly eluted differently from the synthetases acting on 3-methylmargaric, 2-methylpalmitic and palmitic acid . The latter three however, tended to coelute together, although often not in an identical manner.

Trends Biochem Sci, 1994 Sep, 19(9), 368 - 72
The protein import machinery of the mitochondrial inner membrane; Pfanner N et al.; Mitochondria import most of their proteins from the cytosol . Although considerable information is available on the import machineries of the mitochondrial outer membrane and matrix, until recently little was known about the machinery of the inner membrane . Recent studies have identified three mitochondrial inner membrane proteins (MIMs) as essential components of the import machinery . MIM17 and MIM23 seem to form part of a channel, while MIM44, in cooperation with the heat-shock protein Hsp70, binds the preproteins in transit . The electrical membrane potential and ATP are needed to drive protein translocation through the MIM import machinery.

Genes Dev, 1994 Sep 1, 8(17), 2097 - 109
Structure-function analysis of the TBP-binding protein Dr1 reveals a mechanism for repression of class II gene transcription; Yeung KC et al.; Dr1, a repressor of class II genes, regulates transcription by a novel mechanism . Biochemical analyses reveal that Dr1 directly interacts with the multiprotein TFIID complex . By use of the yeast two-hybrid system, we demonstrate that the association of Dr1 with the TATA-binding protein (TBP) subunit of TFIID occurs in vivo . In addition, Dr1 can repress transcription from TATA-containing as well as TATA-less promoters in transient transfection assays . Importantly, Dr1-mediated repression can be reversed by overexpression of TBP in vivo . By use of diverse approaches, we mapped two distinct domains in Dr1 required for repression . One domain is essential for the Dr1-TBP interaction, and the second is rich in alanine residues . The TBP-binding domain of Dr1 cannot be replaced by a heterologous DNA-binding domain in mediating repression . We demonstrate that some, but not all, transcriptional activators can reverse Dr1-mediated repression in vivo.

Comput Chem, 1994 Sep, 18(3), 203 - 5
Inferring genes from open reading frames; Fickett JW; One expects that in DNA without protein coding function, stop codons (which constitute three of the 64 possible codons) should occur frequently in all reading frames, and that a long open reading frame (ORF) can be interpreted as a sign for the existence of a gene . We make a beginning on introducing quantitative measures of confidence into this inference--taking Saccharomyces cerevisiae as a sample case--and show that some common assumptions can reasonably be questioned . In particular we show that statistical support for the biological function of shorter ORFs listed as putative genes in recent papers is in fact very weak . This is an issue of practical as well as theoretical interest, since researching the function of a putative gene is difficult and expensive.

FEMS Microbiol Rev, 1994 Sep, 15(1), 1 - 7
Differential expression of SUC genes: a question of bases; Gozalbo D et al.; Non-coding nucleotide sequences located 5' upstream of the transcriptional start site play an essential role in gene expression as they contain binding sites for transcription and regulatory factors . The yeast SUC gene family is a useful model to study the influence that nucleotide exchanges within the promoter regions have on their expression, since (i) these genes, regulated by glucose repression, are differentially transcribed (invertase activity produced by distinct SUC genes may show variations of about 10-fold); and (ii) promoter sequences of SUC3, SUC4, SUC5 and SUC7 are more than 99% homologous, showing only six base exchanges among all of them . Comparison of these nucleotide exchanges with the expression of each SUC gene (located either on chromosomes or on multicopy and centromeric plasmids) points out that naturally occurring base exchanges as few as one nucleotide modification (G to A transition at position -497 relative to the translational start site, C to T transition at position -460 and insertion/deletion of a T at positions -590, -586 and -435) may have a strong effect on gene expression.

Eur J Biochem, 1994 Sep 1, 224(2), 365 - 71
A quantitative test of long-range correlations and compositional fluctuations in DNA sequences; Chatzidimitriou-Dreismann CA et al.; Recent findings concerning long-range correlations and fractals in intron-containing DNA sequences of living organisms are tested qualitatively and quantitatively . Extending previous studies, we demonstrate that these findings are trivially equivalent to variations of the base-pair composition in different regions of a DNA sequence . It is shown explicitly that a well-defined scaling or fractal exponent does not exist anywhere . Comparisons of natural DNAs with computer-generated, artificial sequences are made . The present study reveals that certain natural DNA sequences (especially those with compact genomes) do have stochastic characteristics which are intrinsically different from artificial sequences . The results for 21 DNA sequences of various types from widely different taxa are reported.

Mol Cell Biol, 1994 Sep, 14(9), 5832 - 9
GSH1, which encodes gamma-glutamylcysteine synthetase, is a target gene for yAP-1 transcriptional regulation; Wu AL et al.; Changes in gene dosage of the YAP1 gene, encoding the yAP-1 transcriptional regulatory protein, cause profound alterations in cellular drug and metal resistance . Previous studies on yAP-1 action in yeast cells have used the AP-1 response element (ARE) from simian virus 40 as an artificial site for yAP-1-mediated transcriptional activation . No authentic yeast target sites for control of gene expression by yAP-1 are known . Here we show that the GSH1 gene, encoding gamma-glutamylcysteine synthetase, is transcriptionally responsive to the yAP-1 protein . GSH1 encodes the rate-limiting step in yeast glutathione biosynthesis and contains within its promoter region a DNA element that matches the ARE in 11 of 12 positions . The GSH1 yAP-1 response element (YRE) was recognized by yAP-1 protein in vitro . Northern (RNA) blot analysis showed that GSH1 mRNA levels were responsive to YAP1 gene dosage . A site-directed mutation in the YRE that blocked yAP-1 binding in vitro prevented the mutant GSH1 promoter from responding to elevation in YAP1 gene dosage . A delta gsh1 mutant strain was constructed and unable to grow in the absence of exogenous glutathione . A mutant GSH1 gene lacking the YRE was unable to confer normal cadmium tolerance, although other yAP-1-mediated phenotypes remained normal . Thus, GSH1 is one of several genes that are transcriptionally controlled by yAP-1 and influence drug resistance.

Eur Cytokine Netw, 1994 Sep-Oct, 5(5), 481 - 7
Murine interleukin-4 production with baculovirus: an easy and rapid method for a small scale production of functional interleukins; Cottrez F et al.; We described a baculovirus expression system for high level production of secreted murine recombinant IL-4 . We have constructed a recombinant baculovirus based on Autographa californica polyhedrosis virus, containing both a synthetic PCR-derived murine IL-4 cDNA under the control of the polyhedrin promoter and the lacZ gene under the control of the P10 promoter to allow an easy detection of recombinant virus . The baculovirus IL-4 was fully functional in biological assay and was present under two glycosylated forms in the supernatants of infected Sf9 cells . We also detected a third unglycosylated intracytoplasmic form resulting from a fusion between the 35 first amino acids of polyhedrin and the murine IL-4 . Finally, confocal microscopy showed that this recombinant protein was secreted along a classical pathway like in mammalian cells.

Hum Mol Genet, 1994 Sep, 3(9), 1537 - 42
p53 tagged sites from human genomic DNA; Tokino T et al.; The product of the tumor suppressor gene p53 binds to DNA and activates transcription from promoters containing its consensus binding site . This activity has been hypothesized to be responsible for its biological effects . However, the total number and nature of human genomic sites with which p53 can functionally interact is unknown . In this paper, we have used a Saccharomyces cerevisiae-based screen to identify human genomic sequences that activate transcription from an adjacent reporter gene in a p53-dependent manner (p53-tagged sites, PTS) . Fifty-seven different PTS were identified, and the total number of such sites in the human genome was predicted to be between 200 and 300 . Almost all contained two adjacent copies of the previously defined consensus 5'-PuPuPuC(A/T)(T/A)GPyPyPy-3' . Spacing between the copies was found to be critical for sequence-specific transcriptional activation in vivo . These results further refine the nature of the genomic sequences likely to be most important for p53-mediated tumor suppression.

Protein Eng, 1994 Sep, 7(9), 1109 - 14
Effect of amino acid deletions in the O-glycosylated region of Aspergillus awamori glucoamylase; Libby CB et al.; Aspergillus awamori glucoamylase (GA) contains globular catalytic and starch-binding domains (residues 1-471 and 509-616, respectively) . A heavily O-glycosylated sequence comprises two parts . The first (residues 441-471) in the crystal structure wraps around an alpha/alpha-barrel formed by residues 1-440 . The second (residues 472-508) is an extended, semi-rigid linker between the two domains . To investigate the functional role of this linker, we made internal deletions to remove residues 466-512 (GA delta 1), 485-512 (GA delta 2) and 466-483 (GA delta 3) . GA delta 2 and GA delta 3 were expressed in Saccharomyces cerevisiae culture supernatants at approximately 60 and 20% the wild-type level, respectively, while GA delta 1 was almost undetectable . Western blots comparing extracellular and intracellular fractions indicated that the region deleted in GA delta 3 was critical for secretion, while the region deleted in GA delta 2 contributed to the production of a stable enzyme structure . The activities of purified GA delta 2 and GA delta 3 on soluble and insoluble starch were similar to those of wild-type GA, indicating that for soluble starch their deletions did not affect the catalytic domain and for insoluble starch the linker does not coordinate the activities of the catalytic and starch-binding domains . The deletions had a significant negative effect on GA delta 2 and GA delta 3 thermostabilities.

J Clin Microbiol, 1994 Sep, 32(9), 2192 - 6
Immunoaffinity purification of baculovirus-expressed rubella virus E1 for diagnostic purposes; Lindqvist C et al.; Three monoclonal antibodies, termed 4E10, 1E11:10, and 2D9:1, were generated against rubella virus . Immunoblot analysis with purified authentic rubella virus or recombinant baculovirus-expressed rubella virus structural proteins E1, E2, and C demonstrated that they were directed against the E1 envelope glycoprotein of the rubella virus particle . By using the yeast Ty virus-like particle system, it was possible to map the binding site of 1E11:10 within amino acids 236 to 286 of the E1 protein and the binding sites of 2D9:1 and 4E10 outside this region . Immunoaffinity purification with these monoclonal antibodies made it evident that they are useful for obtaining large quantities of pure baculovirus-expressed rubella virus envelope protein E1 . The diagnostic potential of this immunoaffinity-purified recombinant rubella virus E1 protein compared with that of authentic rubella virus is demonstrated.

Biotechnol Prog, 1994 Sep-Oct, 10(5), 499 - 502
Reversed micellar extraction of charged fusion proteins; Forney CE et al.; We have investigated the use of charged fusion tails with the enzyme glucoamylase in reversed micellar extraction . The addition of the charged tails increased the fraction of enzymatically active protein recovered at a given pH, with the tails containing the largest number of charges being recovered at the highest level . The series of mutations also allows for investigation of the charge-dependent behavior of reversed micellar extraction . However, in this case, the change in protein charge via fusions had a lesser impact than did the change in charge via a pH change . The difference may be due to the difficulty of partitioning the hydrodynamically larger fusion protein.

Yeast, 1994 Sep, 10(9), 1227 - 34
The sequence of 12.5 kb from the right arm of chromosome II predicts a new N-terminal sequence for the IRA1 protein and reveals two new genes, one of which is a DEAD-box helicase; Zagulski M et al.; We have determined the complete nucleotide sequence of a 12.5 kb segment from the right arm of chromosome II carried by the cosmid alpha 20 . The sequence encodes the 5' end of the IRA1 gene . Two complete new open reading frames and the 3' non-coding region of the SUP1 (SUP45) gene . A comparison of our sequence with the data bank reveals a 154 amino acid extension at the N-terminus of Ira1p compared to the previously predicted sequence . According to the 11th edition of the Saccharomyces cerevisiae genetic map, our sequence should encode the MAK5 gene, which is necessary for the maintenance of dsRNA killer plasmids . One of the two new open reading frames, YBR1119, is predicted to encode an RNA helicase, thus YBR1119 may correspond to the MAK5 gene.

Nat Struct Biol, 1994 Sep, 1(9), 621 - 37
2.1 A resolution refined structure of a TATA box-binding protein (TBP); Nikolov DB et al.; The three-dimensional structure of a TATA box-binding protein (TBP2) from Arabidopsis thaliana has been refined at 2.1 A resolution . TBPs are general eukaryotic transcription factors that participate in initiation of RNA synthesis by all three eukaryotic RNA polymerases . The carboxy-terminal portion of TBP is a unique DNA-binding motif/protein fold, adopting a highly symmetric alpha/beta structure that resembles a molecular saddle with two stirrup-like loops . A ten-stranded, antiparallel beta-sheet provides a concave surface for recognizing class II nuclear gene promoters, while the four amphipathic alpha-helices on the convex surface are available for interaction with other transcription factors . The myriad interactions of TBP2 with components of the transcription machinery are discussed.

Nat Struct Biol, 1994 Sep, 1(9), 605 - 14
Solution structure of the DNA-binding domain of Drosophila heat shock transcription factor; Vuister GW et al.; The solution structure of the DNA-binding domain of the Drosophila heat shock transcription factor, as determined by multidimensional multinuclear NMR, resembles that of the helix-turn-helix class of DNA-binding proteins . The domain comprises a four-stranded antiparallel beta-sheet, packed against a three-helix bundle . The second helix is significantly distorted and is separated from the third helix by an extended turn which is subject to conformational averaging on an intermediate time scale . Helix 3 forms a classical amphipathic helix with polar and charged residues exposed to the solvent . Upon titration with DNA, resonance shifts in the backbone and Asn and Gln side-chain amides indicate that helix 3 acts as the recognition helix of the heat shock transcription factor.

Mol Cell Biol, 1994 Sep, 14(9), 5731 - 40
Ty1 in vitro integration: effects of mutations in cis and in trans; Braiterman LT et al.; Mutations within the TYB gene of Ty1 encoding integrase (IN) as well as alterations in its substrate, a linear DNA molecule, were examined for their effects on in vitro IN activity, using a recently developed physical assay . Five different codon-insertion mutations, two frameshift mutations, and one missense mutation, previously identified as transposition-deficient mutations, were tested . Virus-like particles, the source of IN, from two different protease mutants and a reverse transcriptase mutant exhibited near-normal to normal IN activity . Two frameshift mutations mapping within the phylogenetically variable C-terminal domain of IN resulted in significant in vitro IN activity . In contrast, three mutations within the amino-terminal conserved domain of IN completely abolished IN activity . When the substrate termini were mutated, we found that substrates with as few as 4 bp of Ty1 termini were capable of efficiently generating integration products . Surprisingly, certain substrates that lacked obvious similarity to Ty1 termini were also readily integrated into both linear and circular targets, whereas others were not used as substrates at all . Termini rich in adenosine residues were among the more active substrates; however, certain substrates lacking terminal adenosine residues can form small quantities of integration products, including complete integration reactions.

Biochem Pharmacol, 1994 Aug 30, 48(5), 955 - 66
Induction of hepatic acyl-CoA-binding protein and liver fatty acid-binding protein by perfluorodecanoic acid in rats . Lack of correlation with hepatic long-chain acyl-CoA levels; Sterchele PF et al.; Liver fatty acid-binding protein (L-FABP) and acyl-CoA-binding protein (ACBP) are involved in the intracellular trafficking and compartmentalization of fatty acids and fatty acyl-CoA esters, respectively, in the liver . Both proteins are induced in rat liver by the potent peroxisome proliferator perfluorodecanoic acid (PFDA) . While it is believed that the peroxisome proliferator-activated receptor may mediate the responses to peroxisome proliferators by inducing responsive genes, the ligand(s) of this receptor remains unknown . We hypothesized that induction of L-FABP and ACBP in rat liver by PFDA is secondary to accumulation of long-chain acyl-CoA esters . However, neither dose-response nor time-course effects of PFDA on hepatic long-chain acyl-CoA, L-FABP, or ACBP concentrations confirmed this hypothesis . In a dose-response study, PFDA increased hepatic long-chain acyl-CoA concentrations (7 days after treatment) over the dose range of 20-50 mg/kg, whereas it increased ACBP and L-FABP over the wider dose range of 20-65 mg/kg . In the time-course study, PFDA treatment (50 mg/kg) elevated long-chain acyl-CoA esters in the liver beginning on day 3 post-treatment, yet hepatic L-FABP concentrations were increased earlier beginning on day 2 and ACBP was not induced until day 7 . To determine if this dissociation of increases in hepatic long-chain acyl-CoA concentrations from increases in hepatic L-FABP and ACBP concentrations could be demonstrated under other conditions, control rats fasted for 24-48 hr were used . Fasting increased hepatic long-chain acyl-CoA levels to a greater extent than PFDA treatment, yet neither L-FABP nor ACBP was induced . We conclude that elevated concentrations of hepatic long-chain acyl-CoAs in PFDA-treated rats are not a major contributor to the induction of L-FABP or ACBP by peroxisome proliferators . A more plausible mechanism is that PFDA induces L-FABP and ACBP by activating the peroxisome proliferator receptor directly rather than indirectly through long-chain acyl-CoA esters.

J Chromatogr A, 1994 Aug 26, 678(1), 25 - 34
Immobilized metal-ion affinity partitioning of NAD(+)-dependent dehydrogenases in poly(ethylene glycol)-dextran two-phase systems; Pesliakas H et al.; Affinity partitioning of yeast alcohol dehydrogenase (YADH), lactate dehydrogenase from rabbit muscle (MLDH) and lactate and malate dehydrogenases from pig heart (HLDH and HMDH, respectively) were studied in aqueous two-phase systems containing metal ions (Cu2+, Ni2+, Zn2+ and Cd2+) chelated by iminodiacetate-poly(ethylene glycol) (IDA-PEG) . The partitioning behaviour of the enzymes in the presence of Cu(II)-IDA-PEG was studied as a function of the concentration of NaCl, the pH of the medium and the concentration of added selected agents . It was demonstrated that the partition effect (delta log K) of dehydrogenases in the presence of Cu(II)-IDA-PEG and the affinity of enzymes for immobilized Cu2+ ions increases in the order MLDH > YADH > HMDH > or = HLDH . It was shown that the determined variations in the enzyme affinities for Cu(II)-IDA-PEG might be related to the differences in the content of histidine residues accessible to the solvent.

Nature, 1994 Aug 25, 370(6491), 655 - 8
Structure and transport mechanism of a high-affinity potassium uptake transporter from higher plants; Schachtman DP et al.; Potassium is the most abundant cation in higher plants and is crucial for plant nutrition, growth, tropisms, enzyme homeostasis and osmoregulation . K+ accumulation can be rate-limiting for agricultural production . K+ uptake from soils into roots is largely mediated by high-affinity K+ uptake (Km approximately 10-40 microM) (refs 1, 2, 5-7) . But although K+ channels allow low-affinity K+ uptake, both the transport mechanism and structure of the high-affinity K+ nutrition pathway remain unknown . Here we use expression cloning to isolate a complementary DNA encoding a membrane protein (HKT1) from wheat roots which confers the ability to take up K+ . The substrate affinity, saturation and cation selectivity of HKT1 correspond to hallmark properties of classical high-affinity K+ uptake in plants . The transport mechanism of HKT1 uses K(+)-H+ co-uptake . Expression of HKT1 is localized to specific root and leaf regions which represent primary sites for K+ uptake in plants . HKT1 is important for plant nutrition and could possibly contribute to environmental alkali metal toxicities.

Biochemistry, 1994 Aug 23, 33(33), 10185 - 90
Effects of molecular crowding on protein self-association: a potential source of error in sedimentation coefficients obtained by zonal ultracentrifugation in a sucrose gradient; Cann JR et al.; Theoretical and experimental studies have illustrated a potential source of error in sedimentation coefficients obtained by sucrose density gradient centrifugation of proteins undergoing reversible self-association . The error stems from the excluded volume (molecular crowding) effect of the sucrose on the activity coefficients of monomeric and polymeric states . The consequent displacement of the equilibrium position in favor of polymeric state(s) is a function of sucrose concentration, and can therefore result in failure to detect the equilibrium coexistence of monomer if 5% sucrose suffices to displace the equilibrium completely toward dimer . In less extreme situations, it may result in the evaluation of an average sedimentation coefficient whose magnitude is a function of sucrose concentration and hence of the distance migrated into the sucrose gradient . These features are illustrated by the results of computer-simulated sedimentation of reversibly dimerizing systems in a sucrose gradient, and by conventional sedimentation velocity experiments on yeast enolase.

J Biol Chem, 1994 Aug 19, 269(33), 20995 - 1002
Chimeric enzymes . Structure-function analysis of segments of sn-1,2-diacylglycerol choline- and ethanolaminephosphotransferases; Hjelmstad RH et al.; The Saccharomyces cerevisiae CPT1 and EPT1 genes represent structural genes that encode distinct choline- and choline/ethanolaminephosphotransferases, respectively . To explore the function of linear segments of these enzymes, a series of 14 EPT1-CPT1 chimeric gene constructs and the parental wild-type genes were expressed in a cpt1 ept1 double null mutant background completely devoid of phosphoamino alcohol transferase activity . Eleven of the chimeric genes expressed functional enzymes . The CDP-amino alcohol and sn-1,2-diacylglycerol (DAG) substrate specificities and essential phospholipid cofactor requirements of the parental and chimeric enzymes were investigated using a mixed micellar assay system . Chimeric enzymes exhibited a pattern of CDP-amino alcohol affinities that defined a structural domain sufficient to confer CDP-amino alcohol specificity . When wild-type enzymes were investigated using a chemically defined series of DAGs, each possessed a distinct characteristic pattern of utilization . Chimeric enzymes exhibited DAG acyl chain specificity profiles that either conformed to parental wild-type patterns or represented novel substrate specificities . Correlation of these outcomes with their underlying structural modifications permitted the assignment of an internal, linear region of 218 amino acids sufficient to confer DAG acyl chain specificity; this region contained three predicted transmembrane segments . Neither wild-type enzyme showed significant acyl chain selectivity with respect to phospholipid activation when a homologous series of chemically defined phosphatidylcholines were employed, suggesting that enzyme recognition of the fatty acyl moieties of the DAG substrate and phospholipid activator is fundamentally different . Analysis of chimeric enzymes dependence on phospholipid activators suggested the involvement of discontinuous protein segments participating in the interaction with phospholipid cofactors.

J Biol Chem, 1994 Aug 19, 269(33), 20943 - 51
Mechanistic studies of amsacrine-resistant derivatives of DNA topoisomerase II . Implications in resistance to multiple antitumor drugs targeting the enzyme; Wasserman RA et al.; Wild-type yeast DNA topoisomerase II and three of its amsacrine-resistant derivatives L475A/L480P, L475A/R476G, and A642G, named according to amino acid changes at the codons specified, were overexpressed and purified . Because cells expressing several mutant enzymes missing portions of the carboxyl-terminal domain of the wild-type enzyme were previously found to exhibit amsacrine resistance, a carboxyl-terminal truncation protein Top2(1-1166), which lacks the last 263 amino acids of the wild-type enzyme, was also overexpressed and purified . These purified enzymes were used in the measurement of the turnover numbers of the DNA-dependent hydrolysis of ATP, the transport of one DNA segment through another, and the effects of amsacrine, teniposide or Ca(II) on the formation of the enzyme-DNA covalent intermediate . The results of these studies indicate that mutations leading to cellular resistance to amsacrine may occur by several different mechanisms, including reduction of the nuclear concentration and attenuation of the intrinsic catalytic steps of the enzyme . The significance of this underpinning mechanistic diversity of drug resistance and its relation to the simultaneous development of cellular resistance to chemically distinct drugs that target DNA topoisomerase II are discussed.

Nature, 1994 Aug 18, 370(6490), 578 - 81
Premature translational termination triggers mRNA decapping; Muhlrad D et al.; The degradation of messenger RNA in eukaryotic cells is initiated by endonucleolytic cleavage or by shortening of the poly(A) tail, which for some mRNAs activates a deadenylation-dependent decapping reaction . One type of rapid mRNA degradation in eukaryotes is caused by premature termination of translation . This turnover process prevents the translation of aberrant mRNAs, may affect the abundance and splicing pattern of nuclear transcripts, and may be involved in the aetiology of human genetic disease . Here we show that premature translational termination in yeast triggers decapping, independent of deadenylation, thereby exposing the transcript to 5'-to-3' degradation . Inactivation of the 5'-to-3' exonuclease reveals an additional 3'-to-5' pathway of mRNA turnover . These observations provide in vivo evidence for two new mechanisms of mRNA decay.

Proc Natl Acad Sci U S A, 1994 Aug 16, 91(17), 8180 - 4
A regulatory system for use in gene transfer; Wang Y et al.; We recently have demonstrated that a C-terminal deletion mutant of the human progesterone receptor (hPRB891) fails to bind to progesterone but can bind RU 486 (Mifepristone) and other progesterone antagonists . Most significantly, this mutant receptor activates transcription of a reporter gene containing the progesterone response element in the presence of these antagonists . Taking advantage of this finding and the modular nature of functional domains of steroid receptors, we constructed a chimeric regulator (pGL-VP) by fusing the ligand-binding domain of human progesterone receptor hPRB891 to the yeast transcriptional activator GAL4 DNA-binding domain and the herpes simplex virus protein VP16 activation domain . We demonstrated that this chimeric regulator activates target genes containing the GAL4-binding sites in transient transfection assays in response to RU 486 . In addition, this regulatory system has been validated by ex vivo transplantation of a stable cell line containing both the regulator and a reporter gene into rats . The dosage of RU 486 used is significantly lower than that required for antagonizing progesterone action . The gene-switch system reported here represents a regulatory system, which could be applicable for gene-transfer studies involving animals, as well as humans, in which the delivered gene(s) can be specifically turned on/off in response to an exogenous compound.

Proc Natl Acad Sci U S A, 1994 Aug 16, 91(17), 8142 - 6
Cloning and sequence analysis of the rat augmenter of liver regeneration (ALR) gene: expression of biologically active recombinant ALR and demonstration of tissue distribution; Hagiya M et al.; A full-length cDNA clone encoding a purified augmenter of liver regeneration (ALR) factor prepared from the cytosol of weanling rat livers was isolated . The 1.2-kb cDNA included a 299-bp 5' untranslated region, a 375-bp coding region, and a 550-bp 3' untranslated region . It encoded a protein consisting of 125 amino acids . The molecular weight of ALR calculated from the cDNA was 15,081, which is consistent with the size estimated by SDS/PAGE under reducing conditions . The molecular weight of the purified native ALR estimated by SDS/PAGE under nonreducing conditions was approximately 30,000; thus ALR apparently has a homodimeric structure . The recombinant ALR produced by expression of the cDNA in COS cells was tested in vivo in the canine Eck fistula model and found to have potency equivalent to the purified native ALR . The 125-aa sequence deduced from the rat ALR cDNA shows 50% homology to the amino acid sequence of the gene for oxidative phosphorylation and vegetative growth in the yeast Saccharomyces cerevisiae.

FEBS Lett, 1994 Aug 15, 350(1), 46 - 50
Nystatin changes the properties of transporters for arginine and sugars . An in vitro study; Opekarova M et al.; In ergosterol-containing energized yeast plasma membrane vesicles nystatin (5-10 micrograms/mg total lipid) caused a massive efflux of pre-accumulated arginine while the membrane potential (the principal driving force; -110 mV) decreased by only 10-30 mV . Neither the substrate fluxes nor the membrane potential was influenced by nystatin when the permease was reconstituted in ergosterol-free phospholipid vesicles . The same effect of nystatin was found with the reconstituted sugar transporter from Chlorella kessleri . It is suggested that nystatin binding to ergosterol in the vicinity of the permease releases the transport protein from its coupling to energy and converts it to a facilitator.

Biochem Biophys Res Commun, 1994 Aug 15, 202(3), 1452 - 9
A novel member, PC7, of the mammalian kexin-like protease family: homology to PACE4A, its brain-specific expression and identification of isoforms; Tsuji A et al.; By polymerase chain reaction (PCR) with primers corresponding to the sequences of catalytic domain conserved among the mammalian kexin-like protease family, a cDNA fragment encoding a novel member of the family was obtained from rat pituitary . A cDNA for the novel protease was obtained from three overlapping clones isolated from a rat pituitary cDNA library using the PCR product as a screening probe . The protein, designated as PC7, encoded by this cDNA contained a putative activation site with the sequence RXKR, subtilisin-like catalytic domain and a homo B region which are typical of mammalian kexin-like proteases, and short cysteine-rich region at the carboxyl-terminal end . It exhibited surprising sequence similarity to PACE4A . The PC7 transcript was expressed at high levels in the brain . However it was undetectable in the liver, kidney, heart, and spleen . The presence of PC7 isoforms (PC7A and PC7B) was also shown.

Biochem Biophys Res Commun, 1994 Aug 15, 202(3), 1215 - 21
The tissue distribution of mRNAs for the PACE4 isoforms, kexin-like processing protease: PACE4C and PACE4D mRNAs are major transcripts among PACE4 isoforms; Tsuji A et al.; In the previous study {Biochem . Biophys . Res . Commun . (1994) 200, 943-950} we identified two novel cDNAs (PACE4C and PACE4D) encoding human Kexin-like protease, the PACE4 isoforms . In this study, we examined the expression of PACE4 isoform transcripts in various rat tissues . To detect very low levels and to distinguish among these isoforms, we used the reverse transcriptase-polymerase chain reaction (RT-PCR) . PACE4C and PACE4D transcripts were detected in most tissues like PACE4A transcripts, however their tissue distribution profiles and the extent of expression differ . PACE4C and PACE4D transcripts are expressed at a much higher level than PACE4A transcript . These results indicate that PACE4C and PACE4D mRNAs are major transcripts of PACE4.

Genes Dev, 1994 Aug 15, 8(16), 1920 - 34
Mot1, a global repressor of RNA polymerase II transcription, inhibits TBP binding to DNA by an ATP-dependent mechanism; Auble DT et al.; Basal transcription of many genes in yeast is repressed by Mot1, an essential protein which is a member of the Snf2/Swi2 family of conserved nuclear factors . ADI is an ATP-dependent inhibitor of TATA-binding protein (TBP) binding to DNA that inhibits transcription in vitro . Here we demonstrate that ADI is encoded by the MOT1 gene . Mutation of MOT1 abolishes ADI activity and derepresses basal transcription in vitro and in vivo . Recombinant Mot1 removes TBP from DNA and Mot1 contains an ATPase activity which is essential for its function . Genetic interactions between Mot1 and TBP indicate that their functions are interlinked in vivo . These results provide a general model for understanding the mechanism of action of a large family of nuclear factors involved in processes such as transcription and DNA repair.

Cell, 1994 Aug 12, 78(3), 513 - 23
Purification, cloning, and characterization of a human coactivator, PC4, that mediates transcriptional activation of class II genes; Ge H et al.; Activator-dependent transcription in mammalian cells requires upstream stimulatory activity (USA)-derived cofactors in addition to those present in TFIID . A novel positive cofactor (PC4) purified from the human USA fraction effected a marked enhancement (up to 85-fold) of GAL4-AH-dependent transcription in conjunction with TFIID and other general factors . Isolation of a corresponding cDNA identified PC4 as a 127 residue single-stranded DNA-binding protein with serine-rich regions near the N-terminus . Recombinant PC4 was functionally equivalent to native PC4, and both proteins markedly enhanced activation by diverse activation domains fused to the DNA-binding domain of GAL4 . Recombinant PC4 interacted independently both with free or DNA-bound VP16 activation domains and with free or DNA-bound TFIIA-TBP complexes (but not with TBP alone) . These results indicate that PC4 is a general coactivator that functions cooperatively with TAFs and mediates functional interactions between upstream activators and the general transcriptional machinery.

J Mol Biol, 1994 Aug 12, 241(2), 273 - 4
The crystallization and preliminary X-ray analysis of allosteric chorismate mutase; Xue Y et al.; An allosteric chorismate mutase, the Thr226-->Ile mutant, from the yeast Saccharomyces cerevisiae has been crystallized in space group P6(1)(P6(5)) using the hanging drop vapour diffusion method at room temperature . The cell dimensions are a = b = 95.8 A, c = 157.9 A, alpha = beta = 90 degrees, gamma = 120 degrees . It contains a dimer in the crystallographic asymmetric unit . The crystal diffracts to 2.2 A resolution . A native data set has been collected to 82% completeness at this resolution.

J Biol Chem, 1994 Aug 12, 269(32), 20674 - 81
Negative regulation of Sp1 trans-activation is correlated with the binding of cellular proteins to the amino terminus of the Sp1 trans-activation domain; Murata Y et al.; Sp1 is a well characterized and ubiquitously expressed transcription factor that regulates the constitutive and induced expression of a variety of mammalian genes . It is unclear whether Sp1 activity is regulated in vivo; the mechanism by which Sp1 interacts with the basal transcription complex has not been firmly established . We report the identification of a ubiquitously expressed and evolutionarily conserved nuclear protein, p74, that specifically binds Sp1 in vivo and in vitro . p74 interacts with several portions of the Sp1 trans-activation domain in vitro, and we correlate the binding of p74 to the amino-terminal serine/threonine-rich subdomain of Sp1 with the inhibition of Sp1-mediated transcription in vivo.

Nucleic Acids Res, 1994 Aug 11, 22(15), 3053 - 60
An adenovirus E1A transcriptional repressor domain functions as an activator when tethered to a promoter; Bondesson M et al.; The adenovirus E1A protein contains three well conserved regions, designated conserved region (CR) 1, 2 and 3, which are important for the multiple activities ascribed to E1A . The CR3 domain constitutes a prototypic transcription activator, consisting of a promoter targeting region and a transactivating region . Here we demonstrate the existence of a second transactivating region located within amino acids 28 to 90 (essentially the CR1 domain) of the E1A protein . A fusion protein, containing the Gal4 DNA binding domain linked to CR1, was as efficient as the classical CR3 transactivator in activating transcription from a reporter plasmid containing Gal4 binding sites . However, competition experiments suggest that Gal/CR1 and Gal/CR3 work through different cellular targets . The E1A-243R protein has previously been extensively characterized as a repressor of transcription . Here we show that a Gal4 fusion protein expressing the CR1 domain is indeed sufficient for repression of SV40 enhancer activity . Collectively, our results suggest that CR1 functions as an activator if tethered to a promoter and as a repressor in the absence of promoter association.

Nucleic Acids Res, 1994 Aug 11, 22(15), 2908 - 14
Repression of transcriptional activity at a distance by the evolutionarily conserved KRAB domain present in a subfamily of zinc finger proteins; Pengue G et al.; Sub-families of related zinc finger protein genes have been defined on the basis of evolutionarily conserved structural features found outside the C2-H2 finger repeats . Such elements include the FAX domain found in a large number of Xenopus ZFPs, the evolutionarily conserved KRAB (Kruppel-associated box) and the ZiN (zinc finger N-terminal) domains . Here we describe a new evolutionarily conserved motif within zinc finger proteins which we have named the leucine rich region (LeR) . Since conserved modules in regulatory proteins may specify properties relevant to their action we have determined the functional capabilities of LeR and the KRAB domains in the regulation of gene transcription by fusing relevant regions to a heterologous DNA-binding domain (GAL4 DNA-binding domain) . We found that the KRAB-A domain tethered to RNA polymerase II promoters by a GAL4 DNA-binding domain actively represses transcription in a distance-independent manner . KRAB-mediated repression is dependent on the dose of the GAL4-KRAB-A fusion protein and on the presence of GAL4 binding sites on the DNA . Conversely, the LeR domain did not modulate significantly the transcription . Our results indicate that the KRAB domain present in the non-finger region of many ZFP genes quenches transcription possibly due to specific protein-protein interactions between the KRAB-A domain and components of the proximal transcriptional apparatus.

Nucleic Acids Res, 1994 Aug 11, 22(15), 2882 - 6
A histidine accepting tRNA-like fold at the 3'-end of satellite tobacco mosaic virus RNA; Felden B et al.; A model of secondary structure is proposed for the 3'-terminal sequence of the satellite tobacco mosaic virus (STMV) RNA on the basis of phylogenetic comparisons with tobacco mosaic virus (TMV) genomic RNA . Sequence homologies and compensatory base changes found between the two related viral RNAs imply that the 3'-end of STMV RNA folds into a tRNA-like domain similar to that found in the TMV RNA . Accordingly, functional assays showed that STMV RNA can be aminoacylated in vitro with histidine by yeast histidyl-tRNA synthetase to plateaus reaching 30% . Histidylation properties of STMV RNA were compared to those of TMV RNA and of a canonical yeast tRNA(His) transcript which both are chargeable to nearly 100% plateau levels . Kinetic data indicate an excellent catalytic efficiency of STMV RNA charging expressed as Vmax/Km ratio, quasi-equivalent to that of TMV RNA, and only 17-fold reduced as compared to that of the yeast tRNAHis transcript . Biological implications of the structural mimicry between the tRNA-like regions of TMV and STMV RNAs are discussed in the light of the relationships of a satellite virus with its helper virus . This is the first report on a chargeable tRNA-like structure at the 3'-end of a satellite virus RNA.

Nature, 1994 Aug 11, 370(6489), 481 - 5
Facilitated binding of TATA-binding protein to nucleosomal DNA; Imbalzano AN et al.; BINDING of the TATA-binding protein (TBP) to the TATA box is required for transcription from many eukaryotic promoters in gene expression . Regulation of this binding is therefore likely to be an important determinant of promoter activity . Incorporation of the TATA sequence into nucleosomes dramatically reduces transcription initiation, presumably because of stereochemical constraints on binding of general transcription factors . Biochemical and genetic studies imply that cellular factors such as yeast SWI/SNF are required for activator function and might alter chromatin structure . One step that could be regulated during the activation process is TBP binding in chromatin 12, 13 . We show here that binding of TBP to the TATA sequence is severely inhibited by incorporation of this sequence into a nucleosome . Inhibition can be overcome by ATP-dependent alterations in nucleosomal DNA structure mediated by hSWI/SNF, a putative human homologue of the yeast SWI/SNF complex . Additionally, the orientation of the TATA sequence relative to the surface of the histone core affects the access of TBP . We propose that the dynamic remodelling of chromatin structure to allow TBP binding is a key step in the regulation of eukaryotic gene expression.

Nature, 1994 Aug 11, 370(6489), 477 - 81
Nucleosome disruption and enhancement of activator binding by a human SW1/SNF complex; Kwon H et al.; CHROMATIN structure can affect the transcriptional activity of eukaryotic structural genes by blocking access of sequence-specific activator proteins (activators) to their promoter-binding sites . For example, the DNA-binding domain of the yeast GAL4 protein interacts very poorly with nucleosome cores compared with naked DNA2 (and see below), and binding of other activators is even more strongly inhibited . The way in which activators bind to nucleosomal DNA is therefore a critical aspect of transcriptional activation . Genetic studies have suggested that the multi-component SWI/SNF complex of Saccharomyces cerevisiae facilitates transcription by altering the structure of the chromatin . Here we identify and partially purify a human homologue of the yeast SWI/SNF complex (hSWI/SNF complex) . We show that a partially purified hSWI/SNF complex mediates the ATP-dependent disruption of a nucleosome, thereby enabling the activators, GAL4-VP16 and GAL4-AH, to bind within a nucleosome core . We conclude that the hSWI/SNF complex acts directly to reorganize chromatin structure so as to facilitate binding of transcription factors.

Science, 1994 Aug 5, 265(5173), 806 - 8
An osmosensing signal transduction pathway in mammalian cells; Galcheva-Gargova Z et al.; The osmotic balance between the cytoplasmic and extracellular compartments of cells is critical for the control of cell volume . A mammalian protein kinase, Jnk, which is a distant relative of the mitogen-activated protein kinase group, was activated by phosphorylation on threonine and tyrosine in osmotically shocked cells . The activation of Jnk may be relevant to the biological response to osmotic shock because the expression of human Jnk in the yeast Saccharomyces cerevisiae rescued a defect in growth on hyper-osmolar media . These data indicate that related protein kinases may mediate osmosensing signal transduction in yeast and mammalian cells.

Science, 1994 Aug 5, 265(5173), 808 - 11
A MAP kinase targeted by endotoxin and hyperosmolarity in mammalian cells; Han J et al.; Mammalian cells respond to endotoxic lipopolysaccharide (LPS) by activation of protein kinase cascades that lead to new gene expression . A protein kinase, p38, that was tyrosine phosphorylated in response to LPS, was cloned . The p38 enzyme and the product of the Saccharomyces cerevisiae HOG1 gene, which are both members of the mitogen-activated protein (MAP) kinase family, have sequences at and adjacent to critical phosphorylation sites that distinguish these proteins from most other MAP kinase family members . Both HOG1 and p38 are tyrosine phosphorylated after extracellular changes in osmolarity . These findings link a signaling pathway in mammalian cells with a pathway in yeast that is responsive to physiological stress.

Proc Natl Acad Sci U S A, 1994 Aug 2, 91(16), 7762 - 6
Complexes between STE5 and components of the pheromone-responsive mitogen-activated protein kinase module; Marcus S et al.; We present genetic evidence for complex formation of STE5 and the STE11, STE7, and FUS3 protein kinases, the pheromone-responsive mitogen-activated protein kinase module of Saccharomyces cerevisiae . Interaction between STE5 and STE11 is not dependent on STE7, and interaction between STE5 and STE7 does not require STE11 . The N-terminal regulatory domain of STE11 is both necessary and sufficient for interaction with STE5 . Interaction between STE7 and STE11 is bridged by STE5, suggesting the formation of a multiprotein complex . We also demonstrate biochemical interaction between STE5 and STE11 by using a combination of bacterially expressed fusion proteins and extracts prepared from yeast . Our results suggest that STE5 is a scaffolding protein that facilitates interactions between components of the pheromone-responsive mitogen-activated protein kinase module . We further propose that such scaffolding proteins serve to inhibit cross-talk between functionally unrelated mitogen-activated protein kinase modules within the same cell.

Proc Natl Acad Sci U S A, 1994 Aug 2, 91(16), 7623 - 7
Involvement of the Ku autoantigen in the cellular response to DNA double-strand breaks; Rathmell WK et al.; The Ku autoantigen is a well-characterized heterodimer of 70 and 86 kDa that binds to DNA ends, but its cellular function has been obscure . An electrophoretic mobility-shift assay and Ku antisera were used to show that Ku or a closely related protein was deficient in three mutant hamster cell lines from x-ray-sensitive complementation group 5, which is characterized by defects in DNA double-strand break repair and V(D)J recombination . Furthermore, Ku protein expression was restored when the cells reverted to x-ray resistance . The Ku p86 gene maps to human chromosome 2q33-35, and group 5 cells are rescued by almost precisely the same region, 2q34-36 . Thus, biochemical and genetic evidence suggests that Ku is involved in pathways for DNA recombination and repair . By its association with a DNA-dependent protein kinase activated by DNA ends, Ku may also initiate a signaling pathway induced by DNA damage, perhaps for cell cycle arrest.

Biochim Biophys Acta, 1994 Aug 2, 1218(3), 435 - 8
Primary structure and expression of a gene encoding the cytosolic ribosomal protein S4 from potato; Braun HP et al.; The primary structure of a cDNA clone encoding the S4 protein from the small subunit of 80S ribosomes from potato was determined . Cytosolic ribosomal protein S4 is hydrophilic and has a prevalence for positively charged residues . In potato it is 264 amino acids long and contains a putative nuclear targeting signal close to the N-terminus . Having 65-69% identical amino acids cytosolic ribosomal protein S4 from mammals, fungi and plants belongs to the highly conserved proteins . The S4 gene is transcribed in all potato tissues analysed and has a relatively high expression level in comparison to nuclear genes encoding mitochondrial proteins.

Biochim Biophys Acta, 1994 Aug 2, 1218(3), 308 - 14
Sp1, USF, and GAL4 activate transcription independently of TFIID-downstream promoter interactions; Wang JC et al.; One way specific transcription factors are thought to activate transcription initiation is by facilitating interactions between the general transcription factor TFIID and DNA sequences downstream of the TATA element . Examples supporting this model include transcription activation from the core adenovirus E4 promoter by either the human gene-specific transcription factor ATF or the acidic-domain fusion protein GAL4-AH . In these cases, appearance of downstream promoter binding by TFIID correlated directly with transcription activation by these proteins . Previously we had shown that downstream promoter binding by TFIID involved recognition of the initiator DNA control element and that the extent of this binding correlated directly with initiator-dependent transcription activation in vitro . We now report our use of DNase I footprinting and in vitro transcription to investigate the effects of various stimulatory transcription factors on TFIID binding and transcription efficiency from different initiator-containing promoters . Transcription factors investigated included Sp1, USF, and several GAL4-acidic domain fusion proteins . We found that none of these transcription factors appreciably affected downstream promoter binding by TFIID, whether qualitatively or quantitatively . In fact, all of these transcription factors stimulated transcription in vitro regardless of the strength of the initiator element present . When both elements were present, transcription stimulation mediated by proximally bound transcription factors and by TFIID-initiator interaction seemed to be synergistic . Taken together, our data would suggest that transcription activation by these two means occurs through different steps within the transcriptional process.

Bioessays, 1994 Aug, 16(8), 541 - 7
The establishment of active promoters in chromatin; Becker PB; The organization of eukaryotic genomes as chromatin provides the framework within which regulated transcription occurs in the nucleus . The association of DNA with chromatin proteins required to package the genome into the nucleus is, in general, inhibitory to transcription, and therefore provides opportunities for regulated transcriptional activation . Granting access to the cis-acting elements in DNA, a prerequisite for any further action of the trans-acting factors involved, requires the establishment of local heterogeneity of chromatin and, in some cases, extensive remodeling of nucleosomal structures . Challenging problems relate to the establishment of this heterogeneity at the level of the single nucleosome and to the mechanisms that operate when nucleosomal arrays are reorganized . Recent developments indicate that chromatin reconstitution in cell-free systems allows the biochemical analysis of the interplay between transcription factors and chromatin components that brings about regulated transcription.

Appl Environ Microbiol, 1994 Aug, 60(8), 2779 - 85
The cel3 gene of Agaricus bisporus codes for a modular cellulase and is transcriptionally regulated by the carbon source; Chow CM et al.; A 52-kDa protein, CEL3, has been separated from the culture filtrate of Agaricus bisporus during growth on cellulose . A PCR-derived probe was made, with a degenerate oligodeoxynucleotide derived from the amino acid sequence of a CEL3 CNBr cleavage product and was used to select cel3 cDNA clones from an A . bisporus cDNA library . Two allelic cDNAs were isolated . They showed 98.8% identity of their nucleotide sequences . The deduced amino acid sequence and domain architecture of CEL3 showed a high degree of similarity to those of cellobiohydrolase II of Trichoderma reesei . Functional expression of cel3 cDNA in Saccharomyces cerevisiae was achieved by placing it under the control of a constitutive promoter and fusing it to the yeast invertase signal sequence . Recombinant CEL3 secreted by yeast showed enzymatic activity towards crystalline cellulose . At long reaction times, CEL3 was also able to degrade carboxymethyl cellulose . Northern (RNA) analysis showed that cel3 gene expression was induced by cellulose and repressed by glucose, fructose, 2-deoxyglucose, and lactose . Glycerol, mannitol, sorbitol, and maltose were neutral carbon sources . Nuclear run-on analysis showed that the rate of synthesis of cel3 mRNA in cellulose-grown cultures was 13 times higher than that in glucose-grown cultures . A low basal rate of cel3 mRNA synthesis was observed in the nuclei isolated from glucose-grown mycelia.

DNA Cell Biol, 1994 Aug, 13(8), 857 - 63
Molecular cloning of a chick cochlea cDNA encoding a subunit of DNA replication factor C/activator 1; Oberholtzer JC et al.; A chick cochlea cDNA library was constructed to clone molecules involved in peripheral auditory transduction and in the maintenance and regeneration of the sensory neuroepithelium following damage . Characterization of the library showed it to be of high complexity, to contain a high proportion of full-length cDNA inserts, and to contain a representative proportion of clones derived from hair cell transcripts . A cDNA clone encoding the chick homolog of the 40-kD subunit of the human replication factor C (also called activator 1) was isolated and the complete cDNA sequence determined . The predicted amino acid sequence is about 90% identical to that of the human homolog . Expression of the message for this replication factor was detected in brain and liver as well as in the cochlea . Expression levels in the brain are relatively high and are similar in developing and adult chicken nervous tissue . This suggests that replication factor C message expression, unlike that for the functionally associated factor proliferating cell nuclear antigen (PCNA), may be constitutive rather than cell cycle dependent . Although likely to be involved in DNA replication within the receptor neuroepithelium, expression of this replication factor message is not likely to constitute a marker for proliferation.

Eur J Biochem, 1994 Aug 1, 223(3), 947 - 56
High affinity of ergopeptides for cytochromes P450 3A . Importance of their peptide moiety for P450 recognition and hydroxylation of bromocriptine; Peyronneau MA et al.; The interaction between rat and human liver cytochromes P450 with a series of lysergic acid derivatives and ergopeptide alkaloids was studied by difference visible spectroscopy . Ergopeptides, like bromocriptine, ergocryptine and dihydroergotamine, strongly interacted with rat liver microsomes with the appearance of a difference spectrum which is characteristic of their binding to a protein site close to the heme . The intensity of this spectrum was clearly dependent on the amounts of P450s 3A in the microsomes and was at its maximum in dexamethasone-treated rat microsomes . All the ergopeptides studied exhibited a high affinity for rat P450s 3A (Ks around 1 microM), although lysergic acid derivatives not bearing the tripeptide moiety failed to give significant interactions with these P450s . A cyclic azatripeptide exhibiting a structure very similar to that of the tripeptide moiety of ergopeptides also interacted with P450s 3A with appearance of an intense type I difference spectrum . Very similar results were observed with two allelic forms of human liver P450 3A4, P450 NF25 and P450 hPCN1, produced in yeast . In both cases all the ergopeptides studied showed high affinities for the P450s (Ks 0.6-2.2 microM) and an intense shift from the low-spin to the high-spin state upon substrate binding (60-100% spin shift) . Lysergic acid derivatives not bearing the tripeptide group of ergopeptides also completely failed to interact with P450s 3A4 . Liver microsomes from rats pretreated with dexamethasone, a specific inducer of P450 3A, were found to be particularly active for the hydroxylation of bromocriptine, which occurs at the level of its tripeptide moiety . Human liver microsomes as well as P450 NF25 and P450 hPCN1 also exhibited a high activity for bromocriptine hydroxylation at this level . These results show that ergopeptides exhibit a particularly high affinity for P450s of the 3A subfamily . The tripeptide moiety of ergopeptides is essential for their recognition by P450s 3A and binds at a site close to P450 heme, producing type-I difference spectra . Accordingly, at least one of the studied ergopeptides, bromocriptine, is hydroxylated by P450s 3A at the proline ring of the cyclopeptide moiety . As cyclosporine is known to be a good substrate of P450s 3A, these results suggest that P450s 3A may be especially prone in a general manner to recognize and oxidize peptides or pseudopeptides.

J Cell Biol, 1994 Aug, 126(4), 935 - 43
Sec72p contributes to the selective recognition of signal peptides by the secretory polypeptide translocation complex; Feldheim D et al.; SEC72 encodes the 23-kD subunit of the Sec63p complex, an integral ER membrane protein complex that is required for translocation of presecretory proteins into the ER of Saccharomyces cerevisiae . DNA sequence analysis of SEC72 predicts a 21.6-kD protein with neither a signal peptide nor any transmembrane domains . Antibodies directed against a carboxyl-terminal peptide of Sec72p were used to confirm the membrane location of the protein . SEC72 is not essential for yeast cell growth, although an sec72 null mutant accumulates a subset of secretory precursors in vivo . Experiments using signal peptide chimeric proteins demonstrate that the sec72 translocation defect is associated with the signal peptide rather than with the mature region of the secretory precursor.

FEBS Lett, 1994 Aug 1, 349(2), 222 - 8
The polytopic mitochondrial inner membrane proteins MIM17 and MIM23 operate at the same preprotein import site; Kubrich M et al.; Three proteins of the mitochondrial inner membrane are known that are essential for the viability of yeast and seem to be involved in import of preproteins; the integral membrane proteins MIM17 and MIM23 and the peripheral membrane protein MIM44, MIM17 and MIM23 are homologous to each other in their hydrophobic domain, expose their termini to the intermembrane space, and span the inner membrane up to four times, each . A preprotein in transit across the mitochondrial membrane is specifically cross-linked to MIM17, MIM23, MIM44, and matrix hsp70 . We conclude that MIM17 and MIM23 are integral parts of a preprotein translocation channel and cooperate with MIM44 and hsp70 at the same protein import site.

J Clin Endocrinol Metab, 1994 Aug, 79(2), 372 - 6
Naturally occurring mutations in human steroid 21-hydroxylase influence adrenal autoantibody binding; Asawa T et al.; Human 21-hydroxylase (21-OH) genes containing various mutations, truncations, and deletions were expressed in yeast, and autoantibody binding was studied by Western blotting using patient sera and rabbit antibodies to 21-OH . 21-OH autoantibodies in 13 Addisonian sera showed a marked reduction in their ability to recognize 21-OH mutated at Pro453-->Ser (mean +/- SD, 31 +/- 9% of binding to wild type), whereas the effect on rabbit antibody binding was small (88 +/- 11% of binding to wild type; n = 7) . Mutation at Arg339-->His had a less pronounced effect on autoantibody binding (85 +/- 11% of binding to wild type; n = 13) and caused a small enhancement of rabbit antibody binding (124 +/- 16% of binding to wild type; n = 7) . These studies indicate that Pro453 has a key role in forming an autoantigenic epitope on 21-OH . It is important to note, however, that the Pro453 mutation caused only partial loss of autoantibody binding, i.e . all Addisonian sera studied still reacted with the mutated protein . This may indicate that each serum sample contains at least two different populations of 21-OH autoantibodies, only one of which recognizes a site dependent on Pro453 . A series of more extensive modifications of the 21-OH sequence, including truncations (amino acids 460-494, 448-494, and 418-494) and deletions (amino acids 165-379, 142-240, and 142-280) indicated that most of the sequence of amino acids from 241-494 is important for autoantibody binding . The involvement of such an extensive region of the molecule suggests that the binding sites are generated by three-dimensional folding, with Pro453 having a critical role in forming at least one major autoantigenic epitope.

J Cell Biol, 1994 Aug, 126(3), 619 - 30
Genetic and physical interactions between Srp1p and nuclear pore complex proteins Nup1p and Nup2p; Belanger KD et al.; Nup1p is a yeast nuclear pore complex protein (nucleoporin) required for nuclear protein import, mRNA export and maintenance of normal nuclear architecture . We have used a genetic approach to identify other proteins that interact functionally with Nup1p . Here we describe the isolation of seventeen mutants that confer a requirement for Nup1p in a background in which this protein is normally not essential . Some of the mutants require wild-type Nup1p, while others are viable in combination with specific nup1 alleles . Several of the mutants show nonallelic noncomplementation, suggesting that the products may be part of a hetero-oligomeric complex . One is allelic to srp1 which, although it was identified in an unrelated screen, was shown to encode a protein that is localized to the nuclear envelope (Yano, R., M . Oakes, M . Yamaghishi, J . A . Dodd, and M . Nomura . 1992 . Mol . Cell . Biol . 12:5640-5651) . We have used immunoprecipitation and fusion protein precipitation to show that Srp1p forms distinct complexes with both Nup1p and the related nucleoporin Nup2p, indicating that Srp1p is a component of the nuclear pore complex . The distant sequence similarity between Srp1p and the beta-catenin/desmoplakin family, coupled with the altered structure of the nuclear envelope in nup1 mutants, suggests that Srp1p may function in attachment of the nuclear pore complex to an underlying nuclear skeleton.

J Bacteriol, 1994 Aug, 176(15), 4754 - 6
Halobacterial S9 operon contains two genes encoding proteins homologous to subunits shared by eukaryotic RNA polymerases I, II, and III; McKune K et al.; One key component of the eukaryotic transcriptional apparatus is the multisubunit enzyme RNA polymerase II . We have discovered that two of the subunits shared by the three nuclear RNA polymerases in the yeast Saccharomyces cerevisiae, RPB6 and RPB10, have counterparts among the Archaea.

Mol Cell Biol, 1994 Aug, 14(8), 5547 - 57
Molecular architecture of a Leu3p-DNA complex in solution: a biochemical approach; Remboutsika E et al.; The Leu3 protein (Leu3p) of Saccharomyces cerevisiae is a pleiotropic transregulator that can function both as an activator and as a repressor of transcription . It binds to upstream promoter elements (UASLEU) with the consensus sequence 5'-GCCGGNNCCGGC-3' . The DNA-binding motif of Leu3p belongs to the family of Zn(II)2-Cys6 clusters . The motif is located between amino acid residues 37 and 67 of the 886-residue protein . In this study, we used a recombinant peptide consisting of residues 17 to 147 to explore the interaction between Leu3p and its cognate DNA . We found that the Leu3p(17-147) peptide is a monomer in the absence of UASLEU but assumes a dimeric structure when the DNA is present . Results of protein-DNA cross-linking and methylation and ethylation interference footprinting experiments show that the Leu3p(17-147) dimer interacts symmetrically with two contact triplets separated by 6 bp and suggest that the peptide approaches its target DNA in such a way that each subunit is positioned closer to one DNA strand than to the other . The binding of Leu3p is strongly affected by the spacing between the contact triplets of the UASLEU and by the type of triplet . Binding occurs when the triplets are 6 bp apart (normal spacing) but fails to occur when the triplets are 0, 5, or 8 bp apart . Weak binding occurs when the triplets are 7 bp apart . Binding does not occur when the UASLEU triplets (GCC....GGC) are replaced with triplets found in the UAS elements for Gal4p, Put3p, and Ppr1p (CGG....CCG) . The apparent Kd for the normal Leu3p(17-147)-UASLEU complex is about 3 nM . A mutant form of Leu3p(17-147) in which the histidine at position 50 has been replaced with cysteine binds UASLEU with significantly greater affinity (apparent Kd of about 0.7 nM), even though the interaction between the mutant peptide and target DNA appears to be unchanged . Interestingly, repression of basal-level transcription, which is a hallmark property of the wild-type Leu3p(17-147) peptide, is largely lost with the mutant peptide, indicating that there is no direct correlation between strength of binding and repression.

Mol Cell Biol, 1994 Aug, 14(8), 5290 - 9
A cell-specific factor represses stimulation of transcription in vitro by transcriptional enhancer factor 1; Chaudhary S et al.; Transcription in HeLa cell extracts in vitro was stimulated 8- to 10-fold by a recombinant chimera, GAL-TEF-1, consisting of the DNA-binding domain of GAL4 and the activation function of the HeLa cell activator TEF-1 . In contrast, only a 2- to 3-fold stimulation was obtained with GAL-TEF-1 in extracts from BJA-B lymphoid cells . Stimulation by GAL-TEF-1 in BJA-B extracts was dramatically increased by the addition of immunopurified HeLa cell TFIID, suggesting that BJA-B TFIID lacks or contains lower quantities of a TATA-binding-protein-associated factor(s) required for the activity of the TEF-1 activation function . However, chromatography, immunopurification, and transcriptional reconstitution experiments indicated that BJA-B extracts did not lack the previously identified TATA-binding-protein-associated factors required for TEF-1 activity but rather contained a negatively acting factor(s) which inhibited transactivation by GAL-TEF-1 . These results indicate that the relative lack of activity of the TEF-1 activation function in vitro in BJA-B cell extracts does not result from the absence of positively acting factors from the presence of a cell-specific negatively acting factor(s).

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