Pharmacology, 1991, 43(5), 247 - 55 Studies for the elucidation of the mode of action of the antimycotic hydroxypyridone compound, rilopirox; Kruse R et al.; Rilopirox is a synthetic, fungicidal antimycotic agent with hydrophobic characteristics . Its chemical name is 6-{4-(4-chlorophenoxy)-phenoxy-methyl}-1-hydroxy-4-methyl-2-pyridone and it has a molecular weight of 357.79 . Rilopirox is very soluble in dimethyl sulfoxide (DMSO) and dimethylformamide (DMF) but poorly soluble in water . The amount of antimycotic agent remaining in the solution is dependent on the final concentration of the solvent and the amount of rilopirox used . Complexometric studies show that rilopirox has a high affinity for iron ions {unpubl . data} . Catalase, an iron-containing enzyme, is inhibited by the chelating agent rilopirox . Studies on yeast mitochondria and submitochondrial particles show that rilopirox inhibits the respiratory chain . Complex I (NADH-ubiquinone oxidoreductase) contains iron-sulfur proteins and is the main system which is inhibited. Ann Ist Super Sanita, 1991, 27(1), 105 - 14 Expression vectors and gene transfer; Presutti C et al.; The development of DNA cloning techniques, together with the possibility of reintroducing cloned DNA fragments into the genome of a living organism, has led to an extraordinary growth of our knowledge in molecular biology over the past twenty years . In the present paper a brief overview of the vectors and techniques used in transforming different groups of organisms is given . The importance and applications of genetic engineering for each group (yeasts, plants, Drosophila and mammals) will be discussed. Proteins, 1991, 10(2), 156 - 61 Cu(II)-binding properties of a cytochrome c with a synthetic metal-binding site: His-X3-His in an alpha-helix; Todd RJ et al.; A metal-binding site consisting of two histidines positioned His-X3-His in an alpha-helix has been engineered into the surface of Saccharomyces cerevisiae iso-1-cytochrome c . The synthetic metal-binding cytochrome c retains its biological activity in vivo . Its ability to bind chelated Cu(II) has been characterized by partitioning in aqueous two-phase polymer systems containing a polymer-metal complex, Cu(II)IDA-PEG, and by metal-affinity chromatography . The stability constant for the complex formed between Cu(II)IDA-PEG and the cytochrome c His-X3-His site is 5.3 x 10(4) M-1, which corresponds to a chelate effect that contributes 1.5 kcal mol-1 to the binding energy . Incorporation of the His-X3-His site yields a synthetic metal-binding protein whose metal affinity is sensitive to environmental conditions that alter helix structure or flexibility. Int J Biochem, 1991, 23(7-8), 761 - 8 Further investigations regarding the role of trimethyllysine for cytochrome c uptake into mitochondria; Cessay KJ et al.; 1 . A mutant of the iso-1-cytochrome c gene from Saccharomyces cerevisiae has been constructed which contains an Arg codon, replacing the normal trimethylated Lys at position 77 . 2 . This mutated gene was cloned into a pGem 1 vector and used for the in vitro translation of yeast iso-1-cytochrome c . 3 . Utilizing an in vitro mitochondria binding assay, it was found that the mutant cytochrome c could transverse the yeast mitochondrial membrane, however the amount of protein incorporated was 3-fold less that of the trimethylated wild type . 4 . Omission of the protein methyltransferase from assays containing the wild type cytochrome c caused only a slight reduction (15%) in the amount of protein incorporated . 5 . These results suggest while the lysine residue 77 of apocytochrome c is important for mitochondria uptake, the methylation of this residue seems to play a relatively minor role. SAAS Bull Biochem Biotechnol, 1991 Jan, 4, 76 - 80 Identification of sites of pre-MRNA/spliceosome association; Rymond BC; RNase H and synthetic DNA oligonucleotides were used to analyze the ribonucleoprotein (RNP) structure of the yeast spliceosome and to assay the pre-mRNA sequence requirements for step 1 of splicing . The data suggest that tight, stable contacts between the pre-mRNA and the spliceosome may be limited to the 5' splice site and branch point regions of the intron . A 30 nucleotide segment 3' of the branch point was found to be necessary for spliceosome maturation and essential for step 1 of splicing . Somewhat surprisingly, the 3' splice site was sensitive to nuclease digestion and completely dispensable for step 1 of splicing. Bioseparation, 1991, 2(4), 237 - 46 Enzyme purification using temperature-induced phase formation; Harris PA et al.; A new type of aqueous two-phase system composed of an ethylene oxide and propylene oxide random co-polymer, UCON 50-HB-5100, as the upper phase polymer and either dextran or hydroxypropyl starch as the lower phase polymer has been characterized and used to purify 3-phosphoglycerate kinase (EC 2.7.2.3) and hexokinase (EC 2.7.1.1) from bakers' yeast . The UCON 50-HB-5100 polymer has a cloud point of 55 degrees C at which temperature it phase separates from water . This cloud point can be lowered to 40 degrees C by the addition of 0.2 M sodium sulfate salt . The low cloud point of this UCON polymer makes it possible to obtain the target enzymes in a water and buffer solution, and to recover and recycle the UCON 50-HB-5100 polymer . The phase diagrams for the systems UCON 50-HB-5100/Dextran T500 and UCON 50-HB-5100/hydroxypropyl starch have been determined . Yeast homogenate was first partitioned in a system composed of a top phase containing UCON 50-HB-5100 and a bottom phase containing either dextran or hydroxypropyl starch . The top phase containing the enzyme free of cell debris was removed and the temperature increased above the cloud point of the UCON until a new two phase system composed of water as the top phase and a concentrated liquid UCON 50-HB-5100 bottom phase was formed . The water phase containing the enzyme was removed and the bottom phase containing the UCON 50-HB-5100 could be recycled to perform a second extraction. SAAS Bull Biochem Biotechnol, 1991 Jan, 4, 17 - 9 Eukaryotic gene regulation: simple vs complex models; Swaffield JC et al.; The current generally accepted model of eukaryotic gene regulation is essentially a simple one . Regulatory proteins containing separable DNA binding and transcriptional activation domains, bind to specific DNA sequences in promotors and interact directly or indirectly with the TATA Box binding factor to increase the rate of transcription initiation at selected promotors . Here we present observations suggesting that the process may be more complex. Biochem Biophys Res Commun, 1990 Dec 31, 173(3), 849 - 54 Isoprenoid metabolism in Plasmodium falciparum during the intraerythrocytic phase of malaria; Mbaya B et al.; Products of the isoprenoid metabolism were identified upon incubations of extracts from Plasmodium falciparum infected red blood cells with {14C} mevalonate . Uninfected erythrocytes and wild type yeast Saccharomyces cerevisiae extracts were used as controls . In parasitized red blood cells as well as in yeast extracts, mevalonate was converted into the biosynthetic isoprenoid precursors of sterol pathway until farnesyl pyrophosphate . In contrast, no mevalonate conversion was observed in uninfected erythrocyte extracts . The isoprenoid metabolism appeared stage-dependent as shown by the increase of radiolabelled farnesyl pyrophosphate amount at the beginning of the schizogonic phase (30-36 hours). J Biol Chem, 1990 Dec 25, 265(36), 22243 - 54 Rous sarcoma virus enhancer factor I is a ubiquitous CCAAT transcription factor highly related to CBF and NF-Y; Faber M et al.; We have characterized enhancer factor I (EFI), a trans-acting factor present in avian nuclear extracts which binds to the Rous sarcoma virus long terminal repeat enhancer and promoter . Through deletion and point mutagenesis, we show that EFI is a member of the CCAAT family of transcription factors . Although the CCAAT motif is essential for protein-DNA recognition, EFI shows surprising latitude in the nucleotide sequences flanking the CCAAT motif to which it will bind with high affinity . EFI will cross-bind to the binding sites of a number of previously described CCAAT factors, including CBF, NF-Y, CP2, CP1, CTF/NF-1, and c/EBP, with a range of affinities that is at most 10-fold lower than the high affinity binding site for EFI in the Rous sarcoma virus long terminal repeat . We present evidence, however, that EFI is probably identical or very closely related to CBF and NF-Y . This is based on the fact that EFI in avian nuclear extracts binds with equal or 2-fold greater affinity to the binding sites of NF-Y and CBF, despite less than 50% homology (outside the CCAAT motif) between the EFI, NF-Y, and CBF recognition sequences . Moreover, radiolabeled EFI, NF-Y, or CBF DNAs give rise to identical gel retardation patterns in extracts from a variety of different cell types . EFI, CBF, and NF-Y appear to fractionate identically upon ion exchange chromatography, separating into two heterologous components (A and B) which must be recombined to recover substantial DNA binding activity . Molecular weight estimates for the two heterologous components of EFI, CBF, and a Y-box binding protein (Celada, A., and Maki, R.A . (1989) Mol . Cell . Biol . 9, 3097-3100) are very similar . EFI DNA binding activity has recently been shown to be induced by serum and the oncogene v-src (Dutta, A., Stoeckle, M.Y., and Hanafusa, H . (1990) Genes & Dev . 4, 243-254) . The close relationship or identity between EFI, CBF, and NF-Y, thus has important implications regarding the mechanisms by which serum or the oncogene v-src may affect changes in gene expression. Nucleic Acids Res, 1990 Dec 25, 18(24), 7349 - 55 Site-directed, recombination-mediated mutagenesis of a complex gene locus; Barton MC et al.; We have generated a site-specific 17 bp insertion within a 38 kb chick globin gene cluster by employing the recombination abilities of Saccharomyces cerevisiae . This gene cluster contains four beta-type globin genes which share a high degree of sequence homology . In this procedure, a small fragment of beta A-globin DNA containing a 17 bp insertion is subcloned into a URA3-based yeast integrating vector (YIp) . This mutated globin subclone is introduced into cells that carry the 38 kb globin cluster clone on a single-copy, circular vector derived from a yeast artificial chromosome (YAC) . Insertion of the 17 bp oligomer is achieved by targeted integration of the Ylp subclone . The recombinant contains the normal beta A-globin gene, the mutant gene and Ylp vector sequences between the two copies . Excision of the vector sequences and one copy of the duplicated globin sequences by homologous recombination is required for cell survival when exposed to the selective agent 5-fluoroorotic acid, which is toxic to ura+ yeast cells . Depending upon the point of the cross-over, a ura- yeast cell bearing either a wild-type globin gene or a 17 bp insertion mutation will result . By restriction mapping and in vitro transcription analysis, the beta A-globin gene containing the 17 bp insert has no nonspecific mutations generated during the recombination and selection procedures . Specific mutations of regulatory regions, including protein-DNA binding sites, can be accurately targeted within extensive DNA clones by this method. Biochemistry, 1990 Dec 18, 29(50), 11095 - 100 Tick anticoagulant peptide: kinetic analysis of the recombinant inhibitor with blood coagulation factor Xa; Jordan SP et al.; Tick anticoagulant peptide (TAP) is a 60 amino acid protein which is a highly specific inhibitor of human blood coagulation factor Xa (fXa) isolated from the tick Ornithodoros moubata {Waxman, L., Smith, D . E., Arcuri, K . E., & Vlasuk, G . P . (1990) Science 248, 593-596} . Due to the limited quantities of native TAP, a recombinant version of TAP produced in Saccharomyces cerevisiae was used for a detailed kinetic analysis of the inhibition interaction with human fXa . rTAP was determined to be a reversible, slow, tight-binding inhibitor of fXa, displaying a competitive type of inhibition . The binding of rTAP to fXa is stoichiometric with a dissociation constant of (1.8 +/- 0.02) x 10(-10) M, a calculated association rate constant of (2.85 +/- 0.07) x 10(6) M-1 s-1, and a dissociation rate constant of (0.554 +/- 0.178) x 10(-3) s-1 . Binding studies show that 35S-rTAP binds only to fXa and not to DFP-treated fXa or zymogen factor X, which suggests the active site of fXa is required for rTAP inhibition . That rTAP is a unique serine proteinase inhibitor is suggested both by its high specificity for its target enzyme, fXa, and also by its unique structure. FEBS Lett, 1990 Dec 17, 277(1-2), 281 - 4 Precursor proteins in transit through mitochondrial contact sites interact with hsp70 in the matrix; Ostermann J et al.; We previously reported that hsp70 in the mitochondrial matrix (mt-hsp 70 = Ssclp) is required for import of precursor proteins destined for the matrix or intermembrane space . Here we show that mt-hsp70 is also needed for the import of mitochondrial inner membrane proteins . In particular, the precursor of ADP/ATP carrier that is known not to interact with hsp60 on its assembly pathway requires functional mt-hsp70 for import, suggesting a general role of mt-hsp70 in membrane translocation of precursors . Moreover, a precursor arrested in contact sites was specifically co-precipitated with antibodies directed against mt-hsp70 . We conclude that mt-hsp70 is directly involved in the translocation of many, if not all, precursor proteins that are transported across the inner membrane. J Biol Chem, 1990 Dec 15, 265(35), 21779 - 88 Protein-based asymmetry and protein-protein interactions in FLP recombinase-mediated site-specific recombination; Qian XH et al.; When the FLP recombination target (FRT) is cut in half so that only one FLP protein-binding site is present, FLP protein forms a complex in which two such sites are linked head to head . Although held together exclusively by noncovalent interactions, this complex survives electrophoresis in an agarose gel and exhibits a half-life that can be measured in hours . Characterization of this complex indicates that a very stable, asymmetric dimeric complex of FLP protein monomers bound to the FRT is a likely early intermediate in FLP-mediated site-specific recombination . The apparent asymmetry is a property of the protein components of the complex . Even though the DNA components form a perfect palindrome, only one of the two possible DNA cleavage steps takes place in the course of complex formation . Formation of this complex does not occur with half-FRT site DNA substrates that preclude head to head monomer contact or when a FLP mutant protein is used that binds the FRT site but cannot cleave it . Trimeric and tetrameric complexes are also observed, the latter at very low frequency . These results are discussed in terms of an expanded model for early events in FLP-mediated site-specific recombination. J Biol Chem, 1990 Dec 15, 265(35), 21664 - 9 Ubiquitin-mediated degradation of histone H3 does not require the substrate-binding ubiquitin protein ligase, E3, or attachment of polyubiquitin chains; Haas A et al.; Radioiodinated histone H3 was incubated with ubiquitin, the ubiquitin-activating enzyme E1, and one of three ubiquitin carrier proteins, reticulocyte E2(20K) or E2(32K) or the yeast RAD6 product . Although the resulting ubiquitin-histone conjugates were synthesized in the absence of the substrate-binding protein E3, they were nevertheless degraded by purified rabbit reticulocyte 26 S protease . In contrast, unmodified histone H3 remained intact upon challenge with the 26 S ubiquitin/ATP-dependent enzyme . Conjugates produced by the RAD6 protein were better proteolytic substrates than those formed by reticulocyte E2 unless ubiquitin molecules with altered lysines were used for conjugate synthesis . Substitution of methylated ubiquitin or ubiquitin molecules in which lysine 48 was converted to arginine by site-directed mutation produced histone conjugates that were degraded at slow but measurable rates . Since methylated ubiquitin molecules are incapable of forming branched polyubiquitin chains, these results demonstrate that neither ubiquitin "trees" nor the substrate binding factor E3 is absolutely required for ubiquitin-dependent degradation of histone H3 in vitro. J Biol Chem, 1990 Dec 15, 265(35), 21468 - 75 Structure and function of the mitochondrial bc1 complex . Properties of the complex in temperature-sensitive cor1 mutants; Gatti DL et al.; The properties of the ubiquinol-cytochrome c reductase complex (bc1 complex) have been studied in respiratory defective mutants of Saccharomyces cerevisiae bearing lesions in the core 1 subunit . All the cor1 mutants examined have greatly reduced concentrations of mitochondrial cytochrome b and display succinate-cytochrome c reductase activities near the limits of detection . Two mutants (E576 and C7), however, had 5% of wild type activity when the cells were grown at 23 degrees C, but not at 37 degrees C . The temperature-sensitive phenotype was determined to result from substitution of either Arg or Glu for Gly68 of the core 1 subunit . The respiratory competent revertants E576/R8 and C7/R4 derived from E576 and C7 retain the temperature sensitivity of the original mutants . Both revertants are temperature sensitive in vivo, but only mitochondria isolated from E576/R8 are temperature sensitive in vitro . The bc1 complex of mitochondria isolated from this revertant displays a normal value of the ratio Kcat/Km for cytochrome c and four times higher than the wild type for duroquinol . The succinate-cytochrome c reductase activity of E576/R8 is almost completely abolished after incubation at 37 degrees C for 90 min . It is inferred that the quaternary structure of ubiquinol-cytochrome c reductase complex is more labile at the nonpermissive temperature in the mutant and undergoes an alteration such that cytochrome b is no longer able to receive electrons through either the "o" or the "i" site pathway . The temperature lability and kinetic properties of the mutant enzyme point to a requirement of the core 1 not only for assembly but also for the catalytic activity of the complex. J Immunol, 1990 Dec 15, 145(12), 4222 - 8 Antigenic determinants of the Ku (p70/p80) autoantigen are poorly conserved between species; Porges AJ et al.; The Ku (p70/p80) autoantigen is a DNA-protein complex recognized by sera from certain patients with SLE and related diseases . Although human autoantibodies react with at least eight different epitopes of the human Ku complex, they had little reactivity with rodent Ku Ag on immunoblots . Small amounts of 70- and 80-kDa proteins were immunoprecipitated from murine cell extracts, however, suggesting that the Ku particle is not unique to human cells . This was confirmed by isolating cDNA clones encoding murine Ku Ag by plaque hybridization with a human p70 cDNA probe . The murine p70 cDNA clones had a deduced amino acid sequence 82.9% identical to that of human p70, and comparable amounts of murine and human p70 mRNA were detected in 3T3 and K562 cells, respectively . The poor reactivity of human autoantibodies with murine p70 was attributable to specific amino acid substitutions in an immunodominant conformational epitope located on amino acids 560-609 of human p70 . Several amino acids critical for antigenicity of this region were defined by mutagenesis studies . Other conformational epitopes of Ku were also antigenically poorly conserved among species . Species-specific epitopes recognized by lupus autoantibodies are unusual but not unique to Ku . In general, poorly conserved autoepitopes have been conformational, rather than sequential, suggesting that the antigenicity of conformational epitopes may be particularly sensitive to evolutionary change. FEBS Lett, 1990 Dec 10, 276(1-2), 49 - 53 Complete assignment of the 1H NMR spectrum and secondary structure of the DNA binding domain of GAL4; Gadhavi PL et al.; Complete 1H NMR resonance assignments are presented for the cysteine rich region of the DNA binding domain of the yeast transcriptional activator GAL4 . The protein contains short helical regions between Asp-12 and Leu-19 and between Lys-30 and Trp-36 . It is clearly distinct from the C2H2 class of zinc finger protein typified by the Xenopus laevis transcription factor (TF)IIIA . We also find that the first SP(X)(X) sequence, a recently proposed DNA binding motif (residues 41 to 44), appears to be tightly packed against the metal binding domain. FEBS Lett, 1990 Dec 10, 276(1-2), 123 - 6 Proteolytic activation of a bovine brain protein with phosphatidylinositol transfer activity; van den Akker WM et al.; We have purified a 38 kDa protein from bovine brain which is cross-reactive with an affinity purified antibody against the 35 kDa phosphatidylinositol transfer protein from the same source . Controlled trypsinization of the 38 kDa protein yielded an immunoreactive protein of 35 kDa which displayed a 6-fold increase in phosphatidylinositol transfer activity and a 10-fold higher affinity for this phospholipid . The possibility that the 38 kDa protein is a precursor of the phosphatidylinositol transfer protein is discussed. J Mol Biol, 1990 Dec 5, 216(3), 585 - 610 Modelling of the three-dimensional architecture of group I catalytic introns based on comparative sequence analysis; Michel F et al.; Alignment of the 87 available sequences of group I self-splicing introns reveals numerous instances of covariation between distant sites . Some of these covariations cannot be ascribed to historical coincidences or the known secondary structure of group I introns, and are, therefore, best explained as reflecting tertiary contacts . With the help of stereochemical modelling, we have taken advantage of these novel interactions to derive a three-dimensional model of the conserved core of group I introns . Two noteworthy features of that model are its extreme compactness and the fact that all of the most evolutionarily conserved residues happen to converge around the two helices that constitute the substrate of the core ribozyme and the site that binds the guanosine cofactor necessary for self-splicing . Specific functional implications are discussed, both with regard to the way the substrate helices are recognized by the core and possible rearrangements of the introns during the self-splicing process . Concerning potential long-range interactions, emphasis is put on the possible recognition of two consecutive purines in the minor groove of a helix by a GAAA or related terminal loop. J Biol Chem, 1990 Dec 5, 265(34), 21011 - 5 The cytosolic-binding protein for the immunosuppressant FK-506 is both a ubiquitous and highly conserved peptidyl-prolyl cis-trans isomerase; Siekierka JJ et al.; We have recently isolated an abundant cytosolic protein from human T-cells which specifically binds the immunosuppressive agent, FK-506 . The FK-506-binding protein (FKBP) is a member of a novel class of proteins possessing peptidyl-prolyl cis-trans isomerase activity . These proteins are believed to play an important role in accelerating the rate at which proteins fold into their native conformations . In the present study, we demonstrate that FKBP is not a lymphoid-specific protein, but is widely distributed and phylogenically conserved . FKBP, purified from three sources (a human T-lymphocyte cell line JURKAT, bovine calf thymus, and Saccharomyces cerevisiae) exhibit identical molecular weights, immunological cross-reactivities, and a high degree of NH2-terminal amino acid sequence homology . In addition, FKBP from all sources possesses peptidyl-prolyl cis-trans isomerase activity which can be specifically inhibited by FK-506 . We conclude that FKBP may serve an important biological function in all eukaryotic cells. J Cell Biol, 1990 Dec, 111(6 Pt 2), 2829 - 37 Targeting of a cytosolic protein to the nuclear periphery; Hurt EC; The yeast nuclear envelope protein NSP1 is located at the nuclear pores and mediates its essential function via the carboxy-terminal domain . The passenger protein, cytosolic dihydrofolate reductase from mouse, was fused to the 220 residue long NSP1 carboxy-terminal domain . When expressed in yeast, this chimeric protein was tightly associated with nuclear structures and was localized at the nuclear periphery very similar to authentic NSP1 . Furthermore, the DHFR-C-NSP1 fusion protein was able to complement a yeast mutant lacking a functional NSP1 gene showing that DHFR-C-NSP1 fulfils the same basic function as compared to the endogenous NSP1 protein . These data also show that the NSP1 protein is composed of separate functional moieties: a carboxy-terminal domain that is sufficient to mediate the association with the nuclear periphery and an amino-terminal and middle repetitive domain with an as yet unknown function . It is suggested that heptad repeats found in the NSP1 carboxy-terminal domain, which are similar to those found in intermediate filament proteins, are crucial for mediating the association with the nuclear pores. DNA Cell Biol, 1990 Dec, 9(10), 783 - 8 Defining target sequences of DNA-binding proteins by random selection and PCR: determination of the GCN4 binding sequence repertoire; Mavrothalassitis G et al.; We developed a simple and accurate method to define the sequence recognition properties of DNA-binding proteins . The method employs polymerase chain reaction (PCR) amplification of sequences selected from a mixture of random oligonucleotides by the gel mobility-shift assay . We used this method to define the sequence requirement of the binding domain of the yeast transcriptional activator GCN4 . Using a total of 200 ng of purified protein and four cycles of binding and subsequent amplification, we identified the TGA-(C/G)TCA sequence as the binding consensus of GCN4, which is consistent with the previously reported recognition sequence . In addition, our data indicate that GCN4 can bind with lower affinity to sequences that differ from the optimal sequence in one or even two positions . The most common variation was the C to A at position +2 . The majority of the substitutions that still allowed binding were 3' to the central C residue indicating that the two sides of the palindromic recognition sequence are not equivalent. Proc Natl Acad Sci U S A, 1990 Dec, 87(24), 9761 - 5 In vitro regulation of a SIN3-dependent DNA-binding activity by stimulatory and inhibitory factors; Wang H et al.; The yeast SIN3 gene (also known as SDII, is a known negative regulator of the yeast HO gene . A DNA-binding activity, called SDP1, which binds to the HO promoter, is absent in extracts prepared from sin3 mutants and has been proposed to function as a repressor . We show that SIN3 does not encode SDP1 and that SDP1 DNA-binding activity is modulated in vitro by two factors, an inhibitory factor, I-SDP1, and a stimulatory factor, S-SDP1 . I-SDP1 acts as an in vitro inhibitor of the SDP1 DNA-binding activity . Restoration of the DNA-binding activity is achieved by inclusion of a stimulatory factor, S-SDP1, which copurifies with the SIN3 protein . SDP1 DNA-binding activity was restored by treating a protein fraction containing SDP1 and I-SDP1 with the dissociating agent formamide. Mol Cell Biol, 1990 Dec, 10(12), 6472 - 81 Human cells contain a DNA-activated protein kinase that phosphorylates simian virus 40 T antigen, mouse p53, and the human Ku autoantigen; Lees-Miller SP et al.; HeLa cells contain a serine/threonine protein kinase (DNA-PK) that is strongly activated in vitro by low concentrations of double-stranded DNA (dsDNA) . Activation was specific for dsDNA; both natural DNAs and synthetic oligonucleotides functioned as kinase activators . The fact that DNA-PK activity was rapidly inhibited by incubation with dsDNA and ATP suggests that DNA-PK activity also may be regulated by autophosphorylation . During gel filtration, DNA-PK activity behaved as a 350-kDa protein, and highly purified DNA-PK contained a dsDNA-binding, 350-kDa polypeptide that was phosphorylated in a dsDNA-dependent manner . We conclude that this 350-kDa polypeptide is likely to be DNA-PK . Previously we showed that the dsDNA-activated kinase phosphorylates two threonines at the N terminus of hsp90 alpha (S . P . Lees-Miller and C . W . Anderson, J . Biol . Chem . 264:17275-17280, 1989) . Here we show that DNA-PK also phosphorylates the simian virus 40 large tumor antigen, the mouse tumor-suppressor protein p53, the human Ku autoantigen, and two unidentified HeLa DNA-associated polypeptides of 52 and 110 kDa . Identification of these and other newly identified DNA-binding substrates suggest that the dsDNA-activated kinase may regulate transcription, DNA replication, or cell growth. J Clin Microbiol, 1990 Dec, 28(12), 2733 - 8 Characterization of the sequence of colonization and nosocomial candidemia using DNA fingerprinting and a DNA probe; Reagan DR et al.; The objective of this hospital-based study was to determine the relationship between colonizing and infecting strains of Candida species and Torulopsis glabrata . Surveillance cultures from high-risk patients were paired with subsequent bloodstream isolates . Organisms were typed by using restriction endonuclease digestion of chromosomal DNA with BstNI and EcoRI, followed by Southern hybridization with a DNA probe (pBD4) derived from Saccharomyces cerevisiae . Sixteen patients for whom documented colonization preceded documented bloodstream infection were identified . The mean time between obtainment of surveillance isolates and obtainment of bloodstream isolates was 8 days, with a range of 1 to 423 days . For 15 (94%) of 16 patients, the DNA fingerprint pattern (using BstNI) of the surveillance isolate was identical to that of the bloodstream isolate . Isolates from 13 (81%) of 16 patients were unique to those patients . Typing by Southern hybridization with the pBD4 probe was less discriminating . We conclude that for a well-defined subset of hospitalized patients who were colonized by Candida species before developing nosocomial candidemia, the colonizing and infecting strains were identical, suggesting endogenous acquisition of infection . Restriction endonuclease digestion of chromosomal DNA was shown to be a discriminating and reproducible typing method for Candida species and T . glabrata. J Cell Biol, 1990 Dec, 111(6 Pt 1), 2353 - 63 Import of ADP/ATP carrier into mitochondria: two receptors act in parallel; Steger HF et al.; We have identified the yeast homologue of Neurospora crassa MOM72, the mitochondrial import receptor for the ADP/ATP carrier (AAC), by functional studies and by cDNA sequencing . Mitochondria of a yeast mutant in which the gene for MOM72 was disrupted were impaired in specific binding and import of AAC . Unexpectedly, we found a residual, yet significant import of AAC into mitochondria lacking MOM72 that occurred via the receptor MOM19 . We conclude that both MOM72 and MOM19 can direct AAC into mitochondria, albeit with different efficiency . Moreover, the precursor of MOM72 apparently does not require a positively charged sequence at the extreme amino terminus for targeting to mitochondria. J Cell Physiol, 1990 Dec, 145(3), 514 - 21 Phagocytosis in Acanthamoeba: II . Soluble and insoluble mannose-rich ligands stimulate phosphoinositide metabolism; Allen PG et al.; The generation of second messengers during phagocytosis of yeast by Acanthamoeba castellanii was examined . The kinetics of binding and internalization of yeast by Acanthamoeba were measured and this was compared with the generation of known second messengers . We observed stimulated degradation of PI-4, 5-P2 to 1,4,5 IP3 with kinetics similar to that observed for the binding of yeast to amoeba . Similar production of IP3 could be induced upon treatment with a soluble mannosylated glycoprotein . We propose that the Acanthamoeba mannose receptor stimulates the degradation of PI-4, 5-P2 to 1,4,5 IP3 as an initial event in phagocytosis. Proc Natl Acad Sci U S A, 1990 Dec, 87(24), 9751 - 5 Vitamin D receptor interaction with specific DNA requires a nuclear protein and 1,25-dihydroxyvitamin D3; Liao J et al.; The regulation of osteocalcin gene expression by 1,25-dihydroxyvitamin D3 is mediated by the vitamin D receptor and a cis-acting DNA response element that has been identified within the 5' region of the osteocalcin promoter . In this report, we show that vitamin D receptors derived from nuclear extracts of mammalian cells bind directly to this cis-acting element in vitro and do so in a manner requiring hormone . Vitamin D receptors derived from reticulocyte lysate translations in vitro or from extracts of a Saccharomyces cerevisiae strain that expresses the recombinant protein also bind the osteocalcin responsive element, but only when nuclear extracts of mammalian cells are provided . The vitamin-D-receptor-DNA-binding accessory factor is isolated by salt extraction, labile to temperature, and sensitive to tryptic digestion . These studies suggest that the high-affinity interaction of the vitamin D receptor with the osteocalcin vitamin D response element in vitro requires both 1,25-dihydroxyvitamin D3 and an accessory protein derived from the mammalian cell nucleus. Am J Physiol, 1990 Dec, 259(6 Pt 1), C987 - 94 Synthesis, transfer, and phosphorylation of phosphoinositides in cardiac membranes; Wolf RA; Compartmentation of phosphoinositide synthesis and transfer of endogenous phosphatidylinositol (PI) were characterized in membrane fractions prepared from rabbit myocardium . De novo synthesis of PI was highly enriched in free sarcoplasmic reticulum (551 pmol.mg-1 . min-1) compared with that in sarcolemma (26.8 pmol.mg-1 . min-1) and junctional sarcoplasmic reticulum (178 pmol.mg-1 . min-1) . In contrast, PI phosphorylation was highly enriched in sarcolemma (2.69 nmol.mg-1.min-1) compared with that in free sarcoplasmic reticulum (0.22 nmol.mg-1.min-1) and junctional sarcoplasmic reticulum (0.38 nmol.mg-1.min-1) . Phosphorylation of endogenous phosphatidylinositol 4-phosphate to phosphatidylinositol 4,5-bisphosphate was also enriched in sarcolemma (38.5 pmol.mg-1.min-1) compared with that in free sarcoplasmic reticulum (less than 5.0 pmol.mg-1.min-1) and junctional sarcoplasmic reticulum (6.5 pmol.mg-1.min-1) . Transfer of endogenous PI was characterized as a mechanism to overcome compartmentation of PI synthesis in cardiac membranes . A 29-kDa PI transfer protein was purified 1,500-fold from cytosol of rabbit myocardium . Both cytosol and purified PI transfer protein catalyzed the transfer of endogenous PI from microsomal sites of synthesis to sarcolemma . In conclusion, synthesis of PI is highly enriched in free sarcoplasmic reticulum, whereas phosphorylation of phosphoinositides is highly enriched in sarcolemma . A 29-kDa PI transfer protein in myocardial cytosol can mediate in vitro transfer of de novo-synthesized PI to the sarcolemma. Genes Dev, 1990 Dec, 4(12B), 2264 - 77 Multiple roles for U6 snRNA in the splicing pathway; Madhani HD et al.; U6 is the most highly conserved of the five spliceosomal RNAs . It is associated with U4 by an extensive base-pairing interaction, which is disrupted immediately prior to the first nucleolytic step of splicing . It has been proposed that this event activates catalysis by unmasking U6 . Using a combination of doped synthesis and site-directed mutagenesis to generate point mutations in U6, we have now identified 12 positions, in three domains, at which single nucleotide substitutions or deletions result in lethal or temperature-sensitive phenotypes . Biochemical analysis demonstrates that most of these mutants retain the ability to assemble into U4/U6 and U4/U5/U6 snRNPs . Notably, although mutations at three positions in U6 that base-pair with U4 are lethal, mutations in the complementary residues in U4 are fully viable . Furthermore, compensatory mutations in U4 that restore base-pairing fail to suppress the phenotypes of the U6 mutations . This demonstrates a function for U6 independent of its role in base-pairing . Remarkably, two of the three essential regions in U6 identified genetically correspond to intron insertion points in two yeast species . A temperature-sensitive mutation at one of these sites is defective in the second step of splicing in vitro. Genetics, 1990 Dec, 126(4), 1127 - 38 A polymerization model of chiasma interference and corresponding computer simulation; King JS et al.; A model of chiasma interference is proposed and simulated on a computer . The model uses random events and a polymerization reaction to regulate meiotic recombination between and along chromosomes . A computer simulation of the model generates distributions of crossovers per chromosome arm, position of events along the chromosome arm, distance between crossovers in two-event tetrads, and coincidence as a function of distance . Outputs from the simulation are compared to data from Saccharomyces cerevisiae and the X chromosome of Drosophila melanogaster . The simulation demonstrates that the proposed model can produce the regulation of recombination observed in both genetic and cytological experiments . While the model was quantitatively compared to data from only Drosophila and Saccharomyces, the regulation observed in these species is qualitatively similar to the regulation of recombination observed in other organisms. Proc Natl Acad Sci U S A, 1990 Dec, 87(23), 9148 - 52 cDNA clone encoding Drosophila transcription factor TFIID; Muhich ML et al.; Proper initiation of transcription by RNA polymerase II requires the TATA-consensus-binding transcription factor TFIID . A cDNA clone encoding the Drosophila TFIID protein has been isolated and characterized . The deduced amino acid sequence reveals an open reading frame of 353 residues . The carboxyl-terminal 180 amino acids are approximately 80% identical to yeast TFIID and 88% identical to human TFIID . The amino-terminal portions of the yeast and Drosophila TFIID proteins lack appreciable homology, whereas the Drosophila and human amino termini appear qualitatively similar . In addition, the amino-terminal region of the Drosophila TFIID contains several sequence motifs that are found in other Drosophila proteins which appear to regulate transcription. Mol Cell Biol, 1990 Dec, 10(12), 6578 - 85 Molecular cloning of YPT1/SEC4-related cDNAs from an epithelial cell line; Chavrier P et al.; Molecular analysis of Saccharomyces cerevisiae secretion mutants has led to the identification of two Ras-like GTP-binding proteins, Ypt1p and Sec4p, which are essential for transport along the exocytic route . To study the regulation of membrane traffic in epithelial cells, a set of 11 clones encoding proteins similar to the YPT1/SEC4 products were isolated from an MDCK (Madin-Darby canine kidney) cell cDNA library . Four of these proteins, Rab8, -9, -10, and -11, are novel members of this subfamily of Ras-like proteins, and two of them are closely related to Ypt1p and Sec4p . The ratio of the number of clones isolated over the total number screened reveals a high level of complexity for this subfamily of GTP-binding proteins . This diversity supports their proposed function in controlling different steps in membrane traffic. Genetika, 1990 Dec, 26(12), 2246 - 9 {Toxicogenetic effects of azo- and arylmethane dyes}; Zimina TA et al.; The haploid strain 15B-II4 of Saccharomyces cerevisiae was used to study in an acute experiment the toxic and mutagenic effects of arylmethane dyes Victory Blue (C.I . 44040), Methyl Violet (C.I . 42535), Brilliant Green (C.I . 42040) and cancerogenic aminoazo dye Chrysoidine (C.I . 11270) . High biological activity of all the dyes tested was found, based on such toxic effects as cell killing and growth inhibition . Also, it was shown that the dyes could increase the frequency of appearance of nuclear point mutations and cytoplasmic mutations of respiratory deficiency. Anal Biochem, 1990 Dec, 191(2), 390 - 5 Semiconductor-controlled contour-clamped homogeneous electric field apparatus; Maule J et al.; The design and construction of a transistor-driven hexagonal contour-clamped homogeneous electric field (CHEF) apparatus is discussed in detail . The addition of computer control of pulsed-field timings and experiment duration gives rise to an efficient electrophoresis tool designed to achieve separation of DNA molecules in different size groupings . In particular, pulse time regimes which lead to the monotonic separation of DNA molecules ranging from 90 kbp to over a megabase pair are demonstrated . Theoretical treatment of electric field clamping with transistor-driven multiple electrodes is supported by measurements and by the actual performance of electrophoretic separation of yeast chromosomes . The large sample capacity of gels run in this apparatus coupled with the modest power requirements necessary to provide a homogeneous electric field offer significant advantages over earlier CHEF designs. Genetics, 1990 Dec, 126(4), 851 - 67 Gene conversion tracts stimulated by HOT1-promoted transcription are long and continuous; Voelkel-Meiman K et al.; The recombination-stimulating sequence, HOT1, corresponds to the promoter of transcription by yeast RNA polymerase I . The effect of HOT1 on mitotic interchromosomal recombination was examined in diploid strains carrying a heterozygous URA3 gene on chromosome III . The frequency of Ura- recombinants was increased 20-fold when HOT1 was inserted into the chromosome III copy marked with URA3, at a location 48 kbp centromere-proximal to URA3 . Ura- recombinants were increased only 2-fold when HOT1 and URA3 were on opposite homologues . These results suggest that most HOT1-promoted Ura- recombinants result from gene conversion and that sequences on the HOT1-containing chromosome are preferentially converted . Characterization of Ura- recombinants isolated from strains carrying multiple markers on chromosome III indicates that HOT1-promoted gene conversion tracts are unusually long (often greater than 75 kbp) and almost always continuous . Furthermore, conversion tracts frequently extend to both sides of HOT1 . We suggest that HOT1 promotes the formation of a double-strand break which is often followed by exonucleolytic digestion . Repair of the broken chromosome could then result from gap repair or from replicative repair primed only by the centromere-containing chromosomal fragment. Nature, 1990 Nov 29, 348(6300), 455 - 8 The mitochondrial chaperonin hsp60 is required for its own assembly; Cheng MY et al.; Heatshock protein 60 (hsp60) in the matrix of mitochondria is essential for the folding and assembly of newly imported proteins . Hsp60 belongs to a class of structurally related chaperonins found in organelles of endosymbiotic origin and in the bacterial cytosol . Hsp60 monomers form a complex arranged as two stacked 7-mer rings . This 14-mer complex binds unfolded proteins at its surface, then seems to catalyse their folding in an ATP-dependent process . The question arises as to how such an assembly machinery is itself folded and assembled . Hsp60 subunits are encoded by a nuclear gene and translated in the cytosol as precursors which are translocated into mitochondria and proteolytically processed . In both intact cells and isolated mitochondria of the hsp60-defective yeast mutant mif4, self-assembly of newly imported wild-type subunits is not observed . Functional pre-existing hsp60 complex is required in order to form new, assembled, 14-mer . Subunits imported in vitro are assembled with a surprisingly fast half-time of 5-10 min, indicative of a catalysed reaction . These findings are further evidence that self-assembly may not be the principal mechanism by which proteins attain their functional conformation in the intact cell. FEBS Lett, 1990 Nov 26, 275(1-2), 190 - 4 Polypeptides traverse the mitochondrial envelope in an extended state; Rassow J et al.; Most mitochondrial proteins are synthesized as precursors in the cytosol and imported through contact sites between outer and inner mitochondrial membranes . The molecular mechanism of membrane translocation of precursor proteins is largely unclear . For this report, various hybrid proteins between portions of the precursor of cytochrome b2 and the entire dihydrofolate reductase (DHFR) were accumulated in mitochondrial contact sites . We unexpectedly found that about 50 amino acid residues of the polypeptide chain in transit were sufficient to span both membranes . This suggests a linear translocation of the polypeptide chain and presents evidence for a high degree of unfolding of polypeptides traversing the mitochondrial membranes. J Biol Chem, 1990 Nov 25, 265(33), 20692 - 8 Arginine 127 stabilizes the transition state in carboxypeptidase; Phillips MA et al.; Crystallographic studies suggest that Arg-127 is a key amino acid in the hydrolysis of peptides and esters by carboxypeptidase A . The guanidinium group of Arg-127 is hypothesized to stabilize the oxyanion of the tetrahedral intermediate formed by the attack of water on the scissile carbonyl bond . We have replaced this amino acid in rat carboxypeptidase A1 with lysine (R127K), methionine (R127M), and alanine (R127A), in order to define the role of Arg-127 in carboxypeptidase catalyzed hydrolysis . The wild-type and mutant enzymes were expressed in yeast and purified . Kinetic studies show that Arg-127 substitution decreases kcat for both ester and amide substrates, whereas Km is relatively unchanged; for R127M and R127A this corresponds to a 6 kcal/mol decrease in transition state stabilization of the rate-limiting step . The binding affinity for the phosphonate transition state analog, Cbz-Phe-Ala(P)-OAla, was decreased by 5.4 kcal/mol, whereas binding affinity for the ground state inhibitor, DL-benzylsuccinic acid, was decreased by only 1.7 kcal/mol for R127M . Electrostatic calculations employing a finite difference solution to the Poisson-Boltzmann equation predict that the positive charge of Arg-127 should stabilize the transition state by 6-8 kcal/mol . Therefore, the experimental and theoretical data suggest that the primary role of Arg-127 is stabilization of the transition state through electrostatic interaction with the oxyanion. Biochemistry, 1990 Nov 20, 29(46), 10498 - 503 Phosphonate analogue substrates for enolase; Anderson VE et al.; Phosphonate analogues in which the bridge between C-2 and phosphorus is a CH2 group are slow substrates for yeast enolase . The pH variation of the kinetic parameters for the methylene analogue of 2-phosphoglycerate suggests that the substrate binds as a dianion and that Mg2+ can bind subsequently only if a metal ligand and the catalytic base are unprotonated . Primary deuterium isotope effects of 4-8 on V/KMg, but ones of only 1.15-1.32 on V for dehydration, show that proton removal to give the carbanion intermediate largely limits V/KMg and that a slow step follows which largely limits V (presumably carbanion breakdown) . Since there is a D2O solvent isotope effect on V for the reverse reaction of 5, but not an appreciable one on the forward reaction, it appears that the slow rates with phosphonate analogues result from the fact that the carbanion intermediate is more stable than that formed from the normal substrates, and its reaction in both directions limits V . Increased stability as a result of replacement of oxygen by carbon at C-2 of the carbanion is the expected chemical behavior. J Biol Chem, 1990 Nov 15, 265(32), 19824 - 32 Expression of acid phosphatase-beta-galactosidase hybrid proteins prevents translocation by depleting a soluble factor; Young MR et al.; We have shown that hybrid proteins composed of the yeast repressible acid phosphatase (PHO5) and bacterial beta-galactosidase (lacZ) interfere with secretion of native acid phosphatase (Wolfe, P . B . (1988) J . Biol . Chem . 263, 6908-6915) . We now report that PHO5-LacZ hybrid proteins have a more general effect on secretion and prevent translocation of several secreted proteins . Translocation of both the mating pheromone alpha-factor and the vacuolar protease carboxypeptidase Y is partially blocked when PHO5-LacZ hybrids are expressed . Cell fractionation and protease sensitivity indicate that alpha-factor and carboxypeptidase Y accumulate in precursor form on the cytoplasmic surface of the endoplasmic reticulum . Indirect immunofluorescence with antibody directed against beta-galactosidase supports the localization of hybrid proteins to the endoplasmic reticulum . Analysis of the hybrid protein phenotype in vivo and in vitro suggests that the hybrid proteins deplete a soluble factor required for efficient translocation across the endoplasmic reticulum . First, a decrease in the expression of a hybrid protein in vivo decreases its effect on translocation . Second, an in vitro translation/translocation reaction, prepared from a hybrid-bearing strain, is deficient in its ability to translocate prepro-alpha-factor across yeast microsomal membranes . This deficiency is complemented by addition of cytosol prepared from wild type cells . Finally, the hybrid protein phenotype is shown to be independent of the requirement for SSA gene products. Nucleic Acids Res, 1990 Nov 11, 18(21), 6299 - 304 Field inversion gel electrophoresis with different pulse time ramps; Heller C et al.; The influence of different pulse time ramps on the separation of yeast chromosomes with field inversion gel electrophoresis (FIGE) was investigated by the means of two dimensional gel electrophoresis . The problem of band inversion, which makes it difficult to distinguish DNA molecules of different size, has been solved by using double randomized pulse times . A major disadvantage of the field inversion technique is thereby overcome, making this system comparable to other pulsed field techniques. Nature, 1990 Nov 8, 348(6297), 171 - 3 Gene replacement in parasitic protozoa; Cruz A et al.; Trypanosomatid protozoa frequently cause severe diseases in humans . Many molecules likely to have a role during the infectious cycle have been identified, yet proof of their function is often lacking . We describe studies in Leishmania major of homologous gene targeting, a powerful method for testing gene function in other organisms . Following introduction of a construct containing dihydrofolate reductase-thymidylate synthase (dhfr-ts) flanking sequences fused to neomycin phosphotransferase, 45% of the colonies contained the planned homologous replacement; this frequency rose to nearly 100% in transfections using low amounts of DNA . Integrative transfection in Leishmania thus resembles that of Saccharomyces cerevisiae in giving predominantly homologous events . To facilitate studies of folate metabolism and chemotherapy the sole dhfr-ts copy in a heterozygous deletion line was replaced, yielding lines that were functionally DHFR-TS- . Although most genes are diploid in trypanosomatids, methods exploiting the high frequency of homologous recombination should permit complete replacement of any parasite gene. Nature, 1990 Nov 8, 348(6297), 166 - 8 Reduced levels of hsp90 compromise steroid receptor action in vivo; Picard D et al.; Signalling by steroid hormones is mediated by receptor proteins that bind hormonal ligands and regulate the transcription of specific genes . The heat-shock protein hsp90 seems to associate selectively with unliganded receptors (aporeceptors), but it has not been determined whether this interaction affects receptor function in vivo . To address the role of hsp90, we have taken advantage of the capacity of mammalian steroid receptors to function in yeast . We constructed a strain of Saccharomyces cerevisiae in which hsp90 expression was regulatable and could be reduced more than 20-fold relative to wild type . At low levels of hsp90, aporeceptors seem to be mostly hsp90-free, yet fail to enhance transcription; on hormone addition, the receptors are activated but with markedly reduced efficiency . Thus hsp90 does not inhibit receptor function solely by steric interference; rather, hsp90 seems to facilitate the subsequent response of the aporeceptor to the hormonal signal . This is the first biological evidence that hsp90 acts in the signal transduction pathway for steroid receptors. Biochemistry, 1990 Nov 6, 29(44), 10280 - 8 Amino acid sequence of the cyclic GMP stimulated cyclic nucleotide phosphodiesterase from bovine heart; Trong HL et al.; The complete amino acid sequence of the cyclic GMP stimulated cyclic nucleotide phosphodiesterase (cGS-PDE) of bovine heart has been determined by analysis of five digests of the protein; placement of the C-terminal 330 residues has been confirmed by interpretation of the corresponding partial cDNA clone . The holoenzyme is a homodimer of two identical N alpha-acetylated polypeptide chains of 921 residues, each with a calculated molecular weight of 103,244 . The C-terminal region, residues 613-871, of the cGS-PDE comprises a catalytic domain that is conserved in all phosphodiesterase sequences except those of PDE 1 from Saccharomyces cerevisiae and a secreted PDE from Dictyostelium . A second conserved region, residues 209-567, is homologous to corresponding regions of the alpha and alpha' subunits of the photoreceptor phosphodiesterases . This conserved domain specifically binds cGMP and is involved in the allosteric regulation of the cGS-PDE . This regulatory domain contains two tandem, internal repeats, suggesting that it evolved from an ancestral gene duplication . Common cyclic nucleotide binding properties and a distant structural relationship provide evidence that the catalytic and regulatory domains within the cGS- and photoreceptor PDEs are also related by an ancient internal gene duplication. Anal Biochem, 1990 Nov 1, 190(2), 188 - 92 A filter-based protein kinase assay selective for alkali-stable protein phosphorylation and suitable for acid-labile protein phosphorylation; Wei YF et al.; Alkali-stable phosphorylation of proteins, particularly phosphotyrosine and phosphohistidine, is an important phenomenon in cells . In the case of phosphohistidine and some other phosphoamino acids, the phosphorylation is acid-labile and in these cases studies have been severely limited by the absence of a rapid assay suitable for acid-labile phosphorylation . The assay presented here involves a conventional kinase assay reaction followed by mild alkaline hydrolysis and adsorption of the product to washed Nytran paper at high pH . After further washing, at pH 9, the radioactivity on the papers is determined by liquid scintillation counting . Hence, acid-labile phosphorylation is preserved . The assay is selective for alkali-stable phosphorylation but not fully specific, mainly due to the need to balance the severity of the partial alkaline hydrolysis with the stability of the protein-peptide bonds . The assay has been used for the purification and characterization of a protein histidine kinase from Saccharomyces cerevisiae. Trends Biochem Sci, 1990 Nov, 15(11), 440 - 4 Involvement of aminoacyl-tRNA synthetases and other proteins in group I and group II intron splicing; Lambowitz AM et al.; Group I and group II introns catalyse their own splicing, but depend on protein factors for efficient splicing in vivo . Some of these proteins, termed maturases, are encoded by the introns themselves and may also function in intron mobility . Other proteins are encoded by host chromosomal genes and include aminoacyl-tRNA synthetases and various proteins that function in protein synthesis . The splicing factors identified thus far appear to be idiosyncratic, even in closely related organisms . We suggest that some of these protein-assisted splicing reactions evolved relatively recently, possibly reflecting the recent dispersal of the introns themselves. Trends Biochem Sci, 1990 Nov, 15(11), 420 - 4 Eukaryotic protein elongation factors; Riis B et al.; In eukaryotes, peptide chain elongation is mediated by elongation factors EF-1 and EF-2 . EF-1 is composed of a nucleotide-binding protein EF-1 alpha, and a nucleotide exchange protein complex, EF-1 beta gamma, while EF-2 catalyses the translocation of peptidyl-tRNA on the ribosome . Elongation factors are highly conserved among different species and may be involved in functions other than protein synthesis, such as organization of the mitotic apparatus, signal transduction, developmental regulation, ageing and transformation . Yeast contains a third factor, EF-3, whose structure and function is not yet well understood. Mol Gen Genet, 1990 Nov, 224(2), 269 - 78 Isolation and molecular analysis of the orotidine-5'-phosphate decarboxylase gene (pyrG) of Phycomyces blakesleeanus; Diaz-Minguez JM et al.; The pyrG gene of Phycomyces was isolated from a Phycomyces genomic library, constructed in the cosmid pHS255, by hybridization with a 170 bp fragment of the pyrG gene of Aspergillus niger . This fragment includes a consensus sequence found in almost all species in which the orotidine-5'-phosphate decarboxylase (OMPdecase) gene has been sequenced . The complete nucleotide sequence of the cloned pyrG gene from Phycomyces was determined and the transcription start sites mapped . In the predicted amino acid sequence there are regions of strong homology to the equivalent genes of Saccharomyces cerevisiae, A . niger, Schizophyllum commune and Homo sapiens . Analysis of the sequence revealed the presence of two introns . The precise length and location of these introns was determined by sequencing the pyrG cDNA and comparing it with the genomic clone . Non-coding flanking regions showed obvious homology to the consensus TATA and CAAT boxes, and the polyadenylation signal "AATAAA" . The pyrG gene is the second Phycomyces gene that has been cloned and analysed . This is the first time that introns have been reported in Phycomyces. Proc Natl Acad Sci U S A, 1990 Nov, 87(21), 8192 - 6 Splicing of COB intron 5 requires pairing between the internal guide sequence and both flanking exons; Partono S et al.; Group I introns are characterized by a set of conserved sequence elements and secondary structures . Evidence supporting the pairing of certain of these sequences has come from the comparison of intron sequences and from the analysis of mutations that disrupt splicing by interfering with pairing . One of the structures proposed for all group I introns is an internal guide sequence that base pairs with the upstream and the downstream exons, bringing them into alignment for ligation . We made specific mutations in the internal guide sequence and the flanking exons of the fifth intron in the yeast mitochondrial gene for apocytochrome b (COB) . Mutations that disrupted the pairing between the internal guide sequence and the upstream exon (the P1 pairing) blocked addition of guanosine to the 5' end of the intron during autocatalytic reactions and prevented formation of the full-length circular intron . In contrast, transcripts containing mutations that disrupted the pairing between the guide sequence and the downstream exon (the P10 helix) initiated splicing but failed to ligate exons . Compensatory mutations that restored helices of normal stability mitigated the effects of the original mutations . These data provide direct evidence for the importance of the base pairing between the internal guide sequence and the downstream exon in the splicing of a wild-type group I intron. Mol Cell Biol, 1990 Nov, 10(11), 5945 - 9 Prenylation of mammalian Ras protein in Xenopus oocytes; Kim R et al.; Ras protein requires an intermediate of the cholesterol biosynthetic pathway for posttranslational modification and membrane anchorage . This step is necessary for biological activity . Maturation of Xenopus laevis oocytes induced by an oncogenic human Ras protein can be inhibited by lovastatin or compactin, inhibitors of the synthesis of mevalonate, an intermediate of cholesterol biosynthesis . This inhibition can be overcome by mevalonic acid or farnesyl diphosphate, a cholesterol biosynthetic intermediate downstream of mevalonate, but not by squalene, an intermediate after farnesyl pyrophosphate in the pathway . This study supports the idea that in Xenopus oocytes, the Ras protein is modified by a farnesyl moiety or its derivative . Furthermore, an octapeptide with the sequence similar to the C-terminus of the c-H-ras protein inhibits the biological activity of Ras proteins in vivo, suggesting that it competes for the enzyme or enzymes responsible for transferring the isoprenoid moiety (prenylation) in the oocytes . This inhibition of Ras prenylation by the peptide was also observed in vitro, using both Saccharomyces cerevisiae and Xenopus oocyte extracts . These observations show that Xenopus oocytes provide a convenient in vivo system for studies of inhibitors of the posttranslational modification of the Ras protein, especially for inhibitors such as peptides that do not penetrate cell membranes. Mol Cell Biol, 1990 Nov, 10(11), 5772 - 81 p53 functions as a cell cycle control protein in osteosarcomas; Diller L et al.; Mutations in the p53 gene have been associated with a wide range of human tumors, including osteosarcomas . Although it has been shown that wild-type p53 can block the ability of E1a and ras to cotransform primary rodent cells, it is poorly understood why inactivation of the p53 gene is important for tumor formation . We show that overexpression of the gene encoding wild-type p53 blocks the growth of osteosarcoma cells . The growth arrest was determined to be due to an inability of the transfected cells to progress into S phase . This suggests that the role of the p53 gene as an antioncogene may be in controlling the cell cycle in a fashion analogous to the check-point control genes in Saccharomyces cerevisiae. Virology, 1990 Nov, 179(1), 267 - 75 A DNA ligase gene in the Copenhagen strain of vaccinia virus is nonessential for viral replication and recombination; Colinas RJ et al.; Biochemical and genetic analyses have been conducted to determine whether a vaccinia virus open reading frame (orf) with extensive homology to the Saccharomyces cerevisiae DNA ligase gene encodes a functional ligase activity . This orf in HindIII A, designated A50R, is capable of encoding a 552-amino-acid, 63.4-kDa polypeptide . Full-length A50R mRNA produced in vitro directed the synthesis of a polypeptide with an apparent molecular weight of 57 kDa . Significantly, translation reactions programmed with A50R mRNA were capable of ligating a 3-kb Notl restriction fragment into multimers . DNA ligase activity was not detectable when either truncated sense or full-length antisense mRNA was translated in vitro . In extracts prepared from cells infected with wt vaccinia virus, DNA ligase activity was detected as assayed by the formation of a 57 kDa ligase-AMP adduct which was expressed early in the viral replication cycle . In cells infected with a DNA ligase deletion mutant no equivalent AMP-labeled adduct was detected . Relative to wt virus, the DNA ligase deletion mutant exhibited no significant differences in homologous recombination . These results indicate that the vaccinia orf A50R encodes a functional DNA ligase expressed early in infection, but this DNA ligase is nonessential for either recombination or viral replication. Mol Cell Biol, 1990 Nov, 10(11), 5839 - 48 Molecular analysis of nuc-1+, a gene controlling phosphorus acquisition in Neurospora crassa; Kang S et al.; In response to phosphorus starvation, Neurospora crassa makes several enzymes that are undetectable or barely detectable in phosphate-sufficient cultures . The nuc-1+ gene, whose product regulates the synthesis of these enzymes, was cloned and sequenced . The nuc-1+ gene encodes a protein of 824 amino acids with a predicted molecular weight of 87,429 . The amino acid sequence shows homology with two yeast proteins whose functions are analogous to that of the NUC-1 protein . Two nuc-1+ transcripts of 3.2 and 3.0 kilobases were detected; they were present in similar amounts during growth at low or high phosphate concentrations . The nuc-2+ gene encodes a product normally required for NUC-1 function, and yet a nuc-2 mutation can be complemented by overexpression of the nuc-1+ gene . This implies physical interactions between NUC-1 protein and the negative regulatory factor(s) PREG and/or PGOV . Analysis of nuc-2 and nuc-1; nuc-2 strains transformed by the nuc-1+ gene suggests that phosphate directly affects the level or activity of the negative regulatory factor(s) controlling phosphorus acquisition. Mol Biol Rep, 1990 Nov, 14(4), 265 - 75 Furin is a subtilisin-like proprotein processing enzyme in higher eukaryotes; van de Ven WJ et al.; The human fur gene encodes a protein, designated furin, the C-terminal half of which contains a transmembrane and a cysteine-rich receptor-like domain . The N-terminal half of furin exhibits striking primary amino acid sequence similarity to the catalytic domains of members of the subtilisin family of serine proteases . We here report characteristics of the furin protein and propose a three-dimensional model for its presumptive catalytic domain with characteristics, that predict furin to exhibit an endoproteolytic cleavage selectivity at paired basic residues . This prediction is substantiated by transfection and cotransfection experiments, using COS-1 cells . Full length fur cDNA evokes the specific synthesis of two polypeptides of about 100 kDa and 90 kDa as appeared from Western blot analysis of transfected COS-1 cells using a polyclonal anti-furin antiserum . Functional analysis of furin was performed by cotransfection of fur cDNA with cDNA encoding the 'wild type' precursor of von Willebrand factor (pro-vWF) and revealed an increased proteolytic processing of provWF . In contrast, cotransfection of fur cDNA with a recombinant derivative (provWFgly763), having the arginine residue adjacent to the proteolytic cleavage site (arg-ser-lys-arg) replaced by glycine, revealed that provWFgly763 is not processed by the fur gene product . We conclude that in higher eukaryotes, furin is the prototype of a subtilisin-like class of proprotein processing enzymes with substrate specificity for paired basic residues. Bioessays, 1990 Nov, 12(11), 533 - 6 DNA polymerase epsilon: the latest member in the family of mammalian DNA polymerases; Syvaoja JE; DNA polymerase epsilon is a mammalian polymerase that has a tightly associated 3'----5' exonuclease activity . Because of this readily detectable exonuclease activity, the enzyme has been regarded as a form of DNA polymerase delta, an enzyme which, together with DNA polymerase alpha, is in all probability required for the replication of chromosomal DNA . Recently, it was discovered that DNA polymerase epsilon is both catalytically and structurally distinct from DNA polymerase delta . The most striking difference between the two DNA polymerases is that processive DNA synthesis by DNA polymerase delta is dependent on proliferating cell nuclear antigen (PCNA), a replication factor, while DNA polymerase epsilon is inherently processive . DNA polymerase epsilon is required at least for the repair synthesis of UV-damaged DNA . DNA polymerases are highly conserved in eukaryotic cells . Mammalian DNA polymerases alpha, delta and epsilon are counterparts of yeast DNA polymerases I, III and II, respectively . Like DNA polymerases I and III, DNA polymerase II is also essential for the viability of cells, which suggests that DNA polymerase II (and epsilon) may play a role in DNA replication. Appl Microbiol Biotechnol, 1990 Nov, 34(2), 203 - 7 Synthesis and secretion of hirudin by Streptomyces lividans; Bender E et al.; To examine the secretory production of heterologous proteins by Streptomyces lividans, we fused the DNA encoding the signal peptide of the alpha-amylase inhibitor tendamistat, derived from S . tendae with a synthetic gene encoding the thrombin inhibitor hirudin . The analysis of secretion by immunoblots revealed an efficient translocation of hirudin through the membrane, with no detectable immunoreaction among the cellular proteins . The secreted hirudin was stable in the shaking culture for about 6 days . A comparison of the hirudin secreted by S . lividans and recombinant reference hirudin from yeast by immunoblots and thrombin inhibition assays shows that hirudin from Streptomyces has a lower specific activity, which may be due to a different aminoterminal sequence or to inexact processing of the precursor. Enzyme Microb Technol, 1990 Nov, 12(11), 830 - 5 Application of the enzyme thermistor to the direct estimation of intrinsic kinetics using the saccharose-immobilized invertase system; Stefuca V et al.; The possibility of using the enzyme thermistor (ET) for the direct determination of kinetic parameters (Km, Ki, Vm) of immobilized enzyme (IME) was evaluated using different preparations of invertase conjugated to bead celluloses . Two different ET columns packed with IME were operated in the mode of a differential enzyme reactor (short length, low substrate conversion) . Kinetic parameters of the above IME reactor were computed by a nonlinear curve-fitting procedure . The obtained kinetic parameters were superverified by means of an independent differential reactor (DR) system . This system utilized an indirect postcolumn analytical method based on determination of glucose concentration in the stirred reservoir . Best agreement between the data acquired by direct (ET) and indirect (DR) methods was obtained if the ET column was operated at flow rates within the range of 1.0-1.5 ml min-1 using invertase-cellulose chlorotriazine conjugate . Influence of heat loss and flow nonideality is discussed . The proposed ET method offers a rapid, convenient, and general approach to determination of kinetic constants of IME preparations by omitting postcolumn analytical methods. Biotechnol Prog, 1990 Nov-Dec, 6(6), 430 - 6 Optimal chemostat cascades for periplasmic protein production; Davis RH et al.; This theoretical work predicts the optimal system design for the steady-state production of secreted protein in a chemostat cascade, using bakers' yeast (Saccharomyces cerevisiae) as the host organism . The protein of interest, mutant invertase, is secreted to the periplasmic space instead of the culture medium on account of its large size . This work uses the secretion model developed and tested by Park and Ramirez (1988) . It is shown that the highest productivity is achieved when the chemostat cascade contains two stages, although the improvement over the single-stage productivity is small . When no recycle is used, the advantage of two stages results from the tradeoff between maximizing the cell concentration and maximizing the rate of protein production per cell . When recycle is used, the cell concentration and protein productivity are increased, and the advantage of two stages results from the tradeoff between maximizing the specific protein production rate and maximizing the specific protein secretion rate . Cascades with three stages were also investigated, but these were found to have no improvement over the corresponding two-stage cascades. Science, 1990 Oct 26, 250(4980), 549 - 53 Involvement of the silencer and UAS binding protein RAP1 in regulation of telomere length; Lustig AJ et al.; The yeast protein RAP1, initially described as a transcriptional regulator, binds in vitro to sequences found in a number of seemingly unrelated genomic loci . These include the silencers at the transcriptionally repressed mating-type genes, the promoters of many genes important for cell growth, and the poly{(cytosine)1-3 adenine} {poly(C1-3A)} repeats of telomeres . Because RAP1 binds in vitro to the poly(C1-3A) repeats of telomeres, it has been suggested that RAP1 may be involved in telomere function in vivo . In order to test this hypothesis, the telomere tract lengths of yeast strains that contained conditionally lethal (ts) rap1 mutations were analyzed . Several rap1ts alleles reduced telomere length in a temperature-dependent manner . In addition, plasmids that contain small, synthetic telomeres with intact or mutant RAP1 binding sites were tested for their ability to function as substrates for poly(C1-3A) addition in vivo . Mutations in the RAP1 binding sites reduced the efficiency of the addition reaction. Biochemistry, 1990 Oct 23, 29(42), 9978 - 88 CO dissociation in cytochrome c peroxidase: site-directed mutagenesis shows that distal Arg 48 influences CO dissociation rates; Miller MA et al.; To investigate the molecular basis for the 100-fold slower rate of CO dissociation in ferrous peroxidases relative to myoglobin, CO dissociation rates were measured as a function of pH in the cloned cytochrome c peroxidase from yeast {CCP(MI)} and in several mutants in the heme binding pocket prepared by site-directed mutagenesis . The mutants included Asp 235----Asn; Arg 48----Lys, Leu; and His 181----Gly . Changes in the absorption spectrum with pH are consistent with conversion of the CO-ferrous CCP(MI) complex from acidic to alkaline forms by a two-proton cooperative ionization, with an apparent pKa = 7.6, analogous to that described for CCP from bakers' yeast {Iizuka, T., Makino, R., Ishimura, Y., & Yonetani, T . (1985) J . Biol . Chem . 260, 1407-1412} . The rate of CO dissociation (koff) was increased 11-fold (from 0.7 x 10(-4) to 8.0 x 10(-4) s-1) by conversion of the acidic to the alkaline form . Analogous acidic and alkaline forms of the CO complex were also observed in the mutants of CCP(MI) examined here . In the acidic form, koff was increased 5- and 20-fold when Arg 48 was replaced with Lys and Leu, respectively, while in the acidic form of mutants that possess Arg 48, koff was similar to that observed in CCP(MI) . Conversion of the CO complex from the acidic to alkaline form increased koff in all the mutants, and the pH-dependent increase in koff correlated with a two-proton cooperative ionization, except in the case of His 181----Gly . In this mutant, pH-dependent increase in koff correlated with a single-proton ionization, implicating His 181 as one of the two residues that is deprotonated in the conversion of CO-ferrous CCP(MI) from acidic to alkaline forms . Only a 2.5-fold variation was observed for koff between the alkaline form of CCP(MI) and the Arg 48----Leu mutant, suggesting that the influence of Arg 48 on the rate of CO dissociation is decreased in the alkaline form by a conformational change.(ABSTRACT TRUNCATED AT 400 WORDS) Biochim Biophys Acta, 1990 Oct 23, 1087(2), 137 - 41 The conserved terminal region of Trichoderma reesei cellulases forms a strong antigenic epitope for polyclonal antibodies; Aho S et al.; The specificity of polyclonal antibodies (Pab) raised against Trichoderma reesei cellulases has been studied . cDNAs lacking regions coding for certain functional domains were produced by preparing series of 3'-end deletions from the cDNAs for two cellobiohydrolases, CBH I and CBH II, and an endoglucanase, EG I . The proteins coded by the full length cDNAs and the truncated proteins coded by the deleted cDNAs were expressed in yeast Saccharomyces cerevisiae, under the control of the ADC1 promoter . Each polyclonal antiserum showed cross-reactivity with other cellulases . Pabs for CBH I and CBH II both recognized EG I . Pab for EG I strongly recognized both CBH I and CBH II . By analyzing the truncated proteins, we found that these antibodies were almost entirely directed against the conserved tail of the cellulase enzymes. Gene, 1990 Oct 15, 94(2), 223 - 8 Simplified colorimetric analysis of polymerase chain reactions: detection of HIV sequences in AIDS patients; Kemp DJ et al.; We have previously described a colorimetric test, designated an amplified DNA assay (ADA), for specific segments of DNA amplified by polymerase chain reactions (PCRs), suited to diagnostic applications . This relied on binding the amplified DNA via a sequence in one oligodeoxyribonucleotide (oligo) to the DNA-binding protein GCN4 coated on the wells of a microtiter dish . Avidin-peroxidase was then bound to biotin at the 5' end of the other oligo and detected colorimetrically . Two successive PCRs with nested oligos were utilized . We describe here several modifications that greatly simplify the ADA . First, we bind the DNA to a glutathione S-transferase-GCN4 fused polypeptide (GST-GCN4) and avidin-peroxidase simultaneously, rather than successively . Second, we carry out the two successive PCRs in the one reaction mixture, using the thermal stabilities of oligos of differing lengths to separate the two reactions . Third, PCRs can be performed in the wells of a microtiter dish and the amplified DNA captured and detected via GST-GCN4 immobilized on beads attached to the lid of the microtiter dish . Hence it is only necessary to pipette the DNA sample once, and up to 96 samples can then be handled simultaneously. J Biol Chem, 1990 Oct 15, 265(29), 17911 - 20 Purification and characterization of proximal sequence element-binding protein 1, a transcription activating protein related to Ku and TREF that binds the proximal sequence element of the human U1 promoter; Knuth MW et al.; The promoter structure of the known small nuclear RNA (snRNA) genes contains two major effectors of transcriptional activity, a proximal sequence element (PSE) and a distal sequence element (DSE) . In previous work, methidiumpropyl-EDTA-Fe(II) footprinting was used to demonstrate the existence in human placental extracts of a protein producing footprints within the PSE and the DSE of the human U1 snRNA gene . This protein (PSE1) has now been purified to homogeneity from both human placental extract and K562 cell nuclear extract . PSE1 consists of two subunits, an alpha subunit with an apparent molecular mass of 83 kDa, and a beta subunit with an apparent molecular mass of 73 kDa in K562 nuclear extracts and 63 kDa in placental extracts . Footprinting and UV cross-linking assays indicate that purified PSE1 binds to the PSE and DSE of the U1 gene . Monoclonal antibodies were prepared which specifically recognize the individual subunits of PSE1 . PSE1 is immunologically similar to and shares amino acid sequence with a protein (TREF) which binds the human transferrin receptor (HTFR) promoter . An in vitro transcription system was established for a template consisting of a minimal HTFR promoter placed upstream of the human U1 snRNA-coding region and shown by immunodepletion/addback experiments to specifically require PSE1 . Transcription from the adenovirus 2 major late promoter was unaffected in these experiments . This result supports a functional role of PSE1 as a transcriptional activating protein, but its role in transcription of snRNA genes remains to be established . PSE1 also has an immunological relationship to and shares amino acid sequence with the p70 and p86 subunits of the human Ku autoantigen . Ku, PSE1, and TREF may thus be identical proteins or members of a family of heterodimeric proteins consisting of related subunits . Our results support earlier proposals that Ku may be a transcriptional activator. Biochem Biophys Res Commun, 1990 Oct 15, 172(1), 35 - 41 The primary structure of rat ribosomal protein L12; Suzuki K et al.; The covalent structure of the rat 60S subunit protein L12 which is a component of the ribosomal elongation factor binding domain was deduced from the sequence of nucleotides in a recombinant cDNA and confirmed from the NH2-terminal amino acid sequence of the protein . L12 has 165 amino acids and a molecular weight of 17,834 . Hybridization of the cDNA to digests of nuclear DNA suggests that there are 11-13 copies of the L12 gene . The mRNA for the protein is about 800 nucleotides in length . Rat L12 is homologous to Saccharomyces cerevisiae L15 . The cDNA contains the highly repetitive DNA sequence, R.dre.1, in the 3' noncoding region. Nature, 1990 Oct 11, 347(6293), 575 - 8 Folding transition in the DNA-binding domain of GCN4 on specific binding to DNA; Weiss MA et al.; Protein-DNA recognition is often mediated by a small domain containing a recognizable structural motif, such as the helix-turn-helix or the zinc-finger . These motifs are compact structures that dock against the DNA double helix . Another DNA recognition motif, found in a highly conserved family of eukaryotic transcription factors including C/EPB, Fos, Jun and CREB, consists of a coiled-coil dimerization element the leucine-zipper and an adjoining basic region which mediates DNA binding . Here we describe circular dichroism and 1H-NMR spectroscopic studies of another family member, the yeast transcriptional activator GCN4 . The 58-residue DNA-binding domain of GCN4, GCN4-p, exhibits a concentration-dependent alpha-helical transition, in accord with previous studies of the dimerization properties of an isolated leucine-zipper peptide . The GCN4-p dimer is approximately 70% helical at 25 degrees C, implying that the basic region adjacent to the leucine zipper is largely unstructured in the absence of DNA . Strikingly, addition of DNA containing a GCN4 binding site (AP-1 site) increases the alpha-helix content of GNC4-p to at least 95% . Thus, the basic region acquires substantial alpha-helical structure when it binds to DNA . A similar folding transition is observed on GCN4-p binding to the related ATF/CREB site, which contains an additional central base pair . The accommodation of DNA target sites of different lengths clearly requires some flexibility in the GCN4 binding domain, despite its high alpha-helix content . Our results indicate that the GCN4 basic region is significantly unfolded at 25 degrees C and that its folded, alpha-helical conformation is stabilized by binding to DNA. J Biol Chem, 1990 Oct 5, 265(28), 17110 - 7 Isolation, characterization, and expression of cDNA encoding a rat liver endoplasmic reticulum alpha-mannosidase; Bischoff J et al.; We have isolated a cDNA encoding an endoplasmic reticulum alpha-mannosidase, an asparagine-linked oligosaccharide processing enzyme, from a rat liver lambda gt11 library . Two degenerate oligonucleotides, based on amino acid sequence data from the purified enzyme, were used as primers in the polymerase chain reaction with liver cDNA as a template to generate an unambiguous cDNA probe . The cDNA fragment (524 base pair) obtained was then used to isolate cDNA clones by hybridization . We isolated two overlapping clones which were used to construct a full-length cDNA of 3392 base pairs . A single open reading frame of 1040 amino acids encodes a protein with a molecular mass of 116 kilodaltons containing the six known peptide sequences . The deduced amino acid sequence revealed no classical signal sequence or membrane-spanning domain . The alpha-mannosidase encoding cDNA can be expressed transiently in COS cells using the mammalian expression vector pXM, causing a 400-fold increase in alpha-mannosidase activity as well as a dramatic increase in immunoreactive polypeptide . The rat liver endoplasmic reticulum alpha-mannosidase bears striking homology to the vacuolar alpha-mannosidase from Saccharomyces cerevisiae. Nature, 1990 Oct 4, 347(6292), 491 - 4 RNA polymerase II C-terminal repeat influences response to transcriptional enhancer signals; Scafe C et al.; The large subunit of RNA polymerase II contains a highly conserved and essential heptapeptide repeat (Pro-Thr-Ser-Pro-Ser-Tyr-Ser) at its carboxy terminus . Saccharomyces cerevisiae cells are inviable if their RNA polymerase II large subunit genes encode fewer than 10 complete heptapeptide repeats; if they encode 10 to 12 complete repeats cells are temperature-sensitive and cold-sensitive, but 13 or more complete repeats will allow wild-type growth at all temperatures . Cells containing C-terminal domains (CTDs) of 10 to 12 complete repeats are also inositol auxotrophs . The phenotypes associated with these CTD mutations are not a consequence of an instability of the large subunit; rather, they seem to reflect a functional deficiency of the mutant enzyme . We show here that partial deletion mutations in RNA polymerase II CTD affect the ability of the enzyme to respond to signals from upstream activating sequences in a subset of promoters in yeast . The number of heptapeptide repeats required for maximal response to signals from these sequences differs from one upstream activating sequence to another . One of the upstream elements that is sensitive to truncations of the CTD is the 17-base-pair site bound by the GAL4 transactivating factor. Nature, 1990 Oct 4, 347(6292), 444 - 9 Identification of a receptor for protein import into mitochondria; Pain D et al.; Anti-idiotypic antibodies, prepared using a chemically synthesized signal peptide of a mitochondrial precursor protein, recognized a mitochondrial integral membrane protein (p32) . Fab fragments derived from both anti-idiotypic antibodies and monospecific antibodies against purified p32 inhibited protein import into mitochondria . Moreover, anti-p32 antibodies specifically immunoprecipitated a precursor-p32 complex after detergent solubilization of mitochondria . Immunoelectron microscopy and subfractionation of mitochondria indicate that p32 is located in contact sites between the outer and inner mitochondrial membranes. Mol Cell Biol, 1990 Oct, 10(10), 5177 - 86 Purification and characterization of a novel factor which stimulates rat ribosomal gene transcription in vitro by interacting with enhancer and core promoter elements; Zhang J et al.; Previous studies in our laboratory have characterized a 174-base-pair (bp) enhancer sequence in the rat ribosomal DNA spacer region that exhibits all of the characteristics of a polymerase (Pol) II enhancer . Further studies showed that at least half of the enhancer activity resides in a 37-bp motif (E1) within the 174-bp spacer sequence that is located between positions -2.183 and -2.219 kilobase pairs upstream of the initiation site . To identify the factor(s) that binds specifically to the 37-bp enhancer domain, we fractionated whole-cell extract from rat adenocarcinoma ascites cells by chromatography on a series of columns, including an oligodeoxynucleotide affinity column . The final preparation contained two polypeptides of molecular weights 79,400 and 89,100 and was completely devoid of RNA Pol I activity . Electrophoretic mobility shift analysis showed that the polypeptides in the purified preparation (designated E1BF) interacted with both the enhancer element and the core promoter . To determine whether each polypeptide can separately bind to the core promoter and the enhancer, the individual components were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, renatured, and subjected to gel retardation analysis . This experiment demonstrated that both polypeptides interacted with the two cis-acting sequences . The specificity of the binding was demonstrated by competition with unlabeled 37-bp and core promoter fragments and lack of competition with nonspecific DNAs in the mobility shift assay . The 37-bp enhancer as well as the downstream sequence of the core promoter were protected by E1BF in the DNase I footprinting assay . Addition of E1BF to limiting amounts of fraction DE-B, which contains all factors essential for Pol I-directed transcription, resulted in three- to fourfold stimulation of ribosomal DNA transcription . Comparison of molecular weights and footprinting profiles did not reveal any relationship between E1BF and other Pol I trans-acting factors. Biotechnol Appl Biochem, 1990 Oct, 12(5), 485 - 8 Properties and possible function of phosphatidylinositol-transfer proteins; Wirtz KW et al.; It is proposed that the phosphatidylinositol-transfer protein (PI-TP) may function as a carrier of phosphatidylinositol (PI) in the cell . PI-TP occurs in all mammalian tissues examined and appears to be strongly conserved . Its intracellular distribution was studied by immunoblotting and immunofluorescence techniques . PI-TP displays a dual specificity in that it preferentially transfers PI over phosphatidylcholine (PC) between membranes . Its lipid binding site and transfer characteristics were investigated with fluorescent PI and PC analogues containing parinaroyl- and pyrenylacyl-labeled chains . PI-TP is ideally suited for maintaining PI levels in intracellular membranes, possibly the plasma membrane. FEMS Microbiol Lett, 1990 Oct, 60(1-2), 159 - 62 Do heat shock proteins provide protection against freezing? Komatsu Y, Kaul SC, Iwahashi H, Obuchi K. Yeast cells were frozen by plunging directly into liquid nitrogen (LN2) after exposure at 43 degrees C . Both the cells frozen without prior exposure to heat shock and those treated with cycloheximide showed almost 100% loss of viability during freezing and thawing . Heat exposure prior to freezing and thawing significantly increased the cell viability . This increase in cell viability was associated with the induction of heat shock protein synthesis, which was detected by gel electrophoresis . This protein may act by stabilizing the macromolecules and by increasing the hydrophobic interactions. Cancer Cells, 1990 Oct, 2(10), 311 - 20 Molecular genetics of eukaryotic DNA excision repair; Hoeijmakers JH et al.; DNA repair plays a key role in the prevention of carcinogenesis and mutagenesis . Defective DNA repair has been implicated in various human hereditary disorders that predispose affected individuals to cancer . This article reviews our current understanding of one of major DNA repair systems--the nucleotide excision repair pathway--with special emphasis on the novel findings that have emerged from molecular genetic analysis of yeast and cultured mammalian cells. Antonie Van Leeuwenhoek, 1990 Oct, 58(3), 137 - 46 A review of the role of 70 kDa heat shock proteins in protein translocation across membranes; Craig E et al.; The compartmentalization of essential hsp70 proteins indicates that hsp70s carry out crucial functions in several compartments of the cell . The use of conditional mutants has allowed study of the cellular processes that require hsp70 function . For efficient translocation of proteins across membranes hsp70s are required in the cytoplasm, as well as in the matrix of mitochondria and in the lumen of the endoplasmic reticulum. Proc Natl Acad Sci U S A, 1990 Oct, 87(20), 7978 - 82 The general mitochondrial matrix processing protease from rat liver: structural characterization of the catalytic subunit; Kleiber J et al.; A critical step in the import of nuclear-encoded precursor proteins into mitochondria involves proteolytic cleavage of their amino-terminal leader peptides by processing proteases found in the mitochondrial matrix . We report here the characterization of the general matrix processing protease from rat liver mitochondria . The final enzyme preparation consisted of two polypeptides, a catalytically active 55-kDa subunit and a 52-kDa one . To deduce the complete primary structure of the 55-kDa subunit, we first sequenced its mature amino terminus and several tryptic peptides derived from the pure protein . Next, using mixed oligonucleotide primers that had sequences based on two of these peptides, we synthesized a partial cDNA probe by selective amplification of liver RNA with the polymerase chain reaction . The amplified probe was then used to obtain a nearly full-length clone from a rat liver cDNA library . This cDNA codes for 508 amino acid residues, including 16 residues of an amino-terminal leader peptide, the cleavage site of which is located two polypeptide bonds downstream from an arginine residue . The mature portion has a predicted molecular mass of 55.2 kDa; it shows 36% identity with the mitochondrial processing peptidases of Saccharomyces cerevisiae and Neurospora crassa . A conserved structural feature is a putative, negatively charged alpha-helix, located in the amino-terminal half of the subunit; this element might be important for the recognition of positively charged leader peptides characteristic of mitochondrial precursor proteins. Proc Natl Acad Sci U S A, 1990 Oct, 87(19), 7541 - 5 Identification and preliminary characterization of protein-cysteine farnesyltransferase; Manne V et al.; Ras proteins must be isoprenylated at a conserved cysteine residue near the carboxyl terminus (Cys-186 in mammalian Ras p21 proteins) in order to exert their biological activity . Previous studies indicate that an intermediate in the mevalonate pathway, most likely farnesyl pyrophosphate, is the donor of this isoprenyl group . Inhibition of mevalonate synthesis reverts the abnormal phenotypes induced by the mutant RAS2Val-19 gene in Saccharomyces cerevisiae and blocks the maturation of Xenopus oocytes induced by an oncogenic Ras p21 protein of human origin . These results have raised the possibility of using inhibitors of the mevalonate pathway to block the transforming properties of ras oncogenes . Unfortunately, mevalonate is a precursor of various end products essential to mammalian cells, such as dolichols, ubiquinones, heme A, and cholesterol . In this study, we describe an enzymatic activity(ies) capable of catalyzing the farnesylation of unprocessed Ras p21 proteins in vitro at the correct (Cys-186) residue . This farnesylating activity is heat-labile, requires Mg2+ or Mn2+ ions, is linear with time and with enzyme concentration, and is present in all mammalian cell lines and tissues tested . Gel filtration analysis of a partially purified preparation of protein farnesyltransferase revealed two peaks of activity at 250-350 kDa and 80-130 kDa . Availability of an in vitro protein farnesyltransferase assay should be useful in screening for potential inhibitors of ras oncogene function that will not interfere with other aspects of the mevalonate pathway. J Exp Med, 1990 Oct 1, 172(4), 1049 - 54 The autoantigen Ku is indistinguishable from NF IV, a protein forming multimeric protein-DNA complexes; Stuiver MH et al.; We have isolated a cDNA encoding the 84-kD subunit of NFIV . Tryptic peptide sequences were identified within the coding sequences, confirming its proper identity . The primary sequence of the protein is identical to that of the large subunit of the Ku autoantigen . A missing NFIV peptide sequence was identified within the sequence of the small subunit of Ku . In addition, the proteins are identical in immunological aspects . We suggest that the Ku and NFIV proteins are identical . This connection adds new biochemical data to our knowledge of the Ku autoantigen. J Clin Invest, 1990 Oct, 86(4), 1301 - 5 Cell surface expression of the 70-kD component of Ku, a DNA-binding nuclear autoantigen; Prabhakar BS et al.; The Ku complex, a heterodimer of 86- and 70-kD proteins, is a nuclear DNA-binding autoantigen . However, hydrophobicity analysis of the deduced amino acid sequence of the 70-kD protein had strongly suggested that this might also be a membrane protein . In the present study, using antibodies to synthetic peptides and a polyclonal antiserum to the purified protein, we show that the 70-kD protein of the Ku complex is present in isolated plasma membranes of human cells . By indirect immunofluorescence microscopy and fluorescein-activated cell sorting, we demonstrate that this autoantigen is exposed on the cell surface . In addition, we have identified several domains of the protein that are exposed . Our study provides one of the first demonstrations of a eukaryotic, nuclear DNA-binding protein that is also on the cell membrane . Moreover, our results might help explain how autoantibodies to the Ku autoantigen could target cells for an autoimmune attack. EMBO J, 1990 Oct, 9(10), 3179 - 89 The recognition component of the N-end rule pathway; Bartel B et al.; The N-end rule-based degradation signal, which targets a protein for ubiquitin-dependent proteolysis, comprises a destabilizing amino-terminal residue and a specific internal lysine residue . We report the isolation and functional analysis of a gene (UBR1) for the N-end recognizing protein of the yeast Saccharomyces cerevisiae . UBR1 encodes a approximately 225 kd protein with no significant sequence similarities to other known proteins . Null ubr1 mutants are viable but are unable to degrade the substrates of the N-end rule pathway . These mutants are partially defective in sporulation and grow slightly more slowly than their wild-type counterparts . The UBR1 protein specifically binds in vitro to proteins bearing amino-terminal residues that are destabilizing according to the N-end rule, but does not bind to otherwise identical proteins bearing stabilizing amino-terminal residues. Semin Cell Biol, 1990 Oct, 1(5), 401 - 10 Molecular genetic analysis of cytoskeletal proteins in development and implications for the membrane skeleton; Wolda SL et al.; The membrane skeleton of nonerythroid cells may be involved in a variety of processes, including the formation and maintenance of specific membrane-cytoskeletal domains . Although much has been learned about the ultrastructure and protein chemistry of the membrane skeleton, there are few direct tests of the in vivo functions of the constituent proteins of the membrane skeleton . Recent advances in molecular genetic analysis provide techniques for studying the membrane skeleton and its components in vivo . Considered here in brief detail are a variety of genetic techniques that have already been used to study cytoskeletal proteins . These techniques should also prove useful for future study of the membrane skeleton. New Biol, 1990 Oct, 2(10), 915 - 21 Cell-type control of membrane biogenesis induced by HMG-CoA reductase; Wright R et al.; Quantitative increases in HMG-CoA reductase, the rate-limiting enzyme in sterol biosynthesis, induce membrane biogenesis in both yeast and mammalian cells . The subcellular organization of the resulting membrane differs in the two cell types: mammalian cells generate crystalloid endoplasmic reticulum whereas yeast cells assemble karmellae . We examined the consequences of heterologous expression of HMG-CoA reductase to distinguish features of this response that were cell-type specific from those that were isozyme-specific . This analysis demonstrated that membrane proliferation was induced in both mammalian and yeast cells by HMG-CoA reductase from either organism . However, the morphology of the induced membranes was determined by the cell type rather than the particular isozyme . Thus, both yeast and mammalian HMG-CoA reductase contained functional signals for membrane proliferation that were operational in either cell type, but the qualitative response to those signals was cell-type specific. J Gen Microbiol, 1990 Oct, 136 ( Pt 10), 1991 - 4 Restriction fragment length polymorphisms in isolates of Aspergillus fumigatus probed with part of the intergenic spacer region from the ribosomal RNA gene complex of Aspergillus nidulans; Spreadbury CL et al.; Differences in restriction fragment length polymorphisms (RFLPs) have been detected in isolates of Aspergillus fumigatus . Genomic DNA from 11 isolates was digested with EcoRI, separated by electrophoresis, Southern blotted and probed with DNA from the intergenic spacer or non-transcribed spacer region of the rRNA gene complex of Aspergillus nidulans . Three distinct RFLP patterns were detected which differed from the control patterns observed with A . nidulans, Aspergillus flavus and Aspergillus niger hybridized with the same probe . Furthermore, the differences in RFLP patterns in the A . fumigatus isolates were not detected when probed with DNA coding for the rRNA complex in Saccharomyces cerevisiae . These findings may be of use in the study of the epidemiology and pathogenesis of infections caused by A . fumigatus. New Biol, 1990 Oct, 2(10), 851 - 7 Mx proteins: antiviral proteins by chance or by necessity? Arnheiter H, Meier E. The interferon-inducible Mx1 protein is responsible for inborn resistance of mice to influenza . It is now recognized that this protein is a member of a family of interferon-inducible, putative GTP-binding proteins found in many organisms . Thus, these proteins, called the Mx proteins, are found in species that are naturally infected with influenza virus, and also in species that are not . Some Mx proteins display a broader antiviral profile than the one observed for Mx1 in mice . Others, however, may not be antiviral . Two recently discovered GTP-binding proteins, Vps1p in yeast and dynamin in rat, are also related to Mx1 . These proteins are synthesized constitutively and serve basic cellular functions. Nucleic Acids Res, 1990 Sep 25, 18(18), 5515 - 9 Autonomous replication sequences in an extrachromosomal element of a pathogenic Entamoeba histolytica; Grodberg J et al.; Entamoeba histolytica possesses a 24.5 kilobase plasmid-like molecule which encodes for the organism's ribosomal RNAs . Sequence analysis of this extrachromosomal element revealed the presence of AT rich sequences which show homology to the origin of replication of other lower eucaryotes . An 802 bp fragment containing these sequences was cloned into a yeast shuttle vector lacking the origin of replication and the construct tested for its ability to replicate autonomously in yeast . Mitotic stability tests as well as evidence for plasmid maintenance indicate that the transformed cells contained self-replicating episomes and not stably integrated molecules . The nucleotide sequence of this ARS-containing fragment is presented. Cell, 1990 Sep 21, 62(6), 1177 - 87 Distinct classes of transcriptional activating domains function by different mechanisms; Tasset D et al.; We have previously shown that the two transcriptional activation functions (TAF-1 and TAF-2) of the human estrogen receptor (hER) have synergistic properties different from one another and from those of acidic activating domains (AADs) . Here we compare the transcriptional interference/squelching properties of the hER TAFs with those of the AADs of yeast GAL4 and chimeric GAL-VP16 activators . Our results indicate that AADs interact with a factor(s) that, while required for activation by AADs, is not essential for activation by hER TAFs . In contrast, hER TAFs appear to interact with factors indispensable for mediating both their activation function and that of AADs . Thus, different classes of trans-activators may interact with different factors . In addition, the synergistic and transcriptional interference/squelching properties of the two TAFs of the human glucocorticoid receptor (hGR) indicate that both are composed of acidic and nonacidic activation functions. Nature, 1990 Sep 20, 347(6290), 291 - 4 Sequence homology shared by neurofibromatosis type-1 gene and IRA-1 and IRA-2 negative regulators of the RAS cyclic AMP pathway; Buchberg AM et al.; Neurofibromatosis type-1 (NF-1) is one of the most frequently inherited genetic disorders affecting humans . NF-1 primarily affects cells of neural crest origin and is characterized by patches of skin pigmentation (cafe-au-lait spots) and neurofibromas . Cloning of the human NF-1 gene shows that it encodes an 11-13 kilobase transcript that is frequently disrupted in NF-1 patients . The frequent disruption of the NF-1 gene in NF-1 patients combined with the autosomal dominant mode of inheritance of NF-1 strongly suggest that the NF-1 gene is a tumour-suppressor gene . We have now sequenced a portion of the murine NF-1 gene and show that the predicted amino-acid sequence is nearly the same as the corresponding region of the human NF-1 gene product . Northern blotting identified mouse NF-1 transcripts that are equivalent in size and complexity to those in human tissues, and Southern blotting shows that this region of the NF-1 gene is evolutionarily well conserved . Finally, computer searches identified homology between the mouse NF-1 gene and IRA-1 and IRA-2, two genes identified in Saccharomyces cerevisiae that negatively regulate the RAS-cyclic AMP pathway . These findings provide important new insights into the possible function of the NF-1 gene. Nature, 1990 Sep 20, 347(6290), 256 - 61 Molecular cloning of the microtubule-associated mechanochemical enzyme dynamin reveals homology with a new family of GTP-binding proteins; Obar RA et al.; A complementary DNA encoding the D100 polypeptide of rat brain dynamin--a force-producing, microtubule-activated nucleotide triphosphatase--has been cloned and sequenced . The predicted amino acid sequence includes a guanine nucleotide-binding domain that is homologous with those of a family of antiviral factors, inducible by interferon and known as Mx proteins, and with the product of the essential yeast vacuolar protein sorting gene VPS1 . These relationships imply the existence of a new family of GTPases with physiological roles that may include microtubule-based motility and protein sorting. Biochemistry, 1990 Sep 18, 29(37), 8548 - 54 Affinity of phosphatidylcholine molecular species for the bovine phosphatidylcholine and phosphatidylinositol transfer proteins . Properties of the sn-1 and sn-2 acyl binding sites; Kasurinen J et al.; Both the phosphatidylcholine transfer protein (PC-TP) and the phosphatidylinositol transfer protein (PI-TP) act as carriers of phosphatidylcholine (PC) molecules between membranes . To study the structure of the acyl binding sites of these proteins, the affinity of 32 distinct natural and related PC molecular species was determined by using a previously developed fluorometric competition assay . Marked differences in affinity between species were observed with both proteins . Affinity vs lipid hydrophobicity (determined by reverse-phase HPLC) plots displayed a well-defined maximum indicating that the acyl chain hydrophobicity is an important determinant of binding of a phospholipid molecule by these transfer proteins . However, besides the overall lipid hydrophobicity, steric properties of the individual acyl chains contribute considerably to the affinity, and PC-TP and PI-TP respond differently to modifications of the acyl chain structure . The affinity of PC-TP increased steadily with increasing unsaturation of the sn-2 acyl moiety, resulting in high affinity for species containing four and six double bonds in the sn-2 chain, whereas the affinity of PI-TP first increased up to two to three double bonds and then declined . These data, as well as the distinct effects of sn-2 chain double bond position and bromination, indicate that the sn-2 acyl chain binding sites of the two proteins are structurally quite different . The sn-1 acyl binding sites are dissimilar as well, since variation of the length of saturated sn-1 chain affected the affinity differently . The data are discussed in terms of the structural organization of the sn-1 and sn-2 acyl binding sites of PC-TP and PI-TP.(ABSTRACT TRUNCATED AT 250 WORDS) J Biol Chem, 1990 Sep 15, 265(26), 15776 - 81 Direct effects of radiation on the avidin-biotin system . Absence of energy transfer; Kempner ES et al.; Frozen solutions of biotinylated glucose-6-phosphate dehydrogenase and fluorescently tagged avidin were exposed to high energy ionizing radiation . Parallel experiments with peroxidase coupled to streptavidin and with biotinylated phycoerythrin were also performed . The loss of function of each compound was analyzed according to target theory . Target analysis revealed that the radiation-sensitive mass associated with the enzymatic activity and that associated with the fluorescence were unchanged by irradiation in the strongly coupled state . Therefore the noncovalent bonds between biotin and avidin do not permit the transfer of radiation-deposited energy in amounts sufficient to destroy the activity of apposing molecule. J Biol Chem, 1990 Sep 15, 265(26), 15750 - 7 Intragenic suppressors reveal long distance interactions between inactivating and reactivating amino acid replacements generating three-dimensional constraints in the structure of mitochondrial cytochrome b; di Rago JP et al.; Revertants of nonfunctional cytochrome b mutants were isolated and characterized to determine how specific deleterious mutations in cytochrome b can be suppressed by secondary mutations not restoring a wild type protein . It was recently shown that the cytochrome b function can be recovered following various pseudo-wild type reversions at the level of the original site mutation or adjacent positions (di Rago, J.-P., Netter, P., and Slonimski, P . P . (1990) J . Biol . Chem . 265, 3332-3339) . In the present study, we describe how the cytochrome b function can be recovered by secondary mutations in positions which are removed from the original mutation by up to more than 100 amino acids . Such revertant mutants are useful for the study of the three-dimensional structure of cytochrome b . The results of the analysis of four deficient mutations which affect a short region of the protein (positions 131-138 of the polypeptide chain) lead us to propose a possible mode of interactive combination between the first five putative transmembrane segments of cytochrome b within the membrane. Biochem Biophys Res Commun, 1990 Sep 14, 171(2), 519 - 24 The primary structure of rat ribosomal protein S13; Suzuki K et al.; The covalent structure of the rat 40S ribosomal subunit protein S13 was deduced from the sequence of nucleotides in a recombinant cDNA and confirmed from the NH2-terminal amino acid sequence of the protein . Rat S13 contains 150 amino acids (the NH2-terminal methionine is removed after translation of the mRNA) and has a molecular weight of 17,080 . Hybridization of a S13 cDNA to digests of nuclear DNA suggests that there are 8-10 copies of the gene for the protein . The mRNA for the protein is about 620 nucleotides in length . Rat S13 is related to Saccharomyces cerevisiae YS15 and to Halobacterium marismortui S11 . The protein contains a possible internal duplication of 12 residues. Gene, 1990 Sep 14, 93(2), 177 - 82 Two evolutionarily divergent genes encode a cytoplasmic ribosomal protein of Arabidopsis thaliana; Kim Y et al.; Two clones of Arabidopsis thaliana possessing high sequence identity to the yeast gene encoding ribosomal (r) protein L3 were isolated by heterologous DNA hybridization . The coding regions of these two clones have approx . 63% amino acid (aa) sequence identity to the yeast L3 r-protein and 85% aa sequence identity to each other . Both genes are expressed in shoots . The presence of two divergent genes in A . thaliana raises the possibility that the gene products participate in the formation of functionally distinct ribosomes. Eur J Biochem, 1990 Sep 11, 192(2), 499 - 506 Regulated expression and phosphorylation of the 23-26-kDa ras protein in the sponge Geodia cydonium; Robitzki A et al.; We have cloned, sequenced and examined the sponge Geodia cydonium cDNA encoding a protein homologous to ras proteins . The sponge ras protein has a more conserved N-terminal region and a less conserved C-terminal region, especially in comparison to Dictyostelium discoideum; the similarity to human c-Ha-ras-1 and to Saccharomyces cerevisiae is less pronounced . The sponge ras cDNA comprises five TAG triplets; at the translational level these UAG termination codons are suppressed by a Gln-tRNA . The sponge ras protein was isolated and partially purified (23-26 kDa) and found to undergo phosphorylation at a threonine moiety, when dissociated cells were incubated in the presence of a homologous aggregation factor and insulin . Insulin-mediated phosphorylation of the ras protein resulted in a decrease in its Kd with GTP from 2 microM to 80 nM . The activated ras protein displayed high GTPase activity if the partially purified protein was incubated with homologous lectin and lectin receptor molecules . These results suggest that in the sponge, ras is activated by the insulin/insulin(insulin-like)-receptor system . This transition enables the ras protein to interact with the lectin-receptor/lectin complex, a process which may ultimately lead to an initiation of an intracellular signal-transduction chain. Science, 1990 Sep 7, 249(4973), 1133 - 9 Enzymatic coupling of cholesterol intermediates to a mating pheromone precursor and to the ras protein; Schafer WR et al.; The post-translational processing of the yeast a-mating pheromone precursor, Ras proteins, nuclear lamins, and some subunits of trimeric G proteins requires a set of complex modifications at their carboxyl termini . This processing includes three steps: prenylation of a cysteine residue, proteolytic processing, and carboxymethylation . In the yeast Saccharomyces cerevisiae, the product of the DPR1-RAM1 gene participates in this type of processing . Through the use of an in vitro assay with peptide substrates modeled after a presumptive a-mating pheromone precursor, it was discovered that mutations in DPR1-RAM1 cause a defect in the prenylation reaction . It was further shown that DPR1-RAM1 encodes an essential and limiting component of a protein prenyltransferase . These studies also implied a fixed order of the three processing steps shared by prenylated proteins: prenylation, proteolysis, then carboxymethylation . Because the yeast protein prenyltransferase could also prenylate human H-ras p21 precursor, the human DPR1-RAM1 analogue may be a useful target for anticancer chemotherapy. Cell, 1990 Sep 7, 62(5), 927 - 37 Meiotic gene conversion and crossing over: their relationship to each other and to chromosome synapsis and segregation; Engebrecht J et al.; The yeast mer1 mutant produces inviable spores and is defective in both meiotic recombination and chromosome pairing . A gene called MER2 partially suppresses the mer1 phenotype when present in high copy number . Both gene conversion and chromosome pairing are completely restored in mer1 strains overexpressing MER2; however, reciprocal crossing over and spore viability are not restored . The data presented are consistent with a model in which chromosome pairing is a direct consequence of a homology search mediated through gene conversion . Analysis of random viable spores indicates that the crossovers that occur in mer1 strains overexpressing MER2 are more effective in ensuring meiosis I disjunction than those that occur in mer1 strains . One interpretation of this result is that only those crossovers that occur in the context of the synaptonemal complex