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